ha-1100 and fasudil

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amides; Drug Design; Humans; Intracellular Signaling Peptides and Proteins; Osteogenesis; Osteoporosis; Protein Serine-Threonine Kinases; Pyridines; rho-Associated Kinases

Rho-kinase has been identified as one of the effectors of the small GTP-binding protein Rho. Accumulating evidence has demonstrated that the Rho/Rho-kinase-mediated pathway plays an important role in various cellular functions, not only in vascular smooth muscle contraction but also in actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expressions, all of which may be involved in the pathogenesis of arteriosclerosis/atherosclerosis. Indeed, animal experiments have demonstrated that Rho-kinase inhibitors effectively suppress coronary artery spasm and that long-term inhibition of Rho-kinase inhibits the development of coronary arteriosclerotic lesions and even causes regression of coronary vascular lesions in vivo. Recent clinical studies also have demonstrated the inhibitory effect of a Rho-kinase inhibitor on coronary artery spasm in patients with vasospastic angina and on exercise-induced myocardial ischemia in patients with stable effort angina with adequate safety. It is possible that Rho-kinase is also involved in the pathogenesis of other forms of cardiovascular diseases. Thus, Rho-kinase could be regarded as a novel therapeutic target in treatment of cardiovascular diseases.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amides; Animals; Cardiovascular Diseases; Coronary Vessels; Endothelium, Vascular; Enzyme Inhibitors; Humans; Intracellular Signaling Peptides and Proteins; Muscle, Smooth, Vascular; Protein Serine-Threonine Kinases; Pyridines; rho-Associated Kinases; Signal Transduction; Vasodilator Agents

Multiple sclerosis (MS) is a central nervous system (CNS) disease with complicated etiology. Multifocal demyelination and invasion of inflammatory cells are its primary pathological features. Fasudil has been confirmed to improve experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, Fasudil is accompanied by several shortcomings in the clinical practice. Hydroxyfasudil is a metabolite of Fasudil in the body with better pharmaceutical properties. Therefore, we attempted to study the influence of Hydroxyfasudil upon EAE mice. The results demonstrated that Hydroxyfasudil relieved the symptoms of EAE and the associated pathological damage, reduced the adhesion molecules and chemokines, decreased the invasion of peripheral immune cells. Simultaneously, Hydroxyfasudil modified the rebalance of peripheral T cells. Moreover, Hydroxyfasudil shifted the M1 phenotype to M2 polarization, inhibited inflammatory signaling cascades as well as inflammatory factors, and promoted anti-inflammatory factors in the CNS. In the end, mice in the Hydroxyfasudil group expressed more tight junction proteins, indirectly indicating that the blood-brain barrier (BBB) was protected. Our results indicate that Hydroxyfasudil may be a prospective treatment for MS.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Encephalomyelitis, Autoimmune, Experimental; Mice; Mice, Inbred C57BL; Multiple Sclerosis

1. An investigation on the metabolic mechanism of fasudil to hydroxyfasudil was conducted in vitro using liver subcellular fractions of different species. Hydroxyfasudil was generated in large amounts by rat liver S9 and to a similar extent by human liver S9 but was not detected in dog liver S9 incubations. 2. Studies with various molybdenum hydroxylase inhibitors demonstrated that aldehyde oxidase (AO), but not xanthine oxidase (XO), selectively catalyzed fasudil to hydroxyfasudil in both rat and human liver cytosol. In addition, the oxygen atom incorporated into hydroxyfasudil was derived from water rather than atmospheric oxygen, which further corroborated AO involvement. 3. Enzyme kinetics experiments revealed that fasudil had a higher affinity to human hepatic AO than to rat hepatic AO. Besides, significantly different in vivo pharmacokinetic parameters observed between male and female rats indicated that the AO activity in rats was gender-dependent. 4. The present study provided first evidences that AO causes differences in fasudil metabolism between species.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Aldehyde Oxidase; Animals; Dogs; Female; Humans; Liver; Male; Protein Kinase Inhibitors; Rats; Species Specificity; Xanthine Oxidase

