Signalling mechanisms in the cardiovascular protective effects of estrogen: With a focus on rapid/membrane signalling.

In modern society, cardiovascular disease remains the biggest single threat to life, being responsible for approximately one third of worldwide deaths. Male prevalence is significantly higher than that of women until after menopause, when the prevalence of CVD increases in females until it eventually exceeds that of men. Because of the coincidence of CVD prevalence increasing after menopause, the role of estrogen in the cardiovascular system has been intensively researched during the past two decades in vitro, in vivo and in observational studies. Most of these studies suggested that endogenous estrogen confers cardiovascular protective and anti-inflammatory effects. However, clinical studies of the cardioprotective effects of hormone replacement therapies (HRT) not only failed to produce proof of protective effects, but also revealed the potential harm estrogen could cause. The "critical window of hormone therapy" hypothesis affirms that the moment of its administration is essential for positive treatment outcomes, pre-menopause (3-5 years before menopause) and immediately post menopause being thought to be the most appropriate time for intervention. Since many of the cardioprotective effects of estrogen signaling are mediated by effects on the vasculature, this review aims to discuss the effects of estrogen on vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) with a focus on the role of estrogen receptors (ERα, ERß and GPER) in triggering the more recently discovered rapid, or membrane delimited (non-genomic), signaling cascades that are vital for regulating vascular tone, preventing hypertension and other cardiovascular diseases.

NADPH oxidase in the vasculature: Expression, regulation and signalling pathways; role in normal cardiovascular physiology and its dysregulation in hypertension.

The last 20-25 years have seen an explosion of interest in the role of NADPH oxidase (NOX) in cardiovascular function and disease. In vascular smooth muscle and endothelium, NOX generates reactive oxygen species (ROS) that act as second messengers, contributing to the control of normal vascular function. NOX activity is altered in response to a variety of stimuli, including G-protein coupled receptor agonists, growth-factors, perfusion pressure, flow and hypoxia. NOX-derived ROS are involved in smooth muscle constriction, endothelium-dependent relaxation and smooth muscle growth, proliferation and migration, thus contributing to the fine-tuning of blood flow, arterial wall thickness and vascular resistance. Through reversible oxidative modification of target proteins, ROS regulate the activity of protein tyrosine phosphatases, kinases, G proteins, ion channels, cytoskeletal proteins and transcription factors. There is now considerable, but somewhat contradictory evidence that NOX contributes to the pathogenesis of hypertension through oxidative stress. Specific NOX isoforms have been implicated in endothelial dysfunction, hyper-contractility and vascular remodelling in various animal models of hypertension, pulmonary hypertension and pulmonary arterial hypertension, but also have potential protective effects, particularly NOX4. This review explores the multiplicity of NOX function in the healthy vasculature and the evidence for and against targeting NOX for antihypertensive therapy.

Role of reactive oxygen species and sulfide-quinone oxoreductase in hydrogen sulfide-induced contraction of rat pulmonary arteries.

Application of H2S ("sulfide") elicits a complex contraction in rat pulmonary arteries (PAs) comprising a small transient contraction (phase 1; Ph1) followed by relaxation and then a second, larger, and more sustained contraction (phase 2; Ph2). We investigated the mechanisms causing this response using isometric myography in rat second-order PAs, with Na2S as a sulfide donor. Both phases of contraction to 1,000 µM Na2S were attenuated by the pan-PKC inhibitor Gö6983 (3 µM) and by 50 µM ryanodine; the Ca2+ channel blocker nifedipine (1 µM) was without effect. Ph2 was attenuated by the mitochondrial complex III blocker myxothiazol (1 µM), the NADPH oxidase (NOX) blocker VAS2870 (10 µM), and the antioxidant TEMPOL (3 mM) but was unaffected by the complex I blocker rotenone (1 µM). The bath sulfide concentration, measured using an amperometric sensor, decreased rapidly following Na2S application, and the peak of Ph2 occurred when this had fallen to ~50 µM. Sulfide caused a transient increase in NAD(P)H autofluorescence, the offset of which coincided with development of the Ph2 contraction. Sulfide also caused a brief mitochondrial hyperpolarization (assessed using tetramethylrhodamine ethyl ester), followed immediately by depolarization and then a second more prolonged hyperpolarization, the onset of which was temporally correlated with the Ph2 contraction. Sulfide application to cultured PA smooth muscle cells increased reactive oxygen species (ROS) production (recorded using L012); this was absent when the mitochondrial flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using small interfering RNA. We propose that the Ph2 contraction is largely caused by SQR-mediated sulfide metabolism, which, by donating electrons to ubiquinone, increases electron production by complex III and thereby ROS production.

