Relaxation evoked by extracellular Ca(2+) in rat aorta is nerve-independent and involves sarcoplasmic reticulum and L-type Ca(2+) channell

The perivascular nerve network expresses a Ca(2+) receptor that is activated by high extracellular Ca(2+) concentrations and causes vasorelaxation in resistance arteries. We have verified the influence of perivascular nerve fibers on the Ca(2+)-induced relaxation in aortic rings. To test our hypothesis, either pre-contracted aortas isolated from rats after sensory denervation with capsaicin or aortic rings acutely denervated with phenol were stimulated to relax with increasing extracellular Ca(2+) concentration. We also studied the role of the endothelium on the Ca(2+)-induced relaxation, and we verified the participation of endothelial/nonendothelial nitric oxide and cyclooxygenise-arachidonic acid metabolites. Additionally, the role of the sarcoplasmic reticulum, K(+) channels and L-type Ca(2+) channels on the Ca(2+)-induced relaxation were evaluated. We have observed that the Ca(2+)-induced relaxation is completely nerve independent, and it is potentiated by endothelial nitric oxide (NO). In endothelium-denuded aortic rings, indomethacin and AH6809 (PGF(2 alpha) receptor antagonist) enhance the relaxing response to Ca(2+). This relaxation is inhibited by thapsigargin and verapamil, while was not altered by tetraethylammonium. In conclusion, we have shown that perivascular nervous fibers do not participate in the Ca(2+)-induced relaxation, which is potentiated by endothelial NO. In endothelium-denuded preparations, indomethacin and AH6809 enhance the relaxation induced by Ca(2+). The relaxing response to Call was impaired by verapamil and thapsigargin, revealing the importance of L-type Ca(2+) channels and sarcoplasmic reticulum in this response. (c) 2008 Elsevier Inc. All rights reserved.

5-HT1A receptors of the lateral septum regulate inhibitory avoidance but not escape behavior in rats

Serotonin in the lateral septum (LS) has been implicated in the modulation of defensive behaviors and in anxiety. However, it is currently unknown whether changes in 5-HT mechanisms in this brain area may selectively affect defensive responses associated with specific subtypes of anxiety disorders recognized in clinical settings. To address this question, we evaluated the effect of the intra-LS injection of the 5-HT1A/7 receptor agonist 8-CH-DPAT (0.6, 3.0, 15.0 nmol) in male Wistar rats exposed to the elevated T-maze animal model of anxiety. This test allows the measurement of two behavioral defensive responses in the same rat: inhibitory avoidance and escape behavior. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. The effects of 8-OH-DPAT were compared to those caused by a standard anxiolytic compound, the benzodiazepine receptor agonist midazolam (MDZ, 20 nmol). We also investigated whether the intra-LS injection of the 5-HT1A receptor antagonist WAY-100635 (0.37 nmol) was able to block the effects of 8-OH-DPAT. All animals were also tested in an open field for locomotor activity assessments. Results showed that whereas intra-LS administration of MDZ decreased avoidance latencies, suggesting an anxiolytic action, 8-OH-DPAT caused the opposite effect. Neither drug affected the escape performance. Intra-LS administration of WAY-100635 blocked the anxiogenic effect caused by 8-OH-DPAT. No changes to locomotion were detected in the open field. The data suggests that LS 5-HT1A receptors are involved in the control of inhibitory avoidance behavior and that a failure in this regulatory mechanism may be of importance to the physiopathology of generalized anxiety disorder. (c) 2008 Elsevier Inc. All rights reserved.

Effect of IIb-IIIa Glycoprotein Inhibitors in a Partial Limb Amputation Model Submitted to Warm Ischemia in Rats

Viability and functional results of a segment replantation depend on the prevention of deleterious effects of ischemia. Prolonged ischemia leads to alterations in the microcirculation: thrombosis, edema, production of oxygen free radicals, and platelet aggregation. The effect of IIb-IIIa glycoprotein inhibitors was tested in a partial limb amputation model submitted to warm ischemia. The male Wistar rats were divided into four groups: G1 with 0 hours of ischemia and saline (n = 20), G2 with 6 hours of ischemia and saline (n = 24), G3 with 6 hours of ischemia and abciximab (n = 23), and G4 with 6 hours of ischemia and tirofiban (n = 29). The limbs were observed for 7 days and classified as viable or nonviable. Viability, and mortality rates were obtained and analyzed by Q-square and Fisher exact tests (p < 0.05). The viability rates were 100% (G1), 30% (G2), 77.78% (G3), and 80.95% (G4). G2 was statistically different from G1, G3, and G4. G1, G3, and G4 were not statistically different. Transoperative and postoperative mortalities were not statistically different. The administration of abciximab and tirofiban improved limb salvage after ischemia and reperfusion and did not modify mortality rates significantly.

