bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneity of the early outward current in ventricular cells isolated from normal and hypertrophied rat hearts.
Authors: Bénitah J P , Gomez A M , Bailly P , Da Ponte J P , Berson G , Delgado C , Lorente P
Published: December 2014

1. The nature, magnitude and kinetics of the 4‐aminopyridine‐sensitive early outward current (Ito) were analysed in isolated ventricular myocytes from the septum, the apex and the left ventricular free wall of rat ventricles using the whole‐cell voltage clamp method. The modulatory effect of pressure overload‐induced cardiac hypertrophy on the regional variations of Ito was assessed in each topographical class of cells. 2. Voltage clamp experiments were performed at room temperature (20‐25 degrees C) in the absence of Na+ on both sides of the membrane and in the presence of 3 mM CoCl2. Ito was studied from a holding potential of ‐80 mV and determined by subtraction of total outward currents elicited by the same protocols in the presence of 3 mM 4‐aminopyridine (4‐AP) from those obtained in its absence. 3. In normal hearts, membrane passive properties were very similar in each topographical class of cells. Our results confirmed that the predominant early outward current in rat ventricular cells was 4‐AP‐sensitive, time and voltage dependent, and demonstrated that the magnitude of the current varied on a regional basis: current density of Ito in left ventricular free wall cells (30.1 +/‐ 9.2 pA/pF at +60 mV) was larger than in apex cells (20.2 +/‐ 1.7 pA/pF) or in septum cells (11.9 +/‐ 3.3 pA/pF). We noticed a larger variability in data from left ventricular free wall compared with other regions. 4. No shift in steady‐state voltage dependence of Ito activation and inactivation was found. However, the maximal computed chord conductances were (in microS/pF): 0.18 +/‐ 0.07 for left ventricular free wall cells, 0.13 +/‐ 0.02 for apex cells, and 0.08 +/‐ 0.02 for septum cells. These findings might reflect a differential distribution in functional channel densities. 5. No difference in voltage‐dependent Ito activation kinetics was present with respect to topography. However, inactivation time constants in septum were longer than those of both other groups. 6. Left ventricular hypertrophy was induced by abdominal aortic constriction and its effects compared to the findings from normal rats. Hypertrophied cells had similar resting potentials but higher capacitance values than normal cells. Although Ito magnitude appeared not to be modified, the current density‐voltage curves were slightly shifted to more positive potentials and significantly decreased as compared to normal cells (in pA/pF, at +60 mV): 8.4 +/‐ 5.0 in the left free wall group, 11.6 +/‐ 2.0 in the apex group, and 3.8 +/‐ 1.5 in the septum group.(ABSTRACT TRUNCATED AT 400 WORDS)
On the mechanism of M‐current inhibition by muscarinic m1 receptors in DNA‐transfected rodent neuroblastoma x glioma cells.
Authors: Robbins J , Marsh S J , Brown D A
Published: December 2014

1. Acetylcholine (ACh) produces two membrane current changes when applied to NG108‐15 mouse neuroblastoma x rat glioma hybrid cells transformed (by DNA transfection) to express m1 muscarinic receptors: it activates a Ca(2+)‐dependent K+ conductance, producing an outward current, and it inhibits a voltage‐dependent K+ conductance (the M conductance), thus diminishing the M‐type voltage‐dependent K+ current (IK(M)) and producing an inward current. The present experiments were undertaken to find out how far inhibition of IK(M) might be secondary to stimulation of phospholipase C, by recording membrane currents and intracellular Ca2+ changes with indo‐1 using whole‐cell patch‐clamp methods. 2. Bath application of 100 microM ACh reversibly inhibited IK(M) by 47.3 +/‐ 3.2% (n = 23). Following pressure‐application of 1 mM ACh, the mean latency to inhibition was 420 ms at 35 degrees C and 1.79 s at 23 degrees C. Latencies to inhibition by Ba2+ ions were 148 ms at 35 degrees C and 92 ms at 23 degrees C. 3. The involvement of a G‐protein was tested by adding 0.5 mM GTP‐gamma‐S or 10 mM potassium fluoride to the pipette solution. These slowly reduced IK(M), with half‐times of about 30 and 20 min respectively, and rendered the effect of superimposed ACh irreversible. Effects of ACh were not significantly changed after pretreatment for 24 h with 500 ng ml‐1 pertussis toxin or on adding up to 10 mM GDP‐beta‐S to the pipette solution. 4. The role of phospholipase C and its products was tested using neomycin (to inhibit phospholipase C), inositol 1,4,5‐trisphosphate (InsP3) and inositol 1,3,4,5‐tetrakisphosphate (InsP4), heparin, and phorbol dibutyrate (PDBu) and staurosporin (to activate and inhibit protein kinase C respectively). Both neomycin (1 mM external) and InsP3 (100 microM intrapipette) inhibited the ACh‐induced outward current and/or intracellular Ca2+ transient but did not block ACh‐induced inhibition of IK(M). Intrapipette heparin (1 mM) blocked activation of IK(Ca) and reduced Ach‐induced inhibitions of IK(M), but also reduced inhibition of ICa via endogeneous m4 receptors. PDBu (with or without intrapipette ATP) and staurosporin had no significant effects.(ABSTRACT TRUNCATED AT 400 WORDS)
Two kinetically distinct components of hyperpolarization‐activated current in rat superior colliculus‐projecting neurons.
Authors: Solomon J S , Nerbonne J M
Published: December 2014

