Currents In Single Myocytes Research Articles

NaHS relaxes rat cerebral artery in vitro via inhibition of l-type voltage-sensitive Ca2+ channel

H2S, a gaseous signalling molecule, relaxes blood vessels partly through activation of ATP-sensitive K+ channels. It is however unclear whether H2S or its donors could affect other ion transporting proteins. The present study examined the hypothesis that NaHS, a H2S donor inhibits voltage-sensitive Ca2+ channels and thus relaxes vascular smooth muscle cells (VSMC) in the cerebral arteries. NaHS dilated cerebral arteries from Sprague–Dawley rats with the same potency against pre-contraction by 5-HT and 60mmol/L KCl, which were unaffected by several K+ channel blockers, NG-nitro-l-arginine methyl ester or indomethacin, as assessed in wire myograph under an isometric condition. Likewise, NaHS also dilated cerebral arteries against myogenic constriction in pressurized myograph under an isobaric condition. NaHS concentration-dependently inhibited CaCl2-induced contraction in Ca2+-free, 60mM K+-containing Krebs solution. Patch clamp recordings showed that NaHS reduced the amplitude of l-type Ca2+ currents in single myocytes isolated enzymatically from the cerebral artery. Calcium fluorescent imaging using fluo-4 showed a reduced [Ca2+]i in 60mmol/L KCl-stimulated rat cerebral arteries in response to NaHS. H2S precursor l-cysteine-induced relaxation in cerebral arteries was inhibited by cystathionine γ–lyase (CSE) inhibitor dl-propargylglycine. CSE was expressed in cerebral arteries. In summary, NaHS dilates rat cerebral arteries by reducing l-type Ca2+ currents and suppressing [Ca2+]i of arterial myocyte, indicating that NaHS relaxes cerebral arteries primarily through inhibiting Ca2+ influx via Ca2+ channels

Cardamonin Is a Bifunctional Vasodilator that Inhibits Cav1.2 Current and Stimulates KCa1.1 Current in Rat Tail Artery Myocytes

An in-depth analysis of the effects of cardamonin, 2',4'-dihydroxy-6'-methoxychalcone, on rat tail artery preparations was performed by means of whole-cell patch-clamp recordings of Ca(v)1.2 Ca(2+) [I(Ca(L))] or Ba(2+) [I(Ba(L))] current as well as K(Ca)1.1 currents in single myocytes and by measuring contractile responses in endothelium-denuded isolated rings. At a holding potential (V(h)) of -80 mV, cardamonin decreased both I(Ba(L)) and I(Ca(L)) in a concentration-dependent manner with similar pIC(50) values. The maximum of the I(Ba(L))-voltage relationship was shifted by 10 mV in the hyperpolarizing direction, but threshold remained unaffected. Cardamonin modified both the activation and the inactivation kinetics of I(Ba(L)) and shifted the voltage dependence of both inactivation and activation curves to more negative potentials by 19 and 7 mV, respectively, thus markedly decreasing the Ba(2+) window current. Block of I(Ba(L)) was frequency-dependent, and rate of recovery from inactivation was slowed. Cardamonin increased K(Ca)1.1 currents in a concentration-dependent manner; this stimulation was iberiotoxin- and BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]-sensitive. On the contrary, iberiotoxin did not modify cardamonin-induced relaxation of rings precontracted either with phenylephrine or with (S)-(-)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate [(S)-(-)-Bay K 8644]. The overall effects of cardamonin were incompletely reversed by washout. In conclusion, cardamonin is a naturally occurring, bifunctional vasodilator that, by simultaneously inhibiting I(Ca(L)) and stimulating K(Ca)1.1 current, may represent a scaffold for the design of novel drugs of potential interest for treatment of systemic hypertension.

