The contribution of refractoriness to arrhythmic substrate in hypokalemic Langendorff-perfused murine hearts The clinical effects of hypokalemia including action potential prolongation and arrhythmogenicity suppressible by lidocaine were reproduced in hypokalemic -LRB- 3.0 mM K + -RRB- Langendorff-perfused murine hearts before and after exposure to lidocaine -LRB- 10 μM -RRB- . Novel limiting criteria for local and transmural , epicardial , and endocardial re-excitation involving action potential duration -LRB- at 90 % repolarization , APD90 -RRB- , ventricular effective refractory period -LRB- VERP -RRB- , and transmural conduction time -LRB- Δlatency -RRB- , where appropriate , were applied to normokalemic -LRB- 5.2 mM K + -RRB- and hypokalemic hearts . Hypokalemia increased epicardial APD90 from 46.6 ± 1.2 to 53.1 ± 0.7 ms yet decreased epicardial VERP from 41 ± 4 to 29 ± 1 ms , left endocardial APD90 unchanged -LRB- 58.2 ± 3.7 to 56.9 ± 4.0 ms -RRB- yet decreased endocardial VERP from 48 ± 4 to 29 ± 2 ms , and left Δlatency unchanged -LRB- 1.6 ± 1.4 to 1.1 ± 1.1 ms ; eight normokalemic and five hypokalemic hearts -RRB- . These findings precisely matched computational predictions based on previous reports of altered ion channel gating and membrane hyperpolarization . Hypokalemia thus shifted all re-excitation criteria in the positive direction . In contrast , hypokalemia spared epicardial APD90 -LRB- 54.8 ± 2.7 to 60.6 ± 2.7 ms -RRB- , epicardial VERP -LRB- 84 ± 5 to 81 ± 7 ms -RRB- , endocardial APD90 -LRB- 56.6 ± 4.2 to 63.7 ± 6.4 ms -RRB- , endocardial VERP -LRB- 80 ± 2 to 84 ± 4 ms -RRB- , and Δlatency -LRB- 12.5 ± 6.2 to 7.6 ± 3.4 ms ; five hearts in each case -RRB- in lidocaine-treated hearts . Exposure to lidocaine thus consistently shifted all re-excitation criteria in the negative direction , again precisely agreeing with the arrhythmogenic findings . In contrast , established analyses invoking transmural dispersion of repolarization failed to account for any of these findings . We thus establish novel , more general , criteria predictive of arrhythmogenicity that may be particularly useful where APD90 might diverge sharply from VERP . Introduction Hypokalemia exerts important clinical effects on cardiac function that in some respects resemble those seen in the congenital long-QT syndromes -LRB- LQTS -RRB- . Thus , both conditions result in electrocardiographic QT prolongation -LSB- 12 , 23 -RSB- and premature ventricular depolarizations -LRB- PVDs -RRB- , which may result in the initiation of an arrhythmic activity -LSB- 41 , 52 -RSB- . In contrast to the cardiac effects of hypokalemia , arrhythmic activity in LQTS has been extensively studied and has often been attributed to after-depolarizations occurring against a background of re-entrant substrate -LSB- 2 , 36 , 44 -RSB- . Re-entry may take place as a result of inhomogeneities producing regions of conduction block , which lead to wave-break and circus movement -LSB- 21 , 37 -RSB- or altered repolarization gradients , which lead to wave reflection -LSB- 1 -RSB- . In this situation , depolarization propagates from active cells into previously active adjacent regions , establishing re-entrant circuits . These may become established either locally or over larger regions of the myocardium , such as across the thickness of the myocardial wall . Tendencies to transmural re-entrant excitation in models of LQTS have been previously analyzed in terms of transmural dispersions of repolarization -LRB- TDR -RRB- obtained from the positive part of the difference between respective endocardial and epicardial stimulation to repolarization times -LSB- 36 , 44 , 45 -RSB- . In human LQTS , increases in the interval between the peak and full recovery of electrocardiographic precordial T waves -LRB- Tpeak to Tend -RRB- , previously shown to reflect TDR -LSB- 54 -RSB- , are indeed associated with arrhythmic activity -LSB- 33 -RSB- . Certainly , recent reports correlate Tpeak to Tend to arrhythmic risk more closely than more widely accepted indicators such as corrected QT interval and QT dispersion -LSB- 53 -RSB- . However , such re-excitation may also be limited by recovery from refractoriness ; re-entrant excitation would require this to precede the return of the membrane potential to threshold -LSB- 40 -RSB- . Certainly , class 1 antiarrhythmic drugs such as lidocaine are known to increase ventricular effective refractory period -LRB- VERP -RRB- -LSB- 28 -RSB- . Yet , such use of spatial differences in action potential repolarization times to quantify arrhythmic substrate neither explicitly considers changes in VERP nor applies such criteria to potential local as opposed to transmural re-excitation . This paper associates for the first time the proarrhythmic effect of hypokalemia with a significant decrease in VERP , despite contrasting prolongation of action potentials , in agreement with computer-modeling studies of action potential waveforms using established data on the various effects of hypokalemia on ionic conductivity properties of ventricular myocytes . Furthermore , it associates the antiarrhythmic effects of lidocaine with a significant increase in VERP , despite having little effect on action potential duration , in agreement with clinical observations . Analyses using TDR were insufficiently sensitive to account for any of these arrhythmogenic findings . This study accordingly established more general novel criteria that would provide necessary conditions for local and transmural and epicardial and endocardial re-excitation incorporating not only action potential duration but also VERP and conduction times that may be particularly useful when action potential duration differs sharply from VERP . These criteria successfully accounted for all the arrhythmogenic findings . Materials and methods Experimental animals Mice were housed in an animal facility at 21 ± 1 °C with 12 h light/dark cycles . Animals were fed sterile chow -LRB- RM3 Maintenance Diet , SDS , Witham , Essex , UK -RRB- and had free access to water . Wild-type 129 Sv mice aged 3 -- 6 months were used in the experiments . All procedures complied with UK Home Office regulations -LRB- Animals -LSB- Scientific Procedures -RSB- Act 1986 -RRB- . Solutions All solutions were based on bicarbonate-buffered Krebs-Henseleit solution -LRB- mM : NaCl 119 , NaHCO3 25 , KCl 4 , KH2PO4 1.2 , MgCl2 1 , CaCl2 1.8 , glucose 10 and Na-pyruvate 2 ; pH adjusted to 7.4 -RRB- bubbled with 95 % O2/5 % CO2 -LRB- British Oxygen Company , Manchester , UK -RRB- . Hypokalemic -LRB- 3.0 mM K + -RRB- solutions were prepared by reducing the quantity of KCl added . Lidocaine-containing normokalemic and hypokalemic solutions were prepared by adding lidocaine -LRB- Sigma -- Aldrich , Poole , UK -RRB- to a final concentration of 10 μM . Preparation A Langendorff-perfusion protocol previously adapted for murine hearts -LSB- 4 -RSB- was used . In brief , mice were killed by cervical dislocation -LRB- Schedule 1 : UK Animals -LSB- Scientific Procedures -RSB- Act 1986 -RRB- , and hearts were then quickly excised and placed in ice-cold bicarbonate-buffered Krebs-Henseleit solution . A short section of aorta was cannulated under the surface of the solution and attached to a custom-made 21-gauge cannula filled with the same solution using an aneurysm clip -LRB- Harvard Apparatus , Edenbridge , Kent , UK -RRB- . Fresh Krebs-Henseleit solution was then passed through 200 and 5 μm filters -LRB- Millipore , Watford , UK -RRB- and warmed to 37 °C using a water jacket and circulator -LRB- Techne model C-85A , Cambridge , UK -RRB- before being used for constant-flow retrograde perfusion at 2 -- 2.5 ml/min using a peristaltic pump -LRB- Watson-Marlow Bredel model 505S , Falmouth , Cornwall , UK -RRB- . Hearts were regarded as suitable for experimentation if , on rewarming , they regained a healthy pink colour and began to contract spontaneously . Electrophysiological measurements An epicardial monophasic action potential -LRB- MAP -RRB- electrode -LRB- Hugo Sachs , Harvard Apparatus -RRB- was placed against the basal region of the left ventricular epicardium . In addition , a small access window was created in the interventricular septum to allow access to the left ventricular endocardium -LSB- 9 -RSB- . A custom-made endocardial MAP electrode composing two twisted strands of high-purity Teflon-coated 0.25 mm diameter silver wire -LRB- Advent Research Materials , UK -RRB- was constructed . The Teflon coat was removed from the distal 1 mm of the electrode , which was then galvanically chlorided to eliminate DC offset , inserted and placed against the septal endocardial surface . MAPs were amplified , band-pass filtered -LRB- 0.5 Hz to 1 kHz : Gould 2400S , Gould-Nicolet Technologies , Ilford , Essex , UK -RRB- , and digitized at a sampling frequency of 5 kHz -LRB- micro1401 , Cambridge Electronic Design , Cambridge , UK -RRB- . Analysis of MAPs was performed using Spike II software -LRB- Cambridge Electronic Design -RRB- . Experimental protocol A bipolar platinum stimulating electrode -LRB- 1 mm interpole spacing -RRB- was placed on the basal surface of the right ventricular epicardium . Square-wave stimuli -LRB- Grass S48 stimulator , Grass-Telefactor , Slough , UK -RRB- of 2 