KISS1R Intracellular Trafficking and Degradation: Effect of the Arg386Pro Disease-Associated Mutation

The goal of this study was to investigate how the Arg386Pro mutation prolongs KiSS-1 receptor (KISS1R) responsiveness to kisspeptin, contributing to human central precocious puberty. Confocal imaging showed colocalization of wild-type (WT) KISS1R with a membrane marker, which persisted for up to 5 h of stimulation. Conversely, no colocalization with a lysosome marker was detected. Also, overnight treatment with a lysosome inhibitor did not affect WT KISS1R protein, whereas overnight treatment with a proteasome inhibitor increased protein levels by 24-fold. WT and Arg386Pro KISS1R showed time-dependent internalization upon stimulation. However, both receptors were recycled back to the membrane. The Arg386Pro mutation did not affect the relative distribution of KISS1R in membrane and internalized fractions when compared to WT KISS1R for up to 120 min of stimulation, demonstrating that this mutation does not affect KISS1R trafficking rate. Nonetheless, total Arg386Pro KISS1R was substantially increased compared with WT after 120 min of kisspeptin stimulation. This net increase was eliminated by blockade of detection of recycled receptors, demonstrating that recycled receptors account for the increased responsiveness of this mutant to kisspeptin. We therefore conclude the following: 1) WT KISS1R is degraded by proteasomes rather than lysosomes; 2) WT and Arg386Pro KISS1R are internalized upon stimulation, but most of the internalized receptors are recycled back to the membrane rather than degraded; 3) the Arg386Pro mutation does not affect the rate of KISS1R trafficking-instead, it prolongs responsiveness to kisspeptin by decreasing KISS1R degradation, resulting in the net increase on mutant receptor recycled back to the plasma membrane.(Endocrinology 152: 1616-1626,2011)

Clinical Approaches to Preserve beta-Cell Function in Diabetes

In type 2 diabetes (DM2) there is progressive deterioration in beta-cell function and mass. It was found that islet function was about 50% of normal at the time of diagnosis and reduction in beta-cell mass of about 60% at necropsy (accelerated apoptosis). Among the interventions to preserve the beta-cells, those to lead to short-term improvement of beta-cell secretion are weight loss, metformin, sulfonylureas, and insulin. The long-term improvement was demonstrated with short-term intensive insulin therapy of newly diagnosed DM2, the use of antiapoptotic drugs such as glitazones, and the use of glucagon-like peptide-1 receptor agonists (GLP-1 mimetics), not inactivated by the enzyme dipeptidyl peptidase 4 and/or to inhibit that enzyme (GLP-1 enhancers). The incretin hormones are released from the gastrointestinal tract in response to nutrient ingestion to enhance glucose-dependent insulin secretion from the pancreas and overall maintenance of glucose homeostasis. From the two major incretins, GLP-1 and GIP (glucose-dependent insulinotropic polypeptide), only the first one or its mimetics or enhancers can be used for treatment. The GLP-1 mimetics exenatide and liraglutide as well as the DPP4 inhibitors (sitagliptin and vildagliptin) were approved for treatment of DM2.

