CL- SECRETION BY CULTURED SHARK RECTAL GLAND-CELLS .3. CA2+ REGULATION OF APICAL MEMBRANE CL- CONDUCTANCE

Calcium ionophores (ionomycin and A-23187) were employed to assess the effects of increased intracellular Ca2+ concentration ([Ca2+]i) on apical membrane Cl- conductance (G(a)Cl) and rate of transepithelial Cl- secretion in cultured shark rectal gland (SRG) cells. Apical 2 muM ionomycin induced dramatic changes in cellular electrophysiological properties: the apical membrane electrical potential difference (V(a)) depolarized from -66 mV to -46 mV, the fractional resistance of the apical membrane (fR(a)) decreased from 0.88 to 0.23, and the transepithelial electrical potential difference (V(ab)) increased slightly from +1.2 mV to +1.4 mV. These effects result from increased G(a)Cl because apical low-Cl- shark Ringer (SR) depolarized V(a) by 32 mV and increased fR(a) from 0.23 to 0.36. Ionomycin-stimulated V(ab) or short-circuit current (I(sc)) results largely from increased Cl- secretion because approximately 80% of the increase in I(sc) is Cl- dependent. Establishing the Ca2+ dependence of ionophore activation of G(a)Cl was confounded because apical low-Ca2+ SR [Ca2+ concentration ([Ca2+]) 1 MM to 0.1 muM] alone activated this conductive pathway. To establish the Ca2+ dependence of ionophore action, we assessed the effect of ionomycin on I(sc) in low-Ca2+ SR ([Ca2+] = 0.1 muM) and in SR. In low-Ca2+ SR, apical ionomycin stimulated I(sc) by 14.0 muA/cm2. In SR (normal [Ca2+]), ionomycin increased I(sc) further by 27.0 muA/cm2. Superfusing the basolateral surface with 2 muM ionomycin for 8-16 min failed to activate G(a)Cl. In every case, subsequent superfusion of the apical surface with ionophore for 1.5-2 min activated G(a)Cl. Bilateral 4 muM indomethacin (45-min superfusion) failed to block the ionomycin-induced G(a)Cl. Thus in SRG cells Ca2+ activation of G(a)Cl is not dependent on cyclooxygenase-mediated prostaglandin formation and subsequent generation of adenosine 3',5'-cyclic monophosphate, which strongly activates G(a)Cl. Both the sidedness effects of ionophore action and ionophore stimulation of I(sc) in low-Ca2+ SR suggest that localized increases in [Ca2+]i near the apical membrane activate an apical Cl- conductance in cultured SRG cells.