Expression of receptors for ovarian steroids and prostaglandin E2 in the endometrium and myometrium of mares during estrus, diestrus and early pregnancy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Obesity does not lead to imbalance between myocardial phospholamban phosphorylation and dephosphorylation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of central α1- and α2-adrenoceptors on the dipsogenic and cardiovascular effect of angiotensin II

We investigated the effects of previous central treatment with prazosin (an α1-adrenoceptor antagonist) or clonidine (an α2-adrenoceptor agonist) on the dipsogenic, pressor and tachycardic responses produced by intracerebroventricular (ICV) injection of angiotensin II (AII) in conscious rats. Holtzman rats with a chronic cannula implanted in the lateral ventricle were tested for dipsogenic and cardiovascular (arterial pressure and heart rate) responses in separate experiments. Previous ICV treatment with clonidine (20, 40, 80 and 120 nmol) abolished the pressor, tachycardic and dipsogenic effects of ICV AII. After all doses of prazosin (40, 80 and 120 nmol), AII induced bradycardic responses, but only the 80 and 120 nmol doses of prazosin reduced the pressor responses to AII. Prazosin produced no alteration in the dipsogenic effect of AII. The results show that the periventricular α1-adrenoceptors are involved only in the cardiovascular responses produced by central AII, whereas clonidine acting through α2-adrenergic and/or imidazole receptors can modulate all actions of AII. © 1990.

Role of alpha1 and alpha2-adrenoceptors of the lateral hypothalamic area on urinary excretion caused by centrally administered angiotensin II

To determine whether central α1 and α2-adrenergic mechanisms are involved in urinary sodium and potassium excretion and urine volume induced by angiotensin II (ANGII), these renal parameters were measured in volume-expanded Holtzman rats with cannulas implanted into lateral ventricle (LV) and lateral hypothalamus (LH). The injection of ANGII into LV in rats with volume expansion reduced the sodium, potassium and urine excretion in comparison to the control injections of isotonic saline, whereas prazosin (α1 antagonist) potentiated these effects. Clonidine (α2 agonist) and yohimbine (α2 antagonist) injected into LH previous to injection of ANGII into LV also abolished the inhibitory effect of ANGII. These results suggest that the discharge of central alpha-adrenergic receptors has dual inhibitory and excitatory effect on antinatriuretic, antikaliuretic and antidiuretic effect induced by central ANGII in volume-expanded rats. © 1995.

Role of the alfa1- and alfa2-adrenoceptors of the lateral hypothalamus in the dipsogenic response to central angiotensin II in rats

The present experiments were conducted to investigate the role of the α1- and α2-adrenergic receptors of the lateral hypothalamus (LH) on the drinking response elicited by intracerebroventricular (i.c.v) injections of carbachol and angiotensin II (AII) in rats. Clonidine (an α2-adrenergic agonist) injected into the LH produced a dose-dependent reduction of the drinking responses elicited by i.c.v. administration of carbachol and AII. The α1-adrenergic agonist phenylephrine injected into the LH reduced the dipsogenic response to i.c.v. AII, but not to carbachol. Injection of yohimbine (an α2-adrenergic antagonist) and prazosin (an α1-adrenergic antagonist) into the LH also reduced the water intake produced by i.c.v. injection of AII. Previous injection of α1- or α2-adrenergic antagonists into the LH increased the antidipsogenic effect of clonidine or phenylephrine injected into the same area on the water intake induced by i.c.v. AII. These results show that the α1- and α2-adrenergic receptors of the LH are involved in the control of drinking responses elicited by i.c.v. injection of AII in rats. They also show that clonidine, but not phenylephrine, suppresses the drinking induced by i.c.v. carbachol. The data suggest that the discharge of central α-adrenergic receptors has a dual (inhibitory and excitatory) effect on water intake induced by central AII. © 1991.

