Biomedical subjects

Gervaise Loirand

Publications and source records attributed to Gervaise Loirand.

At least 19 recordsLinked to original sources

PubMed2006

Transglutaminase-dependent RhoA activation and depletion by serotonin in vascular smooth muscle cells.

The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension.

Animals↗

PubMed2006

RhoA activation and interaction with Caveolin-1 are critical for pressure-induced myogenic tone in rat mesenteric resistance arteries.

OBJECTIVE: Myogenic tone, which has a major role in the regulation of local blood flow, refers to the ability of vascular smooth muscle to adapt its contractility to changes in transmural pressure. Although Rho-kinase is involved in myogenic tone, the pathway involved remains unclear, especially concerning translocation to the plasma membrane and activation of RhoA. As caveolae have a key role in the signal transduction of membrane-bound proteins, we tested the hypothesis that RhoA might be activated by pressure and that its activation might involve caveolin-1, which has been shown to be involved in vascular functions. METHODS: Myogenic tone was studied in isolated rat mesenteric resistance arteries (118+/-15 microm internal diameter with a pressure of 75 mmHg) submitted to pressure steps (25, 75, and 150 mmHg). Pharmacological blockade of caveolae or RhoA-Rho-kinase pathway was assessed by confocal microscopy in pressurized arteries to analyze protein co-localization and by co-immunoprecipitation in order to confirm protein interactions. Caveolin-1-deficient mice were used to confirm the role of the protein in myogenic tone. RESULTS: Pressure-induced myogenic tone was significantly reduced by RhoA inactivation with TAT-C3 (90.5% inhibition at 150 mmHg) and by the Rho-kinase inhibitor Y27632 (91.8% inhibition at 150 mmHg). In arteries pressurized at 150 mmHg, RhoA was localized to the plasma membrane (localization by confocal microscopy and increased quantity of RhoA in the membrane fraction after protein extraction). Thus, translocation of RhoA to the plasma membrane was associated with pressure-induced tone. In addition, caveolae disruption with methyl-beta-cyclodextrin reduced myogenic tone by 66% at 150 mmHg. Further, myogenic tone was significantly reduced to 24% of control in caveolin-1-deficient mice (active tone was 32.3+/-2.8 microm and 9.1+/-3.7 microm in +/+ and -/- mice, respectively, n = 5 per group), suggesting a key role of caveolin-1 in myogenic tone. Finally, RhoA and caveolin-1 co-immunoprecipitation and co-localization significantly increased when myogenic tone developed at 150 mmHg (co-localization showed 26+/-13% merging at 25 mmHg versus 97+/-21% at 150 mmHg, n = 5). Co-immunoprecipitation was prevented by TAT-C3 and by methyl beta-cyclodextrin. CONCLUSION: RhoA activation is critical for the development of myogenic tone in resistance arteries. This activation induced translocation of RhoA to the plasma membrane within caveolae, where the interaction of RhoA with caveolin-1 leads selectively to the activation of a Rho-kinase-dependent force development.

Actins↗

PubMed2006

Hyaluronan induces vascular smooth muscle cell migration through RHAMM-mediated PI3K-dependent Rac activation.

OBJECTIVE: Hyaluronan (HA) is an important constituent of the extracellular matrix and is known to regulate cellular events through binding to CD44 and the receptor for HA-mediated motility (RHAMM). Here we investigated the role of these receptors and the signaling pathways involved in HA-mediated effects in arterial smooth muscle cells (ASMC). METHODS: Effects of high-molecular weight HA (1 to 5 mg/ml) were analyzed in cultured ASMC from rat aorta. RESULTS: HA promoted actin stress fiber and lamellipodia formation and dose-dependently induced ASMC migration without effect on proliferation. Pull-down assay of Rho protein activity indicated that HA activated RhoA and Rac. HA-induced ASMC migration was not affected by the RhoA inhibitor Tat-C3 (10 microg/ml), the Rho kinase inhibitor Y-27632 (10 microM) and blocking anti-CD44 antibody ,but was reduced by the non-selective Rho protein inhibitor simvastatin (10 microM), the Rac inhibitor LT-toxin (1 mug/ml), small interfering RNA (siRNA) targeting Rac and the phosphatidyl inositol 3-kinase (PI3K) inhibitor LY294002 (25 microM), which also blocked HA-induced Rac activation. CD44 knockdown by siRNA inhibited HA-mediated RhoA activation without effect on ASMC migration. In contrast, siRNA targeting RHAMM inhibited both HA-induced migration and Rac activation. CONCLUSIONS: High-molecular weight HA independently activates RhoA and Rac through CD44 and RHAMM, respectively. HA-induced migration depends exclusively on RHAMM-mediated PI3K-dependent Rac activation.