1. Whole‐cell and perforated patch recording techniques were used to examine the activation, deactivation and inactivation of the time‐dependent hyperpolarization‐activated inward currents (Ih) in isolated superior colliculus‐projecting (SCP) neurons from rat primary visual cortex. 2. Examination of inward current waveforms revealed the presence of two kinetically distinct components of Ih: one that activates with a time constant of the order of hundreds of milliseconds, and one that activates with a time constant of the order of seconds. We have termed these Ih,f and Ih,s, to denote the fast and slow components, respectively, of current activation. The time constants of activation of both Ih,f and Ih,s decrease with increasing membrane hyperpolarization. 3. Following the onset of hyperpolarizing voltage steps, a delay is evident prior to time‐dependent inward current activation. This delay is voltage dependent and decreases with increasing membrane hyperpolarization. 4. The sigmoidal inward current waveforms are well fitted by the sum of two exponentials in which the faster term, corresponding to the activation of Ih,f, is raised to the power 1.34 +/‐ 0.26 (mean +/‐ S.D.). The non‐integral exponent suggests that Ih,f activation involves at least two energetically non‐equivalent gating transitions prior to channel opening. 5. Over a limited voltage range, tail currents could also be resolved into two distinct components. The faster component, which corresponds to the deactivation of Ih,f, decayed over a single exponential time course with a mean (+/‐ S.D.) time constant of 355 +/‐ 161 ms at ‐70 mV. Ih,s decay also followed a single exponential time course with a mean (+/‐ S.D.) time constant of 2428 +/‐ 1285 ms at ‐70 mV. Both deactivation time constants decreased with increasing depolarization. 6. The separation of inward current activation and deactivation into two distinct components and the lack of correlation between the relative amplitudes of these components suggest that Ih,f and Ih,s reflect the presence of two functionally distinct channel populations. 7. No decrements in time‐dependent hyperpolarization‐activated inward currents were observed during hyperpolarizations lasting up to 18 s, suggesting that neither Ih,f nor Ih,s inactivates from the open state. In addition, 10 s depolarizations to 0 mV prior to activation did not alter the waveforms of the inward currents activated directly from ‐40 mV, suggesting that Ih,f and Ih,s also do not inactivate from closed states. 8. The hyperpolarization‐activated currents in rat SCP neurons are ideally suited to contribute to the control of the resting membrane potential and input resistance.(ABSTRACT TRUNCATED AT 400 WORDS)
Interrelation between baroreceptor and macula densa mechanisms in the control of renin secretion.
Authors: Scholz H , Vogel U , Kurtz A
Published: December 2014

1. It was the aim of this study to examine the interrelation between ‘baroreceptor’ function and the macula densa signal in the control of renin secretion from the kidneys. To this end we investigated the effects of frusemide and bumetanide, two different inhibitors of the macula densa Na(+)‐K(+)‐2Cl‐ cotransport, on pressure‐dependent renin release from isolated perfused rat kidneys. In addition, pressure modulation of renin secretion from hydronephrotic kidneys devoid of macula densa structures was examined. 2. Basal flow rate through isolated kidneys was 13.5 +/‐ 1.0 ml min‐1 g‐1 at a renal artery pressure of 100 mmHg and corresponding renin secretory rates were 5.5 +/‐ 0.5 (ng angiotensin I (Ang I) h‐1) min‐1 g‐1 (mean +/‐ S.E.M., n = 15). 3. Frusemide (10‐100 microM) and bumetanide (5‐50 microM) increased urine flow rates and stimulated urinary sodium excretion in a dose‐dependent fashion from 13.0 +/‐ 2.5 mumol min‐1 g‐1 (n = 10) under control conditions to maximal values of 38.0 +/‐ 5.0 (n = 5) and 37.0 +/‐ 2.0 mumol min‐1 g‐1 (n = 5), respectively. Both drugs also induced concentration‐dependent decreases of the renal vascular resistance. The vasorelaxant effects of frusemide and bumetanide were paralleled by an increase of renin secretion to a maximum of 21 +/‐ 4 (ng Ang I h‐1) min‐1 g‐1 (n = 10). On a molar basis bumetanide was twice as potent as frusemide in stimulating renin secretion. 4. Lowering of the perfusion pressure from 100 to 40 mmHg resulted in a prompt increase of renin release yielding secretion rates of 92 +/‐ 10 (ng Ang I h‐1) min‐1 g‐1 (n = 15). In the presence of frusemide (100 microM) and bumetanide (50 microM) renin secretion rates at 40 mmHg were 97 +/‐ 11 and 133 +/‐ 24 (ng Ang I h‐1) min‐1 g‐1 (n = 6), respectively. Renin release stimulated by bumetanide was significantly reduced to 8.0 +/‐ 1.5 (ng Ang I h‐1) min‐1 g‐1 (n = 5) by elevating the perfusion pressure from 100 to 140 mmHg. 5. Lowering the renal artery pressure from 100 to 40 mmHg in isolated perfused rat hydronephrotic kidneys devoid of tubular structures increased renin secretion rates from 4.5 +/‐ 1.0 to 22.5 +/‐ 2.5 (ng Ang I h‐1) min‐1 (n = 5).(ABSTRACT TRUNCATED AT 400 WORDS)
Organization of motor output in slow finger movements in man.
Authors: Vallbo A B , Wessberg J
Published: December 2014

