bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium in the nerve terminals of chick ciliary ganglia during facilitation, augmentation and potentiation.
Authors: Brain K L , Bennett M R
Published: December 2014

1. The calyciform nerve terminals of chick ciliary ganglia were loaded with the calcium indicators calcium green 1 or fura‐2. These were used to determine the change in calcium concentration in the terminal, [Ca2+]t, following short (10 impulses) and long (600 impulses) trains of high‐frequency (30 Hz) stimulation. 2. Following a single impulse or a short train, the elevated [Ca2+]t declined along two exponentials with time constants similar to slow (F2) facilitation (0.52 s) and augmentation (4.0 s). After a long train elevated [Ca2+]t declined eventually along a single exponential with the time constant of post‐tetanic potentiation (162 s). [Ca2+]t was not elevated through long‐term potentiation. 3. Addition of Ba2+ (0.75 mM) to the extracellular solution slowed only the decline of [Ca2+]t associated with augmentation. The addition of the nitric oxide donor sodium nitroprusside did not affect [Ca2+]t following short or long trains. 4. Removal of extracellular calcium (buffered with EGTA) and the blockade of calcium channels with Cd2+ completely prevented the changes in [Ca2+]t. 5. The soma of ciliary ganglion cells were loaded with calcium green and the postganglionic nerves stimulated with a single impulse or a short train of impulses. Following stimuli, the elevated [Ca2+]t declined along a single exponential with a time constant similar to F2 facilitation with no augmentation component evident. 6. The results are discussed in terms of the hypothesis that each impulse in a train gives an equal increment of residual Ca2+ to a compartment for secretion and that Ca2+ is removed from the compartment by three first‐order kinetics processes associated with F2 facilitation, augmentation and post‐tetanic potentiation.
Urinary bladder distension: its effects on carotid baroreceptor reflex left ventricular inotropic response in the dog.
Authors: Ward J , Daly M B , Wood L M
Published: December 2014

1. The effects of distension of the urinary bladder on heart rate, maximum rate of change of left ventricular pressure (dP/dt max) and hindlimb vascular resistance together with their modulation at different carotid sinus pressures were studied in dogs anaesthetized with a mixture of chloralose and urethane and artificially ventilated. 2. When the carotid sinus mean perfusion pressure was raised in randomly selected steps from 60 to 210 mmHg, it caused a progressive bradycardia, and a reduction in left ventricular dP/dt max and in arterial blood pressure, together with vasodilatation in the perfused hindlimb. Distension of the bladder at each level of carotid sinus pressure resulted in tachycardia, a small but significant increase in left ventricular dP/dt max (160 +/‐ 30 mmHg s‐1) and hindlimb vasoconstriction. 3. When heart rate and arterial blood pressure were held constant to exclude these secondary effects on left ventricular dP/dt max, raising the carotid sinus pressure caused a progressive reduction in left ventricular dP/dt max and hindlimb vasodilatation. Superimposition of tests of bladder distension at each level of sinus pressure resulted in variable responses, but overall there was a significant increase in left ventricular dP/dt max of 190 +/‐ 54 mmHg s‐1. Hindlimb vasocontriction, however, was a consistent finding. 4. The gain of the relationship between the carotid sinus perfusion pressure and left ventricular dP/dt max was unaffected by distension of the bladder. 5. It is concluded that, when changes secondary to increases in heart rate and blood pressure are prevented, distension of the bladder causes a small but significant reflex increase in left ventricular dP/dt max. The responses, however, are variable and the possible reasons for this are discussed.
Mechanism of action of ATP on canine pulmonary vagal C fibre nerve terminals.
Authors: Pelleg A , Hurt C M
Published: December 2014

1. The effects of extracellular adenosine 5'‐triphosphate (ATP) on pulmonary vagal afferent fibres (n = 46) was studied in a canine model in vivo (n = 38). 2. ATP (3‐6 mumol kg‐1), administered as a rapid bolus into the right atrium, elicited a transient burst of action potentials in cervical vagal fibres, which was not affected by either blockade of ganglionic transmission (hexamethonium) or a drop in arterial blood pressure (nitroglycerine). 3. The fibres with ATP‐sensitive terminals were otherwise quiescent with no activity related to either cardiac or respiratory cycles and their conduction velocity was 0.85 +/‐ 0.13 m s‐1 (n = 7). 4. Inflation of the lungs to 2‐3 times the tidal volume triggered brief bursts of action potentials in these fibres. 5. Capsaicin (10 micrograms kg‐1), given as a rapid bolus into the right atrium, elicited a burst of action potentials in these ATP‐sensitive fibres. 6. Smaller amounts of ATP and capsaicin (0.5‐3 mumol kg‐1 and 1‐5 micrograms kg‐1, respectively) had similar effects when the two compounds were given into the right pulmonary artery. 7. Adenosine, adenosine 5'‐monophosphate, or adenosine 5'‐diphosphate did not excite these fibres (n = 30). 8. The non‐degradable analogue of ATP alpha,beta‐methylene ATP (alpha,beta‐mATP) was tenfold more potent than ATP while beta,gamma‐methylene ATP (beta,gamma‐mATP) was in active. 9. The selective P2x‐purinoceptor antagonist pyridoxalphosphate‐6‐azophenyl‐2',4'‐disulphonic acid markedly attenuated the effect of ATP but not of capsaicin. The P2Y‐purinoceptor antagonist Reactive Blue 2 was without effect. 10. Pretreatment with pertussis toxin (PTX) did not affect this action of ATP. 11. In the canine lungs ATP activates vagal C fibre nerve terminals. This action is mediated by P2X‐purinoceptors and is independent of a PTX‐sensitive guanine nucleotide binding protein (G protein).
Role of cytoplasmic calcium concentration in the bleaching adaptation of salamander cone photoreceptors.
Authors: Matthews H R , Fain G L , Cornwall M C
Published: December 2014

