• The Korean Journal of Physiology and Pharmacology
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• v.20 no.3
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• pp.315-324
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• 2016

Human cardiac fibroblasts (HCFs) have various voltage-dependent $K^+$ channels (VDKCs) that can induce apoptosis. Hydrogen peroxide ($H_2O_2$) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether $H_2O_2$ could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of $H_2O_2$ stimulated $Ca^{2+}-activated$ $K^+$ ($K_{Ca}$) currents but not delayed rectifier $K^+$ or transient outward $K^+$ currents, all of which are VDKCs. $H_2O_2-stimulated$ $K_{Ca}$ currents were blocked by iberiotoxin (IbTX, a large conductance $K_{Ca}$ blocker). The $H_2O_2-stimulating$ effect on large-conductance $K_{Ca}$ ($BK_{Ca}$) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated $BK_{Ca}$ currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the $H_2O_2-stimulating$ effect on $BK_{Ca}$ currents. Using RT-PCR and western blot analysis, three subtypes of $K_{Ca}$ channels were detected in HCFs: $BK_{Ca}$ channels, small-conductance $K_{Ca}$ ($SK_{Ca}$) channels, and intermediate-conductance $K_{Ca}$ ($IK_{Ca}$) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to $H_2O_2$, but IbTX decreased $H_2O_2$-induced apoptosis. These data suggest that among the VDKCs of HCFs, $H_2O_2$ only enhances $BK_{Ca}$ currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through $BK_{Ca}$ channels.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.5
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• pp.297-303
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• 2012

This study was designed to elucidate high $K^+$-induced relaxation in the human gastric fundus. Circular smooth muscle from the human gastric fundus greater curvature showed stretch-dependent high $K^+$ (50 mM)-induced contractions. However, longitudinal smooth muscle produced stretch-dependent high $K^+$-induced relaxation. We investigated several relaxation mechanisms to understand the reason for the discrepancy. Protein kinase inhibitors such as KT 5823 (1 ${\mu}M$) and KT 5720 (1 ${\mu}M$) which block protein kinases (PKG and PKA) had no effect on high $K^+$-induced relaxation. $K^+$ channel blockers except 4-aminopyridine (4-AP), a voltage-dependent $K^+$ channel ($K_V$) blocker, did not affect high $K^+$ -induced relaxation. However, N(G)-nitro-L-arginine and 1H-(1,2,4)oxadiazolo (4,3-A)quinoxalin-1-one, an inhibitors of soluble guanylate cyclase (sGC) and 4-AP inhibited relaxation and reversed relaxation to contraction. High $K^+$-induced relaxation of the human gastric fundus was observed only in the longitudinal muscles from the greater curvature. These data suggest that the longitudinal muscle of the human gastric fundus greater curvature produced high $K^+$-induced relaxation that was activated by the nitric oxide/sGC pathway through a $K_V$ channel-dependent mechanism.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.6
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• pp.405-413
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• 2011

This study was designed to elucidate high-$K^+$ induced response of circular and longitudinal smooth muscle from human gastric corpus using isometric contraction. Contraction from circular and longitudinal muscle stripes of gastric corpus greater curvature and lesser curvature were compared. Circular smooth muscle from corpus greater curvature showed high $K^+$ (50 mM)-induced tonic contraction. On the contrary, however, longitudinal smooth muscle strips showed high $K^+$ (50 mM)-induced sustained relaxation. To find out the reason for the discrepancy we tested several relaxation mechanisms. Protein kinase blockers like KT5720, PKA inhibitor, and KT5823, PKG inhibitor, did not affect high $K^+$-induced relaxation. $K^+$ channel blockers like tetraethylammonium (TEA), apamin (APA), glibenclamide (Glib) and barium ($Ba^{2+}$) also had no effect. However, N(G)-nitro-L-arginine (L-NNA) and 1H-(1,2,4) oxadiazolo (4,3-A) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC) and 4-AP (4-aminopyridine), voltage-dependent $K^+$ channel (KV) blocker, inhibited high $K^+$ -induced relaxation, hence reversing to tonic contraction. High $K^+$-induced relaxation was observed in gastric corpus of human stomach, but only in the longitudinal muscles from greater curvature not lesser curvature. L-NNA, ODQ and KV channel blocker sensitive high $K^+$-induced relaxation in longitudinal muscle of higher portion of corpus was also observed. These results suggest that longitudinal smooth muscle from greater curvature of gastric corpus produced high $K^+$-induced relaxation which was activated by NO/sGC pathway and by $K_V$ channel dependent mechanism.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.2
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• pp.149-156
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• 2013

Interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal tract, and histamine is known to regulate neuronal activity, control vascular tone, alter endothelial permeability, and modulate gastric acid secretion. However, the action mechanisms of histamine in mouse small intestinal ICCs have not been previously investigated, and thus, in the present study, we investigated the effects of histamine on mouse small intestinal ICCs, and sought to identify the receptors involved. Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials (in current clamp mode) from cultured ICCs. Histamine was found to depolarize resting membrane potentials concentration dependently, and whereas 2-PEA (a selective H1 receptor agonist) induced membrane depolarizations, Dimaprit (a selective H2-agonist), R-alpha-methylhistamine (R-alpha-MeHa; a selective H3-agonist), and 4-methylhistamine (4-MH; a selective H4-agonist) did not. Pretreatment with $Ca^{2+}$-free solution or thapsigargin (a $Ca^{2+}$-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed histamine-induced membrane depolarization. Furthermore, treatments with U-73122 (a phospholipase C inhibitor) or 5-fluoro-2-indolyl des-chlorohalopemide (FIPI; a phospholipase D inhibitor) blocked histamine-induced membrane depolarizations in ICCs. On the other hand, KT5720 (a protein kinase A inhibitor) did not block histamine-induced membrane depolarization. These results suggest that histamine modulates pacemaker potentials through H1 receptor-mediated pathways via external $Ca^{2+}$ influx and $Ca^{2+}$ release from internal stores in a PLC and PLD dependent manner.

• YAKHAK HOEJI
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• v.47 no.1
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• pp.31-36
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• 2003

To investigate the effect of protein kinase on melanin production via cAMP-dependent pathway, we measured the melanin amount and tyrosinase activity in B16 melanoma cells stimulated by alpha-melanocyte stimulating hormone (MSH), forskolin and 8-Br-cAMP. MSH, forskolin and 8-Br-cAMP significantly increased both melanin production and tyrosinase activity in B16 cells. Melanin production and tyrosinase activity by MSH are significantly inhibited by cyclic AMP-dependent protein kinase inhibitor (KT5720) and protein kinase C down-regulation treated with PMA. Bisindolmaleimide (1$\mu$M), protein kinase C inhibitor, significantly inhibited melanin production and tyrosinase activity stimulated by MSH, forskolin and 8-Br-cAMP with the following order of potency: MSH＞forskolin＞8-Br-cAMP. Tyrosine kinase inhibitor, genistein and DHC, significantly inhibited both, but the inhibitory effect was more potent in 8-Br-cAMP-stimulated B16 cells than MSH-stimulated cells. NFkB inhibitor (parthenolide) significantly inhibited melanin production and tyrosinase activity. Neither melanin production nor tyrosinase activity induced by MSH, forskolin and 8-Br-cAMP were affected by KN-62 (calmodulin-dependent protein kinase II inhibitor), PD098059 (mitogen-activated protein kinase inhibitor, MAPKK) and worthmannin (phosphatidylinositol 3-kinase inhibitor). These results suggest that both protein kinase C and tyrosine kinase are involved in melanin production by cyclic AMP-dependent pathway and NFkB pathway may play an important role in cyclic AMP-dependent melanin production in B16 melanoma cells.