Chronic fluvastatin treatment alters vascular contraction by inhibiting the Rho/Rho-kinase pathway

1. In the present study, we investigated the effects of chronic treatment of stroke-prone spontaneously hypertensive rats (SHRSP) with the statin fluvastatin on vascular Rho/Rho-kinase pathway mediated contraction, which has been shown to be upregulated in hypertension. 2. Contribution of the Rho/Rho-kinase pathway to noradrenaline-induced contraction of arteries from SHRSP was assessed by the inhibitory effect of Y-27632, a Rho/Rho-kinase inhibitor. Stroke-prone spontaneously hypertensive rats were treated with fluvastatin (10 mg/kg per day) for 1 month. 3. Treatment with fluvastatin tended to attenuate the contraction to noradrenaline and significantly decreased the Y-27632-sensitive component of the contraction in controls compared with fluvastatin-treated rats. 4. RhoA, as assessed by western blotting, was also reduced by fluvastatin treatment. 5. These findings suggest that chronic treatment with fluvastatin reduces the contractile response associated with Rho/Rho-kinase in arteries of hypertensive rats.     

Upregulation of Rho-kinase (ROCK-2) expression and enhanced contraction to endothelin-1 in the mesenteric artery from lipopolysaccharide-treated rats

Effects of bacterial lipopolysaccharide (Escherichia coli serotype, 055:B5, 20 mg kg(-1), i.p., for 6 h) and a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate, Y-27632 (10(-9)-10(-5) M) were investigated on the contractile responses of the rat mesenteric artery to phenylephrine (10(-9)-3 x 10(-5) M), angiotensin-2 (10(-10)-10(-6) M) and endothelin-1 (10(-10)-10(-7) M). Moreover, alteration in the level of Rho-kinase (ROCK-2) expression was examined in the superior mesenteric artery obtained from saline- and lipopolysaccharide-treated rats by Western blotting. Endotoxemic rat mesenteric rings exhibited no different contractions to phenylephrine and angiotensin-2 but augmented contractile activity to endothelin-1. In the mesenteric artery obtained from the endotoxemic rats, acetylcholine-induced vasorelaxation did not differ; pD2 value for acetylcholine was 7.85+/-0.12 in the endotoxemic rings; however, it was 7.81+/-0.15 in the control rings (P>0.05). Y-27632 induced relaxation, which was the same in the control arteries as in endotoxemic ones when contracting agent was phenylephrine. However, when endothelin-1 was used to precontract the rings, Y-27632 produced enhanced relaxation in endotoxemic vessels. pD2 values for Y-27632 were, respectively, 7.69+/-0.12 and 8.20+/-0.10 in control and endotoxemic rings precontracted by endothelin-1 (10(-8) M) (P<0.01). Moreover, Y-27632 (10(-5) M) suppressed the contraction induced by angiotensin-2 (10(-10)-10(-6) M). Western blot analysis revealed that Rho-kinase was upregulated significantly in the mesenteric artery obtained from the rats treated with LPS for 6 h. In addition, serum NO2-/NO3- level, which was detected by Griess method, was 10.0+/-1.4 microM in endotoxemic rats; however, it was 6.6+/-0.5 microM in control (P<0.05). Taken together, these results show that the expression of the contractile protein Rho-kinase could be upregulated in endotoxemic mesenteric artery and this upregulation may be coincided with an enhanced contraction to endothelin-1 but not phenylephrine and angiotensin-2.     

Mechanisms Of Endothelin-1-Induced Potentiation Of Noradrenaline Response In Rat Mesenteric Artery

