a 2 - Adrenergic inhibition of Cl - transport by opercular epithelium is mediated by intracellular Ca 2 + (

We isolated the opercular epithelium of seawater killifsh (Fundulus heteroclitus) to study the mediation of catecholamine inhibition of Cl secretion. The receptors are a2-adrenergic, as they have a high affmity for the a2-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 nudeotide-binding proteins (G1 proteins) and prostaglandins (respectively) are not involved. Intracellular pH (pH) of single chloride cells was measured microspectrofluorometrically and resting pHI was 7.22 ± 0.03. However, pH, was unaffected by clonidine; hence pH1 and Na+/H+ exchange are not involved. The lipoxygenase inhibitors nordihydroguaiaretic acid and baicalein and the lipoxygenase products (12S)and (12R)-12hydroxyeicosatetraenoic acid stimulated Clsecretion. 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. lonomycin (1 ,uM) 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 Clsecretion 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 cl-adrenergic receptors commonly act via the Ca2+/inositol trisphosphate pathway, to our knowledge this is the first report of a Ca2+-mediated a2-adrenergic response in a nonmammalian vertebrate. Marine teleosts drink seawater, absorb the water and ions in the intestine, and actively secrete the salt "load" across their gill and skin epithelia using mitochondria-rich "chloride" cells (reviewed in refs. 1-3). Whereas regulation of C1transport in cystic fibrosis has received much attention (e.g., ref. 4), the regulation of anion transport-particularly the hormonal inhibition of Clsecretion-is less well understood. Marine teleost skin and gills are unusual among C--transporting epithelia because even when unstimulated, the chloride cells in isolated epithelia secrete Clat a high rate (50-150 ,uA cm-2) when bathed on both sides with saline (1, 5). The resting transport rate can be inhibited by epinephrine, a-adrenergic agonists, urotensin II, and acetylcholine; it can be stimulated by f3-adrenergic agonists, cAMP, phosphodiesterase inhibitors (e.g., 3-isobutyl-1-methylxanthine), and urotensin I (1-3). The high transport rate in unstimulated tissues indicates that in vivo an inhibitory stimulus must be applied to shut down Clsecretion. This response is physiologically relevant because euryhaline fish that enter freshwater stop secreting Na+ and Cl-. During the acute phase, circulating catecholamines and corticosteroids are increased in vivo (6) and the branchial a-adrenergic response is a The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. reduction in NaCl loss. Physiological levels of epinephrine equivalent to that found in stressed fish (0.1 ,uM epinephrine; ref. 6) inhibit Clsecretion by chloride cells in vitro by about 50% (5). Importantly, the a2-adrenergic receptor action is not blocked by cholinergic antagonists (7), so the receptors appear to be postsynaptic. The intracellular mediator of the a-adrenergic receptor has not been identified for Clsecretion in teleosts. The mediation is not via inhibition of adenylate cyclase and reduction in intracellular cAMP, since a2-receptor activation does not reduce intracellular resting levels of cAMP or cAMP levels that had been augmented by isoproterenol, 3-isobutyl-1methylxanthine, or forskolin (8). There was no effect of nominally Ca2+-free solutions or of the ionophore A23187 on the rate of Clsecretion by Fundulus opercular epithelium (8); hence Ca2+ did not seem to be involved. The a2-receptors in many systems operate via an inhibitory GTP-binding protein (Gi protein) and inhibition of adenylate cyclase (9, 10) or (e.g., in platelets; ref. 11) an activation of Na+/H+ exchange. Van Praag et al. (12) suggested that eicosanoids could be important intracellular regulators in chloride cells. We set out to determine which of several possible intracellular mediators might transduce the a-adrenergic signal in chloride cells. MATERIALS AND METHODS Adult killifish (Fundulus heteroclitus; 4-7 g) from the Antigonish estuary were kept indoors for at least 10 days in full-strength seawater (salinity, 30 g/liter) under natural photoperiod at 20-25°C and were fed daily a marine fish blend (TetraMarine, TetraWerke, Germany) supplemented twice weekly with brine shrimp. The animals were killed by decapitation, and opercular epithelia were dissected and mounted in either standard Ussing membrane chambers with membrane area of 0.125 cm2 (for pharmacological studies) or a microscope stage chamber (surface area, 0.125 cm2) for intracellular pH (pHi) measurements by microspectrofluorometry. In the Ussing chambers, transepithelial conductance (Ge), transepithelial potential (Vt, mucosal side grounded), and short-circuit current (L.., ,uA/cm2) were monitored as described (13). For fluorescence microscopy, the subepithelial chromatophore layer and muscle fibers were microdissected from the epithelium to help identify individual chloride cells and to reduce background autofluorescence. We know ofno previous attempt to measure pH1 ofchloride cells from fish. We employed the fluorophore 2',7'-bis(2carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), a membrane-permeant derivative ofcarboxyAbbreviations: BCECF-AM, 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester; HETE, 12-hydroxy-5-cis-8-cis-10trans-14-cis-eicosatetraenoic acid; NDGA, nordihydroguaiaretic acid; pHi, intracellular pH; Gt, transepithelial conductance; 'Sc, short-circuit current. *To whom reprint requests should be addressed.