Transforming growth factor-ß enhances Rho-kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1.

KEY POINTS: Transforming growth-factor-ß (TGF-ß) and RhoA/Rho-kinase are independently implicated in the airway hyper-responsiveness associated with asthma, but how these proteins interact is not fully understood. We examined the effects of pre-treatment with TGF-ß on expression and activity of RhoA, Rho-kinase and ARHGEF1, an activator of RhoA, as well as on bradykinin-induced contraction, in airway smooth muscle. TGF-ß enhanced bradykinin-induced RhoA translocation, Rho-kinase-dependent phosphorylation and contraction, but partially suppressed bradykinin-induced RhoA activity (RhoA-GTP content). TGF-ß enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF-ß on RhoA and Rho-kinase activity and contraction, respectively. ARHGEF1 expression was also enhanced in airway smooth muscle from asthmatic patients and ovalbumin-sensitized mice. ARHGEF1 is a key TGF-ß target gene, an important regulator of Rho-kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper-responsiveness. ABSTRACT: Transforming growth factor-ß (TGF-ß), RhoA/Rho-kinase and Src-family kinases (SrcFK) have independently been implicated in airway hyper-responsiveness, but how they interact to regulate airway smooth muscle contractility is not fully understood. We found that TGF-ß pre-treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho-kinase (Y27632), but not the SrcFK inhibitor PP2, prevented this enhancement. In cultured human airway smooth muscle cells (hASMCs), TGF-ß pre-treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20 , MYPT-1 and the actin-severing protein cofilin, but not of RhoA, ROCK2 or c-Src. In hASMCs, acute treatment with BK triggered subcellular translocation of ARHGEF1 and RhoA and enhanced auto-phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC20 , but induced de-phosphorylation of cofilin. TGF-ß pre-treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC20 phosphorylation, but suppressed the effects of BK on RhoA-GTP content, SrcFK auto-phosphorylation and cofilin de-phosphorylation. In hASMCs, an ARHGEF1 small interfering RNA suppressed the effects of BK and TGF-ß on RhoA-GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF-ß on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMCs of asthmatic patients and in lungs of ovalbumin-sensitized mice. Our data indicate that TGF-ß enhances BK-induced contraction, RhoA translocation and Rho-kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA-GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper-responsiveness is worthy of further investigation.

ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery: Role of Src-family kinases and ARHGEF1.

The role of reactive oxygen species (ROS) in smooth muscle contraction is poorly understood. We hypothesised that G-protein coupled receptor (GPCR) activation and hypoxia induce Rho-kinase activity and contraction in rat intra-pulmonary artery (IPA) via stimulation of ROS production and subsequent Src-family kinase (SrcFK) activation. The T-type prostanoid receptor agonist U46619 induced ROS production in pulmonary artery smooth muscle cells (PASMC). U46619 also induced c-Src cysteine oxidation, SrcFK auto-phosphorylation, MYPT-1 and MLC20 phosphorylation and contraction in IPA, and all these responses were inhibited by antioxidants (ebselen, Tempol). Contraction and SrcFK/MYPT-1/MLC20 phosphorylations were also inhibited by combined superoxide dismutase and catalase, or by the SrcFK antagonist PP2, while contraction and MYPT-1/MLC20 phosphorylations were inhibited by the Rho guanine nucleotide exchange factor (RhoGEF) inhibitor Y16. H2O2 and the superoxide-generating quinoledione LY83583 both induced c-Src oxidation, SrcFK auto-phosphorylation and contraction in IPA. LY83583 and H2O2-induced contractions were inhibited by PP2, while LY83583-induced contraction was also inhibited by antioxidants and Y16. SrcFK auto-phosphorylation and MYPT-1/MLC20 phosphorylation was also induced by hypoxia in IPA and this was blocked by mitochondrial inhibitors rotenone and myxothiazol. In live PASMC, sub-cellular translocation of RhoA and the RhoGEF ARHGEF1 was triggered by both U46619 and LY83583 and this translocation was blocked by antioxidants and PP2. RhoA translocation was also inhibited by an ARHGEF1 siRNA. U46619 enhanced ROS-dependent co-immunoprecipitation of ARHGEF1 with c-Src. Our results demonstrate a link between GPCR-induced cytosolic ROS or hypoxia-induced mitochondrial ROS and SrcFK activity, Rho-kinase activity and contraction. ROS and SrcFK activate RhoA via ARHGEF1.

