Angiotensin I and II Stimulate Cell Invasion of SARS-CoV-2: Potential Mechanism via Inhibition of ACE2 Arm of RAS.

Angiotensin-converting enzyme 2 (ACE2), one of the key enzymes of the renin-angiotensin system (RAS), plays an important role in SARS-CoV-2 infection by functioning as a virus receptor. Angiotensin peptides Ang I and Ang II, the substrates of ACE2, can modulate the binding of SARS-CoV-2 Spike protein to the ACE2 receptor. In the present work, we found that co incubation of HEK-ACE2 and Vero E6 cells with the SARS-CoV-2 Spike pseudovirus (PVP) resulted in stimulation of the virus entry at low and high micromolar concentrations of Ang I and Ang II, respectively. The potency of Ang I and Ang II stimulation of virus entry corresponds to their binding affinity to ACE2 catalytic pocket with 10 times higher efficiency of Ang II. The Ang II induced mild increase of PVP infectivity at 20 microM; while at 100 microM the increase (129.74+/-3.99 %) was highly significant (p<0.001). Since the angiotensin peptides act in HEK ACE2 cells without the involvement of angiotensin type I receptors, we hypothesize that there is a steric interaction between the catalytic pocket of the ACE2 enzyme and the SARS-CoV-2 S1 binding domain. Oversaturation of the ACE2 with their angiotensin substrate might result in increased binding and entry of the SARS-CoV-2. In addition, the analysis of angiotensin peptides metabolism showed decreased ACE2 and increased ACE activity upon SARS-CoV-2 action. These effects should be taken into consideration in COVID-19 patients suffering from comorbidities such as the over-activated renin-angiotensin system as a mechanism potentially influencing the SARS-CoV-2 invasion into recipient cells.

Cystinyl and pyroglutamyl-beta-naphthylamide hydrolyzing activities are modified coordinately between hypothalamus, liver and plasma depending on the thyroid status of adult male rats.

The hypothalamus determinates metabolic processes in liver through endocrine and autonomic control. Hypothalamic neuropeptides, such as thyrotropin releasing hormone or vasopressin, have been involved in liver metabolism. The thyroid status influences metabolic processes including liver metabolism in modulating those hypothalamic peptides whose functional status is regulated in part by aminopeptidase activities. In order to obtain data for a possible coordinated interaction between hypothalamus, plasma and liver, of some aminopeptidase activities that may partially reflect the hydrolysis of those peptides, pyroglutamyl- (pGluAP) and cystinyl- (CysAP) beta-naphthylamide hydrolyzing activities were determined fluorimetrically, both in their soluble and membrane-bound forms, in eu- hypo- and hyperthyroid adult male rats. Hyperthyroidism and hypothyroidism were induced with daily subcutaneous injections of tetraiodothyronine (300 µg/kg/day) or with 0.03% methimazole in drinking water for 6 weeks. Results demonstrated significant changes depending on the type of enzyme and the thyroid status. The most striking changes were observed for CysAP in liver where it was reduced in hypothyroidism and increased in hyperthyroidism. Significant intra- and inter-tissue correlations were observed. While there were positive inter-tissue correlations between liver, plasma and hypothalamus in eu-and hypothyroid rats, a negative correlation between hypothalamus and liver was observed in hyperthyroidism. These results suggest the influence of thyroid hormones and an interactive role for these activities in the control of liver metabolism. The present data also suggest a role for CysAP and pGluAP activities in liver function linked to their activities in hypothalamus.

Bidirectional asymmetry in the neurovisceral communication for the cardiovascular control: New insights.

The cardiovascular control involves a bidirectional functional connection between the brain and heart. We hypothesize that this connection could be extended to other organs using endocrine and autonomic nervous systems (ANS) as communication pathways. This implies a neuroendocrine interaction controlling particularly the cardiovascular function where the enzymatic cascade of the renin-angiotensin system (RAS) plays an essential role. It acts not only through its classic endocrine connection but also the ANS. In addition, the brain is functionally, anatomically, and neurochemically asymmetric. Moreover, this asymmetry goes even beyond the brain and it includes both sides of the peripheral nervous and neuroendocrine systems. We revised the available information and analyze the asymmetrical neuroendocrine bidirectional interaction for the cardiovascular control. Negative and positive correlations involving the RAS have been observed between brain, heart, kidney, gut, and plasma in physiologic and pathologic conditions. The central role of the peptides and enzymes of the RAS within this neurovisceral communication, as well as the importance of the asymmetrical distribution of the various RAS components in the pathologies involving this connection, are particularly discussed. In conclusion, there are numerous evidences supporting the existence of a neurovisceral connection with multiorgan involvement that controls, among others, the cardiovascular function. This connection is asymmetrically organized.