Actins↗

PubMed2006

Rho kinases in cardiovascular physiology and pathophysiology.

Rho kinases (ROCKs) are the first and the best-characterized effectors of the small G-protein RhoA. In addition to their effect on actin organization, or through this effect, ROCKs have been found to regulate a wide range of fundamental cell functions such as contraction, motility, proliferation, and apoptosis. Abnormal activation of the RhoA/ROCK pathway has been observed in major cardiovascular disorders such as atherosclerosis, restenosis, hypertension, pulmonary hypertension, and cardiac hypertrophy. This review, based on recent molecular, cellular, and animal studies, focuses on the current understanding of ROCK signaling and its roles in cardiovascular physiology and pathophysiology.

Atherosclerosis↗

PubMed2006

Regulation of Rho proteins by phosphorylation in the cardiovascular system.

The small G protein Rho signaling pathways are recognized as major regulators of cardiovascular functions, and activation of Rho proteins appears to be a common component for the pathogenesis of hypertension and vascular proliferative disorders. Rho proteins are tightly regulated, and recent evidence suggests that modulation of Rho protein signaling by phosphorylation of Rho proteins provides an additional simple mechanism for coordinating Rho protein functions. This regulation by phosphorylation is particularly important in the arterial wall, where RhoA protein expressed in vascular smooth muscle cells is controlled by the endothelium through the nitric oxide/cGMP-dependent kinase pathway.

Animals↗

PubMed2005

Regulation of Rho signaling pathways in interleukin-2-stimulated human T-lymphocytes.

Rho GTPases are key regulators of many cellular functions, including cytoskeleton organization which is important for cell morphology and mobility, gene expression, cell cycle progression, and cytokinesis. In addition, it has recently been recognized that Rho GTPase activity is required for development of the immune system, as well as for the specialized functions of the peripheral cells that act in the immune response such as antigen presenting cells and lymphocytes. Stimulation of T lymphocytes with interleukin-2 (IL-2) induces clonal expansion of antigen-specific populations and provides a model to study cell cycle entry and cell cycle progression. We have performed gene expression analysis in a model of human T lymphocytes, which proliferate in response to IL-2. In addition to changes in genes relevant to cell cycling and to the antiapoptotic effects of IL-2, we have analyzed expression and variations of more than 300 genes involved in Rho GTPase signaling pathways. We report here that IL-2 regulates the expression of a number of proteins, which participate in the Rho GTPase pathways, including some of the GTPases themselves, GDP/GTP exchange factors, GTPase activating proteins, as well as GDIs and effectors. Our results suggest that regulation of expression of components of the Rho GTPase pathways may be an important mechanism in assembling specific signal transduction cascades that need to be active at certain times during the cell cycle. Some of our findings may also be relevant to the roles of Rho GTPases in T lymphocyte functions and proliferation.

Blotting, Western↗

PubMed2005

Phosphorylation of serine 188 protects RhoA from ubiquitin/proteasome-mediated degradation in vascular smooth muscle cells.

cAMP and cyclic GMP-dependent kinases (PKA and PKG) phosphorylate the small G protein RhoA on Ser188. We have previously demonstrated that phosphorylation of Ser188 inhibits RhoA-dependent functions and positively regulates RhoA expression, and that the nitric oxide (NO)/cGMP-dependent protein kinase pathway plays an essential role, both in vitro and in vivo, in the regulation of RhoA protein expression and functions in vascular smooth muscle cells. Here we analyze the consequences of Ser188 phosphorylation on RhoA protein degradation. By expressing Ser188 phosphomimetic wild-type (WT-RhoA-S188E) and active RhoA proteins (Q63L-RhoA-S188E), we show that phosphorylation of Ser188 of RhoA protects RhoA, particularly its active form, from ubiquitin-mediated proteasomal degradation. Coimmunoprecipitation experiments indicate that the resistance of the phosphorylated active form of RhoA to proteasome-mediated degradation is because of its cytoplasmic sequestration through enhanced RhoGDI interaction. In rat aortic smooth muscle cells, stimulation of PKG and inhibition of proteasome by lactacystin, induce nonadditive increases in RhoA protein expression. In addition, stimulation of PKG leads to the accumulation of GTP-bound RhoA in the cytoplasm. In vivo stimulation of the NO/PKG signaling by treating rats with sildenafil increased RhoA level and RhoA phosphorylation, and enhanced its association to RhoGDI in the pulmonary artery, whereas opposite effects are induced by chronic inhibition of NO synthesis in N-omega-nitro-L-arginine-treated rats. Our results thus suggest that Ser188 phosphorylation-mediated protection against degradation is a physiological process regulating the level of endogenous RhoA and define a novel function for RhoGDI, as an inhibitor of Rho protein degradation.