Clonidine and phenylephrine injected into the lateral preoptic area reduce water intake in dehydrated rats

In the present study, we investigated the effect of phenylephrine and clonidine (α1- and α2-adrenoceptor agonists, respectively) injected into the lateral preoptic area (LPOA) on the water intake induced by water deprivation in rats. In addition, the effects of prior injections of prazosin and yohimbine (α1- and α2-adrenoceptor antagonists, respectively) into the LPOA on the antidipsogenic action of phenylephrine and clonidine were investigated. After 30 h of water deprivation, the water intake of rats in a control experiment (saline injection) was 10.5 ± 0.8 ml/h. Injection of clonidine (5, 10, 20, and 40 nmol) into the LPOA reduced water intake to 6.3 ± 0.9, 4.9 ± 0.8, 3.6 ± 1.0, and 2.2 ± 0.7 ml/h, respectively. Similar reductions occurred after injection of 80 and 160 nmol phenylephrine into the LPOA (6.2 ± 1.6 and 4.8 ± 1.3 ml/h, respectively). Pretreatment with prazosin (40 nmol) abolished the antidipsogenic action of an 80-nmol dose of phenylephrine (11.3 ± 1.1 ml/h) and reduced the effect of a 20-nmol dose of clonidine (7.4 ± 1.4 ml/h). Yohimbine (20, 40, and 80 nmol), previously injected, produced no significant changes in the effects of either phenylephrine or clonidine. The present results show that phenylephrine and clonidine injected into the LPOA induce an antidipsogenic effect in water-deprived rat. They also suggest an involvement of α1-adrenoceptors in this effect. A possible participation of imidazole receptors in the effect of clonidine should also be taken into account. © 1993.

Role of α1-, and α2- and β-adrenoceptors of the median preoptic area on the water intake, renal excretion, and arterial pressure induced by ANG II

The present experiments were conducted to investigate the role of the α1-, α2- and β-adrenergic receptors of the median preoptic area (MnPO) on the water intake and urinary electrolyte excretion, elicited by central injections of angiotensin II (ANG II). Prazosin (an α1-adrenergic receptor antagonist) and yohimbine (an α2-adrenergic receptor antagonist) antagonized the water ingestion, Na +, K +, and urine excretion induced by ANG II. Administration of propranolol, a β-adrenergic receptor antagonist increased the Na +, K +, and urine excretion induced by ANG II. Previous treatment with prazosin and yohimbine reduced the pressor responses to ANG II. These results suggest that the adrenergic neurotransmission in the MnPO may actively participate in ANG II-induced dipsogenesis, natriuresis, kaliuresis, diuresis and pressor responses in a process that involves α1-, α2-, and β-adrenoceptors.

Antifibrotic Effect of Tamoxifen in a Model of Progressive Renal Disease

Tamoxifen, a selective estrogen receptor modulator, has antifibrotic properties; however, whether it can attenuate renal fibrosis is unknown. In this study, we tested the effects of tamoxifen in a model of hypertensive nephrosclerosis (chronic inhibition of nitric oxide synthesis with L-NAME). After 30 days, treated rats had significantly lower levels of albuminuria as well as lower histologic scores for glomerulosclerosis and interstitial fibrosis than untreated controls. Tamoxifen was renoprotective despite having no effect on the sustained, severe hypertension induced by L-NAME. Tamoxifen prevented the accumulation of extracellular matrix by decreasing the expression of collagen I, collagen III, and fibronectin mRNA and protein. These renoprotective effects associated with inhibition of TGF-beta 1 and plasminogen activator inhibitor-1, and with a significant reduction in a-smooth muscle actin-positive cells in the renal interstitium. Furthermore, tamoxifen abrogated IL-1 beta- and angiotensin-II-induced proliferation of fibroblasts from both kidney explants and from the NRK-49F cell line. Tamoxifen also inhibited the expression of extracellular matrix components and the production and release of TGF-beta 1 into the supernatant of these cells. In summary, tamoxifen exhibits antifibrotic effects in the L-NAME model of hypertensive nephrosclerosis, likely through the inhibition of TGF-beta 1, suggesting that it may have therapeutic use in CKD treatment.