1. In order to study the possible involvement of Ca2+ in the bleaching adaptation of cones isolated from the retina of the salamander Ambystoma tigrinum, changes in cytoplasmic calcium concentration ([Ca2+]i were opposed by exposing the outer segment to a low‐Ca(2+)‐O Na+ solution designed to minimize Ca2+ fluxes across the outer segment membrane. 2. When a cone was exposed in normal Ringer solution to bright light bleaching a significant fraction of the photopigment, the circulating current was initially suppressed completely and then recovered to a maintained value less than the value in darkness before the bleach. When the outer segment of the cone was stepped to low‐Ca(2+)‐O Na+ solution before the bleach was delivered, the circulating current recovered more slowly or (for large bleaches) remained completely suppressed for the duration of the solution exposure. 3. If, during the period for which the current was suppressed in low‐Ca(2+)‐O Na+ solution, the cone outer segment was exposed to the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX), the circulating current was restored. The dim flash response recorded under these conditions exhibited kinetics and integration times similar to those recorded in low‐Ca(2+)‐O Na+ solution in darkness before the bleach. If, instead, the outer segment was returned to Ringer solution after the bleach, thereby allowing [Ca2+]i to fall from its dark‐adapted level to the appropriate bleach‐adapted level, the kinetics of the response in low‐Ca(2+)‐O Na+ solution were greatly accelerated, and the integration time considerably reduced. This was true regardless of whether or not the low‐Ca(2+)‐O Na+ solution included IBMX. 4. The role of Ca2+ in bleaching adaptation appeared to resemble its role in background adaptation, since in both cases exposure to low‐Ca(2+)‐O Na+ solution suppressed the acceleration of response kinetics. Responses recorded from cones in low‐Ca(2+)‐O Na+ solution were nearly identical in waveform and sensitivity during background light or after bleaches, provided that IBMX was used to restore sufficient photocurrent so that responses to flashes could be recorded, and sensitivity was corrected for loss in quantum catch. 5. These results indicate that the fall in [Ca2+]i in cones after a bleach is necessary both for the acceleration of the flash response and the adaptational decrease in sensitivity, as is the case for adaptation by background light.
Changes in quantal parameters of EPSCs in rat CA1 neurones in vitro after the induction of long‐term potentiation.
Authors: Stricker C , Field A C , Redman S J
Published: December 2014

1. Long‐term potentiation (LTP) was induced in EPSCs evoked in CA1 pyramidal neurones of young rats in vitro by extracellular stimulation of stratum radiatum. Low frequency stimulation was paired with postsynaptic depolarization to induce LTP, using whole‐cell recording techniques. 2. Sufficient control and potentiated records were obtained under stable recording conditions to allow a quantal analysis of eleven EPSCs. The fluctuations in amplitude of all eleven EPSCs were quantized before conditioning stimulation, and they remained quantized after LTP induction, usually with an increased quantal variance. 3. Quantal current was increased by conditioning for nine out of eleven EPSCs. The increase in quantal current was correlated with the percentage increase in the EPSC. For only two EPSCs could the entire potentiation be attributed to an increase in quantal current. 4. The amplitude fluctuations of five control EPSCs could be described by binomial statistics, but after conditioning the binomial description held for only one of these EPSCs. For this EPSC, conditioning caused the release probability to increase from 0.39 +/‐ 0.05 to 0.47 +/‐ 0.02. 5. Quantal content was increased by conditioning stimulation for ten out of eleven EPSCs. The increase in quantal content was correlated with the percentage increase in the EPSC. However, for only four EPSCs could the entire potentiation be attributed to an increase in quantal content. 6. Most EPSCs were evoked with a high proportion of response failures. The probability of response failures decreased in eight out of eleven EPSCs following the induction of LTP. There was a negative correlation between the change in the probability of response failures and the amount of LTP. 7. The minimal number of sites at which transmission occurred increased for ten out of eleven EPSCs following LTP induction. Increases in the minimal number of active sites following conditioning were associated with decreases in the probability of response failures for seven out of eleven EPSCs. 8. The induction of LTP usually resulted in changes in the time course of the EPSCs. Cable analysis using a passive compartmental model of a CA1 pyramidal cell suggested that these time course changes were associated with shifts in the average electrotonic location of the active sites following LTP induction, rather than being caused by an increased duration of synaptic current. 9. LTP expression involves postsynaptic modifications to enhance the synaptic current at active sites. New sites are recruited, and our data cannot be used to determine if this is a result of a pre‐ or a postsynaptic change. Evidence for an increase in release probability was found for one EPSC.
Activation of the muscarinic K+ channel by P2‐purinoceptors via pertussis toxin‐sensitive G proteins in guinea‐pig atrial cells.
Authors: Matsuura H , Sakaguchi M , Tsuruhara Y , Ehara T
Published: December 2014

