Comparative assessment of CacyBP/SIP, ß-catenin and cannabinoid receptors in the adrenals of hypertensive rats.

Taking into account homeostatic disorders resulting from arterial hypertension and the key importance of CacyBP/SIP, ß-catenin and endocannabinoids in the functioning of many organs, it was decided to assess the presence and distribution of CacyBP/SIP, ß-catenin, CB1 and CB2 in the adrenal glands of hypertensive rats of various aetiology. The study was conducted on the adrenal glands of rats with spontaneous and renovascular hypertension. The expression of CacyBP/SIP, ß-catenin, CB1 and CB2 was detected by immunohistochemistry and real-time PCR method. The results of the present study revealed both lower gene expression and immunoreactivity of CacyBP/SIP in the adrenal glands of all hypertensive groups compared to the normotensive rats. This study demonstrated a reduction in the immunoreactivity and expression of the ß-catenin, CB1 and CB2 genes in the adrenals of 2K1C rats. While in SHR, the reaction showing ß-catenin and CB1 was very weak or negative, and the expression of CB2 in the adrenal glands of these rats increased. The results of this study show, for the first time, marked differences in the expression of CacyBP/SIP, ß-catenin and CB1 and CB2 cannabinoid receptors in the adrenal glands of rats with primary (SHR) and secondary hypertension (2K1C).

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

Wnt/ß-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/ß-catenin pathway in the adrenal glands under conditions of elevated arterial pressure, here we compare the expression of WNT4, WNT10A, ß-catenin, and GSK-3ß 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, ß-catenin, and GSK-3ß in adrenals of SHR was decreased, compared to control rats. In adrenals of 2K1C rats, intensity of immunohistochemical reaction and expression of WNT4 and ß-catenin was lower, while immunoreactivity and expression of WNT10A and GSK-3ß were higher, compared to normotensive animals. Significantly stronger immunoreaction and expression of WNT4, ß-catenin and GSK-3ß 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.

Canonical Wnt signaling in the kidney in different hypertension models.

There is a close relationship between the kidney and blood pressure. On the one hand, kidney dysfunction causes an increase in blood pressure; on the other hand, high blood pressure causes kidney dysfunction. Wnt/ß-catenin signaling is a key pathway that regulates various cellular processes and tissue homeostasis and is also involved in damage and repair processes. In healthy organs, Wnt/ß-catenin signaling is muted, but it is activated in pathological states. The purpose of the present study was to immunohistochemically evaluate and compare the expression of WNT4, WNT10A, Fzd8, ß-catenin, and GSK-3ß (glycogen synthase kinase 3ß) in the kidneys of rats with essential arterial hypertension (SHR), renal-renal hypertension (2K1C), and DOCA-salt-induced hypertension. The study was performed on five male WKY rats, seven SHRs, and twenty-four (n = 24) young male Wistar rats. The main results showed that during hypertension, there are changes in Wnt/ß-catenin signaling in the kidneys of rats, and the severity of these changes depends on the type of hypertension. This study is the first to assess the levels of some elements of the canonical Wnt/ß-catenin signal transduction pathway in various types of arterial hypertension by immunohistochemistry and may form the basis for further molecular and functional studies of this pathway in hypertension.

Ageing-related changes in the levels of ß-catenin, CacyBP/SIP, galectin-3 and immunoproteasome subunit LMP7 in the heart of men.

Aging is a major risk factor for morbidity and mortality from cardiovascular causes in men. To better understand the cellular processes related to age-related cardiac complications, we undertook research aimed at comparative evaluation of genes expression and distribution of ß-catenin, CacyBP/SIP, galectin-3 and LMP7 in the heart of healthy men in different age groups. The study was conducted on the hearts of 12 men (organ donors) without a history of cardiovascular disease, who were divided into two age groups: men under and men over 45 years of age. On paraffin sections, immunohistochemical reactions were performed to detect ß-catenin, CacyBP/SIP, galectin-3 and immunoproteasome subunit LMP7. The expression of genes coding ß-catenin, CacyBP/SIP, galectin-3 and LMP7 was also evaluated by real-time PCR method. In the heart of men over 45 years old, both gene expression and immunoreactivity of ß-catenin, CacyBP/SIP, galectin-3 and LMP7 were stronger compared to younger individuals. The results of the presented studies suggest that ß-catenin, CacyBP/SIP, galectin-3 and immunoproteasomes might be involved in the internal regulation of heart homeostasis during ageing.

