Rat Adrenal Gland Research Articles

Changes in the Histological Structure of Adrenal Glands and Corticosterone Level after Whey Protein or Bee Pollen Supplementation in Running and Non-Running Rats

Due to the many health-promoting properties of bee pollen and whey protein, both products are widely used as dietary supplements. According to these reports on their health-promoting properties, the aim of our study is to assess whether these products can influence the structure and function of the adrenal glands in rats. Thirty male Wistar rats were divided into six equal groups. Among them, there were three groups which included non-running rats and three groups which included running rats. Both of these running (n = 3) and non-running (n = 3) groups included non-supplemented (control groups), bee-pollen-supplemented groups, and whey-protein-supplemented groups. After 8 weeks, the rats were decapitated, their adrenal glands were collected, and paraffin slides were prepared. Then, staining according to the standard H&E and Masson's trichrome protocols was performed. Fecal and urine samples were collected prior to the end of the study to measure corticosterone levels. In the group of non-running rats, the consumption of bee pollen was noted to be significantly higher when compared to the group of running rats (p < 0.05). The thickness of the particular adrenal cortex layers was similar among all of the groups (p > 0.05). The statistically significant changes in the microscopic structure of the adrenal glands, especially regarding cell nuclei diameter and structure, as well as the architecture of sinusoids, were observed between the groups. Moreover, urine corticosterone concentrations were found to vary between all of the analyzed groups (p < 0.05). These results indicate that both bee pollen and whey protein have limited stress-reducing potential.

PS-B03-5: HIGH-DOSES OF LOSARTAN RATHER ENHANCE CATECHOLAMINE SECRETION FROM THE PERFUSED ADRENAL GLAND

Objective: Previously, we have found that Losartan (5∼50 μM), an AT1 receptor blocker, dose-dependently inhibits secretion of catecholamines (CA) evoked by cholinergic and angiotensin II (Ang II) receptor stimulation from the perfused rat adrenal medulla. The aim of the present study was to examine the characteristic effects of high-dose losartan on the CA secretion from the isolated perfused rat adrenal gland, and also to establish its mechanism of action. Design and Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: The perfusion of high-dose losartan (150 ∼ 600 μM) into an adrenal vein for 90 min resulted in great increases in CA secretions evoked by acetylcholine in a dose-dependent fashion. Also, losartan (300 μM) enhanced the CA release evoked by Ang II, high K+ and DMPP (a selective nicotinic receptor agonist), McN-A-343 (a selective muscarinic M1 receptor agonist). Also, in the presence of losartan (300 μM), the CA secretory responses to veratridine (a selective Na+ channel activator), Bay-K-8644 (a Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly potentiated, respectively. The facilitatory CA-releasing effect of Ach evoked by high-dose losartan (300 μM) was time-dependently depressed by pretreatment with nifedipine (Ca2+ channel blocker), while not by pretreatment with sodium nitroprusside (an NO donor). High-dose olmesartan (150 ∼ 600 μM), one of ARBs, also potentiated Ach-evoked CA release in time-dependent fashion. However, in the simultaneous presence of losartan (300 μM) and CGP42112 (an AT2 receptor agonist), the facilitatory responses of losartan on the CA secretion evoked by ACh were rather significantly reduced in comparison with that of losartan-treatment alone. Conclusion: Conclusively, these results demonstrate that high-dose losartan can potentiate the adrenomedullary CA secretion in a calcium-dependent fashion. It seems that this facilitatory effect of high-dose losartan may be mediated by activation of the Ca2+ influx through L-type Ca2+ channels into adrenomedullary cells without inhibition of NO synthesis, which seems to be relevant to AT2 receptor blockade. It also suggests that high-dose ARBs may be a risk factor in causing hypertensive response.

