Publications by authors named "Steffen-Sebastian Bolz"

47 Publications

Sphingosine-1-phosphate-dependent activation of p38 MAPK maintains elevated peripheral resistance in heart failure through increased myogenic vasoconstriction.

Circ Res 2010 Oct 29;107(7):923-33. Epub 2010 Jul 29.

Toronto General Hospital Research Institute, Toronto, ON, Canada.

Rationale: Mechanisms underlying vasomotor abnormalities and increased peripheral resistance exacerbating heart failure (HF) are poorly understood.

Objective: To explore the role and molecular basis of myogenic responses in HF.

Methods And Results: 10 weeks old C57Bl6 mice underwent experimental myocardial infarction (MI) or sham surgery. At 1 to 12 weeks postoperative, mice underwent hemodynamic studies, mesenteric, cerebral, and cremaster artery perfusion myography and Western blot. Organ weights and hemodynamics confirmed HF and increased peripheral resistance after MI. Myogenic responses, ie, pressure-induced vasoconstriction, were increased as early as 1 week after MI and remained elevated. Vasoconstrictor responses to phenylephrine were decreased 1 week after MI, but not at 2 to 6 weeks after MI, whereas those to endothelin (ET)-1 and sphingosine-1-phosphate (S1P) were increased at all time points after MI. An antagonist (JTE-013) for the most abundant S1P receptor detected in mesenteric arteries (S1P(2)R) abolished the enhanced myogenic responses of HF, with significantly less effect on controls. Mice with genetic absence of sphingosine-kinases or S1P(2)R (Sphk1(-/-); Sphk1(-/-)/Sphk2(+/-); S1P(2)R(-/-)) did not manifest enhanced myogenic responses after MI. Mesenteric arteries from HF mice exhibited increased phosphorylation of myosin light chain, with deactivation of its phosphatase (MLCP). Among known S1P-responsive regulators of MLCP, GTP-Rho levels were unexpectedly reduced in HF, whereas levels of activated p38 MAPK and ERK1/2 (extracellular signal-regulated kinase 1/2) were increased. Inhibiting p38 MAPK abolished the myogenic responses of animals with HF, with little effect on controls.

Conclusions: Rho-independent p38 MAPK-mediated deactivation of MLCP underlies S1P/S1P(2)R-regulated increases in myogenic vasoconstriction observed in HF. Therapeutic targeting of these findings in HF models deserves study.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.110.226464DOI Listing
October 2010

A microfluidic platform for probing small artery structure and function.

Lab Chip 2010 Sep 6;10(18):2341-9. Epub 2010 Jul 6.

Department of Mechanical and Industrial Engineering, University of Toronto, Ontario, Canada.

Although pathologic changes to the structure and function of small blood vessels are hallmarks of various cardiovascular diseases, limitations of conventional investigation methods (i.e. pressure myography) have prohibited a comprehensive understanding of the underlying mechanisms. We developed a microfluidic device to facilitate assessment of resistance artery structure and function under physiological conditions (37 degrees C, 45 mmHg transmural pressure). The platform allows for on-chip fixation, long-term culture and fully automated acquisition of up to ten dose-response sequences of intact mouse mesenteric artery segments (diameter approximately 250 micrometres and length approximately 1.5 mm) in a well-defined microenvironment. Even abluminal application of phenylephrine or acetylcholine (homogeneous condition) yielded dose-response relationships virtually identical to conventional myography. Unilateral application of phenylephrine (heterogeneous condition) limited constriction to the drug-exposed side, suggesting a lack of circumferential communication. The microfluidic platform allows us to address new fundamental biological questions, replaces a manually demanding procedure with a scalable approach and may enable organ-based screens to be routinely performed during drug development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c004675bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753293PMC
September 2010

The phosphorylation motif at serine 225 governs the localization and function of sphingosine kinase 1 in resistance arteries.

Arterioscler Thromb Vasc Biol 2009 Nov 3;29(11):1916-22. Epub 2009 Sep 3.

Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

Objective: The purpose of this study was to characterize a phosphorylation motif at serine 225 as a molecular switch that regulates the pressure-dependent activation of sphingosine kinase 1 (Sk1) in resistance artery smooth muscle cells.

