Agonists such as icilin and menthol can activate the cool temperature-sensitive ion channel TRPM8. However, biological responses to menthol may occur independently of TRPM8 activation. In the rodent urinary bladder, menthol facilitates the micturition reflex but inhibits muscarinic contractions of the detrusor smooth muscle. The site(s) of TRPM8 expression in the bladder are controversial. In this study we investigated the regulation of bladder contractility in vitro by menthol. Bladder strips from wild type and TRPM8 knockout male mice (25-30 g) were dissected free and mounted in organ baths. Isometric contractions to carbachol (1 nM-30 µM), CaCl2 (1 µM to 100 mM) and electrical field stimulation (EFS; 8, 16, 32 Hz) were measured. Strips from both groups contracted similarly in response to both carbachol and EFS. Menthol (300 µM) or nifedipine (1 µM) inhibited carbachol and EFS-induced contractions in both wild type and TRPM8 knockout bladder strips. Incubation with the sodium channel blocker tetrodotoxin (1 µM), replacement of extracellular sodium with the impermeant cation N-Methyl-D-Glucamine, incubation with a cocktail of potassium channel inhibitors (100 nM charybdotoxin, 1 µM apamin, 10 µM glibenclamide and 1 µM tetraethylammonium) or removal of the urothelium did not affect the inhibitory actions of menthol. Contraction to CaCl2 was markedly inhibited by either menthol or nifedipine. In cultured bladder smooth muscle cells, menthol or nifedipine abrogated the carbachol or KCl-induced increases in [Ca2+]i. Intravesical administration of menthol increased voiding frequency while decreasing peak voiding pressure. We conclude that menthol inhibits muscarinic bladder contractions through blockade of L-type calcium channels, independently of TRPM8 activation.
Previous studies from our group have demonstrated the protective effect of S-nitroso-N-acetylcysteine (SNAC) on the cardiovascular system in dyslipidemic LDLr-/- mice that develop atheroma and left ventricular hypertrophy after 15days on a high fat diet. We have shown that SNAC treatment attenuates plaque development via the suppression of vascular oxidative stress and protects the heart from structural and functional myocardial alterations, such as heart arrhythmia, by reducing cardiomyocyte sensitivity to catecholamines. Here we investigate the ability of SNAC to modulate oxidative stress and cell survival in cardiomyocytes during remodeling and correlation with ?2-AR signaling in mediating this protection. Ventricular superoxide (O2(-)) and hydrogen peroxide (H2O2) generation was measured by HPLC methods to allow quantification of dihydroethidium (DHE) products. Ventricular histological sections were stained using terminal dUTP nick-end labeling (TUNEL) to identify nuclei with DNA degradation (apoptosis) and this was confirmed by Western blot for cleaved caspase-3 and caspase-7 protein expression. The findings show that O2(-) and H2O2 production and also cell apoptosis were increased during left ventricular hypertrophy (LVH). SNAC treatment reduced oxidative stress during on cardiac remodeling, measured by decreased H2O2 and O2(-) production (65% and 52%, respectively), and a decrease in the ratio of p-Ser1177 eNOS/total eNOS. Left ventricle (LV) from SNAC-treated mice revealed a 4-fold increase in ?2-AR expression associated with coupling change to Gi; ?2-ARs-S-nitrosation (?2-AR-SNO) increased 61%, while apoptosis decreased by 70%. These results suggest that the cardio-protective effect of SNAC treatment is primarily through its anti-oxidant role and is associated with ?2-ARs overexpression and ?2-AR-SNO via an anti-apoptotic pathway.
The aim of this study was to evaluate the expression of key genes responsible for tendon remodeling of the proximal and distal regions of calcaneal tendon (CT), intermediate and distal region of superficial flexor tendon (SFT) and proximal, intermediate and distal region of deep flexor tendon (DFT) submitted to 7 weeks of jumping water load exercise in combination with AAS administration. Wistar male rats were grouped as follows: sedentary (S), trained (jumping water load exercise) (T), sedentary animals treated with AAS (5 mg/kg, twice a week) and animals treated with AAS and trained (AAST). mRNA levels of COL1A1, COL3A1, TIMP-1, TIMP-2, MMP-2, IGF-IEa, GAPDH, CTGF and TGF-?-1 were evaluated by quantitative PCR. Our main results indicated that mRNA levels alter in different regions in each tendon of sedentary animals. The training did not alter the expression of COL1A1, COL3A, IGF-IEa and MMP-2 genes, while AAS administration or its combination with training reduced their expression. This study indicated that exercise did not alter the expression of collagen and related growth factors in different regions of rat tendon. Moreover, the pattern of gene expression was distinct in the different tendon regions of sedentary animals. Although, the RNA yield levels of CT, SFT and DFT were not distinct in each region, these regions possess not only the structural and biochemical difference, but also divergence in the expression of key genes involved in tendon adaptation.
Pre-osteoblast adhesion attracts increasing interest in both medicine and dentistry. However, how this physiological event alters osteoblast phenotype is poorly understood. We therefore attempted to address this question by investigating key biochemical mechanism that governs pre-osteoblast adhesion on polystyrene surface. Importantly, we found that cofilin activity was strongly modulated by PP2A (Ser/Thr phosphatase), while cell-cycle was arrested. Accordingly, we observed that the profile of cofilin phosphorylation (at Ser03) was similar to phospho-PP2A (at Tyr307). Also, it is plausible to suggest during pre-osteoblast adhesion that PP2A phosphorylation at Y307 was executed by phospho-Src (Y416). In addition, it was observed that MAPKp38, but not MAPK-erk, played a key role on pre-osteoblast adhesion by phosphorylating MAPKAPK-2 and ATF-2 (also called CRE-BP1). Also, the up-modulation of RhoA reported here suggests its involvement at the beginning of osteoblast attachment, while Akt remained active during all periods. Altogether, our results clearly showed that osteoblast adhesion is under an intricate network of signaling molecules, which are responsible to guide their interaction with substrate mainly via cytoskeleton rearrangement.
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