Characterization of vasopressin receptors in cultured cells derived from the region of rat brain circumventricular organs

Abstract

The aim of the present study was to characterize vasopressin receptors within the two circumventricular organs located in the lamina terminalis of the rat brain, namely the organum vasculosum of the lamina terminalis and the subfornical organ. Cells derived from both structures were isolated, cultured and intracellular Ca2+ concentrations were measured in single fura-2 loaded neurons and astrocytes after application of vasopressin and various vasopressin analogues. Subsequent to Ca2+ measurements, the identification of neurons and astrocytes was verified using immunocytochemistry with cell type-specific antibodies. High proportions of subfornical organ (34%) and organum vasculosum laminae terminalis (28%) neurons exhibited increased intracellular Ca2+ concentration after exposure to 1-1000 nM vasopressin. Within single cells, the response was dose-dependent. Similar results were obtained in subfornical organ (62%) and organum vasculosum laminae terminalis (38%) astrocytes with minor differences in the transient amplitude and pattern distribution when compared with neurons. Since omission of extracellular Ca2+ preserved vasopressin responsiveness, it is likely that intracellular stores were the main source of mobilized Ca2+. The preincubation of neurons and astrocytes with the V1 receptor-specific antagonist d(CH2)5[Tyr(Me)2]8-arginine vasopressin (10-100 nM) selectively and reversibly blocked the vasopressin-mediated response. Oxytocin-induced Ca2+ transients (0.32-1000 nM), which were observed in 32% (63%) or organum vasculosum laminae terminalis and in 54% (42%) of subfornical organ neurons (astrocytes), were not affected by the V1-specific antagonist. These data indicate the presence of a V1-like vasopressin receptor and an oxytocin receptor in cultured neurons and astrocytes from both circumventricular organ structures. In addition, the exposure to the highly selective V2 receptor agonist, 1-desamino,8-D-arginine vasopressin, evoked Ca2+ transients almost exclusively in organum vasculosum laminae terminalis neurons (eight of 18 tested). Only 1 (n = 14) subfornical organ neuron and none of the astrocytes tested (n = 26) responded to 1-desamino,8-D-arginine vasopressin. Since 1-desamino,8-D-arginine vasopressin acting via "classical" V2 receptors is not expected to affect the intracellular Ca2+ concentration, these data indicate the tissue and cell type-specific expression of a 1-desamino,8-D-arginine vasopressin-sensitive vasopressin receptor in neurons of the organum vasculosum laminae terminalis. In summary, the results indicate a heterogeneity of neurohypophyseal peptide receptor subtypes in the primary cell culture of both circumventricular structures.(ABSTRACT TRUNCATED AT 400 WORDS)