ms duration and with amplitudes of twice the excitation threshold were initially applied to hearts at a constant cycle length of 125 ms for at least 10 min and until MAPs showed stable baselines , rapid upstroke phases that reached consistent amplitudes and smooth repolarization phases -LSB- 30 -RSB- . Hearts were then exposed to test solutions for 20 min , during which time stimulation was continued , before subsequent recordings were made . Intrinsically evoked MAPs were recorded in the absence of stimulation while action potential duration -LRB- at 90 % repolarization , APD90 -RRB- and stimulation to depolarization latency were determined during regular stimulation at a constant interstimulus interval of 125 ms. Hearts were then subjected to an adapted form of an extrasystolic electrical stimulation procedure previously used to assess arrhythmogenicity and refractoriness in both human -LSB- 43 -RSB- and murine -LSB- 22 -RSB- studies of congenital LQTS , described in detail later . The possibility that events evoked by extrasystolic stimuli rather represented motion artifacts was excluded by their being reproducible between hearts and appearing identical in both electrodes . All data are presented as means ± standard errors of the means and include both the number of repetitions and the number of hearts . Comparisons were made using analysis of variance -LRB- significance threshold set at P ≤ 0.05 -RRB- . Modeling The charge-difference model of Fraser and Huang -LSB- 16 , 18 -RSB- was adapted to permit computational modeling of the murine ventricular cardiac myocyte using ion channel equations and parameters from the model of Bondarenko et al. -LSB- 6 -RSB- with the Na + / K + - ATPase model of Hernandez et al. -LSB- 24 -RSB- . The use of charge-difference modeling allowed the model to reach a true beat-to-beat steady state that was independent of initial intracellular ion concentrations -LSB- 17 -RSB- , thus permitting simulation of the influence of changes in extracellular ion concentrations that are well recognized to influence Na + / K + - ATPase activity , and hence steady-state intracellular ion concentrations . Model cells were studied under normokalemic -LRB- 5.2 mM K + -RRB- conditions with normal ion permeabilities , hypokalemic -LRB- 3 mM K + -RRB- conditions with these same ion permeabilities and hypokalemic -LRB- 3 mM K + -RRB- conditions with the K + permeabilities of channels carrying the repolarizing currents IK1 and Ito reduced by 20 % , replicating the effect of such hypokalemia on transmembrane K + permeabilities observed experimentally by Killeen et al. -LSB- 29 -RSB- . Stimulation was applied at a regular 125 ms interstimulus interval at an amplitude of twice the diastolic threshold , as for the experimental preparations . After beat-to-beat stability was achieved , APD90 was measured under each condition . Refractory periods were then determined using a similar protocol to that used in the experiments , every eighth -LRB- S1 -RRB- stimulus being followed by an extrasystolic -LRB- S2 -RRB- stimulus . S1S2 interval was initially 70 ms and was subsequently decremented by 1 ms with each successive cycle until an S2 stimulus failed to initiate an action potential . Results After-depolarizations initiate arrhythmic activity in bradycardic hypokalemic hearts In initial experiments , isolated perfused hearts were stimulated at a constant interstimulus interval of 125 ms for 20 min after 20 min exposure to test solutions . This demonstrated stable trains of MAPs , under all normokalemic -LRB- 5.2 mM K + , n = 7 ; five hearts -RRB- , hypokalemic -LRB- 3.0 mM K + , n = 8 ; five hearts -RRB- , or lidocaine-treated -LRB- 10 μM -RRB- normokalemic -LRB- n = 6 ; five hearts -RRB- or hypokalemic -LRB- n = 7 ; five hearts -RRB- conditions : After-depolarizations and arrhythmic activity were consistently absent throughout . The subsequent experiments then examined arrhythmic properties at the longer cycle lengths -LRB- between 224 and 271 ms -RRB- that occurred in the absence of extrinsic stimulation -LRB- Fig. 1 -RRB- and that have previously been reported to be proarrhythmic both under hypokalemic conditions and in the congenital LQTS -LSB- 11 -RSB- . Intrinsic cycle length did not differ significantly -LRB- P > 0.05 -RRB- between normokalemic -LRB- 250 ± 21 ms , six hearts -RRB- , hypokalemic -LRB- 253 ± 16 ms , seven hearts -RRB- , lidocaine-treated normokalemic -LRB- 248 ± 22 ms , six hearts -RRB- , and lidocaine-treated hypokalemic -LRB- 260 ± 10 ms , eight hearts -RRB- hearts . Fig. 1After-depolarizations and arrhythmic activity in spontaneously contracting hypokalemic hearts . Epicardial monophasic action potential recordings in the absence of extrinsic stimulation in hearts exposed to normokalemic -LRB- 5.2 mM K + , a -RRB- and hypokalemic -LRB- 3.0 mM K + , b -RRB- test solutions and normokalemic -LRB- c -RRB- and hypokalemic -LRB- d -RRB- test solutions containing lidocaine -LRB- 10 μM -RRB- for 20 min Epicardial MAPs then retained morphologically consistent waveforms and were entirely free of after-depolarization and arrhythmic phenomena through 116 min of recordings over six normokalemic hearts -LRB- Fig. 1a -RRB- . In contrast , 46 ± 7 % of MAPs showed after-depolarizations early in their repolarization phases during 140 min of recordings that led to episodes of arrhythmic activity in 52 ± 3 % of cases in five out of seven hypokalemic hearts -LRB- P < 0.01 as compared to normokalemic hearts , Fig. 1b -RRB- . However , MAPs showed consistent waveforms without such after-depolarizations or arrhythmic activity during 118 min of recordings over six lidocaine-treated normokalemic hearts -LRB- Fig. 1c -RRB- . Finally , 40 ± 9 % of MAPs showed after-depolarizations occurring late in the repolarization phase in 40 ± 9 % of cases more than 98 min of recordings in six out of eight lidocaine-treated hypokalemic hearts -LRB- P > 0.05 as compared to hypokalemic hearts -RRB- . After-depolarizations occurred more frequently in those instances where intrinsic cycle length was long . However , these events were never followed by arrhythmic activity -LRB- P < 0.01 as compared to hypokalemic hearts , Fig. 1d -RRB- . Extrasystolic stimulation immediately after recovery from refractoriness initiates arrhythmic activity in hypokalemic hearts A programmed electrical stimulation protocol recently shown to predict arrhythmogenicity in clinical LQTS -LSB- 43 -RSB- and previously adapted for use in murine models of LQTS -LSB- 22 -RSB- confirmed the above arrhythmogenic tendencies in hypokalemic hearts -LRB- Fig. 2 -RRB- . This comprised regular -LRB- S1 -RRB- stimulation at a constant interstimulus interval of 125 ms interrupted by an extrasystolic -LRB- S2 -RRB- stimulus after every eighth S1 stimulus . The S1S2 interval was decremented in 1 ms steps with each successive stimulus cycle from an initial value of 120 ms until the S2 stimulus either appeared to initiate arrhythmic activity , confirmed during an imposed 250 ms pause , or failed to initiate a MAP suggesting that the VERP has been reached . Accordingly , VERP values are reported to the nearest millisecond . Fig. 2Arrhythmic activity in hypokalemic hearts after extrasystolic stimulation applied close to the refractory period . Epicardial monophasic action potential recordings resulting from application of extrasystolic -LRB- S2 -RRB- stimuli at S1S2 intervals greater than the action potential duration at 90 % repolarization -LRB- A -RRB- , just greater than the ventricular effective refractory period -LRB- VERP ; B -RRB- , and just less than the VERP -LRB- C -RRB- in hearts exposed to normokalemic -LRB- 5.2 mM K + , a -RRB- and hypokalemic -LRB- 3.0 mM K + , b -RRB- test solutions and normokalemic -LRB- c -RRB- and hypokalemic -LRB- d -RRB- test solutions containing lidocaine -LRB- 10 μM -RRB- for 20 min . Single vertical lines indicate the timing of S1 stimuli , and double lines indicate the timing of S2 stimuli Normokalemic hearts -LRB- Fig. 2a , A -- C -RRB- again were consistently free from arrhythmic activity after S2 stimulation after any S1S2 interval -LRB- n = 11 ; eight hearts -RRB- . This also applied to hypokalemic hearts when S2 stimuli were delivered when MAPs had reached 90 % repolarization -LRB- Fig. 2b , A , n = 7 ; five hearts -RRB- . However , S2 stimuli delivered within the period just after recovery from refractoriness consistently initiated arrhythmic activity under these conditions -LRB- Fig. 2b , B -RRB- . In contrast , S2 stimuli delivered before recovery from refractoriness failed to elicit MAPs , and this was followed by the resumption of stable rhythms -LRB- Fig. 2b , C -RRB- . Finally , S2 stimulation did not result in arrhythmic activity in lidocaine-treated hearts whatever the S1S2 interval , whether under normokalemic -LRB- Fig. 2c , A -- C , n = 8 ; six hearts -RRB- or hypokalemic -LRB- Fig. 2d , A -- C , n = 8 ; five hearts -RRB- conditions . When taken together , the presence or absence of arrhythmogenicity in these experiments parallels clinical findings . Arrhythmic tendency in hypokalemia correlates with increased local critical intervals One hypothesis for the tendency towards either local or transmural re-excitation during action potential repolarization