Myocytes Of Guinea-pig Heart Research Articles

Ionic mechanism underlying distinctive excitability in atrium and ventricle of the heart.

Cellular excitability is an important physiological factor in maintaining normal cardiac activity. The present study was designed to investigate the ionic mechanism underlying different excitability in atrial and ventricular myocytes of guinea pig heart using a whole-cell patch configuration. We found that excitability is lower in ventricular myocytes than that in atrial myocytes. Although the density of voltage-gated fast Na(+) current (INa) was lower in ventricular myocytes, it would not correlate to the lower excitability since its availability was greater than that in atrial myocytes around threshold potential. Classical inward rectifier K(+) current (IK1) was greater in ventricular myocytes than that in atrial myocytes, which might contribute in part to the lower excitability. In addition, the transient outward K(+) current with inward rectification (Itoir) elicited by depolarization was greater in ventricular myocytes than that in atrial myocytes and might contribute to the lower excitability. In ventricular myocytes, Ba(2+) at 5 µmol/L significantly inhibited Itoir, enhanced excitability, and shifted the threshold potential of INa activation to more negative, and the effect was independent of affecting INa. Our results demonstrate the novel information that in addition to classical IK1, Itoir plays a major role in determining the distinctive excitability in guinea pig atrial and ventricular myocytes and maintaining cardiac excitability. More effort is required to investigate whether increase of Itoir would be protective via reducing excitability.

Mechanism of activation of the tonic component of contraction in myocytes of guinea pig heart.

Contractions of myocytes of guinea pig heart consist of a phasic component relaxing independently on the voltage and a tonic component relaxing upon repolarization. We found previously that Ca(2+) activating the tonic component is released from the sarcoplasmic reticulum. In this study, we analysed the mechanism of activation and maintenance of this release. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses of the duration of 300 ms to 15-45 s from the holding potential of -40 to +5 mV. [Ca(2+)](i) was monitored as fluorescence of Indo-1 and contractions were recorded with the TV edge-tracking system. Myocytes responded to the short and long pulses with phasic contraction or Ca(2+) transient followed by the sustained contraction or increase in [Ca(2+)](i). Repolarization brought about relaxation. 10 mmol L(-1) Ni(2+) blocking Na(+)/Ca(2+) exchange superfused during the tonic component increased its amplitude. Superfusion of Ca(2+)-free solution during sustained contraction brought about relaxation both in normal cells and in cells superfused with Ni(2+) despite preserved sarcoplasmic reticulum Ca(2+) content assessed with caffeine spritz. Relaxing effect of Ca(2+)-free solution was not affected by carboxyeosin, a blocker of sarcolemmal Ca(2+)-ATPase. Tonic component of contraction and of Ca(2+) transient was inhibited by 200 micromol L(-1) ryanodine, a blocker of Ca(2+) release channels of the sarcoplasmic reticulum and by 20 micromol L(-1) nifedipine, a blocker of L-type I(Ca). Tonic component of contraction results from Ca(2+) release via the sarcoplasmic reticulum Ca(2+) channels activated by sustained, nifedipine-sensitive and Ni(2+)-insensitive Ca(2+) influx. Alternatively, the SR Ca(2+) release is activated by voltage, the dihydropyridine receptors acting like voltage sensors.

Isoflurane decreases ATP sensitivity of guinea pig cardiac sarcolemmal KATP channel at reduced intracellular pH.

Volatile anesthetics can protect the myocardium against ischemic injury by opening the adenosine triphosphate (ATP)-sensitive potassium (K(atp)) channels. However, direct evidence for anesthetic-channel interaction is still limited, and little is known about the role K(atp) channel modulators play in this effect. Because pH is one of the regulators of K(atp) channels, the authors tested the hypothesis that intracellular pH (pHi) modulates the direct interaction of isoflurane with the cardiac K(atp) channel. The effects of isoflurane on sarcolemmal K(atp) channels were investigated at pHi 7.4 and pHi 6.8 in excised inside-out membrane patches from ventricular myocytes of guinea pig hearts. At pHi 7.4, intracellular ATP (1-1,000 microm) inhibited K(atp) channels and decreased channel open probability (Po) in a concentration-dependent manner with an IC(50) of 8 +/- 1.5 microm, and isoflurane (0.5 mm) either had no effect or decreased channel activity. Lowering pHi from 7.4 to 6.8 enhanced channel opening by increasing Po and reduced channel sensitivity to ATP, with IC shifting from 8 +/- 1.2 to 45 +/- 5.6 microm. When applied to the channels activated at pHi 6.8, isoflurane (0.5 mm) increased Po and further reduced ATP sensitivity, shifting IC(50) to 110 +/- 10.0 microm. Changes in pHi appear to modulate isoflurane interaction with the cardiac K(atp) channel. At pHi 6.8, which itself facilitates channel opening, isoflurane enhances channel activity by increasing Po and reduces sensitivity to inhibition by ATP without changing the unitary amplitude of single channel current or the conductance. These results support the hypothesis of direct isoflurane-K(atp) channel interaction that may play a role in cardioprotection by volatile anesthetics.

