Hideo Kanaide

Long-Term Inhibition of RhoA Attenuates Vascular Contractility by Enhancing Endothelial NO Production in an Intact Rabbit Mesenteric Artery

RhoA plays a critical role in regulating NO production in cultured endothelial cells. To determine its role in in situ endothelial cells, we investigated the effects of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors and a RhoA-binding domain of Rho-kinase (RB) on vascular contractility in the isolated rabbit mesenteric artery. Ex vivo treatment of the strips with 3×10-5 mol/L simvastatin and fluvastatin for ≈24 to 30 hours significantly attenuated the contractile response to phenylephrine and high K+ in the presence of endothelium. The addition of N&ohgr;-nitro-l-arginine methyl ester and the removal of endothelium abolished the attenuation of the contractile response. The cotreatment with geranylgeranyl pyrophosphate prevented the statin-induced attenuation of the contractile response, whereas geranylgeranyl transferase inhibitor mimicked the effect of simvastatin. Treatment with simvastatin enhanced the bradykinin-induced endothelium-dependent relaxation in the mesenteric artery, whereas it had no effect on the bradykinin-induced [Ca2+]i elevation in endothelial cells of the aortic valves. Introduction of RB to the strips using a cell-penetrating peptide of Tat protein (TATHA-RB) attenuated the contractile responses in a NO-dependent manner. However, a Rac1/Cdc42-binding fragment of p21-activated protein kinase, RB without Tat peptide or TATHA-protein A had no effect. The in vivo treatment of rabbit with simvastatin and TATHA-RB attenuated the contractility in a NO-dependent manner. Simvastatin and TATHA-RB significantly upregulated eNOS in the rabbit mesenteric artery. The present study provides the first evidence that RhoA plays a physiological role in suppressing NO production in in situ endothelial cells.

Contractile Properties of the Cultured Vascular Smooth Muscle Cells: The Crucial Role Played by RhoA in the Regulation of Contractility

Vascular smooth muscle cells (VSMCs) have a remarkable degree of plasticity and in response to vascular injury, they can change to a dedifferentiated state that can be typically seen in cell cultures. Recently, Y27632, a Rho kinase inhibitor, has been reported to preferentially correct hypertension in a hypertensive rat model. We thus tested the hypothesis that the contraction of the cultured VSMCs might be more dependent on the function of RhoA than the VSMCs in fresh tissue. For this purpose, a tissue-like ring preparation was made using the cultured porcine coronary artery SMCs (CASMCs) and collagen gel (reconstituted ring: R-ring). The R-ring developed an isometric tension on stimulation by high external K+ or various receptor agonists. The phorbol ester (a protein kinase C (PKC) activator)-induced contraction of the intact R-ring was greatly inhibited, while the GTPγS (an activator of RhoA)-induced and Ca2+-independent contraction of permeabilized R-ring was greatly enhanced, in comparison to the fresh coronary artery ring. An immunoblot analysis showed the expression levels of RhoA and myosin phosphatase subunits (MYPT1 and PP1c&dgr;) to be up-regulated, while the levels of CPI-17 (PKC-potentiated protein phosphatase-1 inhibitory protein), h1-calponin and PKC isoforms were downregulated in cultured CASMCs. The knock down of RhoA by RNA interference decreased the contractility of the cultured CASMCs. It is concluded that the contractility of the cultured VSMCs thus appears to be much more dependent on the function of RhoA than VSMCs in fresh tissue. The expression level of RhoA thus plays a crucial role in regulating the contractility of cultured VSMCs.

