Mitochondrial involvement in carbachol-induced intracellular Ca2+ mobilization and contraction in rat gastric smooth muscle

Aims: Mitochondria are important modulators of Ca2+ homeostasis. However, it is not clear if they modulate and participate in smooth muscle signaling and contraction. the aim of the present work was to investigate the role of mitochondria in Ca2+ transients and contraction induced by metabotropic muscarinic receptor activation in rat gastric smooth muscle.Main methods: Carbachol (CC11)-induced contraction was investigated in the absence or presence of increasing concentration of mitochondrial protonophore, carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone (FCCP), in gastric fundus strips. Ca2+ and mitochondrial membrane potential (Delta Psi m) measurements were performed in primarily cultured gastric smooth muscle cells loaded with FURA-2 or TMRE dyes.Key findings: Results show that CCh (1 mu M)-induced contraction was inhibited by FCCP in a concentration-dependent manner. in cultured smooth muscle cells CCh (1 mu M) caused a cytosolic Ca2+ rise. Preincubation with FCCP strongly inhibited CCh-evoked Ca2+ transients indicating that mitochondria shape intracellular Ca2+ Signals. CCh induced elevations of Delta Psi m in 60% of the individual mitochondrion analyzed.Significance: Taken together our results indicate that CCh induces release of Ca2+ from intracellular stores, which may be modulated by mitochondria. Thus, mitochondria participate of the intracellular Ca2+ homeostasis in muscarinic contraction in gastric fundus smooth muscle. (C) 2011 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fed Univ São Paulo UNIFESP EPM, Dept Pharmacol, Paulista Sch Med, São Paulo, BrazilFed Univ Pernambuco UFPE, Dept Pharmaceut Sci, Recife, PE, BrazilFed Univ São Paulo UNIFESP EPM, Dept Pharmacol, Paulista Sch Med, São Paulo, BrazilWeb of Scienc

Papel modulador da inervacao autonomica e do hormonio sexual masculino sobre os canais de calcio voltagem-dependentes e sua importancia no processo contratil em ducto deferente de rato

BV UNIFESP: Teses e dissertaçõe

Evidence for the participation of calcium in non-genomic relaxations induced by androgenic steroids in rat vas deferens

Background and purpose: Androgens cause non-genomic relaxation in several smooth muscle preparations. However, such an effect has not been investigated in rat vas deferens yet. Our purpose was to study the effect of testosterone and derivatives in this tissue.Experimental approach: the influence of androgens was tested on contraction and translocation of intracellular Ca2+ induced by KCl in rat vas deferens in vitro.Key results: the testosterone derivative 5 alpha-dihydrotestosterone produced a rapid and reversible concentration-dependent relaxation of KCl-induced contractions. Other androgens were also effective, showing the following rank order of potency: androsterone >5 beta-dihydrotestosterone >androstenedione >5 alpha-dihydrotestosterone >testosterone. Calcium-induced contractions were also inhibited (about 45%) by 5 alpha-dihydrotestosterone (30 mu M). Moreover 5 alpha-dihydrotestosterone blocked the increase of intracellular Ca2+ induced by KCl, measured by the fluorescent dye fura-2. Relaxation to 5 alpha-dihydrotestosterone was resistant to the K+ channel antagonists glibenclamide, 4-aminopyridine and charybdotoxin. It was not affected by removal of epithelium or by L-NNA (300 mM), an inhibitor of nitric oxide biosynthesis, nor by selective inhibitors of soluble guanylate cyclase, ODQ or LY 83583, indicating that nitrergic or cGMP mediated mechanisms were not involved. the androgen-induced relaxation was also not blocked by the protein synthesis inhibitor cycloheximide (300 mu M) or by the classical androgen receptor flutamide (up to 100 mu M), corroborating that the effect is non-genomic.Conclusions and implications: Testosterone derivatives caused relaxation of the rat vas deferens, that did not involve epithelial tissue, K+ channels, or nitric oxide-dependent mechanisms, but was related to a partial blockade of Ca2+ influx.Universidade Federal de São Paulo, Dept Pharmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, BR-04044020 São Paulo, BrazilWeb of Scienc

Role of noradrenaline on the expression of the Na+/K+-ATPase alpha(2) isoform and the contractility of cultured rat vas deferens