might consider the relationship between the time course of the recovery of membrane voltage and the corresponding time course of recovery of excitability from total refractoriness to a finite threshold for excitation in the myocardial regions concerned . These parameters were approximated by action potential duration at 90 % repolarization -LRB- APD90 -RRB- and VERP , respectively . Both these were measured during the procedures of the kind illustrated in Fig. 2 , allowing for the delay between endocardial and epicardial excitation where appropriate . Firstly , the risk of local reexcitation of either the epicardium or the endocardium would be reflected in a critical interval given by the relevant APD90 -- VERP . Secondly , the risk of transmural re-excitation of either the epicardium by the endocardium -LRB- or the reverse -RRB- would require incorporation of the delay between endocardial and epicardial excitation given by the difference between endocardial and epicardial stimulation to depolarization latencies , Δlatency . This would give critical intervals of -LRB- endocardial APD90 + Δlatency − epicardial VERP -RRB- and -LRB- epicardial APD90 + Δlatency − endocardial VERP -RRB- , respectively . Figures 3a and 4a show typical epicardial and endocardial action potential waveforms during regular stimulation under each of the four above conditions -LRB- A -- D -RRB- . Figures 3b and 4b show the corresponding APD90s -LRB- vertical solid lines and dense hashing -RRB- , VERPs -LRB- vertical broken lines and sparse hashing -RRB- , and local critical intervals -LRB- shading -RRB- . Asterisks indicate values that are significantly -LRB- P < 0.05 -RRB- larger and daggers those that are smaller than those recorded in normokalemic hearts . Neither epicardial -LRB- 46.6 ± 1.2 ms -RRB- nor endocardial -LRB- 58.2 ± 3.7 ms -RRB- APD90s were significantly different -LRB- P > 0.05 -RRB- from the corresponding VERPs -LRB- 41 ± 4 and 48 ± 4 ms -RRB- under normokalemic conditions -LRB- Figs. 3A and 4A , n = 10 ; eight hearts -RRB- . This resulted in local critical intervals taking small positive values of 5.4 ± 4.3 ms in the epicardium and 9.8 ± 5.3 ms in the endocardium . In contrast , epicardial -LRB- 53.1 ± 0.7 ms -RRB- but not endocardial -LRB- 56.9 ± 4.0 ms -RRB- APD90 increased significantly -LRB- P < 0.05 -RRB- , whereas both epicardial -LRB- 29 ± 1 ms -RRB- and endocardial -LRB- 29 ± 2 ms -RRB- VERPs decreased significantly under hypokalemic conditions -LRB- Figs. 3B and 4B , n = 6 ; five hearts -RRB- . This resulted in significant positive shifts -LRB- P < 0.01 -RRB- in both epicardial -LRB- 23.7 ± 1.2 ms -RRB- and endocardial -LRB- 28.5 ± 4.6 ms -RRB- local critical intervals , in fitting with the occurrence of arrhythmic activity under hypokalemic conditions . Fig. 3Changes in epicardial action potential duration , ventricular effective refractory period , and local critical interval after exposure to hypokalemia and to lidocaine . Epicardial MAP waveforms during regular stimulation in hearts exposed to normokalemic -LRB- 5.2 mM K + , A -RRB- and hypokalemic -LRB- 3.0 mM K + , B -RRB- test solutions and normokalemic -LRB- C -RRB- and hypokalemic -LRB- D -RRB- test solutions containing lidocaine -LRB- 10 μM -RRB- for 20 min comparing action potential duration at 90 % repolarization ; APD90 -LRB- vertical solid lines -RRB- , VERP -LRB- vertical broken lines -RRB- , and local critical interval -LRB- shading ; a -RRB- . Action potential duration at 90 % repolarization , APD90 -LRB- dense hashing -RRB- , VERP -LRB- sparse hashing -RRB- and critical interval -LRB- shading -RRB- under these conditions -LRB- b -RRB- . Asterisks indicate values that are significantly -LRB- P < 0.05 -RRB- larger and daggers those that are smaller than those recorded in normokalemic heartsFig . 4Changes in endocardial action potential duration , ventricular effective refractory period , and local critical interval after exposure to hypokalemia and to lidocaine . Endocardial MAP morphologies during regular stimulation in hearts exposed to normokalemic -LRB- 5.2 mM K + , A -RRB- and hypokalemic -LRB- 3.0 mM K + , B -RRB- test solutions and normokalemic -LRB- C -RRB- and hypokalemic -LRB- D -RRB- test solutions containing lidocaine -LRB- 10 μM -RRB- for 20 min comparing action potential duration at 90 % repolarizationl ; APD90 -LRB- vertical solid lines -RRB- , VERP -LRB- vertical broken lines -RRB- , and local critical interval -LRB- shading ; a -RRB- . Action potential duration at 90 % repolarization , APD90 -LRB- dense hashing -RRB- , VERP -LRB- sparse hashing -RRB- , and critical interval -LRB- shading -RRB- under these conditions -LRB- b -RRB- . Asterisks indicate values that are significantly -LRB- P < 0.05 -RRB- larger and daggers those that are smaller than those recorded in normokalemic hearts The opposing effects of hypokalemia on APD90 and VERP can be explained in terms of alterations in conductances of repolarizing K + - channels The above MAP findings concerning APD90 and VERP were in close agreement with the predictions of established ion channel equations and parameters from the model of Bondarenko et al. -LSB- 6 -RSB- with the Na + / K + - ATPase model of Hernandez et al. -LSB- 24 -RSB- using charge-difference modeling in simulated single cells . The model simulated the effects of regular stimulation at a 125 ms interstimulus interval until a steady state was reached , as reflected in beat-to-beat stability . Action potential characteristics were then simulated under normokalemic conditions , hypokalemic conditions with normal K + permeabilities , and hypokalemic condition with the 20 % reduction in the permeabilities of channels carrying the repolarizing K + currents IK1 and Ito -LRB- C -RRB- as reported in recent experimental results -LSB- 29 -RSB- -LRB- Fig. 5 -RRB- . Figure 5a demonstrates the predicted steady-state action potential waveforms under each condition . Figure 5a and b also show APD90 -LRB- vertical solid lines and dense hashing -RRB- , VERP -LRB- vertical broken lines and sparse hashing -RRB- , and critical intervals -LRB- shading -RRB- . Under normokalemic conditions -LRB- A -RRB- , the resting membrane potential was − 83 mV . APD90 -LRB- 22 ms -RRB- was shorter than recorded in the whole hearts in keeping with previous results from microelectrode studies -LSB- 7 , 20 -RSB- . Nevertheless , APD90 was shorter than VERP -LRB- 27 ms -RRB- resulting in a critical interval of − 5 ms. Figures 5B -LRB- a and b -RRB- demonstrate the consequences of an altered Nernst potential for K + alone both upon resting membrane potential and the time course of a subsequent action potential . Hypokalemia hyperpolarized the membrane potential -LRB- − 93 mV -RRB- and shortened both APD90 -LRB- 19 ms -RRB- and VERP -LRB- 25 ms -RRB- , thus having little effect on the limiting criterion for re-excitation -LRB- − 6 ms -RRB- . In contrast , Fig. 5C -LRB- a and b -RRB- additionally demonstrate the combined effects of hypokalemia on both the Nernst potential and K + permeabilities of the respective channels carrying IK1 and Ito . Although there was no additional effect on the resting membrane potential -LRB- − 93 mV -RRB- , hypokalemia increased both APD90 -LRB- 29 ms -RRB- and VERP -LRB- 27 ms -RRB- causing a positive shift in critical interval from − 5 ms to +2 ms -LRB- Fig. 5C -RRB- . Fig. 5Computational modeling of murine ventricular action potentials showing changes in action potential duration , ventricular effective refractory period , and critical interval after exposure to hypokalemia . Action potential morphologies during regular stimulation in cells under normokalemic -LRB- 5.2 mM K + , A -RRB- and hypokalemic -LRB- 3.0 mM K + -RRB- conditions comparing action potential duration at 90 % repolarization ; APD90 -LRB- vertical solid lines -RRB- , VERP -LRB- vertical broken lines -RRB- , and critical interval -LRB- shading ; a -RRB- . In B , permeabilities of ion channel are under normokalemic conditions . In C , permeabilities of ion channels carrying the repolarizing K + currents IK1 and Ito are reduced by 20 % . APD90 -LRB- dense hashing -RRB- , VERP -LRB- sparse hashing -RRB- , and critical interval -LRB- shading -RRB- under these conditions -LRB- b -RRB- The abolition of arrhythmic tendency by lidocaine correlates with negative shifts in local critical intervals Exposure of normokalemic hearts to lidocaine -LRB- Figs. 3 , C and 4C -RRB- significantly increased -LRB- P < 0.05 -RRB- epicardial -LRB- 54.8 ± 2.7 ms -RRB- , although not endocardial -LRB- 56.6 ± 4.2 ms -RRB- , APD90 and significantly increased -LRB- P < 0.01 -RRB- both epicardial -LRB- 84 ± 5 ms -RRB- and endocardial -LRB- 80 ± 2 ms -RRB- VERPs -LRB- n = 5 ; five hearts -RRB- . This resulted in significant -LRB- P < 0.01 -RRB- negative shifts in local critical intervals in both the epicardium -LRB- − 31.7 ± 5.3 ms -RRB- and endocardium -LRB- − 23.4 ± 4.7 ms -RRB- . Lidocaine exerted concordant effects on hypokalemic hearts -LRB- Figs. 3D and 4D -RRB- : Epicardial APD90 increased to 60.6 ± 2.7 ms , and endocardial APD90 remained unchanged -LRB- 63.7 ± 6.4 ms -RRB- , whereas both epicardial -LRB- 81 ± 7 ms -RRB- and endocardial -LRB- 84 ± 4 ms -RRB- VERPs were significantly increased -LRB- P < 0.05 , n = 6 ; five