PS-B03-6: COMPARISON OF SUPPRESSIVE EFFECTS ON CATECHOLAMINE SECRETION BETWEEN OLMESARTAN AND ENALAPRIL

Objective: The present study was designed to compare olmesartan, an angiotensin II receptor blocker (ARB) with enalapril, an angiotensin converting enzyme inhibitor (ACEI) in the suppressive effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both olmesartan (50 μM) and enalapril (50 μM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 μM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 μM), high K+ (a direct membrane-depolarizer, 56 μM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 μM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 μM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 μM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 μM) were significantly reduced. Based on the same concentration (50 μM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan &gt; enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusion: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

Transcriptome Analysis of PC12 Cells Reveals That &lt;i&gt;trans&lt;/i&gt;-Banglene Upregulates &lt;i&gt;RT1-CE1&lt;/i&gt; and Downregulates &lt;i&gt;abca1&lt;/i&gt; in the Neurotrophic Pathway

trans-Banglene and cis(c)-banglene possess neurotrophin-like activity in rat neurons. However, the molecular mechanisms underlying t-banglene-induced neurotrophic activity in rat and human neurons remain unclear. Here, we performed transcriptome analysis in PC12 cells, a rat adrenal gland pheochromocytoma cell line treated with t-banglene, using comprehensive RNA sequencing. The differentially expressed gene analysis of the sequencing data revealed that the expression of RT1 class I, locus CE1 (RT1-CE1) was upregulated, and that of ATP binding cassette subfamily A member 1 (abca1), myosin light chain 6, and hippocampus abundant transcript 1 was downregulated in t-banglene-treated PC12 cells, with statistically significant differences. We also confirmed the RT1-CE1 upregulation and abca1 downregulation in t-banglene-treated PC12 cells by real-time reverse transcription quantitative PCR. RT1-CEl is a major histocompatibility complex class I (MHCI) protein. ABCAl is a major cholesterol transporter that regulates efflux of intracellular cholesterol and phospholipids. Thus, our results suggest an exciting link between MHCI, cholesterol regulation, and neural development.

Abstract 11977: Dapagliflozin Downregulates Adrenal G Protein-Coupled Receptor-Kinase-2 to Exert Sympatholysis in Heart Failure

Introduction: Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor exerting several cardiorenal protective effects independently of blood glucose regulation. Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity, especially in heart failure (HF). GRK2 severely dysregulates adrenal sympatho-inhibitory α 2 -adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Hypothesis: SGLT2 inhibition with dapagliflozin lowers SNS hyperactivity in part by opposing adrenal GRK2 actions. Methods: We used the rat pheochromocytoma PC12 cell line expressing human α 2A AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Results: Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p&lt;.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α 2 AR density was higher in dapagliflozin- vs. vehicle-treated rats(51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p&lt;.05). GRK2 and TH downregulation were also observed in PC12 cells treated with 5 μM dapagliflozin for 24 hours. Importantly, α 2 AR-dependent inhibition of CA secretion was markedly enhanced 24 hrs post-dapagliflozin application. This was due to reduced GRK2-dependent receptor desensitization induced by dapagliflozin, since this effect was abolished in PC12 cells overexpressing GRK2. Conclusions: Dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, thus reducing CA biosynthesis, and GRK2, thus reducing α 2 AR desensitization to lower CA secretion. This adrenal-mediated sympatholysis may be part of the increasingly apparent cardiovascular benefits of SGLT2 inhibitors in human HF irrespective of diabetic status.

Online Detection of Catecholamine Release from the Perfused Rat Adrenal Gland.

Retrogradely perfused adrenal glands have historically served for establishing many of our current knowledge on the stimulus-secretion coupling process. Although the use of intact adrenals has largely been switched to isolated chromaffin cells, adrenal glands are still a very valuable tool to characterize physiological and pharmacological questions. Even more, this is an excellent preparation for studying the splanchnic nerve/chromaffin cell interaction. In this chapter, we will provide the ways to (i) perform retrograde perfusion of isolated rat adrenals, (ii) the method to apply electrical splanchnic nerve stimulation, and (iii) the preparation of adrenals to conduct online electrochemical detection of catecholamine release.

Changes in the Expression of Transcription Factor Oct4 during Postnatal Development of Adrenal Medulla.

We analyzed the expression of the transcription factor Oct4 in the chromaffin cells during the postnatal development of the adrenal glands in rats. Single Oct4+-chromaffin cells with nuclear localization of the protein were found in the medulla, and their number increased in parallel with a decrease in the proliferative activity of cells. In postnatal development, 100% of chromaffin cells demonstrated high expression of tyrosine hydroxylase, which attested to their differentiation and functional activity. It was found that all Oct4+ cells were differentiated chromaffin cells. An increase in the pool of Oct4-expressing cells after the completion of organ growth suggests the formation of a potential source for the physiological and reparative regeneration of the adrenal medulla.