Ryanodine receptor type 2 deficiency changes excitation–contraction coupling and membrane potential in urinary bladder smooth muscle

The possibility that the ryanodine receptor type 2 (RyR2) can function as the major Ca(2+)-induced Ca(2+) release (CICR) channel in excitation-contraction (E-C) coupling was examined in smooth muscle cells (SMCs) isolated from urinary bladder (UB) of RyR2 heterozygous KO mice (RyR2+/-). RyR2 mRNA expression in UB from RyR2+/- was much lower than that in wild-type (RyR2+/+. In single UBSMCs from RyR2+/+, membrane depolarization under voltage clamp initially induced several local Ca(2+) transients (hot spots) in peripheral areas of the cell. Then, Ca(2+) waves spread from Ca(2+) hot spots to other areas of the myocyte. The number of Ca(2+) hot spots elicited by a short depolarization (< 20 ms) in UBSMCs of RyR2+/- was significantly smaller than in those of RyR2+/+. The force development induced either by direct electrical stimulation or by 10 microm acetylcholine in tissue segments of RyR2+/- was smaller than and comparable to those in RyR2+/+, respectively. The frequency of spontaneous transient outward currents in single myocytes and the membrane depolarization by 1 microm paxilline in tissue segments from RyR2+/- were significantly lower and smaller than those in RyR2+/+, respectively. The urination frequency and volume per voiding in RyR2+/- were significantly increased and reduced, respectively, compared with RyR2+/+. In conclusion, RyR2 plays a crucial role in the regulation of CICR during E-C coupling and also in the regulation of resting membrane potential, presumably via the modulation of Ca(2+)-dependent K(+) channel activity in UBSMCs and, thereby, has a pivotal role in the control of bladder activity.

Spontaneous Slow Wave and Contractile Activity of the Guinea Pig Prostate

We characterized the electrical events underlying spontaneous contractions of the stroma of the guinea pig prostate. Membrane potential of the stroma was recorded using standard electrophysiological recording techniques. The structure of the prostate was viewed using confocal or electron microscopy. In stromal cells spontaneous depolarizing membrane transients (12 mV. in amplitude) occurred at 5 minutes-1 and triggered 1 or more spikes. The membrane potential, and frequency and duration of the potential transients were not affected by the calcium channel blocker nifedipine (1 microM. for greater than 5 minutes), or blockers of neuronal propagation (tetrodotoxin), and the effects of cholinergic (atropine), adrenergic (guanethidine or prazosin) and sensory blockers (capsaicin) of neurotransmission. However, the amplitude of the superimposed spikes was significantly reduced by nifedipine. A network of c-Kit immunoreactive cells was evident in the interstitial layer between the stroma and glandular lined lumen. These prostatic interstitial cells contained many morphological features distinguishing interstitial cells of Cajal, the pacemaker cells of the gastrointestinal tract. Prostatic interstitial cells formed close appositions with each other, with neighboring smooth muscle cells and with varicose axon bundles. Movement of the contents of the peripheral prostatic acini into the minor and major prostatic ducts is likely to occur via spontaneous contractions triggered by myogenic slow wave activity in the stromal wall. By analogy with the intestine and urethra prostatic interstitial cells may well act as the pacemaker for prostatic slow waves as well as form an intercellular communication network interacting with the intrinsic nerves and stromal cells.

Glyburide effect on contractility and calcium current insingle myocytes

Glyburide effect on contractility and calcium current insingle myocytes

Quantification of Ca2+ uptake in the sarcoplasmic reticulum of trout ventricular myocytes.

We measured Ca2+ uptake by the sarcoplasmic reticulum (SR) in trout ventricular myocytes, measuring indo 1 fluorescence in permeabilized cells or ionic currents in single myocytes subjected to voltage clamp. Titration of the SR Ca2+ pumps with thapsigargin gave a pump site density of 454 pmol/mg cell protein. Lowering the temperature from 20 degreesC to 10 or 5 degreesC reduced the SR Ca2+ uptake rate in permeabilized myocytes by 50 and 63%, respectively. Surprisingly, Ca2+ leak from the SR also decreased with decreasing temperatures. Exposure of single myocytes to 10 mM caffeine (Caf) induced a cell contracture and an inward ionic current. Neither contracture nor current decreased significantly after rest periods of 120 and 320 s. The inward current was due to Ca2+ extrusion by the Na+/Ca2+ exchanger (NCX), and the time integral of the exchange current (INCX) was used to calculate the SR Ca2+ content. This gave a steady-state SR Ca2+ content of 22.5 +/- 2.8 amol Ca2+/pF or 750 microM. When the SR was loaded by depolarizing the cell to +50 mV, the Ca2+ content increased with increasing length of the depolarization, reaching a maximum of 52.0 +/- 5.9 amol Ca2+/pF. When the cell was depolarized to different voltages for 3 s, a subsequent Caf-induced INCX increased with increasing voltage. At +100 mV, the Ca2+ content was 36.6 +/- 3.8 amol/pF, giving a maximal SR Ca2+ uptake rate of 12.2 +/- 1.2 amol Ca2+. pF-1. s-1 or 417 microM/s. We conclude that maximal SR Ca2+ content and Ca2+ uptake rates can be estimated using specific SR Ca2+ loading protocols. Contrary to the general assumption that contraction in lower vertebrates depends largely on transsarcolemmal Ca2+ fluxes, we found that although the L-type Ca2+ current is insufficient to fully activate contraction, the SR is capable of participating in the regulation of the cytosolic Ca2+ during the excitation-contraction coupling in trout ventricular myocytes.