Alternative renin-angiotensin system pathways in adipose tissue and their role in the pathogenesis of obesity.

Adipose tissue expresses all the renin-angiotensin system (RAS) components that play an important role in the adipogenesis, lipid and glucose metabolism regulation in an auto/paracrine manner. The classical RAS has been found to be over-activated during the adipose tissue enlargement, thus elevated generation of angiotensin II (Ang II) may contribute to the obesity pathogenesis. The contemporary view on the RAS has become more complex with the discovery of alternative pathways, including angiotensin-converting enzyme 2 (ACE2)/angiotensin (Ang)-(1-7)/Mas receptor, (pro)renin receptor, as well as angiotensin IV(Ang IV)/AT4 receptor. Ang-(1-7) via Mas receptor counteracts with most of the deleterious effects of the Ang II-mediated by AT1 receptor implying its beneficial role in the glucose and lipid metabolism, oxidative stress, inflammation, and insulin resistance. Pro(renin) receptor may play a role (at least partial) in the pathogenesis of the obesity by increasing the local production of Ang II in adipose tissue as well as triggering signal transduction independently of Ang II. In this review, modulation of alternative RAS pathways in adipose tissue during obesity is discussed and the involvement of Ang-(1-7), (pro)renin and AT4 receptors in the regulation of adipose tissue homeostasis and insulin resistance is summarized.

Metabolic effects of subchronic peripheral oxytocin administration in lean and obese zucker rats.

Increasing evidence indicates a role of oxytocin in controlling energy metabolism. The aim of his study was to investigate oxytocin effects on obese phenotype in leptin-resistant Zucker fatty rats, focusing on glucose and lipid metabolism. Zucker fatty rats and their lean controls were treated with oxytocin (3.6 µg/100g body weight/day) by osmotic minipumps implanted subcutaneously for 2 weeks. Two-hours intraperitoneal glucose tolerance test was performed in fasting rats. Oxytocin decreased food intake in both phenotypes while body weight gain reduced only in obese animals. In obese rats oxytocin impaired hepatic insulin extraction and enhanced liver triglyceride accumulation. Moreover, in the skeletal muscle of lean rats oxytocin treatment downregulated insulin signal transduction by decreasing of insulin receptor substrate 1 protein level and stimulating of its serine phosphorylation. Concurrently, the gene expression of insulin receptor substrate 1 in the skeletal muscle and adipose tissue was downregulated by oxytocin. In obese rats, oxytocin reduced adipocyte size and normalised mRNA levels of both fatty acid binding protein 4 and fatty acid synthase but attenuated gene expression of glucose transporter 4. The present study in Zucker fatty rats demonstrated ambivalent effects of oxytocin treatment with predominantly negative impact on skeletal muscle insulin pathway in lean animals.

Effect of ivabradine, captopril and melatonin on the behaviour of rats in L-nitro-arginine methyl ester-induced hypertension.

Cardiovascular diseases including hypertension are often associated with behavioural alterations. The aim of this study was to show, whether ivabradine, the blocker of If-channel in sinoatrial node, is able to modify the behaviour of rats in L-nitro-arginine methyl ester (L-NAME)-induced hypertension and to compare the effect of ivabradine with captopril and melatonin. 12-week-old male Wistar rats were divided into the following groups: controls, ivabradine (10 mg/kg/24 h), L-NAME (40 mg/kg/24 h), L-NAME + ivabradine, L-NAME + captopril (100 mg/kg/24 h), L-NAME + melatonin (10 mg/kg/24 h). Systolic blood pressure (SBP) and heart rate (HR) were measured by tail-cuff method once a week. The behaviour of rats was investigated during 23-hours in the phenotyper after four weeks of the treatment. Chronic administration of L-NAME induced hypertension without a change in HR. All tested substances partly prevented the increase of SBP, while ivabradine and melatonin also reduced HR. Ivabradine, captopril and melatonin reduced daily food intake, slightly decreased daily water intake and attenuated body weight gain. In L-NAME group, locomotor activity was enhanced by ivabradine, whereas exploratory behaviour was increased by melatonin and captopril. In conclusion, ivabradine, besides its potentially protective hemodynamic actions, does not seem to exert any disturbing effects on behaviour in L-NAME-induced hypertension in rats, while some of its effects were similar to captopril or melatonin. It is suggested that ivabradine used in cardiovascular indications is harmless regarding the effect on behaviour.