Methods And Results: In isolated hamster gracilis muscle resistance arteries, pressure-dependent activation/translocation of Sk1 by ERK1/2 was critically dependent on its serine 225 phosphorylation site. Specifically, expression of Sk1(S225A) reduced resting and myogenic tone, resting Ca(2+), pressure-induced Ca(2+) elevations, and Ca(2+) sensitivity. The lack of function of the Sk1(S225A) mutant could not be entirely overcome by forced localization to the plasma membrane via a myristoylation/palmitylation motif; the membrane anchor also significantly inhibited the function of the wild-type Sk1 enzyme. In both cases, Ca(2+) sensitivity and myogenic tone were attenuated, whereas Ca(2+) handling was normalized/enhanced. These discrete effects are consistent with cell surface receptor-mediated effects (Ca(2+) sensitivity) and intracellular effects of S1P (Ca(2+) handling). Accordingly, S1P(2) receptor inhibition (1 micromol/L JTE013) attenuated myogenic tone without effect on Ca(2+).

Conclusions: Translocation and precise subcellular positioning of Sk1 is essential for full Sk1 function; and two distinct S1P pools, proposed to be intra- and extracellular, contribute to the maintenance of vascular tone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/ATVBAHA.109.194803DOI Listing
November 2009

RGS4 regulates parasympathetic signaling and heart rate control in the sinoatrial node.

Circ Res 2008 Aug 24;103(5):527-35. Epub 2008 Jul 24.

Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Heart rate is controlled by the opposing activities of sympathetic and parasympathetic inputs to pacemaker myocytes in the sinoatrial node (SAN). Parasympathetic activity on nodal myocytes is mediated by acetylcholine-dependent stimulation of M(2) muscarinic receptors and activation of Galpha(i/o) signaling. Although regulators of G protein signaling (RGS) proteins are potent inhibitors of Galpha(i/o) signaling in many tissues, the RGS protein(s) that regulate parasympathetic tone in the SAN are unknown. Our results demonstrate that RGS4 mRNA levels are higher in the SAN compared to right atrium. Conscious freely moving RGS4-null mice showed increased bradycardic responses to parasympathetic agonists compared to wild-type animals. Moreover, anesthetized RGS4-null mice had lower baseline heart rates and greater heart rate increases following atropine administration. Retrograde-perfused hearts from RGS4-null mice showed enhanced negative chronotropic responses to carbachol, whereas SAN myocytes showed greater sensitivity to carbachol-mediated reduction in the action potential firing rate. Finally, RGS4-null SAN cells showed decreased levels of G protein-coupled inward rectifying potassium (GIRK) channel desensitization and altered modulation of acetylcholine-sensitive potassium current (I(KACh)) kinetics following carbachol stimulation. Taken together, our studies establish that RGS4 plays an important role in regulating sinus rhythm by inhibiting parasympathetic signaling and I(KACh) activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.108.180984DOI Listing
August 2008

Role of sphingosine-1-phosphate phosphohydrolase 1 in the regulation of resistance artery tone.

Circ Res 2008 Aug 26;103(3):315-24. Epub 2008 Jun 26.

Institute of Physiology, Ludwig-Maximilians-University, Munich, Germany.

