• The Korean Journal of Physiology and Pharmacology
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• 제2권6호
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• pp.733-742
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• 1998

Background: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current $(I_{Ca})$ in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of $I_{Ca}}$ by cGMP. However, there is no direct evidence that cGMP-PK can stimulate $I_{Ca}}$ in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK on $I_{Ca}}$ in rabbit ventricular myocytes. Methods and Results: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of $I_{Ca}}$ by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. $I_{Ca}}$ was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal $I_{Ca}}$. cGMP-PK also increased basal $I_{Ca}}$. The stimulation of basal $I_{Ca}}$ by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal $I_{Ca}}$ by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When $I_{Ca}}$ was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of $I_{Ca}}$. In the presence of cGMP-PK, already increased $I_{Ca}}$ was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. Conclusions: We present evidence that cGMP-PK stimulated basal $I_{Ca}}$ by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.

• The Korean Journal of Physiology and Pharmacology
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• 제7권1호
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• pp.15-23
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• 2003

Cellular redox state is known to be perturbed during ischemia and that $Ca^{2+}$ and $K^2$ channels have been shown to have functional thiol groups. In this study, the properties of thiol redox modulation of the ATP-sensitive $K^2$ ($K_{ATP}$) channel were examined in rabbit ventricular myocytes. Rabbit ventricular myocytes were isolated using a Langendorff column for coronary perfusion and collagenase. Single-channel currents were measured in excised membrane patch configuration of patch-clamp technique. The thiol oxidizing agent 5,5'-dithio-bis-(2-nitro-benzoic acid) (DTNB) inhibited the channel activity, and the inhibitory effect of DTNB was reversed by dithiothreitol (disulfide reducing agent; DTT). DTT itself did not have any effect on the channel activity. However, in the patches excised from the metabolically compromised cells, DTT increased the channel activity. DTT had no effect on the inhibitory action by ATP, showing that thiol oxidation was not involved in the blocking mechanism of ATP. There were no statistical difference in the single channel conductance for the oxidized and reduced states of the channel. Analysis of the open and closed time distributions showed that DTNB had no effect on open and closed time distributions shorter than 4 ms. On the other hand, DTNB decreased the life time of bursts and increased the interburst interval. N-ethylmaleimide (NEM), a substance that reacts with thiol groups of cystein residues in proteins, induced irreversible closure of the channel. The thiol oxidizing agents (DTNB, NEM) inhibited of the $K_{ATP}$ channel only, when added to the cytoplasmic side. The results suggested that metabolism-induced changes in the thiol redox can also modulate $K_{ATP}$ channel activity and that a modulatory site of thiol redox may be located on the cytoplasmic side of the $K_{ATP}$ channel in rabbit ventricular myocytes.

• The Korean Journal of Physiology and Pharmacology
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• 제2권5호
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• pp.581-589
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• 1998

A regulating mechanism of the ATP-sensitive potassium channels $(K_{ATP}\;channels)$ is yet to fully explained. This study was carried out to investigate the effects of intracellular application of monocarboxylates (acetate, formate, lactate, and pyruvate) on $K_{ATP}$ channels in isolated rabbit ventricular myocytes. Single channel currents of $K_{ATP}$ channels were recorded using the excised inside-out or permeabilized attached (open-cell) patch-clamp technique at room temperature. Intracellular application of acetate, formate and pyruvate led to an inhibition of channel activity, whereas intracellular application of lactate increased channel activity. These effects were reversible upon washout. Analysis of single channel kinetics showed that monocarboxylates did not affect open-time constant and close-time constant. These results suggest that monocarboxylates participate in modulating $K_{ATP}$ channels activity in cardiac cells and that modulation of $K_{ATP}$ channels activity may resolve the discrepancy between the low $K_i$ in excised membrane patches and high levels of intracellular ATP concentration during myocardial ischemia or hypoxia.

• The Korean Journal of Physiology
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• 제30권1호
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• pp.1-9
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• 1996

The objective of the present study was to characterize the role of adenosine in regulation of ATP-sensitive $K^+\;channel\;(K_{ATP}\;channel)$ activity in isolated rabbit ventricular myocytes using the patch clamp technique. Internal adenosine had little effects on KaTr channel activity. In an outside-out patch with intrapipette GTP and ATP, external adenosine stimulated $K_{ATP}\;channel$ activity. In an inside-out Patch with intrapipette adenosine, ATP reduced $K_{ATP}\;channel$ activity, and GTP stimulated $K_{ATP}\;channel$ activity. Adenosine receptor activation shifted the half-maximal inhibition Of $K_{ATP}\;channel\;from\;70\;to\;241\;{\mu}m$. These results Suggest that activation of adenosine receptors stimulates $K_{ATP}\;channels$ in rabbit ventricular myocytes by reducing the apparent affinity of the channel for ATP. The effect may be important for activating $K_{ATP}\;channels$ during early phase of myocardial ischemia.