Differential regulation of oxytocin receptor in various adipose tissue depots and skeletal muscle types in obese Zucker rats.

Multifunctional peptide oxytocin currently undergoes intensive research due to its proposed anti-obesity properties. Until now, little is known about regulation of oxytocin receptor in metabolically active tissues in obesity. The aim of the present study was to measure expression of oxytocin receptor upon obese phenotype with respect to the variety among adipose tissue and skeletal muscles with distinct anatomical localisation. Total homogenates were prepared from epididymal, retroperitoneal and inguinal adipose tissues as well as quadriceps and soleus muscle from lean and obese Zucker rats. Oxytocin receptor protein was determined by immunoblot. Interestingly, elevated oxytocin receptor was observed in epididymal adipose tissue of obese rats in contrast to its downregulation in subcutaneous and no change in retroperitoneal fat. In lean animals, oxytocin receptor protein was expressed at similar levels in all adipose depots. This uniformity was not observed in the case of skeletal muscle in which fibre type composition seems to be determinant of oxytocin receptor expression. Quadriceps muscle with the predominance of glycolytic fibres exhibits higher oxytocin receptor expression than almost exclusively oxidative soleus muscle. Oxytocin receptor protein levels were decreased in both skeletal muscles analysed upon obese phenotype. The present work demonstrates that even under identical endocrine circumstances, oxytocin receptor is differentially regulated in adipose tissue of obese rats depending on fat depot localisation. These results also imply which tissues may be preferentially targeted by oxytocin treatment in metabolic disease.

Doxorubicin-induced behavioral disturbances in rats: protective effect of melatonin and captopril.

OBJECTIVE: Doxorubicin is a recognized chemotherapeutic agent widely employed in human malignancies. The limiting factor of its use is a number of side effects. The aim of this work was to show, whether administration of doxorubicin could induce behavioral disturbances in rats, and whether angiotensin-converting enzyme inhibitor captopril or melatonin can modify these potential alterations. DESIGN AND METHODS: Four groups of 3-month-old Wistar rats (twelve per group) were treated for 4 weeks: control (placebo-treated), doxorubicin (DOX) (5mg/kg i.v. in a single intravenous dose), DOX rats treated with either melatonin (10mg/kg/24h) or captopril (100mg/kg/24h). Systolic blood pressure (SBP) and the level of oxidative stress were investigated and behavioral tests of anxiety-open field test (OF), elevated plus maze (EPM) and light-dark box (LDB) were accomplished. RESULTS: Doxorubicin increased significantly systolic blood pressure and parameters of oxidative stress. Moreover, doxorubicin enhanced the level of anxiety in the tests of OF, EPM, and LDB. Captopril and melatonin prevented the blood pressure rise and the enhancement of oxidative load. Importantly, both substances reduced the parameters of anxiety. CONCLUSION: Chronic administration of captopril or melatonin has shown anxiolytic effect in the model of doxorubicin-induced anxiety. It does not seem unreasonable to suppose that this protective effect of captopril or melatonin against anxiety development might have been related to the antioxidative effects of both substances.

Aerobic training lasting for 10 weeks elevates the adipose tissue FABP4, Giα, and adiponectin expression associated by a reduced protein oxidation.