Rat vasa deferentia were cultured for 3 days in Dulbecco's modified Eagle's medium in the absence or presence of 1 muM noradrenaline (NA) to investigate if the lack of NA release is the key factor to explain the selective reduction of the Na+/K+-ATPase alpha(2) isoform previously observed after in vivo denervation of this organ (Quintas et al., Biochem Pharmacol 2000;60:741-7). the lack of effects of the indirect sympathomimetic tyramine and the neuronal amine uptake blocker cocaine on NA curves indicated that cultured organs were denervated completely. Organ culture induced supersensitivity, expressed as a 6.3-fold increase of pD(2) and a 42% elevation of maximal contraction for NA but not for Ba2+. Western blotting indicated that the level of the alpha(1) isoform of Na+/K+-ATPase was unchanged after organ culture, but the alpha(2) isoform was down-regulated drastically to levels that were barely detectable. the addition of NA to the culture medium did not prevent the reduction of alpha(2) expression although it did impede NA supersensitivity (in fact a 4-fold decrease of pD(2) and a 32% reduction of maximal response were observed after incubation in the presence of NA). A striking reduction of L-type Ca2+ channel expression also was observed, indicated by an 85% decrease of [H-3]isradipine binding sites. These data suggest that NA is a trophic factor relevant to the control of muscle contraction, mediated by alpha(1)-adrenoceptors,but not to the expression of either Na+/K+-ATPase or the L-type Ca2+ channel. (C) 2002 Elsevier Science Inc. All rights reserved.Fed Univ Rio de Janeiro, Inst Ciencias Biomed, Dept Farmacol Basica & Clin, BR-21941590 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Farmacol, BR-04034970 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Farmacol, BR-04034970 São Paulo, BrazilWeb of Scienc

Time-dependent up-regulation of Ca(2+) channels in vas deferens of newborn rats fed with breast milk of mothers under treatment with nifedipine

Our aim was to check for calcium channel maturation and regulation on newborn rats during breastfeeding by mothers treated with the L-type calcium channel blocker nifedipine. Contractions by KCl and radioligand binding techniques were used to verify if Call Channels are modified in rat vas deferens of 40-day old litters that were breastfed by mothers injected daily with nifedipine during nursery. Injections were applied in the beginning (1st until 8th day), middle (9th until 16th day), or end (17th until 24th day) of nursery, to verify the period of highest susceptibility of newborn to nifedipine receptor regulation. Contractile responses revealed that only after the middle period of treatment of mothers the maximal effects (E(max)) induced in pups by KCl were increased by about 35%, without changes of apparent affinity (pD(2)). Additionally, binding studies with [(3)H] Isradipine in cell membrane preparations showed a greater density (B(max)) of Ca(2+) channels by about 55%, without changes of affinity (K(d)). Changes were not detected after treatment of mothers in the beginning or end of breastfeeding. in addition, in vas deferens of 60-day old litters, the E(max) returned to control values, showing that changes were not persistent. Moreover, body and vas deferens weights and blood testosterone of newborn were never changed. the histology of mammary gland was similar for treated and control mothers, suggesting a stable milk production. It is concluded that nifedipine treatment of mothers, if made during the 9th to 16th day of lactation, produced a short lasting reversible up-regulation of L-type Ca(2+) channels. (C) 2008 Published by Elsevier B.V.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Pharmacol, BR-04034970 São Paulo, SP, BrazilUniv Fed Pernambuco, Dept Pharmacol, Recife, PE, BrazilUniversidade Federal de São Paulo, Dept Med, BR-04034970 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, BR-04034970 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Med, BR-04034970 São Paulo, SP, BrazilWeb of Scienc

Adaptive expression pattern of different proteins involved in cellular calcium homeostasis in denervated rat vas deferens

The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. the function of thapsigargin-sensitive but not thapsigargin-resistant (Ca2+-Mg2+) ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca2+ uptake experiments, was significantly depressed by 30-50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca2+-Mg2+) ATPase type 2 protein expression, without any significant change in plasma membrane (Ca2+-Mg2+)ATPase expression. Finally, [3 H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. the present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens. (c) 2005 Elsevier B.V All rights reserved.Univ Fed Rio de Janeiro, Inst Ciencias Biomed, Dept Farmacol Basica & Clin, BR-21941590 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Farmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Farmacol, BR-04044020 São Paulo, BrazilWeb of Scienc

Gastroprotective Mechanisms of the Monoterpene 1,8-Cineole (Eucalyptol).