Wnt/β-catenin signaling in the adrenal glands of rats in various types of experimental hypertension.

Wnt/β-catenin signaling plays a key role in maintaining homeostasis, which is disturbed in hypertension. Taking into account the lack of literature describing changes in the Wnt/β-catenin pathway in the adrenal glands under conditions of elevated arterial pressure, here we compare the expression of WNT4, WNT10A, β-catenin, and GSK-3β in the adrenal glands of hypertensive rats of various etiologies. The studies were carried out on the adrenal glands of rats with spontaneous hypertension (SHR), renalvascular (2K1C), and deoxycorticosterone acetate (DOCA)-salt. Immunohistochemical and PCR methods were used to identify the molecular components of the canonical signaling pathway and to evaluate gene expression. Immunoreactivity and expression of WNT4, WNT10A, β-catenin, and GSK-3β in adrenals of SHR was decreased, compared to control rats. In adrenals of 2K1C rats, intensity of immunohistochemical reaction and expression of WNT4 and β-catenin was lower, while immunoreactivity and expression of WNT10A and GSK-3β were higher, compared to normotensive animals. Significantly stronger immunoreaction and expression of WNT4, β-catenin and GSK-3β but weaker immunoreactivity and expression of WNT10A were noted in adrenals in DOCA-salt rats, compared to control rats. In conclusion, our data provide new molecular information indicating that the canonical WNT pathway is disrupted in the adrenal glands of hypertensive rats. They show that the dysregulation of the WNT pathway depends on the etiology of hypertension.

Mechanisms of Suppression of Epinephrine Production in Rats by Low-Dose Developmental Exposure to DDT.

We studied the mechanisms underlying decreased production of epinephrine by adrenal glands in rats developmentally exposed to endocrine disruptor DDT was performed on the basis of assessment of morphology, fine structure, and function of the adrenal medulla and medullary cells. It was found that the main mechanisms of disruptive action of DDT leading to a decrease in epinephrine secretion into systemic circulation are a decrease in the growth rate of the adrenal medulla, a decrease in the number of mitochondria in adrenal cells, especially under the outer cytoplasmic membrane, destructive changes in mitochondria, and a progressive decrease in the synthesis of tyrosine hydroxylase. The decrease in the number of mitochondria and suppression tyrosine hydroxylase synthesis in adrenal cells predominate during the pubertal period, while slowdown of the growth of the adrenal medulla and progressive decrease in the synthesis of tyrosine hydroxylase seem to be the most evident mechanisms after puberty.

Морфофункціональні зміни гіпоталамо-адреналової системи щурів під дією наночастинок срібла при ожирінні

The aim of this study was to compare the effect of silver salt and silver nanoparticles on the morphofunctional state of the hypothalamic-adrenal system in obese rats. The experiment was carried out on 24 male rats at the age of 6 months. The rats of the control group received standard food. In other groups of animals, obesity was induced using a diet-induced model of metabolic disorders. The rats, modeled for obesity, were divided into several groups. Animals of different groups received NaCl solution, silver nitrate solution and silver nanoparticle solution for 10 days, respectively. After the end of the experiment, the hypothalamic nuclei and adrenal glands, prepared according to the appropriate histological methods, were taken from the animals. In the paraventricular nucleus of the hypothalamus, a small-cell area of neurons was examined. In the adrenal glands of rats, cells of three zones of the cortex were examined: glomerular, fascicular, and reticular. The results of the study showed that obesity led to the increase of functional activity in the nuclei of neurons in the small-cell region of the PVN of the rat hypothalamus. The cells of the glomerular zone of the adrenal cortex had the maximum increase in functional activity, the fascicular zone – a moderate increase in functional activity, and no significant changes were recorded in the reticular zone. It was shown that the silver salt solution caused the increase in the functional activity of PVN neurons in the hypothalamus of obese rats. In the glomerular zone, the parameters of the functional activity of cells increased significantly, in the fascicular zone, the parameters of cell nuclei decreased to the greater extent, in the reticular zone, mainly the parameters of the nuclei increased. It was investigated that the solution of silver nanoparticles caused the intensification of PVN cells in the hypothalamus of obese rats. In the glomerular zone of the adrenal cortex, a significant increase in cell parameters was observed, in the fascicular zone – the increase in the cytoplasm parameters, in the reticular zone – the decrease mainly in the parameters of cell nuclei. In general, the administration of the solution of silver nanoparticles led to more pronounced activation of PVN cells of the hypothalamus and adrenal cortex in obese rats than the silver salt.