1. Slow finger movements were analysed in normal human subjects with regard to kinematics and EMG activity of the long finger muscles. Surface EMG from the finger extensor and flexor muscles on the forearm was recorded along with angular position and angular velocity during voluntary ramp movements at single metacarpophalangeal joints. Angular acceleration was computed from the velocity record. 2. It was found that movements were not smooth but characterized by steps or discontinuities, often recurring at intervals of 100‐125 ms, yielding velocity and acceleration profiles dominated by 8‐10 Hz cycles. The discontinuities were manifest from the very first trial and thus not dependent on training. Their amplitude and amount varied between subjects but were relatively stable for the individual subject. 3. The 8‐10 Hz cycles were seen with voluntary ramp movements of widely varying velocities, higher velocities being associated with larger steps recurring with the same repetition rate as the small steps of slow voluntary ramps. Maximal step amplitude observed was more than one order of magnitude larger than physiological tremor. 4. The individual 8‐10 Hz cycle was asymmetrical in that decelerations usually reached higher peaks than the preceding acceleration, suggesting that the antagonist contributed with a braking action. Moreover, in very slow voluntary ramps, the movement cycles were often interspaced by periods of zero velocity, providing a highly non‐sinusoidal velocity profile. 5. The EMG of the agonist and the antagonist muscles was modulated in close relation to the accelerations and decelerations respectively of the individual movement cycle. These modulations were present in both extensor and flexor muscles, although they were more consistent and usually more prominent in the former. 6. The findings indicate that a feature of slow finger movements was an 8‐10 Hz periodic output to the muscular system, suggesting that slow finger movements are implemented by a series of biphasic force pulses, involving not only the shortening agonist muscle propelling the movement, but the antagonist muscle as well whose activity increased shortly after the agonist and contributed to a sharp deceleration of the individual step of movement. 7. It is proposed, as a hypothesis, that this biphasic motor output may reflect a similar organization of the descending motor command for slow finger movements. Hence, this command would include a series of biphasic pulses, concatenated at a rate of 8‐10 per second and a pulse‐height regulator capable of setting the size of the pulse and thus the overall speed of the movement.
Human sodium channel myotonia: slowed channel inactivation due to substitutions for a glycine within the III‐IV linker.
Authors: Lerche H , Heine R , Pika U , George A L , Mitrovic N , Browatzki M , Weiss T , Rivet‐Bastide M , Franke C , Lomonaco M
Published: December 2014

1. Three families with a form of myotonia (muscle stiffness due to membrane hyperexcitability) clinically distinct from previously classified myotonias were examined. The severity of the disease greatly differed among the families. 2. Three dominant point mutations were discovered at the same nucleotide position of the SCN4A gene encoding the adult skeletal muscle Na+ channel alpha‐subunit. They predict the substitution of either glutamic acid, valine or alanine for glycine1306, a highly conserved residue within the supposed inactivation gate. Additional SCN4A mutations were excluded. 3. Electrophysiological studies were performed on biopsied muscle specimens obtained for each mutation. Patch clamp recordings on sarcolemmal blebs revealed an increase in the time constant of fast Na+ channel inactivation, tau h, and in late channel openings as compared to normal controls. tau h was increased from 1.2 to 1.6‐2.1 ms and the average late currents from 0.4 to 1‐6% of the peak early current. 4. Intracellular recordings on resealed fibre segments revealed an abnormal tetrodotoxin‐sensitive steady‐state inward current, and repetitive action potentials. Since K+ and Cl‐ conductances were normal, only the increase in the number of non‐inactivating Na+ channels has to be responsible for the membrane hyperexcitability. 5. Length, ramification and charge of the side‐chains of the substitutions correlated well with the Na+ channel dysfunction and the severity of myotonia, with alanine as the most benign and glutamic acid as the substitution with a major steric effect. 6. Our electrophysiological and molecular genetic studies strongly suggest that these Na+ channel mutations cause myotonia. The naturally occurring mutants allowed us to gain further insight into the mechanism of Na+ channel inactivation.
Respiratory sensations, cardiovascular control, kinaesthesia and transcranial stimulation during paralysis in humans.
Authors: Gandevia S C , Killian K , McKenzie D K , Crawford M , Allen G M , Gorman R B , Hales J P
Published: December 2014

1. To determine whether discomfort associated with breathing (dyspnoea) is related to the chemical drive to breath, three subjects were totally paralysed while fully conscious. Subjective responses to a rising CO2 stimulus were obtained during rebreathing, rebreathing with CO2 added, and breath holding. Dyspnoea was measured with a 10‐point Borg scale. 2. Following nasotracheal intubation and ventilation (oxygen saturation, O2,Sat, 98‐100% and end‐tidal CO2, PET,CO2, 30‐40 mmHg), total neuromuscular blockade was induced by a rapid injection of atracurium (> 2.5 mg kg‐1) and complete paralysis was maintained with an infusion (5 mg (kg h)‐1). Paralysis was confirmed by abolition of the compound muscle action potentials of both the diaphragm and abductor hallucis evoked by supramaximal electrical stimulation of the relevant nerves. Communication via finger movement was preserved for the first 20‐30 min following paralysis by inflation of a sphygmomanometer cuff on one arm. 3. Before and during complete paralysis, dyspnoea increased progressively during hypercapnia produced by rebreathing (with or without CO2 added to the circuit at 250 ml min‐1). The mean PET,CO2 eliciting 'severe’ dyspnoea was 46 mmHg during rebreathing, 42 mmHg during ‘breath holding’, and 52 mmHg during rebreathing with added CO2. There were no significant differences between the values obtained during paralysis and in the control study immediately before paralysis. The duration of breath holding was not prolonged by paralysis and the PET,CO2 at the ‘break point’ was not altered by paralysis. 4. Thus, dyspnoea is preserved following total neuromuscular blockade. This suggests that chemoreceptor activity, via the central neuronal activity which it evokes, can lead to discomfort in the absence of any contraction of respiratory muscles. 5. During paralysis, attempted contraction of arm, leg and trunk muscles increased heart rate and blood pressure. For attempted handgrip contractions, the increases in heart rate (range, 7‐15 beats min‐1) and mean arterial pressure (range, 20‐32 mmHg) were similar to those recorded with actual contractions in trials immediately before paralysis. In one subject, graded increases in heart rate and blood pressure occurred for attempted contractions of 45 s duration over a range of intensities (0‐100% maximal effort). 6. During complete paralysis, transcranial electromagnetic stimulation of the motor cortex produced illusory twitch‐like movements of the wrist and digits. This also occurred in separate studies during complete ischaemic paralysis and anaesthesia of the forearm and hand.(ABSTRACT TRUNCATED AT 400 WORDS)
The influence of vasopressin on the arterioles and venules of skeletal muscle of the rat during systemic hypoxia.
Authors: Marshall J M , Lloyd J , Mian R
Published: December 2014