1. Subthreshold concentrations of endothelin (ET)-1 enhance the contractile responses to noradrenaline (NA). We investigated possible mechanisms underlying the ET-1-induced enhancement of vasoconstrictor responses to NA in rat perfused mesenteric arteries. 2. Perfusion of arteries with subpressor dose of ET-1 (3 x 10-10 mol/L) significantly potentiated the pressor responses to NA (10-6, 3 x 10-6 and 10-5 mol/L) and this action of ET-1 was endothelium independent. 3. The protein kinase C (PKC) inhibitors staurosporine (10-8 mol/L) and calphostin C (10-7 mol/L) markedly attenuated the ET-1-induced enhancement of NA responses. Vasoconstrictor responses to NA were potentiated when vessels were perfused with phorbol 12-myristate 13-acetate (10-8 mol/L). 4. The potentiating effect of ET-1 was efficiently suppressed by Y-27632 (10-6 mol/L), a selective Rho-kinase inhibitor. In the presence of both staurosporine and Y-27632, contractile responses to NA alone were decreased markedly and ET-1-induced potentiation was abolished. 5. Both staurosporine and Y-27632 decreased contractile responses to NA in arteries of deoxycorticosterone acetate (DOCA)-salt hypertensive rats to levels observed in normotensive control animals. 6. These findings suggest that ET-1-mediated potentiation of responses to NA occurs through activation of either PKC or Rho-kinase. This mechanism seems to contribute to the enhanced vasoconstrictor responces to NA observed in DOCA-salt hypertensive rats, in which the responses to NA are enhanced tonically by endogenous vascular ET-1.     

The Rho-kinase inhibitor Y-27632 and the soluble guanylyl cyclase activator BAY41-2272 relax rabbit vaginal wall and clitoral corpus cavernosum

The effects of Y-27632, a Rho-kinase inhibitor and BAY41-2272, a soluble guanylyl cyclase activator, on the tone and nitrergic responses of rabbit vaginal wall and clitoral corpus cavernosum were investigated. Y-27632 and BAY41-2272 (10 nM-10 micro M) elicited concentration-dependent relaxation of phenylephrine-induced tone in both tissues. IC(50) values of Y-27632 for vaginal and clitoral tissues were 370+/-30 nM, and 467+/-14 nM, respectively. BAY41-2272 had IC(50) values of 478+/-54 nM and 304+/-38 nM respectively. The effect of the Y-27632 on the tissue tone was not affected by an inhibitor of nitric oxide synthase (L-NAME; 500 micro M). However, L-NAME reduced the potency of BAY41-2272 in the clitoral corpus cavernosum but not in the vaginal wall. BAY41-2272 enhanced nitrergic relaxation responses only in the clitoral corpus cavernosum. Y-27632 had no effect on nitrergic relaxations in either tissue. These results demonstrate that Y-27632 and BAY41-2272 elicit relaxation of the rabbit vaginal wall and clitoral corpus cavernosum.     

Rho-kinase, a potential therapeutic target for the treatment of hypertension

Hypertension is a cardiovascular disorder characterized by increased peripheral vascular resistance and/or vascular structural remodeling. Recently, rapidly growing evidence from hypertensive animal models suggests that small GTPase Rho and its downstream effector, Rho-kinase, play an important role in the pathogenesis of hypertension. Activation of the Rho/Rho-kinase pathway is essential for smooth muscle contractility in hypertension. A greater RhoA expression and an enhanced RhoA activity have been observed in aortas of hypertensive rats, such as genetic spontaneously hypertensive rats and N(omega)-nitro-L-arginine methyl ester-induced hypertension. The enhanced RhoA expression and activity was already observed in young spontaneously hypertensive rats before the onset of hypertension. These results suggest that both genetic factors and blood pressure can upregulate RhoA expression. Moreover, Y-27632 or fasudil, the specific Rho-kinase inhibitors, markedly decreased blood pressure in various hypertensive model rats, but did not in normotensive animals. In addition, Rho-kinase inhibitors have been shown to inhibit hypertensive vascular lesion formation. Therefore, Rho-kinase inhibitors may have a therapeutic potential for the treatment of hypertensive patients.     

Rho-kinase as a potential therapeutic target for the treatment of pulmonary hypertension

Pulmonary hypertension (PH) is a cardiovascular disorder characterized by vasoconstriction and vascular remodeling. Recently, rapidly increasing evidence from various rat models of PH and patients with PH suggest that small GTPase Rho and its downstream effector, Rho-kinase, play a key role in the pathogenesis of PH. Activation of the Rho/Rho-kinase pathway is important for pulmonary endothelial dysfunction, pulmonary vascular smooth muscle cell contractility, proliferation and apoptosis in PH. A greater Rho-kinase expression and an enhanced Rho-kinase activity have been observed in pulmonary arteries of PH rats, such as hypoxia-induced, monocrotaline-induced and genetic spontaneous PH rats. Moreover, Y-27632 or fasudil, the selective Rho-kinase inhibitors, significantly attenuated PH in various pulmonary hypertensive model rats and patients with PH, but did not reduce systemic blood pressure. Therefore, Rho-kinase inhibitors may have therapeutic potential for the treatment of PH.     