OBJECTIVE: The chronic moderate exercise positively alters the systemic glucose homeostasis, enhances the insulin action, and ameliorates the oxidative damage in the skeletal muscle and liver. The aim of this study was to investigate the effect of an intermittent aerobic training on the metabolic parameters of the white adipose tissue in the obese Zucker rats. METHODS: Obese Zucker rats, 8 week old, were subjected to running on a 4-channel treadmill (1 h/day 5 times/week 20 m/min at maximum) for 10 weeks, except the weekends, (Trained Obese Zucker, TOZ) or were placed to the turned-off treadmill (Sedentary Obese Zucker, SOZ) for the same period. The serum insulin, glucose, and triglyceride were determined. The gene expression of the renin-angiotensin system (RAS) components and selected metabolic parameters were quantified by real-time qPCR in the liver and epididymal and retroperitoneal adipose tissues. The content of the protein carbonyl groups was assayed in the liver and epididymal fat depot. RESULTS: The gene expression of the adipocyte fatty acid binding protein 4 (FABP4) was significantly elevated in the epididymal and retroperitoneal adipose tissues of the TOZ rats. The level of the adiponectin mRNA was increased in the retroperitoneal adipose tissue while leptin and inhibitory G-protein α mRNA were elevated in the epididymal adipose tissue after exercise. The aerobic training led to a decrease in the amount of protein carbonyl groups in the epididymal adipose depot. Transcription of the angiotensinogen, angiotensin-converting enzyme (ACE), and AT1 receptor genes in the epididymal adipose tissue was not influenced by the exercise. In the liver, only the AT1 receptor gene expression increased significantly. The serum glucose, insulin, and triglycerides concentrations were not changed in the TOZ rats when compared to SOZ animals. CONCLUSIONS: Data of the present study indicate that an intermittent moderate exercise in the hyperphagic obese Zucker rats lasting for 10 weeks improves some of the morphometric and metabolic parameters of the white adipose tissue and decreases the protein oxidation implying a general beneficial effect of the long-lasting exercising.

Angiotensinogen and angiotensin-converting enzyme mRNA decrease and AT1 receptor mRNA and protein increase in epididymal fat tissue accompany age-induced elevation of adiposity and reductions in expression of GLUT4 and peroxisome proliferator-activated receptor (PPARγ).

Elevated adiposity is one of the accompanying features of increased age in humans and animals. Angiotensin II (Ang II) is considered as growth promoting peptide to be involved in hypertrophic enlargement of adipose tissue. However, systemic renin-angiotensin system (RAS) seems to decrease with increased age of rats. Local adipose tissue RAS might be independent of the systemic one. Therefore we performed a comprehensive study using rats with increased age from 9 to 26 weeks and evaluated angiotensinogen, angiotensin-converting enzyme (ACE) and AT(1) receptor mRNA in epididymal adipose tissue by RT-PCR. In addition, we determined AT(1) receptor protein by Western blotting and Ang II binding. These RAS parameters were correlated with expression of selected adiposity-dependent proteins such as leptin, adiponectin, insulin-dependent glucose transporter (GLUT4) and PPARgamma. Angiotensinogen and ACE expression decreased with increased age and adiposity. On the contrary, AT(1) receptor mRNA and protein was significantly elevated in 26-week-old rats though the Ang II binding was not different between 9 and 26-week-old animals. These results suggest dynamic adaptation of local adipose tissue RAS components to increased age and adiposity most likely by decreasing local Ang II formation which is thereafter compensated by increased expression of AT(1) receptor. However, this increase in AT(1) receptor mRNA and protein is not reflected in increased receptor binding. We believe that this complex regulation of adipose tissue RAS slows down the negative age and adiposity related changes in adipose tissue leptin, adiponectin, GLUT4 and PPARgamma.

Impaired insulin secretion and uptake in patients with diffuse idiopathic skeletal hyperostosis.

OBJECTIVE: So far, high prevalence of metabolic symptoms accompanying diffuse idiopathic skeletal hyperostosis (DISH) appears not definitely elucidated because of their possible origin from other disorders such as diabetes and/or body mass differences. From such reasons this study was aimed to compare non-diabetic DISH patients to a group of age and BMI matched controls in order to distinguish the influence of DISH proper on metabolic parameters free of additional metabolic effects caused by diabetes and/or body weight differences. METHODS: Both groups of patients were subjected to oral glucose tolerance test (OGTT) and fasting serum levels of glucose, insulin, C-peptide, growth hormone, insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGF-BP3) were assayed. Fasting serum total cholesterol, HDL cholesterol, triglycerides, non-esterified fatty acids (NEFA) and uric acid were determined as well. The indices of insulin sensitivity and insulin secretion were calculated. RESULTS: With the exception of decreased NEFA serum level and decreased insulinogenic index and insulin/C-peptide ratio in DISH patients any other significant differences in serum parameters and indices of insulin sensitivity were not found. CONCLUSIONS: The data obtained suggest impaired beta-cell pancreatic stimulation and increased insulin hepatic extraction in DISH. It is assumed that the above mentioned conditions, if persisting for a long time, might lead to decreased ability of insulin to maintain normal serum glucose level and consequently to insulin resistance which is highly prevalent in symptomatic DISH patients.