Recently, our research group identified and reported 1,8-cineole (CIN), a monoterpene that naturally occur in many aromatic plants, as one of the major constituent of the essential oil from leaves of Hyptis martiusii (EOHM), as well as characterized the gastroprotective action of this oil. The aim of this study was to investigate the mechanisms of action involved in the antiulcer and healing activity of CIN, in order to confirm its correlation with the gastroprotective effect of EOHM. Wistar rats were exposed to different protocols (acute ulceration, gastrointestinal motility and antisecretory activity). In addition, were determinated the involvement of nitric oxide and sulphydryl groups; the levels of gastric mucus, lipid peroxidation, sulphydryl groups and myeloperoxidase activity. The healing ability was evaluated by acetic acid-induced chronic ulcer and histological and immunohistochemical analysis (PCNA, Ki-67 and BrdU). The treatment with CIN inhibited ethanol-, ethanol/HCl- and indomethacin-induced gastric lesions. The highest doses of CIN inhibited gastric emptying, but did not affect intestinal transit. CIN (100 mg/kg) reduced the volume of basal but not stimulated acid secretion. CIN increased levels of mucus (89.3%), prevented depletion of -SH groups (62.6%) and reduced the level of lipid peroxidation (55.3%) and myeloperoxidase activity (59.4%) in the gastric mucosa. In chronic ulcer model, CIN reduced in 43.1% the gastric area lesion, promoted significant regeneration and restoration of the levels of mucus in glandular cells as confirmed by histological analysis; and promoted increase in cell proliferation as evidenced by reactivity for PCNA, Ki-67 and BrdU. This findings demonstrate the role of 1,8-cineole as an important ulcer healing agent and indicate the involvement of antioxidant and cytoprotective mechanisms in the gastroprotective effect of compound. This study also provides evidence that 1,8-cineole is related to the gastroprotective effect of the essential oil of Hyptis martiusii

Effect of intraduodenal administration of 1,8-cineole (CIN) on gastric secretion parameters basal or stimulated by histamine (20 mg/kg), bethanechol (2.5 mg/kg) and pentagastrin (400 μg/kg) in Wistar rats subjected to pylorus ligature.

<p>Values are expressed as mean ± S.E.M. for 6 animals. Treatment: control (1% Tween-80 aqueous solution, 0.1 mL/100 g, i.d), CIN (100 mg/kg, i.d.), ranitidine (60 mg/kg, i.d.) and atropine (1 mg/kg, s.c).</p><p>*p < 0.05 vs. control group</p><p>^#p < 0.05 vs. histamine group and</p><p>^†p < 0.05 vs. bethanechol group (ANOVA followed by Tukey’s test).</p><p>Effect of intraduodenal administration of 1,8-cineole (CIN) on gastric secretion parameters basal or stimulated by histamine (20 mg/kg), bethanechol (2.5 mg/kg) and pentagastrin (400 μg/kg) in Wistar rats subjected to pylorus ligature.</p

Effect of 1,8-cineole (CIN) on gastrointestinal motility in Wistar rats.

<p>The values indicate the concentration of phenol red retained in the stomach (gastric emptying) or the percentage of the distance traveled by the marker in relation to the total length of the small intestine (intestinal transit) 30 min after ingestion of the dye. The results represent the mean ± S.E.M. for 6 animals.</p><p>*Statistically different from control group, p < 0.05 (ANOVA followed by Tukey’s test).</p><p>Effect of 1,8-cineole (CIN) on gastrointestinal motility in Wistar rats.</p

Effect of oral administration of 1,8-cineole (CIN) on gastric lesions induced by ethanol in Wistar rats pretreated with L-NAME (N_ω-nitro-L-arginine methyl ester, 70 mg/kg) or NEM (N-ethylmaleimide, 10 mg/kg).

<p>Results are expressed as mean ± S.E.M. for 6 animals.</p><p>*p < 0.05 compared to NaCl solution + control</p><p>^#p < 0.05 compared to L-NAME + control (ANOVA followed by Tukey's test).</p><p>Effect of oral administration of 1,8-cineole (CIN) on gastric lesions induced by ethanol in Wistar rats pretreated with L-NAME (N_ω-nitro-L-arginine methyl ester, 70 mg/kg) or NEM (N-ethylmaleimide, 10 mg/kg).</p