Hypoxia induces hypersensitivity and hyperreactivity to thromboxane receptor agonist in neonatal pulmonary arterial myocytes

PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (Fi(O2) 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+, thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.     

Thromboxane hypersensitivity in hypoxic pulmonary artery myocytes: altered TP receptor localization and kinetics

Hypoxia-induced neonatal persistent pulmonary hypertension (PPHN) is characterized by sustained vasospasm and increased thromboxane (TxA2)-to-prostacyclin ratio. We previously demonstrated that moderate hypoxia induces myocyte TxA2 hypersensitivity. Here, we examined TxA2 prostanoid receptor (TP-R) localization and kinetics following hypoxia to determine the mechanism of hypoxia-induced TxA2 hypersensitivity. Primary cultured neonatal pulmonary artery myocytes were exposed to 10% O2 (hypoxic myocytes; HM) or 21% O2 (normoxic myocytes; NM) for 3 days. PPHN was induced in neonatal piglets by in vivo exposure to 10% FiO2 for 3 days. TP-R was studied in whole lung sections from pigs with hypoxic PPHN- and age-matched controls; intracellular localization was studied by immunocytochemistry. TP-R affinity was studied in cultured myocytes by saturation binding kinetics using 3H-SQ-29548 and competitive binding kinetics by coincubation with U-46619. Phosphorylation and coupling were examined in immunoprecipitated TP-R. We report distal propagation of TP-R expression in PPHN, extending to pulmonary arteries <50 microm. In HM, intracellular TP-R moves towards the perinuclear region, mirroring a change in endoplasmic reticulum (ER) morphology. TP-R kinetics also alter in HM membranes, with decreased Kd and Bmax (maximal binding sites). Additionally, in hypoxia, 3H-SQ-29548 is displaced at lower concentration of U-46619 than in normoxia, suggesting increased agonist affinity. Phosphorylation of serine residues on HM TP-R was significantly decreased compared with NM; this difference correlated with increased Galphaq coupling in hypoxia and was ablated by incubation with PKA. We conclude that the TP-R is normally desensitized in the neonatal pulmonary circuit by PKA-mediated regulatory phosphorylation, decreasing ligand affinity and coupling to Galphaq; this protection is lost following hypoxic exposure. Also, the appearance of TP-R in resistance arteries after development of hypoxic PPHN may contribute to increased pulmonary arterial pressure.     

Simvastatin Attenuates Neuropathic Pain by Inhibiting the RhoA/LIMK/Cofilin Pathway

Neuropathic pain occurs due to deleterious changes in the nervous system caused by a lesion or dysfunction. Currently, neuropathic pain management is unsatisfactory and remains a challenge in clinical practice. Studies have suggested that actin cytoskeleton remodeling may be associated with neural plasticity and may involve a nociceptive mechanism. Here, we found that the RhoA/LIM kinase (LIMK)/cofilin pathway, which regulates actin dynamics, was activated after chronic constriction injury (CCI) of the sciatic nerve. Treatments that reduced RhoA/LIMK/cofilin pathway activity, including simvastatin, the Rho kinase inhibitor Y-27632, and the synthetic peptide Tat-S3, attenuated actin filament disruption in the dorsal root ganglion and CCI-induced neuropathic pain. Over-activation of the cytoskeleton caused by RhoA/LIMK/cofilin pathway activation may produce a scaffold for the trafficking of nociceptive signaling factors, leading to chronic neuropathic pain. Here, we found that simvastatin significantly decreased the ratio of membrane/cytosolic RhoA, which was significantly increased after CCI, by inhibiting the RhoA/LIMK/cofilin pathway. This effect was highly dependent on the function of the cytoskeleton as a scaffold for signal trafficking. We conclude that simvastatin attenuated neuropathic pain in rats subjected to CCI by inhibiting actin-mediated intracellular trafficking to suppress RhoA/LIMK/cofilin pathway activity.

     

LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics

The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. We show that in PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)-LIM kinase (LIMK)-cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways.     

A bronchial epithelium-derived factor reduces pulmonary vascular tone in the newborn rat.