1. Whole‐cell voltage clamp and cell‐attached patch‐clamp techniques were applied to single atrial myocytes enzymatically dissociated from adult guinea‐pig hearts. 2. In whole‐cell clamp conditions, external applications, of ATP activated the muscarinic K+ (KACh) current, identified by its inward rectification, its reversal potential near the calculated K+ equilibrium potential (EK) and its relaxation properties during step changes of whole‐cell membrane potential. Theophylline, an antagonist for Pi‐purinoceptors, did not affect the action of ATP on the KACh current, indicating that the response was evoked through P2‐purinoceptors. 3. The concentration‐response relationship for ATP was well described by a Hill equation with a half‐maximal concentration of 1.84 microM and a Hill coefficient of 0.94. ATP (100 microM) produced a maximal increase of the KACh current to 10.92 microA microF‐1, which corresponds to 44.9 and 80.9% of the maximal increases evoked by ACh (10 microM) and adenosine (100 microM), respectively. 4. The activation of KACh current gradually declined to a steady level despite the continuous presence of ATP (desensitization). Recovery from the desensitization was relatively rapid with a half‐time of approximately 1.5 min. 5. The activation of KACh current by ATP was completely abolished by pre‐incubating myocytes with pertussis toxin (PTX, 5 micrograms ml‐1), indicating that P2‐purinoceptors are coupled to PTX‐sensitive G proteins to activate the KACh channel. 6. In the cell‐attached patch recording, ATP (5 microM) applied to the pipette solution enhanced the activity of a channel with single‐channel conductance of 52.7 +/‐ 0.9 pS (mean +/‐ S.E.M., n = 10), reversal potential near EK and mean open time of 1.1 +/‐ 0.1 ms. These conductance and kinetic properties are identical to those of the KACh channel in the heart. In contrast, ATP applied to the bath solution did not significantly affect the basal activity of KACh channel openings. These observations suggest that the mechanism coupling the P2‐purinoceptor to the activation of the KACh channel involves membrane‐delimited component(s) rather than soluble second messenger(s). 7. These results strongly suggest a direct coupling of the P2‐purinoceptor to the KACh channel through PTX‐sensitive G proteins, analogous to the coupling mechanism of the muscarinic ACh receptor and Pi‐purinoceptor to this channel.
Effect of pentachlorophenol on calcium accumulation in barnacle muscle cells.
Authors: Nwoga J C , Sniffen J C , Peña-Rasgado C , Kimler V A , Rasgado-Flores H
Published: December 2014

1. The effect of extracellularly applied pentachlorophenol (PCP) was studied on the membrane potential (Vm) and Ca2+ uptake in isolated single skeletal muscle cells of Balanus nubilus. 2. When compared with the controls, 0.1 mM PCP induced a significant (P < 0.05) increase in Ca2+ uptake accompanied by membrane depolarization (9 mV at 45 min incubation). This depolarization was reduced by 11% of extracellular Ca2+ (Cao2+) was replaced by Tris+ and by 50% if extracellular Na+ was also replaced by Tris+. 3. The Ca2+ channel blocker, verapamil (0.1 mM), completely inhibited the PCP‐induced Ca2+ uptake as well as the membrane depolarization either in the absence or presence of Cao2+. Experiments on voltage‐clamped cells show that the PCP‐induced Ca2+ uptake was independent of the PCP‐induced depolarization. 4. The results indicate that PCP induces activation of a verapamil‐sensitive Ca2+ influx pathway (presumably L‐type Ca2+ channels) independent of Vm. The permeation of Ca2+, Na+ and Tris+ through this pathway produces membrane depolarization in the following order of effectiveness: Ca2+ > Na+ > Tris+.
Cross‐talk between ATP‐regulated K+ channels and Na+ transport via cellular metabolism in frog skin principal cells.
Authors: Urbach V , Van Kerkhove E , Maguire D , Harvey B J
Published: December 2014

Isolated frog skin epithelium, mounted in an Ussing chamber and bathed in standard NaCl Ringer solution, recycles K+ across the basolateral membrane of principal cells through an inward‐rectifier K+ channel (Kir) operating in parallel with a Na+‐K+‐ATPase pump. Here we report on the metabolic control of the Kir channel using patch clamping, short‐circuit current measurement and enzymatic determination of cellular (ATP (ATPi). 2. The constitutively active Kir channel in the basolateral membrane has the characteristics of an ATP‐regulated K+ channel and is now classed as a KATP channel. In excised inside‐out patches the open probability (Po) of KATP channels was reduced by ATPi with half‐maximum inhibition at an ATPi concentration of 50 microM. 3. ATPi measured (under normal Na+ transport conditions) with luciferin‐luciferase was 1.50 +/‐ 0.23 mM (mean +/‐ S.E.M.; range, 0.4‐3.3 mM n = 11). Thus the KATP channel would be expected to be inactive in intact cells if ATPi was the sole regulator of channel activity. KATP channels which were inactivated by 1 mM ATPi in excised patches could be reactivated by addition of 100 microM ADP on the cytosolic side. When added alone, ADP blocks this channel with half‐maximal inhibition at [ADPi] > 5 mM. 4. Sulphonylureas inhibit single KATP channels in cell‐attached patches as well as the total basolateral K+ current measured in frog skin epithelia perforated with nystatin on the apical side. 5. Na+‐K+‐ATPase activity is a major determinant of cytosolic ATP. Blocking the pump activity with ouabain produced a time‐dependent increase in ATPi and reduced the open probability of KATP channels in cell‐attached membranes. 6. We conclude that the ratio of ATP/ADP is an important metabolic coupling factor between the rate of Na+‐K+ pumping and K+ recycling.
Neuronal expression in cleavage‐arrested ascidian blastomeres requires gap junctional uncoupling from neighbouring cells.
Authors: Saitoe M , Inazawa T , Takahashi K
Published: December 2014

