Received for publication April 22, 2008.
Revised June 30, 2008.
Accepted for publication July 1, 2008.

Levcromakalim and MgGDP activate small conductance K_ATP channels of Kir6.1/SUR2A in pig detrusor smooth muscle cells: Uncoupling of cAMP signal pathways

Abstract

Pharmacological studies had suggested the existence of ATP-sensitive K⁺ channel (K_ATP channel) as a therapeutic target in urinary bladder, however, electrical properties have not yet been shown. Patch clamp techniques were applied to investigate the properties of K_ATP channels in pig detrusor cells. In whole-cell configuration, levcromakalim, a K_ATP channel opener, induced a long-lasting outward current in a concentration-dependent manner. The current-voltage (I-V) curve of the levcromakalim-induced membrane current intersected at around -80 mV. This current was abolished by glibenclamide. Intracellular application of 0.1 mM guanosine 5'-diphosphate (GDP) significantly enhanced the levcromakalim-induced membrane current, while cAMP did not. Furthermore, neurotoransmitters related to cAMP signalling, such as CGRP, VIP, adenosine and somatostatin, had little effect on the levcromakalim-induced membrane current. In cell-attached configuration, levcromakalim activated K⁺ channels with a unitary conductance of approximately 12 pS. When the patch configuration was changed to inside-out mode, the K⁺ channel activity ran down. Subsequent application of 1 mM GDP re-activated the channels. The openings of the 12 pS K⁺ channels in the presence of 1 mM GDP was suppressed by ATP and glibenclamide. In RT-PCR, Kir6.1 and SUR2A were predominant in pig detrusor cells. The 12 pS K⁺ channel activated by levcromakalim in pig detrusor smooth muscle cells is an K_ATP channel. The predominant expression of SUR2A can account for the lack of effect of neurotransmitters related to cAMP.

Key words: ATP, GDP, cAMP, glibenclamide, incontinence, urinary bladder