Rat Adrenal Gland Research Articles

Adrenocortical activity in fetal SHR and WKY rats.

There is increasing evidence that the intrauterine milieu and corticosteroid exposure play a role in the etiology of hypertension. We examined adrenocortical gene expression and circulating corticosteroids in the d 21 fetal spontaneously hypertensive rat (SHR) and its normotensive genetic control, the Wistar-Kyoto (WKY) rat. By RNase protection assays, we found no differences in the relative abundances of mRNAs for P450scc and P450c11beta, and barely detectable P450c11AS mRNA in the adrenals of fetal SHR and WKY rats. P450c11B3 RNA was undetectable by reverse transcription polymerase chain reaction in both SHR and WKY fetuses. The zonal expression of P450c11 mRNA was comparable in SHR and WKY fetuses by in situ hybridization histochemistry. There were no significant differences in peripheral levels of aldosterone and corticosterone by radioimmunoassay in fetal SHR and WKY rats. Based upon the absence of distinct differences in the aspects of adrenocortical activity examined, it is unlikely that they are integral in the programming of hypertension in this model.

Activation of SKca channels inhibits opening of L-type Ca2+ channels in the perfused rat adrenal gland

Activation of SKca channels inhibits opening of L-type Ca2+ channels in the perfused rat adrenal gland

Sik (Salt-Inducible Kinase): Regulation of Acth-Mediated Steroidogenic Gene Expression and Nuclear/Cytosol Re-Distribution

Possible involvement of salt-inducible kinase (SIK), a serine/threonine protein kinase first cloned from high K+-diet treated rat adrenal glands, in the regulation of steroidogenesis was investigated Y-1 cells, when treated with ACTH, underwent a rapid change in SIK's mRNA content. It reached the maximum within a few hours and returned to the base after 8 h. In contrast, the levels of mRNAs for CYP11A and StAR protein reached the maxima after 8 h. The SIK's mRNA induction failed to occur in ACTH-, forskolin- or 8-Br-cAMP-treated Kin-7 cells, a mutant cell line of Y-1 with defective cAMP-dependent protein kinase A (PKA). Y-1 cells that overexpress SIK, when treated with ACTH, had significantly repressed levels of mRNAs for CYP11A and StAR protein. Therefore, SIK might have a negative effect on the CYP11A- and StAR protein-gene expression in the early phase of ACTH-mediated steroidogenesis. To further explore the mechanisms underlying this phenomenon, we examined intracellular distribution of the green fluorescence protein (GFP)-tagged SIK. When GFP-SIK was introduced into HeLa cells, the fluorescent signals were detected in the nucleus. In Y-1 cells GFP-SIK was detected both in the nucleus and cytosol, and the signal in the former moved to the latter after ACTH-treatment. The nuclear/cytosol re-distribution of GFP-SIK was also observed in forskolin- or 8-Br-cAMP-treated Y-1 cells, but not in Kin-7 cells. These results suggest that the intracellular re-distribution of SIK in Y-1 cells may depend on the cAMP/PKA signaling pathway and has an important regulatory role in the ACTH-mediated steroidogenic gene expression.

Rat adrenal gland ultrastructure and catecholamine plasma levels after subchronic exposure to xenon

Rat adrenal gland ultrastructure and catecholamine plasma levels after subchronic exposure to xenon

Distribution, functional role, and signaling mechanism of adrenomedullin receptors in the rat adrenal gland