Effects of nitric oxide donors, S-nitroso-L-cysteine and sodium nitroprusside, on the whole-cell and single channel currents in single myocytes of the guinea-pig proximal colon.

1. The nature of the membrane channels underlying the membrane conductance changes induced by the nitric oxide (NO) donors, S-nitroso-L-cysteine (NOCys) and sodium nitroprusside (SNP) were investigated in single myocytes isolated from the circular muscle layer of the guinea-pig proximal colon, by use of standard whole-cell and single channel recording techniques. 2. Under voltage clamp, depolarizing steps from -60 mV elicited a rapidly-developing, little-inactivating outward K+ current (IK) at potentials positive to -40 mV (at 20-25 degrees C). The steady-state level (ISS) of this K current increased in amplitude as the step potential was made to more positive potentials. If the depolarizing steps were made from a holding potential of -80 mV an additional rapidly activating and inactivating outward K+ current was also elicited, superimposed on IK. 3. At 20-25 degrees C, NOCys (2.5 microM), SNP (100 microM) and 8-bromo-cyclic GMP (500 microM) increased the amplitude of ISS of IK elicited from a holding potential of -60 mV. In contrast, NOCys (2-5 microM) had little effect on ISS at 35 degrees C. Higher concentrations (> or = 5 microM at 20-25 degrees C and > or = 10 microM at 35 degrees C) of NOCys decreased the peak amplitude (I[Peak]) and ISS of IK in a concentration-dependent manner. This blockade of IK with NOCys was always associated with an increase of the holding current (IHold), due to the activation of a membrane conductance with a reversal potential between 0 and + 30 mV and which was reduced approximately 50% upon the addition of Cd2+ (1 mM). 4. NOCys (2.5 to 10 microM) or SNP (100 microM) increased the activity of large conductance Ca2+-activated (BK) K' channels in both cell-attached and excised inside-out patches, bathed in either a symmetrical high K+ (130 mM) or an asymmetrically K+ (6 mMout: 130 mMin) physiological saline. Increases in BK channel activity in NOCys (10 microM) or SNP (100 microM) were associated with an increase in the probability of BK channel opening (N.Po), and with a negative shift of the plots of ln(N.Po) against the patch potential, with little change in the slopes of these plots. In cell-attached patches, the increase in N.Po with NOCys was often associated with a decrease in the BK single channel conductance. 5. In both cell-attached and excised patches, NOCys (2.5 to 10 microM) also activated an additional population of channels which allowed inward current flow at potentials positive to EK. In excised inside-out patches bathed in asymmetrical K+ physiological saline, these single channel currents were 2-3 pA in amplitude at -30 mV and reversed in direction near + 10 mV, even if the NaCl (126 mM) concentration in the pipette solution had been replaced with an equimolar concentration of Na gluconate. 6. Under current clamp, NOCys (2.5 microM) and SNP (100 microM) had variable effects on the membrane potential of colonic myocytes, inducing either a small membrane hyperpolarization of <5 mV, or a slowly-developing membrane depolarization of about 5 mV. In contrast, NOCys (5 microM) produced a transient membrane hyperpolarization which was followed by a large depolarization of the membrane potential to positive potentials. The electrotonic potentials elicited in response to an injection of constant hyperpolarizing current (10 pA for 400 ms) were little changed during the NOCys (5 PM)-induced membrane hyperpolarization, but significantly reduced (to 61% of control) during the periods of membrane depolarization. 7. It was concluded that NOCys and SNP, directly increased the number of active BK channels in the membrane of colonic myocytes which leads to a small rapidly oscillating membrane hyperpolarization. The following rebound depolarization in NOCys arises from both the direct opening of a population of cationic channels and the blockade of voltage- and Ca-activated K+ conductances. Finally, the apamin-sensitive K+channels underlying the initial transient hyperpolarization recorded in the intact proximal colon, in response to nerve-released or directly-applied NO, have yet to be identified at the single channel or whole-cell current level.