Animals↗

PubMed2005

Inhibition of RhoA/Rho kinase pathway is involved in the beneficial effect of sildenafil on pulmonary hypertension.

Inhibition of the type 5 phosphodiesterase and inhibition of Rho kinase are both effective in reducing pulmonary hypertension (PH). Here we investigate whether Rho kinase inhibition is involved in the beneficial effect of the type 5 phosphodiesterase inhibitor sildenafil on PH. Chronic hypoxia-induced PH in rats is associated with an increase in RhoA activity in pulmonary artery that was maximal after 2 days (10.7+/-0.9-fold increase, n=6, P<0.001). The activity of Rho kinase assessed by measuring the level of myosin phosphatase target subunit 1 (MYPT1) phosphorylation was also increased (5.7+/-0.8-fold over control, n=8). Chronic fasudil (30 mg kg(-1) day(-1); 14 days) and sildenafil (25 mg kg(-1) day(-1); 14 days) treatments reduced PH and pulmonary cardiovascular remodelling, and inhibited the MYPT1 phosphorylation in pulmonary artery from hypoxic rats by 82.3+/-3% (n=4) and by 76.6+/-2% (n=4), respectively. The inhibitory effect of sildenafil (10 microM) on MYPT1 phosphorylation was demonstrated by the loss of actin stress fibres in vascular smooth muscle cells. However, in vitro kinase assays indicated that sildenafil had no direct inhibitory action on Rho kinase activity. Sildenafil treatment induced increased RhoA phosphorylation and association to its cytosolic inhibitory protein, guanine dissociation inhibitor (GDI) in pulmonary artery.We propose that sildenafil inhibits RhoA/Rho kinase-dependent functions in pulmonary artery through enhanced RhoA phosphorylation and cytosolic sequestration by GDI. The inhibition of intracellular events downstream of RhoA thus participates in the beneficial effect of sildenafil on PH.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2005

RhoA and resistance artery remodeling.

Resistance arteries are able to adapt to physiological and pathophysiological stimuli to maintain adequate perfusion according to the metabolic demand of the tissue. Although vasomotor control allows rapid adaptation of lumen diameter, vascular remodeling constitutes an active process that occurs in response to long-term alterations of hemodynamic parameters. Unfortunately, this initially adaptive process contributes to the pathology of vascular diseases. Recent studies have demonstrated the participation of Rho protein signaling pathways in several cardiovascular pathologies including hypertension, coronary artery spasm, effort angina, atherosclerosis, and restenosis. Functional analyses have further revealed that RhoA-dependent pathways are involved in excessive contraction, migration, and proliferation associated with arterial diseases. The present review focuses on the role of Rho proteins, in particular RhoA, in vascular smooth muscle cells and the involvement of Rho-dependent signaling pathways in resistance artery remodeling, more particularly in relation to hypertension.

Adaptation, Physiological↗

PubMed2004

Stent implantation activates RhoA in human arteries: inhibitory effect of rapamycin.

In-stent restenosis is a novel pathobiologic process resulting from vascular smooth muscle cell (VSMC) proliferation, migration and excessive matrix production. The present study was designed to assess the activity of RhoA, a major regulator of VSMC proliferation and migration, after stenting and to determine its role in the neointimal formation. Analysis of RhoA activity in an ex vivo organ culture model of human internal mammary arteries demonstrates that stenting induced a time-dependent increase in RhoA activity (4.9 +/- 0.4 vs. 1.2 +/- 0.2 in control at 28 days, n = 4, p < 0.0001) associated with a concomitant decrease in p27 expression. Treatment of stented arteries with the permeant RhoA inhibitor TAT-C3 (10 microg/ml) or Rho-kinase inhibitors (Y-27632, 10 micromol/l; fasudil, 10 micromol/l) inhibited both neointimal formation and decrease in p27 expression. Rapamycin (1 and 10 nmol/l) also inhibited neointimal formation, and induced a loss of RhoA expression. The inhibitory effect of rapamycin on neointimal thickening is prevented by the dominant active form of RhoA. Our study shows that stent implantation induces maintained RhoA activation and demonstrates that the inhibitory action of rapamycin on RhoA expression plays a key role in its antirestenotic effect.

