促肾上腺皮质激素引起大鼠肾上腺c-fos原癌基因表达的免疫组化研究

采用冰冻切片及免疫组化法观察了注射促肾上腺皮质激素(ACTH,75U/kg)、胰岛素及正常对照大鼠中肾上腺各部分c-fos原癌基因表达产物Fos蛋白的出现和分布特点。结果表明注射ACTH后90min,大鼠肾上腺皮质网状带出现Fos蛋白染色阳性细胞,阳性染色物集中于细胞核,肾上腺皮质束状带仅见少数Fos蛋白染色阳性细胞,肾上腺髓质则未见Fos蛋白染色阳性细胞。与注射ACTH相反,注射胰岛素引起肾上腺髓质出现Fos蛋白染色阳性细胞。注射生理盐水对照组动物肾上腺皮质和髓质均未见Fos蛋白染色阳性细胞。上述结果表明,注射ACTH或胰岛素可以引起大鼠肾上腺不同部位c-fos原癌基因表达。     

Dehydration-induced drinking decreases Fos expression in hypothalamic paraventricular neurons expressing vasopressin but not corticotropin-releasing hormone

Water-restricted (WR) rats exhibit a rapid suppression of plasma corticosterone following drinking. The present study monitored Fos-like immunoreactivity (Fos) to assess the effect of WR-induced drinking on the activity of vasopressin (VP)-positive magnocellular and parvocellular neurons and corticotropin-releasing hormone (CRH)-positive parvocellular neurons in the paraventricular nucleus of the hypothalamus. Adult male rats received water for 30 min (WR) in the post meridiem (PM) each day for 6 days and were killed without receiving water or at 1 h after receiving water for 15 min. In WR rats, Fos increased in VP magnocellular and parvocellular neurons but not CRH neurons. After drinking, Fos was reduced in VP magnocellular and parvocellular neurons but did not change in CRH neurons. To assess the severity of osmotic stress, rats were sampled throughout the final day of WR. Plasma osmolality, hematocrit and plasma VP were increased throughout the day before PM rehydration, and plasma ACTH and corticosterone were elevated at 1230 and 1430, respectively, showing that WR activates hypothalamic-pituitary-adrenal activity during the early PM before the time of rehydration. To determine the effects of WR-induced drinking on CRH neurons activated by acute stress, WR rats underwent restraint. Restraint increased plasma ACTH and corticosterone and Fos in CRH neurons; although rehydration reduced plasma ACTH and Fos expression in VP neurons, Fos in CRH neurons was not affected. These results suggest that inhibition of VP magnocellular and parvocellular neurons, but not CRH parvocellular neurons, contributes to the suppression of corticosterone after WR-induced drinking.     

Incorporation of regulatory peptide hormones by individual cells of the adrenal cortex: Prolactin-adrenocorticotrophin differences

Recent refinements in methodology now permit the study of endogenous peptide hormones in their individual target cells. The investigations reported here deal with the question of whether endogenous ACTH can be detected in its target cells in the highly active adrenal gland of the normally lactating rat. This question was examined with immunohistochemistry. ACTH was found in both cytoplasm and nuclei of adrenal glomerulosa cells. In cells of the fasciculata and reticularis layers of the adrenal cortex, it did not appear inside nuclei but was present in the cytoplasm and on the nuclear envelope. The distribution of ACTH was compared with and found to be different from that of PRL. PRL, confirming previous findings, was not detectable at all in glomerulosa cells and, in cells of the inner cortical zones, was present in both cytoplasm and nuclei. In neither case was hormone found in the adrenal medulla. The apparent feasibility of studying peptide regulators such as ACTH and PRL in their individual target cells may be a focal point for an acceleration of our understanding of how these peptides work.     