1. When anterior‐animal (a4‐2) blastomeres isolated from 8‐cell ascidian embryos were cultured under cleavage‐arrested conditions in contact with anterior‐vegetal (A4‐1) blastomeres (a‐A blastomere pairs), the a4‐2 blastomeres differentiated into neuronal cells that expressed Na+ and delayed K+ channels at a time when normal sister embryos became tadpole larvae (after 40 developmental hours at 9 degrees C). When a4‐2 blastomeres were cultured in contact with posterior‐animal (b4‐2) blastomeres (a‐b blastomere pairs), the a4‐2 blastomeres differentiated into epidermal cells expressing Ca2+ channels and tunic on their exterior surface. In these blastomere pairs, we analysed changes in gap junctional communication during neural and epidermal differentiation by using both dye transfer and double voltage clamp. 2. In both types of blastomere pairs, gap junctional communication was detectable at 5 h by double voltage clamp and at 7 h by dye transfer. Gap junctional communication in both types gradually increased until 25 h (equivalent to the neurula stage). However, during 25‐35 h (late neurula or tailbud) in the a‐A pair it decreased and finally disappeared, while it increased steeply in the a‐A pair. When blastomere pairs were treated with a transcription inhibitor, actinomycin D, gap junctional communication also appeared at around 7 h but remained at a plateau level, showing neither a steep increase in a‐b pairs nor a disappearance in a‐A pairs. 3. In blastomere triplets in which an epidermally committed a4‐2 was in contact with both blastomeres of an a‐A pair, the epidermally committed a4‐2 blastomere did, but the neurally committed a4‐2 blastomere did not, communicate through gap junctions with the A4‐1 blastomere, indicating that gap junctional communication is restricted when a4‐2 blastomeres are neurally committed. 4. When a kinase inhibitor, K252a (0.5‐1.0 microM), was applied at 20 h (prior to the disappearance of gap junctional communication), gap junctional communication was maintained in the a‐A pair for more than 40 h. The persistence of gap junctional communication delayed the expression of Na+ and K+ channels in the a4‐2 blastomere. However, channel expression followed an almost normal time sequence in single neuronally committed a4‐2 blastomeres separated from A‐a pairs and treated with K252a. 5. We conclude that the persistence of gap junctions causes a delay in expression of neuronal characteristics, and suggest that one of the functional roles of embryonic gap junctions is to time the expression of neuron‐specific ion channels and other markers in preneuronal cells.
Effect of RNA editing and subunit co‐assembly single‐channel properties of recombinant kainate receptors.
Authors: Swanson G T , Feldmeyer D , Kaneda M , Cull-Candy S G
Published: December 2014

1. Patch‐clamp methods have been used to examine single‐channel properties of recombinant GluR5 and GluR6 kainate‐preferring glutamate receptors which differ in a single amino acid residue as a result of RNA editing at the Q/R (glutamine/arginine) site. Subunits were expressed alone or in combination with the high‐affinity kainate receptor subunit KA ‐ 2 in transfected human embryonic kidney (HEK‐293) cells. 2. In outside‐out patches, unedited homomeric GluR6(Q) receptors exhibited directly resolved domoate‐activated single‐channel conductances of 8, 15 and 25 pS. Variance analysis of GluR6(Q) responses gave a mean conductance of 5.4 pS, while the edited isoform GluR6(R) had an unusually low channel conductance (225 fS). 3. Homomeric channels composed of GluR5(Q) subunits exhibited three conductance states of 5, 9 and 14 pS characterized by prolonged burst activations in the presence of domoate. In contrast, the GluR5(R) subunit, which has not previously been reported to form functional homomeric receptors, had an extremely low conductance (< 200 fS). 4. Heteromeric GluR6(Q)/KA‐2 kainate receptors gave single‐channel events indistinguishible from homomeric GluR6(Q) channels. Conversely, openings produced by GluR5(Q)KA‐2 and GluR5(Q) receptors differed from each other in their kinetic properties. The primary effect of co‐expression of KA‐2 with GluR5(Q) was a dramatic shortening in channel burst length. 5. Spectral and variance analyses were used to estimate mean single‐channel conductances of heteromeric edited receptor‐channels; channel conductances were 950 fS for GluR5(R)KA‐2 receptors and 700 fS for GluR6(R)/KA‐2 receptors. Both receptor types had significantly higher conductances than the respective homomeric channels, GluR5(R) and GluR6(R). 6. We conclude that Q/R site editing dramatically reduces single‐channel conductance. Furthermore, we find similarity between the kainate receptor‐channels described in sensory neurones and the recombinant GluR5(Q) homomeric channel. Characterization of recombinant single‐channel properties could therefore aid identification of the native kainate receptors.
Modulation of Ca2+ channel activity by ATP metabolism and internal Mg2+ in guinea‐pig basilar artery smooth muscle cells.
Authors: McHugh D , Beech D J
Published: December 2014

1. Single smooth muscle cells were isolated from the basilar artery of the guinea‐pig and, within 10 h, inward currents through voltage‐gated Ca2+ channels were recorded using the amphotericin or conventional whole‐cell voltage‐clamp techniques. 2. In amphotericin whole‐cell recordings, bath application of 2,4‐dinitrophenol (DNP, an uncoupler of mitochondrial ATP production) induced an initial stimulation (14% increase in 5 of 11 cells) and then pronounced inhibition (50% decrease in 9 of 11 cells within 9.5 min) of voltage‐dependent Ca2+ current (I(Ca)) elicited by depolarizing to +10 mV in 1.5 mM extracellular Ca2+ solution. By contrast, inhibition of glycolysis by replacing glucose in the bath with 2‐deoxy‐D‐glucose had no effect. 3. Na+ current through Ca2+ channels (I[(Ca)(Na)]) recorded in the absence of extracellular divalent cations also responded to DNP, again with stimulation followed by inhibition of current. The stimulation of I[(Ca)(Na)] was associated with a leftward shift of the Ca2+ channel activation curve which averaged ‐9 mV. A combination of 2‐deoxy‐D‐glucose, mannoheptulose and 3‐0‐methyl‐glucose had only minor effects on I[(Ca)(Na)], whereas rotenone had an effect similar to that of DNP in six of eight cells. 4. The amplitude of I[(Ca)(Na)] in conventional whole‐cell recordings was not different from that in amphotericin whole‐cell recordings, even without ATP in the recording pipette and with metabolic poisons in the bath solution. Furthermore, attempts to dephosphorylate the Ca2+ channels in ATP‐free conditions did not prevent I[(Ca)(Na)], and a high concentration of Mg‐ATP with or without a phosphorylation‐supporting medium in the recording pipette did not increase its amplitude. 5. In the absence of ATP, Mg2+ inhibited whole‐cell I[Ca)(Na)] with a K(d) of about 100 mu M at ‐10 mV and induced a leftward shift of the Ca2+ channel activation curve. When ATP and a phosphorylation‐supporting medium were in the recording pipette the blocking effect of free Mg2+ was reduced but the shift in the Ca2+ channel activation curve was unaffected. 6. From these data it is suggested that inhibition of mitochondrial, but not glycolytic, ATP production has stimulatory and inhibitory effects on voltage‐gated Ca2+ channels of basilar artery smooth muscle cells. Effects of intracellular Mg2+ on the Ca2+ channels were modulated by ATP and mimicked the effects of metabolic poisoning by DNP. A hypothesis is discussed in which the intracellular free Mg2+ concentration may be a key factor coupling ATP production to Ca2+ channels.
Actin microfilament disrupters enhance K(ATP) channel opening in patches from guinea‐pig cardiomyocytes.
Authors: Terzic A , Kurachi Y
Published: December 2014