On the source of Ca(2+) activating the tonic component of contraction of myocytes of guinea pig heart.

Contractions of isolated, single myocytes of guinea pig heart stimulated at 37 degrees C consist of a phasic component and a voltage dependent tonic component. In this study we investigated the source of Ca(2+) activating the tonic component. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped cells were stimulated by the pulses from the holding potential of -40 to +5 mV. [Ca(2+)](i) was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. Superfusion of 5 mmol/l Ni(2+) during 30 s pause did not inhibit subsequent biphasic Ca(2+) transients and contractions despite inhibition of Ca(2+) current and Na(+)/Ca(2+) exchange. KB-R7943 (5 micromol/l) or intracellular dialysis with 0 Na(+) solution, both of which inhibit reversed Na(+)/Ca(2+) exchange, decreased amplitude of Ca(2+) transients and contractions by approximately 40%. The ratio of amplitudes of tonic to phasic component was increased by Ni(2+) and was not changed by KB-R7943 or 0 Na(+)(i). Ryanodine (200 micromol/l) inhibited both components of contractions in cells superfused with Ni(2+). The phasic component but not the tonic component was inhibited by 20 micromol/l nifedipine in cells superfused with Ni(2+). Tonic component of contraction of single myocytes of guinea pig heart is not activated by Ca(2+) current or by the reverse mode Na(+)/Ca(2+) exchange as currently proposed in literature. Rather, it is activated by Ca(2+) released from the sarcoplasmic reticulum. However, kinetics and mechanism of release seem to be quite different from those of Ca(2+) fraction activating the phasic component of contraction.

Restitution of contractility in single ventricular myocytes of guinea pig heart.

Our aim was to assess the extent to which changes in intracellular Ca2+ stores contribute to mechanical restitution in heart muscle. Single, isolated guinea pig ventricular cells were voltage clamped at -45 mV and stimulated continuously at 0.5 or 2 Hz with 200 ms depolarizing pulses (35 degrees C). The recoveries of the peak of contraction force (Fp) and the calcium current (ICa) between beats were measured in contractions interpolated at various intervals (td) after a conditioning twitch. Recovery of SR Ca2+ load was inferred from the peak magnitude (Cp) of similarly interpolated contractures, induced by rapid application of 5 mM caffeine. For a conditioning stimulus rate of 0.5 Hz, both Fp and ICa were very small for small td and recovered along similar time courses with a t1/2 of about 50 ms. Cp was maximal at as early a time after a previous contraction as could be measured, at which time Fp was 56% of maximal. Cp declined throughout the stimulus interval to about 50% of its maximal value. Similar results were obtained for a conditioning stimulus rate of 2 Hz, at which rate both Fp and Cp were increased by a factor of 2. The time course of mechanical restitution is coincident with the recovery of ICa from inactivation. Caffeine-releasable intracellular calcium stores are fully recovered soon after a contraction and well before mechanical restitution is complete.

Cell distension-induced increase of the delayed rectifier K+ current in guinea pig ventricular myocytes.

Single ventricular myocytes of guinea pig heart were distended by applying a positive pressure of 5 to 20 mm Hg in the pipette during the whole-cell voltage clamp. The amplitude of delayed rectifier K+ current (I(K)) was increased by approximately 1.5 times, whereas the inward rectifier K+ current was scarcely affected. The increase of I(K) was reversible by applying a negative pressure of -10 to -30 mm Hg accompanied by shrinkage of the inflated cell. This response of I(K) was largely attributed to the E-4031-insensitive component of I(K). The fully activated current amplitude, measured using long-lasting depolarizing pulses (> 30 seconds) to +60 mV, was increased by the cell distension. The activation time course of I(K) during the long pulse consisted of more than three exponential components, and the slowest time constant was decreased by the distension from control 20.2 +/- 7.7 seconds (n=4) to 7.6 +/- 1.6 seconds (n=5). We failed to detect an involvement of microtubules or microfilaments, protein kinase C, and Ca2+ in the inflation-mediated increase of I(K).

Effects of an extract of Ginkgo biloba On the action potential and associated transmembrane ionic currents in mammalian cardiac myocytes: Inhibition of isoproterenol‐induced chloride current

AbstractVentricular myocytes of guinea pig heart were used to examine the effects of an extract of Ginkgo biolba (EGb 761) on the action potential and individual transmembrane ionic currents. Elecrophysiological events were recorded using the “whole‐cell” configuration of the patch‐clamp technique. A systematic analysis of the data revealed that EGb 761 (5‐50 μg/ml) did not affect the normal action potential or the various ionic currents involved in its generation; i.e., fast inward sodium current (INa), inward calcium currents (ICaT and ICaL), delayed outward potassium current (IK), inward rectifying potassium current (IK1), and ATP‐senstive potassium current (IK‐ATP) evoked by 2,4‐dinitrophenol (2,4‐DNP). However, EGb 761 (≥5 μg/ml) elicited a pronouced concentration‐dependent and reversible inhibiton of isoproternol‐induced Cl− current (ICl), the maximal effect being observed at 50 μg/ml. This current may be significantly involved in sympathetic hyperactivity, hypoxia, and ischemia, pathophysiological conditions for which EGb 761 offers therapeutic benefit. The basic mechanism(s) underlying the inhibitory effect of EGb 761 on ICl and the constituent(s) of EGb 761 responsible for this action remain to be identified, but it seems clear, from results which showed that neither ginkgolide B (50–500 ng/ml) nor biolobalide (150–1,500 ng/ml) influenced this current, that a terpenoid constituent of EGb 761 is probably not involved.

