Rac对失血性休克大鼠血管反应性的调节作用

目的探讨Rac在失血性休克大鼠血管反应性调节中的作用。方法采用SD大鼠复制休克模型,取离体血管环,观察休克早期和晚期血管反应性的变化以及Rac激动剂和特异性抑制剂对休克早期和晚期血管反应性的影响;通过酶消化法培养原代血管平滑肌细胞（vascular smoot hmuscle cell,VSMC）,采用双室培养方式分别观察VSMC缺氧10min和90min后VSMC对去甲肾上腺素（norepinephrine,NE）的收缩反应性变化,同时观察Rac活性调节剂对缺氧后VSMC收缩反应性的影响。结果在休克早期和短暂缺氧后,离体血管环和VSMC对NE收缩反应性均有所升高,Rac的激动剂血小板衍生生长因子（platelet derived growth factor,PDGF）可部分降低休克早期或短暂缺氧后血管反应性,Rac特异性抑制剂NSC23766可拮抗由PDGF所引起的血管反应性的变化,而在休克晚期或长时间缺氧后,离体血管环和VSMC对NE收缩反应性明显降低,NSC23766对休克晚期或长时间缺氧所致血管反应性降低有升高作用。结论休克后血管反应性呈双相变化,休克早期升高,休克晚期降低,Rac参与了休克血管反应性的调节。     

Berberine inhibits platelet-derived growth factor-induced growth and migration partly through an AMPK-dependent pathway in vascular smooth muscle cells

Platelet-derived growth factor (PDGF) is released from vascular smooth muscle cells (VSMCs), endothelial cells, or macrophages after percutaneous coronary intervention and is related with neointimal proliferation and restenosis. Berberine is a well-known component of the Chinese herb medicine Huanglian (Coptis chinensis), and is capable of inhibiting growth and endogenous PDGF synthesis in VSMCs after in vitro mechanical injury. We analyzed the effects of berberine on VSMC growth, migration, and signaling events after exogenous PDGF stimulation in vitro in order to mimic a post-angioplasty PDGF shedding condition. Pretreatment of VSMCs with berberine inhibited PDGF-induced proliferation. Berberine significantly suppressed PDGF-stimulated Cyclin D1/D3 and Cyclin-dependent kinase (Cdk) gene expression. Moreover, berberine increased the activity of AMP-activated protein kinase (AMPK), which led to phosphorylation activation of p53 and increased protein levels of the Cdk inhibitor p21(Cip1). Compound C, an AMPK inhibitor, partly but significantly attenuated berberine-elicited growth inhibition. In addition, stimulation of VSMCs with PDGF led to a transient increase in GTP-bound, active form of Ras, Cdc42 and Rac1, as well as VSMC migration. However, pretreatment with berberine significantly inhibited PDGF-induced Ras, Cdc42 and Rac1 activation and cell migration. Co-treatment with farnesyl pyrophosphate and geranylgeranyl pyrophosphate drastically reversed berberine-mediated anti-proliferative and migratory effects in VSMCs. Based on these findings, we conclude that berberine inhibited PDGF-induced VSMC growth via activation of AMPK/p53/p21(Cip1) signaling while inactivating Ras/Rac1/Cyclin D/Cdks and suppressing PDGF-stimulated migration via inhibition of Rac1 and Cdc42. These observations offer a molecular explanation for the anti-proliferative and anti-migratory properties of berberine.     

Beneficial effect of arginine vasopressin on hemorrhagic shock through improving the vascular reactivity