Renal fibrosis is the major cause of chronic kidney disease, and the Rho/Rho-associated coiled-coil kinase (ROCK) signaling cascade is involved in the renal fibrotic processes. Several studies have reported that ROCK inhibitors attenuate renal fibrosis. However, the mechanism of this process remains to be fully elucidated. The present study assessed the inhibitory effect of fasudil, a ROCK inhibitor using immunohistochemistry, reverse transcription-quantitative polymerase chain reaction and western blot analyses, in vivo and in vitro, to elucidate the mechanisms underlying renal interstitial fibrosis. In mice induced with unilateral ureteral obstruction (UUO), collagen accumulation, the expression of fibrosis‑associated genes and the content of hydroxyproline in the kidney increased 3, 7, and 14 days following UUO. Fasudil attenuated the histological changes, and the production of collagen and extracellular matrix in the UUO kidney. The expression of α‑smooth muscle actin (α‑SMA) and the transforming growth factor‑β (TGFβ)‑Smad signaling pathway, and macrophage infiltration were suppressed by fasudil in the kidneys of the UUO mice. The present study also evaluated the role of intrinsic renal cells and infiltrated macrophages using NRK‑52E, NRK‑49F and RAW264.7 cells. The mRNA and protein expression levels of collagen I and α‑SMA increased in the NRK‑52E and NRK‑49F cells stimulated by TGF‑β1. Hydroxyfasudil, a bioactive metabolite of fasudil, attenuated the increase in the mRNA and protein expression levles of α‑SMA in the two cell types. However, the reduction in the mRNA expression of collagen I was observed in the NRK‑49F cells only. Hydroxyfasudil decreased the mRNA expression of monocyte chemoattractant protein‑1 (MCP‑1) induced by TGF‑β1 in the NRK‑52E cells, but not in the NRK‑49F cells. In the RAW264.7 cells, the mRNA expression levels of MCP‑1, interleukin (IL)‑1β, IL‑6 and tumor necrosis factor α were increased significantly following lipopolysaccharide stimulation, and were not suppressed by hydroxyfasudil. These data suggested that the inhibition of ROCK activity by fasudil suppressed the transformation of renal intrinsic cells into the myofibroblast cells, and attenuated the infiltration of macrophages, without inhibiting the expression or the activation of cytokine/chemokines, in the progression of renal interstitial fibrosis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Animals; Cell Line; Cell Movement; Collagen Type I; Disease Models, Animal; Fibrosis; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Protein Kinase Inhibitors; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1; Up-Regulation; Ureteral Obstruction

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no effective treatment. Fasudil hydrochloride (fasudil), a potent rho kinase (ROCK) inhibitor, is useful for the treatment of ischaemic diseases. In previous reports, fasudil improved pathology in mouse models of Alzheimer's disease and spinal muscular atrophy, but there is no evidence in that it can affect ALS. We therefore investigated its effects on experimental models of ALS.. In mice motor neuron (NSC34) cells, the neuroprotective effect of hydroxyfasudil (M3), an active metabolite of fasudil, and its mechanism were evaluated. Moreover, the effects of fasudil, 30 and 100 mg·kg(-1), administered via drinking water to mutant superoxide dismutase 1 (SOD1(G93A)) mice were tested by measuring motor performance, survival time and histological changes, and its mechanism investigated.. M3 prevented motor neuron cell death induced by SOD1(G93A). Furthermore, M3 suppressed both the increase in ROCK activity and phosphorylated phosphatase and tensin homologue deleted on chromosome 10 (PTEN), and the reduction in phosphorylated Akt induced by SOD1(G93A). These effects of M3 were attenuated by treatment with a PI3K inhibitor (LY294002). Moreover, fasudil slowed disease progression, increased survival time and reduced motor neuron loss, in SOD1(G93A) mice. Fasudil also attenuated the increase in ROCK activity and PTEN, and the reduction in Akt in SOD1(G93A) mice.. These findings indicate that fasudil may be effective at suppressing motor neuron degeneration and symptom progression in ALS. Hence, fasudil may have potential as a therapeutic agent for ALS treatment.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amyotrophic Lateral Sclerosis; Animals; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Male; Mice, Transgenic; Motor Neurons; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; rho-Associated Kinases; Superoxide Dismutase; Superoxide Dismutase-1; Survival Rate