Reciprocal interactions of obstructive sleep apnea and hypertension associated with ACE I/D polymorphism in males

Background: The angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism gene contributes to the genesis of hypertension (HTN) and may help explain the relationship between obstructive sleep apnea (OSA) and HTN. However, ACE is a pleiotropic gene that has several influences, including skeletal muscle and control of ventilation. We therefore tested the hypothesis that ACE polymorphism influences OSA severity. Methods: Male OSA patients (apnea-hypopnea index [AHI] > 5 events/h) from 2 university sleep centers were evaluated by polysomnography and ACE I/D polymorphism genotyping. Results: We studied 266 males with OSA (age = 48 +/- 13y, body mass index = 29 5kg/m(2), AHI = 34 +/- 25events/h). HTN was present in 114 patients (43%) who were older (p < 0.01), heavier (p < 0.05) and had more severe OSA (p < 0.01). The I allele was associated with HTN in patients with mild to moderate OSA (p < 0.01), but not in those with severe OSA. ACE I/D polymorphism was not associated with apnea severity among normotensive patients. In contrast. the only variables independently associated with OSA severity among patients with hypertension in multivariate analysis were BMI (OR = 1.12) and 11 genotype (OR = 0.27). Conclusions: Our results indicate reciprocal interactions between OSA and HTN with ACE I/D polymorphism, suggesting that among hypertensive OSA males, the homozygous ACE I allele protects from severe OSA. (C) 2009 Elsevier B.V. All rights reserved.

Salt-Induced Cardiac Hypertrophy and Interstitial Fibrosis Are Due to a Blood Pressure-Independent Mechanism in Wistar Rats

High salt intake is a known cardiovascular risk factor and is associated with cardiac alterations. To better understand this effect, male Wistar rats were fed a normal (NSD: 1.3% NaCl), high 4 (HSD4: 4%), or high 8 (HSD8: 8%) salt diet from weaning until 18 wk of age. The HSD8 group was subdivided into HSD8, HSD8+HZ (15 mg.kg(-1).d(-1) hydralazine in the drinking water), and HSD8+LOS (20 mg.kg(-1).d(-1) losartan in the drinking water) groups. The cardiomyocyte diameter was greater in the HSD4 and HSD8 groups than in the HSD8+LOS and NSD groups. Interstitial fibrosis was greater in the HSD4 and HSD8 groups than in the HSD8+HZ and NSD groups. Hydralazine prevented high blood pressure (BP) and fibrosis, but not cardiomyocyte hypertrophy. Losartan prevented high BP and cardiomyocyte hypertrophy, but not fibrosis. Angiotensin II type 1 receptor (AT(1)) protein expression in both ventricles was greater in the HSD8 group than in the NSD group. Losartan, but not hydralazine, prevented this effect. Compared with the NSD group, the binding of an AT(1) conformation-specific antibody that recognizes the activated form of the receptor was lower in both ventricles in all other groups. Losartan further lowered the binding of the anti-AT(1) antibody in both ventricles compared with all other experimental groups. Angiotensin II was greater in both ventricles in all groups compared with the NSD group. Myocardial structural alterations in response to HSD are independent of the effect on BP. Salt-induced cardiomyocyte hypertrophy and interstitial fibrosis possibly are due to different mechanisms. Evidence from the present study suggests that salt-induced AT(1) receptor internalization is probably due to angiotensin II binding. J. Nutr. 140: 1742-1751, 2010.

Current management practices for acromegaly: an international survey

To determine whether peer-reviewed consensus statements have changed clinical practice, we surveyed acromegaly care in specialist centers across the globe, and determined the degree of adherence to published consensus guidelines on acromegaly management. Sixty-five acromegaly experts who participated in the 7th Acromegaly Consensus Workshop in March 2009 responded. Results indicated that the most common referring sources for acromegaly patients were other endocrinologists (in 26% of centers), neurosurgeons (25%) and primary care physicians (21%). In sixty-nine percent of patients, biochemical diagnoses were made by evaluating results of a combination of growth hormone (GH) nadir/basal GH and elevated insulin like growth factor-I (IGF-I) levels. In both Europe and the USA, neurosurgery was the treatment of choice for GH-secreting microadenomas and for macroadenomas with compromised visual function. The most widely used criteria for neurosurgical outcome assessment were combined measurements of IGF-I and GH levels after oral glucose tolerance test (OGTT) 3 months after surgery. Ninety-eight percent of respondents stated that primary treatment with somatostatin receptor ligands (SRLs) was indicated at least sometime during the management of acromegaly patients. In nearly all centers (96%), the use of pegvisomant monotherapy was restricted to patients who had failed to achieve biochemical control with SRL therapy. The observation that most centers followed consensus statement recommendations encourages the future utility of these workshops aimed to create uniform management standards for acromegaly.