Hypothalamic activity during altered salt and water balance in the snake <Emphasis Type="Italic">Bothrops jararaca</Emphasis>

The effects of water and salt overload on the activities of the supraoptic and paraventricular nuclei and the adjacent periventricular zone of the hypothalamus of the snake Bothrops jararaca were investigated by measurements of Fos-like immunoreactivity (Fos-ir). Both water and salt overload resulted in changes in body mass, plasma osmolality, and plasma concentrations of sodium, potassium, and chloride. Hyper-osmolality increased Fos immunoreactivity in the rostral supraoptic nucleus (SON), the paraventricular nucleus (PVN), and adjacent periventricular areas. Both hyper- and hypo-osmolality increased Fos immunoreactivity in the intermediate SON, but not in other areas of the hypothalamus. Immunostaining was abundant in cerebrospinal fluid (CSF)-contacting tanycyte-like cells in the ependymal layer of the third ventricle. These data highlight some features of regional distribution of Fos immunoreactivity that are consistent with vasotocin functioning as a hormone, and support the role of hypothalamic structures in the response to disruption of salt and water balance in this snake.     

Vagal-Central Nervous System Interactions Modulate the Feeding Response to Peripheral Enterostatin

Enterostatin selectively inhibits the intake of dietary fat after both peripheral and central administration. We have investigated the role of the hepatic vagus nerve in modulating the peripheral response to enterostatin in Sprague-Dawley rats adapted to a high fat (HF) diet. Intraperitoneal (ip) enterostatin reduced intake of HF diet after overnight starvation. This response was abolished by selective vagal hepatic branch transection. Immunohistochemical techniques were used to identify the location of Fos protein in brain nuclei after ip enterostatin. Fos protein was evident in the nucleus tractus solitarius (NTS), parabrachial, paraventricular and supraoptic nuclei. The pattern of expression of Fos-like immunoreactivity differed from that induced by the lipoprivic agent β-mercaptoacetate. Transection of the hepatic vagus blocked the central Fos responses to ip enterostatin. We conclude that afferent hepatic vagal nerve activity is required for the feeding response to peripheral enterostatin.     

Adrenocortical responses to adrenocorticotrophin in the rat

The time course of plasma adrenocorticotrophin (ACTH), adrenal cyclic AMP, adrenal corticosterone, and plasma corticosterone was measured in male Sprague-Dawley rats whose endogenous release of ACTH had been blocked (1) following rapid injections of 100 and 300 ng ACTH/100 g body weight, i.v., (2) during prolonged infusions at rates of 1, 2, and 4 ng ACTH/min per 100 g body weight, and (3) after termination of 30-min infusions at rates extending from 0.06 to 8 ng ACTH/min per 100 g body weight. Following injections, the time course of the variables is similar to the one simulated from our models of adrenal cortical secretion, including the simulation of an intermediate variable of our models of the adrenal cortex cell which was presumed to correspond to cyclic AMP. However, during prolonged infusions there is an unexpected overshoot of adrenal cyclic AMP content whereas adrenal and plasma corticosterone concentrations rise to a steady-state value without overshoot. The total amount of cyclic AMP gradually increases following the three increasing infusion rates of ACTH whereas similar levels of plasma corticosterone concentrations are reached at steady state; therefore the saturation of the adrenal cortical secretion is due to a step ulterior to cyclic AMP formation in the steroidogenesis. After 30-min infusions, plasma corticosterone concentration reaches its maximal value following a rate of ACTH input which evokes only a 4-fold increase in adrenal cyclic AMP content; however, there is a 250-fold increase of adrenal cyclic AMP with respect to control value following the higher rates of infusion of ACTH.     

辣椒素引起脑干内心血管活动相关核团中c-fos的表达

在１６只切断两侧缓冲神经的大鼠,观察颈总动脉注射辣椒素对脑干内心血管活动相关核团ｃ－ｆｏｓ原癌基因表达的影响。在剂对照组大鼠脑干,仅见少数Ｆｏｓ蛋白样免疫反应（ＦＬＩ）神经元。与对照组相比,颈总动脉注射辣椒素（１０μｍｏｌ,０．１ｍｌ）时,脑干内巨细胞旁外侧核（ＰＧＬ）、蓝斑（ＬＣ）、最后区（ＡＰ）和孤束核（ＮＴＳ）等部位的ＦＬＩ神经元显著增加,而中脑中央灰质（ＰＡＧ）和中缝核群（ＲＮ）的ＦＬＩ神     

Action sites of adrenomedullin in the rat brain: functional mapping by Fos expression.

