Jitendra R. Dave

Chronic ethanol ingestion decreases vasopressin mRNA in hypothalamic and extrahypothalamic nuclei of mouse brain.

Endogenous arginine vasopressin was previously shown to modulate the rate of loss of functional (CNS) tolerance to ethanol, suggesting that chronic ethanol ingestion might alter vasopressin synthesis and/or release. Since extrahypothalamic vasopressin is believed to be involved in the CNS effects of the peptide, we determined the effect of ethanol on vasopressin mRNA in the bed nucleus of the stria terminalis (BST), as well as in several hypothalamic nuclei. Chronic ethanol ingestion, that produced functional tolerance and physical dependence in mice, resulted in decreased vasopressin mRNA levels in all areas examined. In contrast, as expected, dehydration resulted in increases in vasopressin mRNA in the BST and in all hypothalamic nuclei except the suprachiasmatic nucleus. In the BST, both ethanol ingestion and dehydration affected cells in the central region of the nucleus, while cells in the caudal portion were only affected by ethanol treatment. The results indicate that chronic ethanol ingestion generally reduces the synthesis of vasopressin, and that increased vasopressin synthesis is not necessary in order for the peptide to affect ethanol tolerance.

Arginine vasopressin induces the expression of c-fos in the mouse septum and hippocampus

Arginine vasopressin is a neuropeptide that has been shown to modulate functional ethanol tolerance and memory processes. These actions of vasopressin in the CNS have been shown by us and others to be mediated by V1 receptors. Intracerebroventricular injection of vasopressin in mice resulted in a substantial increase in mRNA for the proto-oncogene c-fos in septum and hippocampus, but no increase in cerebral cortex. A V1-selective agonist also increased septal c-fos mRNA levels, while a V2-selective agonist was less effective. Similarly, the response to vasopressin was more effectively blocked by a V1- than a V2-selective antagonist. These results indicate that vasopressin acts specifically at V1 receptors in mouse septum and hippocampus to increase c-fos mRNA. The vasopressin metabolite, AVP(4-9), also increased c-fos mRNA levels in septum and hippocampus, while the response to oxytocin, which has different effects from vasopressin on memory and tolerance, was greater in hippocampus than in septum. Nerve growth factor, in contrast to the other peptides, had a more pronounced effect on c-fos mRNA levels in cerebral cortex than in the other brain areas. Increased c-fos expression has been hypothesized to play a role in neuroadaptation, and these results suggest that modulation of septal c-fos expression could be important for vasopressin effects on ethanol tolerance and/or memory.

Hypothalamic vasopressin mRNA levels in mice are decreased after chronic ethanol ingestion

Vasopressin mRNA levels in the supraoptic and paraventricular nuclei of the hypothalamus, measured by in situ hybridization with a 35S-labeled RNA probe, were decreased by nearly 50% in C57BL/6NCR mice that had ingested an ethanol-containing diet for 7 days, and were tolerant to and physically dependent on ethanol. At 24 h after withdrawal, vasopressin mRNA levels in the supraoptic nucleus were still decreased, while levels in the paraventricular nucleus returned toward control values. Although plasma osmolality was increased in ethanol-fed mice, there was no increase in plasma vasopressin levels, possibly as a result of the effect of chronic ethanol ingestion to decrease vasopressin synthesis. In contrast, in mice that were dehydrated, but not fed ethanol, plasma osmolality, plasma vasopressin levels, and hypothalamic vasopressin mRNA all increased, as expected. The data suggest that chronic ethanol ingestion interferes with the synthesis and secretion of vasopressin, and may result in the reduced ability of an individual to respond to physiological stimuli for vasopressin secretion.

Arachidonic acid, bradykinin and phospholipase A2 modify both prolactin binding capacity and fluidity of mouse hepatic membranes