Using a cellular approach, the present study examined whether fasudil and active metabolite hydroxyfasudil, Rho-kinase inhibitors, exert a direct protective effect on endothelin-induced cardiac myocyte hypertrophy in vitro. Treatment with endothelin (10nM) caused significant hypertrophy of cultured neonatal rat cardiomyocytes by a 21.2% increase in cell surface area. Fasudil (1-10 μM) and hydroxyfasudil (0.3-10 μM) significantly prevented endothelin-induced cardiomyocyte hypertrophy. The present results suggest that inhibition of cardiac hypertrophy by fasudil is, at least in part, due to direct protection of cardiomyocytes from hypertrophy.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cardiomegaly; Cells, Cultured; Cytoprotection; Endothelins; Myocytes, Cardiac; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; rho-Associated Kinases

The aim of this study was to investigate the possible effects of the Rho-kinase inhibitor, fasudil, on the lysophosphatidic acid (LPA)-induced neurite retraction in N1E-115 cells. In cultured N1E-115 cells, LPA produced a marked increase in the population of rounded cells. Fasudil or hydroxyfasudil, an active metabolite of fasudil, blocked cell rounding in a concentration-dependent manner at levels between 1 and 10 μM, with IC₅₀ values of 1.7 or 1.6 μM, respectively. Fasudil or hydroxyfasudil concentration-dependently inhibited phosphorylation of the myosin binding subunit of myosin phosphatase in N1E-115 cells. These results indicate that Rho-kinase is essential for LPA-induced neurite retraction in N1E-115 cells and that inactivation of Rho-kinase by a Rho-kinase inhibitor, such as fasudil, eliminates cell rounding and promotes neurite outgrowth, thus improving neurological function in the brain damage.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cell Line; Humans; Lysophospholipids; Mice; Neurites; Neurons; Protein Kinase Inhibitors; rho-Associated Kinases

We investigated the neuroprotective effects of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, in a rat stroke model in which endothelial damage and subsequent thrombotic occlusion were selectively induced in perforating arteries. By examining the effects on the endothelial damage/dysfunction, we thought to explore the mechanism of Rho-kinase inhibitors. Hydroxyfasudil (10mg/kg, i.p., once daily for 3 days) significantly improved neurological functions and reduced the size of the infarct area produced by internal carotid artery injection of sodium laurate in a rat cerebral microthrombosis model. Treatment with fasudil or hydroxyfasudil concentration-dependently inhibited tumor necrosis factor alpha-induced tissue factor expression on the surface of cultured human umbilical vein endothelial cells. They also inhibited thrombin-induced endothelial hyperpermeability. The present findings suggest that hydroxyfasudil is efficacious in preventing brain damage associated with cerebral ischemia, and is partially responsible for fasudil's cytoprotective potential. The results also suggest that the therapeutic benefits against ischemic injury of Rho-kinase inhibitors are attributed, at least in part, to activity upon endothelial damage/dysfunction.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Brain; Brain Ischemia; Capillary Permeability; Cells, Cultured; Disease Models, Animal; Endothelium; Enzyme Inhibitors; Humans; In Vitro Techniques; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Stroke; Thromboplastin; Tumor Necrosis Factor-alpha; Umbilical Veins

A fast and sensitive method to quantify fasudil hydrochloride (FH) and its active metabolite hydroxyfasudil (M3) in human plasma using HPLC-MS/MS has been developed and validated in present study. The method involved simple sample preparation with methanol as protein precipitation (3:1, v/v) and ranitidine as an internal standard (IS). The analytes and IS were separated using a gradient elution procedure on the analytical column ZORBAX StableBond-C18 (5 microm, 150 mm x 4.6mm). Detection was performed by an AB 3200 QTRAP tandem mass spectrometer equipped with a Turbo IonSpray ionization source set in positive ion mode. Multiple reaction monitoring (MRM) using the precursor to product ion was m/z 292.2/99.2 for fasudil, m/z 308.2/99.2 for M3 and m/z for 315.3/176.2 for IS. The linear range of the method was from 0.4 to 250 ng/mL for both fasudil and M3. The lower limit of quantification was 0.4 ng/mL for both fasudil and M3. The intra- and inter-day relative standard deviation over the entire concentration range was less than 7.11% for fasudil and 10.6% for M3, respectively. The validated method was successfully applied for the evaluation of pharmacokinetic of fasudil hydrochloride after administration of 30 mg fasudil hydrochloride by continuous intravenous infusion over 30 min in 12 healthy Chinese volunteers.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Chromatography, Liquid; Female; Humans; Male; Molecular Structure; Quality Control; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Time Factors