1. To determine whether actin filament networks are associated with the regulation of ATP‐sensitive K+ (K(ATP)) channel activity, single channel currents were measured in the inside‐out configuration, and cytoskeletal disrupters applied to the internal side of patches excised from guinea‐pig ventricular myocytes. 2. Treatment of patches with DNase I (10‐200 micrograms ml(‐1)), which forms complexes with G‐actin and prevents actin filament formation, antagonized the ATP‐induced inhibition of K(ATP) channels. 3. In the absence of ATP, DNase I did not increase K(ATP) channel activity. 4. When denatured by boiling or co‐incubated with purified actin subunits (200 mu g ml(‐1)), DNase I(100 mu g ml(‐1)) did not antagonize the ATP‐induced inhibition of K(ATP) channels. 5. The DNase I‐induced decrease in the sensitivity of K(ATP) channels towards ATP‐induced inhibition was partially restored by addition of purified actin subunits (200 micrograms ml(‐1)). 6. Cytochalasin B (10 microM), another actin filament disrupter, but neither taxol nor nocodazole (30‐100 microM), two antimicrotubule agents, enhanced K(ATP) channel activity in the presence of ATP. 7. Hence, actin filament disrupters can attenuate the ATP‐dependent inhibitory gating of K(ATP) channels. This suggests that subsarcolemmal actin filament networks may be associated with the regulation of cardiac K(ATP) channels.
Origin of concurrent ATPase activities in skinned cardiac trabeculae from rat.
Authors: Ebus J P , Stienen G J
Published: December 2014

1. To determine the rate of ATP turnover by the sarcoplasmic reticulum (SR) Ca2+ pump in cardiac muscle, and to assess the contributions of other ATPase activities to the overall ATP turnover rate, ATPase activity and isometric force production were studied in saponin‐skinned trabeculae from rat. ATP hydrolysis was enzymatically coupled to the oxidation of NADH; the concentration of NADH was monitored photometrically. All measurements were performed at 20 +/‐ 1 degrees C and pH 7.0. Resting sarcomere length was adjusted to 2.1 microns. All solutions contained 5 mM caffeine to ensure continuous release of Ca2+ from the SR. 2. The Ca(2+)‐independent ATPase activity, determined in relaxing solution (pCa 9), amounted to 130 +/‐ 13 microM s‐1 (mean +/‐ S.E.M., n = 7) at the beginning of an experiment. During subsequent measurements in relaxing solution, a decrease in ATPase activity was observed, indicative of loss of membrane‐bound ATPase activity. The steady‐state Ca(2+)‐independent (basal) ATPase activity was 83 +/‐ 5 microM s‐1 (n = 66). 3. Treatment of saponin‐skinned preparations with Triton X‐100 abolished 50 microM s‐1 (60%) of the basal ATPase activity. Addition of ouabain (1 mM) suppressed 14 +/‐ 5% of the basal activity, whereas 8 +/‐ 3% was suppressed by 20 microM cyclopiazonic acid (CPA). It is argued that 31 microM s‐1 of the basal ATPase activity may be associated with MgATPase from the transverse tubular system. 4. The maximal Ca(2+)‐activated ATPase activity, i.e. the total ATPase activity (determined in activating solution, pCa 4.3) corrected for basal ATPase activity, was found to be 409 +/‐ 15 microM s‐1 (n = 66). Experiments with CPA indicated that at least 9 +/‐ 6% of the maximal Ca(2+)‐activated ATPase activity originates from the sarcoplasmic Ca2+ pump. These experiments indicate that the rate of ATP consumption by the SR Ca2+ transporting ATPase amounts to at least 37 microM s‐1. 5. Treatment of preparations with Triton X‐100 abolished 15 +/‐ 3% of the maximal Ca(2+)‐activated ATPase activity, indicating that 15 +/‐ 3% of the maximal Ca(2+)‐activated ATPase activity is membrane bound. 6. Variation of free [Ca2+] indicated that apart from the actomyosin ATPase activity a second Ca(2+)‐dependent ATPase activity contributed to the overall ATP turnover rate. This activity was half‐maximal at pCa 6.21, and probably reflects the SR Ca2+ transporting ATPase. It constituted 18 +/‐ 3% of the Ca(2+)‐dependent ATPase activity, yielding an upper limit for the SR Ca2+ transporting ATPase activity of 74 microM s‐1.
Substance P contributes to rapidly adapting receptor responses to pulmonary venous congestion in rabbits.
Authors: Bonham A C , Kott K S , Ravi K , Kappagoda C T , Joad J P
Published: December 2014

