dinoprost and fasudil

We have previously reported that prostaglandin F(2alpha) (PGF(2alpha)) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in the PGF(2alpha)-stimulated IL-6 synthesis in MC3T3-E1 cells. PGF(2alpha) time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced the PGF(2alpha)-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed the PGF(2alpha)-stimulated IL-6 synthesis. Y27632 and fasudil failed to affect the PGF(2alpha)-induced phosphorylation of p44/p42 MAP kinase. SB203580 and BIRB0796, potent inhibitors of p38 MAP kinase, suppressed the IL-6 synthesis induced by PGF(2alpha). While SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), failed to reduce the synthesis. Y27632 as well as fasudil attenuated the PGF(2alpha)-induced phosphorylation of p38 MAP kinase. These results strongly suggest that Rho-kinase regulates PGF(2alpha)-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3T3 Cells; Animals; Dinoprost; Enzyme Inhibitors; Interleukin-6; Mice; Myosin-Light-Chain Kinase; Myosin-Light-Chain Phosphatase; Osteoblasts; p38 Mitogen-Activated Protein Kinases; rho-Associated Kinases

We have shown recently that fasudil, a Rho-kinase inhibitor, has renoprotective effects in salt-sensitive hypertensive rats. We hypothesized that activation of Rho-kinase is involved in the pathogenesis of glomerulosclerosis in malignant hypertensive rats. To test this hypothesis, we studied the following 4 groups: control Wistar-Kyoto rats, untreated deoxycorticosterone-acetate salt spontaneously hypertensive rats (DOCA-SHR), low-dose fasudil-treated DOCA-SHR, and high-dose fasudil-treated DOCA-SHR. After 3 weeks of treatment, the effects of fasudil were examined. DOCA-SHR was characterized by increased blood pressure (BP); increased kidney weight; decreased renal function; increased proteinuria; abnormal histological findings; increased monocyte/macrophage infiltration; increased urinary 8-isoprostran levels; increased gene expression of collagen I, collagen III, transforming growth factor-beta, and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits (p40phox, p47phox, and p67phox); and decreased gene expression of endothelial NO synthase (eNOS) in the renal cortex as compared with Wistar-Kyoto rats. Long-term high-dose fasudil treatment significantly improved renal function and histological findings without changing BP, as compared with untreated DOCA-SHR. Interestingly, long-term fasudil treatment significantly decreased monocyte/macrophage infiltration and urinary 8-isoprostran excretion, in association with decreased mRNA levels of transforming growth factor-beta, collagen I, collagen III, and NADPH oxidase subunits (p40phox, p47phox, and p67phox), and increased mRNA levels of eNOS in the renal cortex. Long-term low-dose fasudil treatment tended to improve these variables slightly but did not affect most of them significantly. Our results suggest that long-term fasudil treatment provides renoprotective effects independent of BP-lowering activity. These renoprotective effects are associated with inhibition of extracellular matrix gene expression, monocyte/macrophage infiltration, oxidative stress, and upregulation of eNOS gene expression.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Desoxycorticosterone; Dinoprost; Dose-Response Relationship, Drug; Drug Administration Schedule; Ectodysplasins; Gene Expression; Hypertension, Malignant; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Cortex; Membrane Proteins; NADP; Nitric Oxide Synthase Type III; Protein Isoforms; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Rats; Rats, Inbred SHR; Rats, Inbred WKY; rho-Associated Kinases; Tumor Necrosis Factors