Rac1 Regulation of Surface Expression of Protease-Activated Receptor-1 and Responsiveness to Thrombin in Vascular Smooth Muscle Cells

Objective—Protease-activated receptor-1 (PAR1) mediates the thrombin-induced proliferation and hypertrophy of vascular smooth muscle cells. A role of Rac1 in the regulation of PAR1 expression was investigated. Methods and Results—Treatment with simvastatin, a hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor, for 24 hours attenuated the transient [Ca2+]i elevation induced by thrombin. Immunofluorescence staining revealed that simvastatin decreased the surface expression of PAR1 in a manner dependent on protein geranylgeranylation. Introduction of a Rac1/Cdc42 inhibitory fragment but not a RhoA inhibitory fragment using a cell-penetrating peptide also attenuated the response to thrombin and decreased the surface expression of PAR1. Finally, downregulation of Rac1, but not RhoA, using an RNA interference technique attenuated the thrombin-induced [Ca2+]i elevation. However, the level of PAR1 mRNA and the total amount of PAR1 protein remained unchanged. Conclusions—Here, we provide for the first time 3 lines of evidence that Rac1 plays a critical role in maintaining the surface expression of PAR1 and the responsiveness to thrombin in vascular smooth muscle cells. Rac1 is suggested to regulate the constitutive trafficking of PAR1 and thereby regulate the surface expression of PAR1.

A critical period requiring Rho proteins for cell cycle progression uncovered by reversible protein transduction in endothelial cells

The time‐specific requirement of Rho proteins for the S phase progression of vascular endothelial cells was determined by reversibly introducing inhibitor proteins with a cell‐penetrating peptide. We found evidence of the reversibility of protein transduction. The removal of extracellular protein caused the transduced protein to decay in a manner sensitive to low temperatures. The time required for a 50% decay correlated with the protein size. The time‐specific transduction of the inhibitor proteins uncovered a critical period requiring Rho proteins in the G1–S transition phase. Reversible protein transduction may thus be a powerful tool to investigate the time‐specific role of signaling proteins.

Transduction of the N-Terminal Fragments of MYPT1 Enhances Myofilament Ca2+ Sensitivity in an Intact Coronary Artery

Objective—The region of the 110 kDa regulatory subunit (MYPT1) of smooth muscle myosin phosphatase involved in the regulation of contraction was determined under physiological conditions. Methods and Results—Using HIV Tat protein-mediated protein transduction, the N-terminal fragments of MYPT1 were introduced to the intact porcine coronary arterial strips. Pre-incubation with 3 &mgr;mol/L TAT-MYPT11–374, a construct containing the Tat peptide and the residues 1 to 374 of MYPT1, for 15 minutes augmented (2.4-fold) the subsequent contraction induced by adding 1.25 mmol/L of extracellular Ca2+ under 118 mmol/L K+ depolarization, with no augmentation of the [Ca2+]i elevation. The deletion of the Tat peptide, MYPT11–374, abolished the augmenting effect. TAT-MYPT11–296 demonstrated a weaker but significant augmentation (1.7-fold). However, TAT-MYPT11–171, TAT-MYPT139–374, TAT-MYPT139–296, and TAT-MYPT1297–374 had no augmenting activity. The myosin light chain phosphorylation level as a function of extracellular Ca2+ concentrations was shifted to the left in the strips pretreated with TAT-MYPT11–374 compared with the control. Conclusions—Region 1 to 296 was the minimal region involved in the enhancement of contraction, and region 297 to 374 played a supplemental role. These results suggested that the interaction mainly between catalytic subunit and MYPT1 play a critical role in the regulation of the endogenous myosin phosphatase in intact smooth muscle.

Functional role of PKC in contraction of cultured human prostatic stromal cells

The contractile activity of prostatic stromal cells contributes to symptoms of benign prostatic hyperplasia (BPH). However, the mechanisms for this contraction have not yet been fully elucidated. In this study, we investigated the role of protein kinase C (PKC) in prostatic contraction by measuring the isometric tension development of cultured human prostatic stromal cells (CHPSCs) derived from BPH patients. Fresh human BPH tissue was used only in a Western blot analysis. A ring preparation made of CHPSCs and collagen gel could develop an isometric tension during activation with various agonists. Phorbol 12,13 dibutyrate (PDBu), a PKC activator, induced a relaxation. A Western blot analysis revealed the expression of PKC‐potentiated protein phosphatase‐1 inhibitory protein (CPI‐17) in both CHPSCs and fresh human BPH tissue to be much lower than that in the rabbit aorta. When CPI‐17 was over‐expressed, PDBu induced a large contraction, but the agonist‐induced contraction did not become larger than expected. In α‐toxin permeabilized preparations, PDBu induced a relaxation in control CHPSCs, while it induced a contraction at a constant [Ca2+]i in CPI‐17 over‐expressing CHPSCs. These results indicated that the activation of PKC in CHPSCs induces a relaxation probably due to low expression level of CPI‐17 and also that the PKC‐CPI‐17 pathway does not appear to play a major role in the agonist‐induced contraction even when CPI‐17 was over‐expressed.