Amiodarone blocks L-type calcium current in single myocytes isolated from the rabbit atrioventricular node

1. 1. Single myocytes were isolated from the rabbit atrioventricular node (AVN) and whole cell patch clamped, using a Cs +-based internal dialysis solution. Depolarizing voltage clamps from a holding potential of −40 mV were applied to investigate the effects of the antiarrhythmic agent amiodarone on L-type calcium current (I Ca,L). 2. 2. The current-voltage (I–V) relation for I Ca,L was bell shaped in normal Tyrode's solution, with a peak at +10 mV. After a 1-min exposure to 10 μM amiodarone, I Ca,L at all potentials between −30 mV and +60 mV was largely and reversibly blocked ( n=4). In 13 cells, peak I Ca,L was blocked by 85% after amiodarone application. 3. 3. The time course of the blocking effect was monitored during continuous pulsing (at 0.33 Hz) from −40 mV to + 10 mV to observe the time course of I Ca,L blockade. This could be described by a single exponential function with a rate constant of 0.21 per second. In other cells, amiodarone was applied for 30 sec without any stimulation; and, when stimulation was resumed, the I Ca,L amplitude with the first pulse was 22% of the control amplitude ( n=4), indicating that a substantial portion of the blockade of I Ca,L by amiodarone was tonic in nature. 4. 4. In addition to the effect of amiodarone on the I Ca,L channel, when the β adrenergic agonist isoprenaline (1 μM) was applied to cells that had been pretreated with amiodarone, I Ca,L amplitude was increased by 44.5±10.8% ( n=4). In cells that had received no such pretreatment with amiodarone, isoprenaline increased the I Ca,L amplitude by 101.5±5.9% ( n=4). Thus, isoprenaline produced a larger increase in I Ca,L in the absence of amiodarone than in its presence ( P<0.001). 5. 5. It is concluded that, in single AVN cells, amiodarone exerts a direct I Ca,L blocking action. In addition, the attenuated increase in I Ca,L with isoprenaline in amiodarone pretreated cells appears consistent with an antagonism of β adrenergic stimulation of I Ca,L.

Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes.

1. Whole-cell and amphotericin-perforated patch-clamp techniques have been used to study the effects of hydrogen peroxide (H2O2) on action potentials and underlying ionic currents in single myocytes from the ventricles of adult rat hearts. 2. The results obtained differed markedly depending on the recording method utilized. Conventional whole-cell recordings, in which the myoplasm is dialysed with the contents of the pipette, failed to show any significant effects of H2O2 on the action potential or cell shortening. In contrast, when action potentials were recorded with the amphotericin-perforated patch method, H2O2 (50-200 microM) produced a marked prolongation of the action potential and an increase in cell shortening. 3. Voltage-clamp recordings with the amphotericin-perforated patch method showed that H2O2 caused no significant changes in either the Ca(2+)-independent transient outward K+ current (Ito) or the inwardly rectifying K+ current (IK1). 4. Application of tetrodotoxin (TTX; 8 x 10(-6) M), a Na+ channel blocker, largely inhibited the effects of H2O2 on the action potential. Moreover, anthopleurin A (4 x 10 (-7) M), which augments Na+ current (INa) by slowing its inactivation, mimicked the effects of H2O2 on the action potential of ventricular myocytes. These effects on INa were also blocked almost completely by TTX. 5. The hypothesis that H2O2 can augment INa by slowing its kinetics of inactivation was tested directly using ensemble recordings from cell-attached macropatches. These results demonstrated a significant enhancement of late opening events when H2O2 (200 microM) was included in the recording pipette. A corresponding slowing of inactivation of the ensemble INa was observed. 6. The possibility that protein kinase C (PKC) is an intracellular second messenger for the observed effects of H2O2 was examined using the blocker bisindolylmaelimide (BIS; 10(-7) M). Bath application of BIS prior to H2O2 exposure significantly delayed and also attenuated the development of the action potential prolongation. 7. These results demonstrate marked electrophysiological effects of H2O2 in rat ventricle. The dependence of these effects on recording methods suggests involvement of an intracellular second messenger, and the results with the PKC inhibitor, BIS, support this possibility. The most prominent effect of H2O2 on the ionic currents which underlie the action potential is a slowing of inactivation of the TTX-sensitive INa. Recent molecular studies have demonstrated a PKC phosphorylation site on the rat cardiac Na+ channel isoform and have also shown that PKC activation can slow inactivation of INa.

