Characterization of the ATP-sensitive potassium channels (KATP) expressed in guinea pig bladder smooth muscle cells

ATP-sensitive K+ (KATP) channels play an important role in the regulation of smooth muscle membrane potential. To investigate the properties of KATP channels in guinea pig urinary bladder smooth muscle cells, fluorescence-based assays were carried out with the membrane potential-sensitive probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)]. The prototypical channel openers, including pinacidil, (-)-cromakalim, and diazoxide, elicited concentration-dependent decreases in membrane potential that were attenuated by glyburide. Similar responses were evoked by a reduction in intracellular ATP levels by metabolic inhibition. The observed rank order potency (EC50) for evoking membrane potential changes by potassium channel openers, P1075 (53 nM) approximately Bay X 9228 > (-)-cromakalim approximately ZD6169 approximately pinacidil > Bay X 9227 approximately ZM244085 > diazoxide (59 microM), showed a good correlation with that of bladder smooth muscle relaxation, as assessed by isolated tissue bath studies. The maximal efficacies of (-)-cromakalim, pinacidil, Bay X 9228, and ZD6169 were comparable with the response achieved by the reference activator P1075. Whole cell currents in bladder smooth muscle cells were increased in both inward and outward directions by P1075 and were reversed by glyburide to control levels. The molecular composition assessed by reverse transcriptase-polymerase chain reaction analysis using subunit-specific primers revealed the presence of mRNA for inward rectifying potassium channel (KIR6.2) and sulfonylurea receptors (SUR)2B and SUR1. The subunit profile together with pharmacological properties suggests that the KATP channel in bladder smooth muscle cells could be composed of SUR2B associated with a single inward rectifier, KIR6.2. In summary, these studies have characterized the pharmacological profile using fluorescent imaging plate reader-based membrane potential techniques and provide evidence for the molecular identity of KATP channels expressed in guinea pig bladder smooth muscle cells.