Activation of hypothalamic NPY, AgRP, MC4R, AND IL-6 mRNA levels in young Lewis rats with early-life diet-induced obesity.

OBJECTIVE: Obesity represents a low-grade inflammatory disease and appears a risk factor for insulin resistance, but little is known on whether this may contribute to the development of autoimmune inflammatory diseases. The aim of this work was to study the early-life diet-induced obesity in Lewis rats which are known to be highly susceptible to autoimmunity. METHODS: Obesity was induced by reduced litter size (4 pups per litter) followed by high-fat diet (SHF rats). Control rats (8 pups per litter) were fed with standard diet (CN rats). Oral glucose tolerance test (3 g glucose per kg b.w.) was performed by intra-gastric tube in conscious rats after 12 h fast. Adipocyte size was assessed by light microscope after collagenase digestion. Hypothalamic arcuate (ARC) and paraventricular nuclei (PVN) were isolated by the punching technique. Target mRNAs were quantified by real-time PCR with the use of TaqMan probes and primers. Serum hormones (leptin, ghrelin, adiponectin, visfatin and insulin) were assayed by specific RIAs . RESULTS: During the experimental period SHF rats had the same body weight gain and caloric intake as CN rats. At the age of 8 weeks SHF rats showed increased epididymal fat mass and adipocyte volume, impaired glucose tolerance, normal basal fasting insulin, visfatin, and ghrelin level, but decreased adiponectin and high leptin level. In the ARC, the SHF rats showed increased expression of mRNA for orexigenic neuropeptide Y (NPY), agouti-related protein (AgRP) and anorexigenic pro-inflammatory cytokine IL-6. In the PVN, the SHF rats showed increased expression of mRNA for anorexigenic melanocortin 4 receptor (MC4R) and IL-6. CONCLUSION: Overexpression of orexigenic NPY and AgRP in the ARC indicates leptin resistance in SHF rats. The increased expression of MC4R in PVN points to the activation of melanocortin anorexigenic system which, along with increased hypothalamic IL-6, might prevent the animals from overfeeding. Higher adiposity in these rats results from the high fat-diet composition and not from increased caloric intake. Furthermore, enhanced leptin production appears the main factor indicating the predisposition to autoimmunity in these overfed rats.

Rats with monosodium glutamate-induced obesity and insulin resistance exhibit low expression of Galpha(i2) G-protein.

In order to test the potential role of inhibitory G-proteins in mechanisms of insulin resistance in adipose tissue of obese animals we determined the content of Galpha(i1) and Galpha(i2) proteins and an extent of protein tyrosine phosphorylation in epididymal fat tissue cell membranes using immunoblot. Monosodium glutamate-induced obese rats displayed adipose tissue hypertrophy, elevated levels of insulin, leptin and slightly elevated serum glucose. We found significantly decreased protein content of Galpha(i2) in adipose tissue plasma membranes of obese rats. This was in accordance with lower protein tyrosine phosphorylation noticed in adipose tissue cell homogenate of glutamate-treated animals. Our results confirm the role of Galpha(i2) in development of insulin resistance by crosstalk between the reduced level of inhibitory G-protein and insulin receptor mediated most likely by activation of phosphotyrosine protein dephosphorylation.

Hormone response to stress in rat strains of different susceptibility to immunologic challenge.

OBJECTIVE: The aim of this study was to compare the changes in plasma levels of hormones involved in modulation of the immune system function after exposure to stress in two rat strains with different susceptibility to immunoantigens. METHODS: Adult rat males of Lewis (LEW) and Fischer 344 (FIS) strains were exposed to restrain stress for 2 hours and blood samples were collected during stress exposure. Other groups of animals were exposed to restrain stress for 2 hours and sacrificed 3 hours later for blood and organ collection. Corticosterone, testosterone, dehydroepiandrosterone, 17beta-estradiol and progesterone were estimated by radioimmunoassay, epinephrine and norepinephrine levels were determined by radioenzymatic method. RESULTS: The levels of plasma corticosterone and catecholamines were significantly higher during stress exposure in FIS as compared to LEW rats. Greater decrease of testosterone levels and higher levels of estradiol were noted after exposure to stress in LEW rats. Higher values of progesterone plasma levels were noted in FIS rats after stress. CONCLUSIONS: These results demonstrated the differences in the response of catecholamines, adrenal and gonadal steroids after exposure to stress in LEW and FIS rats with lower levels of hormones with anti-inflammatory action in LEW rats.