Adrenomedullin (ADM) is a hypotensive peptide, highly expressed in the mammalian adrenal medulla, which belongs to a peptide superfamily including calcitonin gene-related peptide (CGRP) and amylin. Quantitative autoradiography demonstrated the presence of abundant [ 125I]ADM binding sites in both zona glomerulosa (ZG) and adrenal medulla. ADM binding was selectively displaced by ADM(22–52), a putative ADM-receptor antagonist, and CGRP(8–37), a ligand that preferentially antagonizes the CGRP1-receptor subtype. ADM concentration-dependently inhibited K +-induced aldosterone secretion of dispersed rat ZG cells, without affecting basal hormone production. Both ADM(22–52) and CGRP(8–37) reversed the ADM effect in a concentration-dependent manner. ADM counteracted the aldosterone secretagogue action of the voltage-gated Ca 2+-channel activator BAYK-8644, and blocked K +- and BAYK-8644-evoked rise in the intracellular Ca 2+ concentration of dispersed ZG cells. ADM concentration-dependently raised basal catecholamine (epinephrine and norepinephrine) release by rat adrenomedullary fragments, and again the response was blocked by both ADM(22–52) and CGRP(8–37). ADM increased cyclic-AMP release by adrenal-medulla fragments, but not capsule-ZG preparations, and the catecholamine response to ADM was abolished by the PKA inhibitor H-89. Collectively, the present findings allow us to draw the following conclusions: (1) ADM modulates rat adrenal secretion, acting through ADM(22–52)-sensitive CGRP1 receptors, which are coupled with different signaling mechanisms in the cortex and medulla; (2) ADM selectively inhibits agonist-stimulated aldosterone secretion, through a mechanism probably involving the blockade of the Ca 2+ channel-mediated Ca 2+ influx; (3) ADM raises catecholamine secretion, through the activation of the adenylate cyclase/PKA signaling pathway.

Distribution of P2X receptors in the rat adrenal gland

Distribution of P2X receptors in the rat adrenal gland

Sympathetic‐related neurons in the preoptic region of the rat identified by viral transneuronal labeling

The viral transneuronal labeling method was used to localize sympathetic-related neurons in the preoptic region following pseudorabies virus (PRV) injections into either the superior cervical ganglion, stellate ganglion, celiac ganglion, or adrenal gland of rats. A general pattern of infection was detected. First, neuronal labeling was found in the medial preoptic area, medial preoptic nucleus, median preoptic nucleus, and lateral preoptic area, and then it spread to the anteroventral periventricular, anteroventral preoptic, and parastrial nuclei. Finally, the forebrain circumventricular organs: organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO) became infected. Neuropeptide-containing preoptic neurons were analyzed following PRV injections in the stellate ganglion. Some thyrotropin-releasing hormone and neurotensin neurons were labeled, but none of the calcitonin gene-related peptide, cholecystokinin, corticotropin-releasing factor, galanin, luteinizing hormone-releasing hormone, enkephalin, substance P, or tyrosine hydroxylase neurons were PRV infected. Two major sympathetic networks appear to be represented in the preoptic region. One is linked to the OVLT, SFO, and anteroventral third ventricular (AV3V) region, sites previously implicated in fluid and electrolyte balance as well as cardiovascular control. The other descending sympathetic pathway appears to target the medial preoptic nucleus as its key nodal point, receiving inputs from infralimbic cortex and limbic regions, such as the lateral septum, medial nucleus of the amygdala, subiculum, and amygdalohippocampal area, and then, projecting caudally to the hypothalamus and brainstem. This second sympathetic network may subserve affiliative, defensive and sexual behaviors. J. Comp. Neurol. 414:361–378, 1999. © 1999 Wiley-Liss, Inc.

The effects of exercise duration on adrenal HSP72/73 induction in rats.

This study investigated the effects of varying durations of exercise training on heat shock proteins 72 and 73 (HSP72/73) induction in the rat adrenal gland. Female Sprague-Dawley rats (120 days old) were assigned to either a sedentary control group (C) or one of the three endurance exercise training groups. Trained animals ran on a treadmill at approximately 75% VO2max for 10 weeks (4-5 days week-1) at one of the three different exercise durations (30 min day-1=T30, 60 min day-1=T60, or 90 min day-1=T90). All durations of exercise training resulted in an increase in the HSP72 levels (P < 0.0001). The magnitude of the training-induced elevation in the HSP72 levels in the adrenal gland increased as a function of the training duration ( approximately 60-fold increase in T90; approximately 40-fold increase in T60; and approximately 15-fold increase in T30). Longer exercise durations (>60 min day-1), also resulted in small but significant increase in HSP73 level ( approximately 1.2-fold increase in both T60 and T90; P < 0.05). These results demonstrate that the physiological stress created by chronic treadmill running can induce both HSP72 and HSP73 in rat adrenal gland. Increased levels of adrenal HSP72/73 expression during rigorous exercise, may be one of the adaptive mechanisms of the adrenals to cope with an increased dose of stress.