Inhibition of dephosphorylation of the 20 kDa myosin light chain (MLC(20)) is an important mechanism for the Ca(2+)-induced sensitization of vascular smooth muscle contraction. We investigated whether this mechanism operates in prostaglandin F(2alpha) (PGF(2alpha))-induced contraction of rabbit aortic smooth muscle and, if so, whether protein kinase C (PKC) or rho-associated kinase (rho kinase) contribute to the inhibition of dephosphorylation. In normal medium, PGF(2alpha) (10 microM) increased the phosphorylation of MLC(20) and developed tension. The rho-kinase inhibitors fasudil and hydroxyfasudil inhibited these changes, despite having no effect on a phorbol-ester-induced MLC(20) phosphorylation. After treatment with verapamil or chelation of external Ca(2+) with EGTA, PGF(2alpha) increased the MLC(20) phosphorylation and tension without an increase in [Ca(2+)](i), all of which were sensitive to fasudil and hydroxyfasudil. ML-9, a MLC kinase inhibitor, quickly reversed the KCl-induced MLC(20) phosphorylation and contraction to the resting level. However, fractions of PGF(2alpha)-induced contraction and MLC(20) phosphorylation were resistant to ML-9 but were sensitive to fasudil. Ro31-8220 (10 microM), a PKC inhibitor, did not affect the phosphorylation of MLC(20) and the tension caused by PGF(2alpha), thus excluding the possibility of the involvement of PKC in the PGF(2alpha)-induced MLC(20) phosphorylation. PGF(2alpha) increased phosphorylation at Thr654 of the myosin binding subunit (MBS) of myosin phosphatase, which is a target of rho kinase, and fasudil decreased the phosphorylation. These data suggest that the PGF(2alpha)-induced contraction is accompanied by the inhibition of MLC(20) dephosphorylation through rho kinase-induced MBS phosphorylation, leading to Ca(2+) sensitization of contraction. An actin-associated mechanism may also be involved in the PGF(2alpha)-induced sensitization.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Aorta; Azepines; Calcium; Dinoprost; Enzyme Inhibitors; In Vitro Techniques; Indoles; Intracellular Signaling Peptides and Proteins; Isoenzymes; Molecular Weight; Myosin Light Chains; Myosin-Light-Chain Phosphatase; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Protein Serine-Threonine Kinases; Rabbits; rho-Associated Kinases; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

Using the specific antibodies pLC1 and pLC2 for mono- and diphosphorylated 20-kDa myosin light chain (MLC20) at Ser19 and at both Thr18 and Ser19, respectively, we visualized the dynamics of the MLC20 phosphorylation in rabbit aortic smooth muscle cells (cell line SM-3) stimulated with PGF2alpha. In the resting state, the diphosphorylated form was located in the peripheral region of the cell, such as the leading edge or the adhesion plaque, and the monophosphorylated form was located not only in the peripheral region but also on a discontinuous fibrillary structure along the long axis of the cell. After stimulation with 30 microM PGF2alpha, although localization of the monophosphorylated form changed little, the content of the diphosphorylated form increased and the distribution spread along the fibrillary structure to an extent the same as or similar to that of the monophosphorylated form, which colocalized with actin filament bundles. The diphosphorylation of MLC20 was more sensitive to protein kinase inhibitors, HA-1077, HA-1100, staurosporine, wortmannin, and ML-9, than was the monophosphorylation. In light of these observations, we propose that MLC20 diphosphorylation and monophosphorylation are regulated by different mechanisms.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Androstadienes; Animals; Aorta; Azepines; Blotting, Western; Cell Line; Dinoprost; Enzyme Inhibitors; Kinetics; Mice; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Myosin Light Chains; Myosin-Light-Chain Kinase; Phosphorylation; Phosphoserine; Phosphothreonine; Rabbits; Staurosporine; Wortmannin

We examined the effects of HA1077, a potent inhibitor of protein kinases in vitro, on the relationship between tension and myosin-light chain (MLC20) phosphorylation in the initial phase of contraction of the rabbit aorta. The dose-response curve of HA1077 for MLC20 phosphorylation was to the left of the tension curve produced by 40 mM K+. In contrast, the potassium dose-response (15-100 mM) curves for tension and MLC20 phosphorylation were virtually identical. The nifedipine dose-response (1-3000 nM) curves for tension and MLC20 phosphorylation after 40 mM K(+)-stimulation were much the same. HA1077 inhibited the contraction induced by 30 microM prostaglandin F2 alpha (ED50 = 50 microM). Stimulation with prostaglandin F2 alpha induced both mono (MLC-P) and diphosphorylation (MLC-P2) of MLC20. In the presence of various concentrations of HA1077 (1-300 microM), the dose-response curves for MLC-P and MLC-P2 were also to the left of the tension curve. HA1077 inhibited MLC-P2 (ED50 less than microM) more effectively than it inhibited MLC-P (ED50 = 2.1 microM). These observations indicate that the relationship between tension and MLC20 phosphorylation involves inhibition of protein kinases by HA1077. The mechanism underlying the formation of MLC-P2 induced by prostaglandin F2 alpha may differ from that underlying MLC-P formation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Aorta, Thoracic; Dinoprost; In Vitro Techniques; Isoquinolines; Male; Muscle Contraction; Muscle, Smooth, Vascular; Myosin-Light-Chain Kinase; Myosins; Phosphorylation; Potassium; Protein Kinase Inhibitors; Rabbits