1. In rats anaesthetized with Saffan, the spinotrapezius muscle was prepared for in vivo microscopy. Systemic hypoxia (breathing 8% O2 for 3 min) induced a fall in arterial pressure and tachycardia, together with constriction in some arterioles and venules of each section of the vascular tree and dilatation in others. 2. The vasopressin V1‐receptor antagonist d(CH2)5Tyr(Me)‐arginine vasopressin (20 mg kg‐1 I.V.) preferentially attenuated constrictor responses induced by hypoxia in both arterioles and venules, but had no significant effect on the dilator responses. Analysis of responses in individual sections of the vascular tree suggested that the V1‐receptor antagonist reduced hypoxia‐induced constrictor responses in proximal arterioles (> 13 microns diameter) though not in terminal arterioles (< 13 microns), but reduced hypoxia‐induced constrictor responses in both the proximal and distal venules (9‐130 microns). 3. Infusion of vasopressin at 1.4, 2.8, 5.7 and 11.4 ng min‐1 kg‐1 i.v. for 3 min, expected to produce plasma concentrations within the range 28‐228 pg ml‐1, evoked rises in arterial pressure together with decreases in heart rate. There was also vasoconstriction in the proximal arterioles of spinotrapezius that was graded with vasopressin concentration (5‐35% decrease in diameter). 4. Infusion of vasopressin at 1.4 mg min‐1 kg‐1 i.v. for 3 min with the intention of producing a plasma concentration likely to be reached or exceeded during 8% O2, evoked constriction of all proximal arterioles, though not of terminal arterioles, and constriction of all venous vessels. The magnitude of the constriction induced by vasopressin in vessels that dilated during hypoxia was just as great as in those that constricted during hypoxia. 5. We propose that vasopressin released during systemic hypoxia exerts a constrictor influence upon the proximal arterioles and all sections of the venous tree of skeletal muscle. In individual arterioles and venules the constrictor influence of vasopressin and catecholamines may be overcome by the influence of locally released vasodilator metabolites.
Cytoplasmic Ca2+ oscillation in rat megakaryocytes evoked by a novel type of purinoceptor.
Authors: Uneyama C , Uneyama H , Akaike N
Published: December 2014

1. The responses of megakaryocytes isolated from rat bone marrow to externally applied adenosine triphosphate (ATP) were investigated in the whole‐cell mode by the use of nystatin perforated patch‐clamp technique. 2. ATP at 1‐100 microM evoked periodic outward currents at a holding potential of ‐40 mV. The reversal potential of the currents was close to K+ equilibrium potential (EK) and the K+ channel blockers such as quinine and quinidine suppressed the currents, indicating that the outward currents are predominantly carried by K+. 3. Since it has been reported that adenosine diphosphate (ADP) evoked monophasic K+ current using a conventional whole‐cell recording, we compared the results obtained by perforated and conventional patch‐clamp techniques. The crucial difference between our results and previous results was due to the intracellular perfusion with internal solution containing a high concentration of EGTA by which both current shape and concentration response were modified. 4. The membrane permeable Ca2+ chelator, 1,2‐bis(O‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid (acetoxy methyl ester; BAPTA AM), inhibited the K+ current concentration dependently, suggesting that ATP‐induced oscillatory K+ currents are caused by changes in cytoplasmic free Ca2+ concentration ([Ca2+]i). 5. With increasing ATP concentration, the frequency and the maximum amplitude of K+ current oscillation increased and the latency of current, which is the period required to activate the first K+ current after ATP application, decreased. 6. ADP, 2‐methylthio‐ATP and ATP‐gamma‐S could also evoke the periodic K+ currents, but adenosine, uridine triphosphate (UTP) and alpha‐beta‐methylene adenosine 5'‐triphosphate (AMP‐CPP) failed. 2‐Methylthio‐ATP was the most potent agonist; next was ADP which showed a 10‐30 times stronger effect than ATP. Cross‐desensitization was observed between ATP and ADP, but not between ATP or ADP and thrombin. 7. Extracellular Ca2+ was not required for the ATP‐induced K+ current activation, indicating that Ca2+ released from intracellular pools induced the oscillatory response. In addition, the agonist potency increased when extracellular Ca2+ concentration ([Ca2+]o) decreased, suggesting that the principal agonists might be ATP4‐ and ADP3‐. 8. The results suggest the presence of a novel subtype of purinoceptor in the megakaryocyte plasma membrane which induces cytoplasmic Ca2+ oscillation and evokes periodic K+ current flux.
Cholinergic neuromuscular transmission in the longitudinal muscle of the guinea‐pig ileum.
Authors: Cousins H M , Edwards F R , Hirst G D , Wendt I R
Published: December 2014