1. This study tested the hypothesis that substance P stimulates rapidly adapting receptors (RARs), contributes to the increase in RAR activity produced by mild pulmonary congestion, and evokes an augmented response from RARs when combined with near‐threshold levels of pulmonary congestion. 2. RAR activity, peak tracheal pressure, arterial blood pressure and left atrial pressure were measured in paralysed, anaesthetized and ventilated rabbits. Substance P was given i.v. in one‐half log incremental doses to a maximum of 3 micrograms kg‐1. Mild pulmonary congestion was produced by inflating a balloon in the left atrium to increase left atrial pressure by 5 mmHg. Near‐threshold levels of pulmonary congestion were produced by increasing left atrial pressure by 2 mmHg. 3. Substance P produced dose‐dependent increases in RAR activity. The highest dose given increased the activity from 1.3 +/‐ 0.5 to 11.0 +/‐ 3.1 impulses bin‐1. Increases in left atrial pressure of 5 mmHg increased RAR activity from 3.8 +/‐ 1.4 to 14.7 +/‐ 3.9 impulses bin‐1. Blockade of NK1 receptors with CP 96345 significantly attenuated RAR responses to substance P and to mild pulmonary congestion. 4. Doses of substance P, which alone had no effect, stimulated the RARs when delivered during near‐threshold levels of pulmonary congestion. 5. The findings suggest that substance P augments the stimulatory effect of mild pulmonary congestion on RAR activity, most probably by enhancing hydraulically induced microvascular leak.
Activation of delayed rectifier potassium channels in canine proximal colon by vasoactive intestinal peptide.
Authors: Shuttleworth C W , Koh S D , Bayginov O , Sanders K M
Published: December 2014

1. Vasoactive intestinal peptide (VIP) inhibits phasic contractions and tone of gastrointestinal smooth muscles. This study examines electrical mechanisms that may mediate the inhibitory actions of VIP. 2. Electrical slow waves were recorded from canine proximal colon circular muscles. VIP (0.1 microM) decreased basal slow wave frequency but had no effect on amplitude or duration. When slow waves were enhanced with Bay K 8644 (1 microM), VIP decreased slow wave duration and inhibited contractions. 3. VIP inhibited slow waves and phasic contractions stimulated by tetraethylammonium chloride (TEA; 10 mM), but did not significantly reduce events stimulated by 4‐amino‐pyridine (4‐AP; 10 mM). 4. Whole‐cell outward currents were recorded from isolated myocytes, using the amphotericin B perforated patch technique. VIP (1 microM) increased charybdotoxin‐insensitive outward currents. 5. Single voltage‐dependent K+ channels were recorded in cell‐attached patches. VIP increased reversibly the open probability, mean open time and mean burst duration of 4‐AP‐sensitive, charybdotoxin‐insensitive K+ channels (KDR1). Two additional 4‐AP‐ and charybdotoxin‐insensitive K+ channels (approximately 90 pS and < 4 pS) were also observed in these patches, but were not significantly affected by VIP. 6. In summary, the effects of VIP on electrical slow waves may be due, in part, to activation of 4‐AP‐sensitive, ‘delayed rectifier’ K+ channels. Activation of these channels may contribute to premature slow wave repolarization, reduced Ca2+ entry, and inhibition of contractile force.
Binding constants determined from Ca2+ current responses to rapid applications and washouts of nifedipine in frog cardiac myocytes.
Authors: Méry P F , Hove‐Madsen L , Mazet J L , Hanf R , Fischmeister R
Published: December 2014

1. A fast perfusion system was used to analyse the kinetics of the response of L‐type calcium current (ICa) to rapid applications and washouts of the dihydropyridine antagonist nifedipine in whole‐cell patch‐clamped frog ventricular myocytes. 2. Both the inhibition of ICa induced by nifedipine and the recovery from inhibition upon washout of the drug behaved as mono‐exponential functions of time. 3. During application or washout of 100 nM nifedipine, only the peak amplitude of ICa varied but not its time course of activation or inactivation. 4. The rate constant of the onset of ICa inhibition increased with the concentration of nifedipine. However, the time course of the recovery from inhibition was independent of drug concentration. 5. Both rate constants were strongly sensitive to the holding potential but insensitive to the test potential. 6. Using simple rate equations and a one‐binding‐site analysis it was possible to determine the rate constants for association (k1) and dissociation (k‐1) and the equilibrium dissociation constant (KD) of the reaction between nifedipine and Ca2+ channels. KD values for nifedipine were identical to IC50 values obtained from classical steady‐state experiments. 7. With depolarized holding potentials, KD decreased strongly due to a large reduction in k‐1 and a modest increase in k1. Assuming that these changes result from the distribution of Ca2+ channels between resting and inactivated states, a low‐affinity binding to the resting state (R) and a high‐affinity binding to the inactivated state (I) were obtained with the binding constants: k1R = 1.0 x 10(6) M‐1 S‐1, k‐1R = 0.077 S‐1, and KDR = 77 nM for the resting state; k1I = 4.47 x 10(6) M‐1 S‐1, k‐1I = 7.7 x 10(‐4) S‐1, and KDI = 0.17 nM for the inactivated state. 8. Rapid application/washout experiments provide a unique way to determine, in an intact cell and in a relatively short period (2‐4 min), the binding rate constants and the KD value of the reaction between a dihydropyridine antagonist and the Ca2+ channels.
Identification of a native low‐conductance NMDA channel with reduced sensitivity to Mg2+ in rat central neurones.
Authors: Momiyama A , Feldmeyer D , Cull-Candy S G
Published: December 2014