Arterial Occlusive Diseases↗

PubMed2004

Expression of sialyl-Tn epitopes on beta1 integrin alters epithelial cell phenotype, proliferation and haptotaxis.

Sialyl-Tn (STn) is a tumor-associated carbohydrate antigen overexpressed in various carcinomas. To obtain its expression, murine carcinoma cells were transfected with the cDNA encoding ST6GalNAc I, a glycosyltransferase that acts exclusively on O-glycans. Overexpression of this enzyme led to the expected expression of cell surface STn epitopes. Surprisingly, the transfectants (STn+ cells) presented dramatic morphological changes and altered behavior. These STn+ cells lost the epithelial appearance of parental cells, became larger, more elongated and presented disorganized actin stress fibers. Additionally, their proliferation was impaired and their ability to migrate on fibronectin and hyaluronic acid was severely reduced. By contrast their adhesion on fibronectin remained unchanged. The major glycoprotein carrying the STn epitope was shown to be the integrin beta1 subunit. Anti-STn antibodies could restore migration of STn+ cells on fibronectin. A constitutively active permeant form of RhoA (TAT-RhoA(Val-14)) also restored motility on fibronectin of STn+ cells as well as a parental STn-cellular phenotype. These observations indicate that overexpression of ST6GalNAc I leads to a major change of the O-glycosylation of the integrin beta1 chain which in turn impairs the integrin-mediated signalling and leads to major alterations in morphology and cell behavior.

Actins↗

PubMed2003

Rho-associated protein kinase contributes to early atherosclerotic lesion formation in mice.

Members of the Rho family of small GTPases have been recently implicated in inflammatory signaling. We examined the effect of in vivo inhibition of Rho kinase on atherogenesis in mice. Low-density lipoprotein receptor (LDLR) knockout (KO) mice fed a cholate-free high-fat diet received daily intraperitoneal injection of saline (n=8, control group) or Y-27632 (30 mg/kg, n=9), a specific Rho kinase inhibitor. After 9 weeks, Y-27632 treatment resulted in significant in vivo inhibition of Rho kinase activity (P=0.004). Body weights, arterial blood pressures, and plasma cholesterol levels were comparable in both groups. Atherosclerotic lesion size in the aortic sinus and thoracic aorta of mice treated with Y-27632 was reduced by respectively 35% and 29% in comparison with the saline-treated animals (P=0.006 and P=0.03, respectively). This was associated with a significant reduction in T lymphocyte accumulation (P=0.035) and expression of p65 subunit of NF-kappaB within plaques (P<0.05). In vitro, treatment with Y-27632 inhibited p65 phosphorylation and degradation of IkappaBalpha in mouse peritoneal macrophages and significantly inhibited concanavalin A-induced proliferation of spleen-derived T cells (P<0.001). In conclusion, inhibition of Rho kinase significantly limits early atherosclerotic plaque development in the LDLR KO mice. This study identifies Rho kinase inhibitors as potential candidates for the treatment of atherosclerosis.

Amides↗

PubMed2003

Sildenafil prevents change in RhoA expression induced by chronic hypoxia in rat pulmonary artery.