MiRNA-99a alleviates inflammation and oxidative stress in lipopolysaccharide-stimulated PC-12 cells and rats post spinal cord injury

ABSTRACT Spinal cord injury (SCI) is caused by spinal fracture after the displacement of the spine or broken bone fragments protruding into the spinal canal, resulting in different degrees of injury to the spinal cord or spinal nerves. Expression levels of miR-99a and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in cerebrospinal fluid of SCI patients were analyzed. Rat adrenal gland pheochromocytoma cell line PC-12 were stimulated with lipopolysaccharide (LPS) to mimic the in vitro environment of SCI. A rat mode of SCI was established by laminectomy. Reactive oxygen species (ROS) levels were measured by 2’,7’-Dichlorodihydrofluorescein diacetate staining assay. Western blot was conducted to evaluate the expression levels of apoptotic indexes and proinflammatory cytokines. The interaction between miR-99a and NOX4 was verified by dual-luciferase reporter assay. The expression level of miR-99a was reduced while NOX4 expression was upregulated in cerebrospinal fluid of SCI patients and LPS-treated PC-12 cells. LPS impeded cell viability and promoted inflammation, apoptosis and ROS levels of PC-12 cells. Overexpression of miR-99a significantly promoted cell viability and reduced inflammation, apoptosis and oxidative stress in LPS-stimulated PC-12 cells. Dual-luciferase reporter assays verified that NOX4 was a target of miR-99a. Moreover, the expression of NOX4 was reduced in PC-12 cells after transfection with miR-99a mimic. Overexpression of NOX4 partly abolished the protective effect of miR-99a in LPS-treated PC-12 cells. To sum up, miR-99a suppresses NOX4 expression to relieve the LPS-induced inflammation, apoptosis and the progression of oxidative stress in SCI.

Novel regulation mechanism of adrenal cortisol and DHEA biosynthesis via the endogen ERAD inhibitor small VCP-interacting protein

Endoplasmic reticulum-associated degradation (ERAD) is a well-characterized mechanism of protein quality control by removal of misfolded or unfolded proteins. The tight regulation of ERAD is critical for protein homeostasis as well as lipid metabolism. Although the mechanism is complex, all ERAD branches converge on p97/VCP, a key protein in the retrotranslocation step. The multifunctionality of p97/VCP relies on its multiple binding partners, one of which is the endogenous ERAD inhibitor, SVIP (small VCP-interacting protein). As SVIP is a promising target for the regulation of ERAD, we aimed to assess its novel physiological roles. We revealed that SVIP is highly expressed in the rat adrenal gland, especially in the cortex region, at a consistently high level during postnatal development, unlike the gradual increase in expression seen in developing nerves. Steroidogenic stimulators caused a decrease in SVIP mRNA expression and increase in SVIP protein degradation in human adrenocortical H295R cells. Interestingly, silencing of SVIP diminished cortisol secretion along with downregulation of steroidogenic enzymes and proteins involved in cholesterol uptake and cholesterol biosynthesis. A certain degree of SVIP overexpression mainly increased the biosynthesis of cortisol as well as DHEA by enhancing the expression of key steroidogenic proteins, whereas exaggerated overexpression led to apoptosis, phosphorylation of eIF2α, and diminished adrenal steroid hormone biosynthesis. In conclusion, SVIP is a novel regulator of adrenal cortisol and DHEA biosynthesis, suggesting that alterations in SVIP expression levels may be involved in the deregulation of steroidogenic stimulator signaling and abnormal adrenal hormone secretion.