hearts -RRB- . The resulting significant negative shifts -LRB- P < 0.01 -RRB- in local critical intervals in both epicardium -LRB- − 20.4 ± 7.5 ms -RRB- and endocardium -LRB- − 20.7 ± 7.6 ms -RRB- paralleled the antiarrhythmic effect of lidocaine . Arrhythmic tendency in hypokalemia also correlates with increased transmural critical intervals Figures 6a and 7a show epicardial and endocardial action potential waveforms during regular stimulation under each condition . Figures 6b and 7b show APD90 -LRB- vertical solid lines and dense hashing -RRB- and VERPs -LRB- vertical dotted lines and sparse hashing -RRB- , as detailed above , together with Δlatencies -LRB- horizontal arrows and horizontal shading -RRB- and transmural critical intervals -LRB- shading -RRB- under the four conditions studied . Asterisks indicate values that are significantly -LRB- P < 0.05 -RRB- larger and daggers those that are smaller than recorded in normokalemic hearts . Figure 6 thus compares endocardial APD90s with epicardial VERPs , allowing for Δlatency to describe critical intervals for epicardial re-excitation . In contrast , Fig. 7 compares epicardial APD90s with endocardial VERPs , allowing for Δlatency to describe critical intervals for endocardial re-excitation . Fig. 6Changes in endocardial action potential duration , epicardial ventricular effective refractory period , transmural conduction time , and epicardial transmural critical interval after exposure to hypokalemia and to lidocaine . Epicardial and endocardial MAP morphologies during regular stimulation cross-comparing epicardial and endocardial waveforms and the relationship between the action potential duration at 90 % repolarization ; APD90 -LRB- vertical solid lines -RRB- and VERP -LRB- vertical broken lines -RRB- of one waveform and the decay of the other , indicating critical intervals -LRB- shading -RRB- . Hearts were exposed to normokalemic -LRB- 5.2 mM K + , A -RRB- and hypokalemic -LRB- 3.0 mM K + , B -RRB- test solutions and normokalemic -LRB- C -RRB- and hypokalemic -LRB- D -RRB- test solutions containing lidocaine -LRB- 10 μM -RRB- for 20 min . Horizontal arrows indicate the time taken for depolarization to spread from epicardium to endocardium -LRB- a -RRB- . APD90 -LRB- dense hashing -RRB- , VERP -LRB- sparse hashing -RRB- , transmural conduction time -LRB- horizontal hashing -RRB- , and critical interval -LRB- shading -RRB- under these conditions -LRB- b -RRB- . Asterisks indicate values that are significantly -LRB- P < 0.05 -RRB- larger , and daggers those that are smaller than those recorded in normokalemic heartsFig . 7Changes in epicardial action potential duration , endocardial ventricular effective refractory period , transmural conduction time , and endocardial transmural critical interval after exposure to hypokalemia and to lidocaine . Epicardial and endocardial MAP morphologies during regular stimulation cross-comparing epicardial and endocardial waveforms and the relationship between the action potential duration at 90 % repolarization ; APD90 -LRB- vertical solid lines -RRB- and VERP -LRB- vertical broken lines -RRB- of one waveform and the decay of the other , indicating critical intervals -LRB- shading -RRB- . Hearts were exposed to normokalemic -LRB- 5.2 mM K + , A -RRB- and hypokalemic -LRB- 3.0 mM K + , B -RRB- test solutions and normokalemic -LRB- C -RRB- and hypokalaemic -LRB- D -RRB- test solutions containing lidocaine -LRB- 10 μM -RRB- for 20 min . Horizontal arrows indicate the time taken for depolarization to spread from epicardium to endocardium -LRB- a -RRB- . APD90 -LRB- dense hashing -RRB- , VERP -LRB- sparse hashing -RRB- , transmural conduction time -LRB- horizontal hashing -RRB- , and critical interval -LRB- shading -RRB- under these conditions -LRB- b -RRB- . Asterisks indicate values that are significantly -LRB- P < 0.05 -RRB- larger and daggers those that are smaller than those recorded in normokalemic hearts Epicardial -LRB- 21.0 ± 0.82 ms -RRB- and endocardial -LRB- 58.2 ± 3.7 ms -RRB- latencies were statistically indistinguishable in normokalemic hearts giving a Δlatency of 1.6 ± 1.4 ms. This contributed to significant -LRB- P < 0.05 -RRB- negative shifts in transmural critical intervals in both the epicardium and endocardium -LRB- − 14.3 ± 6.1 and − 3.3 ± 4.2 ms , respectively , Figs. 6A and 7A -RRB- . Hypokalemia had no significant effect -LRB- P > 0.05 -RRB- on latencies or on Δlatency -LRB- 1.1 ± 1.1 ms -RRB- but resulted in significant -LRB- P > 0.01 -RRB- positive shifts in transmural critical intervals in both the epicardium -LRB- 23.6 ± 2.6 ms -RRB- and endocardium -LRB- 29.2 ± 6.0 ms ; Figs. 6B and 7B -RRB- . The abolition of arrhythmic tendency by lidocaine also correlates with significantly decreased transmural critical intervals In contrast , exposure of normokalemic hearts to lidocaine significantly increased both epicardial and endocardial latencies -LRB- 33.8 ± 2.6 and 46.3 ± 5.6 ms , respectively -RRB- but still did not significantly alter -LRB- P > 0.05 -RRB- Δlatency -LRB- 12.5 ± 6.2 ms -RRB- . However , transmural critical intervals became significantly negative in both the epicardium -LRB- − 15.2 ± 6.6 ms -RRB- and endocardium -LRB- − 16.6 ± 8.8 ms ; Figs. 6C and 7C -RRB- . This was also true when hypokalemic hearts were exposed to lidocaine : Both epicardial and endocardial latencies were significantly increased -LRB- 26.3 ± 3.2 and 33.8 ± 1.3 ms , respectively -RRB- but Δlatency was not significantly altered -LRB- P > 0.05 , 7.6 ± 3.4 ms -RRB- . Again , transmural critical intervals were significantly decreased -LRB- P < 0.05 -RRB- in both the epicardium -LRB- − 12.8 ± 5.9 ms -RRB- and endocardium -LRB- − 10.4 ± 1.7 ms ; Figs. 6D and 7D -RRB- . Arrhythmogenesis occurs in the absence of significant alterations in the TDR The analysis above thus established four critical intervals , which , when taken together , provided a clear prediction of arrhythmogenicity under these circumstances when changes in APD90 did not correspond to changes in VERP . In contrast , an analysis in terms of TDR , previously shown to predict arrhythmogenicity in both congenital and acquired forms of the LQTS -LSB- 2 -RSB- , gave insufficiently sensitive predictions . TDR was calculated as the time between stimulation and 90 % repolarization in the endocardium minus the time between stimulation and 90 % repolarization in the epicardium . TDR values did not significantly alter with the presence or otherwise of arrhythmogenicity -LRB- Fig. 8 -RRB- . Thus , in normokalemic hearts -LRB- Fig. 8 , A -RRB- epicardial repolarization time -LRB- 67.6 ± 1.5 ms -RRB- was significantly shorter -LRB- P < 0.05 -RRB- than endocardial repolarization time -LRB- 77.6 ± 3.9 ms -RRB- , giving a TDR of 10.0 ± 4.2 ms -LRB- n = 10 ; eight hearts -RRB- . Epicardial and endocardial repolarization times , as well as the resulting TDR , remained unchanged under hypokalemic conditions -LRB- Fig. 8 , B -RRB- . Finally , although treatment with lidocaine significantly increased -LRB- P < 0.05 -RRB- epicardial and endocardial repolarization times under both normokalemic -LRB- Fig. 8 , C , to 86.1 ± 2.8 and 103.0 ± 7.0 ms , respectively , n = 6 ; five hearts -RRB- and hypokalemic -LRB- Fig. 8 , D , to 86.9 ± 4.1 and 97.6 ± 6.6 ms , respectively , n = 5 ; five hearts -RRB- conditions , in neither case did it affect TDR . Fig. 8Changes in transmural dispersion of repolarization after exposure to hypokalemia and to lidocaine . Epicardial -LRB- up-sloping hashing -RRB- and endocardial -LRB- down-sloping hashing -RRB- stimulation to repolarization times , and the difference between these values giving transmural dispersion of repolarization -LRB- open bars -RRB- in hearts exposed to normokalemic -LRB- 5.2 mM K + , A -RRB- and hypokalemic -LRB- 3.0 mM K + , B -RRB- test solutions and normokalemic -LRB- C -RRB- and hypokalemic -LRB- D -RRB- test solutions containing lidocaine -LRB- 10 μM -RRB- for 20 min . Asterisks indicate values that are significantly -LRB- P < 0.05 -RRB- larger than those recorded in normokalemic hearts These findings indicate that despite similarities , criteria that have been established to predict arrhythmogenicity in LQTS do not necessarily apply in hypokalemia . However , explicit inclusion of refractory behavior yields novel criteria , which may constitute more sensitive general predictors of arrhythmogenicity and may prove particularly useful in situations where APD90 diverges sharply from VERP . Further , these novel criteria provide a physiological basis for the participation of refractoriness in arrhythmogenicity . Discussion In clinical situations , hypokalemia is associated with arrhythmogenesis initiated by PVDs -LSB- 52 -RSB- and accompanied by prolongation of the electrocadiographic QT interval , reflecting increased action potential duration -LSB- 23 -RSB- . Furthermore , class 1 antiarrhythmic agents are effective in suppressing arrhythmic activity in hypokalemic patients -LSB- 42 -RSB- . These features thus resemble corresponding characteristics of the congenital LQTS -LSB- 12 , 41 -RSB- , where arrhythmic activity is thought to result from re-entrant excitation -LSB- 2 -RSB- . This has been attributed to the propagation of depolarization from active cells to