Divergent modulation of Rho-kinase and Ca(2+) influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle.

BACKGROUND AND PURPOSE: The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src-family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR-mediated ASM contraction and associated signalling events. EXPERIMENTAL APPROACH: Contraction was recorded in intact or α-toxin permeabilized rat bronchioles. Phosphorylation of SrcFK, FAK, myosin light-chain-20 (MLC20 ) and myosin phosphatase targeting subunit-1 (MYPT-1) was evaluated in cultured human ASM cells (hASMC). [Ca(2+) ]i was evaluated in Fura-2 loaded hASMC. Responses to carbachol (CCh) and bradykinin (BK) and the contribution of SrcFK and FAK to these responses were determined. KEY RESULTS: Contractile responses in intact bronchioles were inhibited by antagonists of SrcFK, FAK and Rho-kinase, while after α-toxin permeabilization, they were sensitive to inhibition of SrcFK and Rho-kinase, but not FAK. CCh and BK increased phosphorylation of MYPT-1 and MLC20 and auto-phosphorylation of SrcFK and FAK. MYPT-1 phosphorylation was sensitive to inhibition of Rho-kinase and SrcFK, but not FAK. Contraction induced by SR Ca(2+) depletion and equivalent [Ca(2+) ]i responses in hASMC were sensitive to inhibition of both SrcFK and FAK, while depolarization-induced contraction was sensitive to FAK inhibition only. SrcFK auto-phosphorylation was partially FAK-dependent, while FAK auto-phosphorylation was SrcFK-independent. CONCLUSIONS AND IMPLICATIONS: SrcFK mediates Ca(2+) -sensitization in ASM, while SrcFK and FAK together and individually influence multiple Ca(2+) influx pathways. Tyrosine phosphorylation is therefore a key upstream signalling event in ASM contraction and may be a viable target for modulating ASM tone in respiratory disease.

Sphingosylphosphorylcholine potentiates vasoreactivity and voltage-gated Ca2+ entry via NOX1 and reactive oxygen species.

AIMS: Sphingosylphosphorylcholine (SPC) elicits vasoconstriction at micromolar concentrations. At lower concentrations (≤1 µmol/L), however, it does not constrict intrapulmonary arteries (IPAs), but strongly potentiates vasoreactivity. Our aim was to determine whether this also occurs in a systemic artery and to delineate the signalling pathway. METHODS AND RESULTS: Rat mesenteric arteries and IPAs mounted on a myograph were challenged with ∼25 mmol/L [K+] to induce a small vasoconstriction. SPC (1 µmol/L) dramatically potentiated this constriction in all arteries by ∼400%. The potentiation was greatly suppressed or abolished by inhibition of phospholipase C (PLC; U73122), PKCε (inhibitory peptide), Src (PP2), and NADPH oxidase (VAS2870), and also by Tempol (superoxide scavenger), but not by inhibition of Rho kinase (Y27632). Potentiation was lost in mesenteric arteries from p47(phox-/-), but not NOX2(-/-), mice. The intracellular superoxide generator LY83583 mimicked the effect of SPC. SPC elevated reactive oxygen species (ROS) in vascular smooth muscle cells, and this was blocked by PP2, VAS2870, and siRNA knockdown of PKCε. SPC (1 µmol/L) significantly reduced the EC50 for U46619-induced vasoconstriction, an action ablated by Tempol. In patch-clamped mesenteric artery cells, SPC (200 nmol/L) enhanced Ba2+ current through L-type Ca2+ channels, an action abolished by Tempol but mimicked by LY83583. CONCLUSION: Our results suggest that low concentrations of SPC activate a PLC-coupled and NOX1-mediated increase in ROS, with consequent enhancement of voltage-gated Ca2+ entry and thus vasoreactivity. We speculate that this pathway is not specific for SPC, but may also contribute to vasoconstriction elicited by other G-protein coupled receptor and PLC-coupled agonists.

