ha-1100 and Disease-Models--Animal

This experimental study evaluated the anti-asthmatic potential of the Rho-kinase inhibitor hydroxyfasudil in the settings of allergen-induced allergen-induced experimental asthma.. Chronic allergic airway inflammation was caused by 28 days-sensitisation of guinea pigs with ovalbumin (OVA). Hydroxyfasudil was administered intraperitoneally in two doses for the last two weeks (1 mg/kg b.w.; 10 mg/kg b.w.). The degree of allergic inflammation was determined based on concentrations of inflammatory Th2 cytokines (IL-4, IL-13), Th1 cytokines (TNF-α and IFN-γ) in the lung homogenate and leukocyte count in the bronchoalveolar lavage fluid (BALF). The markers of remodelling and fibrosis, the growth factors (TGF-β1, EGF), EGF receptor, collagen type III and V were estimated in lung homogenate. The changes in specific airway resistance (sRaw) were used as an in vivo bronchial hyperreactivity parameter.. Hydroxyfasudil administration at both doses significantly reduced sRaw after a week of therapy. We observed a decline of IL-13, TNF-α and IFN-γ in lung homogenate and a lower presence of lymphocytes in BALF after 14 days of hydroxyfasudil administration at both tested doses. Hydroxyfasudil 14 days-treatment at both doses effectively reduced the concentrations of TGF-β1, EGF receptors, collagen type III and V in BALF and modulated EGF levels.. These findings indicate that RhoA/Rho-kinase is involved in the pathophysiology of allergic airway inflammation and suggest that Rho-kinase inhibitor hydroxyfasudil has therapeutic potential for asthma management.

Topics: Allergens; Animals; Anti-Asthmatic Agents; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Epidermal Growth Factor; Guinea Pigs; Inflammation; Interleukin-13; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; rho-Associated Kinases; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

The Rho kinase (ROCK) pathway is implicated in the pathogenesis of several conditions, including neurological diseases. In Huntington's disease (HD), ROCK is implicated in mutant huntingtin (HTT) aggregation and neurotoxicity, and members of the ROCK pathway are increased in HD mouse models and patients. To validate this mode of action as a potential treatment for HD, we sought a potent, selective, central nervous system (CNS)-penetrant ROCK inhibitor. Identifying a compound that could be dosed orally in mice with selectivity against other AGC kinases, including protein kinase G (PKG), whose inhibition could potentially activate the ROCK pathway, was paramount for the program. We describe the optimization of published ligands to identify a novel series of ROCK inhibitors based on a piperazine core. Morphing of the early series developed in-house by scaffold hopping enabled the identification of a compound exhibiting high potency and desired selectivity and demonstrating a robust pharmacodynamic (PD) effect by the inhibition of ROCK-mediated substrate (MYPT1) phosphorylation after oral dosing.

Topics: Animals; Brain; Disease Models, Animal; Huntingtin Protein; Huntington Disease; Mice; Protein Kinase Inhibitors; rho-Associated Kinases

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Mutations in PKD1 (encoding for polycystin-1 [PC1]) are found in 80%-85% of patients with autosomal dominant polycystic kidney disease (ADPKD). We tested the hypothesis that changes in actin dynamics result from PKD1 mutations through dysregulation of compartmentalized centrosomal RhoA signaling mediated by specific RhoGAP (ARHGAP) proteins resulting in the complex cellular cystic phenotype. Initial studies revealed that the actin cytoskeleton was highly disorganized in cystic cells derived from patients with PKD1 and was associated with an increase in total and centrosomal active RhoA and ROCK signaling. Using cilia length as a phenotypic readout for centrosomal RhoA activity, we identified ARHGAP5, -29, and -35 as essential regulators of ciliation in normal human renal tubular cells. Importantly, a specific decrease in centrosomal ARHGAP35 was observed in PKD1-null cells using a centrosome-targeted proximity ligation assay and by dual immunofluorescence labeling. Finally, the ROCK inhibitor hydroxyfasudil reduced cyst expansion in both human PKD1 3D cyst assays and an inducible Pkd1 mouse model. In summary, we report a potentially novel interaction between PC1 and ARHGAP35 in the regulation of centrosomal RhoA activation and ROCK signaling. Targeting the RhoA/ROCK pathway inhibited cyst formation in vitro and in vivo, indicating its relevance to ADPKD pathogenesis and for developing new therapies to inhibit cyst initiation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Animals; Cell Line; Centrosome; Cilia; Disease Models, Animal; Guanine Nucleotide Exchange Factors; Humans; Mice, Transgenic; Mutation; Polycystic Kidney, Autosomal Dominant; Protein Kinase C; Repressor Proteins; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; TRPP Cation Channels