The aim of this study was to investigate the influence of delayed Rho-kinase inhibition with fasudil on second ischemic injury in a rat cerebral thrombosis model. Cerebral ischemia was induced in rats by injecting 150 mug of sodium laurate into the left internal carotid artery on day 1. In the ischemic group, the regional cerebral blood flow (rCBF) was significantly decreased 6.5 h after the injection. Fasudil (3 mg/kg/30 min i.v. infusion) significantly increased rCBF. The viscosity of whole blood was significantly increased 48 h after the injection of sodium laurate. Fasudil (10 mg/kg, i.p.) significantly decreased blood viscosity. To clarify the therapeutic time window of fasudil, rats received their first i.p. administration of fasudil (10 mg/kg) 6 h after an injection of sodium laurate. Administration of fasudil twice daily was continued until day 4. Fasudil prevented the accumulation of neutrophils within the brain as seen from measurements taken on day 3, and improved neuronal functions and reduced the infarction area as seen on day 5. Fasudil and hydroxyfasudil, an active metabolite of fasudil, concentration-dependently inhibited phosphorylation of myosin binding subunit of myosin phosphatase in neutrophils. The present results indicate that inhibition of Rho-kinase activation with fasudil is effective for the treatment of ischemic brain damage with a wide therapeutic time window by improving hemodynamic function and preventing the inflammatory responses. These results suggest that fasudil will be a novel and efficacious approach for the treatment of acute ischemic stroke.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antipyrine; Blood Flow Velocity; Brain Ischemia; Cerebral Infarction; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Intracranial Thrombosis; Lauric Acids; Male; Myosins; Protein Binding; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Regional Blood Flow; rho-Associated Kinases; Time Factors

ROCK or Rho-associated kinase, a serine/threonine kinase, is an effector of Rho-dependent signaling and is involved in actin-cytoskeleton assembly and cell motility and contraction. The ROCK protein consists of several domains: an N-terminal region, a kinase catalytic domain, a coiled-coil domain containing a RhoA binding site, and a pleckstrin homology domain. The C-terminal region of ROCK binds to and inhibits the kinase catalytic domains, and this inhibition is reversed by binding RhoA, a small GTPase. Here we present the structure of the N-terminal region and the kinase domain. In our structure, two N-terminal regions interact to form a dimerization domain linking two kinase domains together. This spatial arrangement presents the kinase active sites and regulatory sequences on a common face affording the possibility of both kinases simultaneously interacting with a dimeric inhibitory domain or with a dimeric substrate. The kinase domain adopts a catalytically competent conformation; however, no phosphorylation of active site residues is observed in the structure. We also determined the structures of ROCK bound to four different ATP-competitive small molecule inhibitors (Y-27632, fasudil, hydroxyfasudil, and H-1152P). Each of these compounds binds with reduced affinity to cAMP-dependent kinase (PKA), a highly homologous kinase. Subtle differences exist between the ROCK- and PKA-bound conformations of the inhibitors that suggest that interactions with a single amino acid of the active site (Ala215 in ROCK and Thr183 in PKA) determine the relative selectivity of these compounds. Hydroxyfasudil, a metabolite of fasudil, may be selective for ROCK over PKA through a reversed binding orientation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amides; Amino Acid Sequence; Binding Sites; Binding, Competitive; Cyclic AMP-Dependent Protein Kinases; Dimerization; Enzyme Inhibitors; Humans; Intracellular Signaling Peptides and Proteins; Ligands; Molecular Sequence Data; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Pyridines; rho-Associated Kinases; Structure-Activity Relationship