The mechanism underlying the contractile effect of a chemotactic peptide, formyl-Met-Leu-Phe on the guinea-pig Taenia coli

1 The contractile mechanism of N‐formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine (fMLP) was investigated in the guinea‐pig Taenia coli, by simultaneously monitoring the changes in the cytosolic Ca2+ concentration ([Ca2+]i) and force. 2 fMLP induced a significant elevation of [Ca2+]i and force at concentrations higher than 10u2003nM. The maximal response was obtained at a concentration of higher than 1u2003μM. 3 fMLP (10u2003μM) augmented the force development induced by a stepwise increment of the extracellular Ca2+ concentration during 60u2003mM K+ depolarization, while it had no effect on the [Ca2+]i elevation, and thus produced a greater force for a given elevation of [Ca2+]i than 60u2003mM K+ depolarization. 4 The removal of extracellular Ca2+ completely abolished the fMLP‐induced contraction. The fMLP‐induced [Ca2+]i elevation was inhibited substantially but not completely by 10u2003μM diltiazem, partly by 10u2003μM SK&F 96365, and completely by their combination. 5 Y27632, a specific inhibitor of rho‐kinase, had no significant effect on the fMLP‐induced [Ca2+]i elevation and force development. 6 Chenodeoxycholic acid, a formyl peptide receptor antagonist, specifically abolished the fMLP‐induced contraction but not high K+‐ or carbachol‐induced contractions. 7 A dual lipoxygenase/cyclooxygenase inhibitor, a 5‐lipoxygenase inhibitor, a nonselective leukotriene receptor antagonist, and a selective type 1 cysteinyl‐containing leukotriene receptor antagonist specifically reduced the fMLP‐induced contraction. 8 We suggest that the low‐affinity‐type fMLP receptor and lipoxygenase metabolites of arachidonic acid are involved in the fMLP‐induced contraction in the guinea‐pig T. coli. This contraction mainly depends on the [Ca2+]i elevation due to Ca2+ influx and the enhancement of Ca2+ sensitivity in the contractile apparatus.

Endothelium-Dependent and Independent Enhancement of Vascular Contractility in the Ovariectomized Rabbit

Objective: Estrogen suppresses contractile response and increases vasodilator response, partly by modulating endothelial function. However, the effect of estrogen on the contractility of vascular smooth muscle remains to be elucidated. We investigated the effect of a long-term estrogen deficiency on vascular contractility and the Ca2+ sensitivity of the contractile apparatus in arterial smooth muscle. Methods: Female rabbits were divided into the following three groups: control group, an ovariectomized group (OVX), and a group supplemented with 17β-estradiol after ovariectomy (OVX+E2). Twelve weeks later, the mesenteric artery was isolated, and the vascular contractility was evaluated. Results: In OVX, the contractile responses to phenylephrine and 118 mM postassium were enhanced, and the basal release of nitric oxide decreased in the strips with endothelium compared with either OVX+E2 or control. An enhancement of contraction was also observed in the strips without endothelium. However, the extent of enhancement was smaller than that observed in the presence of endothelium. The simultaneous measurement of calcium ([Ca2+]i) and tension revealed no significant difference in the [Ca2+]i elevations induced by phenylephrine among the three groups. In the α-toxin permeabilized strips, the Ca2+-tension relationships obtained both with and without phenylephrine and guanosine triphosphate were similar among the three groups. No difference in the myosin expression and the histology of vascular tissue was observed among the three groups. Conclusion: Long-term estrogen deficiency increased the vascular tone mainly by enhancing smooth muscle contractility. Endothelial dysfunction is considered to play a minor role in the augmentation of vascular tone.