Rho-kinase expression and its contribution to the control of perfusion pressure in the isolated rat mesenteric vascular bed

Rho-kinase expression was investigated in the rat mesenteric artery and the effects of its inhibitors, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and fasudil (HA-1077), were examined on the increase in perfusion pressure induced by two different receptor agonists, namely the alpha-adrenoceptor agonist, phenylephrine and, the endothelin ET(A) and ET(B) receptor agonist, endothelin-1. Y-27632 and fasudil produced a concentration-dependent decrease in perfusion pressure. There was no difference between the concentration-response lines of these two inhibitors. The maximum decrease in the perfusion pressure induced by 10(-5) M Y-27632 was 85.8+/-3.7% when the tone was increased by phenylephrine. However, it was 48.1+/-5.4% (P<0.001) when the perfusion pressure was elevated by endothelin-1. Saponin perfusion (100 mg l(-1), for 10 min), which abolished acetylcholine-induced relaxation, did not significantly modify the Y-27632-elicited relaxation. Western blot analysis revealed that rat mesenteric artery expresses Rho-kinase protein with a molecular weight of approximately 160 kDa. These results show that Rho-kinase enzyme is expressed in rat mesenteric artery and that it contributes to the control of vascular resistance. Moreover, endothelium removal had no marked effect on the vasodilatation induced by Y-27632. In addition, the endothelin-1-induced vasoconstriction was more resistant to the Rho-kinase inhibitors than was that induced by phenylephrine, probably because excitatory endothelin receptors are associated with this signal transduction pathway at a different level from that of alpha-adrenoceptors.     

Long-term effects of benidipine on cerebral vasoreactivity in hypertensive rats

We tested the hypothesis that long-term application of a Ca2+ channel blocker would ameliorate the functional and morphological deterioration of the cerebral arteries during hypertension. Male spontaneously hypertensive rats (SHR) were fed a standard rat chow, containing a low (3 mg/kg/day) or high dose (6 mg/kg/day) of benidipine, a Ca2+ channel blocker, for 2 months. Using a cranial window, we examined responses of the basilar artery to acetylcholine, sodium nitroprusside, (-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-3-ol (Y-26763; an opener of ATP-sensitive K+ channels), and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632; an inhibitor of Rho-associated kinase). Mean arterial pressure of the control group was 193+/-5 mm Hg (mean+/-S.E.M.), while that of the low-dose benidipine group was 183+/-5 mm Hg and that of the high-dose group was 159+/-4 mm Hg. Dilator responses of the basilar artery to acetylcholine and Y-26763 were impaired in SHR compared with those of normotensive Wistar-Kyoto (WKY) rats and treatment with benidipine enhanced the vasodilator responses to acetylcholine and Y-26763 in SHR. Y-27632-induced dilatation of the basilar artery was enhanced in SHR compared to that in WKY rats and the vasodilatation was reduced by benidipine in SHR. Sodium nitroprusside caused similar dilatation of the basilar artery, in both WKY rats and the SHR control group, and benidipine did not affect nitroprusside-induced dilatation of the artery in SHR. The wall of the basilar artery was significantly thicker in SHR than in WKY rats and benidipine treatment reduced the wall thickness of the artery in SHR. These findings suggest that chronic treatment with a Ca2+ channel blocker may enhance the dilator capacity and reduce contractility of the basilar artery during hypertension. Benidipine may also ameliorate the morphological changes of the basilar artery in hypertension.     