Sphingosine-1-phosphate (S1P), which mediates pleiotropic actions within the vascular system, is a prominent regulator of microvascular tone. By virtue of its S1P-degrading function, we hypothesized that S1P-phosphohydrolase 1 (SPP1) is an important regulator of tone in resistance arteries. Hamster gracilis muscle resistance arteries express mRNA encoding SPP1. Overexpression of SPP1 (via transfection of a SPP1(wt)) reduced resting tone, Ca2+ sensitivity, and myogenic vasoconstriction, whereas reduced SPP1 expression (antisense oligonucleotides) yielded the opposite effects. Expression of a phosphatase-dead mutant of SPP1 (SPP1(H208A)) had no effect on any parameter tested, suggesting that catalytic activity of SPP1 is critical. The enhanced myogenic tone that follows overexpression of S1P-generating enzyme sphingosine kinase 1 (Sk1(wt)) was functionally antagonized by coexpression with SPP1(wt) but not SPP1(H208A). SPP1 modulated vasoconstriction in response to 1 to 100 nmol/L exogenous S1P, a concentration range that was characterized as S1P2-dependent, based on the effect of S1P(2) inhibition by antisense oligonucleotides and 1 mumol/L JTE013. Inhibition of the cystic fibrosis transmembrane regulator (CFTR) (1) restored S1P responses that were attenuated by SPP1(wt) overexpression; (2) enhanced myogenic vasoconstriction; but (3) had no effect on noradrenaline responses. We conclude that SPP1 is an endogenous regulator of resistance artery tone that functionally antagonizes the vascular effects of both Sk1(wt) and S1P2 receptor activation. SPP1 accesses extracellular S1P pools in a manner dependent on a functional CFTR transport protein. Our study assigns important roles to both SPP1 and CFTR in the physiological regulation of vascular tone, which influences both tissue perfusion and systemic blood pressure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.108.173575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2746908PMC
August 2008

Cardioprotective and vasodilatory actions of glucagon-like peptide 1 receptor are mediated through both glucagon-like peptide 1 receptor-dependent and -independent pathways.

Circulation 2008 May 21;117(18):2340-50. Epub 2008 Apr 21.

Heart and Stroke Richard Lewar Centre of Excellence for Cardiovascular Research, University of Toronto, Ontario, Canada M5G-1C4.

Background: The glucagon-like peptide 1 receptor (GLP-1R) is believed to mediate glucoregulatory and cardiovascular effects of the incretin hormone GLP-1(7-36) (GLP-1), which is rapidly degraded by dipeptidyl peptidase-4 (DPP-4) to GLP-1(9-36), a truncated metabolite generally thought to be inactive. Novel drugs for the treatment of diabetes include analogues of GLP-1 and inhibitors of DPP-4; however, the cardiovascular effects of distinct GLP-1 peptides have received limited attention.

Methods And Results: Here, we show that endothelium and cardiac and vascular myocytes express a functional GLP-1R as GLP-1 administration increased glucose uptake, cAMP and cGMP release, left ventricular developed pressure, and coronary flow in isolated mouse hearts. GLP-1 also increased functional recovery and cardiomyocyte viability after ischemia-reperfusion injury of isolated hearts and dilated preconstricted arteries from wild-type mice. Unexpectedly, many of these actions of GLP-1 were preserved in Glp1r(-/-) mice. Furthermore, GLP-1(9-36) administration during reperfusion reduced ischemic damage after ischemia-reperfusion and increased cGMP release, vasodilatation, and coronary flow in wild-type and Glp1r(-/-) mice, with modest effects on glucose uptake. Studies using a DPP-4-resistant GLP-1R agonist and inhibitors of DPP-4 and nitric oxide synthase showed that the effects of GLP-1(7-36) were partly mediated by GLP-1(9-36) through a nitric oxide synthase-requiring mechanism that is independent of the known GLP-1R.

Conclusions: These data describe cardioprotective actions of GLP-1(7-36) mediated through the known GLP-1R and novel cardiac and vascular actions of GLP-1(7-36) and its metabolite GLP-1(9-36) independent of the known GLP-1R. Our data suggest that the extent to which GLP-1 is metabolized to GLP-1(9-36) may have functional implications in the cardiovascular system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.107.739938DOI Listing
May 2008

The emerging role of Ca2+ sensitivity regulation in promoting myogenic vasoconstriction.

Cardiovasc Res 2008 Jan;77(1):8-18

University of Rostock, Institute of Physiology, PSF 100888, D-18055 Rostock, Germany.

