Greg A. Knock

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.

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 constricts rat pulmonary arteries via Rho-kinase-mediated Ca2+ 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 Ca2+ sensitization and changes in intracellular Ca2+ concentration ([Ca2+]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 (MLC20) 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). [Ca2+]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 MLC20 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 [Ca2+]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, Ca2+ sensitization, and [Ca2+]i responses are depressed by srcFK inhibitors and/or siRNA knockdown, suggesting a central role of srcFKs in HPV.

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

Abstract 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 F2α (PGF2α) in α-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 PGF2α were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF2α enhanced phosphorylation of three srcFK proteins at tyr-416. In α-toxin-permeabilized IPAs, PGF2α 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. PGF2α 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 PGF2α-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF2α triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656. Conclusions srcFK are activated by PGF2α in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1.

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 Ca2+ concentration, including transient receptor potential channels, voltage‐gated Ca2+ 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.

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 Ca2+ sensitization, ion channels, and intracellular Ca2+ ([Ca2+]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 [Ca2+]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 [Ca2+]i were abolished and LY83583 constricted both artery types. The KV 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 KV current in isolated smooth muscle cells of both artery types. Conclusions Superoxide only causes Rho-kinase-dependent Ca2+ sensitization in pulmonary artery, resulting in constriction, and whilst it opens KV channels in both artery types, this only results in relaxation in mesenteric.

Effects of the 5-lipoxygenase activating protein inhibitor MK886 on voltage-gated and Ca2+-activated K+ currents in rat arterial myocytes.

The effects on the voltage‐gated (IK) and Ca2+ activated (IK,Ca) K+ currents in rat arterial myocytes of the 5‐lipoxygenase activating protein (FLAP) inhibitor MK886, and its inactive analogue L583,916 were evaluated. In rat pulmonary arterial myocytes (RPAMs), MK886 caused a concentration‐dependent reduction of the IK, with little obvious change in the kinetics of the current. Half maximal current block was observed at 75 nM MK886. MK886 application led to a concentration‐dependent increase in the amplitude of the TEA‐sensitive IK,Ca current and single channel activity in RPAMs in whole cell and inside‐out configurations, respectively. The threshold concentration for this effect was approximately 300 nM and a maximal 4–5 fold increase was observed at 10 μM MK886. MK886 also increased IK,Ca in rat mesenteric arterial myocytes (RMAMs). L538,916, an analogue of MK886 which does not block FLAP, had no effect on either IK or IK,Ca at a concentration of 10 μM. Leukotriene C4 (100 nM) had no effect on either IK or IK,Ca in RPAMs. MK886 produced its usual increase in IK,Ca and also blocked IK, in the presence of leukotriene C4. Similarly, leukotriene E4 (100 nM) did not alter the amplitude of IK. Also, the nonselective leukotriene receptor antagonist ICI 198,615 (3 μM) did not affect IK in RPAMs, and did not affect the response to MK886. Arachidonic acid (10 μM) enhanced IK,Ca in both RPAMs and RMAMs. The results show that MK886 markedly affects both IK and IK,Ca in a manner similar to that of arachidonic acid and independent of the endogenous production of leukotrienes. It is therefore possible that MK886, which is thought to compete with arachidonic acid for its binding to FLAP, may similarly occupy arachidonic acid binding sites on these K+ channels, and mimic its effects. Alternatively, MK886 might act via non‐selective effects on other arachidonic acid metabolites which could modify K+ channel function.

Low Concentrations of Sphingosylphosphorylcholine Enhance Pulmonary Artery Vasoreactivity The Role of Protein Kinase Cδ and Ca2+ Entry

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC50 is ≈100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (≤1 &mgr;mol/L) modulated vasoreactivity of rat intrapulmonary arteries using myography and measurement of intracellular [Ca2+]. SPC (1 &mgr;mol/L) had no effect on force or intracellular [Ca2+] on its own, but dramatically potentiated constrictions induced by ≈25 mmol/L [K+], such that at 40 minutes, force and intracellular [Ca2+] (Fura PE3 340/380 ratio) were increased by 429±96% and 134±26%, respectively. The potentiation was stereospecific, apparent at concentrations >100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane–sensitive Ca2+ 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&dgr; inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC&dgr;. The potentiation could be attributed to enhancement of Ca2+ entry. SPC also potentiated the responses to prostaglandin F2&agr; 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 &mgr;mol/L) caused translocation of PKC&dgr; 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 Ca2+ entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC&dgr;. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.