• The Korean Journal of Physiology and Pharmacology
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• 제9권4호
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• pp.187-193
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• 2005

Several signal transduction pathways have been implicated in ischemic preconditioning induced by the activation of ATP-sensitive $K^+$ $(K_{ATP})$ channels. We examined whether protein kinase C (PKC) modulated the activity of $K_{ATP}$ channels by recording $K_{ATP}$ channel currents in rabbit ventricular myocytes using patch-clamp technique and found that phorbol 12,13-didecanoate (PDD) enhanced pinacidil-induced $K_{ATP}$ channel activity in the cell-attached configuration; and this effect was prevented by bisindolylmaleimide (BIM). $K_{ATP}$ channel activity was not increased by $4{\alpha}-PDD$. In excised insideout patches, PKC stimulated $K_{ATP}$ channels in the presence of 1 mM ATP, and this effect was abolished in the presence of BIM. Heat-inactivated PKC had no effect on channel activity. PKC-induced activation of $K_{ATP}$ channels was reversed by PP2A, and this effect was not detected in the presence of okadaic acid. These results suggest that PKC activates $K_{ATP}$ channels in rabbit ventricular myocytes.

• The Korean Journal of Physiology and Pharmacology
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• 제5권2호
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• pp.165-175
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• 2001

Background: Recent in vivo experimental evidence suggests that isoflurane-induced cardioprotection may involve $K_{ATP}$ channel activation. However, it was demonstrated that isoflurane inhibited $K_{ATP}$ channel activities in the inside-out patch mode. To explain this discrepancy, the present investigation tested the hypothesis that a metabolite of isoflurane, trifluoroacetic acid (TFA), contributes to isoflurnae-induced cardioprotection via $K_{ATP}$ channel activation during myocardial ischemia and reperfusion. Methods: Single ventricular myocytes were isolated from rabbit hearts by an enzymatic dissociation procedure. Patch-clamp techniques were used to record single-channel currents. $K_{ATP}$ channel activities were assessed before and after the application of TFA with the inside-out patch mode. Results: TFA enhanced channel activity in a concentration-dependent fashion. The concentration of TFA for half-maximal activation and the Hill coefficient were 0.03 mM and 1.2, respectively. TFA did not affect the single channel conductance of $K_{ATP}$ channels. Analysis of open and closed time distributions showed that TFA increased burst duration and decreased the interburst interval without changes in open and closed time distributions shorter than 5 ms. TFA diminished ATP sensitivity of $K_{ATP}$ channels in a concentration-response relationship for ATP. Conclusions: TFA, a metabolite of isoflurane, enhanced $K_{ATP}$ channel activity in a concentration-dependent fashion. These results imply that TFA could mediate isoflurane-induced cardioprotection via $K_{ATP}$ channel activation during myocardial ischemia and reperfusion.

• The Korean Journal of Physiology
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• 제27권2호
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• pp.151-162
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• 1993

In order to investigate the effect of intracellular cyclic GMP on calcium current the whole-cell patch clamp technique with internal perfusion method was used in isolated ventricular myocytes of the rabbit. Cyclic GMP, 8-bromo-cyclic GMP, cyclic AMP, isoprenaline and forskolin were perfused into cells and their effects on calcium current were analysed by applying depolarizing step pulses of + 10 mV in amplitude far 300 msec from holding potential of - 40 mV. Not only cyclic AMP $(100\;{\mu}M)$ but also cyclic GMF $(100\;{\mu}M)$ increased the basal calcium current. 8-Bromo-cyclic GMP $(100\;{\mu}M)$, a good stimulator of the cyclic GMP-dependent protein kinase, also increased the basal calcium current and its peak amplitude of calcium current was larger than that in the presence of cyclic AMP or cyclic GMP alone. In the presence of $100\;{\mu}M$ cyclic GMP or $100\;{\mu}M$ 8-bromo-cyclic GMP, already augmented calcium current was potentiated by intracellular application of $100\;{\mu}M$ cyclic AMP or $1\;{\mu}M$ isoprenaline or $1\;{\mu}M$ forskolin. In the presence of cyclic GMP, acetylcholine reduced the calcium current only when the calcium current was increased by isoprenaline. From the above results it could be concluded that intracellular perfusion with cyclic GMP increases the basal calcium current via a mechanism involving a cyclic GMP-dependent protein kinase.

