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Vasopressin in Neuronal Cultures From Neonatal Rat Brain

This study was performed to characterize and quantify vasopressin in neuron-enriched primary cultures of whole brains from 1-day-old rats and to compare such cultures between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Vasopressin was extracted from cells and evaluated by radioimmunoassay and high-pressure liquid chromatography. Radioimmunoassay of high-pressure liquid chromatography fractions from cell extracts showed that the major peak of immunoreactivity comigrated with synthetic vasopressin. Cell vasopressin content increased in a graded manner when one to six dishes (plated 8xlO6 cells/ dish) were pooled (8.7±0.5 to 67.7±8.2 pg/dish). When the number of plated cells per dish was increased (2 to 16xlO6 cells), there was also a graded rise in dish vasopressin content (3.4±0.4 to 15.3±3.9 pg). Treatment of cultures with 100 aM to 1 nM of angiotensin II for 5 minutes caused a dose-dependent decrease in cell vasopressin content. Furthermore, the decrease in cell vasopressin content of cultures treated with 1 nM angiotensin II (12.6±0.8 to 7.0±1.0 pg/106 cells, p < 0.05) corresponded with the increase in medium vasopressin concentration (3.8±0.5 to 7.5±2.3 pg/ml) and this vasopressin-releasing effect of angiotensin II was blocked by [Sar1, Thr8]angiotensin II. Treatment of cultures with potassium chloride (56 mM) and acetylcholine chloride (5.5 μM) also resulted in significant decreases in cell vasopressin content. Five to 15-day-old cultures from SHR had significantly smaller amounts of vasopressin than those from WKY rats (32.6+5.2 vs. 82.5±12.7 pg/106 cells, respectively, p < 0.01). Hence, our data show that vasopressin is present in primary neuronal cultures and is released by angiotensin II and other substances known to release vasopressin in vivo. Furthermore, there may be differences in the synthesis or release of vasopressin between SHR and WKY rats.