Short term 13-cis-retinoic acid treatment at therapeutic doses elevates expression of leptin, GLUT4, PPARgamma and aP2 in rat adipose tissue.

Temporary defects in the plasma lipid and glucose homeostasis are frequent complication accompanying chronic treatment with 13-cis-retinoic acid (13cRA). White adipose tissue acts as an endocrine organ producing a variety of hormones (adipocytokines) including leptin, adiponectin, tumor-necrosis factor alpha (TNFalpha) and angiotensin II (Ang II), which influence lipid metabolism, systemic insulin sensitivity and inflammation. To study the effect of a short-term 13cRA administration on metabolism of epididymal fat tissue, we treated Wistar rats with five identical therapeutic doses of 13cRA (0.8 mg/kg b.w.) by gavage during a period of 10 days. Expression of adiponectin, leptin, TNFalpha and selected proteins such as adipocyte fatty acid binding protein (aP2), insulin-dependent glucose transporter GLUT4, peroxisome proliferator-activated receptor gamma (PPARgamma) and retinoid X receptors (RXRs) was investigated using RT-PCR. Short-term treatment with therapeutic doses of 13cRA caused significant increase of the aP2, PPARgamma and moderately RXRalpha gene expression. Similarly, the relative amount of mRNA for leptin and GLUT4 was increased, while the TNFa transcript was decreased after treatment with 13cRA. The gene expression and plasma concentration of adiponectin were without any significant changes. Since local adipose renin-angiotensin system (RAS) has been presumed to be involved in the regulation of fat tissue metabolism, we also investigated the gene expression of RAS components in epididymal fat depot. Our data has shown that 13cRA elevated Ang II receptor type 1 (AT(1) receptor)--at both, mRNA and protein level. Thus, our results demonstrate that short-term 13cRA treatment is inducing alterations in fat tissue metabolism in relation to stimulated adipogenesis.

Oxytocin levels in the posterior pituitary and in the heart are modified by voluntary wheel running.

We hypothesized that voluntary wheel running results in increased secretion of oxytocin, a peptide involved in the stress response. An additional hypothesis was that prolonged exercise affects oxytocin levels in the heart, which is in line with the potential role of oxytocin in cardiovascular functions. Voluntary wheel running lasted 3 weeks and daily running distances increased progressively reaching maximum levels about 8 km (Sprague-Dawley rats) and 4 km (Lewis strain). The exercise resulted in significant reduction of epididymal fat, slight increase in glucose transporter GLUT4 mRNA levels and significant enhancement of plasma density. Voluntary exercise failed to influence plasma oxytocin levels either in Lewis or Sprague-Dawley rats, but it resulted in a significant decrease of oxytocin concentrations in the posterior pituitary. Plasma oxytocin concentrations were not modified even if the measurements were made in the dark phase of the day. In voluntary wheel running Sprague-Dawley rats, the content of oxytocin in the right heart atrium was lower than in controls. Thus, the present findings demonstrate that prolonged voluntary wheel running results in a decrease in pituitary oxytocin content without evident changes in hormone concentrations in peripheral blood. However, prolonged exercise used has a significant impact on oxytocin levels in the heart.

Thyrotropin-releasing hormone in rat heart: effect of swelling, angiotensin II and renin gene.

AIM: This study was performed to examine thyrotropin-releasing hormone (TRH) secretion and regulation in rat heart. METHODS: Expression of prepro-TRH gene in left atrium and left ventricle was studied by RT-PCR. TRH secretion from slices of left auricle and left ventricle in response to cell swelling (induced by hypotonic medium or ethanol in isosmotic medium), angiotensin II and losartan and their combinations was studied. RESULTS: RT-PCR revealed two times higher prepro-TRH expression in left auricle than left ventricle. In transgenic rats with extra copy of mouse renin gene a marked increase of prepro-TRH expression in the heart was noted but the relative difference between left atrium and left ventricle persisted. The swelling stimulated TRH release from both left auricle and left ventricle and this stimulation could not be inhibited by bumetanide. Angiotensin II (10 nmol L-1) added into medium significantly decreased basal secretion of TRH. The inhibiting effect of Angiotensin II was prevented by 1 micromol L-1 losartan, an angiotensin II AT1 receptor blocker. When angiotensin II and hypotonicity were applied simultaneously, swelling-induced secretion persisted. CONCLUSION: TRH secretion from heart slices has attributes of regulated secretion--depending on the stimulus it could be either stimulated or inhibited. Renin positively affects prepro-TRH expression in the heart. Angiotensin II inhibits TRH secretion from heart tissue by a mechanism involving AT1 receptors. Swelling-induced TRH secretion overrides inhibitory effect of angiotensin II. Swelling could be a useful tool when natural or pharmacological secretagogue is unknown. Peptides and proteins released by swelling could be mediators of local and remote ischaemic preconditioning protecting from subsequent ischaemia.