The vascular reactivity and calcium sensitivity were decreased following hemorrhagic shock. Arginine vasopressin (AVP) was beneficial to endotoxic, infectious/spetic and hemorrhagic shock. Our previous studies found that Rho kinase played an important role in the occurrence of calcium desensitization following shock. It was reported that AVP was with stimulation effect of Rho kinase. So we hypothesized that AVP might have beneficial effect on shock via activation of Rho kinase to regulate the calcium sensitivity and vascular reactivity. Hemorrhagic shock (40 mmHg for 2 h) Wistar rats in vivo were adopted to observe the effects of small dose of AVP on hemodynamics, 24-h survival rate, the pressor effect of norepinephrine (NE) and the contractility of superior mesenteric artery (SMA). Isolated SMAs from hemorrhagic shock rats were adopted to observe the effects of AVP on vascular reactivity and calcium sensitivity and its relationship to Rho kinase with an isolated organ perfusion system. The results show that AVP at the concentration of 0.1 U/kg and 0.4 U/kg significantly improved the hemodynamic parameters and the 24-h survival rate of hemorrhagic shock rats. Meanwhile, these dosages of AVP significantly increased the pressor effect of NE and the contractile response of SMA to NE. Y-27632 (3 μg/kg), a Rho kinase specific inhibitor, abolished the beneficial effects of AVP. In vitro, the calcium sensitivity and vascular reactivity of SMA to calcium and NE were significantly decreased following hemorrhagic shock. AVP at the concentration of 0.5 nmol/L and 5 nmol/L significantly increased the calcium sensitivity and vascular reactivity. These effects of AVP were abolished by Y-27632 (10 μmol/L). Taken together, the results suggest that AVP at 0.1 U/kg and 0.4 U/kg is beneficial to hemorrhagic shock by improving the vascular reactivity, which involves activation of Rho kinase.     

The role of Rho-associated kinase in differential regulation by statins of interleukin-1beta- and lipopolysaccharide-mediated nuclear factor kappaB activation and inducible nitric-oxide synthase gene expression in vascular smooth muscle cells

An optimal level of NO has protective effects in atherosclerosis, whereas large amounts contribute to septic shock. To study how statins, the potent inhibitors of cholesterol synthesis, regulate NO in the vascular wall, we determined their effects on interleukin-1beta (IL-1beta)- and lipopolysaccharide (LPS)-induced NO production in aortic vascular smooth muscle cells (VSMCs). Compared with the large amounts of NO and inducible NO synthase (iNOS) protein expression induced by LPS, the responses of IL-1beta were modest. Various statins were found to inhibit LPS-induced iNOS expression and NO production, although they potentiated IL-1beta responses. In addition, fluvastatin increased IL-1beta-induced p65 nuclear translocation and nuclear factor kappaB (NF-kappaB) activity, although it inhibited those induced by LPS. To address the role of small G proteins in statin's actions, farnesyl transferase inhibitors [alpha-hydroxyfarne-sylphosphonic acid and (2S)-2-[[(2S)-2-[(2S,3S)-2-[(2R)-2-amino-3-mercaptopropyl]amino]-3-methylpentyl]oxy]-1-oxo-3-phenylpropyl]amino]-4-(methylsulfonyl)-butanoic acid 1-methylethyl ester (L-744382)], Rac inhibitor (NSC23766), and Rho-associated kinase (ROCK) inhibitor [N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride (Y-27632)] were used. We found that Y-27632 potentiated IL-1beta-induced iNOS expression, p65 nuclear translocation, IkappaB kinase (IKK), and NF-kappaB activation, whereas it had minimal effects on LPS-induced responses. In contrast, farnesyl transferase inhibitors blocked iNOS protein expression induced by LPS and IL-1beta, whereas NSC23766 had no effect. Further studies showed that LPS down-regulated Rho and ROCK activity, whereas IL-1beta increased them, suggesting a negative role of Rho and ROCK signaling, which is regulated in contrary manners by IL-1beta and LPS, in IKK/NF-kappaB activation. Through abrogating this negative signaling, statins differentially regulate iNOS expression induced by LPS and IL-1beta in VSMCs. These differential actions of statins on iNOS gene regulation might provide an additional explanation for the pleiotropic beneficial effects of statins.     

缝隙连接及其连接蛋白对血管加压素诱导的失血性休克大鼠血管收缩的作用研究

摘要：目的 观察缝隙连接（GJ）及其组成亚单位连接蛋白（Cx）在血管加压素（AVP）诱导失血性休克大鼠血管收缩中的作用。方法 采用失血性休克大鼠模型和缺氧培养血管平滑肌细胞（VSMC）,观察GJ阻断剂CBX和octanol、以及各Cx亚型反义寡核苷酸（AODN）对AVP诱导的血管收缩反应的影响,随后进一步观察参与AVP作用的Cx37和Cx43对AVP调节休克血管钙敏感性和缺氧VSMC内钙离子浓度的影响。结果 GJ阻断剂CBX和octanol明显抑制了AVP诱导的休克血管的收缩反应。在所有血管中表达的连接蛋白中,Cx37AODN和Cx43AODN明显抑制了AVP的血管收缩作用。进一步结果显示,Cx43AODN、而不是Cx37AODN,可拮抗AVP升高休克血管钙敏感性的作用。此外, AVP处理和干扰Cx37及Cx43对缺氧VSMC内钙离子浓度无明显影响。结论 缝隙连接在休克后AVP介导的血管收缩调节中有重要作用,Cx37和Cx43参与了这一过程,其中Cx43可能通过影响AVP介导的血管钙敏感性调节途径来发挥作用,而Cx37可能通过其它机制来参与AVP的血管调节作用。     