Growing evidence suggests that mechanisms which regulate the Ca2+ sensitivity of the contractile apparatus in vascular smooth muscle cells form the backbone of pressure-induced myogenic vasoconstriction. The modulation of Ca2+ sensitivity is suited to partially uncouple intracellular Ca2+ from constriction, thereby allowing the maintenance of tone with fully conserved function of other Ca2+-dependent processes. Following a brief review of 'classical' Ca2+-dependent signalling pathways involved in the myogenic response, the present review describes the emerging mechanisms that promote myogenic vasoconstriction via modulation of Ca2+ sensitivity. For the purpose of this review, Ca2+ sensitivity reflects the dynamic equilibrium between myosin light-chain kinase and myosin light-chain phosphatase activities in terms of its impact on vascular tone. Several signalling pathways (PKC, RhoA/Rho kinase, ROS) which have been identified as prominent regulators of Ca2+ sensitivity will be discussed. Although Ca2+ sensitivity modulation is clearly an important component of the myogenic response, attempts to integrate it into existing mechanistic models resulted in a two-phase model, with a predominant Ca2+-dependent 'initiation/trigger' phase followed by a Ca2+-independent 'maintenance' phase. We propose that the two-phase model is rather simplistic, because the literature reviewed here demonstrates that Ca2+-dependent and -independent mechanisms do not operate in isolation and are important at all stages of the response. The regulation of Ca2+ sensitivity, as an equal and complimentary partner of Ca2+-dependent processes, significantly enhances our understanding of the complex array of signalling pathways, which ultimately mediate the myogenic response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cardiores.2007.07.018DOI Listing
January 2008

Deafness and stria vascularis defects in S1P2 receptor-null mice.

J Biol Chem 2007 Apr 6;282(14):10690-6. Epub 2007 Feb 6.

Genetics of Development and Disease Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1821, USA.

The S1P(2) receptor is a member of a family of G protein-coupled receptors that bind the extracellular sphingolipid metabolite sphingosine 1-phosphate with high affinity. The receptor is widely expressed and linked to multiple G protein signaling pathways, but its physiological function has remained elusive. Here we have demonstrated that S1P(2) receptor expression is essential for proper functioning of the auditory and vestibular systems. Auditory brainstem response analysis revealed that S1P(2) receptor-null mice were deaf by one month of age. These null mice exhibited multiple inner ear pathologies. However, some of the earliest cellular lesions in the cochlea were found within the stria vascularis, a barrier epithelium containing the primary vasculature of the inner ear. Between 2 and 4 weeks after birth, the basal and marginal epithelial cell barriers and the capillary bed within the stria vascularis of the S1P(2) receptor-null mice showed markedly disturbed structures. JTE013, an S1P(2) receptor-specific antagonist, blocked the S1P-induced vasoconstriction of the spiral modiolar artery, which supplies blood directly to the stria vascularis and protects its capillary bed from high perfusion pressure. Vascular disturbance within the stria vascularis is a potential mechanism that leads to deafness in the S1P(2) receptor-null mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M700370200DOI Listing
April 2007

Sphingosine-1-phosphate modulates spiral modiolar artery tone: A potential role in vascular-based inner ear pathologies?

Cardiovasc Res 2006 Apr 13;70(1):79-87. Epub 2006 Mar 13.

ENT-Department, Technical University of Munich, Ismaninger Str. 22, D-81675 Munich, Germany.

Objective: The mechanisms regulating spiral modiolar artery (SMA) tone are not known, yet their characterization is pivotal for understanding inner ear blood flow regulation. Sphingosine-1-phosphate (S1P), known to stimulate vasoconstriction in several vascular beds, is a candidate regulator of SMA tone with potential pathophysiological relevance.

Methods: Gerbil SMAs were isolated, cannulated and pressurized (30 mm Hg transmural) for experimentation under near-in vivo conditions. For functional experiments, vascular diameter and intracellular Ca2+ were simultaneously measured. Standard RT-PCR and immunohistochemical techniques were also employed.

Results: mRNA transcripts encoding sphingosine kinase, S1P phosphohydrolase and three S1P receptors (S1P(1-3)) were detected in the SMA. S1P induced dose-dependent vasoconstriction of the SMA (EC50 = 115 nmol/L), and enhanced the apparent Ca2+-sensitivity of the contractile apparatus. Noradrenaline did not elicit vasoconstriction. The Rho kinase inhibitor Y27632 (1 micromol/L) reversed S1P-induced vasoconstriction and the S1P-mediated enhancement of Ca2+-sensitivity. RhoA was observed to translocate to the plasma membrane in response to stimulation with 30 micromol/L S1P.