1. We have identified a new type of NMDA channel in rat central neurones that express mRNA for the NR2D subunit. We have examined single NMDA channels in cerebellar Purkinje cells (which possess NR1 and 2D), deep cerebellar nuclei (NR1, 2A, 2B and 2D) and spinal cord dorsal horn neurones (NR1, 2B and 2D). 2. In Purkinje cells, NMDA opened channels with a main conductance of 37.9 +/‐ 1.1 pS and a subconductance of 17.8 +/‐ 0.7 pS, with frequent transitions between the two levels. 3. NMDA activated low‐conductance (‘38/18 pS’) events (along with high‐conductance‐‐'50/40 pS'‐‐openings) in some patches from deep cerebellar nuclei and dorsal horn neurones. Our evidence suggests that 38/18 pS and 50/40 pS events arose from distinct types of NMDA receptors. 4. The transitions for 38/18 pS events were asymmetrical: steps from 38 to 18 pS were more frequent (72.2%) than steps from 18 to 38 pS. This feature appeared common to the 38/18 pS events in all three cell types, suggesting similarity in the low‐conductance channels. 5. The 38/18 pS channels in Purkinje cells exhibited characteristic NMDA receptor properties, including requirement for glycine, antagonism by D‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5) and 7‐chlorokynurenic acid, and voltage‐dependent block by extracellular Mg2+. 6. The mean open time for the 38 pS state (0.74 +/‐ 0.07 ms) was significantly briefer than that for the 18 pS state (1.27 +/‐ 0.18 ms). 7. Mg2+ block of low‐conductance NMDA channels in Purkinje cells was less marked than block of 50/40 pS channels in cerebellar granule cells. 8. The time course of appearance of 38/18 pS NMDA channels matched the expression of mRNA for the NR2D subunit. Thus 38/18 pS events were present in > 70% of Purkinje cell patches in 0‐ to 8‐day‐old animals, and absent by postnatal day 12. 9. We propose that the 38/18 pS NMDA channels identified here (associated with the NR2D subunit), and the other low‐conductance NMDA channel associated with the NR2C subunit, may together constitute a functionally distinct subclass of native NMDA receptors.
Inhibition of nitric oxide synthase does not prevent ocular dominance plasticity in kitten visual cortex.
Authors: Ruthazer E S , Gillespie D C , Dawson T M , Snyder S H , Stryker M P
Published: December 2014

1. The neural messenger molecule nitric oxide (NO) has been shown to be involved in several forms of plasticity including hippocampal long‐term potentiation. We examined the effects of chronic intracortical infusion of inhibitors of NO synthase (NOS) activity on the plasticity of visual cortical responses following monocular lid suture during the critical period. 2. Single unit recordings (618 cells) made in both the NOS inhibitor‐treated (30 mM NG‐methyl‐L‐arginine (L‐NMMA), or 22 or 2 mM nitro‐L‐arginine (L‐NOArg)) and saline‐treated control hemispheres of barbiturate‐anaesthetized, critical‐period kittens (n = 8) revealed a profound shift in favour of the non‐deprived eye. Shifts were of similar magnitude in hemispheres in which NOS was inhibited and in saline control hemispheres. 3. Subsequent analysis of NOS activity in the same cortical tissue in which recordings had been made showed a pronounced decrease in NOS activity in inhibitor‐treated hemispheres. In the region in which all the single unit recordings were made (< 3 mm from the infusion cannula), 22 mM L‐NOArg resulted in a reduction of NOS activity to 5.55 +/‐ 5.33% of control hemisphere NOS activity levels. L‐NOArg (2 mM) and L‐NMMA (30 mM) also produced clear, but smaller, inhibition of NOS activity. 4. These findings demonstrate that NOS activity is not essential for ocular dominance plasticity in visual cortex.
Mechanisms underlying orientation selectivity of neurons in the primary visual cortex of the macaque.
Authors: Sato H , Katsuyama N , Tamura H , Hata Y , Tsumoto T
Published: December 2014

1. Effects of blocking intracortical inhibition by microiontophoretic administration of bicuculline methiodide (BMI), a selective antagonist for GABAA receptors, on orientation selectivity of 109 neurones were studied in the primary visual cortex (V1) of anaesthetized and paralysed monkeys. 2. The averaged orientation tuning of visual responses of cells was poor in cytochrome oxidaserich blobs of layer II/III and in layer IVc beta, moderate in layers IVb, IVc alpha and V, and sharp in the interblob region of layer II/III and in layers IVa and VI. 3. Iontophoretic administration of BMI reduced the sharpness of orientation tuning of cells to a varying extent in each layer. In most cells, furthermore, the originally ineffective stimuli induced visual responses during the BMI administration, suggesting that excitatory inputs evoked by the non‐optimally oriented stimuli were masked by GABAergic inhibition. Nevertheless, the maximal facilitation was observed in the response to the optimally or near‐optimally oriented stimuli. 4. There was a difference in such an effect of BMI among layers. Orientation selectivity of cells in interblobs in layer II/III and in layer IVb was sensitive to BMI whereas that of cells in layer VI was relatively insensitive to BMI, suggesting a larger contribution of excitatory mechanisms to the orientation selectivity in this layer. 5. In the orientation‐selective cells, an analysis of the magnitude of excitation and inhibition evoked by stimuli at various orientations suggests that both inputs tune around the optimal orientation and their magnitudes are almost proportional to each other except at the optimal orientation. This analysis also indicates that the orientation tuning of inhibition had a less prominent peak around the optimal orientation than that of excitation. This dominance of excitation over inhibition around the optimal orientation may function to accentuate the response to the optimally oriented stimulus. 6. These results suggest that, in the monkey V1, the orientation selectivity of cells is largely dependent on the orientation‐biased excitatory and inhibitory inputs which have a broader tuning profile, covering from the optimal to null‐orientation, than that observed in extracellularly recorded responses at the control level.
Natriuretic effect of non‐pressor doses of endothelin‐1 in conscious dogs.
Authors: Sandgaard N C , Bie P
Published: December 2014

