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.

Fig. 1

Aging reduces guinea pig gallbladder myogenic response. Strips were exposed to increasing concentrations of a bethanechol (BE, 10 nM to 10 mM) and b cholecystokinin (CCK, 10 pM to 1 μM). Note a significant reduction in the contractile response in gallbladders from aged animals. In (a) and (b), contractile responses are expressed as mean ± SEM. n = 19–27 strips (***P < 0.0001, adult vs. aged). c Original Western blots on gallbladder smooth muscle from adult (Ad) and aged (Ag) guinea pigs for phospho-MLC20 and total MLC20 in unstimulated (control) or agonist-treated gallbladder samples. After normalization to actin content, the optic density of phosphorylated protein vs. total protein was calculated for control and treated samples in both age groups. Then, the resulting ratio for treated samples was expressed as fold increase respect to unstimulated conditions. Note that protein phosphorylations were decreased in the aged group The histograms (mean ± SEM) show summarized data of changes in MLC20 phosphorylation for 8–10 blots (*P < 0.05, adult vs. aged)

Fig. 2

Aging reduces the sensitivity of bethanechol-induced gallbladder contractions to Y-27632 and GF109203X, inhibitors of ROCK and PKC, respectively. Effects of 5 μM Y-27632 (a and b) and 5 μM GF109203X (c and d) on bethanechol (BE) evoked contractions in gallbladder strips from adult and aged guinea pigs. Note that the effects of the inhibitors are lower in aged than adult animals indicative of a lower participation of calcium sensitization pathways. Data are expressed as mean ± SEM. n = 10–15 strips (**P < 0.01 and ***P < 0.001, adult vs. aged)

Fig. 3

Aging reduces the sensitivity of CCK-induced contractile response to Y-27632 and GF109203X, inhibitors of ROCK and PKC, respectively. Effects of 5 μM Y-27632 (a and b) and 5 μM GF109203X (c and d) on CCK-evoked contractions in gallbladder strips from adult and aged guinea pigs. Note that the effects of the inhibitors are smaller in aged than adult animals indicative of a lower participation of calcium sensitization pathways. Data are expressed as mean ± SEM. n = 6–9 strips (**P < 0.01 and ***P < 0.001, adult vs. aged)

Fig. 4

Aging decreases the expression of proteins involved in Rho A/ROCK pathway. Original Western blots on gallbladder smooth muscle from adult (Ad) and aged (Ag) guinea pigs for a Rho A, b ROCK I, c ROCK II and d MYPT-1. There are not differences in the expression of Rho A while aging reduces the expression of ROCK I, ROCK II and MYPT-1. Actin was used as loading control and proteins expression was calculated respect to actin expression. Histograms summarize proteins expression (mean ± SEM) for 6–8 blots (*P < 0.05 and ** P < 0.01, adult vs. aged)

Fig. 5

Aging decreases the expression of proteins involved in PKC/CPI-17 pathway. Original Western blots on gallbladder smooth muscle from adult (Ad) and aged (Ag) guinea pigs for a phospho-PKC and b CPI-17. Note that aging reduces the expression of phospho-PKC. Actin was used as loading control and proteins expression was calculated respect to actin expression. Histograms summarize protein expression (mean ± SEM) for 6–8 blots (** P < 0.01, adult vs. aged)

Fig. 6

Aging impairs activation of Ca²⁺ sensitization pathways. a Western blots using anti-p-Thr853-MYPT-1 and anti-MYPT-1 in unstimulated (control) or CCK-treated gallbladder samples from adult (Ad) and aged (Ag) guinea pigs. c Changes in phospho-PKC expression in response to CCK for both groups of animals. e Representative Western blots using anti-p-Thr38-CPI-17 and anti-CPI-17 in unstimulated (control) or CCK-treated tissues. The histograms show summarized data of changes in b MYPT-1, d PKC and f CPI-17 phosphorylation. After normalization to actin content, the optic density of phosphorylated protein vs. total protein was calculated for control and treated samples in both age groups. Then, the resulting ratio for treated samples was expressed as fold increase respect to unstimulated conditions. Note that protein phosphorylations were decreased in the aged group for all the proteins studied and for both challenges. n = 6–8 experiments. (*P < 0.05, ** P < 0.01, adult vs. aged)

Fig. 7

Melatonin normalizes contractile responses and calcium sensitization contribution to contraction in aged gallbladders. Histograms represent maximal contractile response to a bethanechol and b cholecystokinin in aged animals (gray bars) and aged animals treated with melatonin (2.5 mg kg⁻¹ day⁻¹) for 28 days (empty bars). Gallbladder contractility and the inhibitory effects of Y-27632 and GF109203X were increased after melatonin treatment. Data are expressed as percentage of adult values. n = 8–15 strips (*P < 0.05 and **P < 0.01, aged vs. aged treated with melatonin)

Fig. 8

Melatonin restores the expression and activation of proteins involved in calcium sensitization pathways in aged gallbladders. Histograms represent protein expression of proteins involved in the a Rho A/ROCK and b PKC/CPI-17 calcium sensitization pathways in aged animals (gray bars) and aged animals treated with melatonin (empty bars). c BE-induced and d CCK-induced phosphorylation of MYPT-1 (left), PKC (middle) and CPI-17 (right) in aged animals (gray bars) and aged animals treated with melatonin (empty bars). Note the significant increase in protein expression and activation after melatonin treatment. Data are expressed as percentage of adult values. n = 6–8 blots (*P < 0.05 and **P < 0.01, aged vs. aged treated with melatonin)

Fig. 9

Aging decreases the expression of COX-2. Original Western blots on gallbladder smooth muscle from adult (Ad), aged (Ag) and aged animals treated with melatonin (Mel) guinea pigs for COX-2. Note that aging increases the expression of COX-2 and that melatonin normalizes this expression pattern. Actin was used as loading control and protein expression was calculated respect to actin expression. Histograms summarize protein expression (mean ± SEM) for 10–12 blots (* P < 0.05, aged vs. adult and aged treated with melatonin)