Control of vascular smooth muscle function by Src-family kinases and reactive oxygen species in health and disease.

Reactive oxygen species (ROS) are now recognised as second messenger molecules that regulate cellular function by reversibly oxidising specific amino acid residues of key target proteins. Amongst these are the Src-family kinases (SrcFKs), a multi-functional group of non-receptor tyrosine kinases highly expressed in vascular smooth muscle (VSM). In this review we examine the evidence supporting a role for ROS-induced SrcFK activity in normal VSM contractile function and in vascular remodelling in cardiovascular disease. VSM contractile responses to G-protein-coupled receptor stimulation, as well as hypoxia in pulmonary artery, are shown to be dependent on both ROS and SrcFK activity. Specific phosphorylation targets are identified amongst those that alter intracellular Ca(2+) concentration, including transient receptor potential channels, voltage-gated Ca(2+) channels and various types of K(+) channels, as well as amongst those that regulate actin cytoskeleton dynamics and myosin phosphatase activity, including focal adhesion kinase, protein tyrosine kinase-2, Janus kinase, other focal adhesion-associated proteins, and Rho guanine nucleotide exchange factors. We also examine a growing weight of evidence in favour of a key role for SrcFKs in multiple pro-proliferative and anti-apoptotic signalling pathways relating to oxidative stress and vascular remodelling, with a particular focus on pulmonary hypertension, including growth-factor receptor transactivation and downstream signalling, hypoxia-inducible factors, positive feedback between SrcFK and STAT3 signalling and positive feedback between SrcFK and NADPH oxidase dependent ROS production. We also discuss evidence for and against the potential therapeutic targeting of SrcFKs in the treatment of pulmonary hypertension.

Redox regulation of protein kinases as a modulator of vascular function.

Reactive oxygen species (ROS) are continuously generated in vascular tissues by various oxidoreductase enzymes. They contribute to normal cell signaling, and modulate vascular smooth muscle tone and endothelial permeability in response to physiological agonists and to various cellular stresses and environmental factors, such as hypoxia. While concentrations of ROS are normally tightly controlled by cellular redox buffer systems, if produced in excess they may contribute to vascular disease. Protein kinases are essential components of most cell signaling pathways, including those involving ROS. The functioning of several members of this highly diverse group of enzymes, which include receptor and nonreceptor tyrosine kinases, protein kinase C, mitogen-activated kinases, and Rho-kinase, are modified by ROS, either through direct oxidative modification or indirectly through modification of associated proteins such as tyrosine phosphatases and monomeric G proteins. In this review, we discuss the molecular mechanisms of redox modification of these proteins, the downstream pathways affected, the often complex interaction between major kinase pathways, and feedback to ROS production itself. We also discuss complicating factors such as differential actions of superoxide anion and hydrogen peroxide, questions concerning concentration dependence, and the significance of signaling microdomains.

Superoxide differentially controls pulmonary and systemic vascular tone through multiple signalling pathways.

AIMS: the aim of this study was to determine the relative importance of Ca(2+) sensitization, ion channels, and intracellular Ca(2+) ([Ca(2+)](i)) in the mixed constrictor/relaxation actions of superoxide anion on systemic and pulmonary arteries. METHODS AND RESULTS: pulmonary and mesenteric arteries were obtained from rat. Superoxide was generated in arteries and cells with 6-anilino-5,8-quinolinequinone (LY83583). Following pre-constriction with U46619, 10 µmol/L LY83583 caused constriction in pulmonary and relaxation in mesenteric arteries. Both constrictor and relaxant actions of LY83583 were inhibited by superoxide dismutase and catalase. LY83583 caused Rho-kinase-dependent constriction in α-toxin-permeabilized pulmonary but not mesenteric arteries. Phosphorylation of myosin phosphatase-targeting subunit-1 (MYPT-1; as determined by western blot), was enhanced by LY83583 in pulmonary artery only. However, in both artery types, changes in tension were closely correlated with changes in phosphorylation of the 20 kDa myosin light chain as well as changes in [Ca(2+)](i) (as measured with Fura PE-3), with LY83583 causing increases in pulmonary and decreases in mesenteric arteries. When U46619 was replaced by 30 mmol/L K(+), all changes in [Ca(2+)](i) were abolished and LY83583 constricted both artery types. The K(V) channel inhibitor 4-aminopyridine abolished the LY83583-induced relaxation in mesenteric artery without affecting constriction in pulmonary artery. However, LY83583 caused a similar hyperpolarizing shift in the steady-state activation of K(V) current in isolated smooth muscle cells of both artery types. CONCLUSIONS: superoxide only causes Rho-kinase-dependent Ca(2+) sensitization in pulmonary artery, resulting in constriction, and whilst it opens K(V) channels in both artery types, this only results in relaxation in mesenteric.

