Aging impairs Ca2+ sensitization pathways in gallbladder smooth muscle.
ABSTRACT Calcium sensitization is an important physiological process in agonist-induced contraction of smooth muscle. In brief, calcium sensitization is a pathway that leads to smooth muscle contraction independently of changes in [Ca(2+)](i) by mean of inhibition of myosin light chain phosphatase. Aging has negative impacts on gallbladder contractile response due to partial impairment in calcium signaling and alterations in the contractile machinery. However, information regarding aging-induced alterations in calcium sensitization is scanty. We hypothesized that the calcium sensitization system is negatively affected by age. To investigate this, gallbladders were collected from adult (4 months old) and aged (22-24 months old) guinea pigs. To evaluate the contribution of calcium sensitization pathways we assayed the effect of the specific inhibitors Y-27632 and GF109203X on the "in vitro" isometric gallbladder contractions induced by agonist challenges. In addition, expression and phosphorylation (as activation index) of proteins participating in the calcium sensitization pathways were quantified by Western blotting. Aging reduced bethanechol- and cholecystokinin-evoked contractions, an effect associated with a reduction in MLC20 phosphorylation and in the effects of both Y-27632 and GF109203X. In addition, there was a drop in ROCK I, ROCK II, MYPT-1 and PKC expression and in the activation/phosphorylation of MYPT-1, PKC and CPI-17 in response to agonists. Interestingly, melatonin treatment for 4 weeks restored gallbladder contractile responses due to re-establishment of calcium sensitization pathways. These results demonstrate that age-related gallbladder hypocontractility is associated to alterations of calcium sensitization pathways and that melatonin treatment exerts beneficial effects in the recovery of gallbladder contractility.
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ABSTRACT: Background & Aims: Internal anal sphincter (IAS) tone plays an important role in rectoanal incontinence (RI). The IAS tone may be compromised during aging leading to RI in certain patients. Herein, we examined the influence of oxidative stress in the aging-associated decrease in the IAS tone (AADI). Methods: Using two different age groups of adult (4- 6 months (Ad)) vs. aging (24 to 30 months (Ag)) rats, we determined the effect of oxidative stress on IAS tone, and the regulatory RhoA/ROCK signal transduction cascade. We determined the effect of an oxidative stress inducer LY83583 (that produces superoxide anions or O2(-)), on the basal and stimulated IAS tone, before and after O2(-) scavenger superoxide dismutase (SOD) using intact smooth muscle (SM) strips and SMCs. Results: Data showed that AADI was associated with decrease in RhoA/ROCK expression at the transcriptional and translational levels. Oxidative stress with LY83583-mediated decrease in the IAS tone and relaxation of the IAS SMCs were associated with the decrease in RhoA/ROCK signal transduction, reversible by SOD. In addition, LY83583 caused a significant decrease in the IAS contraction produced by the RhoA activator, and a known RhoA/ROCK agonist U46619, also reversible by SOD. The inhibitory effects of LY83583 and ROCK inhibitor Y27632 on U46619-induced increase in the IAS tone were similar. Conclusion: An increase in oxidative stress plays an important role in the AADI in the elderly, and may be one of the underlying mechanisms for the RI in certain aging patients.AJP Gastrointestinal and Liver Physiology 04/2014; 306(11). DOI:10.1152/ajpgi.00087.2014 · 3.74 Impact Factor
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ABSTRACT: BACKGROUND; Decreased gallbladder smooth muscle (GBSM) contractility is a hallmark of cholesterol gallstone disease, but the interrelationship between lithogenicity, biliary stasis, and inflammation are poorly understood. We studied a mouse model of gallstone disease to evaluate the development of GBSM dysfunction relative to changes in bile composition and the onset of sterile cholecystitis. BALB/cJ mice were fed a lithogenic diet for up to 8 weeks, and tension generated by gallbladder muscle strips was measured. Smooth muscle Ca(2+) transients were imaged in intact gallbladder. Key Lipid composition of bile was altered lithogenically as early as 1 week, with increased hydrophobicity and cholesterol saturation indexes; however, inflammation was not detectable until the fourth week. Agonist-induced contractility was reduced from weeks 2 through 8. GBSM normally exhibits rhythmic synchronized Ca(2+) flashes, and their frequency is increased by carbachol (3 μm). After 1 week, lithogenic diet-fed mice exhibited disrupted Ca(2+) flash activity, manifesting as clustered flashes, asynchronous flashes, or prolonged quiescent periods. These changes could lead to a depletion of intracellular Ca(2+) stores, which are required for agonist-induced contraction, and diminished basal tone of the organ. Responsiveness of Ca(2+) transients to carbachol was reduced in mice on the lithogenic diet, particularly after 4-8 weeks, concomitant with appearance of mucosal inflammatory changes. These observations demonstrate that GBSM dysfunction is an early event in the progression of cholesterol gallstone disease and that it precedes mucosal inflammation.Neurogastroenterology and Motility 05/2012; 24(7):e313-24. DOI:10.1111/j.1365-2982.2012.01935.x · 3.42 Impact Factor