Abstract The objective of this study was to determine if arachidonic acid, a precursor of prostaglandin synthesis, bradykinin, a decapeptide known to stimulate membrane phospholipid methylation, arachidonic acid release and prostacyclin synthesis, and enzyme phospholipase A 2 , capable of liberating arachidonic acid, alter the fluidity of hepatic membranes which could in turn modify the functionality of prolactin receptors. Liver homogenates of adult C 3 H female mice incubated at 28°C for various times with 1–20 μg/ml arachidonic acid, 1–100 μg/ml bradykinin or 0.26–0.00026 U/ml phospholipase A 2 provided the 100,000 × g membrane pellets for subsequent ovine prolactin binding and membrane fluidity studies. Membrane microviscosity was determined by fluorescence polarization techniques using the lipid probe 1,6 diphenylhexatriene. Arachidonic acid, bradykinin and phospholipase A 2 stimulated specific oPRL binding, in a dose-related fashion, with maximum increases of 73%, 21% and 46%, at 4 μg/ml arachidonic acid, 5 μg/ml bradykinin and 0.026 U/ml PLA 2 , respectively. This induction, occurring within 30 min of incubation, was found to be due to an increase in the number of receptor sites. Under the same conditions, arachidonic acid, bradykinin and PLA 2 induced 22%, 16%, and 18% decreases in membrane microviscosity, respectively. These data suggest that prostaglandin synthesis modifying agents may modulate the number of prolactin receptors in vivo by changing the lipid fluidity of the target cell membranes by either of their known effects: arachidonic acid release from the phospholipid matrix, synthesizing appropriate prostaglandins at correct concentration or methylation of membrane phospholipids.

Prolactin modifies the prostaglandin synthesis, prolactin binding and fluidity of mouse liver membranes.

Abstract The objective of these studies was to determine if prolactin, known to induce its own receptors, alters the prostaglandin (PG) synthesis which could, in turn, modify the fluidity of the membrane and thus alter the functionality of prolactin receptors. Adult male C3H mice were injected subcutaneously with 100 μg of oPRL every 4 h for 0, 24 or 48 h and sacrificed 8 h after receiving the last injection. Liver 100,000 × g membrane pellets were used in the measurement of these parameters. The amount of binding of prolactin to these membranes increased with the duration of injections, the values being 179 and 244% of control values after 24 and 48 h of injections, respectively. The amounts of PGF2α and PGE synthesized also increased after these injections, the values being 127 and 270% of control for PGF2α and 634 and 695% of control values for PGE after 24 and 48 h of injections, respectively. Fluorescence polarization, an index of microviscosity, was decreased by 14 and 20% after 24 and 48 h of PRL administration, respectively. Previous studies have demonstrated simultaneous in vitro effects of prostaglandin on both prolactin receptors and membrane fluidity. The current data are in agreement with those observations and suggest that prolactin may modulate its own receptor by increasing the fluidity of the membrane in which it exists by alterations within the PG cascade. Such biochemical changes may then modify existing restraints and allow the hormone receptor to assume a more functional configuration.

The Role of Arginine Vasopressin in Alcohol Tolerance

Administration of the neuropeptide, arginine vasopressin, to animals that have acquired functional tolerance to ethanol will maintain such tolerance, even in the absence of further ethanol ingestion by the animals. In mice, this action of the peptide is mediated by central nervous system V1 receptors and requires intact brain noradrenergic systems. Autoradiographic studies have shown that some V1 receptors are localized presynaptically on catecholaminergic neuronal terminals in the mouse lateral septum, suggesting that vasopressin may act via modulation of catecholamine release. In addition, vasopressin has been found to increase mRNA levels for the proto-oncogene, c-fos, in septum and hippocampus, possibly by an action at postsynaptic receptors. Expression of c-fos, which has been hypothesized to play a role in central nervous system neuroadaptation, could transform short-term actions of vasopressin into long-term effects on ethanol tolerance. Studies with vasopressin antagonists indicate that the endogenous peptide influences tolerance, and therefore the effect of chronic ethanol ingestion on vasopressin synthesis and release was studied. In mice and rats, hypothalamic vasopressin mRNA is decreased by chronic ethanol exposure, although effects on plasma vasopressin levels differ in the two species. The effect of ethanol on extrahypothalamic vasopressin synthesis in brain is under investigation. The results suggest mechanisms by which vasopressin can produce long-term changes in central nervous system function, and provide evidence for a disturbance of vasopressin regulation during chronic ethanol ingestion.

Prolactin modifies the fluidity of rat liver membranes.