Rho-kinase plays an important role in hypertension and is reported to interfere with insulin signaling through serine phosphorylation of insulin receptor substrate-1 (IRS-1) in cultured vascular smooth muscle cells. We therefore examined the role of Rho-kinase in the development of insulin resistance in Zucker obese rats. In skeletal muscles and aortic tissues of Zucker obese rats, activation of RhoA/Rho-kinase was observed. Long-term Rho-kinase inhibition by 4 wk treatment with fasudil (a Rho-kinase inhibitor) not only reduced blood pressure but corrected glucose and lipid metabolism, with improvement in serine phosphorylation of IRS-1 and insulin signaling in skeletal muscles. Direct visualization of skeletal muscle arterioles with an intravital CCD videomicroscope demonstrated that both acetylcholine- and sodium nitroprusside-induced vasodilations were blunted, which were restored by the fasudil treatment. Furthermore, both fasudil and Y-27632 prevented the serine phosphorylation of IRS-1 induced by insulin and/or tumor necrosis factor-alpha in skeletal muscle cells. Collectively, Rho-kinase is responsible for the impairment of insulin signaling and may constitute a critical mediator linking between metabolic and hemodynamic abnormalities in insulin resistance.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetylcholine; Animals; Arterioles; Cell Line; Glucose Tolerance Test; Hypertension; Insulin; Insulin Resistance; Intracellular Signaling Peptides and Proteins; Muscle, Skeletal; Nitroprusside; Protein Serine-Threonine Kinases; Protein Transport; Rats; Rats, Zucker; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Tumor Necrosis Factor-alpha

Endothelium-derived nitric oxide (NO) plays a pivotal role in vascular protection. The Rho kinase (ROCK) inhibitor, hydroxyfasudil, prevents the downregulation of endothelial NO synthase (eNOS) under hypoxic conditions. However, it is unknown whether inhibition of ROCK can attenuate ischemia-induced endothelial dysfunction and tissue damage in vivo.. Human vascular endothelial cells were treated with increasing concentrations of hydroxyfasudil (0.1 to 100 micromol/L) and eNOS expression and activity were measured. To determine the physiological relevance of eNOS regulation by ROCK, we administered fasudil, which is metabolized to hydroxyfasudil in vivo, to mice for 2 days before subjecting them to middle cerebral artery occlusion. Cerebral blood flow, cerebral infarct size, and neurologic deficit were measured.. In a concentration-dependent manner, hydroxyfasudil increased eNOS mRNA and protein expression, resulting in a 1.9- and 1.6-fold increase, respectively, at 10 micromol/L (P<0.05 for both). This correlated with a 1.5- and 2.3-fold increase in eNOS activity and NO production, respectively (P<0.05 for both). Fasudil increased cerebral blood flow to both ischemic and nonischemic brain areas, reduced cerebral infarct size by 33%, and improved neurologic deficit score by 37% (P<0.05). This correlated with inhibition of brain and vascular ROCK activity and increased eNOS expression and activity. Another ROCK inhibitor, Y-27632, also showed similar effects. The neuroprotective effects of fasudil were absent in eNOS-deficient mice.. These findings indicate that the neuroprotective effect of ROCK inhibition is mediated by endothelium-derived NO and suggest that ROCK may be an important therapeutic target for ischemic stroke.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amides; Analysis of Variance; Animals; Antihypertensive Agents; Aorta; Blotting, Northern; Blotting, Western; Brain Ischemia; Cattle; Cell Line; Cells, Cultured; Cerebrovascular Circulation; Dose-Response Relationship, Drug; Down-Regulation; Endothelium, Vascular; Enzyme Inhibitors; Humans; Hypoxia; Infarction, Middle Cerebral Artery; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Protein Serine-Threonine Kinases; Pyridines; Regional Blood Flow; rho-Associated Kinases; RNA, Messenger; Stroke; Time Factors; Umbilical Veins; Up-Regulation

Primary pulmonary hypertension is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells (VSMCs), and migration of inflammatory cells, for which no satisfactory treatment has yet been developed. We have recently demonstrated that intracellular signaling pathway mediated by Rho-kinase, an effector of the small GTPase Rho, is involved in the pathogenesis of arteriosclerosis. In the present study, we examined whether the Rho-kinase-mediated pathway is also involved in the pathogenesis of fatal pulmonary hypertension in rats. Animals received a subcutaneous injection of monocrotaline, which resulted in the development of severe pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions in 3 weeks associated with subsequent high mortality rate. The long-term blockade of Rho-kinase with fasudil, which is metabolized to a specific Rho-kinase inhibitor hydroxyfasudil after oral administration, markedly improved survival when started concomitantly with monocrotaline and even when started after development of pulmonary hypertension. The fasudil treatment improved pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions with suppression of VSMC proliferation and macrophage infiltration, enhanced VSMC apoptosis, and amelioration of endothelial dysfunction and VSMC hypercontraction. These results indicate that Rho-kinase-mediated pathway is substantially involved in the pathogenesis of pulmonary hypertension, suggesting that the molecule could be a novel therapeutic target for the fatal disorder.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Intracellular Signaling Peptides and Proteins; Male; Monocrotaline; Muscle, Smooth, Vascular; Protein Serine-Threonine Kinases; Pulmonary Artery; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Survival Rate; Time Factors

