cAMP-activated Cl- channels in primary cultures of spiny dogfish (Squalus acanthias) rectal gland.
Whole cell and single-channel patch-clamp techniques were used to identify and characterize the Cl- currents responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the rectal gland of the spiny dogfish (Squalus acanthias). During whole cell recordings, in cultured rectal gland cells forskolin (10 microM) and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) stimulated a 28-fold increase in Cl- conductance (n = 10). This cAMP-activated conductance pathway had a linear current-voltage (I-V) relationship that was time and voltage independent. Substitution of 235 meq Cl- with I- in the bath inhibited the cAMP-activated current at both positive and negative voltages (64%). Glibenclamide (60 microM) abolished the cAMP-stimulated current, and its effect was irreversible (n = 3). During cell-attached recording, increased cellular cAMP activated single Cl- channels in nine previously quiet patches. These channels had a linear I-V relationship with an average single-channel conductance of 5.1 +/- 0.2 pS (n = 6). Similar properties were observed in excised inside-out patches, permitting further characterization of the single-channel properties. Excised quiescent patches could be activated by the addition of ATP and protein kinase A. Replacing bath Cl- with I- inhibited both inward and outward currents (n = 3). In three inside-out patches, glibenclamide (300 microM) reversibly reduced open probability by 74%, with no effect on single-channel current amplitude. Similar results were obtained in four outside-out recordings. These results suggest that increased cellular cAMP in dogfish rectal gland activates a small linear Cl- channel that resembles human cystic fibrosis transmembrane conductance regulator in its biophysical and pharmacological properties.
Devor
D C
DC
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294.
Forrest
J N
JN
Suggs
W K
WK
Frizzell
R A
RA
D000818:Animals; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004284:Dogfish / Q000502:physiology*; D004594:Electrophysiology; D005905:Glyburide / Q000494:pharmacology; D007454:Iodides / Q000494:pharmacology; D012491:Salt Gland / Q000166:cytology / Q000378:metabolism*
D018118:Chloride Channels; D007454:Iodides; D005576:Colforsin; D000242:Cyclic AMP; D005905:Glyburide