previously active adjacent regions subsequently triggering spread of excitation and thus establishing re-entrant circuits -LSB- 1 , 3 , 37 -RSB- . We sought to study the physiological basis for the arrhythmogenicity observed under hypokalemic conditions , particularly the extent to which this resembles or differs from the corresponding features of LQTS , in intact isolated perfused murine hearts . Epicardial and endocardial MAPs were first recorded from hypokalemic hearts , confirming that stimulation at a regular interstimulus interval of 125 ms -LRB- S1 stimulation -RRB- resulted in stable rhythms . In contrast , at the long intrinsic cycle lengths occurring in the absence of extrinsic stimulation , frequent after-depolarizations were observed and were often followed by the initiation of an arrhythmic activity . This is consistent with the known proarrhythmic effect of bradycardia -LSB- 10 , 13 , 46 , 51 -RSB- . Previous studies correlating results from single-cell and whole-heart preparations have attributed such after-depolarizations to inward currents flowing through reactivated voltage-operated Ca2 + channels -LSB- 27 , 35 -RSB- , Ca2 + - coupled inward Na + currents via the Na + -- Ca2 + exchanger -LSB- 47 -RSB- , or Ca2 + - induced Ca2 + - release from intracellular stores -LSB- 26 -RSB- . In the presence of the class 1b antiarrhythmic agent lidocaine , after-depolarizations persisted . These events were especially common in hearts where intrinsic cycle length was particularly long , again in agreement with the known proarrhythmic effect of bradycardia -LSB- 10 , 13 , 46 , 51 -RSB- . However , in the presence of lidocaine , these events were never followed by the initiation of arrhythmic activity . Thus , the experiments demonstrated that hypokalemic murine hearts showed arrhythmogenic properties in agreement with clinical findings and established conditions in which arrhythmogenicity was and was not observed . A quantitative assessment of MAP waveforms and refractory characteristics associated with this arrhythmogenicity was then performed using an extrasystolic stimulation -LRB- S2 -RRB- procedure previously established in the assessment of arrhythmogenicity in both clinical -LSB- 43 -RSB- and murine -LSB- 22 -RSB- studies of LQTS . S2 stimulation reproducing the effect of after-depolarizations immediately after recovery from refractoriness failed to initiate arrhythmic activity in normokalemic hearts . However , such stimulation consistently resulted in arrhythmic activity in hypokalemic hearts . This is in agreement with previous reports that after-depolarizations and S2 stimulation early during action potential repolarization are particularly arrhythmogenic -LSB- 14 , 38 -RSB- and also parallels clinical observations that PVDs coincident with T-waves frequently initiate arrhythmic activity -LSB- 48 -RSB- . In contrast , S2 stimuli did not elicit arrhythmic activity in lidocaine-treated hearts , whether studied under normokalemic or hypokalemic conditions . These are consistent with after-depolarizations having occurred late in action potential repolarization , and thence , failing to initiate arrhythmic activity . Previous studies in murine -LSB- 30 -RSB- , canine -LSB- 15 -RSB- , and human -LSB- 31 -RSB- ventricles have consistently reported that maneuvers , which alter action potential duration , also produce corresponding changes in refractory period , with the notable exception of exposure to class 1 antiarrhythmic drugs -LSB- 39 -RSB- . However , the effect of isolated reduction in -LSB- K + -RSB- o on these parameters has not been studied . Exploration of the effect of varying S1S2 interval demonstrated for the first time that although action potential duration -LRB- quantified at 90 % repolarization , APD90 -RRB- was increased in hypokalemia , VERP was decreased . Furthermore , exposure to lidocaine had no effect on APD90 in the epicardia or endocardia of normokalemic hearts and significantly increased APD90 only in the endocardia of hypokalemic hearts , despite significantly increasing VERP in all cases . Thus , the proarrhythmic effect of hypokalemia was associated with recovery from refractoriness occurring earlier in action potential repolarization , whereas the antiarrhythmic effect of lidocaine was associated with recovery from refractoriness occurring later in action potential repolarization . Reduction of -LSB- K + -RSB- o might be expected to increase outward K + currents and thereby decrease APD90 . Indeed , computer modeling of single ventricular myocytes confirmed that reduction of -LSB- K + -RSB- o per se resulted in decreased APD90 . Furthermore , the Nernst equation would predict that reduction of -LSB- K + -RSB- o should hyperpolarize the resting membrane potential , thereby increasing the proportion of sodium channels available for activation -LSB- 19 -RSB- and decreasing the VERP : our model replicated this effect . However , incorporation of recent data from our group demonstrating that reduction of -LSB- K + -RSB- o decreases the repolarizing K + currents IK1 and Ito -LSB- 29 -RSB- resulted in VERP returning to its normokalemic value and APD90 being increased beyond its normokalemic value . Thus , although reduction in K + permeability compensates for the change in VERP , it overcompensates for the change in APD90 , rendering the APD90 longer than the VERP , in fitting with experimental results and with the proarrhythmic effect of hypokalemia . Previous studies have reported that although exposure to lidocaine increases VERP through an effect on the gating of fast Na + - channels -LSB- 32 , 34 -RSB- , it has a proportionately smaller effect on action potential duration -LSB- 5 -RSB- . Exposure to lidocaine is thus established to result in postrepolarization refractoriness -LSB- 39 -RSB- , in fitting with experimental results and with the antiarrhythmic effect of lidocaine . We then applied an analytical scheme to provide a simple physiological explanation for these findings . Subject to electrotonic coupling between cells -LSB- 25 -RSB- , the simplest condition for local re-excitation between adjacent cells within either epicardium or endocardium would require membrane potential to exceed threshold at some point during action potential repolarization , the recovery of membrane potential lagging behind the recovery of excitability . Recovery of excitability was approximated by the VERP , measured using a standard stimulus of consistent amplitude and duration . Recovery of membrane potential was approximated by the action potential duration at 90 % repolarization -LRB- APD90 -RRB- . Accordingly , APD90 − VERP gives a critical interval that reflects tendency towards local re-excitation and arrhythmogenicity . Positive shifts in this interval would reflect a relatively proarrhythmic state , whereas negative shifts in this interval would reflect an antiarrhythmic state . The corresponding analytical condition for transmural re-excitation across the thickness of the myocardial wall with a transmural conduction time given by Δlatency -LSB- 40 -RSB- yields transmural critical intervals of -LRB- endocardial APD90 + Δlatency − epicardial VERP -RRB- for the epicardium and -LRB- epicardial APD90 + Δlatency − endocardial VERP -RRB- for the endocardium , again subject to electrotonic spread of current between cells -LSB- 25 -RSB- . Although exposure to lidocaine increased both epicardial and endocardial stimulation to depolarization latencies , attributable to its established effect on conduction velocity -LSB- 8 -RSB- , it had no significant effect on Δlatency . Hypokalemia resulted in significant positive shifts in the magnitude of all four critical intervals , whereas exposure to lidocaine resulted in significant negative shifts , in precise agreement with the presence or absence of arrhythmogenicity . Modelling of single ventricular myocytes predicted shorter APD90s than were recorded from whole-heart preparations , in agreement with previous experimental observations in such single-cell preparations -LSB- 7 , 20 , 30 -RSB- . Nevertheless , hypokalemia when similarly modeled for such single-cell preparations resulted in a positive shift in critical interval , in common with our experimental observations from whole hearts . Although changes in all four critical intervals correlated with arrhythmogenicity , TDR , previously shown to predict arrhythmogenicity in LQTS -LSB- 36 , 44 , 45 -RSB- , proved an insufficiently sensitive predictor . This finding may be attributable to sharp differences between APD90 and VERP : it is possible that APD90 and VERP were in close agreement in previous studies on LQTS -LSB- 49 , 50 -RSB- . In the latter event , the present analysis would have yielded identical results to one adopting TDR . Thus , we establish for the first time that hypokalemia decreases VERP despite increasing APD90 and attribute this surprising finding to effects of reduced -LSB- K + -RSB- o on ion channel gating . Secondly , we establish novel indices incorporating VERP as general criteria for re-entrant arrhythmogenicity that additionally provide a physiological basis for the association between changes in epicardial and endocardial VERP and APD90 and susceptibility to arrhythmogenesis . Thirdly , we establish that such analyses provide more sensitive indications of arrhythmogenicity than previous analyses invoking TDR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I I O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