1. Renal, endocrine and haemodynamic responses to separate intravenous infusions of three doses of endothelin‐1 (40, 400 and 4000 fmol kg‐1 min‐1) were investigated in conscious dogs. 2. Administration of 40, 400 and 4000 fmol kg‐1 min‐1 endothelin‐1 for 120 min increased plasma endothelin‐1 levels by two‐, seven‐ and 250‐fold, respectively. 3. The two low doses did not have measurable effects on mean arterial blood pressure and heart rate but tended to increase glomerular filtration rate. The high dose increased mean arterial blood pressure (MABP; from 104 +/‐ 4 to 138 +/‐ 4 mmHg, P < 0.05) and decreased heart rate (from 71 +/‐ 4 to 46 +/‐ 3 beats min‐1, P < 0.05) as well as glomerular filtration rate (from 47 +/‐ 3 to 19 +/‐ 5 ml min‐1, P < 0.05). 4. At rates of 40 and 400 fmol kg‐1 min‐1, endothelin‐1 increased sodium excretion about five‐ and eightfold, respectively. Relative changes in fractional sodium excretion were very similar. The high dose was markedly antinatriuretic (reducing sodium excretion from 8.3 +/‐ 1.1 to 1.2 +/‐ 0.2 mumol min‐1, P < 0.05). 5. Diuresis increased during the administration of the two lower doses, which did not change plasma atrial natriuretic peptide or vasopressin concentrations. Urine flow increased after termination of the infusion of the pressor dose despite elevated plasma vasopressin and subnormal glomerular filtration rate. 6. Infusion of endothelin‐1 at 40 fmol kg‐1 min‐1 did not change the concentrations of angiotensin II and atrial natriuretic peptide in plasma. Infusion of 400 fmol kg‐1 min‐1 was associated with a decrease in plasma angiotensin II, while plasma atrial natriuretic peptide was unchanged. The high dose of endothelin‐1 markedly increased plasma levels of both hormones. 7. It is concluded that endothelin‐1 at low plasma concentrations increases sodium excretion while a higher pressor dose of endothelin‐1 is antinatriuretic. However, increases in plasma endothelin‐1 seem to elicit diuresis over a wide concentration range, although possibly by different mechanisms.
Growth and differentiation of fast and slow muscles in fetal sheep, and the effects of hypophysectomy.
Authors: Javen I , Williams N A , Young I R , Luff A R , Walker D
Published: December 2014

1. Isometric contractile characteristics of fast‐twitch (flexor digitorum longus, FDL; medial gastrocnemius, MG) and slow‐twitch (soleus) muscles were determined in pentobarbitone‐anaesthetized fetal sheep between 90 and 140 days gestation. Five fetuses were hypophysectomized (HPX) at 90‐95 days gestation and then studied at 138‐140 days. 2. At 90‐95 days gestation the time to peak of single twitch contractions for the soleus, MG and FDL were not significantly different from each other; the mean value (+/‐S.E.M.) for all the muscles at this age was 77.6 +/‐ 9.0 ms. At 120‐125 days gestation the MG and FDL contracted significantly faster (44.0 +/‐ 0.9 and 40.8 +/‐ 1.8 ms, respectively) than at 90‐95 days, and did not change significantly thereafter. In contrast, the soleus muscle contracted more slowly (111.9 +/‐ 6.6 ms) at 138‐140 days than at 90‐95 days and 120‐125 days gestation. 3. Soleus muscle consisted of type I fibres at all gestational ages. There was no significant change with gestational age in the relative numbers of type I and II fibres in the MG and FDL, but in the diaphragm the number of type I fibres increased and the number of type II fibres decreased between 125 and 138 days gestation. 4. HPX abolished the normal increase of soleus weight relative to body weight between 125 and 138 days but did not alter the change of twitch contraction time with age. HPX significantly prolonged twitch time to peak and time to half‐relaxation of MG and time to half‐relaxation of FDL at 138 days. 5. The maximum rate of rise of the isometric tetanic contraction was unchanged by HPX in all three hindlimb muscles, but fatigue of MG and FDL was increased. 6. The relative proportions of different fibre types in the hindlimb muscles and the diaphragm were unchanged by HPX, but there was a significant decrease in mean areas of type I and II fibres in the FDL and MG of the HPX fetuses.
Open channel and competitive block of the embryonic form of the nicotinic receptor of mouse myotubes by (+)‐tubocurarine.
Authors: Bufler J , Wilhelm R , Parnas H , Franke C , Dudel J
Published: December 2014

1. Embryonic‐like nicotinic channels were studied in mouse myotubes. Channel currents were measured by patch clamping outside‐out excised patches to which pulses of agonists and drugs could be applied by a liquid filament switch. The holding potential of the patches was generally around‐10 to‐40 mV. 2. Pulses of 100 microM or 1 mM acetylcholine (ACh) elicited average channel currents which reached a maximum open probability of 0.93 within 0.5‐1.0 ms, decayed with a time constant of desensitization of 20‐80 ms, and fell rapidly to zero at the end of the pulse. When such pulses together with increasing concentrations of (+)‐tubocurarine (TC) were applied to outside‐out patches, the time constant of current decay, tau, decreased beginning at concentrations of TC added to the test solution of > 10 microM, and the peak amplitude of the current decreased markedly at concentrations of TC of > 30 microM due to an open channel block of nicotinic channels by TC. 3. When the outside‐out patches were pre‐incubated with TC, the peak current elicited by pulses of 100 microM ACh or 1 mM ACh + TC decreased markedly, beginning with concentrations of TC > 30 nM due to a competitive block. 4. The results could be quantitatively modelled by computer calculations based on a circular reaction scheme containing desensitization. TC blocked the open state as well as the unliganded closed state of the embryonic‐like nicotinic receptors of mouse myotubes. Also the blocked open channel was subject to desensitization. 5. The rates of block and unblock of the open channel were 3 x 10(6) M‐1 S‐1 and 0.8 S‐1, respectively, and those of the competitive block were 0.5 x 10(6) M‐1 S‐1 and 0.1 S‐1, respectively (at 20 degrees C).