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system characterized by oligodendrocyte loss and progressive neurodegeneration. The cuprizone (CPZ)-induced demyelination is widely used to investigate the demyelination/remyelination. Here, we explored the therapeutic effects of Hydroxyfasudil (HF), an active metabolite of Fasudil, in CPZ model. HF improved behavioral abnormality and reduced myelin damage in the corpus callosum. Splenic atrophy and myelin oligodendrocyte glycoprotein (MOG) antibody were observed in CPZ model, which were partially restored and obviously inhibited by HF, therefore reducing pathogenic binding of MOG antibody to oligodendrocytes. HF inhibited the percentages of CD4

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Behavior, Animal; Brain; CD4-Positive T-Lymphocytes; Cuprizone; Cytokines; Demyelinating Diseases; Disease Models, Animal; Macrophages; Male; Mice, Inbred C57BL; Motor Activity; Spleen

Cystitis is defined as an inflammation of the bladder caused by a bacterial infection, and it can be dangerous and painful when it spreads through the internal organs. In this study, antioxidant effects of hydroxylfasudil (HF) at the enzymatic and molecular level on kidney and liver tissues in cystitis rat model, which is caused by inflammation of the rat bladder with a protamine sulphate (PS), was examined. Quantitative changes of reduced glutathione (GSH) and lipid peroxidation (LPO) levels, which are a marker for oxidative stress, were determined in rat kidney and liver tissues for each groups. And then molecular and biochemical impact of HF treatment on antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) in cystitis model were studied. The results suggest that HF could be beneficial to the renal and hepatic antioxidant system. Thus, HF might be used as a novel therapeutics agent to eliminate interstitial cystitis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antioxidants; Catalase; Cystitis; Disease Models, Animal; Glutathione; Kidney; Lipid Peroxidation; Liver; Protamines; Rats; Superoxide Dismutase

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

We previously demonstrated that inactivation of Rho-kinase by hydroxyfasudil could impact N-methyl-d-aspartate (NMDA) excitatory interneurons in the hippocampus and attenuate the spatial learning and memory dysfunction of rats caused by chronic forebrain hypoperfusion ischemia. Complementary interactions between the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter GABA form the molecular basis of synaptic plasticity and cognitive performance. However, whether the GABAergic inhibitory interneurons are involved in the mechanisms underlying these processes remains unclear. Here, we further examined the role of GABAergic interneurons in the neuroprotective effect of the Rho-kinase inhibitor. Chronic forebrain ischemia was induced in Wistar rats by bilateral common carotid artery occlusion (BCAO). The general synaptic transmission and long-term potentiation (LTP) of hippocampal CA3 neurons were evaluated at 30 days after sham surgery or BCAO. Real-time PCR and Western blot analyses were conducted to determine the effect of the Rho-kinase inhibitor hydroxyfasudil on GABAergic inhibitory interneuron expression and function after ischemia. Hydroxyfasudil showed no significant effect on general synaptic transmission, but it could abolish the inhibition of LTP induced by chronic forebrain ischemia. Moreover, the mRNA and protein levels of GABAA and GABAB in three brain regions after ischemia were markedly decreased, and hydroxyfasudil could up-regulate all mRNA and protein expression levels in these areas except for GABAA mRNA in the cerebral cortex and striatum. Using phosphorylation antibodies against specific sites on the GABAA and GABAB receptors, we further demonstrated that hydroxyfasudil could inhibit GABAergic interneuron phosphorylation triggered by the theta burst stimulation. In summary, our results indicated that the inactivation of Rho-kinase could enhance GABAA and GABAB expressions by different mechanisms to guarantee the induction of hippocampal LTP, and it could decrease the phosphorylation level of GABAergic inhibitory interneurons to promote the LTP induction rate and magnitude, hence improving the cognitive deficit suffered after chronic forebrain ischemia.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Brain Ischemia; Chronic Disease; Disease Models, Animal; Interneurons; Long-Term Potentiation; Male; Phosphorylation; Prosencephalon; Protein Kinase Inhibitors; Random Allocation; Rats, Wistar; Receptors, GABA-A; Receptors, GABA-B; rho-Associated Kinases; RNA, Messenger; Up-Regulation