The effects of intracerebroventricular (icv) administration of adrenomedullin (AM) and proadrenomedullin NH2-terminal 20 peptide (PAMP) on the expression of Fos in the central nervous system (CNS) were examined in conscious rats, using immunohistochemistry. Fos-like immunoreactivity (LI) was detected in various brain areas of the rats, including the supraoptic nucleus, the paraventricular nucleus, the locus coeruleus, the area postrema and the nucleus of the tractus solitarius 90 min after icv administration of AM. Few cells with Fos-LI were found in the CNS 90 min after icv administration of saline. Fos-LI was also detected in the various hypothalamic areas after icv administration of PAMP. These results suggest that centrally administered AM and PAMP may cause physiological responses through the activation of a neural network in the hypothalamus and the brainstem.     

Involvement of 3',5'-cyclic adenosine monophosphate-dependent protein kinase in regulation of Fos expression and tyrosine hydroxylase levels during morphine withdrawal in the hypothalamic paraventricular nucleus and medulla oblongata catecholaminergic cell groups

Morphine withdrawal stimulates the hypothalamic-pituitary-adrenocortical axis activity by activation of nucleus tractus solitarius (NTS)/ventrolateral medulla (VLM) noradrenergic pathways innervating the hypothalamic paraventricular nucleus (PVN). We investigated whether cAMP-dependent protein kinase (PKA) plays a role in this process by estimating changes in PKA immunoreactivity and the influence of inhibition of PKA on Fos protein expression and tyrosine hydroxylase (TH) immunoreactivity levels in the PVN and NTS/VLM during morphine withdrawal. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg s.c.). When opioid withdrawal was precipitated, an increase in PKA immunoreactivity levels was observed 90 min after naloxone administration in the PVN and NTS/VLM areas. Morphine withdrawal induced expression of Fos in the PVN and NTS/VLM, indicating an activation of neurones in those nuclei. TH immunoreactivity in NTS/VLM was increased 90 min after induction of morphine withdrawal, whereas there was a decrease in TH levels in the PVN at the same time point. When the selective PKA inhibitor HA-1004 was infused it greatly diminished the Fos expression observed in morphine-withdrawn rats. Furthermore, the changes in TH immunoreactivity were significantly modified by infusion of HA-1004. The present findings suggest that an up-regulated PKA-dependent transduction pathway might contribute to the activation of the hypothalamic-pituitary-adrenocortical axis in response to morphine withdrawal.     

CCK enhances response to gastric distension by acting on capsaicin-insensitive vagal afferents

Capsaicin treatment destroys vagal afferent C fibers and markedly attenuates reduction of food intake and induction of hindbrain Fos expression by CCK. However, both anatomical and electrophysiological data indicate that some gastric vagal afferents are not destroyed by capsaicin. Because CCK enhances behavioral and electrophysiological responses to gastric distension in rats and people, we hypothesized that CCK might enhance the vagal afferent response to gastric distension via an action on capsaicin-insensitive vagal afferents. To test this hypothesis, we quantified expression of Fos-like immunoreactivity (Fos) in the dorsal vagal complex (DVC) of capsaicin-treated (Cap) and control rats (Veh), following gastric balloon distension alone and in combination with CCK injection. In Veh rats, intraperitoneal CCK significantly increased DVC Fos, especially in nucleus of the solitary tract (NTS), whereas in Cap rats, CCK did not significantly increase DVC Fos. In contrast to CCK, gastric distension did significantly increase Fos expression in the NTS of both Veh and Cap rats, although distension-induced Fos was attenuated in Cap rats. When CCK was administered during gastric distension, it significantly enhanced NTS Fos expression in response to distension in Cap rats. Furthermore, CCK's enhancement of distension-induced Fos in Cap rats was reversed by the selective CCK-A receptor antagonist lorglumide. We conclude that CCK directly activates capsaicin-sensitive C-type vagal afferents. However, in capsaicin-resistant A-type afferents, CCK's principal action may be facilitation of responses to gastric distension.     

