Neurohypophysial hormone receptors were studied in primary cultures of sea bass (Dicentrarchus labrax) gill respiratory-like cells grown on permeable supports. This preparation was previously shown to provide a functional model for investigating the hormonal regulation of Cl- secretion. Under control conditions, the cultured monolayered epithelium had a short-circuit current (ISC) of 3.5+/-1.1 micro A x cm(-2). This current had previously been identified as an active Cl- secretion. The addition of increasing concentrations of the fish neurohypophysial hormones, arginine vasotocin (AVT) or isotocin (IT), elicited a concentration-dependent stimulation of the ISC. Maximal increases of 60.9+/-12.1% and 117.7+/-28.0% above the basal ISC value were obtained for 10(-7) M AVT and IT respectively. Half-maximal effects were obtained for 3.1 x 10(-9) M AVT and for 1.4 x 10(-9) M IT. Mucosal application of 1.0 mM diphenylalamine-2-carboxylic acid (a specific blocker of Cl- channels) after serosal addition of 5 x 10(-8) M AVT or IT inhibited not only the basal but also the stimulated current, revealing a correlation with a hormone-dependent Cl- transport. Specific V1 or V2 receptor analogues of vasopressin (mammalian hormone) were used to characterize the type of neurohypophysial hormone receptors pharmacologically. While the V1 agonist [Phe2,Orn8]-oxytocin stimulated the basal Cl- secretion with a similar profile to that of AVT or IT, the V2 agonist [Deamino1,Val4,d -Arg8]-vasopressin had no effect. The V1 antagonist [d(CH2)5 1,O-Me-Tyr2,Arg8]-vasopressin used at a concentration of 5 x 10(-7) M totally reversed the 10-8 M AVT-stimulated Cl- secretion, whereas the V2 antagonist [d(CH2)5 1,d -Ile2,Ile4,Arg8,Ala9]-vasopressin used at the same concentration had no significant effect. In contrast, similar experiments carried out in the presence of 10(-8) M IT showed that both antagonists significantly reduced the IT-stimulated Cl- secretion, with the efficiency of the V1 receptor antagonist being significantly greater than that of the V2. This study provides evidence for neurohypophysial hormone control of Cl- secretion in fish cultured gill respiratory cells. It suggests that on a physiological basis the hormonal effect is shared by the two peptides present in fish neurohypophysis (AVT and IT), acting by means of two distinct, although pharmacologically similar, V1-type receptors (according to the mammalian classification). These specific receptors are expected to play an important role in controlling ion homeostasis in seawater fish.

Abstract

We analysed the effects of specific neurohypophysial analogues for pharmacological characterization of the type of vasotocin receptor involved in the control of the adrenocorticotrophin hormone (ACTH) release from the perifused pituitary in the rainbow trout, Oncorhynchus mykiss.

Mammalian corticotrophin releasing factor (CRF) and teleostean neurohypophysial peptides (arginine vasotocin (AVT) and isotocin (IT)) stimulated ACTH release. Analysis of concentrations giving half-maximal effects (D₅₀) showed that these peptides affected ACTH release in the following order of potency: CRF (8 × 10⁻¹³ m) >AVT (2 × 10⁻¹⁰ m)>IT (10⁻⁷ m). Maximal responses (D_max) were obtained for hormonal concentrations of 10⁻¹⁰ m, 10⁻⁸ m and 10⁻⁶ m respectively. This suggests that AVT and IT have different roles in the control of ACTH release. The values obtained for AVT and IT were in agreement with the circulating levels we previously found for these peptides.

Specific V₁ or V₂ agonists or antagonists (with reference to vasopressin in mammals) were used to define the specificity of the neurohypophysial peptide receptor involved in this stimulation. The V₁ agonist, [Phe², Orn⁸]-oxytocin, stimulated ACTH release while the V₂ agonist, [deamino¹, Val⁴, d-Arg⁸]-vasopressin, had no such effect. Maximal and half-maximal responses were obtained in the presence of the V₁ agonist with 10⁻⁷ m and 7 × 10⁻⁹ m respectively, and were in the range of values obtained with natural peptides. The V₁ antagonist, [d(CH₂)₅ ¹, O-Me-Tyr², Arg⁸]-vasopressin, and the V₂ antagonist, [d(CH₂)₅ ¹, d-Ile², Ile⁴, Arg⁸, Ala⁹]-vasopressin, maximally reversed the 10⁻⁹ m AVT-stimulated ACTH release by 60% and 25% respectively, for a 5 × 10⁻¹⁰ m concentration of the analogues and a D₅₀ approximately 2 × 10⁻¹¹ m.

These results demonstrated the presence of only one V₁-type receptor in fish pituitary, with some of the structural and functional peculiarities typically displayed by the mammalian V_1a-type receptor, but distinct from it. In this sense, the fish pituitary vasotocin receptor may represent a novel type of neurohypophysial hormone receptor, more closely related to the mammalian V_1b-type.

Journal of Endocrinology (1996) 149, 109–115

ABSTRACT

Cyclic AMP levels were measured in freshly isolated hepatocytes of the rainbow trout. Compared with basal values, the average levels were increased up to 60 times in a dose-dependent manner either by mammalian glucagon (concentration range 1 nmol– 1 μmol/l; dose giving half maximum response (EC₅₀) 0· 18 μmol/l) or by forskolin (concentration range 0·1–100 μmol/l; EC₅₀ about 10 μmol/l). These stimulatory effects were partially inhibited by fish or mammalian neurohypophysial hormones used at relatively high concentrations (1–5 μmol/l). It is suggested that these results are evidence for the presence of V₁-type receptors in fish hepatocytes. Together with previous results obtained with gills on the hormonal inhibition of adenylate cyclase activity, they suggest that teleost fish may possess only V₁-type receptors (or two V₁-related types), while the V₂ receptors have evolved (or have become functional) in higher vertebrates.

J. Endocr. (1988) 119, 439–445