The Role of Rac1 on Carbachol‐induced Contractile Activity in Detrusor Smooth Muscle from Streptozotocin‐induced Diabetic Rats

This study was designed to determine the role of the small GTPase Rac1 on carbachol‐induced contractile activity in detrusor smooth muscle using small inhibitor NSC 23766 in diabetic rats. Rac1 expression in bladder tissue was also evaluated. In the streptozotocin (STZ)‐induced diabetic rat model, three study groups were composed of control, diabetic and insulin‐treated diabetic subjects. The detrusor muscle strips were suspended in organ baths at the end of 8–12 weeks after STZ injection. Carbachol (CCh) (10^?9–10^?4 M) concentration–response curves were obtained both in the absence and in the presence of Rac1 inhibitor NSC 23766 (0.1, 1 and 10 μM). Diabetes‐related histopathological changes and Rac1 expressions were assessed by haematoxylin and eosin staining and immunohistochemical staining, respectively. CCh caused dose‐dependent contractile responses in all the study groups. Rac1 inhibitor NSC 23766 inhibited CCh‐induced contractile responses in all groups, but this inhibition seen in both diabetes groups was greater than in the control group. Histological examination revealed an increased bladder wall thickness both in the diabetes and in the insulin‐treated diabetes groups compared to the control group. In immunohistochemical staining, expression of Rac1 was observed to be increased in all layers of bladder in both diabetic groups compared to the control group. In the diabetic bladders, increased expression of Rac1 and considerable inhibition of CCh‐induced responses in the presence of NSC 23766 compared to those of the control group may indicate a specific role of Rac1 in diabetes‐related bladder dysfunction, especially associated with cholinergic mediated detrusor overactivity.     

Structure-function based design of small molecule inhibitors targeting Rho family GTPases

Rho GTPases of the Ras superfamily are involved in the regulation of multiple cell functions and have been implicated in the pathology of various human diseases including cancer. They are attractive drug targets in future targeted therapy. A wealth of structure-function information made available by high resolution structures and mutagenesis studies has laid out the foundation for the derivation of a mechanism-based targeting strategy. Here we describe the rational design and characterizations of a first generation Rac-specific small molecule inhibitor. Based on the structure-function information of Rac interaction with GEFs, in a computer based Virtual Screening we have identified NSC23766, a highly soluble and membrane permeable compound, as a specific inhibitor of a subset of GEF binding to Rac and therefore Rac activation. In fibroblast cells NSC23766 inhibited Rac1 GTP-loading without affecting Cdc42 or RhoA activity and suppressed the Rac-GEF, Tiam1, and oncogenic Ras induced cell growth and transformation. NSC23766 also potently inhibited the prostate PC-3 cancer cell proliferation and invasion induced by Rac hyperactivation. Intraperitoneal administration of NSC23766 to laboratory mice resulted in effective Rac GTPase suppression and hematopoietic stem cell mobilization from the bone marrow to the peripheral blood, similar to the effects of genetically targeted disruption of Rac GTPases in the animals. A co-crystal structure of NSC23766 bound to Rac1 provided further insight for future medicinal chemistry modification and improvement of this lead Rac-specific inhibitor. Thus, structure-function based rational design may represent a new avenue for generating lead small molecule inhibitors of Ras superfamily GTPases that are useful for modulating pathological conditions in which the small GTPase deregulation may play a role.     

Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic β-cells

The phagocytic NADPH oxidase [NOX] has been implicated in the generation of superoxides in the pancreatic β-cell. Herein, using normal rat islets and clonal INS 832/13 cells, we tested the hypothesis that activation of the small G-protein Rac1, which is a member of the NOX holoenzyme, is necessary for palmitate [PA]-induced generation of superoxides in pancreatic β-cells. Incubation of isolated β-cells with PA potently increased the NOX activity culminating in a significant increase in the generation of superoxides and lipid peroxides in these cells; such effects of PA were attenuated by diphenyleneiodonium [DPI], a known inhibitor of NOX. In addition, PA caused a transient, but significant activation [i.e., GTP-bound form] of Rac1 in these cells. NSC23766, a selective inhibitor of Rac1, but not Cdc42 or Rho activation, inhibited Rac1 activation and the generation of superoxides and lipid peroxides induced by PA. Fumonisin B-1 [FB-1], which inhibits de novo synthesis of ceramide [CER] from PA, also attenuated PA-induced superoxide and lipid peroxide generation and NOX activity implicating intracellularly generated CER in the metabolic effects of PA; such effects were also demonstrable in the presence of the cell-permeable C2-CER. Further, NSC23766 prevented C2-CER-induced Rac1 activation and production of superoxides and lipid peroxides. Lastly, C2-CER, but not its inactive analogue, significantly reduced the mitochondrial membrane potential, which was prevented to a large degree by NSC23766. Together, our findings suggest that Tiam1/Rac1 signaling pathway regulates PA-induced, CER-dependent superoxide generation and mitochondrial dysfunction in pancreatic β-cells.     

5,8-Dimethoxy-2-Nonylamino-Naphthalene-1,4-Dione Inhibits Vascular Smooth Muscle Cell Proliferation by Blocking Autophosphorylation of PDGF-Receptor β

As the abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis and vascular restenosis, a candidate drug with antiproliferative properties is needed. We investigated the antiproliferative action and underlying mechanism of a newly synthesized naphthoquinone derivative, 5,8-dimethoxy-2-nonylamino-naphthalene-1,4-dione (2-nonylamino-DMNQ), using VSMCs treated with platelet-derived growth factor (PDGF). 2-Nonylamino-DMNQ inhibited proliferation and cell number of VSMCs induced by PDGF, but not epidermal growth factor (EGF), in a concentration-dependent manner without any cytotoxicity. This derivative suppressed PDGF-induced [³H]-thymidine incorporation, cell cycle progression from G₀/G₁ to S phase, and the phosphorylation of phosphor-retinoblastoma protein (pRb) as well as the expression of cyclin E/D, cyclin-dependent kinase (CDK) 2/4, and proliferating cell nuclear antigen (PCNA). Importantly, 2-nonylamino-DMNQ inhibited the phosphorylation of PDGF receptorβ(PDGF-Rβ) enhanced by PDGF at Tyr⁵⁷⁹, Tyr⁷¹⁶, Tyr⁷⁵¹, and Tyr¹⁰²¹ residues. Subsequently, 2-nonylamino-DMNQ inhibited PDGF-induced phosphorylation of STAT3, ERK1/2, Akt, and PLCγ1. Therefore, our results indicate that 2-nonylamino-DMNQ inhibits PDGF-induced VSMC proliferation by blocking PDGF-Rβ autophosphorylation, and subsequently PDGF-Rβ-mediated downstream signaling pathways.     

Inhibition of prostate smooth muscle contraction and prostate stromal cell growth by the inhibitors of Rac,NSC23766 and EHT1864

Background and Purpose

Medical therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) targets smooth muscle contraction in the prostate, or prostate growth. However, current therapeutic options are insufficient. Here, we investigated the role of Rac in the control of smooth muscle tone in human prostates and growth of prostate stromal cells.

Experimental Approach

Experiments were performed using human prostate tissues from radical prostatectomy and cultured stromal cells (WPMY-1). Expression of Rac was examined by Western blot and fluorescence staining. Effects of Rac inhibitors (NSC23766 and EHT1864) on contractility were assessed in the organ bath. The effects of Rac inhibitors were assessed by pull-down, cytotoxicity using a cell counting kit, cytoskeletal organization by phalloidin staining and cell growth using an 5-ethynyl-2′-deoxyuridine assay.

Key Results

Expression of Rac1–3 was observed in prostate samples from each patient. Immunoreactivity for Rac1–3 was observed in the stroma, where it colocalized with the smooth muscle marker, calponin. NSC23766 and EHT1864 significantly reduced contractions of prostate strips induced by noradrenaline, phenylephrine or electrical field stimulation. NSC23766 and EHT1864 inhibited Rac activity in WPMY-1 cells. Survival of WPMY-1 cells ranged between 64 and 81% after incubation with NSC23766 (50 or 100 μM) or EHT1864 (25 μM) for 24 h. NSC23766 and EHT1864 induced cytoskeletal disorganization in WPMY-1 cells. Both inhibitors impaired the growth of WPMY-1 cells.