Differential Expression of c-fos mRNA and Fos Protein in the Rat Brain After Restraint Stress or Pentylenetetrazol-Induced Seizures

1. c-fos mRNA expression and Fos protein expression were investigated by in situ hybridization and immunohistochemistry after 30 min of forced restraint stress or pentylenetetrazol (PTZ; 64 mg/kg, i.p.)-induced seizures.2. Forced restraint stress and PTZ-induced seizures generated c-fos mRNA expression of distinct intensities, but in similar brain regions, including the hippocampus, the amygdala, the piriform cortex, the paraventricular hypothalamic nucleus, the habenula, and parts of the cerebral cortex.3. The distribution of Fos-like immunoreactivity induced by stress or seizures only partially overlap. No Fos-like expression was found in the hippocampus or the habenula after restraint stress. Nevertheless, both areas presented Fos-like expression after PTZ-induced seizures.4. Our results support the suggestion that immediate early gene expression in vivo may exhibit both region- and stimulus-specific expression.     

侧脑室注射肾上腺髓质素增加大鼠心血管相关核团中c-fos的表达并激活脑内一氧化氮神经元

本研究利用Fos蛋白和一氧化氮合酶(nNOS)双重免疫组化方法,观察侧腑脑室注射肾上腺髓质素(adrenomedullin,ADM)对大鼠心血管相关核中c-fos表达及一氧化氮神经元的影响,以探讨ADM在中枢的作用部位并研究其在中枢的作用是否有NO神经元参与。侧脑室注射ADM(1nmol／kg,3nmol／kg)诱发脑干的孤束核、最后区、蓝斑核、臂旁核和外侧巨细胞旁核,下丘脑的室旁核、视上核才腹内侧核以及前脑的中央杏仁核和外侧缰核等多个部位的心血管中枢出现大量Fos样免疫反应神经元。侧脑室注射ADM(3nmol／kg),引起脑干的孤束核、外侧巨细胞旁核,下丘脑的室旁核、视上核内的Fos-nNOS双标神经元增加;ADM(1nmol／kg)亦可引起室旁核、视上核内的Fos-nNOS双标神经元增加,而对孤束核、外侧巨细胞旁核内的Fos-nNOS双标神经元无影响。降钙素基因相关肽(calcitonin gene—related peptide,CGRP)受体拈抗剂CGRP8-37(30nmol／kg)可明显减弱此效应。以上结果表明,ADM可兴奋脑内多个心血管相关核闭的神经元并激活室旁核、视上核、孤束核及外侧巨细胞核内一氧化氮神经元,此效应可能部分山CGRP受体介导。     

Effect of angiotensin II on secretion of adrenal androgens

To assess the effect of angiotensin II (A II) on the secretion of human adrenal androgens (AA), plasma dehydroepiandrosterone (DHEA), DHEA sulfate (DS) and delta 4-androstenedione (delta 4-A) were measured in eight normal men 60 and 120 min after stimulation of endogenous A II by a bolus injection of 40 mg frusemide, and the direct effect of A II on the secretion of adrenal androgens was examined in cultured human adrenocortical cells in the presence of a low concentration of ACTH. The administration of frusemide led to a significant increase in the plasma DHEA and DS concentration as well as plasma renin activity (PRA) and aldosterone concentration (PAC), but did not change plasma cortisol and delta 4-A. In the culture of human adrenocortical cells, 10(-9)-10(-5) M A II or 10(-13) M ACTH alone did not stimulate the secretion of DHEA, DS and delta 4-A, while 10(-7) and 10(-5) M A II in the presence of 10(-13) M ACTH caused a significant increase in DHEA and DS secretion with no change in delta 4-A. These results suggest that the activated renin-angiotensin system stimulates the secretion of adrenal androgens by a direct effect of A II on adrenal cortical cells.     

