Chronic stress effects on the rat adrenal cortex

Under the influence of a chronic permanent stress, the adrenal function as well as the entire hypothalamic-pituitary-adrenal axis (HPAA) suffered an adaptation process that resulted in the normalization of the studied stress hormones (ACTH, corticosterone and aldosterone) with the exception of plasma renin activity which first diminished and at the end increased. ACTH receptors exhibited a dual response since after 14 days of permanent stress MC2-R showed a slight reduction while MC5-R was still up-regulated.     

Actions of vasoactive intestinal peptide on the rat adrenal zona glomerulosa

Previous studies, by this group and others, have shown that vasoactive intestinal peptide (VIP) stimulates aldosterone secretion, and that the actions of VIP on aldosterone secretion by the rat adrenal cortex are blocked by beta adrenergic antagonists, suggesting that VIP may act by the local release of catecholamines. The present studies were designed to test this hypothesis further, by measuring catecholamine release by adrenal capsular tissue in response to VIP stimulation. Using intact capsular tissue it was found that VIP caused a dose-dependent increase in aldosterone secretion, with a concomitant increase in both adrenaline and noradrenaline release. The effects of VIP on aldosterone secretion were inhibited by atenolol, a beta1 adrenergic antagonist, but not by ICI-118,551, a beta2 adrenergic antagonist. Binding studies were carried out to investigate VIP receptors. It was found that adrenal zona glomerulosa tissue from control rats contained specific VIP binding sites (Bmax 853+/-101 fmol/mg protein; Kd 2.26+/-0.45 nmol/l). VIP binding was not displaced by ACTH, angiotensin II or by either of the beta adrenergic antagonists. The response to VIP in adrenals obtained from rats fed a low sodium diet was also investigated. Previous studies have found that adrenals from animals on a low sodium diet exhibit increased responsiveness to VIP. Specific VIP binding sites were identified, although the concentration or affinity of binding sites in the low sodium group was not significantly different from the controls. In the low sodium group VIP was found to increase catecholamine release to the same extent as in the control group, however, in contrast to the control group, the adrenal response to VIP was not altered by adrenergic antagonists in the low sodium group. These data provide strong support for the hypothesis that VIP acts by the local release of catecholamines in adrenal zona glomerulosa tissue in normal animals. It does not appear that VIP acts through the same mechanism in animals maintained on a low sodium diet. The mechanism by which VIP stimulates aldosterone in this group remains to be determined.     

Zonal differentiation in the rat adrenal cortex

The factors that establish and maintain adrenocortical zonation are poorly understood. The capsular adrenal gland of the rat has been shown to develop into a functionally zoned tissue in autotransplanted glands in vivo. To examine this in vitro, capsular gland preparations (largely glomerulosa (zg) with some fasciculata (zf) were cultured in vitro in Eagles MEM (3.6mM K+) for 14 days. Zonal differentiation was determined by immunocytochemical localisation of inner zone antigen (IZA, zf/reticularis specific) and Pref-1 (zg specific). In the absence of further additions these preparations invariably maintained a good zonal arrangement of zg and zf over the whole period, though without significant cellular proliferation. Neither the daily addition of the stimulants, maximally 8.3mM potassium, 1nM ACTH, or 100nM angiotensin II (AII), or the AII type 1 receptor antagonist losartan (10microM) had any significant effect on the glands intrinsic capacity to maintain zonation in vitro. Aldosterone output declined rapidly under control conditions (3.6mM K+), but was stimulated by AII, or high K+ reaching a maximum after 7 days, and thereafter declined. However at higher K+ conditions (5.6mM) aldosterone was not supported by angiotensin II. Corticosterone secretion increased autonomously after 2 days in 3.6mM K+ then declined. At higher K+ conditions corticosterone rapidly declined. The factors studied had no effect on the inherent property of the adrenal gland to express the zg or zf phenotype. However the functional steroidogenic capacity of the adrenocortical cells was affected in a highly specific and complex manner by the added stimulants.     

Prevention of renal hypertension in the rat by neuropeptide Y

Neuropeptide Y is known to enhance blood pressure responsiveness to various constrictors, including angiotensin II, and to suppress renin secretion. This study was undertaken to assess the effect of neuropeptide Y on the development of two-kidney, one clip renal hypertension. Normotensive rats either had a silver clip placed on the left renal artery or were sham-operated upon. An osmotic minipump, which was connected via a catheter to a jugular vein, was implanted subcutaneously in all rats. These pumps delivered either neuropeptide Y (0.001 microgram/min) or saline intravenously. Eight days later, an intra-arterial catheter was inserted and the rats were studied while not anesthetized on the following day. Neuropeptide Y did not affect body weight. In clipped rats, neuropeptide Y prevented the development of hypertension and suppressed renin secretion. Neuropeptide Y significantly decreased blood pressure also in sham-operated rats, although it had no effect on plasma renin activity. These data indicate that prolonged neuropeptide Y infusion may lower blood pressure by different mechanisms, one of which is probably a suppression of renin release.     