Corticotropin increases protein tyrosine phosphatase activity by a cAMP-dependent mechanism in rat adrenal gland.

Corticotropin signal transduction pathway involves serine/threonine protein phosphorylation. Recent reports suggest that protein tyrosine dephosphorylation may also be an integral component of that pathway. The present study was performed to investigate the role played by protein tyrosine phosphatases (PTPs) on acute response to corticotropin and the hypothetical regulation of PTPs by this hormone. We have used two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), in order to examine the relevance of PTP activity on hormone-stimulated and 8-bromo-adenosine 3',5'-phosphate (8Br-cAMP is a permeant analogue of adenosine 3',5'-phosphate)-stimulated steroidogenesis in adrenal zona fasciculata (ZF) cells. In both cases, PAO and PV inhibited the steroid production in a dose-dependent fashion, and had no effect on steroidogenesis supported by a permeant analogue of cholesterol. The effect of hormonal stimulation on PTP activity was analyzed in rat adrenal ZF. In vivo corticotropin treatment reduced phosphotyrosine content in endogenous proteins and produced a transient increase of PTP activity in the cytosolic fraction, reaching a maximum (twofold) after 15 min. Incubation of adrenal ZF with 8Br-cAMP also produced PTP activation, suggesting that it can be mediated by cAMP-dependent protein kinase (PKA)-dependent phosphorylation. Detection of PTP activity in an in-gel assay showed three corticotropin-stimulated soluble PTPs with molecular masses of 115, 80 and 50 kDa. In summary, we report for the first time a hormone-dependent PTP activation in a steroidogenic tissue and provide evidence that PTP activity plays an important role in corticotropin signal pathway, acting downstream of PKA activation and upstream of cholesterol transport across the mitochondrial membrane.

Distribution of P2X receptors in the rat adrenal gland.

The distribution of each of the seven subtypes of ATP-gated P2X receptors was investigated in the adrenal gland of rat utilizing immunohistochemical techniques with specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. A small number of chromaffin cells showed positive immunoreaction for P2X5 and P2X7, with the relative occurrence of P2X7-immunoreactive chromaffin cells exceeding that of P2X5. The preganglionic nerve fibres that form terminal plexuses around some chromaffin cells showed P2X1 immunoreactivity. Intrinsic adrenal neurones were observed to be positively stained for P2X2 and P2X3 receptors. P2X2 immunoreactivity occurred in several neurones found singly or in groups in the medulla, while only a small number of neurones were immunoreactive for P2X3. Adrenal cortical cells were positively immunostained for P2X4-7. Immunoreactivity for P2X4 was confined to the cells of the zona reticularis, while P2X5-7 immunoreactivities occurred in cells of the zona fasciculata. The relative occurrence of immunoreactive cortical cells of the zona fasciculata was highest for P2X6, followed by P2X7 and then P2X5. The smooth muscle of some capsular and subcapsular blood vessels showed P2X2 immunoreactivity. The specific and widespread distribution of P2X receptor subtypes in the adrenal gland suggests a significant role for purine signalling in the physiology of the rat adrenal gland.

Sympathetic-related neurons in the preoptic region of the rat identified by viral transneuronal labeling