Fatty acid amide hydrolase inhibitor (URB597) as a regulator of myocardial lipid metabolism in spontaneously hypertensive rats.

Pressure overload, which is typical of hypertension, is known to evoke alterations not only in the morphology of the heart but also in the preference of myocardial energetic substrates usage. Nowadays, the endocannabinoid system (ECS) serves as a potential therapeutic target for cardiovascular disorders and, simultaneously, affects whole body metabolism homeostasis. Therefore, an open question is whether ECS, apart from decreasing blood pressure, also affects cardiac muscle metabolism in hypertensive conditions. All experiments were conducted on a genetic model of primary hypertension i.e. spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKY) served as a normotensive control. ECS was chronically activated by 2-weeks intraperitoneal injections of fatty acid amide hydrolase (FAAH) inhibitor - URB597. Lipid analyses in the left ventricle and serum were based on ex vivo heart perfusion in Langendorff perfusion system, thin layer chromatography, and gas liquid chromatography. The total expression of selected proteins was determined using Western blot as well as immunohistochemical techniques. As expected, URB597 markedly reduced systolic as well as mean blood pressures in SHRs. Moreover, prolonged FAAH inhibition resulted in stimulation of 3H-palmitate uptake and incorporation into different lipid fractions in cardiomyocytes in the hypertensive as well as normotensive conditions. An increase in fatty acid oxidation caused by URB597 treatment was observed only in WKY rats, but not SHRs, and was accompanied by an elevation in peroxisome proliferator-activated receptor alpha (PPARα) and ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) expressions. Chronic activation of ECS significantly upregulates palmitate uptake and its esterification but not oxidation in the SHR's myocardium.

The Endocannabinoid System Affects Myocardial Glucose Metabolism in the DOCA-Salt Model of Hypertension.

BACKGROUND/AIMS: Recent interest in the use of cannabinoids as therapeutic agents has revealed the involvement of the endogenous cannabinoid system (ECS) in the regulation of the cardiovascular system in hypertension. Abnormalities in glucose metabolism and insulin action are commonly detected in hypertensive animals. Thus, potential antihypertensive drugs should be investigated with respect to modulation of glucose homeostasis. Therefore, the aim of the present study was to evaluate the effects of the ECS activation after chronic fatty acid amide hydrolase inhibitor (URB597) administration on plasma glucose and insulin concentrations as well as parameters of myocardial glucose metabolism in the deoxycorticosterone acetate (DOCA)-salt hypertensive rats, an animal model of secondary hypertension. METHODS: Hypertension was induced by DOCA (25mg/kg) injections and addition of 1% NaCl in the drinking water for six weeks. Chronic activation of the ECS was performed by URB597 (1mg/kg) injections for two weeks. We examined fasting plasma levels of insulin (ELISA), glucose and intramyocardial glycogen (colorimetric method). Expressions of glucose transporters (GLUT1, 4) and selected proteins engaged in GLUT translocation as well as glucose metabolism were determined using Western blotting. RESULTS: Hypertension induced hypoinsulinemia with concomitant lack of significant changes in glycemia, reduced intramyocardial glycogen content and increased pyruvate dehydrogenase (PDH) expression in the cardiac muscle. Importantly, chronic URB597 administration in the hypertensive rats increased insulin concentration, elevated plasmalemmal GLUT1 and GLUT4 expression and concomitantly improved myocardial glycogen storage. CONCLUSION: Chronic administration of fatty acid amide hydrolase (FAAH) inhibitor has potential protective properties on myocardial glucose metabolism in hypertension.

