Receptor-independent activation of Rho-kinase-mediated calcium sensitisation in smooth muscle

Abstract

1. The aim of this work was to determine whether Rho-kinase-mediated calcium sensitisation contributes to contractions of the mouse anococcygeus smooth muscle and, if so, whether the process was activated by receptor-dependent or receptor-independent mechanisms. 2. The Rho-kinase inhibitor Y27632 produced concentration-dependent decreases in tone raised by either the muscarinic receptor agonist carbachol (CCh), or the sarco-endoplasmic reticulum calcium ATPase inhibitor thapsigargin (Tg) (EC(50) values against CCh and Tg of 8.4+/-3.3 (n=6) and 6.1+/-2.1 (n=7) micro M, respectively). Pretreatment of tissues with Y27632 also inhibited contractions produced by 65 mM external potassium (69+/-7% (n=4) inhibition using 10 micro M Y27632). Y27632 had no effect on contractions produced by the inhibitor of smooth muscle myosin light-chain phosphatase, calyculin-A. 3. In beta-escin-permeabilised preparations, both CCh and Tg produced significant increases in tone over-and-above that produced by a combination of calcium (1 micro M) and GTP (100 micro M). These responses to CCh and Tg were inhibited by Y27632 (10 micro M). 4. Western blot analysis of fractionated tissue samples probed for RhoA immunoreactivity, indicated that both CCh and Tg were able to induce translocation of RhoA from the cytosol to the membrane. 5. These findings suggest that Rho-kinase-mediated calcium sensitisation is activated by both receptor-dependent and receptor-independent mechanisms in the mouse anococcygeus.

Figure 1

Diagram illustrating RhoA-mediated calcium sensitisation and sites at which Y27632 and calyculin act to interfere with the sensitisation process. Bold, solid arrows indicate stimulatory effects; bold broken arrows indicate inhibitory effects. See text for further details.

Figure 2

Effects of Y27632 on contractile responses in the mouse anococcygeus to CCh and Tg. Panel (a) shows example traces illustrating the inhibitory effect of Y27632 against CCh- and Tg-induced tone (upper and lower panels, respectively). Increasing concentrations of Y27632 (1, 2, 4, 10, 30, 100 and 200 μM) were applied cumulatively at the time points indicated by the diamond symbols. CCh and Tg were applied where indicated by the arrows. Panel (b) shows full, cumulative concentration–response curves for the inhibitory effects of Y27632 against CCh- and Tg-induced tone (n=6 and 7 tissues, respectively).

Figure 3

Y27632 is ineffective against contractions produced by the phosphatase inhibitor calyculin-A. Panel (a) shows the inhibitory effect of Y27632 (10 μM for 10 min) against contractions produced by either CCh (50 μM; n=4), potassium (65 mM; n=4) or calyculin-A (1 μM; n=3). *indicates significant difference (P<0.05) from matching control. As shown in panel (b), the differential effect of Y27632 against CCh and calyculin-A was in marked contrast to the general smooth muscle relaxant papaverine, which was equally effective at relaxing tone raised by either CCh (50 μM; n=6) or calyculin-A (1 μM; n=6).

Figure 4

Y27632 inhibits CCh and Tg induced tone in β-escin-permeabilised muscles. Experiments were performed in the presence of 1 μM free calcium. Contractile responses to GTP, CCh and Tg are expressed as a percentage of those seen in the presence of calcium alone. Application of GTP (100 μM) produced a significant increase in muscle tone. This was further increased by the addition of either CCh (50 μM; n=6) or Tg (100 nM; n=5). The increases in tension produced by both CCh and Tg were inhibited by a subsequent addition of Y27632 (10 μM). *Indicates significantly different (P<0.05) from paired contraction to GTP alone. ^#Indicates significantly different (P<0.05) from paired contraction to GTP plus CCh or Tg.

Figure 5

Both CCh (50 μM) and Tg (100 nM) cause RhoA translocation in mouse anococcygeus. The left-hand side of the figure shows representative Western blots of RhoA immunoreactivity in the cytosolic (C) and membrane (M) fractions obtained from tissues treated with either vehicle control, CCh or Tg. The histogram shows pooled data from four control tissues, four tissues treated with CCh and three treated with Tg. Data are presented as the percentage of the total RhoA immunoreactivity detected in the membrane fraction. *indicates P<0.05 and **P<0.01 when compared to control values.