Pathways for K+ efflux in isolated surface and crypt colonic cells. Activation by calcium.

K+ -conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring (86)Rb efflux. In crypt cells, basal K+ efflux (rate constant: 0.24 +/- 0.044 min(-1), span: 24 +/- 1.3%) was inhibited by 30 mM TEA and 5 mM Ba2+ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 microM) stimulated K+ efflux, increasing the rate constant to 0.65 +/- 0.007 min(-1) and the span to 83 +/- 3.2%. Ionomycin-induced K+ efflux was inhibited by clotrimazole (IC(50) of 25 +/- 0.4 microM) and charybdotoxin (IC(50) of 65 +/- 5.0 nM) and was insensitive to TEA, Ba2+, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca2+-activated intermediate-conductance K+ channels (IK(ca)). Absence of extracellular Ca2+ did neither affect basal nor ionomycin-induced K+ efflux. However, intracellular Ca2+ depletion totally inhibited the ionomycin-induced K+ efflux, indicating that the activation of these K+ channels mainly depends on intracellular calcium liberation. K+ efflux was stimulated by intracellular Ca(2+) with an EC(50) of 1.1 +/- 0.04 microM. In surface cells, K+ efflux (rate constant: 0.17 +/- 0.027 min(-1); span: 25 +/- 3.4%) was insensitive to TEA and Ba2+. However, ionomycin induced K+ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK(Ca) channels but only crypt cells have TEA- and Ba2+-sensitive conductive pathways, which would determine their participation in colonic K+ secretion.

beta-adrenergic agonists stimulate Na+-K+-Cl- cotransport by inducing intracellular Ca2+ liberation in crypt cells.

Epinephrine and beta-adrenergic agonists (beta1 and beta2 for isoproterenol, beta1 for dobutamine, beta2 for salbutamol) stimulated K+ (or 86Rb) influx mediated by the Na+-K+-2Cl- cotransporter and the Na+-K+ pump in isolated colonic crypt cells. Preincubation with bumetanide abolished the epinephrine effect on the Na+-K+ pump, suggesting that the primary effect is on the cotransporter. Maximal effect was obtained with 1 microM epinephrine with an EC50 of 91.6 +/- 9.98 nM. Epinephrine-induced K+ transport was blocked by propranolol with an IC50 of 134 +/- 28.2 nM. alpha-Adrenergic drugs did not modify K+ transport mechanisms. Neither Ba2+ nor tetraethylammonium nor DIDS modified the adrenergic enhancement on the cotransporter. In addition, epinephrine did not affect K+ efflux. Dibutyryl cAMP did not alter K+ transport. Reduction of extracellular Ca2+ to 30 nM did not influence the response to epinephrine. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM abolished epinephrine-induced K+ transport. Ionomycin increased Na+-K+-2Cl- cotransport activity. Moreover, epinephrine increased intracellular Ca2+ concentration in a process inhibited by propranolol. In conclusion, epinephrine stimulated the Na+-K+-2Cl- cotransporter in a process mediated by beta1- and beta2-receptors and modulated by intracellular Ca2+ liberation.

K+ transport in isolated guinea pig colonocytes: evidence for Na(+)-independent ouabain-sensitive K+ pump.

K+ transport mechanisms in epithelial cells isolated from guinea pig distal colon have been studied using 86Rb as a tracer. A transport pathway has been identified that is proposed to be identical to the mechanism mediating transepithelial K+ absorption. Guinea pig colonocytes take up K+ through at least three separate mechanisms: 1) a Na(+)-dependent, ouabain-sensitive influx that is consistent with the Na(+)-K+ pump, 2) a Na(+)-dependent bumetanide-sensitive influx consistent with the Na(+)-K(+)-2Cl- cotransporter, and 3) a Na(+)-independent ouabain-sensitive influx, consistent with an apical colonic K+ pump. These transport mechanisms are sensitive to metabolic inhibition by rotenone and to vanadate, a blocker of type P adenosinetriphosphatase (ATPases). SCH-28080, an inhibitor of gastric K(+)-H(+)-ATPase, was without effect. Measurements of net K+ fluxes revealed that isolated colonocytes concentrated K+ by two processes: 1) a Na(+)-dependent ouabain-sensitive mechanism, which is compatible with the Na(+)-K+ pump and 2) a Na(+)-independent ouabain-sensitive mechanism consistent with the proposed absorptive K+ pump. These concentrative mechanisms were also inhibited by rotenone and vanadate, but not by SCH-28080. The Na(+)-independent ouabain-sensitive K+ pump was present in the distal colon, but absent in the proximal colon and the small intestine of guinea pig. It is proposed that this Na(+)-independent ouabain-sensitive K+ pump mediates K+ absorption and is related to the luminal K(+)-ATPase.