refractory period
conduction time
action potential duration
transmural dispersion of repolarization
critical intervals


Calcium-dependent release of adenosine and uridine nucleotides from A549 cells Extracellular nucleotides play an important role in lung defense , but the release mechanism and relative abundance of different nucleotide species secreted by lung epithelia are not well defined . In this study , to minimize cell surface hydrolysis , we used a low-volume , flow-through chamber and examined adenosine and uridine nucleotide concentrations in perfusate aliquots of human lung A549 cells challenged by 50 % hypotonic shock . Adenosine triphosphate -LRB- ATP -RRB- , adenosine diphosphate -LRB- ADP -RRB- , adenosine monophosphate -LRB- AMP -RRB- , and adenosine -LRB- Ado -RRB- were quantified in high-performance liquid chromatography -LRB- HPLC -RRB- analysis of fluorescent etheno derivatives , and uridine triphosphate -LRB- UTP -RRB- and uridine diphosphate -LRB- UDP -RRB- were measured using HPLC-coupled radioenzymatic assays . After the onset of hypotonic shock , ATP , ADP , UTP , and UDP in the perfusates increased markedly and peaked at approximately 2.5 min , followed by a gradual decay in the next 15 -- 20 min ; peak changes in Ado and AMP were relatively minor . The peak concentrations and fold increment -LRB- in parentheses -RRB- were : 34 ± 13 nM ATP -LRB- 5.6 -RRB- , 11 ± 5 nM ADP -LRB- 3.7 -RRB- , 3.3 ± 1.2 nM AMP -LRB- 1.4 -RRB- , 23 ± 7 nM Ado -LRB- 2.1 -RRB- , 21 nM UTP -LRB- > 7 -RRB- , and 11 nM UDP -LRB- 27 -RRB- . Nucleotide release was almost completely abolished from cells loaded with the calcium chelator 1,2-bis -LRB- 2-aminophenoxy -RRB- ethane-N , N , N ' , N ' - tetraacetic acid -LRB- BAPTA -RRB- . Under isotonic conditions , elevation of intracellular calcium with the calcium ionophore ionomycin -LRB- 5 μM , 3 min -RRB- also released nucleotides with kinetics and relative abundance as above , albeit less robust . ADP : ATP -LRB- 1:3 -RRB- and UDP : UTP -LRB- 1:2 -RRB- ratios in perfusates from stimulated cells were markedly higher than the cytosolic ratios of these species , suggesting that a nucleotide diphosphate -LRB- NDP -RRB- - rich compartment , e.g. , the secretory pathway , contributed to nucleotide release . Laser confocal microscopy experiments illustrated increased FM1-43 uptake into the plasma membrane upon hypotonic shock or ionomycin treatment , consistent with enhanced vesicular exocytosis under these conditions . In summary , our results strongly suggest that calcium-dependent exocytosis is responsible , at least in most part , for adenosine and uridine nucleotide release from A549 cells . Introduction Extracellular nucleotides control a diverse range of physiological processes by interacting with a large group of cell-surface P2Y and P2X receptor families -LSB- 1 -- 3 -RSB- . In the lungs , extracellular nucleotides regulate airway surface liquid homeostasis and mucociliary clearance by modulating epithelial ion and fluid transport as well as ciliary beating -LSB- 4 -RSB- . In alveoli , adenosine triphosphate -LRB- ATP -RRB- is a potent secretagogue that stimulates type II cell-surfactant secretion . Nucleotide levels on airway surfaces , measured in vitro and ex vivo , show dynamic changes due to the combination of basal and stimulated release and their rapid metabolism by several groups of membrane-associated and membrane-secreted soluble ectoenzymes . These ectoenzymes extend the signaling potential of ATP by converting it to adenosine -LRB- Ado -RRB- , a ligand of the A1 -- 3 family of G-protein-coupled receptors -LSB- 1 , 4 -- 7 -RSB- . Nucleotide release is stimulated by cell mechanical perturbations , such as shear stress , membrane stretch , medium change , hyposmotic swelling , and hypoxia -LSB- 1 , 8 -RSB- . It is now recognized that mechanosensitive ATP release occurs from healthy cells via physiological processes , which do not involve cell damage . Numerous reports have suggested that the cystic fibrosis transmembrane conductance regulator -LRB- CFTR -RRB- and other members of the superfamily of ATP-binding cassette-transport proteins serve as a conductive pathway for ATP release or regulate an associated ATP channel . However , other groups using patch-clamp , lipid-bilayer and luminometry techniques have not found any detectable CFTR-mediated or CFTR-regulated ATP release -LRB- reviewed in -LSB- 1 -RSB- -RRB- . Volume-regulated anion channels -LRB- VRAC -RRB- and voltage-dependent anion channels -LRB- VDAC , porins or maxi-Cl − channels -RRB- are known to have considerable permeability to cations and large organic anions , and several laboratories have implicated these channels in cell-swelling-induced ATP release and the autocrine regulation of cell volume . However , recent studies have demonstrated that conductive pathways , including VRAC , VDAC , and stretch-activated channels , are not involved in cell-swelling-induced ATP release from A549 cells -LSB- 1 , 9 , 10 -RSB- . Finally , connexin hemichannels also have been proposed to mediate ATP release , but their role in this process in a physiological setting is not always clear -LSB- 1 -RSB- . Whereas conductive release mechanisms are still being debated , exocytotic ATP release is well established in excitatory cells , blood platelets , and chromaffin cells . Furthermore , there is growing experimental evidence supporting such a release mechanism also by epithelial and other nonexcitatory cells . In particular , we recently demonstrated that cell-swelling-induced ATP release from A549 and 16HBE14o − epithelial cells and 3T3 fibroblasts is tightly correlated with intracellular Ca2 + elevations ; it is abolished in cells loaded with the calcium -LRB- Ca2 + -RRB- chelator 1,2-bis -LRB- 2-aminophenoxy -RRB- ethane-N , N , N ' , N ' - tetraacetic acid -LRB- BAPTA -RRB- or by low temperature , strongly suggesting the involvement of Ca2 + - dependent exocytosis -LSB- 10 -RSB- . Whether release of other nucleotides from epithelial cells also involves Ca2 + - dependent exocytosis remains incompletely explored . Such studies , however , are complicated by nucleotide hydrolysis at the airway surface , which makes it difficult to assess the magnitude and relative abundance of different nucleotide species released . Therefore , we used a flow-through chamber to minimize cell surface hydrolysis . We found that hypotonic shock markedly increased ATP , adenosine diphosphate -LRB- ADP -RRB- , uridine triphosphate -LRB- UTP -RRB- , and uridine diphosphate -LRB- UDP -RRB- concentrations in perfusates , which peaked at approximately 2.5 min . Nucleotide release was almost completely abolished from cells loaded with BAPTA and , under isotonic conditions , could be evoked by elevation of intracellular calcium with the calcium ionophore ionomycin . High nucleotide diphosphate -LRB- NDP -RRB- concentrations in perfusates of stimulated cells suggested that an NDP-rich compartment , e.g. , the secretory pathway , contributed to this release . Together with real-time FM1-43 fluorescence experiments , our results strongly indicate that calcium-dependent exocytosis is a major mechanism of adenosine and uridine nucleotide release from A549 cells . Materials and methods Cells Human lung carcinoma A549 cells were grown in Dulbecco 's modified eagles medium -LRB- DMEM -RRB- supplemented with 10 % fetal bovine serum -LRB- FBS -RRB- , 2 mM L-glutamine , 56 U/ml penicillin-G and 56 μg/ml streptomycin sulfate . All constituents of the culture media were from GIBCO-BRL -LRB- Burlington , ON , Canada -RRB- . ATP efflux was measured from cell monolayers grown to confluency on 24 × 60-mm glass coverslips . Fura-2 calcium imaging and FM1-43 microscopy experiments were performed on cells grown on circular 15-mm diameter no. 1 glass coverslips . Nucleotide efflux assay To measure nucleotide efflux during hypotonic challenge with high temporal resolution , we used a custom-designed , low-volume -LRB- 300 μl -RRB- , flow-through chamber , as described previously -LSB- 10 -RSB- . Briefly , 24 × 60-mm glass coverslips with confluent cell monolayers of ∼ 500 cells/mm2 were mounted in the chamber and perfused with warm -LRB- 37 °C / in-line heater ; Warner Instrument , Hamden , CT , USA -RRB- Ringer solution at the rate of 1.3 ml/min . The Ringer solution contained -LRB- in mM -RRB- : 110.5 NaCl , 24 NaHCO3 , 1.3 KCl , 1 MgCl2 , 1 CaCl2 , 2.5 Na2HPO4 , 2.5 KH2PO4 , 1.2 K2HPO4 , and 10 glucose ; pH 7.4 was maintained by bubbling with 5 % CO2 . After an equilibration period in isotonic solution -LRB- 5 -- 15 min -RRB- , a 50 % hypotonic solution was applied , and the perfusate was continuously collected for 30-s intervals during the initial burst of ATP secretion -LRB- 0 -- 5 min -RRB- and during 1 min elsewhere . Nucleotide concentrations in the samples were evaluated by high-performance liquid chromatography -LRB- HPLC0 analysis , as described below . For studies in absence of extracellular calcium , CaCl2 was omitted , and the solutions were supplemented with 0.1 mM