The factors accounting for the maintenance of a low pulmonary vascular resistance postnatally are not completely understood. The aim of this study was to test the hypothesis that bronchial epithelium produces a factor capable of relaxing adjacent pulmonary arterial smooth muscle. We studied fourth-generation intralobar pulmonary arteries and bronchi of 4- to 8-day-old rats. Arteries were mounted on a wire myograph, alone or with the adjacent bronchus. The presence of the attached bronchus significantly reduced pulmonary artery force generation induced by the thromboxane analog (U-46619) or KCl whether the endothelium was present or absent (P < 0.01). The converse was not true in that bronchial force generation was not affected when studied with the adjacent pulmonary artery. Mechanical removal of the bronchial epithelium or addition of the nitric oxide (NO) synthase (NOS) nonspecific (N(G)-monomethyl-l-arginine) or the specific neuronal NOS (7-nitroindazole) inhibitors increased arterial force generation to levels comparable to the isolated artery preparation. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, significantly decreased (P < 0.01) NO release of pulmonary arteries only when the adjacent bronchus was present. We conclude that bronchial epithelium in the newborn rat produces a factor capable of lowering pulmonary vascular muscle tone. This relaxant effect can be suppressed by NOS and phosphatidylinositol 3-kinase kinase inhibition, suggesting an action via NOS phosphorylation and NO release. We speculate that such a mechanism may be operative in vivo and plays an important role in control of pulmonary vascular resistance in the early postnatal period.     

Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase

LIM-kinase 1 (LIMK1) and LIM-kinase 2 (LIMK2) regulate actin cytoskeletal reorganization via cofilin phosphorylation downstream of distinct Rho family GTPases. We report our findings that ROCK, a downstream protein kinase of Rho, specifically activates LIMK2 but not LIMK1 downstream of RhoA. LIMK1 and LIMK2 activities toward cofilin phosphorylation were stimulated by co-expression with the active form of ROCK (ROCK-Delta3), whereas full-length ROCK selectively activates LIMK2 but not LIMK1. Activation of LIMK2 by RhoA was inhibited by Y-27632, a specific inhibitor of ROCK, but Rac1-mediated activation of LIMK1 was not. ROCK directly phosphorylated the threonine 505 residue within the activation segment of LIMK2 and markedly stimulated LIMK2 activity. A LIMK2 mutant with replacement of threonine 505 by valine abolished LIMK2 activities for cofilin phosphorylation and actin cytoskeletal changes, whereas replacement by glutamate enhanced the protein kinase activity and stress fiber formation by LIMK2. These results indicate that ROCK directly phosphorylates threonine 505 and activates LIMK2 downstream of RhoA and that this phosphorylation is essential for LIMK2 to induce actin cytoskeletal reorganization. Together with the finding that LIMK1 is regulated by Pak1, LIMK1 and LIMK2 are regulated by different protein kinases downstream of distinct Rho family GTPases.     

Ceramide inhibits Kv currents and contributes to TP-receptor-induced vasoconstriction in rat and human pulmonary arteries

Neutral sphingomyelinase (nSMase)-derived ceramide has been proposed as a mediator of hypoxic pulmonary vasoconstriction (HPV), a specific response of the pulmonary circulation. Voltage-gated K(+) (K(v)) channels are modulated by numerous vasoactive factors, including hypoxia, and their inhibition has been involved in HPV. Herein, we have analyzed the effects of ceramide on K(v) currents and contractility in rat pulmonary arteries (PA) and in mesenteric arteries (MA). The ceramide analog C6-ceramide inhibited K(v) currents in PA smooth muscle cells (PASMC). Similar effects were obtained after the addition of bacterial sphingomyelinase (SMase), indicating a role for endogenous ceramide in K(v) channel regulation. K(v) current was reduced by stromatoxin and diphenylphosphine oxide-1 (DPO-1), selective inhibitors of K(v)2.1 and K(v)1.5 channels, respectively. The inhibitory effect of ceramide was still present in the presence of stromatoxin or DPO-1, suggesting that this sphingolipid inhibited both components of the native K(v) current. Accordingly, ceramide inhibited K(v)1.5 and K(v)2.1 channels expressed in Ltk(-) cells. Ceramide-induced effects were reduced in human embryonic kidney 293 cells expressing K(v)1.5 channels but not the regulatory subunit K(v)β2.1. The nSMase inhibitor GW4869 reduced the thromboxane-endoperoxide receptor agonist U46619-induced, but not endothelin-1-induced pulmonary vasoconstriction that was partly restored after addition of exogenous ceramide. The PKC-ζ pseudosubstrate inhibitor (PKCζ-PI) inhibited the K(v) inhibitory and contractile effects of ceramide. In MA ceramide had no effect on K(v) currents and GW4869 did not affect U46619-induced contraction. The effects of SMase were also observed in human PA. These results suggest that ceramide represents a crucial signaling mediator in the pulmonary vasculature.     

