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Immunoreactive levels of vasopressin (VP) and oxytocin (OT) were quantitated in the rat pineal gland in the middle of August, when nonapeptide levels have been reported to peak annually. The pineal levels of both VP and OT were found to be substantially elevated when sampled in August, compared to sampling in July and September. mRNA levels for OT and VP in hypothalamic nuclei (supraoptic, paraventricular, and suprachiasmatic nuclei) showed no such increases during August. A lesioning of the paraventricular nuclei did not suppress pineal VP and OT levels. Finally, the injection of colchicine into the third ventricle caused pineal VP and OT levels to increase substantially. Together, these results affirm the occurrence of a summertime rise in pineal VP and OT levels and suggest that such increases do not derive from sites of VP and OT cell bodies in the hypothalamus. Rather, they indicate that the source of these pineal nonapeptides may be the pineal itself.

The course of the hypothalamo-infundibular somatostatin containing pathway was visualized by immunocytochemistry (the unlabeled antibody peroxidase-antiperoxidase complex method) combined with various knife cuts in the hypothalamus. Somatostatin immunoreactive fibers left the perikarya in the anterior hypothalamic-preoptic periventricular cell mass in a lateral direction. After a loop-like path through the lateral hypothalamus, fibers enter the medial basal hypothalamus mainly in the lateral retrochiasmatic area near the ventral surface and project to the ipsilateral half of the median eminence and the pituitary stalk. Somatostatin immunoreactivity almost completely disappeared from the stalk-median eminence 3-7 days following a complete anterolateral transection while transections dorsal or posterior to the lateral retrochiasmatic area failed to influence it. 7 weeks after anterolateral deafferentation an increased density of somatostatin containing terminals and immunopositive perikarya were seen in the arcuate and ventromedial nuclei but these neurones failed to reinnervate the stalk-median eminence. Thus there appears to be a neuron system containing somatostatin-like immunoreactivity within the medial basal hypothalamus, cell bodies being most abundant in the posterior divisions of the ventromedial and arcuate nuclei. However, most if not all of the somatostatin immunoreactivity in the stalk median eminence originates from outside of the medial-basal hypothalamus. Further, we have demonstrated the importance of the careful histological evaluation of each individual operation if functional studies are to be carried out after hypothalamic deafferentation.

In an in vitro system, we have demonstrated concomitant release of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) from the median eminence (ME) of normal and adrenalectomized rats. The ME were incubated in a Krebs-Ringer bicarbonate medium, CRF and AVP released into the medium were measured by radioimmunoassay. The release of both neuropeptides was stimulated by increasing concentrations of potassium (28-56 mM) in the incubation medium, or by addition of veratridine (5-20 microM). In both cases the release process was dependent on the presence of calcium in the incubation medium. Interestingly, potassium-induced release was found to be relatively insensitive to calcium channel antagonists that are potent inhibitors in smooth and cardiac muscle. The sodium channel antagonist, tetrodotoxin (1 microM), completely blocked the effect of veratridine, while no change was seen in the response to potassium. Adrenalectomy increased the ratio of AVP:CRF release from 2:1 in ME removed from sham-operated rats to 8:1 in ME from the adrenalectomized group. We suggest that this ratio of AVP:CRF release may also pertain in vivo, and that AVP could be the predominant corticotropic stimulus in adrenalectomized rats.

In this study we have characterized binding sites for ovine corticotropin-releasing factor (oCRF) in the rat anterior pituitary gland, and have investigated whether the site of interaction of vasopressin and oCRF is at the plasma membrane level. The binding 125I-oCRF to anterior pituitary membranes was shown to be dependent on temperature, pH and cation concentration. Magnesium was essential for the binding reaction to take place. Two binding sites of Kd 7.63 X 10(-10) and 3.39 X 10(-8) M were found. Activity of adenylate cyclase in the membrane preparation increased in the presence of oCRF. The activity of the enzyme as well as the binding of 125I-oCRF was found to be influenced by guanosine-5'-triphosphate. Several hypothalamic neurohormones, including vasopressin, failed to alter the binding of 125I-oCRF to anterior pituitary membranes. Moreover, vasopressin failed to influence the stimulation of adenylate cyclase activity induced by oCRF. Preincubation of anterior pituitary segments with oCRF desensitized the corticotropin (ACTH) response to oCRF and decreased the amount of 125I-oCRF bound to membranes prepared from similarly treated pituitaries. The ACTH response to vasopressin remained unchanged. Following a preincubation of anterior pituitary segments with vasopressin, oCRF stimulated ACTH secretion, as well as the binding of 125I-oCRF to pituitary membranes was normal, while the ACTH response to vasopressin was markedly reduced. These results show that separate receptors mediate the action of vasopressin and oCRF. Moreover, the ACTH response to vasopressin and oCRF may be modulated separately.

The ligand specificities of sites binding tritium-labeled arginine vasopressin (3H-AVP) were investigated in membrane preparations of rat anterior pituitary gland and liver. Labeled AVP interacted with high-affinity, low-capacity binding sites in liver as well as in pituitary membrane fractions. Binding displacement studied showed that AVP was a potent ligand for liver (KI = 0.23 nM) and pituitary (KI = 0.4 nM) receptors. Oxytocin and the antidiuretic (V2) agonist dDAVP had low affinities (KI greater than 10 nM) both for liver and pituitary binding sites. The pressor (V1) antagonist d(CH2)5Tyr(Me)AVP was equipotent with AVP in liver membranes, but was 1,000-fold less potent on pituitary receptors. Another V1 antagonist, dPenTyr(Me)AVP, displaced 3H-AVP with KI values of 3 and 8 nM from pituitary and liver receptors, respectively. These data are in reasonable agreement with recent studies of the in vitro biological activities of the analogs tested, and suggest that the ligand specificity of rat pituitary AVP receptors is distinct from those of previously characterized V1, V2 and oxytocin binding sites in the rat.

The aim of this study was to investigate further the possible role of nerve cells of the arcuate nucleus in the regulation of growth hormone (GH) and prolactin (PRL) secretion. Newborn male rats were treated with monosodium-L-glutamate (MSG) (4 g/kg body weight subcutaneously) or NaCl (0.5 g/kg) on alternate days for the first 10 days of life, and were used 8-9 weeks afterwards. beta-Endorphin-like immunoreactivity in the arcuate nucleus decreased by 54% after MSG treatment. The dopamine content of the median eminence was reduced to 55% of that in NaCl-treated litter mates. No significant change of dopamine content was found in the other hypothalamic regions examined. Basal levels of plasma PRL were unaltered. Intravenous injection of morphine to urethane-anesthetized animals was 3 times more potent in stimulating PRL secretion in MSG-treated rats, while it was 2.5 times less potent in releasing GH. These data suggest that lesioning of the arcuate nucleus by MSG treatment damages neurons inhibiting PRL secretion, as well as neurons facilitating GH secretion.