Blockade of AT1 receptors by losartan did not affect renin gene expression in kidney medulla.

This study was designed to determine particular changes in the renin gene expression and activity in renal cortex and medulla after AT(1) receptor blockade. It was found that two-week-treatment with AT(1) blocker losartan induced an increase in tissue renin activity in both parts of kidney causing subsequent elevation of plasma renin activity. Renin mRNA in losartan-treated rats was increased only in cortex, suggesting cortex origin of elevated renin activity in medulla. Medullary renin mRNA indicated local synthesis of renin within the whole kidney and supported the idea of the presence of tissue renin-angiotensin system. Our results show that gene expression of renin in kidney medulla is insensitive to AT(1) receptor blockade and this points out that the regulation of kidney renin-angiotensin system probably differs from that in cortex.

Effect of neonatal streptozotocin and thyrotropin-releasing hormone treatments on insulin secretion in adult rats.

Neonatal STZ (nSTZ) treatment results in damage of pancreatic B-cells and in parallel depletion of insulin and TRH in the rat pancreas. The injury of B-cells is followed by spontaneous regeneration but dysregulation of the insulin response to glucose persists for the rest of life. Similar disturbance in insulin secretion was observed in mice with targeted TRH gene disruption. The aim of present study was to determine the role of the absence of pancreatic TRH during the perinatal period in the nSTZ model of impaired insulin secretion. Neonatal rats were injected with STZ (90 microg/g BW i.p.) and the effect of exogenous TRH (10 ng/g BW/day s.c. during the first week of life) on in vitro functions of pancreatic islets was studied at the age 12-14 weeks. RT-PCR was used for determination of prepro-TRH mRNA in isolated islets. Plasma was assayed for glucose and insulin, and isolated islets were used for determination of insulin release in vitro. The expression of prepro-TRH mRNA was only partially reduced in the islets of adult nSTZ rats when compared to controls. nSTZ rats had normal levels of plasma glucose and insulin but the islets of nSTZ rats failed to response by increased insulin secretion to stimulation with 16.7 mmol/l glucose or 50 mmol/l KCl. Perinatal TRH treatment enhanced basal insulin secretion in vitro in nSTZ animals of both sexes and partially restored the insulin response to glucose stimulation in nSTZ females.

Effect of immobilization on in vitro thyrotropin-releasing hormone release from brain septum in wild-type and corticotropin-releasing hormone knock-out mice.

There is considerable evidence linking alcohol consumption, sedation, and thyrotropin-releasing hormone (TRH) in the brain septum. We have shown that ethanol in clinically relevant concentrations can in vitro induce TRH release from the septum by a mechanism involving neuronal swelling. Corticotropin-releasing hormone-deficient (CRH-KO) mice serve as an interesting model to help us understand the role of CRH in the regulation of different neuroendocrine systems. The aim of this study was to compare TRH release activity in the brain septum at basal and stress conditions in CRH-KO mice and their wild-type (WT) littermates. Experimental mice were decapitated immediately or 3 h after single (2 h) or repeated (seven times for 2 h daily) immobilization stress. The brain septum was immediately cut out and incubated to measure basal-, ethanol-, and hyposmosis-stimulated TRH release in vitro. Ethanol in isosmotic medium or hyposmotic medium stimulated TRH release from mice septal explants from WT and CRH-KO mice. The response was disturbed immediately after immobilization and recovered 3 h later. Our results show that immobilization stress transiently affects the TRH system in brain septum. Inborn absence of CRH does not affect septal TRH and its response to ethanol before and 3 h after immobilization.