Summary The objective of this study was to determine the effect of prolactin upon the fluidity of hepatic membranes and subsequent modification of the prolactin receptors. Hypophysectomized, immature, female rats (HIFR) having subcutaneous diethyl stillbesterol implants were treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). In addition, graded doses of 0, 0.25, 0.5, 1.0, 2.5 or 50 μg oPRL were injected hourly for the 48 hours prior to sacrifice at 28 days of age. Both the lipid fluidity as determined by 1,6-diphenylhexatriene (DPH) fluorescence polarization and phospholipid/cholesterol (P/C) ratio were measured in hepatic microsomal membranes. A biphasic response was observed in both of these parameters as the dose of oPRL was increased. P/C values were 91,113, 117, 88, and 93% of control values for the 0.25, 0.5, 1.0, 2.5 and 50 μg oPRL groups, respectively, the maximal value being significantly different from the saline injected controls. These ratios were inversely proportional to the corresponding values of fluidity which also varied in a biphasic manner, with fluorescence polarization equal to 97, 90, 82, 100 and 101% of control values. The value for the 1.0 μg group was, again, significantly different from control. No significant specific 125 I-oPRL binding was detectable in any of the groups after treating the membranes with distilled water, however, pretreatment of membrane preparations with 4 M MgCl 2 for 5 min resulted in a significant increase in the oPRL-binding in the 0.5 and 1.0 μg oPRL groups. The values being 273 and 521% of control, respectively in these two groups. These data demonstrate that prolactin modifies the viscosity of hepatic membranes and suggests that this phenomenon may be responsible for the autoregulation of detectable prolactin receptors.

Barium distinguishes separate calcium targets for synthesis and secretion of peptides in neuroendocrine cells

The effect of barium and potassium on the secretion and biosynthesis of enkephalin in bovine chromaffin cells, and prolactin and beta-endorphin in rat anterior pituitary cells, was examined to determine whether calcium-dependent secretion and biosynthesis are mediated by the same or by different calcium targets within the neuroendocrine cell. In the presence of 1.8 mM calcium, barium and potassium stimulated the secretion of all three peptides over 30 min, and increased the levels of proenkephalin and prolactin mRNA in 24 hr. These effects were inhibited by the calcium channel blocker D600. When the extracellular calcium concentration was lowered to 0.1 mM or less, secretion elicited by potassium was blocked, whereas secretion elicited by barium was enhanced, indicating that barium wholly substitutes for extracellular calcium in mediating peptide secretion. On the other hand, stimulation of proenkephalin and prolactin mRNA by both potassium and barium was inhibited when the extracellular calcium concentration was reduced. We conclude that calcium acts at two different intracellular targets to activate secretion versus biosynthesis of both enkephalin and prolactin. This appears to be the first report in which two different calcium-dependent processes in the intact cell are distinguished by a calcium ion agonist. Calcium-dependent processes such as protein phosphorylation, protein translocation, and enzyme activation may thus be related to events in the intact cell such as peptide synthesis and secretion on the basis of selective stimulation by barium.

Changes in the prolactin-binding capacity of mouse hepatic membranes with development and aging.

The objective of this study was to document the developmental changes in the prolactin-binding capacity of hepatic membranes of the female mouse. Prolactin-binding capacity was measured in the microsomal membranes of liver obtained from C3H female mice at various ages. Binding capacity of these membranes remained low until 21 days of age after which it increased and reached the adult levels by 44 days of age. Additional studies were made to observe this parameter in male and female mice during aging. Both female and male mice at 450-470 days of age had values of prolactin binding that were 66% and 79% that of the 77-day-old animals, respectively. A significant increase in membrane lipid microviscosity was observed in animals from both sexes at 450-470 days of age. This was in agreement with earlier studies that showed that prolactin receptors of hepatic membranes are modulated by changes in the membrane lipid microviscosity. These changes suggest that such modifications of cellular membranes are interrelated and that changes in the membrane microviscosity with aging may be a factor modulating cellular responses in older animals.

Chronic ethanol exposure uncouples vasopressin synthesis and secretion in rats

To assess the chronic effect of ethanol on vasopressin release and synthesis, hypothalamic vasopressin mRNA, plasma vasopressin levels and plasma osmolality were measured in control rats and rats exposed chronically to ethanol by vapor inhalation for 8 days. The level of hypothalamic vasopressin mRNA (vasopressin synthesis) was unchanged or significantly decreased in ethanol-treated rats, even when these animals displayed increased plasma osmolality and vasopressin levels. The results suggest that chronic ethanol exposure produces a down-regulation of vasopressin synthesis and/or a disruption of vasopressin synthesis-secretion coupling. These findings may have important implications for evaluation of the hydration state of chronic alcoholics.