Expression and functional role of Rho-kinase in rat urinary bladder smooth muscle

Abstract

(1) The involvement of Rho-kinase (ROCK) in the contractile mechanisms mediating smooth muscle contraction of the rat urinary bladder was investigated using expression studies and the ROCK inhibitor Y-27632. (2) Both isoforms of ROCK (ROCK I and ROCK II) were detected in high levels in rat urinary bladder. (3) Y-27632 (10 micro M) significantly attenuated contractions of rat urinary bladder strips evoked by the G-protein coupled receptor agonists carbachol (58.1+/-10.5% at 0.3 micro M) and neurokinin A (68.6+/-12.7% at 1 micro M) without affecting contractions to potassium chloride (10-100 mM). In addition, basal tone was reduced by 47.8+/-2.0% by 10 micro M Y-27632 in the absence of stimulation. (4) Contractions of urinary bladder strips evoked by the P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-mATP; 10 micro M) were also attenuated by Y-27632 (30.0+/-7.2% at 10 micro M). (5) Y-27632 (10 micro M) significantly attenuated contractions evoked by electrical field stimulation (2-16 Hz). The effect of Y-27632 on the tonic portion of the neurogenic response (4-16 Hz) was not significantly different from the effect of atropine (1 micro M) alone. (6) While the mechanism underlying the ability of Y-27632 to inhibit alpha,beta-mATP-evoked contractions remains undetermined, the results of the present study clearly demonstrate a role for ROCK in the regulation of rat urinary bladder smooth muscle contraction and tone.

Figure 1

Expression of ROCK I and ROCK II in rat tissues from Western blotting experiments. Tissue homogenates were subjected to SDS–PAGE and immunoblotted with anti-ROCK I, II or GADPH. Each lane represents results from different rat tissues: heart (H), bladder (Bl), brain (Br), aorta (A), liver (Li), kidney (K) and skeletal muscle (diaphragm; M). Results are representative of tissue samples from five animals.

Figure 2

Expression of ROCK I and ROCK II in rat tissues from RT–PCR experiments. ROCK I or ROCK II specific message was normalized to GAPDH specific message and the relative amount of mRNA of each sample was compared to that of heart while arbitrarily setting heart mRNA level as 1.0 unit. Each bar represents the mean value of four tissue samples from separate animals and vertical bars show s.e.mean.

Figure 3

Effects of 10 μM Y-27632, 10 μM HA-1077 and 10 mM GF 109203X on baseline bladder tension in rat urinary bladder strips. Data is expressed as a percentage of pre-treatment tension. Each point represents the mean value and vertical bars show s.e.mean of five experiments. ^*P<0.05 compared to vehicle.

Figure 4

Effect of 10 μM Y-27632 on carbachol-evoked contractions of rat urinary bladder strips. Typical traces are shown in (A), while summary data (n=4–6 for each point) is shown in (B). Responses are expressed as a percentage of the response evoked by 100 m MCl. Vertical bars indicate s.e.mean. ^*P<0.05 compared to vehicle.

Figure 5

Effect of 10 μM Y-27632 on NKA-evoked contractions of rat urinary bladder strips. Responses are expressed as a percentage of the response evoked by 100 mM KCl (n=4–5 for each point). Vertical bars indicate s.e.mean. ^*P<0.05 compared to vehicle.

Figure 6

Effect of 10 μM Y-27632 on KCl-evoked contractions of rat urinary bladder strips. Responses are expressed as a percentage of the response evoked by 100 mM KCl (n=5 for each point). Vertical bars indicate s.e.mean.

Figure 7

Effects of Y-27632 and HA-1077 on α,β-mATP evoked contractions of rat urinary bladder strips. (A) Typical traces of responses to 10 μM α,β-mATP in the absence and presence of 10 μM Y-27632 or 10 μM HA-1077. Shaded circles indicate addition of α,β-mATP to the tissue strips. Summary data (n=4–6 for each) is displayed in (B). Vertical bars indicate s.e.mean. ^*P<0.05 compared to vehicle.

Figure 8

Effects of 10 μM Y-27632, 10 μM HA-1077 and 10 μM GF 109203X on PDBu-evoked contractions of rat urinary bladder strips. Responses are expressed as a percentage of the response evoked by 100 mM KCl (n=4–6 for each point). Vertical bars indicate s.e.mean. ^*P<0.05 compared to vehicle.

Figure 9

Effects of 10 μM Y-27632, 1 μM TTX, 10 μM atropine, desensitization of P2X receptors with 10 μM α,β-mATP and 10 μM MEN 10,367 on electrically-evoked contractions of rat urinary bladder strips. Data are expressed as percentage changes from control responses in the amplitude (A) and area under the curve (B) of EFS-evoked contractions at 2 (a), 4 (b), 8 (c) and 16 (d) Hz stimulation, following incubation with vehicle or antagonists. Each bar represents the mean value of 4–5 experiments and vertical bars show s.e.mean. ^*P<0.05 compared to vehicle.