Expression of Rho-kinase (ROCK-1 and ROCK-2) and its substantial role in the contractile activity of the sheep ureter

Expression of two isoforms of Rho-kinase (ROCK) and its functional role in the physiological control of smooth muscle contraction in the sheep ureter were investigated. Helical strips of the ureteric smooth muscle were stimulated by electrical field stimulation (EFS, 60 V, 1 mS, 2, 4, 8, 16 and 32 Hz, for 20 S), KCl (80 mm), carbachol (CCh, 10(-8)-10(-4) m) or phenylephrine (Phe, 10(-8)-10(-4) m). EFS produced a reproducible contractile activity, which was abolished by tetrodotoxin (3 x 10(-6) m), a Na(+) channel blocker. A muscarinic receptor antagonist, atropine (2 x 10(-6) m), and an adrenergic neuron blocker, guanethidine (10(-5) m), significantly suppressed the contraction induced by EFS. However, this contraction was augmented in the presence of N(G)-nitro-l-arginine (l-NA, 10(-4) m), a nitric oxide synthase inhibitor. Two Rho-kinase inhibitors, Y-27632 (5 x 10(-5) m) and fasudil (5 x 10(-5) m), markedly attenuated the EFS-elicited contraction. CCh and Phe produced concentration-dependent contraction in the sheep ureter. pD(2) values for Phe and CCh were 5.04+/-0.11 and 5.00+/-0.22, respectively. Y-27632 (5 x 10(-5) m) and fasudil (5 x 10(-5) m) also significantly inhibited CCh- and Phe-induced contractions. Moreover, these ROCK inhibitors produced relaxations in the KCl-elicited contraction in a concentration-dependent manner. pD(2) values for Y-27632 and fasudil were, respectively, 5.17+/-0.07 and 4.58+/-0.08 (P<0.001). Furthermore, the influences of these agents were also tested on spontaneous phasic contractions of the tissue. Among Y-27632, fasudil, TTX, l-NA, guanethidine and atropine, only the ROCK inhibitors (10(-6)-10(-5) m) were able to suppress the spontaneous contractile activity. Western blot analysis has revealed that both isoforms of Rho-kinase (ROCK-1 and ROCK-2) are expressed in the sheep ureter. Densitometric analysis has indicated that these enzymes are less expressed in the sheep ureter than are in the sheep aorta in a significant manner. These results show that a contractile enzyme, Rho-kinase, is expressed, and it mediates agonist- and EFS-induced contractions as well as spontaneous contractile activity of the isolated sheep ureter. Since Y-27632 and fasudil depressed the contractions, it seems plausible to postulate that Rho-kinase inhibitors may be beneficial in the treatment of renal colic.     

Augmented sphingosylphosphorylcholine-induced Ca2+-sensitization of mesenteric artery contraction in spontaneously hypertensive rat

Sphingosylphosphorylcholine (SPC) is a vasoconstricting lysosphingolipid, and the RhoA/Rho-kinase pathway plays an important role in SPC-induced contraction. Since RhoA/Rho-kinase-mediated signaling is involved in the generation and/or maintenance of hypertension, we compared the effect of SPC on the contractility of endothelium-denuded small mesenteric arteries in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Fura-2 Ca²⁺ signals, contractile responses, and phosphorylation of 20-kDa myosin light chains (MLC₂₀) were measured. Ten μM SPC induced a gradual and sustained vasoconstriction, which was greater in arteries of the SHR (82.5±4.3%, n=9) than in those of the WKY (26.7±4.5%, n=10). In Ca²⁺-free media, SPC gradually increased vascular tone in the SHR, but caused little vasoconstriction in the WKY. In the SHR and WKY, SPC evoked a greater vasoconstriction than did high K⁺depolarization at a given Ca²⁺ ratio, and the Ca²⁺ ratio–tension curve induced by SPC was significantly shifted to the left compared with that induced by high K⁺ depolarization. However, the magnitude of shift to the left was greater in the SHR than in the WKY. The Rho-kinase inhibitor Y-27632 significantly inhibited SPC-induced contractions, but neither the protein kinase C inhibitor calphostin-C nor PD98059, which inhibits activation of some mitogen-activated protein kinases, had any effect on the SHR or the WKY. SPC significantly increased the phosphorylation of MLC₂₀ in both the SHR and the WKY, and Y-27632 inhibited the SPC-induced increase in MLC₂₀ phosphorylation in the SHR. Our results suggest that SPC induces greater vascular tone in the SHR than in the WKY. Furthermore, our results indicate that activation of the Rho-kinase pathway plays an important role in the SPC-induced Ca²⁺ sensitization in the SHR.     

Differential Rho-kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. Whereas treatment with Y-27632 (1 microM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4+/-2.8 and 50.4+/-7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.     

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

(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.     