The Influence of Chitosan Cross-linking on the Properties of Alginate Microparticles with Metformin Hydrochloride-In Vitro and In Vivo Evaluation.

Sodium alginate is a polymer with unique ability to gel with different cross-linking agents in result of ionic and electrostatic interactions. Chitosan cross-linked alginate provides improvement of swelling and mucoadhesive properties and might be used to design sustained release dosage forms. Therefore, the aim of this research was to develop and evaluate possibility of preparing chitosan cross-linked alginate microparticles containing metformin hydrochloride by the spray-drying method. In addition, influence of cross-linking agent on the properties of microparticles was evaluated. Formulation of microparticles prepared by the spray drying of 2% alginate solution cross-linked by 0.1% chitosan was characterized by good mucoadhesive properties, high drug loading and prolonged metformin hydrochloride release. It was shown that designed microparticles reduced rat glucose blood level, delayed absorption of metformin hydrochloride and provided stable plasma drug concentration. Additionally, histopathological studies of pancreas, liver and kidneys indicated that all prepared microparticles improved degenerative changes in organs of diabetic rats. Moreover, no toxicity effect and no changes in rats behavior after oral administration of chitosan cross-linked alginate microparticles were noted.

Renovascular hypertensive decrease immunoreactivity of cells containing chromogranin A and pancreastatin in the pancreas of rats.

Hypertension significantly increases the risk of hyperglycemia in patients. It is known that chromogranin (CgA) and pancreastatin (PST) are involved in regulation of blood pressure and endocrine function of the pancreas. However, still little is known about the physiology of these hormones' secretion in hypertension. The objective of the study was to examine the effects of hypertension on pancreatic islet cells containing CgA and PST in rats. The studies were carried out on the pancreas of rats. After 6 week period of the renal artery clipping procedure, eight 2K1C rats developed stable hypertension. Cells containing CgA and PST were detected using immunohistochemical method. The hypertension significantly decreased the number of pancreatic endocrine cells immunoreactive to CgA and PST antisera. The differences between the hypertensive and normotensive rats concerned not only the number of endocrine cells but also intensity of reactions. In conclusion, the research results indicate that hypertension causes the diminished biosynthesis of CgA and PST in the pancreas of rats and suggests the participation of those peptides in pancreatic disorders occurring in a state of elevated blood pressure.

The Toxicokinetic Profile of Dex40-GTMAC3-a Novel Polysaccharide Candidate for Reversal of Unfractionated Heparin.

Though protamine sulfate is the only approved antidote of unfractionated heparin (UFH), yet may produce life threatening side effects such as systemic hypotension, catastrophic pulmonary vasoconstriction or allergic reactions. We have described 40 kDa dextrans (Dex40) substituted with glycidyltrimethylammonium chloride (GTMAC) as effective, immunogenically and hemodynamically neutral inhibitors of UFH. The aim of the present study was to evaluate in mice and rats toxicokinetic profile of the most promising polymer-Dex40-GTMAC3. Polymer was rapidly eliminated with a half-time of 12.5 ± 3.0 min in Wistar rats, and was mainly distributed to the kidneys and liver in mice. The safety studies included the measurement of blood count and blood biochemistry, erythrocyte osmotic fragility and the evaluation of the histological alterations in kidneys, liver and lungs of mice and rats in acute and chronic experiments. We found that Dex40-GTMAC3 is not only effective but also very well tolerated. Additionally, we found that protamine may cause overt hemolysis with appearance of permanent changes in the liver and kidneys. In summary, fast renal clearance behavior and generally low tissue accumulation of Dex40-GTMAC3 is likely to contribute to its superior to protamine biocompatibility. Intravenous administration of therapeutic doses to living animals does not result in the immunogenic, hemodynamic, blood, and organ toxicity. Dex40-GTMAC3 seems to be a promising effective and safe candidate for further clinical development as new UFH reversal agent.