Rho-kinase has been implicated as an important regulator of inflammatory responses mediated by cytokines and chemokines. Because proinflammatory cytokines play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), we examined whether long-term blockade of Rho-kinase suppresses LV remodeling in a mouse model of MI in vivo.. Mice underwent ligation of the left coronary artery and were treated with a Rho-kinase inhibitor, fasudil (100 mg x kg(-1) x d(-1) in tap water), for 4 weeks, starting 1 day after the surgery. At 4 weeks, LV infarct size was histologically comparable between the 2 groups. LV cavity dilatation and dysfunction evaluated by echocardiography were significantly suppressed in the fasudil group (P<0.05, n=15 to 28). The beneficial effects of fasudil were accompanied by suppression of cardiomyocyte hypertrophy and interstitial fibrosis (both P<0.01, n=6). The expression of inflammatory cytokines, including transforming growth factor (TGF)-beta2, TGF-beta3, and macrophage migration inhibitory factor, was upregulated in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (both P<0.05, n=10 to 11). Rho-kinase activity as evaluated by the extent of phosphorylation of the ERM family, a substrate of Rho-kinase, was significantly increased in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (P<0.05, n=5).. These results indicate that Rho-kinase is substantially involved in the pathogenesis of LV remodeling after MI associated with upregulation of proinflammatory cytokines, suggesting a therapeutic importance of the molecule for the prevention of post-MI heart failure.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; DNA-Binding Proteins; Drug Administration Schedule; Enzyme Inhibitors; Fibrosis; Gene Expression Regulation; Hypertrophy, Left Ventricular; Intramolecular Oxidoreductases; Macrophage Migration-Inhibitory Factors; Male; Mice; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Organ Size; Phosphorylation; Protein Processing, Post-Translational; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta2; Transforming Growth Factor beta3; Ultrasonography; Ventricular Dysfunction, Left; Ventricular Remodeling

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

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Blood Vessels; Cardiovascular Diseases; Hypertension; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Male; Protein Serine-Threonine Kinases; Rats; Rats, Inbred SHR; Rats, Inbred WKY; rho-Associated Kinases; Signal Transduction; Vascular Resistance; Vasoconstriction; Vasodilator Agents

This study was designed to investigate possible effects of the Rho-kinase inhibitor, fasudil, on the progression of renal failure in rats with unilateral ureteral obstruction. The renal failure markers monitored were the extent of renal interstitial fibrosis and that of macrophage infiltration. In kidneys with unilateral ureteral obstruction, interstitial fibrosis was observed, using Sirius-Red staining, on day 16 after unilateral ureteral obstruction. Macrophage infiltration was observed by immunohistochemistry, using the antibody, ED1. Interstitial fibrosis and macrophage infiltration were significantly attenuated in fasudil-treated animals. The migration of monocytes in vitro elicited by N-formyl-methionyl-leucyl-phenylalanine was potently inhibited by fasudil and its active metabolite, hydroxyfasudil. These results suggest that inhibition of Rho-kinase produces a reduction of macrophage infiltration and represents a new therapeutic strategy for renal fibrosis, a major factor in the progression to end-stage renal failure.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Chemotaxis; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibrosis; Kidney; Macrophages; Male; Monocytes; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Ureteral Obstruction