Exposure to chronic hypoxia (CH) induces a sustained pulmonary hypertension associated with structural and functional changes in the pulmonary arterial bed, including alterations of contractile properties. The small G-protein RhoA and its effector Rho kinase play a major role in the sustained rise in tension induced by vasoconstrictors. The aim of this study was to analyze the effect of CH on the RhoA/Rho kinase signaling pathway in the rat pulmonary artery. Maximal contraction of pulmonary artery rings to endothelin-1, noradrenaline, and the thromboxane A2 analog U46619 was markedly decreased in rats exposed to CH (10% O2, 2 weeks). This CH-induced decrease response to agonists was attributable to the abolition of RhoA-mediated Ca2+ sensitization of the contraction. Real-time reverse transcriptase-polymerase chain reaction and Western blot analysis revealed a decrease in RhoA mRNA (79.4+/-6.0%, n=4) and RhoA (81.1+/-8.0%, n=4) expression in the main pulmonary artery from CH rats, whereas RhoA expression was not modified in arterial smooth muscle cells and arteries exposed to hypoxia and high intraluminal pressure, respectively. Treatment of rats with sildenafil (25 mg/kg per day) throughout 2 weeks of exposure to CH prevented CH-induced downregulation of RhoA, reduction of contraction, and pulmonary artery remodeling. These findings indicate that CH-induced downregulation of RhoA expression, leading to the abolition of RhoA/Rho kinase-mediated Ca2+ sensitization of contraction, is responsible for the decreased responses to contracting agonists in the pulmonary artery of CH rats. These alterations are prevented by sildenafil, indicating a major role of the NO/cyclic GMP pathway in CH-induced altered RhoA signaling in the pulmonary artery.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2003

Nitric oxide-induced decrease in calcium sensitivity of resistance arteries is attributable to activation of the myosin light chain phosphatase and antagonized by the RhoA/Rho kinase pathway.

BACKGROUND: NO-induced dilations in resistance arteries (RAs) are not associated with decreases in vascular smooth muscle cell Ca2+. We tested whether a cGMP-dependent activation of the smooth muscle myosin light chain phosphatase (MLCP) resulting in a Ca2+ desensitization of the contractile apparatus was the underlying mechanism and whether it could be antagonized by the RhoA pathway. METHODS AND RESULTS: The Ca2+ sensitivity of RA was assessed as the relation between changes in diameter and [Ca2+]i in depolarized RA (120 mol/L K+) exposed to stepwise increases in Ca2+ex (0 to 3 mmol/L). Effects of 10 micromol/L sodium nitroprusside (SNP) on Ca2+ sensitivity were determined before and after application of the soluble guanylate cyclase inhibitor ODQ (1 micromol/L) and the MLCP inhibitor calyculin A (120 nmol/L) and in presence of the RhoA-activating phospholipid sphingosine-1-phosphate (S1P, 12 nmol/L). SNP-induced dilations were also studied in controls and in RAs pretreated with the Rho kinase inhibitor Y27632 or transfected with a dominant-negative RhoA mutant (N19RhoA). Constrictions elicited by increasing Ca2+ex were significantly attenuated by SNP, which, however, left associated increases in [Ca2+]i unaffected. This NO-induced attenuation was blocked by ODQ, calyculin A, and S1P. The S1P-induced translocation of RhoA indicating activation of the GTPase was not reversed by SNP. Inhibition of RhoA/Rho kinase by N19RhoA or Y27632 significantly augmented SNP-induced dilations. CONCLUSIONS: NO dilates RA by activating the MLCP in a cGMP-dependent manner, thereby reducing the apparent Ca2+ sensitivity of the contractile apparatus. MLCP inactivation via the RhoA/Rho kinase pathway antagonizes this Ca2+-desensitizing effect that, in turn, can be restored using RhoA/Rho kinase inhibitors.

ADP Ribose Transferases↗

PubMed2003

RhoA expression is controlled by nitric oxide through cGMP-dependent protein kinase activation.

The small G protein RhoA is a convergence point for multiple signals that regulate smooth muscle cell functions. NO plays a major role in the structure and function of the normal adult vessel wall, mainly through modulation of gene transcription. This study was thus performed to analyze in vitro and in vivo the effect of NO signaling on RhoA expression in arterial smooth muscle cells. In rat or human artery smooth muscle cells, sodium nitroprusside or 8-(2-chlorophenylthio)-cGMP induced a rise in RhoA mRNA and protein expression, which was inhibited by the cGMP-dependent protein kinase (PKG) inhibitor (R(p))-8-bromo-beta-phenyl-1,N(2)-ethenoguanosine 3':5'-phosphorothioate. The NO/PKG stimulation of RhoA expression involved both an increase in RhoA protein stability and stimulation of rhoA gene transcription. Cloning and functional analysis of the human rhoA promoter revealed that the effect of NO/PKG involved phosphorylation of ATF-1 and subsequent binding to the cAMP-response element. Chronic inhibition of NO synthesis in N(omega)-nitro-l-arginine-treated rats induced a strong decrease in RhoA mRNA and protein expression in aorta and pulmonary artery associated with inhibition of RhoA-mediated Ca(2+) sensitization. These effects were prevented by oral administration of the cGMP phosphodiesterase inhibitor sildenafil. These results show that NO/PKG signaling positively controls RhoA expression and suggest that the basal release of NO is necessary to maintain RhoA expression and RhoA-dependent functions in vascular smooth muscle cells.