Superoxide constricts rat pulmonary arteries via Rho-kinase-mediated Ca(2+) sensitization.

Reactive oxygen species play a key role in vascular disease, pulmonary hypertension, and hypoxic pulmonary vasoconstriction. We investigated contractile responses, intracellular Ca(2+) ([Ca(2+)](i)), Rho-kinase translocation, and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light chain (MLC(20)) in response to LY83583, a generator of superoxide anion, in small intrapulmonary arteries (IPA) of rat. LY83583 caused concentration-dependent constrictions in IPA and greatly enhanced submaximal PGF(2alpha)-mediated preconstriction. In small femoral or mesenteric arteries of rat, LY83583 alone was without effect, but it relaxed a PGF(2)alpha-mediated preconstriction. Constrictions in IPA were inhibited by superoxide dismutase and tempol, but not catalase, and were endothelium and guanylate cyclase independent. Constrictions were also inhibited by the Rho-kinase inhibitor Y27632 and the Src-family kinase inhibitor SU6656. LY83583 did not raise [Ca(2+)](i), but caused a Y27632-sensitive constriction in alpha-toxin-permeabilized IPA. LY83583 triggered translocation of Rho-kinase from the nucleus to the cytosol in pulmonary artery smooth muscle cells and enhanced phosphorylation of MYPT-1 at Thr-855 and of MLC(20) at Ser-19 in IPA. This enhancement was inhibited by superoxide dismutase and abolished by Y27632. Hydrogen peroxide did not activate Rho-kinase. We conclude that in rat small pulmonary artery, superoxide triggers Rho-kinase-mediated Ca(2+) sensitization and vasoconstriction independent of hydrogen peroxide.

Constriction of pulmonary artery by peroxide: role of Ca2+ release and PKC.

Reactive oxygen species are implicated in pulmonary hypertension and hypoxic pulmonary vasoconstriction. We examined the effects of low concentrations of peroxide on intrapulmonary arteries (IPA). IPAs from Wistar rats were mounted on a myograph for recording tension and estimating intracellular Ca2+ using Fura-PE3. Ca2+ sensitization was examined in alpha-toxin-permeabilized IPAs, and phosphorylation of MYPT-1 and MLC(20) was assayed by Western blot. Peroxide (30 microM) induced a vasoconstriction with transient and sustained components and equivalent elevations of intracellular Ca2+. The transient constriction was strongly suppressed by indomethacin, the TP-receptor antagonist SQ-29584, and the Rho kinase inhibitor Y-27632, whereas sustained constriction was unaffected. Neither vasoconstriction nor elevation of intracellular Ca2+ was affected by removal of extracellular Ca2+, whereas dantrolene suppressed the former and ryanodine abolished the latter. Peroxide-induced constriction of permeabilized IPAs was unaffected by Y-27632 but abolished by PKC inhibitors; these also suppressed constriction in intact IPAs. Peroxide caused translocation of PKCalpha, but had no significant effect on MYPT-1 or MLC(20) phosphorylation. We conclude that in IPAs peroxide causes transient release of vasoconstrictor prostanoids, but sustained constriction is associated with release of Ca2+ from ryanodine-sensitive stores and a PKC-dependent but Rho kinase- and MLC(20)-independent constrictor mechanism.

Role of src-family kinases in hypoxic vasoconstriction of rat pulmonary artery.

