Adenosine 3′,5′-cyclic monophosphate (cAMP) is known to stimulate exogenous IsK channel current in the Xenopus oocyte expression system. The present study was performed to determine whether elevation of cytosolic cAMP in a native mammalian epithelium known to secrete K+ through endogenously expressed IsK channels would stimulate K+ secretion through these channels. The equivalent short circuit current (I sc ) across vestibular dark cell epithelium in gerbil was measured in a micro-Ussing chamber and the apical membrane current (I IsK ) and conductance (g IsK ) of IsK channels was recorded with both the on-cell macro-patch and nystatin-perforated whole-cell patch-clamp techniques. It has previously been shown that I sc can be accounted for by transepithelial K+ secretion and that the apical IsK channels constitute a significant pathway for K+ secretion. The identification of the voltage-dependent whole-cell currents in vestibular dark cells was strengthened by the finding that a potent blocker of IsK channels, chromanol 293B, strongly reduced I IsK from 646 ± 200 to 154 ± 22 pA (71%) and g IsK from 7.5 ± 2.6 to 2.8 ± 0.4 nS (53%). Cytoplasmic cAMP was elevated by applying dibutyryl cyclic AMP (dbcAMP), or the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-20-1724. dbcAMP (1 mm) increased I sc and I IsK from 410 ± 38 to 534 ± 40 μA/cm2 and from 4.3 ± 0.8 to 11.4 ± 2.2 pA, respectively. IBMX (1 mm) caused transient increases of I sc from 415 ± 30 to 469 ± 38 μA/cm2 and Ro-20-1724 (0.1 mm) from 565 ± 43 to 773 ± 58 μA/cm2. IBMX increased I IsK from 5.5 ± 1.5 to 16.9 ± 5.8 pA in on-cell experiments and from 191 ± 31 to 426 ± 53 pA in whole-cell experiments. The leak conductance due to all non-IsK channel sources did not change during dbcAMP and IBMX while 293B in the presence of dbcAMP reduced I IsK by 84% and g IsK by 62%, similar to unstimulated conditions. These results demonstrate that the cAMP pathway is constitutively active in vestibular dark cells and that the cAMP pathway stimulates transepithelial K+ secretion by increasing IsK channel current rather than by altering another transport pathway.
The Journal of Membrane Biology – Springer Journals
Published: Mar 1, 1997
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