1. Brief transmural stimuli, 0.5‐1 ms, initiated contractions of the longitudinal muscle taken from the guinea‐pig ileum that were recorded isometrically. In separate preparations similar stimuli were found to initiate excitatory junction potentials which were recorded using intracellular recording electrodes. All of these responses were abolished by either tetrodotoxin, omega‐conotoxin or hyoscine. 2. The contractions produced by increasing [K+]o were blocked by nifedipine, 1 x 10(‐7) M; nicardipine, 1 x 10(‐7) M; verapamil, 1 x 10(‐5) M or diltiazem, 1 x 10(‐5) M. In these solutions brief stimuli continued to initiate contractions: this indicates that neuronally released acetylcholine continues to trigger a contraction when muscle voltage‐dependent calcium channels appear to have been blocked. 3. When membrane potential recordings were made from the smooth muscle layer, brief transmural stimuli initiated excitatory junction potentials that triggered muscle action potentials. Although muscle action potentials were abolished by low concentrations of a range of organic calcium antagonists, excitatory junction potentials persisted and continued to initiate contractions of reduced amplitude. 4. When the internal concentration of calcium ions, [Ca2+]i, was measured using fura‐2, brief transmural stimuli caused an increase in [Ca2+]i. Part of this response, which occurred at a time corresponding to the unblocked excitatory junction potential, persisted in the presence of the organic calcium antagonist nifedipine. 5. Two explanations appear possible. Neuronally released acetylcholine may simultaneously activate non‐selective cation channels and cause the release of Ca2+ from an internal store. Alternatively, neuronally released acetylcholine may cause an increase in [Ca2+]i which is separate from that which accompanies the activation of voltage‐dependent calcium channels. At this stage there is little other anatomical or electrophysiological evidence to support this view.
Modulation of L‐type Ca2+ current by extracellular ATP in ferret isolated right ventricular myocytes.
Authors: Qu Y , Campbell D L , Strauss H C
Published: December 2014

1. The effects of extracellular adenosine triphosphate (ATP) on the basal L‐type Ca2+ current (ICa) were investigated in ferret isolated right ventricular myocytes using the gigaohm seal voltage clamp in the whole‐cell and cell‐attached configurations. 2. Micromolar levels of extracellular ATP reversibly inhibited ICa in a concentration‐dependent manner, without any significant changes in the voltage dependence of either the peak ICa I‐V relationship or steady‐state activation curve. 3. In contrast, micromolar levels of extracellular ATP did significantly alter the inactivation characteristics of ICa. Ten micromolar ATP: (i) increased the degree of steady‐state inactivation of ICa; (ii) altered the time constants of ICa inactivation at 0 mV; and (iii) decreased the time constant of ICa recovery from inactivation at ‐70 mV. 4. The inhibitory effect of ATP on ICa was not blocked by atropine, a muscarinic cholinergic receptor antagonist, or CPDPX (8‐cyclopentyl‐3,4‐dipropylxanthine), an A1 adenosine receptor antagonist. In contrast, the inhibitory effect of 10 microM ATP could be nearly completely antagonized by 100 microM suramin, a purinergic P2 receptor antagonist. 5. The potency order of ATP analogues in inhibiting ICa was 2‐methyl‐thio‐ATP > ATP > alpha,beta‐methylene‐ATP, indicating involvement of a P2Y‐type ATP receptor. 6. Pretreatment of cells with pertussis toxin (PTX) did not prevent the ATP‐induced decrease in ICa. However, (i) ATP produced an irreversible decrease of ICa in the presence of intracellular GTP gamma S, and (ii) the inhibitory effect was significantly attenuated in the presence of intracellular GDP beta S, indicating the involvement of a PTX‐insensitive G protein in the P2Y receptor‐coupling process. 7. Neither (i) replacing extracellular Ca2+ with 1 mM Ba2+, nor (ii) intracellular perfusion of 10 mM BAPTA for at least 30 min attenuated the inhibitory effect of ATP on the current through Ca2+ channels, suggesting that the inhibitory effect was not obligatorily dependent upon influx of Ca2+ or changes in [Ca2+]i. 8. Ensemble‐average current behaviour constructed from cell‐attached patch recordings of single L‐type Ca2+ channels (110 mM BaCl2) demonstrated that when 10 microM ATP was added to the superfusate on the outside of the patch electrode the inhibition of ICa was still observed, providing evidence for the involvement of intracellular diffusible second messenger(s).(ABSTRACT TRUNCATED AT 400 WORDS)
Relationship between intracellular pH and metabolite concentrations during metabolic inhibition in isolated ferret heart.
Authors: Smith G L , Donoso P , Bauer C J , Eisner D A
Published: December 2014

1. Intracellular pH (pHi) and phosphorus metabolites were measured in isolated ferret hearts with 31P nuclear magnetic resonance (NMR). 2. The application of cyanide (to mimic hypoxia) produced a fall of the concentration of phosphocreatine ([PCr]) and a rise of those of inorganic phosphate ([Pi]) and sugar phosphates. These were accompanied by an intracellular acidosis. 3. If glycolysis was partly inhibited by prior exposure to a glucose‐free solution then the application of cyanide also produced a fall of [ATP]. The acidosis was similar to that observed in the presence of glucose. 4. If glycolysis was completely inhibited by iodoacetate then the acidosis produced by subsequent addition of cyanide developed more quickly. 5. The results are reproduced by a model which incorporates lactic acid production as well as the effects of protons released and absorbed by the changes in metabolite concentrations. The results suggest that the acidosis produced by cyanide (without inhibition of glycolysis) is largely due to lactic acid production. When glycolysis is partly inhibited (glucose‐free solution) the acidosis produced by cyanide is partly due to protons released by ATP breakdown. Finally, when glycolysis is entirely inhibited the acidosis is completely due to ATP breakdown. There is no need to postulate a contribution on this time scale from inhibition of pH regulating mechanisms.
Occurrence of intracellular pH transients during spontaneous contractions in rat uterine smooth muscle.
Authors: Taggart M J , Wray S
Published: December 2014