Conclusion: We conclude that all key signalling pathway constituents are present at the mRNA level for S1P to act as an endogenous regulator of SMA tone. S1P stimulates potent, RhoA/Rho kinase-dependent SMA vasoconstriction and Ca2+ sensitization. The high sensitivity to S1P suggests that SMA vasoconstriction is likely to occur under pathological conditions that increase intramural S1P concentrations (i.e., inflammation). From a clinical perspective, the present study identifies new potential therapeutic targets for the treatment of vascular-based, "stroke-like" inner ear pathologies: the enzymes responsible for S1P bioavailability and the S1P receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cardiores.2006.01.011DOI Listing
April 2006

Sphingosine kinase functionally links elevated transmural pressure and increased reactive oxygen species formation in resistance arteries.

FASEB J 2006 Apr 13;20(6):702-4. Epub 2006 Feb 13.

Institute of Physiology, Ludwig-Maximilians-University, Munich, Germany.

Myogenic vasoconstriction, an intrinsic response to elevated transmural pressure (TMP), requires the activation of sphingosine kinase (Sk1) and the generation of reactive oxygen species (ROS). We hypothesized that pressure-induced Sk1 signaling and ROS generation are functionally linked. Using a model of cannulated resistance arteries isolated from the hamster gracilis muscle, we monitored vessel diameter and smooth muscle cell (SMC) Ca2+i (Fura-2) or ROS production (dichlorodihydrofluorescein). Elevation of TMP stimulated the translocation of a GFP-tagged Sk1 fusion protein from the cytosol to the plasma membrane, indicative of enzymatic activation. Concurrently, elevation of TMP initiated a rapid and transient production of ROS, which was enhanced by expression of wild-type Sk1 (hSk(wt)) and inhibited by its dominant-negative mutant (hSk(G82D)). Exogenous sphingosine-1-phosphate (S1P) also stimulated ROS generation is isolated vessels. Chemical (1 micromol/L DPI), peptide (gp91ds-tat/gp91ds), and genetic (N17Rac) inhibition strategies indicated that NADPH oxidase was the source of the pressure-induced ROS. NADPH oxidase inhibition attenuated myogenic vasoconstriction and reduced the apparent Ca2+ sensitivity of the SMC contractile apparatus, without affecting Ca2+-independent, RhoA-mediated vasoconstriction in response to exogenous S1P. Our results indicate a mandatory role for Sk1/S1P in mediating pressure-induced, NADPH oxidase-derived ROS formation. In turn, ROS generation appears to increase Ca2+ sensitivity, necessary for full myogenic vasoconstriction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.05-4075fjeDOI Listing
April 2006

Impaired calmodulin binding of myosin-7A causes autosomal dominant hearing loss (DFNA11).

Hum Mutat 2004 Sep;24(3):274-5

Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Butenfeld 42, Hamburg, Germany.

Both myosin 7A (MYO7A) and calmodulin (CaM) are required for transduction and adaptation processes in inner ear hair cells. We identified a novel heterozygous missense mutation (c.2557C>T; p.R853C) in a family with autosomal dominant non-syndromic hearing loss that changes an evolutionarily invariant residue of the fifth IQ motif (IQ5), a putative calmodulin (CaM) binding domain, of MYO7A. Functional effects of the p.R853C mutation were investigated in a physiological cellular environment by expressing MYO7A IQ5-containing peptides in smooth muscle cells of microarteries, in which overexpression of wildtype IQ5 (with intact calmodulin binding) would be expected to compete with myosin light chain kinase (MLCK) for CaM binding. Indeed, analysis of calmodulin-dependent vasoconstriction suggests constitutive binding of CaM to the wildtype, but not the p.R853C-mutated IQ5 motif at all physiologically relevant Ca2+ concentrations. Thus our data suggest a disturbed CaM/MYO7A binding of the p.R853C mutant, this amino acid change may result in impaired adaptation to environmental stimuli and progressive deterioration of hearing transduction in heterozygotes. A defect in CaM/MYO7A interaction represents a novel pathomechanism for genetic hearing loss. It provides an attractive molecular target for therapeutic interventions aimed to delay or prevent the onset of hearing loss in families with mutations in myosin IQ domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.9272DOI Listing
September 2004

Highly effective non-viral gene transfer into vascular smooth muscle cells of cultured resistance arteries demonstrated by genetic inhibition of sphingosine-1-phosphate-induced vasoconstriction.