Corticotropin-releasing factor in humans. I. CRF stimulation in normals and CRF radioimmunoassay

The biological activity of ovine (o) and human (h) corticotropin-releasing factor (CRF) in normal volunteers was investigated, using bolus injections with different CRF dosages. There was a significant increase of ACTH, beta-endorphin and cortisol after the injection of all dosages. Repetitive stimulation and continuous infusion of hCRF lead to repetitive release of identical amounts of ACTH or constant elevation of ACTH levels. oCRF and hCRF serum immunoreactivity was measured with specific radioimmunoassays after bolus injection, pulsatile administration and infusion of CRF. The half-time of serum disappearance after acute injection studies was calculated as 9 min for hCRF dand 18 min for oCRF. The 'metabolic clearance' of hCRF calculated using the infusion study was 2.72 ml/min X kg. Endogenous CRF immunoreactivity was detectable in 14 patients during insulin hypoglycemia and in 86 out of 97 pregnant females. Furthermore, CRF could be extracted from human placenta. The chromatographic pattern of extracted placenta CRF, pregnancy serum CRF and CRF standard preparation was identical. Furthermore, CRF immunoreactivity was detectable in some patients with different causes of ACTH hypersecretion.     

Ultrastructural and biochemical evidence for a steroid-containing secretory organelle in the perfused cat adrenal gland

A correlative study of the ultrastructural and biochemical effects of ACTH on fasciculata cells was carried out on the isolated cat adrenal gland perfused in situ with Locke's solution. The outstanding morphologic feature of cortical cells exposed to microunit ACTH concentrations for 40 min was the abundance of electron-dense granules (0.2-0.4 mum). These organelles were observed in small groups in close proximity to the Golgi region and to the cell membrane. Morphometric and biochemical analysis of control and ACTH-treated glands demonstrated that ACTH stimulation was associated with a fourfold increase in the number of these granules and a comparable increase in the corticosteroid content of the gland. By contrast, ACTH failed to augment cortical lysosomal enzyme activity. These findings, which link steroid release to the appearance of intracellular granules, extend further the parallels between the mechanism of release of newly synthesized steroid and the release of preformed hormones stored in secretory organelles. These results also lend support to the concept that a process related to exocytosis may be the underlying mechanism for extruding steroid from the cortical cell.     

Differential effects of stress and amphetamine administration on Fos-like protein expression in corticotropin releasing factor-neurons of the rat brain

Corticotropin releasing factor (CRF) appears to be critical for the control of important aspects of the behavioral and physiological response to stressors and drugs of abuse. However, the extent to which the different brain CRF neuronal populations are similarly activated after stress and drug administration is not known. We then studied, using double immunohistochemistry for CRF and Fos protein, stress and amphetamine-induced activation of CRF neurons in cortex, central amygdala (CeA), medial parvocellular dorsal, and submagnocellular parvocellular regions of the paraventricular nucleus of the hypothalamus (PVNmpd and PVNsm, respectively) and Barrington nucleus (Bar). Neither exposure to a novel environment (hole-board, HB) nor immobilization (IMO) increased Fos-like immunoreactivity (FLI) in the CeA, but they did to the same extent in cortical regions. In other regions only IMO increased FLI. HB and IMO both failed to activate CRF+ neurons in cortical areas, but after IMO, some neurons expressing FLI in the PVNsm and most of them in the PVNmpd and Bar were CRF+. Amphetamine administration increased FLI in cortical areas and CeA (with some CRF+ neurons expressing FLI), whereas the number of CRF+ neurons increased only in the PVNsm, in contrast to the effects of IMO. The present results indicate that stress and amphetamine elicited a distinct pattern of brain Fos-like protein expression and differentially activated some of the brain CRF neuronal populations, despite similar levels of overall FLI in the case of IMO and amphetamine.