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 iden tify 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 recording s, in cultured rectal gland cells forskolin (10 mu M) and 8-(4-chlorop henylthio)adenosine 3',5'-cyclic monophosphate (400 mu M) Stimulated a 28-fold increase in Cl- conductance (n = 10). This cAMP-activated con ductance pathway had a linear current-voltage (I-V) relationship that was time and voltage independent. Substitution of 235 meg Cl- with I- in the bath inhibited the cAMP-activated current at both positive and negative voltages (64%). Glibenclamide (60 mu M) abolished the cAMP-st imulated current, and its effect was irreversible (n = 3). During cell -attached recording, increased cellular cAMP activated single Cl- chan nels 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 p atches, permitting further characterization of the single-channel prop erties. Excised quiescent patches could be activated by the addition o f ATP and protein Kinase A. Replacing bath Cl- with I-inhibited both i nward and outward currents (n = 3). In three inside-out patches, glibe nclamide (300 mu M) reversibly reduced open probability by 74%, with n o effect on single-channel current amplitude. Similar results were obt ained in four outside-out recordings. These results suggest that incre ased cellular cAMP in dogfish rectal gland activates a small linear Cl - channel that resembles human cystic fibrosis transmembrane conductan ce regulator in its biophysical and pharmacological properties.