AIMS: We investigated the role of src-family kinases (srcFKs) in hypoxic pulmonary vasoconstriction (HPV) and how this relates to Rho-kinase-mediated Ca(2+) sensitization and changes in intracellular Ca(2+) concentration ([Ca(2+)](i)). METHODS AND RESULTS: Intra-pulmonary arteries (IPAs) were obtained from male Wistar rats. HPV was induced in myograph-mounted IPAs. Auto-phosphorylation of srcFKs and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and myosin light-chain (MLC(20)) in response to hypoxia were determined by western blotting. Translocation of Rho-kinase and effects of siRNA knockdown of src and fyn were examined in cultured pulmonary artery smooth muscle cells (PASMCs). [Ca(2+)](i) was estimated in Fura-PE3-loaded IPA. HPV was inhibited by two blockers of srcFKs, SU6656 and PP2. Hypoxia enhanced phosphorylation of three srcFK proteins at Tyr-416 (60, 59, and 54 kDa, corresponding to src, fyn, and yes, respectively) and enhanced srcFK-dependent tyrosine phosphorylation of multiple target proteins. Hypoxia caused a complex, time-dependent enhancement of MYPT-1 and MLC(20) phosphorylation, both in the absence and presence of pre-constriction. The sustained component of this enhancement was blocked by SU6656 and the Rho-kinase inhibitor Y27632. In PASMCs, hypoxia caused translocation of Rho-kinase from the nucleus to the cytoplasm, and this was prevented by anti-src siRNA and to a lesser extent by anti-fyn siRNA. The biphasic increases in [Ca(2+)](i) that accompany HPV were also inhibited by PP2. CONCLUSION: Hypoxia activates srcFKs and triggers protein tyrosine phosphorylation in IPA. Hypoxia-mediated Rho-kinase activation, Ca(2+) sensitization, and [Ca(2+)](i) responses are depressed by srcFK inhibitors and/or siRNA knockdown, suggesting a central role of srcFKs in HPV.

Low concentrations of sphingosylphosphorylcholine enhance pulmonary artery vasoreactivity: the role of protein kinase C delta and Ca2+ entry.

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC(50) is approximately 100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane-sensitive Ca(2+) entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC delta inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC delta. The potentiation could be attributed to enhancement of Ca(2+) entry. SPC also potentiated the responses to prostaglandin F(2 alpha) and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 micromol/L) caused translocation of PKC delta to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca(2+) entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC delta. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.

Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery.

AIMS: We investigated the role of src family kinases (srcFK) in agonist-mediated Ca2+-sensitization in pulmonary artery and whether this involves interaction with the rho/rho-kinase pathway. METHODS AND RESULTS: Intra-pulmonary arteries (IPAs) and cultured pulmonary artery smooth muscle cells (PASMC) were obtained from rat. Expression of srcFK was determined at the mRNA and protein levels. Ca2+-sensitization was induced by prostaglandin F(2 alpha) (PGF(2 alpha)) in alpha-toxin-permeabilized IPAs. Phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light-chain-20 (MLC20) and translocation of rho-kinase in response to PGF(2 alpha) were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF(2 alpha) enhanced phosphorylation of three srcFK proteins at tyr-416. In alpha-toxin-permeabilized IPAs, PGF(2 alpha) enhanced the Ca2+-induced contraction (pCa 6.9) approximately three-fold. This enhancement was inhibited by the srcFK blockers SU6656 and PP2 and by the rho-kinase inhibitor Y27632. Y27632, but not SU6656 or PP2, also inhibited the underlying pCa 6.9 contraction. PGF(2 alpha) enhanced phosphorylation of MYPT-1 at thr-697 and thr-855 and of MLC20 at ser-19. This enhancement, but not the underlying basal phosphorylation, was inhibited by SU6656. Y27632 suppressed both basal and PGF(2 alpha)-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF(2 alpha) triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656. CONCLUSIONS: srcFK are activated by PGF(2 alpha) in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1.

Effect of ceramide on the contractility of pregnant rat uterus.