Rho-kinase inhibitor, Y-27632, has an antinociceptive effect in mice

The possible antinociceptive effect of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), was investigated in mice by using the hot-plate and abdominal constriction response (writhing) tests. In addition, the expression of Rho-kinase protein (ROCK-2) was studied in the mouse brain and spinal cord by Western blotting. Male balb/c mice (n=8, for each group) were used in the experiment. Hot-plate latency and the number of writhes were recorded in control and in Y-27632-treated (1-5 mg/kg, i.p.) groups. Y-27632 (1 mg/kg) did not affect hot-plate latency; however, it considerably diminished the number of writhes, from 89+/-12 in control to 30+/-6 in the mice treated with 1 mg/kg Y-27632 (P=0.001). At a higher dose (5 mg/kg), Y-27632 prolonged the hot-plate latency from 8.7+/-1.0 s to 14.4+/-1.7 s (P=0.005) and decreased the number of writhes from 80+/-8 to 24+/-7 (P=0.002). Western blot analysis revealed that mouse spinal cord and brain homogenates expressed ROCK-2 protein. These results indicate that Rho-kinase may be involved in nociception and that its inhibitors, such as Y-27632, may represent a new type of antinociceptive drug.     

Microtubule depolymerization facilitates contraction of rat aorta via activation of Rho-kinase

This study tests the hypothesis that microtubule (MT) depolymerization facilitates contraction of rat aorta via activation of Rho-kinase. Aortic rings from Sprague-Dawley rats were placed in a muscle bath for the measurement of isometric force generation. Bath temperature was decreased from 37 to 10-20 degrees C (30 min), inducing MT depolymerization. Some vessels were treated with nocodazole (10(-5) M) or colchicine (10(-8)-10(-5) M) to stabilize the MTs in the depolymerized state, and the remaining vessels were treated with dimethyl sulfoxide (DMSO: vehicle). Warming of vessels to 37 degrees C induced a significantly greater contraction in nocodazole- and colchicine-treated vessels as compared with controls, and this increase was blocked by pretreatment with taxol (10(-5) M; a MT stabilizing agent) [force (mg): NOC 1159 +/- 93; COL 1138 +/- 69; DMSO 578 +/- 14; TAX + NOC 526 +/- 43; TAX + COL 538 +/- 90]. Following the sustained contraction in response to rewarming, Rho-kinase inhibition with Y-27632 (10(-5) M) relaxed nocodazole- and colchicine-treated rings to a significantly greater extent as compared to DMSO-treated vessels (percent relaxation: NOC 64 +/- 2; COL 65 +/- 5; DMSO 33 +/- 5). These results support the hypothesis that MT depolymerization facilitates contraction of rat aorta via activation of Rho-kinase.     

Effects of Y-27632 on acetylcholine-induced contraction of intact and permeabilized intrapulmonary bronchial smooth muscles in rats.

In the present study, the effects of a selective Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor, Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] on acetylcholine-induced contraction and Ca(2+) sensitization of rat bronchial smooth muscle were examined. Intact and beta-escin-permeabilized muscles of the third branch of intrapulmonary bronchi were used. In intact muscles, Y-27632 (10(-6)-10(-4) M) concentration-dependently inhibited acetylcholine-induced contractile responses. In acetylcholine (10(-3) M)-precontracted intact muscles, the maximal relaxation (about 50% inhibition of contraction) was obtained by a concentration of 10(-4) M Y-27632, which had no effect on the resting tone. In beta-escin-permeabilized muscles, addition of acetylcholine (10(-5)-10(-3) M) plus GTP (100 microM) induced a further contraction, i.e., Ca(2+) sensitization at a constant Ca(2+) concentration of pCa=6.0. The acetylcholine-induced Ca(2+) sensitization was completely blocked in the presence of 10(-4) M Y-27632, whereas the Ca(2+)-induced contraction itself was not affected by Y-27632. Immunoblot study revealed the expression of ROCK-I and ROCK-II proteins in the intrapulmonary bronchi of rats. These findings suggest that Y-27632 dilates acetylcholine-mediated contraction of rat bronchial smooth muscle by inhibiting RhoA/ROCK-mediated Ca(2+) sensitization.     

