Small columns packed with octadecasilyl-silica were used to extract arginine-vasopressin in femtomole amounts from biological fluids for radioimmunoassay. This method is effective in isolating the peptide from substances in plasma and serum which interfere with its quantitation. Consistent and reproducible results and recoveries > 80% were obtained with this procedure. High pressure liquid chromatography and serial dilution of extracted samples confirm the identity of the extracted product.

The influence of vasopressin on renal hemodynamics was assessed by treating conscious Brattleboro homozygotes (DI rats) both acutely and chronically with physiologic doses of vasopressin. Intravenous infusions of vasopressin for 1 h, resulting in plasma vasopressin concentrations of less than 1.25, 2.3, and 8.0 pg/ml, respectively, failed to change glomerular filtration rate (GFR) or effective renal blood flow (ERBF) significantly, nor were there significant changes during 5 h of infusion. Body weight was not altered during these infusions. When synthetic vasopressin was given by osmotic minipumps for 10 days, with the rats gaining weight and thus changing the volume of their body fluids, GFR and ERBF increased significantly, by approximately 45 and 55%, respectively. Acute administration of volume alone, as well as acute vasopressin plus acute administration of volume, did not alter GFR or ERBF significantly. The data are compatible with the view that vasopressin, in physiologic plasma concentrations, exerts an influence on renal hemodynamics, that may be mediated through the long-term alteration of body fluid volumes. Alternatively or additionally, prolonged exposure of DI rats to vasopressin may increase their renal hemodynamics through tubuloglomerular feedback.

Despite the absence of vasopressin, Brattleboro homozygous (DI) rats concentrate their urine to hypertonic levels when deprived of drinking water for 24 h. Glomerular filtration rate (GFR) falls concurrently and might contribute to the increased concentrating ability. The present studies concerned the time course of the changes in concentrating ability and GFR during the early hours of dehydration. Experiments were performed in 10 chronically catheterized conscious DI rats in the normally hydrated control state and during 3 h of fluid deprivation. Urine osmolality (Uosmol) increased from 97 +/- 6 (SE) to 325 +/- 11 mosmol/kg H2O at 3 h. Averaged over the 3 h, neither GFR nor effective renal blood flow changed significantly (103 +/- 2 and 106 +/- 4% of control, respectively). Fractional excretion of sodium (FENa) rose markedly from 0.3 +/- 0.1 to 1.3 +/- 0.1% at its peak. Clearly, a fall in GFR cannot explain the rise in Uosmol during the first 3 h. Plasma oxytocin (OT) increased from 5.6 +/- 0.8 to 36.4 +/- 4.5 pg/ml after 3 h of dehydration. In additional experiments, d(CH2)5-D-Phe-VAVP, an antidiuretic antagonist (anti-ADH), was administered to eight DI rats after 3-h dehydration. Control, 3-h dehydration, and post-anti-ADH values were, respectively: for Uosmol, 102 +/- 7, 347 +/- 14, 145 +/- 11 mosmol/kg H2O; for GFR, 1,003 +/- 43, 1,042 +/- 59, 866 +/- 54 microliter X min-1 X 100 g body wt-1; for FENa, 0.4 +/- 0.1, 1.4 +/- 0.1, 0.5 +/- 0.1%. The decreases following anti-ADH were all statistically significant. We conclude that OT is released during the early hours of dehydration in the DI rat and has at least three renal effects. It causes a natriuresis, it maintains renal hemodynamics and GFR during the volume contraction, and it elicits a weak antidiuretic response.

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.

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