Structure and expression of the rat neuropeptide Y gene.

Neuropeptide Y is a 36-amino acid peptide that is abundant throughout the mammalian nervous system. It belongs to the same family of carboxyl-terminally amidated peptides as pancreatic polypeptide and peptide YY. We describe here the gene encoding the rat neuropeptide Y precursor. The gene spans 7.2 kilobase pairs and contains four exons. The exon organization is identical to the pancreatic polypeptide gene, although the amino acid sequences of the neuropeptide Y and pancreatic polypeptide precursors differ extensively. The predicted amino acid sequence of mature rat neuropeptide Y is identical to the human sequence. Also the sequence of the 30-amino acid carboxyl-terminal peptide of preproneuropeptide Y is highly conserved, which suggests that it is functionally important. Two neuropeptide Y alleles were found to differ at nine positions in 2.5 kilobase pairs at the 5' portion of the gene. No exon difference was found. One nucleotide substitution close to the gene promoter may influence the regulation of expression. Neuropeptide Y mRNA was found in all rat brain subregions tested, which shows that neuropeptide Y is synthesized throughout the brain. Developmentally, mRNA was detected in the rat brain as early as embryonic day 16 and increased rapidly to adult levels. The level of neuropeptide Y mRNA was also studied in several rat peripheral organs. Unexpectedly high levels were observed in heart and spleen. This mRNA may be synthesized in intrinsic ganglia and non-neuronal cells, respectively.     

Cocaine-induced reduction of brain neuropeptide Y synthesis dependent on medial prefrontal cortex.

Repeated administration of cocaine elicits substantial, long-lasting, but reversible reductions in neuropeptide Y (NPY) and NPY mRNA in the rat cerebral cortex and nucleus accumbens. The NPY reduction appears to be mediated through a decrease in NPY biosynthesis, occurring transneuronally, perhaps in response to changes in synaptic dopamine associated with mesolimbic and mesocortical dopamine neurons. The medial prefrontal cortex appears necessary for maintenance of cocaine's action on this neuronal network since excitotoxic lesions of this area prevented (lesion before cocaine) and reversed (lesion after cocaine) the reductions in NPY elicited by the cocaine. NPY may be a sensitive marker for chronic cocaine use. Its decrease may relate to the anxiety and depression associated with cocaine withdrawal in humans.     

Cardiovascular effects of neuropeptide Y in rat brainstem nuclei

Central catecholaminergic neurons are involved in cardiovascular regulation. Neuropeptide Y (NPY) coexists with adrenaline and noradrenaline in the rat brain, and interactions among these substances have been studied. The purpose of this study was to investigate the possible role of NPY in central cardiovascular control. Male Sprague-Dawley rats were anesthetized with urethane, and blood pressure was monitored intra-arterially. Intramedullary microinjection (60 nl) of NPY (0, 46.5 fmol, 465 fmol, 1.5 pmol, and 4.65 pmol) was made into the nucleus tractus solitarii (NTS), into the area postrema, and into the C1 area in the rostroventrolateral medulla. Injection site was identified by L-glutamate administration and confirmed histologically. Unilateral injection of NPY into the NTS produced a prominent dose-related decrease in heart rate and systolic and diastolic blood pressure (-106 +/- 8 beats/min, -56 +/- 2 mm Hg, and -33 +/- 2 mm Hg, respectively after 4.65 pmol NPY, n = 7, p less than 0.001). Maximal changes occurred at 30 seconds and recovered in 10 minutes for blood pressure and 20 minutes for heart rate. Injection into the area postrema produced an initial increase in heart rate and mean blood pressure (+23 +/- 2 beats/min and +18 +/- 2 mm Hg) followed by a prolonged decrease in heart rate and mean blood pressure (-14 +/- 4 beats/min and -15 +/- 2 mm Hg, respectively, n = 7, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of neuropeptide Y binding sites in the zona glomerulosa of the bovine adrenal gland

We studied neuropeptide Y (NPY) binding sites in the bovine adrenal gland by incubating tissue sections with [125I]-Bolton Hunter NPY, and then by measuring the number and affinity of binding sites in the tissue with quantitative autoradiography. Specific NPY binding sites were localized exclusively in the zona glomerulosa. These binding sites have an apparent dissociation constant (Kd) of 0.45 +/- 0.06 nM and a binding capacity (Bmax of 134 +/- 15 fmol mg-1 protein. Our results suggest that NPY may directly affect the release of aldosterone in the zona glomerulosa of the adrenal gland.     