• Journal of Chest Surgery
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• 제25권4호
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• pp.364-382
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• 1992

In order to investigate the effect of intracellular cyclic GMP on the calcium channel, whole cell patch clamp technique with internal perfusion method was used in the single ventricular myocytes of the rabbit. Cyclic GMP, cGMP analogues, cAMP, isopernaline and forskolin were perfused into cells and their effects on the calcium current were analysed by applying depolarizing step pulse of 10 mV in amplitude for 200 msec from holding potential of -40 mV. Calcium currents usually activated from -30 mV and then reached a peak at +10 mV. Amplitude of the calcium current was standardized with membrane capacitance, 50 pF. Peak amplitude at +10 mV in control was -0.15 nA/50pF. When 100 mM cAMP was applied from the pipette, peak amplitude of calcium current increased to -0.32 nA and addition of 1 mM isoprenaline further increased its amplitude. In the presence of cGMP it alone also produced an increase of the calcium current to -0.52 nA/50pF and addition of isoprenaline or forskolin increased its magnitude to -[0.55~0.95] nA/50pF. Simultaneous application of cGMP and cAMP increased the calcium current to -0.67 nA/50pF. Among the cGMP analogues, 8-Br-cGMP was the most potent stimulant for the calcium current activation. From the above results it could be concluded tlat cGMP increases the calcium current not through cAMP dependent protein kinase nor cAMP dependent phosphodiesterase pathway, but through independent phosphorylation pathway, possibly cGMP dependent protein kinase pathway.

• The Korean Journal of Physiology
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• 제26권1호
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• pp.27-35
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• 1992

We used the whole cell patch clamp technique to examine the ionic basis for the tail current after depolarizing pulse in single atrial myocytes of the rabbit. We recorded the tail currents during various repolarizations after short depolarizing pulse from a holding potential of -70 mV. The potassium currents were blocked by external 4-aminopyridine and replacement of internal potassium with cesium. The current was reversed to the outward direction above +10 mV. High concentrations of intracellular calcium buffer inhibited the activation of the current. Diltiazem and ryanodine blocked it too. These data suggest that the current is activated by intracellular calcium released from sarcoplasmic reticulumn. When the internal chloride concentration was increased, the inward tail current was increased. The current was partially blocked by the anion transport blocker niflumic acid. The current voltage curve of the niflumic acid sensitive current component shows outward rectification and is well fitted to the current voltage curve of the theoretically predicted chloride current calculated from the constant field equation. The currents recorded in rabbit atrial myocytes, with the method showing isolated outward Na Ca exchange current in ventricular cells of the guinea pig, suggested that chloride conductance could be activated with the activation of Na/ca exchange current. From the above results it is concluded that a chloride sensitive component which is activated by intracellular calcium contributes to tail currents in rabbit atrial cells.

• The Korean Journal of Physiology
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• 제26권2호
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• pp.99-111
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• 1992

Permissive action of thyroid hormone at the level of Ca channel and responsible mechanisms underlying thyroid hormone-induced change in myocardial contractile state and $T_3-induced$ arrhythmias were investigated in rabbit ventricular or atrial myocytes using whole cell patch clamp technique. Single cells were isolated by Langendorff perfusion with collagenase. Cardiac myocytes were incubated in $low-Cl^-,$, $high-K^+$ medium containing $1_{\mu}M\;L-triiodothyronine\;(T_3)$ at $4^{\circ}C$ for 2.10 hours. The calcium currrent $(I_{Ca})$ was increased in $T_3$ loaded cells, however, the shape of current voltage curve and reverse potential did not altered. Cyclic AMP, cyclic GMP, isoprenaline and 3-isobutyl-1-methyl-xanthine increased $I_{Ca}$ in euthyroid and hyperthyroid conditions, and acetylcholine blocked the increase of $I_{Ca}\;in\;T_3$ loaded cells. The amplitude of $I_{Ca}$ was much larger after perfusing cGMP than cGMP in both conditions, whereas the degree of increase of $I_{Ca}$ was greater after perfusing cAMP than cGMP in $T_3$ loaded cells. The degree of increase of $I_{Ca}$ after perfusing isoprenaline or IBMX also was greater in $T_3$ loaded cells than in control cells. Background current induced by isoprenaline also increased in $T_3$ loaded cells. The Ca release dependent inward current was increased in amplitude but its activation and inactivation time course was not changed in $T_3$ loaded cells. Activation of Na pump current was not changed in $T_3$ loaded cells. From the above results it is suggested that thyroid hormone induced increase in the contractile state of cardiac myocytes are accompanied by augmented $I_{Ca}$ and the increase of Ca release from sarcoplasmic reticulum and the permissive action of thyroid hormone to catecholamines could induce arrhythmias through the increase of $I_{Ca}$ and background current.