Pharmacology-based toxicity assessment of molsidomine and ATP-LONG combination with singular and repetitive injections under experimental conditions

Abstract. The aim of the work was to study toxic properties of the new combined drug which comprise nitrovasodilator molsidomine and adenosine- 5’-triphosphate in a form of coordination compound with histidine, magnesium, and potassium (ATP-LONG). The drug was examined for its acute and subacute toxicity on Balb/c mice and Wistar rats of reproductive age with peroral (p/o) and sublingual (s/l) administrations. It has been established that LD50 of the substance contains over 10000 mg/kg (p/o) and 5010 mg/kg (s/l), which corresponds to the category of Practically non-toxic substances. The repetitive administrations within a 28 day period of the conditionally therapeutic dose of 260 mg/hg (s/l) did not cause any negative impact on physiological, biochemical, histological values in male and female rats. In doses 1300 and 2080 mg/kg, which exceed conditionally therapeutic doses by 5 and 8 times, the combination was not changing clinical laboratory urine and blood values but induced histological changes such as dilation and plethora of capillaries along with edema of smooth muscle cells of the brain, myocardium, liver, spleen, kidneys, and adrenal glands in rats. Additionally, the particular dosages of the combined substance provoked irritation of the mucous membrane of the tongue. Detected effects of the drug do not carry any pathological character and can be viewed as a specific reaction of the organism to high doses of nitrovasodilator. However, the duration and reversibility of unwanted consequences of molsidomine overdose, particularly in its combined form, need further investigation. Keywords: combination of molsidomine and ATP-LONG, acute and subacute toxicity

Therapeutic Effect of Curcumin on Scanning Electron Microscopy of Rat Adrenal Gland in Experimental Fluorosis

Aim: The present study was designed to investigate the therapeutic role of Curcumin against fluoride induced toxicity on adrenal gland of rats by using scanning electron microscopy (SEM).&#x0D; Methodology: Wistar albino rats were divided randomly into six groups The group I was administered with 1 ml of deionized water/kg b.w./day orally for 40 days. The Groups II and III were given 300 and 600 mg of NaF/kg b.w./day for the same period, respectively. The group IV was given 200 mg/kg b.w. of Curcumin for 20 days. The Groups V and VI were treated with 300 and 600 mg of NaF/kg b.w./day for 40 days respectively were post-treated with 200 mg of Curcumin for next 20 days. The animals were excised and adrenal tissue was taken out and processed for SEM.&#x0D; Results: The results revealed that rats exposed to 300 mg/kg b.w./day of NaF showed rough edges, numerous microvilli and damaged surface with crystal depositions. Also, numerous granules were distributed all over the surface. The rats treated with 600 mg/kg b.w./day of NaF showed decellularized adrenal tissue along with network of collagen fibres. Moreover, adrenal gland surface displayed abrasions and distorted cuboidal cells. The filopodia were prominent on the surface and wall of cavity possessed rough outline. After post-treatment with Curcumin, fluoridated adrenal gland of rats showed normal structure, reappearance of cuboidal cells on the surface as well as less number of microvilli and filopodia.&#x0D; Conclusion: The post-treatment with Curcumin possess therapeutic potential against NaF induced toxicity in adrenal gland of rats.

Obesogenic and histopathological effect of monosodium glutamate on the adrenal gland of maternal and postnatal albino rats

Monosodium glutamate (MSG) is a flavoring food additive that’s found in many foods like chips, and fast foods. The aim of this study was to demonstrate the injurious effects of MSG on the adrenals of the female albino rats and its potentiality to cross the placental barrier carrying its injurious effects to their pups. Twenty-five female albino rats weighing about (200) g were mated then separated into two groups. The control group (10 rats), and MSG-treated Group(15 rats) received 10 g/kg MSG orally, dissolved in water at a concentration of 5 g/100 ml, starting on day 7 to 20 day of gestation.A day after delivery, animals were weighed, blood samples collected then sacrificed, and the adrenals were collected and examined by the light and electron microscope. Statistically, there was a significant increase in the body weight and decrease in adrenal weight of MSG-treated mothers and their pups, as well as a decrease in the corticosterone level in MSG-treated mothers. Histologically, MSG-treated mothers and pups showed many degenerative changes, with thinning of the capsule that was confirmed by Masson trichrome stain, a disorganized structure of Zona Golmeruloza and Zona Fasciculata, and areas of shedding and hemorrhage were noticed. Shrunken nuclei and degenerated mitochondria with disrupted cristae and penetration of the lipid droplets to form “myelin-like” structures were noticed by E/M examination. The study concluded that MSG had an unwholesome effect on the structure of the adrenal tissue of both pregnant rats and their pups

Microscopic features of the rat adrenal gland associated with chronic codeine phosphate administration.