Effects of losartan, in monotherapy or in association with hydrochlorothiazide, in chronic nephropathy resulting from losartan treatment during lactation

Fanelli C, Fernandes BH, Machado FG, Okabe C, Malheiros DM, Fujihara CK, Zatz R. Effects of losartan, in monotherapy or in association with hydrochlorothiazide, in chronic nephropathy resulting from losartan treatment during lactation. Am J Physiol Renal Physiol 301: F580-F587, 2011. First published June 8, 2011; doi:10.1152/ajprenal.00042.2011.-We recently standardized a model (L(Lact)) of severe chronic kidney disease based on impaired nephrogenesis by suppression of angiotensin II activity during lactation (Machado FG, Poppi EP, Fanelli C, Malheiros DM, Zatz R, Fujihara CK. Am J Physiol Renal Physiol 294: F1345-F1353, 2008). In this new study of the L(Lact) model, we sought to gain further insight into renal injury mechanisms associated with this model and to verify whether the renoprotection obtained with the association of the angiotensin II receptor blocker losartan (L) and hydrochlorothiazide (H), which arrested renal injury in the remnant kidney model, would provide similar renoprotection. Twenty Munich-Wistar dams, each nursing six pups, were divided into control, untreated, and L(Lact) groups, given losartan (L; 250 mg.kg(-1).day(-1)) until weaning. The male LLact offspring remained untreated until 7 mo of age, when renal functional and structural parameters were studied in 17 of them, used as pretreatment control (L(Lact)Pre), and followed no further. The remaining rats were then divided among groups L(Lact) + V, untreated; L(Lact) + L, given L (50 mg.kg(-1).day(-1)) now as a therapy; L(Lact) + H, given H (6 mg.kg(-1).day(-1)); and L(Lact) + LH, given L and H. All parameters were reassessed 3 mo later in these groups and in age-matched controls. At this time, L(Lact) rats exhibited hypertension, severe albuminuria, glomerular damage, marked interstitial expansion/inflammation, enhanced cell proliferation, myofibroblast infiltration, and creatinine retention. L monotherapy normalized albuminuria and prevented hypertension and the progression of renal injury, inflammation, and myofibroblast infiltration. In contrast to the remnant model, the LH combination promoted only slight additional renoprotection, perhaps because of a limited tendency to retain sodium in L(Lact) rats.

Regression of glomerular injury by losartan in experimental diabetic nephropathy

Many features of chronic kidney disease may be reversed, but it is unclear whether advanced lesions, such as adhesions of sclerotic glomerular tufts to Bowman`s capsule (synechiae), can resolve during treatment. We previously showed, using a renal ablation model, that the renoprotective effect of the AT-1 receptor blocker, losartan, is dose-dependent. Here we determined if moderate and advanced glomerular lesions, associated with streptozotocin-induced diabetes, regress with conventional or high-dose losartan treatment. Using daily insulin injection for 10 months, we maintained diabetic adult male Munich-Wistar rats in a state of moderate hyperglycemia. Following this period, some rats continued to receive insulin with or without conventional or high-dose losartan for an additional 2 months. Diabetic rats pretreated with insulin for 10 months and age-matched non-diabetic rats served as controls. Mesangial expansion was found in the control diabetic rats and was exacerbated in those rats maintained on only insulin for an additional 2 months. Conventional and high-dose losartan treatments reduced this mesangial expansion and the severity of synechiae lesions below that found prior to treatment; however, the frequency of the latter was unchanged. There was no dose-response effect of losartan. Our results show that regression of mesangial expansion and contraction of sclerotic lesions is feasible in the treatment of diabetes, but complete resolution of advanced glomerulosclerosis may be hard to achieve.