Rho-kinase inhibitors show promise in pulmonary hypertension

In pulmonary arterial hypertension, it is necessary to obtain a vasodilation that is selective for the pulmonary circulation. Ras human orthologue (Rho)/Rho-kinase-mediated Ca2+ sensitisation plays a central role in mediating the sustained vasoconstriction and increased vasoreactivity in the rat hypoxic model of pulmonary hypertension. Rho-kinase inhibitors (Y-27632 and/or fasudil) have been shown to reduce pulmonary arterial pressure in three rat models of pulmonary hypertension. The first clinical study to report the effects of a Rho-kinase inhibitor in pulmonary hypertension enrolled nine patients with severe pulmonary hypertension. Fasudil hydrochloride 30 mg for 30 min i.v. caused a slight decrease in the mean pulmonary artery pressure and increase in the cardiac index but neither of these responses was significant. However, fasudil caused a significant decrease in pulmonary vascular resistance. Rho-kinase inhibitors may be useful in pulmonary hypertension, and should undergo further development for this indication.     

Allergic sensitization enhances the contribution of Rho-kinase to airway smooth muscle contraction

1. Repeated allergen challenge has been shown to increase the role of Rho-kinase in airway smooth muscle (ASM) contraction. We considered the possibility that active allergic sensitization by itself, that is, without subsequent allergen exposure, could be sufficient to enhance Rho-kinase-mediated ASM contraction. 2. Guinea pigs were actively IgE-sensitized to ovalbumin (OA), using Al(OH)(3) as adjuvant. Contractile responsiveness to G(q)-coupled receptor agonists (methacholine, histamine or PGF(2alpha)) was investigated in tracheal rings. No effect of sensitization was observed on basal- and methacholine-induced myogenic tone. In contrast, potency of histamine and PGF(2alpha) increased, that is, EC(50) decreased, after OA-sensitization by 2.6- and 4.7-fold, respectively, without effect on maximal contraction (E(max)). 3. Basal tone in preparations from both control and OA-sensitized animals was strongly decreased in the presence of the Rho-kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632) (1 microm). In control preparations, the E(max) and potency of histamine were unaffected by Y-27632, but were decreased for PGF(2alpha) (by 38.2% and 2.0-fold, respectively). However, in preparations from OA-sensitized animals, Y-27632 induced a significant reduction in E(max) (33.5%) and potency (2.3-fold) of histamine and of PGF(2alpha) (48.3% and 6.6-fold, respectively), normalizing the OA-sensitization-induced increase in sensitivity toward these agonists. 4. We also investigated the contribution of Rho-kinase in vivo by measuring airway responsiveness toward inhaled histamine in permanently instrumented, unanaesthetized control and OA-sensitized guinea pigs. Treatment with Y-27632 by inhalation (5 mm, nebulizer concentration) decreased airway responsiveness toward histamine both in control and OA-sensitized animals. However, the histamine PC(100) ratio pre/post Y-27632 inhalation was significantly smaller in OA-sensitized animals as compared to control animals, indicating an enhanced contribution of Rho-kinase. 5. Expression of RhoA, an upstream activator of Rho-kinase, was significantly increased (2.6-fold) in lung homogenates of OA-sensitized guinea pigs compared to control animals, as determined by Western analysis. 6. In conclusion, the results show a receptor-dependent role of Rho-kinase in agonist-induced ASM contraction. The contribution of Rho-kinase to contractile airway responsiveness, both in vivo and ex vivo, is augmented after active allergic sensitization, as a consequence of increased expression of RhoA presumably. Inhibition of the RhoA/Rho-kinase pathway may be considered a useful pharmacotherapeutical target in allergy and asthma.     

Evaluation of Y-27632, a rho-kinase inhibitor, as a bronchodilator in guinea pigs

To evaluate (+)-(R)-trans-4-(l-Aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate (Y-27632), a selective Rho-kinase inhibitor, as a novel bronchodilator in vivo and in vitro, we investigated the effect of Y-27632 on the acetylcholine- or ovalbumin-induced increase in lung resistance (R(L)) in non-sensitized or passively sensitized guinea pigs, and the relaxant effects of salbutamol, Y-27632 and theophylline on acetylcholine- or ovalbumin-induced contraction of isolated trachea. Y-27632 inhalation (1 mM, 2 min) inhibited acetylcholine- or ovalbumin-induced increase in R(L) without changes in mean blood pressure, and the effect persisted for at least 3 h. Salbutamol, Y-27632 and theophylline each completely reversed the acetylcholine- or ovalbumin-induced contraction of isolated trachea with rank order of potency, salbutamol>Y-27632>theophylline. The relaxant effect of Y-27632 was not affected by propranolol. We conclude that, although Y-27632 is not as potent as a beta-adrenoceptor agonist, Y-27632 may become an alternative inhaled bronchodilator, because Y-27632 is more potent than theophylline, and the relaxant effect is independent of beta-adrenoceptors.     