J Vasc Res 2003 Jul-Aug;40(4):399-405. Epub 2003 Aug 8.

Institute of Physiology, Ludwig Maximilians University, Munich, Germany.

The linkage of vascular genes to specific functions will lead to a better understanding of cardiovascular pathophysiology. We developed an experimental model that enables the introduction of one or multiple gene(s) into vascular smooth muscle cells (VSMCs) of isolated resistance arteries. Exposure of the arteries to a green fluorescent protein (GFP)-encoding plasmid in combination with the transfectant Effectene((R)) for 20 h resulted in the expression of GFP in virtually all VSMCs in the arterial wall at fully preserved vascular function. For functional validation of the model, plasmids encoding the specific RhoA inhibitors C3 transferase or N19RhoA were transfected. In subsequent functional tests, inhibition of RhoA-dependent constriction induced by sphingosine-1-phosphate was similar to that in arteries treated with exogenous C3 transferase protein or the Rho kinase inhibitor Y27632. Responses to norepinephrine remained unaffected. This novel transfection technique enables gene function to be assessed in direct conjunction with signalling pathways in vascular tissue and provides, therefore, a new tool for microvascular proteomics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000072830DOI Listing
October 2003

Sphingosine kinase modulates microvascular tone and myogenic responses through activation of RhoA/Rho kinase.

Circulation 2003 Jul 7;108(3):342-7. Epub 2003 Jul 7.

Institute of Physiology, Ludwig Maximilians University, Schillerstrasse 44, 80336 Muenchen, Germany.

Background: RhoA and Rho kinase are important modulators of microvascular tone.

Methods And Results: We tested whether sphingosine kinase (Sphk1) that generates the endogenous sphingolipid mediator sphingosine-1-phosphate (S1P) is part of a signaling cascade to activate the RhoA/Rho kinase pathway. Using a new transfection model, we report that resting tone and myogenic responses of isolated resistance arteries increased with forced expression of Sphk1 in smooth muscle cells of these arteries. Overexpression of a dominant negative Sphk1 mutant or coexpression of dominant negative mutants of RhoA or Rho kinase together with Sphk1 completely inhibited development of tone and myogenic responses.

Conclusions: The tone-increasing effects of a Sphk1 overexpression suggest that Sphk1 may play an important role in the control of peripheral resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/01.CIR.0000080324.12530.0DDOI Listing
July 2003

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.

Circulation 2003 Jun 9;107(24):3081-7. Epub 2003 Jun 9.

Physiologisches Institut der Ludwig Maximilians Universität, Schillerstrasse 44, 80336 München, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/01.CIR.0000074202.19612.8CDOI Listing
June 2003

Oxidized LDL and its compound lysophosphatidylcholine potentiate AngII-induced vasoconstriction by stimulation of RhoA.

J Am Soc Nephrol 2003 Jun;14(6):1471-9

Department of Medicine, Julius-Maximilians Universität, Würzburg, Germany.

RhoA stimulates vascular tone by increasing smooth muscle Ca(2+) sensitivity, e.g., in atherosclerosis. This study was an investigation of the influence of oxidized LDL (OxLDL), which accumulates in atherosclerotic plaques, on vascular tone induced by angiotensin II (AngII), with particular emphasis on the RhoA pathway. OxLDL had no influence on unstimulated vascular tone of isolated rabbit aorta, but it potentiated contractile responses induced by AngII. The Ca(2+)-antagonist felodipin partially prevented potentiation of contractile responses, whereas the AT(1) receptor antagonist losartan blunted AngII responses in presence and in absence of OxLDL. Rho-kinase inhibition by Y27632 abolished potentiation of contractile responses, and RhoA inhibition by C3-like transferase partially prevented it, suggesting that OxLDL activated RhoA. Activation of RhoA was further analyzed by detection of its translocation to the cell membrane after stimulation with OxLDL. Western blot analysis of aorta homogenates, as well as direct visualization in cultured smooth muscle cells using confocal laser scan microscopy, revealed that OxLDL potently activated RhoA. The effect of OxLDL was mimicked by its compound lysophosphatidylcholine, and C3 inhibited both lysophosphatidylcholine and OxLDL-induced RhoA stimulation. In conclusion, OxLDL stimulates the RhoA pathway, resulting in potentiation of AngII-induced vasoconstriction. Lysophosphatidylcholine mimics the OxLDL effect, consistent with a causal role of this OxLDL compound. Stimulation of RhoA by OxLDL may contribute to vasospasm in atherosclerotic arteries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/01.asn.0000067412.18899.9bDOI Listing
June 2003

