Radioimmunoassays for individual rat neurophysins.

Abstract

Radioimmunoassays are described for vasopressin-associated rat neurophysin (VP-RNP), oxytocin-associated rat neurophysin (OT-RNP) and a major metabolic derivative of oxytocin-associated rat neurophysin (OT-RNP′). The cross-reactions in the radioimmunoassay for VP-RNP with preparations of OT-RNP and OT-RNP′ were 4 and 2%. However, the very small values found for VP-RNP using this radioimmunoassay in concentrated extracts of neural lobes from rats homozygous for the condition of hypothalamic diabetes insipidus (DI) indicate that the sum of the cross-reactions of OT-RNP and OT-RNP′ in the assay is <0·02%. The radioimmunoassay for OT-RNP showed a 100% cross-reaction with OT-RNP′ and a 4% cross-reaction with VP-RNP, while cross-reactions in the radioimmunoassay for OT-RNP′ with OT-RNP and VP-RNP were 17 and 3%. All three radioimmunoassays had a range of measurement from 20 to 1280 pg protein (2–132 fmol). The radioimmunoassays for VP-RNP and OT-RNP were used to measure neurophysin levels in the neural lobes and serum of Long–Evans rats and rats homozygous and heterozygous for DI. Neurophysin values in neural lobes were compared with values for oxytocin and vasopressin obtained by radioimmunoassay. On a molar basis the storage levels of vasopressin in Long–Evans rats were similar to those of VP-RNP; oxytocin levels were similar to the levels of total OT-RNP (OT-RNP + OT-RNP′). The storage levels in oxytocinergic and vasopressinergic neurones were similar. Total OT-RNP and oxytocin levels were similar in homozygous DI rats, while vasopressin and VP-RNP in these animals were <0·013 and <0·1% of the oxytocin and OT-RNP levels respectively. Long–Evans rats given 2% NaC1 for 96 h had drastically reduced storage of all four neurohypophysial peptides, but there was no significant change in their subcellular distribution when compared with control animals. This is taken as evidence that the readily releasable pool is not extragranular. Oxytocin and total OT-RNP were reduced to about 50% by depriving homozygous DI rats of water for 24 h. The storage levels of oxytocin and OT-RNP in female homozygous DI rats were twice those of their male counterparts. Normal serum values of VP-RNP and OT-RNP were 221±41 and 312±43 ng/l for males, and 280 ± 40 and 462±116 ng/l for females. Heterozygous DI rats (all female) had serum values of 165 ± 5 ng VP-RNP/1 and 1130±136 ng OT-RNP/1. In the serum of homozygous DI rats VP-RNP levels were below detectable limits; OT-RNP levels of rats with free access to water were 442± 37 ng/l for males and 959 ± 121 ng/l for females. These values are approximately twice those in Long–Evans rats. In water-deprived homozygous DI rats values of OT-RNP were increased threefold to 1202± 87 ng/l in males and twofold to 1822± 259 ng/l in females. Data indicate that female DI rats have twice the production/release rate of OT-RNP as males.