1. Intracellular pH (pHi) and contraction have been measured simultaneously in isolated longitudinal rat myometrium. Tissue was loaded with the pH‐sensitive fluorophore carboxy‐SNARF and superfused with oxygenated, buffered salt solution at 37 degrees C and pH 7.4. 2. Small (0.04 pH unit) transient intracellular acidifications followed each spontaneous uterine contraction. The pHi transients lagged contraction by 15 s. Removal of external Ca2+ abolished both spontaneous contractions and the pHi changes. 3. Increasing uterine contraction by depolarization (40 mM K+) or agonist (50 microM carbachol) produced a larger acidification (0.18 pH unit). 4. The acidification with contraction does not appear to be due to increased glycolysis as no increase in lactate efflux occurred with high‐K+ stimulation. 5. Uterine contraction is known to be sensitive to changes in pHi. Comparable pHi changes to the pHi transients seen during spontaneous contraction were induced by application and withdrawal of weak acids and bases. The resulting pHi changes (0.02‐0.08 pH unit) produced effects on contraction frequency and amplitude, alkalinization augmenting and acidification depressing contractile activity, suggesting that the pHi transients with contraction are functionally important.
Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones.
Authors: Chard P S , Bleakman D , Christakos S , Fullmer C S , Miller R J
Published: December 2014

1. We have examined the ability of the Ca(2+)‐binding proteins (CABP) calbindin D28k and paravalbumin to modulate increases in the intracellular free Ca2+ concentration ([Ca2+]i), produced by brief depolarizations, in rat dorsal root ganglion (DRG) neurones. 2. In order to obtain good voltage control, we replated DRG neurones prior to performing these experiments. Immunocytochemical staining of these cells revealed that approximately 10% stained for CABPs. 3. Using fluorescently labelled parvalbumin, we demonstrated that in the whole‐cell voltage clamp mode the protein freely entered the cell soma with a mean half‐life t0.5 of 6 min 22 s +/‐ 54 s. 4. Analysis of the effects of calbindin D28k (370 microM) and parvalbumin (1 mM) on Ca2+ currents in the whole‐cell voltage clamp mode, revealed that neither protein changed the rate of inactivation of the Ca2+ current or its rate of run‐down. 5. Introducing either calbindin D28k (370 microM) or parvalbumin (1 mM) into the cell soma did not significantly alter the basal [Ca2+]i when compared to control cells. 6. Compared to control cells, both CABPs significantly reduced the peak [Ca2+]i obtained for a Ca2+ influx of an equivalent charge density, whereas lysozyme (1 mM), a protein with low affinity for Ca2+, failed to do so. 7. Calbindin D28k caused an 8‐fold decrease in the rate of rise in [Ca2+]i and altered the kinetics of decay of [Ca2+]i to a single slow component. Parvalbumin also slowed the rate of rise in [Ca2+]i. Parvalbumin selectively increased a fast component in the decay of the Ca2+ signal. 8. These data demonstrate that both calbindin D28k and paravalbumin effectively buffer Ca2+ in a cellular environment and may therefore regulate Ca(2+)‐dependent aspects of neuronal function.
Effects of membrane potential and muscarine on potassium M‐channel kinetics in rat sympathetic neurones.
Authors: Selyanko A A , Brown D A
Published: December 2014

1. Using cell‐attached patch pipettes, sustained activity of single potassium M‐channels was recorded from dissociated rat superior cervical ganglion neurones. Previous results indicated that this activity, consisting of three main levels of open‐channel conductance (congruent to 7, congruent to 12 and congruent to 19 pS) was activated by membrane depolarization and inhibited by muscarine added outside the patch. Consequently, a kinetic analysis was undertaken in order to identify M‐channel states sensitive to muscarine and membrane potential. 2. Channel activity recorded at 30 mV positive to the resting membrane potential level (congruent to ‐60 mV) showed three shut and two open times. Mean shut times were: tau s1 = 8.0 +/‐ 2.2 ms; tau s2 = 71.3 +/‐ 8.6 ms and tau s3 = 740 +/‐ 220 ms. Mean open times were: tau o1 = 10.6 +/‐ 1.9 ms and tau o2 = 59.3 +/‐ 8.7 ms. When bursts of channel openings were determined as those including tau s1, two exponential components were evident in burst duration distributions (tau b1 = 11.0 +/‐ 0.9 ms and tau b2 = 80.4 +/‐ 11.0 ms). 3. Membrane hyperpolarization significantly lengthened all three shut times and shortened both open times. It also slightly enhanced the relative contribution of high‐conductance channels and decreased the relative contribution of low‐conductance channels to overall activity. 4. All three shut times of the M‐channels were lengthened by 10 microM muscarine without significantly affecting their open times. 5. It is concluded that both open and shut states of the M‐channel are voltage sensitive while only shut states are sensitive to muscarine.
Block by amiloride and its derivatives of mechano‐electrical transduction in outer hair cells of mouse cochlear cultures.
Authors: Rüsch A , Kros C J , Richardson G P
Published: December 2014