Conclusions and Implications

Rac may be a link connecting the control of prostate smooth muscle tone with proliferation of smooth muscle cells. Improvements in LUTS suggestive of BPH by Rac inhibitors appears possible.     

Listeria monocytogenes induced Rac1-dependent signal transduction in endothelial cells

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. Small GTPases of the Rho family act as molecular switches in signal transduction. We tested the hypothesis that Rho GTPases contribute to the regulation of cytokine expression following L. monocytogenes infection. L. monocytogenes induced release of distinct CC and CXC, as well as Th1 and Th2 cytokines and growth factors by endothelial cells and activated RhoA and Rac1. Inhibition of Rac1 by inhibitor Nsc23766 reduced cytokine expression, and slightly yet significantly the uptake of bacteria. Blocking of Rho proteins by Clostridium difficile toxin B-10463 (TcdB) reduced Listeria-dependent cytokine expression, whereas activating Rho proteins by Escherichia coli CNF1 increased it. We analyzed regulation of IL-8 expression in more detail: Listeria-induced IL-8 release was reduced by inhibition of RhoA, Rac1 and Cdc42 (TcdB) or Rac1 while blocking of RhoA/B/C by Clostridium limosum C3 fusion toxin (C3FT) or Rho kinase by Y27632 reduced cytokine expression only slightly. Activation of RhoA, Rac1 and Cdc42 (CNF1), but not of RhoA alone (CNF(Y)), enhanced Listeria-dependent IL-8 release significantly. Furthermore, inhibition of RhoA, Rac1 and Cdc42 (TcdB) and Rac1 (Nsc23766), but not of RhoA (C3FT) reduced Listeria-related recruitment of NF-kappaB/p65 and RNA polymerase II to the il8 promoter, as well as acetylation of histone H4 and Ser10/Lys14-phosphorylation/acetylation of histone H3 at the il8 gene promoter in HUVEC. In conclusion, Rac1 contributed to L. monocytogenes-induced cytokine expression by human endothelial cells.     

Differential effect of simvastatin on various signal transduction intermediates in cultured human smooth muscle cells

The underlying mechanism of the antiproliferative effect of S (simvastatin), a HMG-CoA reductase inhibitor, in vascular smooth muscle cells (SMC) is still poorly understood. In the present study, we used synchronized human SMC, isolated from left interior mammary artery, as an in vitro model to test the effects of S on platelet-derived growth factor (PDGF)-induced DNA synthesis, extracellular-regulated kinase 1/2 (ERK1/2), p38/stress-activated protein kinase 2 (SAPK2), RhoA and Rac1 activation. ERK1/2 phosphorylation was triggered within 2 min of PDGF stimulation (early G1 phase) and was blocked by PD98059, a specific inhibitor of the ERK1/2 pathway, which also strongly inhibited PDGF-induced DNA synthesis (IC(50) = 10 micromol/L). PDGF quickly induced p38 phosphorylation (early G1 phase) and SB203580, a specific inhibitor of the p38/SAPK2 pathway, also blocked PDGF-induced DNA synthesis (IC(50) = 0.3 micromol/L). Translocation to the plasma membrane of small GTPases, such as RhoA and Rac1, could not be detected within 15 min of stimulation with PDGF or lysophosphatidic acid (LPA) (early G1 phase), but occurred after 24 hr of PDGF stimulation (late G1/S phase). S inhibited PDGF-induced DNA synthesis (IC(50) = 3.5 micromol/L), and this effect was dependent on intracellular mevalonate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate availability. The critical time period for the reversal of the S effect by mevalonate comprised both the early and late G1 phase of the SMC cycle. PDGF-induced ERK1/2 phosphorylation and PDGF-induced p38 phosphorylation were not markedly affected by S during the whole G1 phase. However, S treatment blocked the PDGF- and LPA-induced membrane translocation of RhoA that occurred during the late G1/S phase. In the case of Rac1, the same process was also inhibited by S treatment. We concluded from these results that, in SMC, the early events associated with ERK1/2 and p38 signal transduction pathways, recruited for PDGF-mediated DNA synthesis, were insensitive to S action, whereas the mevalonate-dependent, posttranslational modification of RhoA and Rac1 molecules, required for PDGF-induced membrane translocation, was blocked by this drug. These results suggest that the antiproliferative effect of S can be explained not only by the blockage of RhoA-mediated signaling events but also by Rac1-mediated signaling events.     