The role of sarcoplasmic reticulum and NaCa exchange in Ca extrusion from the resting single myocytes of guinea-PIG heart

The role of sarcoplasmic reticulum and NaCa exchange in Ca extrusion from the resting single myocytes of guinea-PIG heart

On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart.

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.

Contraction and sarcoplasmic reticulum Ca2+ content in single myocytes of guinea pig heart: effect of ryanodine

The relationship between the ability of sarcoplasmic reticulum (SR) to accumulate and retain Ca2+ and the electrically stimulated contractions (ESCs) of isolated cells from guinea pig ventricular myocardium was investigated. Caffeine contractures or rapid cooling contractures were used as a relative measure of the SR Ca2+ content. Depletion of SR Ca2+ by short exposure to caffeine (15 mM) or by prolonged rest resulted in a reduction of the amplitude of the ESCs by 83 +/- 14 and 65 +/- 11% (means +/- SD), respectively. This result points to SR as a major source of the Ca2+ that activates contraction. However, depriving the SR of the ability to retain Ca2+ by means of prolonged (up to 75 min) exposure to 0.1 microM ryanodine (as shown by the absence of contractile response to caffeine or cooling) did not prevent an ESC of nearly normal amplitude (81 +/- 24% control), albeit with a reduced contraction velocity and a time to peak contraction prolonged by 51 +/- 11%. Additionally, while rest decay of ESCs was present after ryanodine treatment, the time for the ESCs to recover their steady-state amplitude was prolonged at least twofold. Thus, in contrast with the normal guinea pig cells, ESCs of the myocytes exposed to ryanodine are controlled by sarcolemmal processes. This change in the state of excitation-contraction coupling results mainly in modification of the time course of the ESCs and of the time course of the response of the cells to the change in the rate of stimulation.

Voltage-dependent inhibition of the delayed outward potassium current by OPC-8490, a novel positive inotropic agent, in isolated atrial myocytes of guinea-pig heart

To elucidate the ionic mechanisms underlying prolongation of the action potential by OPC-8490, a novel positive inotropic agent, we examined effects of the drug on the membrane currents in isolated atrial myocytes of guinea-pig heart. The tight-seal whole cell clamp technique was used. In the current clamp condition, OPC-8490 prolonged the atrial action potential in a concentration-dependent fashion. In the voltage clamp condition, OPC-8490 affected neither the conventional L type Ca current nor the outward IK1 current significantly. The drug blocked the delayed outward K current (IK) in a concentration-dependent fashion. At a constant concentration of the drug, inhibition of IK became more marked as the membrane potential became more negative. The tail current of IK could be fitted by a sum of two exponentials (fast and slow components). OPC-8490 blocked the fast component more prominently than the slow one. Time constants of both components were not affected significantly by the drug. In conclusion, OPC-8490 prolongs the cardiac action potential mainly by blocking IK in a voltage-dependent and time-dependent fashion.

Glibenclamide Specifically Blocks ATP-Sensitive K

Effects of glibenclamide on the control membrane ionic currents, acetylcholine or adenosine-induced K+ current, and nicorandil-induced K+ current were examined in single atrial myocytes of guinea pig heart. The nystatin-whole cell clamp technique was used. Nicorandil evoked the time-independent K+ current which is probably the current through the ATR-sensitive K+ channel. Glibenclamide inhibited this current in a concentration-dependent fashion, although it had no effect on the other currents. We concluded that glibenclamide specifically inhibits the ATP-sensitive K+ channel current in cardiac myocytes.

On the mechanism of muscarinic inhibition of the cardiac Ca current

The mechanism of muscarinic inhibition of the Ca-current (ICa) was studied in ventricular myocytes of guinea pig hearts and the following results were obtained. Acetylcholine (ACh) in concentrations up to 10(-4) M had little effect, if any, on ICa in control cells. ACh reduced the isoprenaline (ISP)-induced increase of ICa. The dose-response-relation (ISP concentration vs. ICa density) was shifted by ACh towards higher ISP concentrations. But both, at low and high ISP concentrations ACh had nor or little effect. ACh was ineffective when ICa was increased by dialysing the cell with catalytic subunit of cAMP-dependent protein kinase or cAMP. ACh reduced ICa enhanced by isobutylmethylxanthine or by forskolin. ACh did not depress ICa when the cell was dialysed with the non-hydrolysable GTP-derivative, GMP-PNP. In this condition the beta-adrenergic enhancement of ICa was also absent. Pertussis toxin, which is known to inhibit the inhibitory transducer protein (Ni), abolished the ACh response. We concluded from these results that ACh depresses ICa by inhibiting, via Ni, the cAMP production.