Nobuyuki Moriki

Activation of RhoA and Inhibition of Myosin Phosphatase as Important Components in Hypertension in Vascular Smooth Muscle

Abstract— Two mechanisms are proposed to account for the inhibition of myosin phosphatase (MP) involved in Ca2+ sensitization of vascular muscle, ie, phosphorylation of either MYPT1, a target subunit of MP or CPI-17, an inhibitory phosphoprotein. In cultured vascular aorta smooth muscle cells (VSMCs), stimulation with angiotensin II activated RhoA, and this was blocked by pretreatment with 8-bromo-cGMP. VSMCs stimulated by angiotensin II, endothelin-1, or U-46619 significantly increased the phosphorylation levels of both MYPT1 (at Thr696) and CPI-17 (at Thr38). The angiotensin II-induced phosphorylation of MYPT1 was completely blocked by 8-bromo-cGMP or Y-27632 (a Rho-kinase inhibitor), but not by GF109203X (a PKC inhibitor). In contrast, phosphorylation of CPI-17 was inhibited only by GF109203X. Y-27632 dramatically corrected the hypertension in N&ohgr;-nitro-l-arginine methyl ester (L-NAME)-treated rats, and this hypertension also was sensitive to isosorbide mononitrate. The level of the active form of RhoA was significantly higher in aortas from L-NAME-treated rats. Expression of RhoA, Rho-kinase, MYPT1, CPI-17, and myosin light chain kinase were not significantly different in aortas from L-NAME-treated and control rats. Activation of RhoA without changes in levels of other signaling molecules were observed in three other rat models of hypertension, ie, stroke-prone spontaneously hypertensive rats, renal hypertensive rats, and DOCA-salt rats. These results suggest that independent of the cause of hypertension, a common point in downstream signaling and a critical component of hypertension is activation of RhoA and subsequent activation of Rho-kinase.

Rho/Rho-Kinase Pathway Contributes to C-Reactive Protein–Induced Plasminogen Activator Inhibitor-1 Expression in Endothelial Cells

Objective—Rho/Rho-kinase pathway plays pivotal roles in cardiovascular diseases including arteriosclerosis and hypertension. Recently it has become evident that C-reactive protein (CRP), a powerful marker for cardiovascular events, has direct proatherothrombotic effects on vascular cells. However, its molecular mechanism has not been fully investigated. We examined the involvement of Rho/Rho-kinase signaling in CRP-induced plasminogen activator inhibitor-1 (PAI-1) expression in bovine aortic endothelial cells (BAECs). Methods and Results—PAI-1 expression was determined by Western blotting. RhoA activation was determined by an affinity pull-down assay using Rho-binding fragment of rhotekin. NF-&kgr;B activity was determined using the luciferase reporter gene. Incubation of BAECs with human recombinant CRP (≥25 &mgr;g/mL) induced a significant increase in PAI-1 expression. Stimulation of BAECs with CRP significantly increased RhoA activation. Pretreatment with TAT-C3 (a membrane-permeable RhoA inhibitor) and Y-27632 (Rho-kinase inhibitor) significantly inhibited CRP-induced PAI-1 expression. NF-&kgr;B activity was markedly enhanced by CRP and pretreatment with Y-27632 inhibited its activation. Parthenolide, SN50, and BAY 11-7082 (NF-&kgr;B inhibitors) significantly blocked CRP-mediated PAI-1 expression. Conclusions—These data suggested that CRP activates Rho/Rho-kinase signaling, which in turn activates NF-&kgr;B activity, resulting in PAI-1 expression in BAEC. These observations provide evidence for the possible involvement of Rho/Rho-kinase signaling in CRP-induced atherothrombogenesis.

The targeted disruption of the MYPT1 gene results in embryonic lethality

Myosin phosphatase (MP) is a major phosphatase responsible for the dephosphorylation of the regulatory light chain of myosin II. MYPT1, a target subunit of smooth and nonmuscle MP, is responsible for activation and regulation of MP. To identity the physiological roles of MP, we have generated MYPT1-deficient mice by gene targeting. The heterozygous mice showed no changes in expression levels of MYPT1 and no distinct phenotype compared to wild-type mice was observed. None of the F2 mice were homozygous for the MYPT1 deletion, indicating that the targeted disruption of the MYPT1 gene resulted in embryonic lethality. The point of embryonic lethality is before 7.5 dpc. These findings indicate that MYPT1 is essential for mouse embryogenesis.

A Combination of Two Simultaneous Tachycardias in the Right Atrium Close to the Atrio-Ventricular Node and Within the Coronary Sinus in a Post-Operative Cor Triatriatum Patient

A 71-year-old male was referred to another hospital for dizziness. A bradycardia -tachycardia syndrome and Cor triatriatum were detected, and an operation to resect the membrane in the left atrium and implant a pacemaker epicardially was performed. However, no suitable site could be found on either atria and therefore, a single chamber ventricular pacemaker was implanted. In the electrophysiological study performed in our hospital, we could not detect any atrial potentials in either atria, excluding the region close to the His bundle (HB) and within coronary sinus (CS), in spite of extensive catheter mapping. A regular atrial rhythm with a cycle length of 820 ms, which was synchronous with the rate of the QRS complex on the surface ECG, was recorded only at the HB. Meanwhile, the CS catheter recording exhibited regular focal activity with a cycle length of 150 ms, and this focal activity did not conduct to the atrium close to the HB. Furthermore, this activity was dissociated from the ventricular activity recorded from the CS catheter. During an isoproterenol infusion, an atrial tachycardia with a cycle length of 380 ms was recorded only at the HB, and the twelve-lead ECG exhibited a regular tachycardia with the same cycle length as this tachycardia. Meanwhile, the focal activity within the CS persisted without any change in the cycle length. These findings suggested that there was dissociation between the right atrium (RA) and CS. Furthermore, partial atrial standstill was observed in both atria, excluding the RA close to the atrio-ventricular (AV) node and area within the CS. These rare electrophysiological features were considered to play an important role in the genesis of a simultaneous combination of the two tachycardias at their respective sites.