1. The effects of amiloride and amiloride derivatives on mechano‐electrical transducer currents in outer hair cells of the cultured neonatal mouse cochlea were examined under whole‐cell voltage clamp. 2. At ‐84 mV transducer currents were reversibly blocked by the extracellular application of the pyrazinecarboxamides amiloride, benzamil, dimethylamiloride, hexamethyleneiminoamiloride, phenamil and methoxynitroiodobenzamil with half‐blocking concentrations of 53, 5.5, 40, 4.3, 12 and 1.8 microM, respectively. Hill coefficients were determined for all but the last of these compounds and were 1.7, 1.6, 1.0, 2.2 and 1.6, respectively, suggesting that two drug molecules co‐operatively block the transducer channel. 3. Both the structure‐activity sequence for amiloride and its derivatives and the mechanism of the block of the transducer channel appear to be different from those reported for the high‐affinity amiloride‐sensitive epithelial Na+ channels but similar to those of stretch‐activated channels in Xenopus oocytes. 4. The block by all pyrazinecarboxamides was voltage dependent with positive membrane potentials releasing the block. The form of the voltage dependence is consistent with a voltage‐independent binding of the drug to a site that is accessible at hyperpolarized but not at depolarized potentials, suggesting that the transducer channel undergoes a voltage‐dependent conformational change. The channel was not blocked by 1 mM amiloride from the intracellular side at either negative or positive membrane potentials. 5. The kinetics of the block were studied using force steps or voltage jumps. The results suggest that the drug binding site is only accessible when the transducer channel is open (open‐channel block) and that the channel cannot close when the drug molecules are bound. 6. The time dependence and voltage dependence of the block together reveal that the transducer channel has at least two open conformational states, the transition between which is voltage dependent.
Activity‐dependent regulation of inwardly rectifying potassium currents in non‐myelinating Schwann cells in mice.
Authors: Konishi T
Published: December 2014

1. Voltage‐gated potassium currents were recorded from freshly dissociated non‐myelinating Schwann cells of sural and sympathetic nerves from 1‐ to 12‐week‐old mice using the whole‐cell or a single channel variation of the patch‐clamp technique. 2. All sural cells from 2‐week‐old mice showed inwardly rectifying potassium (Kir+) currents in whole‐cell recordings. Kir+ currents were virtually undetectable in sural cells from mice more than 6 weeks old, which also showed depolarization of the resting membrane potential. On the other hand, the magnitude of Kir+ currents increased in cervical sympathetic trunk (CST) cells in parallel with an increase of cell capacitance 1‐6 weeks after birth. The density of Kir+ currents in CST cells increased 1‐4 weeks after birth and then stayed constant for up to 12 weeks. 3. The unitary conductance of a single Kir+ channel in CST cells was 30 pS 2‐12 weeks after birth; this was recorded in a cell‐attached configuration with 154 mM K+ in the pipette. The steady‐state open channel probability of single Kir+ channels in CST cells decreased with membrane hyperpolarization, but was not markedly changed 2‐12 weeks after birth. 4. Conduction block of CST for 5 days induced by local application of tetrodotoxin (TTX) resulted in a significant decrease in both the magnitude and the density of Kir+ currents in whole‐cell recordings in CST cells rostral to the sites of TTX block. Similar changes of Kir+ currents in whole‐cell recordings were observed in cells in the inferior postganglionic branch of a superior cervical ganglion after 5 days of TTX block of CST. 5. These results suggest that neuronal activity regulates the expression of functional Kir+ channels in non‐myelinating Schwann cells in adult nerves. The activity‐dependent regulation of the expression of glial potassium channels could play an important role in the regulation of the potassium microenvironment around active axons to maintain impulse conduction in unmyelinated fibres.
Topographical organization of the cerebellar cortical projection to nucleus interpositus anterior in the cat.
Authors: Garwicz M , Ekerot C F
Published: December 2014

1. A new methodological approach for detailed study of the organization of the cerebellar corticonuclear projection was evaluated in barbiturate‐anaesthetized cats. Extracellular field potentials were simultaneously recorded in nucleus interpositus anterior and in the forelimb area of the C3 zone, at the cerebellar surface. On electrical and natural stimulation of the forelimb skin, the evoked positive field potentials in the nucleus and the climbing fibre field potentials in the cerebellar cortex had similar characteristics, indicating that the nuclear potentials were related to climbing fibre activity. 2. Application of a local anaesthetic to the cerebellar surface reversibly diminished the positive field potentials in the nucleus, demonstrating that the potentials were dependent on cerebellar cortical activity. It was thus concluded that the positive field potentials were mainly generated by climbing fibre‐activated Purkinje cells and reflected synaptic inhibitory potentials in nuclear neurones. Accordingly, the positive field potentials in the nucleus could be used to reveal the termination area of Purkinje cells activated by a specific climbing fibre input evoked on peripheral stimulation. 3. The topographical organization of the cerebellar cortical projection to the forelimb part of nucleus interpositus anterior was then investigated by systematically mapping the cutaneous tactile and nociceptive ‘receptive fields’ of the positive field potentials at different sites in the nucleus. Five groups of receptive fields were distinguished and tentatively divided into a total of nineteen subgroups. 4. Each group of receptive fields corresponded to one or two of the previously described receptive field classes of climbing fibres to the C1, C3 and Y zones and was represented in a single area of the nucleus. Within each area there was an orderly representation of different receptive fields. The results suggest that microzones in the C1, C3 and Y zones with similar climbing fibre input project to a common set of neurones in nucleus interpositus anterior. 5. We propose a modular organization for the cerebellar control of forelimb movements through the rubrospinal tract. Each module would consist of a set of neurones in nucleus interpositus anterior and their afferent microzones in the C1, C3 and Y zones. A module would control a specific group of muscles and receive a homogeneous climbing fibre input related to the movement controlled.
Accommodation mediated by enteric inhibitory reflexes in the isolated guinea‐pig small intestine.
Authors: Waterman S A , Costa M , Tonini M
Published: December 2014

