α 1 -Adrenoceptors antagonize activated chloride conductance of amphibian skin epithelium

Abstract

The effects of adrenoceptor agonists on the transepithelial Cl- conductance (GCl) in the skin of several amphibian species, both toads and frogs, were studied. Epinephrine (Epi) from the serosal side selectively and reversibly inhibited the voltage-activated GCl in toad skin and the short-circuit GCl in frog skin. The main effects of activation of the adrenoceptors must reside in the skin epithelium and not in the glands, since measurements were made both from intact skins and split epithelia with essentially the same results. Effective concentrations of Epi were variable among individual tissues. GCl was reduced to 34+/-17% (n=46) with 1 micromol/l Epi, but in some tissues 0.1 micromol/l inhibited more than 80% of GCl, whereas some preparations were little influenced at >3 micromol/l Epi. The affected receptor type was identified by the use of the alpha1-agonist phenylephrine, which mimicked the response of Epi at concentrations above 30 micromol/l, whereas the alpha2-agonists xylazine and iodoclonidine had no effect at supramaximal concentrations. Prazosin, a specific alpha1-antagonist, reduced or eliminated the inhibition by Epi, but the response pattern suggests a low affinity. The alpha2-antagonist yohimbine, at concentrations </=0.3 micromol/l, had a minimal effect, but reduced the inhibition by Epi at concentrations of 1-10 micromol/l. This might indicate affinity to alpha1-adrenoceptors in amphibian skin. Activation of beta-adrenoceptors by isoproterenol (0.1-5 micromol/l) led to a transient increase of the baseline inactivated component of GCl with a slight reduction of the voltage-activated GCl at the higher concentrations, but the inhibitory effect of Epi was not altered. Epi, on the other hand, neither prevented nor reversed the induction of a voltage-insensitive GCl in toad skin caused by application of cAMP at supramaximal concentrations (>100 micromol/l CPT-cAMP). Preincubation of the serosal medium with Ca2+-free solution (in the presence of 2 mmol/l EGTA) for extended periods of time (>30 min) eliminated the response to Epi. It is concluded that alpha1-adrenoceptors participate in the physiological control of voltage-activated Cl- conductance in amphibian skin epithelium via modulation of intracellular Ca2+, presumably by efflux from intracellular stores.

Selective inhibition of Cl− conductance in toad skin by blockers of Cl− channels and transporters

“…In brief, skin pieces were mounted in an Ussing-type chamber with a 0.5-cm 2 aperture and were continuously perfused with Ringer solution at 2-5 ml/min on both sides. For the analysis of serosal effects and some experiments in the presence of 8-(4-chlorophenylthio)adenosine 3Ј,5Ј-cyclic monophosphate (CPT-cAMP), split epithelia devoid of the connective tissue layer and glands were prepared by the collagenase method as described recently (24). The collagenase solution was supplemented with 0.1 mg/ml trypsin inhibitor to avoid interference from traces of trypsin (25).…”

“…22). The epithelial glands can also be excluded from a role in voltageactivated or cAMP-induced G Cl , because split epithelia devoid of glands show a magnitude and response pattern identical to those of intact tissues (12,24). This implies that the route involves the mitochondria-rich (MR) cells, a minor fraction only of the epithelial cells, or the paracellular path.…”

Transepithelial chloride conductance in amphibian skin: regulatory mechanisms and localization

“…A new aspect in the regulatory control of G Cl has emerged from the observation that stimulation of α 1 -adrenoceptors inhibits the activation of G Cl by voltage, but not the increase in conductance induced by cAMP [57]. This inhibitory effect is presumably transmitted by changes in intracellular Ca 2+ due to efflux from intracellular stores and could be mediated by protein kinase C (PKC).…”

“…Larsen proposed the presence of voltage-activated, cAMP-regulated Cl -channels in the apical membrane of MRC. Since then, much new information has been gathered on this topic, addressing in particular the regulatory control mechanisms [23,25,57] and the localization of the pathway [61,70,74,88]. These analyses cast doubts on the general validity of the view that MRC are the sole pathway for activated Cl -transport.…”