The viral transneuronal labeling method was used to localize sympathetic-related neurons in the preoptic region following pseudorabies virus (PRV) injections into either the superior cervical ganglion, stellate ganglion, celiac ganglion, or adrenal gland of rats. A general pattern of infection was detected. First, neuronal labeling was found in the medial preoptic area, medial preoptic nucleus, median preoptic nucleus, and lateral preoptic area, and then it spread to the anteroventral periventricular, anteroventral preoptic, and parastrial nuclei. Finally, the forebrain circumventricular organs: organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO) became infected. Neuropeptide-containing preoptic neurons were analyzed following PRV injections in the stellate ganglion. Some thyrotropin-releasing hormone and neurotensin neurons were labeled, but none of the calcitonin gene-related peptide, cholecystokinin, corticotropin-releasing factor, galanin, luteinizing hormone-releasing hormone, enkephalin, substance P, or tyrosine hydroxylase neurons were PRV infected. Two major sympathetic networks appear to be represented in the preoptic region. One is linked to the OVLT, SFO, and anteroventral third ventricular (AV3V) region, sites previously implicated in fluid and electrolyte balance as well as cardiovascular control. The other descending sympathetic pathway appears to target the medial preoptic nucleus as its key nodal point, receiving inputs from infralimbic cortex and limbic regions, such as the lateral septum, medial nucleus of the amygdala, subiculum, and amygdalohippocampal area, and then, projecting caudally to the hypothalamus and brainstem. This second sympathetic network may subserve affiliative, defensive and sexual behaviors.

Role of calcium channels in catecholamine secretion in the rat adrenal gland.

1. We elucidated the contribution of voltage-dependent Ca2+ channels to cholinergic control of catecholamine secretion in the isolated perfused rat adrenal gland. 2. Nifedipine (0.3-3 microM) inhibited increases in noradrenaline output induced by transmural electrical stimulation (1-10 Hz) and acetylcholine (6-200 microM), whereas it only slightly inhibited the adrenaline output responses. Nifedipine also inhibited the catecholamine output response induced by 1, 1-dimethyl-4-phenyl-piperazinium (DMPP; 5-40 microM) but not by methacholine (10-300 microM). 3. omega-Conotoxin MVIIC (10-1000 nM) inhibited the catecholamine output responses induced by electrical stimulation but not by acetylcholine, DMPP and methacholine. 4. omega-Conotoxin GVIA (50-500 nM) had no inhibitory effect on the catecholamine output responses. 5. These results suggest that L-type Ca2+ channels are responsible for adrenal catecholamine secretion mediated by nicotinic receptors but not by muscarinic receptors, and that their contribution to noradrenaline secretion may be greater than that to adrenaline secretion. P/Q-type Ca2+ channels may control the secretion at a presynaptic site.

Renin in thymus, gut, hindlimb, and adrenal of (mRen-2)27 and normal rats: secretion and content studies.

Thymic ablation and assay of organ renin revealed that one-third of the increasing plasma level of active renin after removal of kidneys and adrenals from Ren-2 rats originates from the thymus. Splanchnic arteriovenous difference and renin content indicate that gut can account for the remainder. Secretion of active renin from these sites correlated significantly with increasing plasma potassium. Prorenin was not secreted from these sites or from hindlimb in amounts sufficient to raise the plasma level, and yet plasma prorenin remained higher than active renin throughout the 12-h protocol. The source of prorenin that accounts for the high plasma prorenin phenotype of the intact conscious Ren-2 rat was not specifically identified. When sensitive assays were used, a low level of active renin secretion from thymus and gut was also apparent 12 h after removal of kidneys and adrenals in normal Sprague-Dawley rats, and plasma prorenin was at this time higher than active renin. A likely source of this extrarenal, extra-adrenal renin is the macrophage.

Pharmacological and molecular characterization of 5-hydroxytryptamine(7) receptors in the rat adrenal gland.

Serotonin (5-hydroxytryptamine; 5-HT) is a potent stimulator of aldosterone secretion in the rat adrenal gland but the type of receptor involved in the mechanism of action of 5-HT remains unknown. The aim of the present study was to determine the pharmacological profile and to clone the receptor responsible for the corticotropic effect of 5-HT in rat glomerulosa cells. A series of 10 serotonergic receptor agonists and 12 receptor antagonists was used to characterize the receptor mediating the effect of 5-HT on aldosterone secretion from perifused rat adrenocortical slices. Correlation analysis between the potencies of the different compounds in our model and those previously reported for various recombinant 5-HT receptors showed that the rat adrenal 5-HT receptor exhibits the same pharmacological profile as the 5-HT(7) receptor transiently expressed in COS-7 cells (r = 0.82 for agonists, p <.05; r = 0.83 for antagonists, p <.01). Polymerase chain reaction with specific primers revealed the expression of 5-HT(7) receptor mRNA in the rat adrenal gland. Cloning of the polymerase chain reaction product confirmed that the amplified DNA corresponded to the 5-HT(7) receptor cDNA sequence. Western blot analysis showed the presence of a protein with an apparent molecular mass of 66 kDa in the adrenal cortex but not in the medulla. Taken together, these data demonstrate that the rat adrenal glomerulosa expresses functional 5-HT(7) receptors. Rat glomerulosa cells will thus provide a robust and sensitive bioassay for future studies on native 5-HT(7) receptors.