Fasudil hydrochloride (AT877, hexahydro-1-(5-isoquinolinesulfonyl)-1H-1,4-diazepine hydrochloride, identical to HA1077) inhibits cerebral vasospasm after subarachnoid hemorrhage in experimental animals and humans. In the current study, the vasorelaxing mechanism of hydroxyfasudil, a hydroxylated metabolite of fasudil hydrochloride, was determined in the rabbit basilar artery. The effects of hydroxyfasudil on tension, intracellular Ca2+ concentration ([Ca2+]i), and phosphorylation of the myosin light chain were examined using the isolated and intact or permeabilized rabbit basilar artery without endothelium in vitro. In the intact rabbit basilar artery, hydroxyfasudil elicited a concentration-dependent relaxation of the artery precontracted with 1 nmol/L endothelin-1 (ET-1) plus 20 mmol/L KCl without any significant decrease in [Ca2+]i as determined by fura-2 microfluorometry (IC50: 5.1 +/- 4.6 micromol/L). The relaxation induced by hydroxyfasudil was accompanied with dephosphorylation of the myosin light chain. In the permeabilized preparation, hydroxyfasudil inhibited the contraction induced by ET-1, guanosine 5'-O-(3-thiotriphosphate), or the catalytic subunit of rho-associated kinase, but it did not inhibit Ca2+-induced contraction under the condition of inhibited myosin light chain phosphatase. Hydroxyfasudil showed a greater relaxant effect under decreased adenosine triphosphate (ATP) levels. The present study indicated that hydroxyfasudil relaxes the rabbit basilar artery mainly by disinhibiting myosin light chain phosphatase through the inhibition of rho-associated kinase and that this effect depends on the intracellular ATP concentration.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Animals; Basilar Artery; Calcium; Endothelin-1; Enzyme Activation; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Myosin-Light-Chain Phosphatase; Phorbol 12,13-Dibutyrate; Phosphoprotein Phosphatases; Protein Kinase C; Rabbits; Vasodilator Agents

Long-term inhibition of nitric oxide (NO) synthesis with N(omega)-nitro-L-arginine methyl ester (L-NAME) induces coronary vascular remodeling in rats. To determine the pathogenic mechanism involved in vascular remodeling, we examined the effects of fasudil, a Rho-kinase inhibitor, on vascular lesion formation. In rats treated with L-NAME at 10 mg/kg/day, vascular remodeling was evident in both large and small coronary arteries at the fourth week. Fasudil (3 mg/kg, p.o., twice daily) markedly prevented the development of vascular remodeling in small coronary arteries. Coronary flow was measured in Langendorff perfused isolated heart preparations. Long-term treatment with L-NAME caused a significant decrease in coronary flow, which was significantly inhibited by fasudil. Fasudil suppressed the structural and functional changes in coronary arteries by chronic blockade of NO synthesis. Thus, the Rho-kinase pathway may be substantially involved in the pathogenesis of vascular remodeling in this rat model.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Administration, Oral; Animals; Area Under Curve; Coronary Circulation; Coronary Vessels; Enzyme Inhibitors; Heart; Heart Rate; Heart Ventricles; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Protein Serine-Threonine Kinases; Rats; Rats, Inbred WKY; rho-Associated Kinases

1. The effects of Rho-kinase inhibitor, fasudil, and of a more specific Rho-kinase inhibitor, hydroxyfasudil, on pacing-induced myocardial ischaemia were determined in anaesthetized open-chest dogs. 2. The dogs were subjected to left anterior descending coronary artery (LAD) stenosis producing a sufficient ischaemia as measured by ST-segment depression on electrocardiograms only when the hearts were paced 60 beats min(-1) above the baseline. After a recovery (nonpacing) period, drugs or saline were infused intravenously over 30 min. The animals were again subjected to 5 min of pacing 25 min after the initiation of the treatment. 3. Hydroxyfasudil (0.1 and 0.3 mg kg(-1)) and fasudil (0.3 mg kg(-1)) suppressed the ST-segment depression. Hydroxyfasudil and fasudil also increased the regional blood flow of the LAD perfused endomyocardium region in the canine model of effort angina. 4. To determine the flow profile for hydroxyfasudil in dogs, blood flow in three vascular beds was measured. Hydroxyfasudil (0.3 mg kg(-1) for 30 min) significantly increased coronary blood flow and vertebral blood flow, without significantly changing the femoral blood flow. 5. Hydroxyfasudil had no inotropic or chronotropic effect on the isolated hearts of guinea-pigs. Hydroxyfasudil (2 mg kg(-1) for 20 min) did not affect the PR or QTc interval in anaesthetized dogs. 6. Inhibition of Rho-kinase appears to protect myocardium subjected to pacing-induced ischaemia through the increase in the regional myocardial blood flow. Hydroxyfasudil may be categorized as a novel type of anti-anginal drug, without any inotropic or chronotropic effects.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Anesthesia; Angina Pectoris; Animals; Cardiac Pacing, Artificial; Coronary Circulation; Coronary Stenosis; Dogs; Dose-Response Relationship, Drug; Electrocardiography; Enzyme Inhibitors; Female; Guinea Pigs; Heart Atria; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Male; Myocardial Ischemia; Physical Exertion; Protein Serine-Threonine Kinases; Regional Blood Flow; rho-Associated Kinases; Vasodilator Agents