Alterations of rat stomach endocrine cells under renovascular hypertension.

PURPOSE: The aim of the present study was to perform immunohistochemical and ultrastructural analysis of gastrin-, synaptophysin (SY)- and atrial natriuretic peptide (ANP)-positive cells in the pylorus of "two kidney, one clip" (2K1C) renovascular hypertension model in rats. MATERIAL/METHODS: In order to identify neuroendocrine (NE) cells, immunohistochemical reactions were performed with the use of specific antibodies against gastrin, SY and ANP. Gastric NE cells were also examined using an electron microscope. RESULTS: The present study revealed a twofold increase in the number of gastrin- and SY-positive cells and a significant decrease in the number of ANP-immunoreactive (IR) cells in the pyloric mucosa of 2K1C rats. Test results obtained with an electron microscope confirmed a change in the activity of the stomach endocrine cells of hypertensive rats. CONCLUSIONS: Immunohistochemical and ultrastructural investigations demonstrated the impact of renovascular hypertension on the neuroendocrine system in the rat stomach. The changes in the total number and ultrastructure of DNES cells proved their undeniable role in the modulation of gastric dysfunction, as a consequence of deregulation of homeostasis-maintaining systems.

Effects of CP 55,940--agonist of CB1 cannabinoid receptors on ghrelin and somatostatin producing cells in the rat pancreas.

Cannabinoids participate in the modulation of numerous functions in the human organism, increasing the sense of hunger, affecting carbohydrate and lipid metabolism, and controlling systemic energy balance mechanisms. Moreover, they influence the endocrine system functions, acting via two types of receptors, CB1 and CB2. The aim of the present study was to examine the number, distribution and activity of ghrelin and somatostatin producing endocrine cells in the pancreas of rats after a single administration of selective CP 55,940 agonist of CB1 receptor. The study was performed on 20 rats. Neuroendocrine cells were identified by immunohistochemical reactions, involving specific antibodies against ghrelin and somatostatin. The distribution and number of ghrelin- and somatostatin-immunoreactive cells were separately studied in five pancreas islets of each section. A performed analysis showed a decreased number of somatostatin-immunoreactive cells and a weak immunoreactivity of ghrelin and somatostatin containing neuroendocrine cells in the pancreatic islets of experimental rats, compared to control animals. The obtained results suggest that a single administration of a selective CP 55,940 agonist of CB1 receptor influences the immunoreactivity of endocrine cells with ghrelin and somatostatin expression in the pancreas islets.

Distribution pattern of CART-containing neurons and cells in the human pancreas.

Cocaine- and amphetamine-regulated transcript (CART) has been shown to play a critical role in appetite suppression, cell survival, thermoregulation, glucose sensing, stimulation of hormone secretion, as well as for the regulatory function of the islets of Langerhans. Although the principal site of CART synthesis has already been reported, our knowledge of the subject is mainly based on and limited to research conducted on animals owing to difficulties in obtaining human samples. Therefore, the primary goal of the reported study was an attempt to identify and localize CART in healthy human pancreas. Nineteen deceased subjects (donors of organs) with normal pancreas and alimentary tract were used in the study. After determination of brain death and confirmation of death by the relevant doctors committee, pancreas samples, about 1cm long, were collected from each corpse (the same part of the pancreas) after the organs were harvested for transplantation. Paraffin sections were made and stained with hematoxylin and eosin and then subjected to CART immunohistochemistry. In the normal pancreas of human adults, CART is mainly present in both nerve fibers and in nerve cell bodies in pancreatic ganglia. In addition to pancreatic neurons, immunoreactivity to CART was also seen in islet endocrine cells. This is the first report on the presence of CART-IR structures in the normal human pancreas. CART should be now added to the numerous regulatory peptides that are involved in the complex regulation of pancreatic endocrine and exocrine processes.