Endophenotypes and serotonergic polymorphisms associated with treatment response in obsessive-compulsive disorder

OBJECTIVES: Approximately 40-60% of obsessive-compulsive disorder patients are nonresponsive to serotonin reuptake inhibitors. Genetic markers associated with treatment response remain largely unknown. We aimed (1) to investigate a possible association of serotonergic polymorphisms in obsessive-compulsive disorder patients and therapeutic response to selective serotonin reuptake inhibitors and (2) to examine the relationship between these polymorphisms and endocrine response to intravenous citalopram challenge in responders and non-responders to serotonin reuptake inhibitors and in healthy volunteers. METHODS: Patients with obsessive-compulsive disorder were classified as either responders or non-responders after long-term treatment with serotonin reuptake inhibitors, and both groups were compared with a control group of healthy volunteers. The investigated genetic markers were the G861C polymorphism of the serotonin receptor 1D beta gene and the T102C and C516T polymorphisms of the serotonin receptor subtype 2A gene. RESULTS: The T allele of the serotonin receptor subtype 2A T102C polymorphism was more frequent among obsessive-compulsive disorder patients (responders and non-responders) than in the controls (p<0.01). The CC genotype of the serotonin receptor subtype 2A C516T polymorphism was more frequent among the non-responders than in the responders (p<0.01). The CC genotype of the serotonin receptor subtype 1D beta G681C polymorphism was associated with higher cortisol and prolactin responses to citalopram (p<0.01 and p<0.001, respectively) and with a higher platelet-rich plasma serotonin concentration among the controls (p<0.05). However, this pattern was not observed in the non-responders with the same CC genotype after chronic treatment with serotonin reuptake inhibitors. This CC homozygosity was not observed in the responders.

Cerebral amyloid angiopathy impact on endothelium

Cerebral amyloid angiopathy (CAA) is an age-associated disease characterized by amyloid deposition in cerebral and meningeal vessel walls. CAA is detected in the majority of the individuals with dementia and also in a large number of non-demented elderly individuals. In addition, CAA is strongly associated with Alzheimer's disease (AD) pathology. Mechanical consequences including intra-cerebral or subarachnoid hemorrhage remains CAA most feared complication, but only a small fraction of CAA results in severe bleeding. On the hand the non-mechanical consequences in cerebrovascular regulation are prevalent and may be even more deleterious. Studies of animal models have provided strong evidence linking the vasoactive A beta 1-40, the main species found in CAA, to disturbances in endothelial-dependent factors, disrupting cerebrovascular regulation Here, we aimed to review experimental findings regarding the non-mechanical consequences of CAA for cerebrovascular regulation and discuss the implications of these results to clinical practice. (C) 2012 Elsevier Inc. All rights reserved.

Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells

Objective: The aim of this study was to investigate the effects of PRP on SAOS-2 cells in terms of cytokine expression, cell activity and oxidative stress. Design: Cell line SAOS-2 (1 x 10(5) cells/mL) were grown in culture medium alpha-MEM with 10% FBS for 24 h and stimulated (or not) with PRP at concentrations of 3, 10 and 20%, LPS (E. coli, 10 g/mL) and IL-1 beta (1 mg/mL) for 24 h. The supernatant was collected and analyzed for the expression of cytokines in a panel array, ALP using a commercial kit and NO2- with Griess reaction method. Also, the cells were analyzed using Western blot for RANKL and slot blotting for nitrotyrosine expression. Result: There were no significant differences amongst the groups in terms of NO2-, protein nitrotyrosine content and RANKL expression. However, all stimuli increased ALP activity and in case of PRP, it was in a dose-dependent manner (p < 0.001). Also, all stimuli induced an increase in cytokines and chemokines expression, but only PRP promoted an increase of component C5, sICAM-1 and RANTES expression. Whilst IL-1 receptor antagonist (IL-1ra) expression was down-regulated by PRP, both LPS and IL-1 beta caused up-regulation of this cytokine. Conclusions: PRP can stimulate osteoblast activity and cytokine/chemokine release, as well as indicate some of the mediators that can (and cannot) be involved in this activation. (C) 2012 Elsevier Ltd. All rights reserved.

Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-alpha, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this beta-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen.

Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

Background: Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance. Methods: Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. Results: GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose response study. The highest tested concentration of NE (10 (-7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (-8) M), a non-selective beta-adrenergic antagonist. Conclusions: The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

Activation of central α(2)-adrenoceptors mediates salivary gland vasoconstriction

OBJECTIVE: Peripheral treatment with the cholinergic agonist pilocarpine increases salivary gland blood flow and induces intense salivation that is reduced by the central injection of moxonidine (α(2)-adrenoceptors/imidazoline agonist). In the present study, we investigated the effects of the intracerebroventricular (i.c.v.) injection of pilocarpine alone or combined with moxonidine also injected i.c.v. On submandibular/sublingual gland (SSG) vascular resistance. In addition, the effects of these treatments on arterial pressure, heart rate and on mesenteric and hindlimb vascular resistance were also tested. DESIGN: Male Holtzman rats with stainless steel cannula implanted into lateral ventricle and anaesthetized with urethane+α-chloralose were used. RESULTS: Pilocarpine (500nmol/1μl) injected i.c.v. Reduced SSG vascular resistance and increased arterial pressure, heart rate and mesenteric vascular resistance. Contrary to pilocarpine alone, the combination of moxonidine (20nmol/1μl) and pilocarpine injected i.c.v. Increased SSG vascular resistance, an effect abolished by the pre-treatment with the α(2)-adrenoceptor antagonist yohimbine (320nmol/2μl). The increase in arterial pressure, heart rate and mesenteric resistance was not modified by the combination of moxonidine and pilocarpine i.c.v. CONCLUSION: These results suggest that the activation of central α(2)-adrenoceptors may oppose to the effects of central cholinergic receptor activation in the SSG vascular resistance.

Use of bioassays and biomarkers in Daphnia magna to assess the effect of pharmaceutical residuals in freshwater ecosystems

Widespread occurrence of pharmaceuticals residues has been reported in aquatic ecosystems. However, their toxic effects on aquatic biota remain unclear. Generally, the acute toxicity has been assessed in laboratory experiments, while chronic toxicity studies have rarely been performed. Of importance appears also the assessment of mixture effects, since pharmaceuticals never occur in waters alone. The aim of the present work is to evaluate acute and chronic toxic response in the crustacean Daphnia magna exposed to single pharmaceuticals and mixtures. We tested fluoxetine, a SSRI widely prescribed as antidepressant, and propranolol, a non selective β-adrenergic receptor-blocking agent used to treat hypertension. Acute immobilization and chronic reproduction tests were performed according to OECD guidelines 202 and 211, respectively. Single chemicals were first tested separately. Toxicity of binary mixtures was then assessed using a fixed ratio experimental design with concentrations based on Toxic Units. The conceptual model of Concentration Addition was adopted in this study, as we assumed that the mixture effect mirrors the sum of the single substances for compounds having similar mode of action. The MixTox statistical method was applied to analyze the experimental results. Results showed a significant deviation from CA model that indicated antagonism between chemicals in both the acute and the chronic mixture tests. The study was integrated assessing the effects of fluoxetine on a battery of biomarkers. We wanted to evaluate the organism biological vulnerability caused by low concentrations of pharmaceutical occurring in the aquatic environment. We assessed the acetylcholinesterase and glutathione s-transferase enzymatic activities and the malondialdehyde production. No treatment induced significant alteration of biomarkers with respect to the control. Biological assays and the MixTox model application proved to be useful tools for pharmaceutical risk assessment. Although promising, the application of biomarkers in Daphnia magna needs further elucidation.