1. The aim of the present study was to investigate whether the guinea‐pig small intestine shows accommodation to infused fluid, similarly to other regions of the gastrointestinal tract. Tetrodotoxin, papaverine and transmitter antagonists were used to establish the existence of reflex pathways and the nature of the neurotransmitters involved. 2. Compliance, measured as the change in volume of infused fluid divided by the intraluminal pressure change, was reduced by tetrodotoxin (0.6 microM), indicating that there is an overall neurally mediated relaxation of the circular muscle in response to low rates of distension. Papaverine (10 microM) did not have any significant effect on compliance at the low rates of distension, suggesting that the circular muscle is fully relaxed. 3. At each rate of distension, 400 microM N omega‐nitro‐L‐arginine methyl ester (L‐NAME, a nitric oxide synthase inhibitor) significantly decreased the compliance of the intestinal wall, indicating that the circular muscle was relaxed by a nitric oxide‐mediated mechanism. Apamin (0.5 microM), which blocks a component of inhibitory transmission, did not have a significant effect. 4. In control preparations, the intestinal wall was less compliant when distended by fluid at a fast rate, compared with the lower rates of distension. This was not due to changes in passive components of the intestinal wall or a myogenic response to rapid stretch. 5. When the intestine was distended rapidly, 1 microM hyoscine and 100 microM hexamethonium increased intestinal compliance. However, they had no detectable effect on compliance with low rates of distension.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of classically conditioned eyeblink responses by stimulation of the cerebellar cortex in the decerebrate cat.
Authors: Hesslow G
Published: December 2014

The purpose of the present study was to test the hypothesis that neurones in the anterior interpositus nucleus, under the control of Purkinje cells in the c1 and c3 zones of the cerebellar cortex, exert some control over classically conditioned responses. In particular, the experiments were designed to determine whether the cerebellar control of conditioned and unconditioned responses is different. The experiments were performed on cats decerebrated rostral to the red nucleus under halothane anaesthesia. The cats were conditioned using either a 1000 Hz tone or trains of stimuli through the skin of the proximal forelimb as the conditioned stimulus, and periorbital electrical stimulation as the unconditioned stimulus. A large proportion of the animals acquired conditioned responses at normal rates. It could be shown that these were true conditioned responses and did not result from sensitization or pseudoconditioning. For instance, unpaired presentations of conditioned and unconditioned stimuli caused rapid extinction. Cerebellar areas controlling eyeblink were identified by recording climbing fibre responses in the cerebellar cortex and recording EMG activity in the eyelid evoked by stimulation of the cerebellar cortex. When single shocks of 40‐70 microA were applied to these areas during the emission of conditioned eyeblink responses, the latter were strongly inhibited. The inhibition had a latency of about 10 ms and a duration of 25‐75 ms. It was shown that this inhibition of the conditioned responses was topographically specific and could only be evoked from cortical sites identified as controlling eyeblink. Stimulation of the periphery of an eyeblink area caused little or no inhibition. The effect of cortical stimulation on unconditioned reflex responses in the orbicularis oculi muscle was also tested. Some inhibition of unconditioned responses was observed, but quantitative analysis showed that this inhibition was considerably weaker than the corresponding inhibition of conditioned responses. The magnitude of the inhibition was determined for unconditioned responses of different sizes including responses which were weaker than the conditioned responses. It is concluded that conditioned eyeblink responses are under strong cerebellar control from areas in the c1 and c3 zones receiving climbing fibre input from the periorbital area. This effect is not likely to be due to a reduction in the background facilitation of facial motoneurones. In contrast, the weak inhibition of the unconditioned response was probably due to this mechanism. The results, therefore, suggest that the conditioned responses are dependent on the cerebellum in a way that is not true of unconditioned responses.
An obligatory role for nitric oxide in autonomic control of mammalian heart rate.
Authors: Han X , Shimoni Y , Giles W R
Published: December 2014

Cholinergic modulation of heart rate in isolated spontaneously beating single cells from the rabbit sino‐atrial node was investigated by measuring transmembrane ionic currents using the nystatin‐perforated patch whole‐cell voltage‐clamp technique. Carbamylcholine (CCh), a stable analogue of acetylcholine (ACh), significantly inhibited L‐type calcium currents (Ica(L) which had been augmented by beta‐adrenergic stimulation. In addition, CCh activated a potassium outward current (IK(ACh)). Both effects were blocked by atropine. The possible involvement of nitric oxide (NO) in these responses was evaluated by inhibiting NO synthesis. In the presence of NG‐monomethyl‐L‐arginine (L‐NMMA, 100 microM) or nitro‐L‐arginine methyl ester (L‐NAME, 1 mM), two specific inhibitors of nitric oxide synthase (NOS), CCh no longer inhibited ICa(L). IK(ACh) could still be activated. Co‐incubation of cells in L‐NAME or in L‐NMMA with arginine (the endogenous substrate of NOS) restored the CCh‐induced attenuation of ICa(L), indicating that L‐NAME or L‐NMMA did not interfere directly with the muscarinic action of CCh on ICa(L). Effects of the NO‐releasing agent molsidomine (SIN‐1) on CCh‐induced changes in ICa(L) were also investigated. After ICa(L) had been augmented by beta‐adrenergic stimulation, SIN‐1 (0.1 mM) inhibited ICa(L); however, SIN‐1 had no further inhibitory effect after a maximal CCh concentration had been applied. These findings suggest that NO generation is an obligatory process in cholinergic inhibition of ICa(L) in mammalian cardiac pacemaker tissue.