Modulation of chloride, potassium and bicarbonate transport by muscarinic receptors in a human adenocarcinoma cell line

1 Short-circuit current (I-sc) responses to carbachol (CCh) were investigat ed in Colony 1 epithelia, a subpopulation of the HCA-7 adenocarcinoma cell line. In Krebs-Henseleit (KH) buffer, CCh responses consisted of three I-sc components: an unusual rapid decrease (the 10 s spike) followed by an upwa rd spike at 30 s and a slower transient increase (the 2 min peak). This res ponse was not potentiated by forskolin; rather, CCh inhibited cyclic AMP-st imulated I-sc. 2 In HCO3- free buffer, the decrease in forskolin-elevated I-sc after CCh w as reduced, although the interactions between CCh and forskolin remained at best additive rather than synergistic. When Cl- anions were replaced by gl uconate, both Ca2+- and cyclic AMP-mediated electrogenic responses were sig nificantly inhibited. 3 Basolateral Ba2+ (1-10 mM) and 293B (10 mu M) selectively inhibited forsk olin stimulation of I-sc, without altering the effects of CCh. Under Ba2+- or 293B-treated conditions, CCh responses were potentiated by pretreatment with forskolin. 4 Basolateral charybdotoxin (50 nM) significantly increased the size of the 10 s spike of CCh responses in both KH and HCO3- free medium, without affe cting the 2 min peak. The enhanced 10 s spike was inhibited by prior additi on of 5 mM apical Ba2+. Charybdotoxin did not affect forskolin responses. 5 In epithelial layers prestimulated with forskolin, the muscarinic antagon ists atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, both at 100 nM) abolished subsequent 10 mu M CCh responses. Following addit ion of p-fluoro hexahydro-sila-difenidol (pF-HHSiD, 10 mu M) or pirenzepine (1 mu M), qualitative changes in the CCh response time-profile also indica ted a rightward shift of the agonist concentration-response curve; however, 1 mu M gallamine had no effect. These results suggest that a single M-3-li ke receptor subtype mediates the secretory response to CCh. 6 It is concluded that CCh and forskolin activate discrete populations of b asolateral K+ channels gated by either Ca2+ or cyclic AMP, but that the Cl- permeability of the apical membrane may limit their combined effects on el ectrogenic Cl- secretion. In addition, CCh activates a Ba2+-sensitive apica l K+ conductance leading to electrogenic K+ transport. Both agents may also modulate HCO3- secretion through a mechanism at least partially dependent on carbonic anhydrase.