ABSTRACT
Gliclazide, a sulfonylurea that is widely used to treat type II-diabetes, specifically blocks KATP channels and recombinant smooth muscle (SUR2B/Kir6.1) KATP channels with high potency. Furthermore, it exerts antioxidant properties and inhibits tumor cell proliferation. In this study, we investigated the inhibitory effect of gliclazide on vascular smooth muscle cell (VSMC) proliferation and tried to identify the underlying signaling pathway. We first investigated the effect of gliclazide-induced AMP-activated protein kinase (AMPK) activation on the proliferation of VSMCs. Gliclazide induced phosphorylation of AMPK in a dose- and time-dependent manner and inhibited VSMC proliferation following stimulation by platelet-derived growth factor (PDGF). However, KATP channel openers and Kir6.1 siRNA prevented gliclazide-mediated inhibition of VSMC proliferation. Gliclazide also increased the levels of Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß), an upstream kinase of AMPK. These findings suggested that the effects of KATP channels on AMPK activity were mediated by the regulation of intracellular Ca2+ levels. Oral administration of 2mg/kg gliclazide resulted in the activation of CaMKKß and AMPK in vivo, suggesting that gliclazide suppressed VSMC proliferation via the CaMKKß-AMPK signaling pathway. Taken together, our observations indicated that gliclazide-induced AMPK activation may act to prevent diabetes-associated atherosclerosis.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Cell Proliferation/drug effects , Gliclazide/pharmacology , KATP Channels/antagonists & inhibitors , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Potassium Channel Blockers/pharmacology , Sulfonylurea Receptors/antagonists & inhibitors , Animals , Calcium/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme Activation , KATP Channels/genetics , KATP Channels/metabolism , Male , Mice, Inbred C57BL , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/pathology , Phosphorylation , RNA Interference , Rats, Sprague-Dawley , Signal Transduction/drug effects , Sulfonylurea Receptors/genetics , Sulfonylurea Receptors/metabolism , Time Factors , TransfectionABSTRACT
This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive K+ channel blocker). However, neither N(G)-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive K+ channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.