Regulatory roles for Tiam1, a guanine nucleotide exchange factor for Rac1, in glucose-stimulated insulin secretion in pancreatic beta-cells

Using various biochemical, pharmacological and molecular biological approaches, we have recently reported regulatory roles for Rac1, a small G-protein, in glucose-stimulated insulin secretion (GSIS). However, little is understood with respect to localization of, and regulation by, specific regulatory factors of Rac1 in GSIS. Herein, we investigated regulatory roles for Tiam1, a specific nucleotide exchange factor (GEF) for Rac1, in GSIS in pancreatic beta-cells. Western blot analysis indicated that Tiam1 is predominantly cytosolic in distribution. NSC23766, a specific inhibitor of Tiam1-mediated activation of Rac1, markedly attenuated glucose-induced, but not KCl-induced insulin secretion in INS 832/13 cells and normal rat islets. Further, NSC23766 significantly reduced glucose-induced activation (i.e. GTP-bound form) and membrane association of Rac1 in INS 832/13 cells and rat islets. Moreover, siRNA-mediated knock-down of Tiam1 markedly inhibited glucose-induced membrane trafficking and activation of Rac1 in INS 832/13 cells. Interestingly, however, in contrast to the inhibitory effects of NSC23766, Tiam1 gene depletion potentiated GSIS in these cells; such a potentiation of GSIS was sensitive to extracellular calcium. Together, our studies present the first evidence for a regulatory role for Tiam1/Rac1-sensitive signaling step in GSIS. They also provide evidence for the existence of a potential Rac1/Tiam1-independent, but calcium-sensitive component for GSIS in these cells.     

Regulatory roles for Tiam1, a guanine nucleotide exchange factor for Rac1, in glucose-stimulated insulin secretion in pancreatic β-cells

Using various biochemical, pharmacological and molecular biological approaches, we have recently reported regulatory roles for Rac1, a small G-protein, in glucose-stimulated insulin secretion (GSIS). However, little is understood with respect to localization of, and regulation by, specific regulatory factors of Rac1 in GSIS. Herein, we investigated regulatory roles for Tiam1, a specific nucleotide exchange factor (GEF) for Rac1, in GSIS in pancreatic β-cells. Western blot analysis indicated that Tiam1 is predominantly cytosolic in distribution. NSC23766, a specific inhibitor of Tiam1-mediated activation of Rac1, markedly attenuated glucose-induced, but not KCl-induced insulin secretion in INS 832/13 cells and normal rat islets. Further, NSC23766 significantly reduced glucose-induced activation (i.e. GTP-bound form) and membrane association of Rac1 in INS 832/13 cells and rat islets. Moreover, siRNA-mediated knock-down of Tiam1 markedly inhibited glucose-induced membrane trafficking and activation of Rac1 in INS 832/13 cells. Interestingly, however, in contrast to the inhibitory effects of NSC23766, Tiam1 gene depletion potentiated GSIS in these cells; such a potentiation of GSIS was sensitive to extracellular calcium. Together, our studies present the first evidence for a regulatory role for Tiam1/Rac1-sensitive signaling step in GSIS. They also provide evidence for the existence of a potential Rac1/Tiam1-independent, but calcium-sensitive component for GSIS in these cells.     

TAS-301 blocks receptor-operated calcium influx and inhibits rat vascular smooth muscle cell proliferation induced by basic fibroblast growth factor and platelet-derived growth factor