Alcohol withdrawal increases neuropeptide Y immunoreactivity in rat brain

BACKGROUND: Neuropeptide Y (NPY) is widely expressed in the brain and is known to affect consummatory behaviors including drinking alcohol as well as to play a role in seizures. We investigated the effects of a 4 day binge ethanol treatment model that is known to induce physical dependence and withdrawal seizures to determine the effects of ethanol dependence and withdrawal on NPY expression. METHODS: Male Sprague Dawley rats were treated with ethanol or control nutritionally complete diets by intragastric treatment three times per day for 2 or 4 days with an average daily dose of approximately 8 g/kg ethanol per day. Ethanol-fed rats treated for 4 days and then withdrawn for 24, 72, and 168 hr also were studied. Brains were perfused and sectioned for immunohistochemistry for NPY, phospho-cyclic adenosine monophosphate responsive element binding (pCREB), and other proteins. RESULTS: NPY immunoreactivity (NPY-IR) was found in several brain regions, with the hippocampus and cerebral cortex showing the most pronounced changes. NPY-IR was reduced by ethanol treatment in hippocampus and cortex, although at 72 hr of withdrawal there was a dramatic increase in NPY-IR in the hilus of the dentate gyrus and in CA3 and CA2 fields of hippocampus. Ethanol withdrawal seizures occurred around 12 to 24 hr of withdrawal, preceding the changes in NPY-IR at 72 hr. pCREB immunoreactivity (pCREB-IR) tended to decrease during ethanol treatment but showed a dramatic increase in dentate gyrus at 72 hr of withdrawal. Parvalbumin immunoreactivity indicated that some of the pCREB-IR and NPY-IR were within inhibitory interneuron basket cells of the hippocampal hilus. NPY-IR returned to control levels by 168 hr of withdrawal. CONCLUSIONS: These studies suggest that hippocampal NPY is reduced during the development of ethanol dependence. Ethanol withdrawal seizures precede a dramatic increase in hippocampal NPY-IR. Previous studies have suggested that NPY in the hippocampus reduces seizure activity and that NPY is induced by seizure activity. Thus, the increase in NPY-IR at 72 hr of withdrawal after binge ethanol treatment may be protective against prolonged withdrawal seizure activity.     

Biochemical and immunocytochemical characterization of neuropeptide Y in the immature rat ovary

Neuropeptide Y (NPY)-like immunoreactivity has been found in nerves that innervate the rat ovary. In this study, we used immunohistochemical and biochemical methods to identify NPY in the prepubertal rat ovary. The normal distribution of NPY-containing nerve fibers and the route by which these nerves enter the ovary were analyzed with indirect immunofluorescence techniques. In ovaries with intact nerves, a profuse network of NPY-labeled fibers was observed surrounding blood vessels. Immunoreactive fibers were also seen in the interstitial tissue and coursing between follicles. Occasionally some fibers appeared to enter the follicles. Surgical transection of the superior ovarian nerve had no effect on NPY immunoreactivity; however, transection of the plexus nerve completely eliminated NPY-labeled nerve fibers in all ovarian compartments. The nature of this immunoreactivity was examined in extracts of pooled ovaries that were subjected to reverse phase HPLC and then analyzed by RIA. The major peak of NPY immunoreactivity in each extract eluted at the same time or slightly before synthetic porcine NPY. Two additional peaks of NPY-like immunoreactivity that eluted much earlier than porcine NPY were found in each extract. We conclude that the plexus nerve carries NPY afferents to the ovary and that the ovary contains NPY-like peptides, one of which has a retention time on reverse phase HPLC nearly identical to that of porcine NPY, whereas two others elute with earlier retention times. While the identity and composition of these substances remain to be determined, the presence of peptides that display NPY-like immunoreactivity in the ovary as well as the profuse network of NPY-containing fibers strongly imply a physiological involvement of NPY in the regulation of ovarian function.     

Development of functional zonation in the rat adrenal cortex.

In an attempt to elucidate the mechanism(s) through which the functional adrenal cortex is established, we analyzed immunohistochemically the expression of various markers for the adrenocortical zones, i.e. the zona glomerulosa (zG), the zona fasciculata (zF), and the zona reticularis (zR), as well as markers for the medulla, and further examined the distribution and behavior of DNA-synthesizing cells in rat adrenal glands during development. The results showed that 1) separation of the cortex and medulla, and the development of functional zonation in the cortex began at around the time of birth, 2) at fetal stages when cortical zonation was not established, DNA-synthesizing cells were found scattered throughout the gland, where they proliferated without significant migration, and 3) after birth in the adrenal cortex with established cortical zonation, DNA-synthesizing cells were localized near the undifferentiated zone between zG and zF, and then they migrated centripetally. Cell death appeared to occur in the innermost portion of the cortex, where many resident macrophages are present. These findings illustrate basic processes underlying adrenal development and suggest that the undifferentiated region is apparently the stem cell zone of the adrenal cortex that maintains the cortical zonation.