ALPHA-2-ADRENERGIC INHIBITION OF CL- TRANSPORT BY OPERCULAR EPITHELIUM IS MEDIATED BY INTRACELLULAR CA2+

We isolated the opercular epithelium of seawater killifish (Fundulus heteroclitus) to study the mediation of catecholamine inhibition of Cl- secretion. The receptors are alpha2-adrenergic, as they have a high affinity for the alpha2-adrenergic agonist clonidine over phenylephrine and clonidine action is blocked by yohimbine. Pertussis toxin and indomethacin did not block the clonidine effect; hence inhibitory guanine nucleotide-binding proteins (G(i) proteins) and prostaglandins (respectively) are not involved. Intracellular pH (pH(i)) of single chloride cells was measured microspectrofluorometrically and resting pH(i) was 7.22 +/- 0.03. However, pH(i) was unaffected by clonidine; hence pH(i) and Na+/H+ exchange are not involved. The lipoxygenase inhibitors nordihydroguaiaretic acid and baicalein and the lipoxygenase products (12S)- and (12R)-12-hydroxyeicosatetraenoic acid stimulated Cl- secretion. Protein kinase C is an unlikely site of action because the diacylglycerol kinase inhibitor R59022 had no effect alone and did not block the clonidine effect. Ionomycin (1 muM) in normal but not low-Ca2+ solutions mimicked the action of clonidine and both inhibitions were reversible by isoproterenol. Thapsigargin, a releaser of intracellular Ca2+, inhibited Cl- secretion and this effect was reduced in low-Ca2+ solutions. Low-Ca2+ solutions also blunted but did not block entirely the clonidine response, indicating that the primary Ca2+ release was from intracellular stores. Whereas alpha1-adrenergic receptors commonly act via the Ca2+/inositol trisphosphate pathway, to our knowledge this is the first report of a Ca2+-mediated alpha2-adrenergic response in a nonmammalian vertebrate.