Ceramide and other sphingolipid mediators have emerged as a novel class of lipid second messengers in cell signaling. We assessed the effect of C(2)-ceramide (a membrane permeable analog of ceramide) on spontaneous and agonist-induced contractile responses of uterus, isolated from 19-day pregnant rats. Ceramide (3, 10 microM) moderately, but significantly inhibited the amplitude of spontaneous rhythmic contractions. However, a variable effect was seen on agonist-induced contractions. While 5-HT-induced contractions were markedly inhibited at 3 and 10 microM ceramide, oxytocin and carboprost (a PGF(2)alpha analogue)-induced contractions were not affected by the sphingolipid. Ceramide (10 microM) also markedly inhibited CaCl(2)-induced contractions elicited in K(+)-depolarized tissues. Further, in rabbit portal vein myocytes, which display robust L-type calcium channel current, ceramide inhibited the I(Ba) in a dose-dependent manner. Therefore, it is suggested that the inhibitory effect of ceramide on uterine contractility may involve a decrease in the influx of Ca(2+) through voltage-dependent L-type Ca(2+) channels, such that contractile responses that are primarily dependent on extracellular Ca(2+), like rhythmic and serotonin contractions, were inhibited by ceramide. Further study is required to establish the role of endogenous ceramide and other sphingolipids in regulating uterine tone during gestation and at term.

Dietary soy modulates endothelium-dependent relaxation in aged male rats: Increased agonist-induced endothelium-derived hyperpolarising factor and basal nitric oxide activity.

We examined the effects of dietary soy on the contributions of endothelium-derived hyperpolarising factor (EDHF), nitric oxide (NO), and oxidative stress to vascular tone in isolated aortic rings and small mesenteric and pulmonary arteries in vitro. Male Wistar rats were either continuously fed a soy-deficient diet (SD) or switched from a soy-deficient diet to a soy-rich one for 6 months (SW). Contractile responses were generally smaller in arteries from SW rats. In mesenteric arteries, this difference was blunted by L-NAME, but not by charybdotoxin and apamin. Preconstricted SW mesenteric arteries were more sensitive to acetylcholine (ACh) than SD ones. This difference was unaffected by L-NAME but was abolished by charybdotoxin and apamin. Exogenous superoxide dismutase (SOD) and catalase induced powerful relaxations in aortic rings, which were smaller in those from SW rats. In mesenteric and pulmonary arteries, however, they partially inhibited ACh-mediated relaxation, and enhanced PGF(2alpha)-mediated contraction, respectively. Our results suggest that feeding aging male rats a soy-rich diet results in improved agonist-mediated EDHF production and a generalized reduction in contractile force, which is partly due to elevated basal NO. Our data also suggest a prorelaxant role for endogenous H(2)O(2) in small arteries, which is modulated by a soy diet.

Dietary soy isoflavone induced increases in antioxidant and eNOS gene expression lead to improved endothelial function and reduced blood pressure in vivo.

Epidemiological evidence suggests that populations consuming large amounts of soy protein have a reduced incidence of coronary heart disease (1-5). The cardiovascular risks associated with conventional hormone replacement therapy in postmenopausal women (5-7) have precipitated a search for alternative estrogen receptor modulators. Here we report that long-term feeding of rats with a soy protein-rich (SP) diet during gestation and adult life results in decreased oxidative stress, improved endothelial function, and reduced blood pressure in vivo measured by radiotelemetry in aged male offspring. Improved vascular reactivity in animals fed an SP diet was paralleled by increased mitochondrial glutathione and mRNA levels for endothelial nitric oxide synthase (eNOS) and the antioxidant enzymes manganese superoxide dismutase and cytochrome c oxidase. Reduced eNOS and antioxidant gene expression, impaired endothelial function, and elevated blood pressure in animals fed a soy-deficient diet was reversed after refeeding them an SP diet for 6 months. Our findings suggest that an SP diet increases eNOS and antioxidant gene expression in the vasculature and other tissues, resulting in reduced oxidative stress and increased NO bioavailability. The improvement in endothelial function, increased gene expression, and reduced blood pressure by soy isoflavones have implications for alternative therapy for postmenopausal women and patients at risk of coronary heart disease.

Modulation of PGF2alpha- and hypoxia-induced contraction of rat intrapulmonary artery by p38 MAPK inhibition: a nitric oxide-dependent mechanism.