Effects of acute restraint stress on endogenous adrenomedullin levels.

Adrenomedullin (ADM) is a 52 amino acid peptide, with a potent hypotensive/vasodilatory action. Levels of ADM are significantly elevated in patients with hypertension, and it has been postulated that such stressor-related increases may serve a regulatory or protective function. The current study assessed the effects of acute restraint stress on ADM levels in regions of the brain, plasma and peripheral tissue including heart, lung and the adrenal glands of rats. This stressor, known to stimulate sympathetic activity as well as the hypothalamic-pituitary adrenal (HPA) axis, produced a significant increase in ADM levels in the pituitary gland, plasma and adrenal glands, all of which are key components of the HPA axis. The results suggest a regulatory and/or protective role for ADM in countering HPA activation following a variety of physiological and psychological stressors.

DNA damage and p21(WAF1/CIP1/SDI1) in experimental injury of the rat adrenal cortex and trauma-associated damage of the human adrenal cortex.

In vivo models are needed to study the reactions of tissues to DNA damage, such as the induction of the cyclin-dependent kinase inhibitor p21, indicating potential repair of the damage, versus apoptosis, indicating the elimination of the damaged cells. Damage to DNA occurs in tissues during shock, sepsis, and other critical medical conditions. Previous studies have found evidence of damage to the cortex of adrenal glands from organ donors who had undergone severe trauma prior to death. The present experiment studied rats under experimental interventions of clinical relevance to patients with conditions that put them at risk for damage to the adrenal glands. These interventions comprised ischaemia and reperfusion injury, sepsis following caecal ligation and puncture, acute pancreatitis, and administration of chemical agents (zymosan and acrylonitrile). All the interventions caused an increase in p21 mRNA as assessed by northern blotting and in situ hybridization. Increased nuclear p21 protein was shown by immunohistochemistry. All the interventions caused damage to DNA, as shown by labelling of available 3' termini of single-strand breaks with terminal transferase. The number of cells undergoing apoptosis, visualized by ligation of a hairpin oligonucleotide probe to double-strand breaks in DNA, was much lower. In rat adrenal glands, apoptotic cells were infrequent under all the conditions studied. They were more abundant in human organ donor adrenal glands that were previously shown to have extensive DNA damage accompanied by induction of p21. The similarity of the effects of a wide variety of surgical interventions and chemical agents suggest a common pathophysiological mechanism which is not specific to the initiating injury. Experimental injury of the rat adrenal cortex provides a model for investigating the role of organ DNA damage and of mediators of the response to DNA damage, such as p21.

Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade

Orexins-A and B are two novel hypothalamic peptides, which, like leptin and neuropeptide-Y (NPY), are involved in the central regulation of feeding. Since leptin and NPY were found to modulate adrenal function, we have examined whether orexins are able to directly affect rat adrenal steroid secretion. Both orexin-A and orexin-B raised basal corticosterone secretion of dispersed rat zona fasciculata–reticularis (ZF/R) cells, their maximal effective concentration being 10 −8 M. In contrast, orexins did not affect either maximally ACTH (10 −9 M)-stimulated corticosterone production by ZF/R cells or the basal and agonist-stimulated aldosterone secretion of dispersed zona glomerulosa cells. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10 −6 M) annulled corticosterone response of ZF/R cells to ACTH (10 −9 M), but not to orexins (10 −8 M). Orexins (10 −8 M) enhanced cyclic-AMP release by ZF/R cells, and the selective inhibitor of protein-kinase A (PKA) H-89 (10 −5 M) abolished corticosterone responses to both ACTH (10 −9 M) and orexins (10 −8 M). A subcutaneous injection of both orexins (5 or 10 nmol/kg) evoked a clear-cut increase in the plasma concentration of corticosterone (but not aldosterone), the effect of orexin-A being significantly more intense than that of orexin-B. Collectively, these findings suggest that orexins exert a selective and direct glucocorticoid secretagogue action on the rat adrenals, acting through a receptor-mediated activation of the adenylate cyclase/PKA-dependent signaling pathway.

