Gramicidin-perforated patch analysis on HCO3- secretion through a forskolin-activated anion channel in rat parotid intralobular duct cells.
Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3- secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 microM), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. The anion current was markedly suppressed by addition of 1 mM methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3-. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3- significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl- channel located in luminal membranes) and production of cytosolic HCO3- induce the inward anion current and resulting depolarization. Inhibition of the Na(+)-K(+)-2Cl- cotransporter and the Cl(-)-HCO3- exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl- via the Na(+)-K(+)-2Cl- cotransporter or the Cl(-)-HCO3- exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3- produced intracellularly, but not of Cl- due to lack of active Cl- transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.
Hirono
C
C
Department of Oral Physiology, Hiroshima University School of Dentistry, Hiroshima 734-8553, Japan.
Nakamoto
T
T
Sugita
M
M
Iwasa
Y
Y
Akagawa
Y
Y
Shiba
Y
Y
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000838:Anions; D001639:Bicarbonates / Q000378:metabolism*; D002034:Bumetanide / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin; D005905:Glyburide / Q000494:pharmacology; D006096:Gramicidin; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials; D008704:Methazolamide / Q000494:pharmacology; D010306:Parotid Gland / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar
D000838:Anions; D001639:Bicarbonates; D002712:Chlorides; D002034:Bumetanide; D006096:Gramicidin; D005576:Colforsin; C039465:5-dimethylamiloride; D000584:Amiloride; D005905:Glyburide; D008704:Methazolamide