The Nef protein of HIV-1 binds to and induces apoptotic cytolysis of uninfected but activated human peripheral blood mononuclear cells (PBMC) and various cell line cells derived from CD4+ T, CD8+ T and B lymphocytes, macrophages, and neutrophils. The Nef-induced apoptosis also occurs with blood cells not expressing CD95 (Fas). The Nef-induced apoptosis as well as Fas-mediated apoptosis was inhibited by acetyl-Try-Val-Ala-Asp-CHO, an IL-1beta converting enzyme (ICE) inhibitor. On the other hand, serine/threonine protein kinase (PK) inhibitors, H-7, fasudil hydrochloride and M3, inhibited the Nef-induced apoptosis, and not the Fas-mediated one, without affecting the cell-binding activity of Nef and Nef-binding capacity of the activated cells. Preincubation of the cells with the drugs before being bound by Nef was required for the inhibition of apoptosis. These results suggest that the PK inhibitors specifically act on a cellular protein involved in the upper stream of signal transduction pathway of the Nef-induced apoptosis, which is different from the Fas-mediated pathway but meets it upstream of ICE. In addition, the drugs suppressed the cellular activation-associated cell surface expression of a putative Nef-binding protein in PBMC, although they had no influence on its expression in cell line cells. These findings suggest the feasibility of clinical use of the PK inhibitors to prevent the development of AIDS by inhibiting the Nef-induced apoptosis of uninfected blood cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acquired Immunodeficiency Syndrome; Adult; Anti-HIV Agents; Apoptosis; Enzyme Inhibitors; fas Receptor; Feasibility Studies; Gene Products, nef; HIV-1; Humans; nef Gene Products, Human Immunodeficiency Virus; Protein Serine-Threonine Kinases; Receptors, HIV; T-Lymphocytes; Tumor Cells, Cultured

Calponin is a thin filament-associated protein which has been implicated in the modulation of the contractile state of smooth muscle via its interaction with actin and inhibition of the actin-activated myosin Mg-ATPase. This inhibitory effect is alleviated by phosphorylation of calponin at Ser175 in vitro by protein kinase C. The issue of calponin phosphorylation in intact smooth muscle in response to agonists that activate protein kinase C is controversial. We have produced a monoclonal antibody that specifically recognizes calponin phosphorylated at Ser175 and used it to analyze calponin phosphorylation in porcine coronary arterial smooth muscle stimulated with prostaglandin F2alpha or phorbol 12,13-dibutylate (PDB). Calponin phosphorylation increased rapidly in response to prostaglandin F2alpha concomitant with the increase in tension. Calponin was then dephosphorylated while force was maintained. Tension development in response to PDB was significantly slower, but again calponin phosphorylation paralleled force development. In this case, calponin dephosphorylation was very slow, consistent with prolonged activation of protein kinase C. The protein kinase inhibitors, HA1077 (1-5-(isoquinoline sulfonyl)-homopiperazine HCl) and HA1100 (1-hydroxy HA1077; 1-(hydroxy-5-isoquinoline sulfonyl-homopiperazine), inhibited tension development and calponin phosphorylation in a concentration-dependent manner with similar ED50 values in response to prostaglandin F2alpha and PDB. These results support physiological roles for calponin in force development in smooth muscle in response to agonists which trigger protein kinase C activation and in the latch state, i.e., force maintenance at low energy cost. Furthermore, the vasodilator effect of HA1077 and HA1100 is more likely due to inhibition of protein kinase C than of myosin light chain kinase.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Arteries; Calcium-Binding Proteins; Calponins; Carrier Proteins; Coronary Vessels; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Microfilament Proteins; Muscle Contraction; Muscle, Smooth, Vascular; Phosphorylation; Protein Kinase C; Serine; Swine; Vasodilator Agents