CART peptide analysis by Western blotting.

CART peptides have been implicated in leptin-regulated feeding, reward and reinforcement, neurotropism, and other processes. In this Western blotting study, at least six different CART peptides varying from 4 to 14 kD were found in rat brain, pituitary, gut, and adrenal gland. The peptides may be processed differently in different tissues and one species found in the rat was not found in human hypothalamus. The higher molecular weight species are likely to include preproCART and proCART, while lower molecular weight peptides may be CART 55-102 and 62-102, physiologically active fragments.

Calcium channel subtypes and exocytosis in chromaffin cells: a different view from the intact rat adrenal

In the intact rat adrenal gland perfused with an oxygenated Krebs-bicarbonate solution at 37 degrees C, the electrical field stimulation of splanchnic nerves (100 V, 0.5 ms duration, 10 Hz during 10 s) produced transient catecholamine release peaks that were reproduced in subsequent stimuli, applied at 8-min intervals. Omega-Conotoxin GVIA (0.3 microM) caused only a modest inhibition of the secretory response, suggesting that the N-subtype of voltage-dependent Ca2+ channels are scarcely involved in such a response. Both omega-conotoxin MVIIC (1 microM) and furnidipine (1 microM) halved the secretion, suggesting that the L- and P/Q-types of Ca2+ channels were involved. N-type Ca2+ channels appear to be involved in the maintenance of secretion in response to sustained stimulus since omega-conotoxin GVIA (0.3 microM) reduced the catecholamine output to 28%. When secretion was elicited by acetylcholine (10 microM), furnidipine reduced the catecholamine release by 50% and omega-conotoxin MVIIC by 40%, whereas omega-conotoxin GVIA did not modify the response. The K+-induced secretory responses (23.6 mM K+, 15 s) were reduced 75% by furnidipine and 45% by omega-conotoxin MVIIC, indicating that this type of stimulation preferentially recruited L-type channels. These data show that electrical stimulation recruits Ca2+ channel subtypes different from those recruited by direct depolarization of chromaffin cells.

Aldehyde dehydrogenase from rat intestinal mucosa: purification and characterization of an isozyme with high affinity for γ-aminobutyraldehyde

In rat adrenal gland and gastric mucosa putrescine is efficiently oxidized to GABA via γ-aminobutyraldehyde (ABAL) by action of diamine oxidase and aldehyde dehydrogenase. Having turned our attention on the rat intestinal mucosa, where putrescine uptake and diamine oxidase are active, we have purified and characterized an aldehyde dehydrogenase optimally active on γ-aminobutyraldehyde. A dimer with a subunit molecular weight of 52,000, the native enzyme binds ABAL and NAD + with high affinity: at pH 7.4, K m values are equal to 18 and 14 μM, respectively. Affinity for betaine aldehyde is much lower ( K m=285 μM), but the efficiency is equally good, thanks to a high value of V. Unaffected by disulfiram and Mg 2+, the enzyme is activated by high NAD + concentrations ( V nn=1.6× V n) and is competitively inhibited by NADH. According to the best fitting model, the dimeric enzyme only binds one NADH and the mixed complex enzyme–NAD +–NADH is inactive. The increase of activity promoted by NAD + can therefore be ascribed to an allosteric effect, rather than to the activation of a second reaction center. Highly stable at pH 6.8 in the presence of dithiothreitol and high phosphate concentrations, ABALDH is inactivated by ion-exchange resins and by cationic buffers. Our results show that the enzyme can be effectively involved in the metabolism of biogenic amines and, with a K m for ABAL lower than 20 μM, in the synthesis of GABA.