Ca2+ currents in single myocytes from human mesenteric arteries: evidence for a physiological role of L-type channels.

1. Voltage-gated Ca2+ currents (ICa) in isolated human mesenteric arterial cells were characterized in solutions containing normal (1.5 mM) Ca2+ and elevated concentrations of divalent cations using the conventional whole-cell patch clamp technique. 2. In normal Ca2+ solution, depolarization beyond -40 mV elicited a slowly decaying ICa which reached a maximum at +10 mV and appeared to reverse between +40 and +50 mV. The amplitude of this current in a group of cells correlated with cell membrane capacitance. 3. In two of thirty-three cells a small transient component of inward current was detected in the voltage range between -40 and -10 mV when cells were held at -80 mV. This current was abolished at a holding potential of -40 mV, while the current at 10 mV was not affected. These currents were referred to as T- and L-type Ca2+ respectively. 4. Elevation of the extracellular Ca2+ concentration to 20 mM shifted the voltage dependencies of Ca2+ current activation and inactivation by approximately +20 mV; a small T-current component was then observed in seven of nine cells held at -60 mV. 5. Replacement of 1.5 mM Ca2+ with 10 mM Ba2+ increased the amplitude of the current elicited at +10 mV by a factor of 3.7 and a small barium current (IBa) through T-type Ca2+ channels was also observed in most cells studied. Activation and steady-state inactivation curves for L-type current were found to be almost identical in both solutions. The steady-state inactivation for the T-type IBa was, however, more than 30 mV more negative (half-inactivation potential of -62.6 mV) of that for L-current in 1.5 mM Ca2+ and 10 mM Ba2+ solutions (-30.4 and -24.9 mV respectively). 6. A sustained inward Ca2+ channel current was recorded in the presence of normal Ca2+ and high divalent cation concentrations during 30 s depolarizations. The amplitude of this sustained current was found to be similar to the theoretical 'window current' predicted by the overlap of the activation and inactivation functions in these solutions. 7. Examination of the inactivation of the L-type current using a two-pulse protocol with a 240 ms prepulse revealed a U-shaped potential dependency for ICa, but not for IBa, suggesting the presence of a Ca(2+)-dependent component of the inactivation process. 8. These cells resemble other arterial smooth muscle cells previously studied in that they demonstrate both T- and L-components of ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological effects of calcitonin gene‐related peptide in bull‐frog and guinea‐pig atrial myocytes.

