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\m='\). The cross-reactions in the radioimmunoassay for VP-RNP with preparations of OT-RNP and OT-RNP\m='\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\ x=req-\ RNP and OT-RNP\m='\in the assay is <0\m=.\02%. The radioimmunoassay for OT-RNP showed a 100% cross-reaction with OT-RNP\m='\and a 4% cross-reaction with VP-RNP, while cross\x=req-\ reactions in the radioimmunoassay for OT-RNP\m='\with OT-RNP and VP-RNP were 17 and 3%. All three radioimmunoassays had a range of measurement from 20 to 1280pg protein (2\p=n-\132 f mol ) . The radioimmunoassays for VP-RNP and OT-RNP were used to measure neurophysin levels in the neural lobes and serum of Long\p=n-\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\p=n-\Evans rats were similar to those of VP-RNP; oxytocin levels were similar to the levels of total OT-RNP (OT-RNP + OT-RNP\m='\). 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\m=.\013and <0\m=.\1% of the oxytocin and OT-RNP levels respectively. Long\p=n-\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\m=+-\41and 312\m=+-\43ng/l for males, and 280 \ m=+-\ 40 and 462\m=+-\116ng/l for females. Heterozygous DI rats (all female) had serum values of 165 \ m=+-\ 5 ng VP-RNP/1 and 1130\m=+-\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\m=+-\37 ng/1 for males and 959 \ m=+-\121 ng/1 for females. These values are approximately twice those in Long\p=n-\Evans rats. In water-deprived homozygous DI rats values of OT-RNP were increased threefold to 1202\m=+-\87 ng/1 in males and twofold to 1822\m=+-\259 ng/1 in females. Data indicate that female DI rats have twice the production/release rate of OT-RNP as males.

Secretion of Vasopressin, Oxytocin, and Two Neurophysins from Rat Hypothalamo-Neurohypophyseal Explants in Organ Culture

“…Accurate measurements were obtained in all cases. The inter-and intraassay variabilities were the same as those reported previously [9,II,12].…”

“…However, a 7-fold difference between the level of OT-RNP and OT was noted. This may indicate that most of the OT present was in the precursor form, resulting in masking of hypothalamic OT to measurement by our antibodies [ 12]. …”

“…The polyethylene glycol pellets were washed and counted in a Packard gamma counter (model 5260/545). For iodinations of hormones and neurophysins the lactoperoxidase method was used [21] and was followed by column chromatography as previously described [11,12]. The radioimmu noassay for AVP was accomplished using equilibrium conditions (48 h. 0-4 C) with a final antiserum dilution of 1: 12.000.…”

“…AM-08469-16, AM 07301-4, and NRSA CA06921. cent study from this laboratory [12], highly specific radio immunoassays for AVP, OT, VP-RNP, and OT-RNP were used to determine comparative levels of these peptides in plasma and neural lobes of Long-Evans and Brattleboro homozygous rats under normal conditions or osmotic stress. While others have used the explants to examine se cretion of AVP in culture, our aim in this study was to esta blish whether HNS explants could be effectively used as an in vitro model system to measure control mechanisms involved in the secretion of VP-RNP, OT.…”

Effects of Acute Increases in Plasma Osmolality on Plasma Vasopressin-Associated Neurophysin in Conscious Rats: Implications for Osmoregulation

“…The following measurements were made for each blood sam ple: ( I ) plasma [VP-RNP] by radioimmunoassay using a previously described method [9]; (2) …”