Effect of Extended Exposure to Hypertonicity on Vasopressin Messenger Ribonucleic Acid Content in Hypothalamo-Neurohypophyseal Explants*

Abstract

The feasibility of using organ-cultured explants of the rat hypothalamo-neurohypophyseal system (HNS) to study the mechanisms regulating the vasopressin (VP) mRNA content of the HNS was examined by evaluating the effect of exposure to hypertonicity on the VP mRNA content of these explants. Different effects were observed after a step increase in osmolality and a gradual increase in the same amount over 24 h. The VP mRNA content of control HNS explants determined from a RNA protection assay was 22 +/- 6 pg. It gradually decreased to 23% and 9% of the control value during 24 and 48 h in culture, respectively. Northern blot analysis revealed a single band of VP mRNA approximately 700 bases long in explants cultured for 36 h. Explants exposed to the step increase in osmolality were maintained in static culture. The control explants were placed directly into isotonic medium (299 mosmol/kg H2O). The explants exposed to the step increase were placed directly into hypertonic medium (greater than 304 mosmol/kg H2O). After 24 h in culture, basal VP release was measured, and all explants were then exposed to a further acute 15 mosm/kg H2O increase in osmolality. The highest basal release of VP was observed in the explants maintained under isotonic conditions (299 mosm/kg H2O). These explants significantly increased VP release in response to the acute increase in osmolality. Basal VP release was lower in explants maintained in hypertonic medium (greater than 304 mosmol/kg H2O), and these explants did not respond to the acute hypertonic pulse. VP mRNA content was significantly decreased in explants maintained for 24 or 48 h in hypertonic medium compared to that in explants maintained in isotonic medium (47 +/- 10% and 57 +/- 6%, respectively; P less than 0.01). No significant difference existed in the VP content of the posterior pituitary between the groups. To achieve a slow increase in osmolality, explants were perifused in individual chambers with medium at 2.1 ml/h. A gradual increase in osmolality (16 mosmol/kg H2O medium) was achieved by increasing the NaCl concentration in the perifusion medium. In response to this stimulus there was a significant increase in VP release, which was sustained for 9 h. VP mRNA content in the hypertonic group was 165 +/- 19% of that in control explants (P less than 0.001), but no difference existed in VP content in the posterior pituitary compared to that in time control explants.(ABSTRACT TRUNCATED AT 400 WORDS)