• The Korean Journal of Physiology and Pharmacology
• /
• v.6 no.4
• /
• pp.183-186
• /
• 2002

Ischemic damage is one of the most serious problems. The openers of KATP channel have been suggested to have an effect to limit the ischemic damage. However, it is not yet clear how KATP channels of a cell correspond to hypoxic damage. To address the question, N2a cells were exposed to two different hypoxic conditions as follows: 6 hours hypoxia followed by 3 hours reperfusion and 12 hours hypoxia followed by 3 hours reperfusion. As the results, 6 hours hypoxic treatment increased glibenclamide- sensitive basal $K_{ATP}$ current activity (approximately 6.5-fold at 0 mV test potential) when compared with nomoxic condition. In contrast, 12 hours hypoxic treatment induced a relatively smaller change in the $K_{ATP}$ current density (2.5-fold at 0 mV test potential). Additionally, in experiments where $K_{ATP}$ channels were opened using diazoxide, the hypoxia for 6 hours significantly increased the current density in comparison to control condition (p＜0.001). Interestingly, the augmentation in the $K_{ATP}$ current density reduced after exposure to the 12 hours hypoxic condition (p＜0.001). Taken together, these results suggest that $K_{ATP}$ channels appear to be recruited more in cells exposed to the 6 hours hypoxic condition and they may play a protective role against hypoxia-reperfusion damage within the time range.

• The Korean Journal of Physiology and Pharmacology
• /
• v.11 no.1
• /
• pp.15-20
• /
• 2007

To investigate whether hydrogen peroxide($H_2O_2$) affects intestinal motility, pacemaker currents and membrane potential were recorded in cultured interstitial cells of Cajal(ICC) from murine small intestine by using a whole-cell patch clamp. In whole cell patch technique at $30^{\circ}C$, ICC generated spontaneous pacemaker potential under current clamp mode(I=0) and inward currents(pacemaker currents) under voltage clamp mode at a holding potential of -70 mV. When ICC were treated with $H_2O_2$ in ICC, $H_2O_2$ hyperpolarized the membrane potential under currents clamp mode and decreased both the frequency and amplitude of pacemaker currents and increased the resting currents in outward direction under voltage clamp mode. Also, $H_2O_2$ inhibited the pacemaker currents in a dose-dependent manner. Because the properties of $H_2O_2$ action on pacemaker currents were same as the effects of pinacidil(ATP-sensitive $K^+$ channels opener), we tested the effects of glibenclamide(ATP-sensitive $K^+$ channels blocker) on $H_2O_2$ action in ICC, and found that the effects of $H_2O_2$ on pacemaker currents were blocked by co- or pre- treatment of glibenclamide. These results suggest that $H_2O_2$ inhibits pacemaker currents of ICC by activating ATP-sensitive $K^+$ channels.

• The Korean Journal of Physiology and Pharmacology
• /
• v.5 no.2
• /
• pp.157-163
• /
• 2001

The effects of dimethyl sulfoxide (DMSO) were studied in two groups of Xenopus oocytes, one expressing ATP sensitive $K^+\;(K_{ATP})$ channel comprised of sulfonylurea receptor SUR1 and inwardly rectifying $K^+$ channel subunit Kir6.2, and the other expressing renal $K_{ATP}$ channel ROMK2. At concentrations of $0.3{\sim}10%$ (vol/vol) DMSO inhibited whole cell Kir6.2/SUR1 currents elicited by bath application of sodium azide (3 mM) in a concentration-dependent manner. The inhibition constant and Hill coefficient were 2.93% and 1.62, respectively. ROMK2 currents, however, was not affected significantly by DMSO. The results support the idea that DMSO inhibits $K_{ATP}$ channel expressed in Xenopus oocyte through a protein-specific mechanism(s) that remains to be further elucidated.

• The Korean Journal of Physiology and Pharmacology
• /
• v.8 no.4
• /
• pp.201-206
• /
• 2004

Mitochondrial ATP-sensitive potassium $(mitoK_{ATP})$ channels play a role in early and late ischemic preconditioning. Nevertheless, the subunit composition of $mitoK_{ATP}$ channels remains unclear. In this study, we investigated the subunit composition of $mitoK_{ATP}$ channels in mitochondria isolated from rat cardiac myocytes. Mitochondria were visualized using the red fluorescence probe, Mitrotracker Red, while $mitoK_{ATP}$ channels were visualized using the green fluorescence probe, glibenclamide-BODIPY. The immunofluorescence confocal microscopy revealed the presence of Kir6.1, Kir6.2 and SUR2 present in the cardiac mitochondria. Western blot analysis was carried to further investigate the nature of $mitoK_{ATP}$ channels. For SUR proteins, a 140-kDa immunoreactive band that corresponded to SUR2, but no SUR1 was detected. For Kir6.2, three bands $({\sim}44,\;{\sim}46,\;and\;{\sim}30\;kDa)$ were detected, and a specific ${\sim}46-kDa$ immunoreactive band corresponding to Kir6.1 was also observed. These observations suggest that the subunits of $mitoK_{ATP}$ channels in rat myocytes include Kir6.1, Kir6.2, and a SUR2-related sulfonylurea-binding protein.