The purpose of this study was to determine the effect of a recently synthesized drug, TAS-301 [3-bis(4-methoxyphenyl)methylene-2-indolinone], on vascular smooth muscle cell (VSMC) proliferation and the intracellular signal transduction pathways involved in VSMC proliferation. In an in vitro assay, TAS-301 inhibited the proliferation of rat VSMCs stimulated by platelet-derived growth factor (PDGF)-BB, basic fibroblast growth factor, or 2% fetal bovine serum in a concentration-dependent manner. TAS-301 dose-dependently inhibited the PDGF-induced Ca2+ influx; the concentration for the inhibition of Ca2+ influx was nearly identical to that for inhibition of VSMC proliferation. The Ca2+ influx induced by PDGF was also attenuated by NiCl2 but not by nifedipine, suggesting that PDGF-induced Ca2+ influx would be mediated by some non-voltage-dependent mechanisms. Furthermore, TAS-301 inhibited PDGF-induced activation of protein kinase C (PKC) and the phorbol 12-myristate 13-acetate-mediated induction of activator protein 1 (AP-1) in a concentration-dependent manner. These findings indicate that TAS-301 inhibited the proliferation of VSMCs by blocking voltage-independent Ca2+ influx and downstream signals such as the Ca2+/PKC signaling pathway, leading to AP-1 induction.     

Lysophosphatidylcholine induces azurophil granule translocation via Rho/Rho kinase/F-actin polymerization in human neutrophils

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.     

Targeting of myosin light chain kinase in smooth muscle cell

We constructed a plasmid vector to have a 1.4 kb insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells (VSMCs), producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaf1p4++ motility to the platelet derived growth factor (PDGF), which was supported by the membrane ruffling. However, the transfectants showed neither chemotaxic motility nor developed membrane ruffling, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by demonstrating that Rho-kinase activity, which also phosphorylates the myosin light chain (MLC), was well preserved in both SM3 and the transfectants. In spite of this importance of MLCK, PDGF failed to induce MLC phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to myosin-binding property of MLCK (Ye et al. Proc Natl. Acad. Sci. USA 96 6666-6671, 1999).     

Current topics in the regulatory mechanism underlying the Ca2+ sensitization of the contractile apparatus in vascular smooth muscle

The Ca2+ signal is the primary determinant of the contraction of the vascular smooth muscle. However, the alteration of the Ca2+ sensitivity of the contractile apparatus also plays an essential role. The regulation of the myosin light chain phosphatase (MLCP) activity is considered to be the most important mechanism underlying the regulation of Ca2+ sensitivity. The investigations during the last 15 years have identified many proteins that participate in the regulation of the MLCP activity. Recently, the Ca2+ signal has also been shown to cross-talk with the mechanisms regulating the Ca2+ sensitivity. Consequently, Rho kinase, protein kinase C, CPI-17, and MYPT1 have all been suggested to play a physiologically important role in the regulation of the MLCP activity. We are now close to elucidating the major rules regulating the MLCP activity and the Ca2+ sensitivity during vascular contractions. This article will give an overview of the current understanding of the biochemical basis for the regulation of the MLCP activity, while also discussing their functional roles from a physiological point of view. I hope this article will help to develop new pharmacological strategies for the prevention and treatment of the pathological vasoconstriction often seen in vascular diseases.     

Pitavastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, blocks vascular smooth muscle cell populated-collagen lattice contraction

Constrictive arterial remodeling plays a major role in lumen narrowing following angioplasty. We investigated the effect of pitavastatin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, on vascular smooth muscle cell (SMC)-populated collagen lattice contraction, an in vitro model of vascular contraction. Type I collagen gel contraction by SMCs, which are cultured in collagen gel, was used as a model of vascular remodeling. Pitavastatin pretreatment inhibited 10% serum- or platelet-derived growth factor-BB (PDGF)-induced SMC-mediated collagen lattice contraction in a concentration-dependent manner. The effect of pitavastatin was prevented by mevalonate or geranylgeranyl pyrophosphate, but not by squalene, a precursor of cholesterol, or farnesyl pyrophosphate. The serum- or PDGF-induced SMC-mediated collagen gel contraction was inhibited by GGTI-298, a geranylgeranyltransferase inhibitor, C3 exoenzyme, an inhibitor of Rho, or Y27634, a Rho kinase inhibitor, but not by FTI-277, a farnesyltransferase inhibitor. Serum or PDGF treatment increased the stress fiber organization in SMCs, which was blocked by the pitavastatin pretreatment. Pitavastatin had no effect on the serum- and PDGF-induced lamelliopodia extension of SMC. These results may suggest that pitavastatin attenuates SMC-mediated collagen gel contraction probably via an inhibition of geranylgeranylated Rho protein and a disruption of actin cytoskeletal reorganization.