Lack of vascular connexin 40 is associated with hypertension and irregular arteriolar vasomotion.

Physiol Genomics 2003 Apr 16;13(2):169-77. Epub 2003 Apr 16.

Physiologisches Institut, Ludwig-Maximilians-Universität München, 80336 Munich, Germany.

Gap-junctional communication coordinates the behavior of individual cells in arterioles. Gap junctions are formed by connexins 40 (Cx40), Cx43, Cx37, and Cx45 in the vasculature. Previously, we have shown that lack of Cx40 impairs conduction of dilatory signals along arterioles. Herein, we examined whether hypertension is present in conscious animals and whether this is a direct effect or due to secondary mechanisms. Mean arterial pressure was elevated by 20-25 mmHg in conscious Cx40-deficient mice (Cx40(-/-)) compared with wild-type controls in both sexes. Differences in heart rate were not observed. Blockade of NO synthase increased pressure equally in both genotypes. Conversely, the angiotensin AT(1)-receptor antagonist, candesartan, decreased pressure to similar extents in Cx40(-/-) and wild-type mice. Acetylcholine and sodium nitroprusside (0.05-15 nmol) were equally potent and effective in decreasing pressure and inducing dilatory responses in the microcirculation. However, in contrast to wild type, Cx40(-/-) arterioles exhibited spontaneous, irregular vasomotion leading temporarily to complete vessel closure. We conclude that loss of Cx40 is associated with hypertension independent of the action of angiotensin II. It is also not related to an altered efficacy of NO or other endothelial dilators. However, the observed irregular vasomotion suggests that peripheral vascular resistance is affected.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/physiolgenomics.00169.2002DOI Listing
April 2003

Depolarization of endothelial cells enhances platelet aggregation through oxidative inactivation of endothelial NTPDase.

Arterioscler Thromb Vasc Biol 2002 Dec;22(12):2003-9

Institute of Physiology, München, Germany.

Objective: The objective of this study was to investigate whether depolarization of cultured endothelial cells (human umbilical vein endothelial cells, HUVECs) affects their ectonucleotidase activity through superoxide (O2-) production.

Methods And Results: Depolarization by the cation channel gramicidin (100 nmol/L) or tetrabutylammonium chloride (1 mmol/L) induced O2- release from HUVECs (n=4), which was decreased by superoxide dismutase (SOD, 500 U/mL). The activity of endothelial ectonucleotidases was assessed by the production of inorganic phosphate from ADP, which was decreased by chronic depolarization by 25% (n=6, P<0.05) and the amount of AMP derived from ADP in the presence of the 5'-nucleotidase inhibitor alpha,beta-methylene-5'-diphosphate (100 micromol/L). AMP was decreased by chronic depolarization from 0.54+/-0.16 to 0.39+/-0.11 micromol/min/mg protein (n=6, P<0.05). This was abolished in the continuous presence of SOD (n=6). NTPDase protein was predominantly expressed in HUVECs (n=4). Protein abundance, viability of cells, and apoptosis rates were not altered by depolarization (n=10). Only in the presence of depolarized HUVECs, but not with control cells or with HUVECs depolarized in the presence of SOD, did 5 micromol/L of ADP cause irreversible platelet aggregation. Increases in transmural pressure induced endothelial depolarization in intact hamster small arterioles.

Conclusions: Depolarization causes the endothelial production of O2-, which inhibits the activity of endothelial ectonucleotidases. Increases in transmural pressure induce endothelial depolarization. In chronically hypertensive diseases, depolarization might favor platelet aggregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/01.atv.0000043454.08172.51DOI Listing
December 2002
-->