The mechanisms through which p38 mitogen-activated protein kinase (p38 MAPK) is involved in smooth muscle contraction remain largely unresolved. We examined the role of p38 MAPK in prostaglandin F(2alpha) (PGF(2alpha))-induced vasoconstriction and in hypoxic pulmonary vasoconstriction (HPV) of rat small intrapulmonary arteries (IPA). The p38 MAPK inhibitors SB-203580 and SB-202190 strongly inhibited PGF(2alpha)-induced vasoconstriction, with IC(50)s of 1.6 and 1.2 microM, whereas the inactive analog SB-202474 was approximately 30-fold less potent. Both transient and sustained phases of HPV were suppressed by SB-203580, but not by SB-202474 (both 2 microM). Western blot analysis revealed that PGF(2alpha) (20 microM) increased phosphorylation of p38 MAPK and of heat shock protein 27 (HSP27), and this was abolished by SB-203580 but not by SB-202474 (both 2 microM). Endothelial denudation or blockade of endothelial nitric oxide (NO) synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly suppressed the relaxation of PGF(2alpha)-constricted IPA by SB-203580, but not by SB-202474. Similarly, the inhibition of HPV by SB-203580 was prevented by prior treatment with L-NAME. SB-203580 (2 microM), but not SB-202474, enhanced relaxation-induced by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in endothelium-denuded IPA constricted with PGF(2alpha). In alpha-toxin-permeabilized IPA, SB-203580-induced relaxation occurred in the presence but not the absence of the NO donor sodium nitroprusside (SNP); SB-202474 was without effect even in the presence of SNP. In intact IPA, neither PGF(2alpha)- nor SNAP-mediated changes in cytosolic free Ca(2+) were affected by SB-203580. We conclude that p38 MAPK contributes to PGF(2alpha)- and hypoxia-induced constriction of rat IPA primarily by antagonizing the underlying Ca(2+)-desensitizing actions of NO.

Euhydric hypercapnia increases vasoreactivity of rat pulmonary arteries via HCO3- transport and depolarisation.

OBJECTIVE: To examine whether altered PCO2 or HCO3- at normal pH potentiate agonist-induced vasoconstriction of small pulmonary arteries, and if so to determine the mechanism. METHODS: Small intrapulmonary arteries (IPA) from rats were mounted on a myograph and PGF2alpha (3 microM)-induced tension recorded before and 40 min after replacing normal bath solution (5% CO2, 24 mM [HCO3-], pH 7.4) with one containing either normal [HCO3-] (24 mM) gassed with 10% CO2 (pH 7.12; hypercapnic acidosis) or high [HCO3-] (48 mM) gassed with 10% CO2 (pH 7.4; euhydric hypercapnia). RESULTS: Hypercapnic acidosis had no significant effect on the response of IPA to PGF2alpha. Euhydric hypercapnia however caused a substantial approximately 5.5-fold potentiation of the response (n=17, p<0.001) in the majority of preparations, whilst 20% of IPA (11 of 58) developed a slow spontaneous vasoconstriction after approximately 20 min. No equivalent responses to euhydric hypercapnia were observed in either mesenteric or renal arteries. Both the potentiation of PGF2alpha-induced vasoconstriction and the spontaneous vasoconstriction in IPA were inhibited by the L-type channel blocker diltiazem (10 microM). The potentiation was also suppressed by DIDS, an inhibitor of anion transporters, removal of extracellular Na+, and anthracene-9-carboxylic acid (A9C; 200 microM), reported to inhibit Ca2+-activated Cl- channels. Inhibition of nitric oxide synthase with L-NAME (100 microM) did not prevent potentiation. Depolarisation with 20 mM [K+] mimicked the effect of euhydric hypercapnia in that it also potentiated the response to PGF(2alpha) (>sixfold, n=6). CONCLUSIONS: Euhydric hypercapnia increases vasoreactivity of IPA, but not mesenteric or renal arteries, via a mechanism involving Na+-dependent HCO3- transport, activation of Ca2+-dependent Cl- channels, and subsequent depolarisation. These results may have consequences for patients with CO2-retaining chronic respiratory disease where plasma [HCO3-] is raised following renal compensation, and could explain the increased propensity to pulmonary hypertension and increased mortality in such patients.