ethyleneglycoltetraacetic acid -LRB- EGTA -RRB- . The 50 % hypotonic solution was prepared by appropriate reduction of salt concentration , whereas divalent cation concentrations were kept constant . HPLC quantification of adenine and uridine nucleotides UTP concentrations were quantified by UDP-glucose pyrophosphorylase-based reaction -LSB- 11 -RSB- . Briefly , 100-μl samples were incubated in the presence of 0.5 U/ml UDPglucose pyrophosphorylase , 0.5 U/ml inorganic pyrophosphatase , 1.6 mM CaCl2 , 2 mM MgCl2 , 25 mM hydroxyethylpiperazine ethanesulfonic acid -LRB- HEPES -RRB- -LRB- pH 7.4 -RRB- , and ∼ 100,000 cpm 1 μM -LSB- 14C -RSB- glucose-1P . Incubations lasted 1 h at 30 °C . Reactions were terminated by heating the samples at 95 °C for 2 min . Conversion of -LSB- 14C -RSB- glucose-1P to -LSB- 14C -RSB- UTP was determined by HPLC -LRB- Shimadzu -RRB- via a Nova Pack C18 column and ion-pairing mobile phase . Radioactivity was measured on-line with a Packard Flo-One detector . Assay sensitivity was 1 pmol -LRB- 3 nM in 100-μl samples -RRB- . UDP was quantitatively phosphorylated in the presence of -LSB- γ32P -RSB- ATP using nucleoside diphosphokinase -LRB- NDPK -RRB- . Briefly , 100-μl samples containing 0.1 U/ml NDPK , 0.1 μCi 60 nM -LSB- γ32P -RSB- ATP , 1.6 mM CaCl2 , 2 mM MgCl2 , and 25 mM HEPES -LRB- pH 7.4 -RRB- were incubated for 5 min at 30 °C . Reactions were terminated by heating the samples at 95 °C for 2 min . The resulting conversion of -LSB- γ32P -RSB- ATP to -LSB- γ32P -RSB- UTP was monitored by HPLC -LSB- 12 -RSB- . A calibration curve employing known amounts of UDP was chartered in parallel during each assay . This assay allows UDP quantification with sensitivity of 20 fmol -LRB- 0.2 nM in 100-μl samples -RRB- . For etheno -LRB- ɛ -RRB- derivatization , samples -LRB- 200 μl -RRB- were derivatized for 30 min at 72 °C in the presence of 1.0 M chloroacetaldehyde and 25 mM Na2HPO4 -LRB- pH 4.0 -RRB- . The resulting fluorescent 1,N6-ethenoadenine derivatives were analyzed by HLPC -LRB- Waters -RRB- in a Hamilton PRP - × 100 anion exchange column , as described previously -LSB- 4 -RSB- . ɛ-ATP , ɛ-ADP , ɛ-AMP , and ɛ-Ado were quantified with a sensitivity of 200 fmol -LRB- 1 nM in 200-μl samples -RRB- . FM1-43 studies Bulk exocytosis was quantified in A549 cells as changes in the fluorescence intensity of FM1-43 incorporated into the plasma membrane -LSB- 13 , 14 -RSB- and recorded by real-time confocal microscopy . Cells were washed with Hank 's balanced salt solution + 20 mM HEPES + 2 mM MgCl2 and 1.6 mM CaCl2 -LRB- HBSSH -RRB- or with HBSSH devoid of calcium -LRB- HBSSH 0 Ca -RRB- . They were mounted onto the stage of a Leica SP2 AOBS confocal microscope equipped with HCX Apo L63x NA 0.9 immersion Leica lens and a 488-nm Argon laser . Experiments were initiated by incubation of the cells with 3 μM FM1-43 . Cells were incubated for at least 10 min , and hypotonic shock was applied by decreasing salt concentration 33 % while maintaining calcium , magnesium , and FM1-43 concentrations constant . Alternatively , cells incubated with FM1-43 in HBSSH or HBSSH 0 Ca were stimulated with 5 - to 10-μM ionomycin . Real-time recording was performed by laser scanning in thexz axes with a galvostage , initially every 10 s and then every 30 s for the time periods indicated in the figures . Overall fluorescence intensity changes associated with the plasma membrane were estimated by measuring the intensity value associated with each pixel through time . The entire apical membrane compartment displayed in a confocal plane and five random regions of basolateral and subapical domains were analyzed , normalized to basal values -LRB- time = 0 -RRB- , and averaged for each region . Cell swelling was estimated as a change of cell height in thexz plane at different time points , normalized to basal values , and averaged . Fura-2 calcium measurements To load Fura-2 , cells were incubated -LRB- 1 h , 37 °C , 5 % CO2 -RRB- in physiological solution containing 25 μM Fura-2-AM + 0.02 % Pluronic F127 and 2.5 mM probenecid . This was followed by 30 min deesterification period in physiological solution containing probenecid . The physiological saline solution consisted of -LRB- in mM -RRB- : 140 NaCl , 5 KCl , 1 MgCl2 , 1 CaCl2 , 10 glucose and 10 HEPES , pH 7.4 , adjusted with sodium hydroxide -LRB- NaOH -RRB- . Fifty percent hypotonic medium was prepared by reducing the salt concentration while keeping divalent cation concentration constant . For calcium imaging , coverslips with Fura-2-loaded cells were mounted in the imaging/perfusion chamber attached to the heated platform -LRB- Warner Instruments -RRB- on the stage of an inverted microscope -LRB- Nikon TE300 -RRB- . The cells were exposed to alternate -LRB- 200 ms -RRB- illumination at 340 nm and 380 nm with a high-pressure mercury lamp -LRB- 100 W -RRB- via interference filters -LRB- Chroma Technology , Brattleboro , VT , USA -RRB- mounted on a filter wheel -LRB- Sutter Lambda 10-C , Sutter Instrument , Novato , CA , USA -RRB- and a dichroic mirror -LRB- 510/540 nm , Chroma Technology -RRB- . Fluorescence images were recorded at 15 - to 60-s intervals with the digital camera and stored for later analysis . Chemicals For calcium-imaging experiments , Fura-2-AM was obtained from Molecular Probes , Invitrogen Corp. -LRB- Burlington , ON , Canada -RRB- . Probenicid , Pluronic F127 and all other reagents were from Sigma Aldrich -LRB- Oakville , ON , Canada -RRB- . Results Kinetics of nucleotide release Figure 1 shows an example of the time course of nucleotide release induced by 50 % hypotonic shock . For clarity , the release of adenine and uridine nucleotides appears on separate graphs : a and b , respectively . The kinetics of release were remarkably similar for all nucleotides . After the onset of hypotonic shock , nucleotide concentration increased rapidly , peaking at ∼ 2.5 min , followed by a gradual decay in the next 10 -- 15 min . The average peak values from several separate experiments are shown in Fig. 1c . Interestingly , ATP was the major species at the peak of stimulated release , whereas for basal release , Ado was the predominant species . The rank order of nucleotide abundance at the peak was : ATP > Ado ≥ UTP > ADP ≈ UDP > AMP . Relative increases of nucleotide conconcentrations at the peak were also the highest for nucleotide triphosphates -LRB- NTPs -RRB- and nucleotide diphosphates -LRB- NDPs -RRB- -LRB- ATP 5.6-fold , ADP 3.7-fold , UTP > 7-fold , UDP 27-fold -RRB- , whereas the increase was smaller for AMP and Ado -LRB- 1.4-fold and 2.1-fold , respectively -RRB- . Fig. 1Transient nucleotide release from A549 cells induced by 50 % hypotonic shock . Time course of adenosine -LRB- a -RRB- and uridine -LRB- b -RRB- nucleotide release observed in response to 50 % hypotonic shock . A representative experiment is shown out of four performed under the same conditions . Hypotonic shock was applied at t = 0 min and was preceded by 15-min equilibration in isotonic solution . Basal -LRB- t = 0 min -RRB- and peak -LRB- t = 2.5 min -RRB- nucleotide concentrations detected in perfusates -LRB- c -RRB- . Average values -LSB- ± standard deviation -LRB- SD -RRB- -RSB- are from three to four experiments , such as in a and b , except for uridine triphosphate -LRB- UTP -RRB- and uridine diphosphate -LRB- UDP -RRB- , which are from a single experiment . * Due to the limited sensitivity of UTP evaluations -LRB- ≈ 3 nM -RRB- , the basal level of UTP was found to be below the detection limit and was not shown Role of intracellular calcium -LRB- -LSB- Ca +2 -RSB- i -RRB- To investigate the role of intracellular calcium -LRB- -LSB- Ca +2 -RSB- i -RRB- in adenine nucleotide release , we tested the effect of the Ca2 + ionophore ionomycin . In the absence of hypotonic shock , exposure to 5 μM ionomycin for 3 min induced transient nucleotide release from A549 cells -LRB- Fig. 2a -RRB- . This release reached a peak at approximately 1.5 min , i.e. , slightly earlier and at somewhat lesser absolute peak amplitude compared with that induced by 50 % hypotonic shock . Otherwise , the kinetics and relative nucleotide abundance were similar for both stimuli . Almost complete inhibition of swelling-induced adenine nucleotide release was observed for A549 cells loaded with the Ca2 + chelator BAPTA , compared with control untreated cells of the same batch tested in parallel experiments -LRB- Fig. 2b , c , respectively -RRB- . Fura-2 fluorescence -LSB- Ca +2 -RSB- i measurements confirmed a dramatic reduction of the -LSB- Ca +2 -RSB- i response to 50 % hypotonic shock in BAPTA-loaded cells -LRB- Fig. 2d -RRB- . These experiments demonstrate that elevation of -LSB- Ca +2 -RSB- i was required to trigger adenine nucleotide release from A549 epithelial cells . Fig. 2Effect of intracellular calcium -LRB- -LSB- Ca2 + i -RSB- -RRB- modulators on nucleotide release . -LRB- a -RRB- . In the absence of hypotonic shock , application of 5 μM ionomycin resulted in transient nucleotide release with kinetics similar to that induced by hypotonic shock . An example , out of two similar experiments , is shown . Loading A549 cells with the Ca2 + chelator 1,2-bis -LRB- 2-aminophenoxy -RRB- ethane-N , N , N ' , N ' - tetraacetic acid -LRB- BAPTA -RRB- almost completely inhibited hypotonic-stress-induced nucleotide release compared with controls that were run in parallel -LRB- b and c , respectively -RRB- -LRB- n = 2 -RRB- . Effect of BAPTA on the -LSB- Ca2 + -RSB- i response -LRB- d -RRB- . The two traces represent changes of the Fura-2 fluorescence ratio at two excitation wavelengths , λ340/λ380 , in response to 50 % hypotonic shock , applied at t = 0 min , in control and BAPTA-loaded cells . Note that the rapid peak of the -LSB- Ca2 + -RSB- i response was abolished in BAPTA-loaded cells . Similar responses were observed in four experiments FM1-43 fluorescence changes implicate vesicular exocytosis Strong calcium dependence of nucleotide release may indicate the involvement of vesicular exocytosis . To further explore this possibility , bulk exocytosis was examined by real-time confocal microscopy in cells bathed in FM1-43 . Fluorescence intensity associated with the plasma membrane increased rapidly by incorporation of the soluble probe into the membrane and remained almost constant after 5 min in nonstimulated cells . Plasma-membrane-associated fluorescence intensity rose rapidly -LRB- ∼ 20 s -RRB- with hypotonic shock stimulation . This change was accompanied by increased cell volume seen as increment of cell height -LRB- Fig. 3a , b -RRB- . Cell volume peaked at about 3 min , followed by a regulatory volume decrease in the next 10 -- 15 min -LRB- data not shown -RRB- . A similar plasma-membrane-associated FM1-43 fluorescence intensity increase was observed in the absence of hypotonic shock when the cells were stimulated with ionomycin in the presence but not in the absence of calcium in the bathing solution -LRB- Fig. 4 -RRB- . These data indicate that both hypotonic-shock - and ionomycin-stimulated bulk exocytosis in A549 cells . Fig. 3Effect of hypotonic shock on FM1-43 surface membrane staining . Confocal FM1-43 fluorescence images of confluent A549 cells after 20 min of dye loading in isotonic solution , followed by 50 % hypotonic shock stimulation -LRB- a -RRB- . The images depict the xz scans of the cell monolayer at different time points . Note the increase in cell size and fluorescence intensity consistent with hypotonic-shock-induced cell swelling and heightened bulk exocytosis . Time course of FM1-43 plasma membrane fluorescence and cell height changes during hypotonic shock -LRB- b -RRB- Fig. 4Effect of ionomycin on FM1-43 surface-membrane staining . Confocal FM1-43 fluorescence images of confluent A549 cells prior to and after 4-min exposure to 5 μM ionomycin in the presence of extracellular calcium . -LRB- a -RRB- . The images show the xz scans of the cell monolayer at different time points . Note the increase in fluorescence intensity after ionomycin stimulation , indicating enhanced exocytosis . Time course of FM1-43 plasma membrane fluorescence changes of unstimulated cells -LRB- Δ CON -RRB- and cells during ionomycin treatment in the presence -LRB- ▪ ION -RRB- or absence -LRB- • ION 0 Ca -RRB- of extracellular calcium -LRB- b -RRB- Discussion In this study , we used the flow-through chamber and an etheno derivatization measurement technique to examine the kinetics of nucleotide release from A549 lung epithelial cells induced by hypotonic shock . We demonstrated that ATP , ADP , AMP , Ado , UTP , and UDP appeared in the perfusates with the same kinetics , peaking at ∼ 2.5 min -LRB- Fig. 1 -RRB- , which coincided with the peak of -LSB- Ca +2 -RSB- i elevation evoked by hypotonic shock -LRB- Fig. 2d -RRB- . Similar tight temporal correlation between the -LSB- Ca +2 -RSB- i signal and ATP release , measured by luciferase-luciferin luminescence assay , was reported previously for A549 , 16HBE14o − epithelial cells and NIH3T3 fibroblasts -LSB- 10 -RSB- . Such a close temporal association suggests that hypotonic-shock-induced elevation of -LSB- Ca +2 -RSB- i is a trigger for the release of nucleotides . This was supported by the strong inhibitory effect of BAPTA-AM and the induction of nucleotide release in the absence of hypotonic shock solely by the rise of -LSB- Ca +2 -RSB- i with ionomycin . These data demonstrate tight Ca2 + dependence of nucleotide release , and point to a Ca2 + - dependent exocytotic mechanism . It should be noted that the bulk of -LSB- Ca +2 -RSB- i changes shown in Fig. 2d likely represent superposition of spatially separated , distinct -LSB- Ca +2 -RSB- i responses , one evoked directly by hypotonic shock and the other due to autocrine/paracrine actions of the released nucleotides on P2Y receptors . The latter effects might be minimized , at least partially , by continuous perfusion , which reduced nucleotide concentration in the bulk of the chamber far below the IC50 of P2Y receptor activation ; however , this possibility requires further study . Consistent with a Ca2 + - dependent exocytotic mechanism , FM1-43 fluorescence studies revealed enhanced incorporation of the probe into the plasma membrane induced by hypotonic shock or ionomycin treatment . Increased plasma membrane-associated FM1-43 fluorescence intensity was a measure of the cumulative amount of membrane added by exocytosis -LSB- 13 -RSB- . FM1-43 fluorescence increased rapidly during the first 20 s of hypotonic shock -LRB- Fig. 3 -RRB- . Interestingly , the initial fluorescence increment exceeded the cell-surface increase estimated from xz confocal scans . In our previous study using a dual-image 3D cell reconstruction technique -LSB- 15 -RSB- , we observed only an 11 -- 30 % surface increase of single substrate-attached A549 cells swollen in 50 % hypotonic solution -LSB- 10 -RSB- . This difference could be attributed in part to different methods of cell-height and surface evaluation in that study , as well as differences in swelling responses of single isolated cells compared with confluent cell monolayer . However , a similar discrepancy between FM1-43 fluorescence , cell-surface and membrane electrical capacitance has been also reported by others , e.g. , with pituitary lactotrophs , where the dense granules docked into the plasma membrane were intensely stained by FM1-43 in addition to the membrane added to the cell surface -LSB- 16 -RSB- . Therefore , the divergence of fluorescence and the cell-surface changes seen in our study with A549 cells may also result in part from FM1-43 staining of the lipophilic content of fused vesicles in addition to the staining of fused vesicular membranes . The FM1-43 dye is often employed to fluorescently marked surfactant-containing lamellar bodies in alveolar type II -LRB- ATII -RRB- cells -LSB- 17 , 18 -RSB- and A549 cells , a model of ATII cells , also contain granules enriched in surface-active phospholipids -LSB- 19 -RSB- . However , the exact contribution of such granules to increased FM1-43 fluorescence in stimulated A549 cells was out of the scope of the present investigation and will require future direct study . Vesicle exocytosis may be mechanistically associated with nucleotide release in two ways : nucleotides can be delivered to the extracellular medium as cargo molecules within exocytotic vesicles or , alternatively , secreted from the cytosol via vesicle-associated nucleotide-conducting channels or transporters transiently expressed at the plasma membrane as a consequence of vesicle-plasma membrane fusion . Analysis of the relative abundance of different nucleotide species that appear in the extracellular medium may help to distinguish between these two mechanisms . ATP and UDP sugars accumulate in the lumen of the secretory pathway up to 20 - to 50-fold above their cytosolic concentrations , and luminal utilization of these molecules generates ADP and UDP . UDP is the major uridine nucleotide detected in ER/Golgi fractions -LSB- 20 -RSB- and , eventually , a UDP-selective apyrase -LRB- UDPase -RRB- converts UDP to urodine monophosphate -LRB- UMP -RRB- -LSB- 21 -RSB- . ER/Golgi ADP and UMP are exchanged for cytosolic ATP and UDP glucose via specific transporters -LSB- 22 -- 25 -RSB- . As intraluminal nucleotides are not subject to the mechanisms that retrieve resident ER/Golgi proteins , they are predicted to remain within trafficking vesicles and to be released to the extracellular space from the secretory pathway . Our results suggest that this mechanism likely accounted for UDP release from A549 cells . Figure 1c illustrates that hypotonic stimulation promoted a sharp increase in UDP levels , with a UTP : UDP concentration ratio of 2:1 . As the cytosolic UTP : UDP ratio is > 10:1 -LSB- 26 -RSB- , our results suggest , at least in part , a vesicular rather than a cytosolic source of UDP and , as a corollary , of ATP and ADP . In summary , our results provide strong evidence for Ca2 + - dependent vesicular exocytosis as a major mechanism of adenosine and uridine nucleotide release from A549 epithelial cells induced by hypotonic stress . Part of this release involves vesicles of the protein secretory pathway . Further investigations are needed to clarify the origin and contribution of other vesicular pools as well as the mechanisms and sources of intracellular Ca2 + elevations that evoke nucleotide secretion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O

exocytosis
hypotonic shock
lung epithelial cells
nucleotide secretion