Role of Ca2+-sensitization in attenuated carbachol-induced contraction of the colon in a rat model of colitis

Inflammatory bowel disease is associated with reduced colonic smooth muscle contractility. However the underlying mechanism responsible for the decrease in contractility is not fully understood. In this study we investigated the role of Ca(2+)-sensitization in reduced carbachol-induced contraction of colonic segments from rats treated with trinitrobenzenesulphonic acid (TNBS). Functional alterations in RhoA/Rho-kinase and protein kinase C (PKC) pathways were examined using specific antagonists, Y-27632 and GF-109203X respectively. In this study, TNBS-induced colitis was associated with a decrease in the maximum response but not sensitivity to carbachol. Permeabilized inflamed colonic segments showed greater sensitivity to Ca(2+) as compared to controls, indicating greater Ca(2+)-sensitivity of the myofilaments. In contrast, carbachol-induced increase in Ca(2+)-sensitization was reduced in these tissues suggesting that the reduced carbachol-induced contraction could be due to decreased Ca(2+)-sensitization. Y-27632, a Rho-kinase inhibitor, induced significantly greater relaxation in colon strips from TNBS-treated rats indicating higher basal tone in these tissues. This is consistent with increased expression of Rho-kinase in the inflamed colon. Y-27632 concentration-dependently inhibited carbachol-induced contractions in control and TNBS-treated rats. However its effect was not significantly different between the two groups. GF-109203X, a PKC antagonist, produced concentration-dependent reduction in carbachol-induced contractions in control and TNBS-treated rats. GF-109203X was less effective in reducing carbachol-induced contractions of colonic segments from TNBS-treated rats suggesting a defect in PKC activation. Western blotting analysis showed reduced expression of total PKC in inflamed colonic smooth muscle. Carbachol-induced phosphorylation of CPI-17 was also reduced in colonic segments from TNBS-treated rats. These findings suggest that Ca(2+)-sensitization in rat colon involves both the PKC and the Rho-kinase pathways and that the reduced carbachol-induced contraction in colitis was due to inflammation-induced changes in Ca(2+)-sensitization involving a defect in the PKC pathway.     

Rho-kinase (ROCK-1 and ROCK-2) upregulation in oleic acid-induced lung injury and its restoration by Y-27632

The possible contribution of Rho/Rho-kinase signalling in oleic acid (100 mg kg-1, i.v., for 4 h)-induced lung injury was investigated in rats. Furthermore, the possible protective effect of the administration of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 0.5-5 mg kg-1, i.v., 15 min before the administration of oleic acid), was also examined. Western blot analysis as well as histopathological examination revealed that Rho-kinase (ROCK-1 and ROCK-2) was upregulated in lungs obtained from oleic acid-administrated rats. In addition, the markers of oxidative and nitrosative stress, i.e., malondialdehyde, myeloperoxidase, 3-nitro-L-tyrosine and nitrite/nitrate, in serum and lung tissue were also increased in the injury group. Treatment of rats with 5 mg kg-1 Y-27632 reversed the oleic acid-induced lung damage, which was demonstrated by histopathological assessment and confirmed in Western blot experiments: ROCK-blots were more intense in the oleic acid group than in control and Y-27632 treatment reversed ROCK upregulation. In addition, malondialdehyde, myeloperoxidase, 3-nitro-L-tyrosine and nitrite/nitrate were also normalized after the administration of Y-27632 (0.5 mg kg-1 and 5 mg kg-1). These findings suggest that ROCK-1 and ROCK-2 are involved in oleic acid-induced lung damage in rats, and that inhibition of this enzyme by Y-27632 may have a protective effect against such damage. Consequently, Rho kinase inhibitors may be potential therapeutic agents in the treatment of acute respiratory distress syndrome (ARDS).