3T3 Cells↗

PubMed2003

Rho kinase blockade prevents inflammation via nuclear factor kappa B inhibition: evidence in Crohn's disease and experimental colitis.

BACKGROUND & AIMS: Rho proteins are involved in the regulation of several cellular functions. Data from in vitro studies suggest that RhoA could be involved in the inflammatory response. We investigated the role of RhoA and its downstream effector Rho kinase in intestinal inflammation. METHODS: Activation of RhoA was assessed by pull-down assays. A specific inhibitor of Rho kinase, Y-27632, was used to examine the role of Rho kinase in inflammatory response in vivo and in vitro by molecular biology and by immunological and biochemical approaches. RESULTS: Increased activation of RhoA was found in inflamed intestinal mucosa of patients with Crohn's disease and of rats with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Oral administration of Y-27632 in rats significantly reduced the colonic inflammation. In vitro, activation of RhoA alone was sufficient to induce tumor necrosis factor production. Y-27632 inhibited production of tumor necrosis factor-alpha and interleukin-1 beta by lamina propria and peripheral blood mononuclear cells. Rho kinase inhibition prevented nuclear factor kappa B activation and I-kappa B phosphorylation and degradation. We showed that Rho kinase associates with and activates I-kappa B kinase alpha and that Y-27632 prevents I-kappa B kinase activation. CONCLUSIONS: Our study provides the first evidence that Rho kinase activates I-kappa B kinase and, thus, nuclear factor kappa B, suggesting a key role of Rho kinase in inflammatory responses and intestinal inflammation. Specific inhibition of Rho kinase may be a promising approach for the treatment of patients with Crohn's disease.

Adolescent↗

PubMed2003

Structure-activity relationships of human urotensin II and related analogues on rat aortic ring contraction.

The sequence of human urotensin II (UII) has been recently established as H-Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH, and it has been reported that UII is the most potent mammalian vasoconstrictor peptide identified so far. A series of UII analogues was synthesized, and the contractile activity of each compound was studied in vitro using de-endothelialised rat aortic rings. Replacement of each amino acid by an L-alanine or by a D-isomer showed that the N- and C-terminal residues flanking the cyclic region of the amidated peptide were relatively tolerant to substitution. Conversely, replacement of any residue of the cyclic region significantly reduced the contractile activity of the molecule. The octapeptide UII(4-11) was 4 times more potent than UII, indicating that the C-terminal region of the molecule possesses full biological activity. Alanine or D-isomer substitutions in UII(4-11) or in UII(4-11)-NH2, respectively, showed a good correlation with the results obtained for UII-NH2. Disulfide bridge disruption or replacement of the cysteine residues by their D-enantiomers markedly reduced the vasoconstrictor effect of UII and its analogues. In contrast, acetylation of the N-terminal residue of UII and UII-NH2 enhanced the potency of the peptide. Finally, monoiodination of the Tyr6 residue in UII(4-11) increased by 5 fold the potency of the peptide in the aortic ring bioassay. This structure-activity relationship study should provide useful information for the rational design of selective and potent UII receptor agonists and antagonists.

Amino Acid Sequence↗

PubMed2003

Synthesis and pharmacological study of Rho-kinase inhibitors: pharmacomodulations on the lead compound Fasudil.

With a view to specifying structure-activity relationships we have synthesised a new series of analogues of the Rho-kinase inhibitor 1-(5-isoquinolinesulfonyl)-homopiperazine (Fasudil). The structural modifications concerned the isoquinolinyl heterocycle and the sulfonyl group which are the two main features of this lead compound. These analogues were evaluated on the actin cytoskeleton and on the enzymatic activity of Rho-kinase. Most of the chemical modifications result in a loss of activity showing that interactions of Fasudil with the catalytic domain of Rho-kinase seem to be particularly definite and sensitive to structural variations. The presence of an isoquinolinyl nitrogen and a basic amino group separated by a spacer bearing a sulfonamide function are of utmost importance. Only the tetra-hydroisoquinoline analogue 3 shows the same activity as Fasudil. Moreover, this compound is unable to inhibit PKC biological activity contrary to Fasudil. The loss of the aromatic property could increase the selectivity level in favour of compound 3.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