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

We investigated the anti-vasospastic potential of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, after subarachnoid hemorrhage (SAH) and also its effect on hemorheological abnormalities following cerebral ischemia. Chronic cerebral vasospasm was produced using a two-hemorrhage canine model. On day 7, angiographic vasospasm was observed in all animals, and intravenous administration of hydroxyfasudil (3 mg·kg(-1)·30 min(-1)) significantly reversed the vasospasm (predose diameter of the basilar artery, 57.9% ± 2.0% of the baseline before the injection of blood; postdose diameter, 64.5% ± 1.9%). The viscosity of whole blood was significantly increased 24 h after 1 h middle cerebral artery occlusion in rats. Hydroxyfasudil (3 and 10 mg/kg, i.p.) significantly decreased blood viscosity. The specificity of hydroxyfasudil was examined against a panel of 17 protein kinases using ELISA analysis. Hydroxyfasudil inhibited Rho-kinase α and β at a concentration of 10 µM by 97.6% and 97.7%, respectively. No other protein kinase was inhibited with 10 µM hydroxyfasudil by over 40%. The present results indicate hydroxyfasudil is a selective inhibitor of Rho-kinase. The results also suggest that hydroxyfasudil contributes to the potency of fasudil to prevent cerebral vasospasm and hyperviscosity and suggest the potential utility of hydroxyfasudil as a therapeutic agent for patients with SAH.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Blood Viscosity; Brain Ischemia; Disease Models, Animal; Dogs; Female; Hematocrit; Male; Protein Kinase Inhibitors; Rats; Rats, Wistar; rho-Associated Kinases; Subarachnoid Hemorrhage; Vasospasm, Intracranial

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

To investigate the effects of the rho-kinase inhibitor hydroxyfasudil on bladder overactivity in cyclophosphamide (CYP)-induced cystitis.. Female Sprague-Dawley rats received a single intraperitoneal injection of CYP (200 mg/kg). Four days later, bladder function was evaluated by: (i) monitoring micturition behavior in metabolic cages between hydroxyfasudil- and vehicle-treated animals; (ii) measuring changes in continuous cystometrograms in response to intravenous hydroxyfasudil under anesthesia; and (iii) conducting a functional study examining the effect of hydroxyfasudil on the concentration-response curves to carbachol in bladder tissue strips.. Intraperitoneal injection of hydroxyfasudil (10 mg/kg) significantly increased both the average and maximal voided volumes. Hydroxyfasudil significantly decreased the maximal detrusor pressure, whereas the intercontraction interval was not significantly affected. After administration of 0.1, 0.3, 1, and 3 microM hydroxyfasudil, the maximal contraction of the concentration-response curves to carbachol was significantly reduced to 74.5 +/- 4.2%, 55.2 +/- 5.6%, 29.4 +/- 5.6%, and 21.6 +/- 8.2% of the control values, respectively.. The present findings indicate that hydroxyfasudil might be a new treatment option for CYP-induced detrusor overactivity.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Disease Models, Animal; Female; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Urinary Bladder, Overactive