Effect of academic psychological stress in post-graduate students: The modulatory role of cortisol on superoxide release by neutrophils

Experimental and clinical evidence shows that neutrophils play an important role in the mechanism of tissue injury in immune complex diseases through the generation of reactive oxygen species. In this study, we examined the influence of academic psychological stress in post-graduate students on the capacity of their blood neutrophils to release superoxide when stimulated by immune complexes bound to nonphagocytosable surfaces and investigated the modulatory effect of cortisol on this immune function. The tests were performed on the day before the final examination. The state-trait anxiety inventory questionnaire was used to examine whether this stressful event caused emotional distress. In our study, the psychological stress not only increased plasma cortisol concentration, but it also provoked a reduction in superoxide release by neutrophils. This decrease in superoxide release was accompanied by diminished mRNA expression for subunit p47(phox) of the phagocyte superoxide-generating nicotinamide adenine dinucleotide phosphate-oxidase. These inhibitory effects were also observed by in vitro exposure of neutrophils from control volunteers to 10(-7) M hydrocortisone, and could be prevented by the glucocorticoid receptor antagonist RU-486. These results show that in a situation of psychological stress, the increased levels of cortisol could inhibit superoxide release by neutrophils stimulated by IgG immune complexes bound to nonphagocytosable surfaces, which could attenuate the inflammatory state.

Inhibition of the renin-angiotensin system prevents seizures in a rat model of epilepsy

The RAS (renin angiotensin system) is classically involved in BP (blood pressure) regulation and water electrolyte balance, and in the central nervous system it has been mostly associated with homoeostatic processes, such as thirst, hormone secretion and thermoregulation. Epilepsies are chronic neurological disorders characterized by recurrent epileptic seizures that affect 1-3% of the world`s population, and the most commonly used anticonvulsants are described to be effective in approx. 70% of the population with this neurological alteration. Using a rat model of epilepsy, we found that components of the RAS, namely ACE (angiotensin-converting enzyme) and the AT(1) receptor (angiotensin II type I receptor) are up-regulated in the brain (2.6- and 8.2-fold respectively) following repetitive seizures. Subsequently, epileptic animals were treated with clinically used doses of enalapril, an ACE inhibitor, and losartan, an AT(1) receptor blocker, leading to a significant decrease in seizure severities. These results suggest that centrally acting drugs that target the RAS deserve further investigation as possible anticonvulsant agents and may represent an additional strategy in the management of epileptic patients.

Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins

In the kallikrein-kinin and renin-angiotensin systems the main receptors, B-1 and B-2 (kinin receptors) and AT(1) and AT(2) (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B, agonists Des-Arg(9)-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg(9)-BK or Des-Arg(10)-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT, agonist (Asp-Arg-Val-Tyr-lle-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B-1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT, receptor. To investigate this hypothesis, we replaced Arg(212) for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B, receptor by the 32 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg(212) in the TM V and a region of TM VI of rat B, receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII. (c) 2007 Elsevier B.V. All rights reserved.

Effect of NF kappa B Inhibition by CAPE on Skeletal Muscle Ischemia-Reperfusion Injury

Background/Aims. Nuclear factor kappa B (NF kappa B) plays important role in the pathogenesis of skeletal muscle ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent NF kappa B inhibitor, exhibits protective effects on I/R injury in some tissues. In this report, the effect of CAPE on skeletal muscle I/R injury in rats was studied. Methods. Wistar rats were submitted to sham operation, 120-min hindlimb ischemia, or 120-min hindlimb ischemia plus saline or CAPE treatment followed by 4-h reperfusion. Gastrocnemius muscle injury was evaluated by serum aminotransferase levels, muscle edema, tissue glutathione and malondialdehyde measurement, and scoring of histological damage. Apoptotic nuclei were determined by a terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay. Muscle neutrophil and mast cell accumulation were also assessed. Lipoperoxidation products and NF kappa B were evaluated by 4-hydroxynonenal and NF kappa B p65 immunohistochemistry, respectively. Results. Animals submitted to ischemia showed a marked increase in aminotransferases after reperfusion, but with lower levels in the CAPE group. Tissue glutathione levels declined gradually during ischemia to reperfusion, and were partially recovered with CAPE treatment. The histological damage score, muscle edema percentage, tissue malondialdehyde content, apoptosis index, and neutrophil and mast cell infiltration, as well as 4-hydroxynonenal and NF kappa B p65 labeling, were higher in animals submitted to I/R compared with the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. Conclusions. CAPE was able to protect skeletal muscle against I/R, injury in rats. This effect may be associated with the inhibition of the NF kappa B signaling pathway and decrease of the tissue inflammatory response following skeletal muscle I/R. (C) 2009 Elsevier Inc. All rights reserved.