• The Korean Journal of Physiology and Pharmacology
• /
• v.4 no.1
• /
• pp.25-31
• /
• 2000

ATP-sensitive potassium channels ($K_{ATP}$ channels) play an important role in insulin secretion from pancreatic beta cells. We have investigated the effect of propofol on $K_{ATP}$ channels in cultured single pancreatic beta cells of rats. Channel activity was recorded from membrane patches using the patch-clamp technique. In the inside-out configuration bath-applied propofol inhibited the $K_{ATP}$ channel activities in a dose-dependent manner. The half-maximal inhibition dose (ED50) was $48.6{\pm}8.4\;{\mu}M$ and the Hill coefficient was $0.73{\pm}0.11.$ Single channel conductance calculated from the slope of the relationship between single channel current and pipette potential $(+20{\sim}+100\;mV)$ was not significantly altered by propofol $(control:\;60.0{\pm}2.7\;pS,\;0.1\;mM\;propofol:\;58.7{\pm}3.5\;pS).$ However, mean closed time was surely increased. Above results indicate that propofol blocks the $K_{ATP}$ channels in the pancreatic beta cells in the range of its blood concentrations during anesthesia, suggesting a possible effect on insulin secretion and blood glucose level.

• The Korean Journal of Physiology and Pharmacology
• /
• v.5 no.2
• /
• pp.165-175
• /
• 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 and Pharmacology
• /
• v.3 no.3
• /
• pp.293-303
• /
• 1999

The influences of specific protein phosphatase and protein kinase inhibitors on the ATP-sensitive $K^+\;(K_{ATP})$ channel-opening effect of pinacidil were investigated in single rat ventricular myocytes using patch clamp technique. In cell-attached patches, pinacidil $(100\;{\mu}M)$ induced the opening of the $K_{ATP}$ channel, which was blocked by the pretreatment with H-7 $(100\;{\mu}M)$ whereas enhanced by the pretreatment with genistein $(30\;{\mu}M)$ or tyrphostin A23 $(10\;{\mu}M)$. In inside-out patches, pinacidil $(10\;{\mu}M)$ activated the $K_{ATP}$ channels in the presence of ATP (0.3 mM) or AMP-PNP (0.3 mM) and in a partial rundown state. The effect of pinacidil $(10\;{\mu}M)$ was not affected by the pretreatment with protein tyrosine phosphatase 1B $(PTP1B,\;10\;{\mu}g\;ml^{-1}),$ but blocked by the pretreatment of protein phosphatase 2A $(PP2A,\;1\;U\;ml^{-1})$. In addition, pinacidil $(10\;{\mu}M)$ could not induce the opening of the reactivated $K_{ATP}$ channels in the presence of H-7 $(100\;{\mu}M)$ but enhanced it in the presence of ATP (1 mM) and genistein $(30\;{\mu}M).$ These results indicate that the $K_{ATP}$ channel-opening effect of pinacidil is not mediated via phosphorylation of $K_{ATP}$ channel protein or associated protein, although it still requires the phosphorylation of serine/threonine residues as a prerequisite condition.

• The Korean Journal of Physiology and Pharmacology
• /
• v.9 no.4
• /
• pp.209-213
• /
• 2005

Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. In the present study, we investigated the effect of mitochondrial ATP-sensitive potassium (mitoKATP) channel on pacemaking activity in cultured ICCs from murine small intestine by using whole-cell patch clamp techniques. Under current clamp mode, at 10μM glibenclamide, there was no change in pacemaking activity of ICCs. At $30{\mu}M$ glibenclamide, an inhibitor of the ATP sensitive $K^+$ channels, we could find two examples. If pacemaking activity of ICCs was irregulating, pacemaking activity of ICCs was changed into regulating and if in normal conditions, membrane potential amplitude was increased. At $50{\mu}M$ glibenclamide, the resting membrane potential was depolarized. At 3mM 5-HDA, an inhibitor of the mitoKATP channels, inhibited the pacemaking activity of ICCs. Both the amplitude and the frequency were decreased. At 5 mM 5-HDA, both the amplitude and the frequency were completely abolished. Diazoxide, an opener of the mitoKATP channels, was applied to examine its effect on pacemaking activity of ICCs. At $50{\mu}M$ concentration, the pacemaking activity of ICCs was inhibited. Both the amplitude and the frequency were decreased. At 1 mM concentration, both the amplitude and the frequency were completely abolished and the resting membrane potential was shaked.These results indicate that mitoKATP channel has an important role in pacemaking activity of ICCs.

• The Korean Journal of Physiology and Pharmacology
• /
• v.8 no.2
• /
• pp.95-100
• /
• 2004

Ischemic preconditioning (IPC) has been accepted as a heart protection phenomenon against ischemia and reperfusion (I/R) injury. The activation of ATP-sensitive potassium $(K_{ATP})$ channels and the release of myocardial nitric oxide (NO) induced by IPC were demonstrated as the triggers or mediators of IPC. A common action mechanism of NO is a direct or indirect increase in tissue cGMP content. Furthermore, cGMP has also been shown to contribute cardiac protective effect to reduce heart I/R-induced infarction. The present investigation tested the hypothesis that $K_{ATP}$ channels attenuate DNA strand breaks and oxidative damage in an in vitro model of I/R utilizing rat ventricular myocytes. We estimated DNA strand breaks and oxidative damage by mean of single cell gel electrophoresis with endonuclease III cutting sites (comet assay). In the I/R model, the level of DNA damage increased massively. Preconditioning with a single 5-min anoxia, diazoxide $(100\;{\mu}M)$, SNAP $(300\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) $(100\;{\mu}M)$ followed by 15 min reoxygenation reduced DNA damage level against subsequent 30 min anoxia and 60 min reoxygenation. These protective effects were blocked by the concomitant presence of glibenclamide $(50\;{\mu}M)$, 5-hydroxydecanoate (5-HD) $(100\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-8-pCPT-cGMP) $(100\;{\mu}M)$. These results suggest that NO-cGMP-protein kinase G (PKG) pathway contributes to cardioprotective effect of $K_{ATP}$ channels in rat ventricular myocytes.

• The Korean Journal of Physiology and Pharmacology
• /
• v.7 no.1
• /
• pp.15-23
• /
• 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.