Codeine is an opioid analgesic and antitussive that has been widely abused. Some adverse effects noted with its abuse include adrenocortical insufficiency and activation of the hypothalamic-pituitary-adrenal axis. The structural basis for these dysfunctions is not clearly understood. Twenty-five adult male rats were used for the study. They were divided into intervention and control groups that were administered 40 mg/kg of codeine phosphate and normal saline respectively by gavage daily for 50 days. Subsequently, both groups were given normal saline for a further fourteen days to note recovery changes. At day 0, 50 and 64, rats were randomly selected from both groups, euthanized and adrenal glands harvested for histological processing and analysis. At day 50 of codeine administration, the adrenal glands demonstrated an increase in zona fasciculata thickness but a decrease in zona reticularis thickness. Lower values were noted in the volume density of zona reticularis and cells count of the medulla in the experimental compared to the control groups (P-value<0.05). The experimental group also showed an increase in vascularization and connective tissue in the glands. After 14 days of recovery, most of the changes observed in experimental animals were reversed and the adrenal glands in both groups had similar features. A decrease in cell count of the adrenal medulla was however observed (P-value<0.05). In conclusion administration of codeine phosphate causes discernible changes in the microscopic structure of the adrenal gland, most of which appear to be reversed after two weeks recovery period.

High-throughput rat immunoglobulin G N-glycosylation profiling revealed subclass-specific changes associated with chronic stress

Immunoglobulin G (IgG) glycosylation corresponds well with immune system changes, so it can potentially be used as a biomarker for the consequences of chronic stress such as low-grade inflammation and enhanced immunosenescence in older animals. Here we present a high-throughput glycoproteomic workflow, including IgG enrichment, HILIC glycopeptide purification, and nano-LC-MS analysis of tryptic glycopeptides applied for the analysis of rat IgG. A cohort of 80 animals was exposed to seven stressors in a customized chronic stress protocol with blood and tissue sampling in three timepoints. Young female rats experienced an increase in agalactosylated glycoforms on IgG2a and IgG2c accompanied by a decrease in monogalactosylation. Among old females, increased galactosylation was observed in the IgG2b subclass, pointing to an anti-inflammatory activity of IgG. Additionally, IgG Fc N-glycosylation patterns in Sprague Dawley rats were analyzed, quantified, and reported for the first time. Our findings emphasize age-, sex- and subclass-dependent differences in IgG glycosylation related to chronic stress exposure, confirming the relevance of newly developed methods for further research in glycobiology of rodent immune response. SignificanceIn this study, we showed that a high-throughput streamlined methodology based on protein L 96-well monolithic plates for efficient rat IgG immunoaffinity enrichment from blood plasma, paired with appropriate tryptic glycopeptide preparation, HILIC-SPE enrichment, and nano-LC-MS methods was suitable for quick processing of large sample sets. We report a subclass-specific profiling and changes in rat IgG Fc galactosylation and adrenal gland immunohistochemistry of male and female animals exposed to a customized chronic stress protocol.

Dapagliflozin Exerts Adrenal Sympatholysis via G protein-Coupled Receptor Kinase-2 &amp; Tyrosine Hydroxylase Downregulation