1. Electrophysiological effects of calcitonin gene-related peptide (CGRP) on action potentials and corresponding transmembrane currents in single myocytes from bull-frog and guinea-pig atria were studied using a whole-cell voltage-clamp method. 2. CGRP at relatively low concentrations increased the height of the action potential plateau in a dose-dependent manner in both bull-frog and guinea-pig myocytes. In addition, in bull-frog cells CGRP accelerated the early phase of repolarization, thus shortening the overall duration of the action potential. In contrast, in guinea-pig myocytes CGRP prolonged the action potential duration at all concentrations that were studied. 3. Voltage-clamp measurements demonstrated that CGRP increased transmembrane calcium current (ICa) in guinea-pig myocytes without a significant change in its voltage dependence. The ED50 value for this effect on ICa was 1.28 +/- 0.55 X 10(-8) M (n = 4). The time course of the inactivation of ICa was not affected by CGRP. 4. CGRP increased the delayed rectifier K+ current (IK) at relatively low concentrations in bull-frog atria, whereas relatively high concentrations were needed to increase IK in guinea-pig myocytes. This effect was observed even after complete inhibition of ICa. 5. CGRP had no significant effect on the inwardly rectifying background K+ current, IK1, even at very high concentrations. 6. Comparison of the time course of ICa augmentation in bull-frog and guinea-pig myocytes revealed an important difference in the effect of CGRP in these two types of cells. CGRP at maximal concentrations increased ICa transiently in bull-frog myocytes, whereas this response was sustained in guinea-pig myocytes. Isoprenaline (Iso) induced sustained increase in ICa in both species. When ICa was fully activated by Iso, CGRP at high concentrations strongly inhibited ICa in the bull-frog, whereas it had little effect on ICa in guinea-pig myocytes. 7. Intracellular application of GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) 10(-4) M) greatly potentiated the CGRP effect on ICa; in contrast, GDP beta S (guanosine 5'-O-(2-thiodiphosphate), 2 x 10(-3) M) partially inhibited the CGRP-induced augmentation of ICa. Taken together, these results indicate that the stimulation of ICa by CGRP is mediated by a GTP-binding protein. 8. The observed dose-dependent changes in ICa and IK in bull-frog and guinea-pig myocytes can explain the different patterns of CGRP-induced changes in action potential shape in these two myocyte preparations.

Slow inward tail currents in rabbit cardiac cells.

1. A whole-cell gigaseal suction microelectrode voltage-clamp technique has been used to study slow inward tail currents in single myocytes obtained by enzymatic dispersion of rabbit ventricle and atrium. A variety of stimulation protocols, Tyrode solutions and pharmacological agents have been used to test three hypotheses: (a) that the slow inward tail current is generated by an electrogenic Na(+)-Ca2+ exchanger; (b) that a rise in [Ca2+]i, due to release from the sarcoplasmic reticulum can modulate the activity of this exchanger; and (c) that the uptake of calcium by the sarcoplasmic reticulum is a major determinant of the time course of the tail current. 2. As shown previously in amphibian atrium and guinea-pig ventricle, slow inward tail currents can be observed consistently under conditions in which action potentials and ionic currents are recorded using microelectrode constituents which only minimally disturb the intracellular milieu. 3. In ventricular cells, the envelope of these tail currents obtained by varying the duration of the preceding depolarizations shows that (a) the tail currents are activated by pulses as short as 10 ms, and reach a maximum for pulse durations of 100-200 ms, (b) the rate of decay of the tail current gradually increases as the activating depolarizations are prolonged, and (c) the tails cannot be due to deactivation of calcium currents, in agreement with other studies in frog heart. 4. When the mean level of [Ca2+]i is raised following inhibition of the Na(+)-K+ pump by strophanthidin (10(-5) M) or reductions in [K+]o (0.5 mM), the slow inward tail grows in size prior to the onset of a contracture or other signs of calcium-induced toxicity. 5. In a number of different preparations, replacement of [Ca2+]o with BaCl2 markedly or completely inhibits the Na(+)-Ca2+ exchanger, whereas Sr2+ replacement does not have this effect. In myocytes from rabbit ventricle the slow inward tails are reduced significantly and decay more slowly in 0.5-2.2 mM-BaCl2 Tyrode solution, while in 2.2 mM SrCl2 these tails are not altered. 6. The slow inward tail also shows a dependence on [K+]o, corresponding to previous data on Na(+)-Ca2+ exchange in other tissues. Increasing [K+]o in the Tyrode solution to a final concentration of 10-15 mM results in a marked inhibition of the slow tails. This effect cannot be accounted for by changes in the inwardly rectifying potassium current, IK1. 7. The slow tail currents were changed significantly by increasing the temperature of the superfusing Tyrode solution.(ABSTRACT TRUNCATED AT 400 WORDS)