(1) The role of activation of Rho-kinase in the pathogenesis of cognitive deficit and neuronal damage caused by chronic global ischemia is not clear. In this study, hydroxyfasudil, a Rho-kinase inhibitor, was found to improve the learning and memory performance significantly in rats with ischemia induced by chronic cerebral hypoperfusion after permanent bilateral carotid artery ligation (BCAL). This was observed by the administration of hydroxyfasudil (1 mg/kg or 10 mg/kg, once per day for 30 days) to ischemic rats and the measurements of escape latency and time spent in the target quadrant among the ischemic, sham, and ischemic plus hydroxyfasudil rats by the method of Morris water maze. (2) In electrophysiological study, hydroxyfasudil abolished the inhibition of long-term potentiation (LTP) in rats with ischemia. Morphologically, it also markedly reduced pathological changes such as neuronal cells loss and nuclei shrinkage in cortex and hippocampus of ischemic rats. Biochemical analysis showed that the inhibition of Rho-kinase by hydroxyfasudil reduced the amount of MDA and increased the activities of SOD and GPx in ischemic rats that had increased MDA and decreased SOD and GPx activities. (3) To explore mechanism (s) of the beneficial effects of hydroxyfasudil in ischemia, we performed immunohistochemistry and RT-PCR analyses of NMDA NR2B subunit and for the first time found that hydroxyfasudil increased the expression of NR2B in cortex and hippocampus at both protein and mRNA levels. (4) Taken together, our data further support the notion that the inhibition of Rho-kinase provides neuroprotective effects in cerebral ischemia.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Brain Infarction; Brain Ischemia; Cerebral Cortex; Chronic Disease; Cognition Disorders; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hippocampus; Long-Term Potentiation; Male; Maze Learning; Nerve Degeneration; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; rho-Associated Kinases; Superoxide Dismutase; Superoxide Dismutase-1; Treatment Outcome; Up-Regulation

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

We have recently demonstrated that protein kinase C (PKC) and Rho-kinase play important roles in coronary vasospasm in a porcine model. However, it remains to be examined whether there is an interaction between the two molecules to cause the spasm.. A segment of left porcine coronary artery was chronically treated with IL-1beta-bound microbeads in vivo. Two weeks after the operation, phorbol ester caused coronary spasm in vivo and coronary hypercontractions in vitro at the IL-1beta-treated segment; both were significantly inhibited by hydroxyfasudil, a specific Rho-kinase inhibitor. Guanosine 5'-[gamma-thio]triphosphate (GTPgammaS), which activates Rho with a resultant activation of Rho-kinase, enhanced Ca2+ sensitization of permeabilized vascular smooth muscle cells, which were resistant to the blockade of PKC by calphostin C. The GTPgammaS-induced Ca2+ sensitization was greater in the spastic segment than in the control segment. Western blot analysis revealed that only PKCdelta isoform was activated during the hypercontraction.. These results demonstrate that PKC and Rho-kinase coexist on the same intracellular signaling pathway, with PKC located upstream on Rho-kinase, and that among the PKC isoforms, only PKCdelta may be involved. Thus, the strategy to inhibit Rho-kinase rather than PKC may be a more specific and useful treatment for coronary spasm.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Blotting, Western; Calcium; Capillary Permeability; Coronary Vasospasm; Coronary Vessels; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Guanosine 5'-O-(3-Thiotriphosphate); In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Male; Monomeric GTP-Binding Proteins; Muscle Contraction; Muscle, Smooth, Vascular; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein Serine-Threonine Kinases; Protein Transport; rho-Associated Kinases; Swine