Abstract The sodium-glucose co-transporter (SGLT)-2 inhibitor dapagliflozin was recently reported to reduce renal tyrosine hydroxylase (TH) and norepinephrine levels, lowering blood pressure and preventing endothelial dysfunction, in a murine model of neurogenic hypertension. This suggested that dapagliflozin may combat sympathetic nervous system (SNS) hyperactivity, which is known to accompany and aggravate chronic heart failure (CHF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and SNS hyperactivity, especially in CHF. GRK2 severely dysregulates adrenal sympatho-inhibitory α 2-adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Therefore, we hypothesized herein that SGLT2 inhibition with dapagliflozin may lower SNS hyperactivity in the adrenal gland by antagonizing GRK2 actions on α 2ARs in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α 2AAR, as well as freshly isolated adrenal glands from adult male Sprague-Dawley rats treated with dapagliflozin in vivo. We measured circulating norepinephrine (NE) in vivo via RIA, GRK2 &amp; TH expression levels via real-time PCR and immunoblotting, adrenal α 2AR density (Bmax) via saturation radioligand binding with [methyl-3H]-rauwolscine, and G protein activation via the GTPγS assay. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p&amp;lt;.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and TH mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α 2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p&amp;lt;.05). These results (i.e. GRK2 and TH downregulation) were completely recapitulated in PC12 α 2AAR-expressing cells in culture, treated with 5 μM dapagliflozin (or vehicle) for 24 hours. Importantly, α 2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced at 24 hrs post-dapagliflozin application in PC12 cells. This was the result of reduced GRK2-dependent receptor desensitization, since dapagliflozin lacked this effect in cells co-transfected with a GRK2-encoding adenovirus to acutely overexpress GRK2. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, which reduces CA biosynthesis, and GRK2, which reduces α 2AR desensitization in favor of enhanced inhibition of CA secretion.

COMPARISON OF INHIBITORY EFFECTS EVOKED BY OLMESARTAN AND ENALAPRIL ON CATECHOLAMINE SECRETION

Objective: The present study was designed to compare enalapril, an angiotensin converting enzyme inhibitor (ACEI) with olmesartan an angiotensin II receptor blocker (ARB) in the inhibitory effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and method: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both enalapril (50 microM) and olmesartan (50 microM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 microM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 microM), high K+ (a direct membrane-depolarizer, 56 mM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 microM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 microM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 microM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 microM) were significantly reduced. Based on the same concentration (50 microM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan&gt; enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusions: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

HIGH-DOSES OF LOSARTAN RATHER ACCELERATE THE SECRETION OF ADRENAL CATECHOLAMINES

Objective: Previously, we have found that Losartan (5∼50 microM), an AT1 receptor blocker, dose-dependently inhibits secretion of catecholamines (CA) evoked by cholinergic and angiotensin II (Ang II) receptor stimulation from the perfused rat adrenal medulla. The aim of the present study was to examine the characteristic effects of high-dose losartan on the CA secretion from the isolated perfused rat adrenal gland, and also to establish its mechanism of action. Design and method: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: The perfusion of high-dose losartan (150 ∼ 600 microM) into an adrenal vein for 90 min resulted in great increases in CA secretions evoked by acetylcholine in a dose-dependent fashion. Also, losartan (300 microM) enhanced the CA release evoked by Ang II, high K+ and DMPP (a selective nicotinic receptor agonist), McN-A-343 (a selective muscarinic M1 receptor agonist). Also, in the presence of losartan (300 microM), the CA secretory responses to veratridine (a selective Na+ channel activator), Bay-K-8644 (a Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly potentiated, respectively. The facilitatory CA-releasing effect of Ach evoked by high-dose losartan (300 microM) was time-dependently depressed by pretreatment with nifedipine (Ca2+ channel blocker), while not by pretreatment with sodium nitroprusside (an NO donor). High-dose olmesartan (150 ∼ 600 microM), one of ARBs, also potentiated Ach-evoked CA release in time-dependent fashion. However, in the simultaneous presence of losartan (300 microM) and CGP42112 (an AT2 receptor agonist), the facilitatory responses of losartan on the CA secretion evoked by ACh were rather significantly reduced in comparison with that of losartan-treatment alone. Conclusions: Conclusively, these results demonstrate that high-dose losartan can potentiate the adrenomedullary CA secretion in a calcium-dependent fashion. It seems that this facilitatory effect of high-dose losartan may be mediated by activation of the Ca2+ influx through L-type Ca2+ channels into adrenomedullary cells without inhibition of NO synthesis, which seems to be relevant to AT2 receptor blockade. It also suggests that high-dose ARBs may be a risk factor in causing hypertensive response.