5,6-EET-induced contraction of intralobar pulmonary arteries depends on the activation of Rho-kinase.

The mechanism mediating epoxyeicosatrienoic acid (EET)-induced contraction of intralobar pulmonary arteries (PA) is currently unknown. EET-induced contraction of PA has been reported to require intact endothelium and activation of the thromboxane/endoperoxide (TP) receptor. Because TP receptor occupation with the thromboxane mimetic U-46619 contracts pulmonary artery via Rho-kinase activation, we examined the hypothesis that 5,6-EET-induced contraction of intralobar rabbit pulmonary arteries is mediated by a Rho-kinase-dependent signaling pathway. In isolated rings of second-order intralobar PA (1-2 mm OD) at basal tension, 5,6-EET (0.3-10 microM) induced increases in active tension that were inhibited by Y-27632 (1 microM) and HA-1077 (10 microM), selective inhibitors of Rho-kinase activity. In PA in which smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) was increased with KCl (25 mM) to produce a submaximal contraction, 5,6-EET (1 microM) induced a contraction that was 7.0 +/- 1.6 times greater than without KCl. 5,6-EET (10 microM) also contracted beta-escin permeabilized PA in which [Ca(2+)](i) was clamped at a concentration resulting in a submaximal contraction. Y-27632 inhibited the 5,6-EET-induced contraction in permeabilized PA. 5,6-EET (10 microM) increased phosphorylation of myosin light chain (MLC), increasing the ratio of phosphorylated MLC/total MLC from 0.10 +/- 0.03 to 0.30 +/- 0.02. Y-27632 prevented this increase in MLC phosphorylation. These data suggest that 5,6-EET induces contraction in intralobar PA by increasing Rho-kinase activity, phosphorylating MLC, and increasing the Ca(2+) sensitivity of the contractile apparatus.     

Molecular mechanism of thromboxane A(2)-induced platelet aggregation. Essential role for p2t(ac) and alpha(2a) receptors.

Thromboxane A(2) is a positive feedback lipid mediator produced following platelet activation. The G(q)-coupled thromboxane A(2) receptor subtype, TPalpha, and G(i)-coupled TPbeta subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T(AC), coupled to G(q) and G(i), respectively. We investigated whether the stable thromboxane A(2) mimetic, (15S)-hydroxy-9, 11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G(q) and G(i), through co-activation of TPalpha and TPbeta receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T(AC) receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an alpha(2A)-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G(i) pathway by selective activation of either the P2T(AC) receptor or the alpha(2A)-adrenergic receptor. We conclude that whereas thromboxane A(2) causes intracellular calcium mobilization and shape change independently, thromboxane A(2)-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G(i)-coupled receptors.     

Differential effects of losartan and candesartan on vasoconstrictor responses in the rat

Losartan has been reported to have inhibitory effects on thromboxane (TP) receptor-mediated responses. In the present study, the effects of 2 nonpeptide angiotensin II (AT1) receptor antagonists, losartan and candesartan, on responses to angiotensin II, the thromboxane A2 mimic, U46619, and norepinephrine were investigated and compared in the pulmonary and systemic vascular beds of the intact-chest rat. In this study, intravenous injections of angiotensin II, U46619, and norepinephrine produced dose-related increases in pulmonary and systemic arterial pressure. Losartan and candesartan, in the doses studied, decreased or abolished responses to angiotensin II. Losartan, but not candesartan, and only in a higher dose, produced small, but statistically significant, reductions in pressor responses to U46619 and to norepinephrine in the pulmonary and systemic vascular beds. Furthermore, losartan significantly reduced arachidonic acid-induced platelet aggregation, whereas candesartan had no effect. Pressor responses to angiotensin II were not changed by thromboxane and alpha-adrenergic receptor antagonists, or by cyclooxygenase and NO synthase inhibitors. These results show that losartan and candesartan are potent selective AT1 receptor antagonists in the pulmonary and systemic vascular beds and that losartan can attenuate thromboxane and alpha-adrenergic responses when administered at a high dose, whereas candesartan in the highest dose studied had no effect on responses to U46619 or to norepinephrine. The present data show that the effects of losartan and candesartan on vasoconstrictor responses are different and that pulmonary and systemic pressor responses to angiotensin II are not modulated or mediated by the release of cyclooxygenase products, activation of TP receptors, or the release of NO in the anesthetized rat.     

LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.     

Effect of long-term high-altitude hypoxia on fetal pulmonary vascular contractility.

Hypoxia in the fetus and/or newborn is associated with an increased risk of pulmonary hypertension. The present study tested the hypothesis that long-term high-altitude hypoxemia differentially regulates contractility of fetal pulmonary arteries (PA) and veins (PV) mediated by differences in endothelial NO synthase (eNOS). PA and PV were isolated from near-term fetuses of pregnant ewes maintained at sea level (300 m) or high altitude of 3,801 m for 110 days (arterial Po(2) of 60 Torr). Hypoxia had no effect on the medial wall thickness of pulmonary vessels and did not alter KCl-induced contractions. In PA, hypoxia significantly increased norepinephrine (NE)-induced contractions, which were not affected by eNOS inhibitor N(G)-nitro-l-arginine (l-NNA). In PV, hypoxia had no effect on NE-induced contractions in the absence of l-NNA. l-NNA significantly increased NE-induced contractions in both control and hypoxic PV. In the presence of l-NNA, NE-induced contractions of PV were significantly decreased in hypoxic lambs compared with normoxic animals. Acetylcholine caused relaxations of PV but not PA, and hypoxia significantly decreased both pD(2) and the maximal response of acetylcholine-induced relaxation in PV. Additionally, hypoxia significantly decreased the maximal response of sodium nitroprusside-induced relaxations of both PA and PV. eNOS was detected in the endothelium of both PA and PV, and eNOS protein levels were significantly higher in PV than in PA in normoxic lambs. Hypoxia had no significant effect on eNOS levels in either PA or PV. The results demonstrate heterogeneity of fetal pulmonary arteries and veins in response to long-term high-altitude hypoxia and suggest a likely common mechanism downstream of NO in fetal pulmonary vessel response to chronic hypoxia in utero.     

Hypoxic pulmonary hypertension: role of superoxide and NADPH oxidase (gp91phox)

Chronic exposure to low-O2 tension induces pulmonary arterial hypertension (PAH), which is characterized by vascular remodeling and enhanced vasoreactivity. Recent evidence suggests that reactive oxygen species (ROS) may be involved in both processes. In this study, we critically examine the role superoxide and NADPH oxidase plays in the development of chronic hypoxic PAH. Chronic hypoxia (CH; 10% O2 for 3 wk) caused a significant increase in superoxide production in intrapulmonary arteries (IPA) of wild-type (WT) mice as measured by lucigenin-enhanced chemiluminescence. The CH-induced increase in the generation of ROS was obliterated in NADPH oxidase (gp91phox) knockout (KO) mice, suggesting that NADPH oxidase was the major source of ROS. Importantly, pathological changes associated with CH-induced PAH (mean right ventricular pressure, medial wall thickening of small pulmonary arteries, and right heart hypertrophy) were completely abolished in NADPH oxidase (gp91phox) KO mice. CH potentiated vasoconstrictor responses of isolated IPAs to both 5-hydroxytryptamine (5-HT) and the thromboxane mimetic U-46619. Administration of CuZn superoxide dismutase to isolated IPA significantly reduced CH-enhanced superoxide levels and reduced the CH-enhanced vasoconstriction to 5-HT and U-46619. Additionally, CH-enhanced superoxide production and vasoconstrictor activity seen in WT IPAs were markedly reduced in IPAs isolated from NADPH oxidase (gp91phox) KO mice. These results demonstrate a pivotal role for gp91phox-dependent superoxide production in the pathogenesis of CH-induced PAH.     

Chronic hypoxia increases MCA contractile response to U-46619 by reducing NO production and/or activity.

Chronic hypoxia alters contractile sensitivity of isolated arteries to alpha-adrenergic stimulation and other agonists. However, most studies have been performed in thoracic aortas or other large vessels making little contribution to vascular resistance in their respective circulations. To determine the effect of chronic hypoxia on the vasoconstrictor response in a small, resistance-sized vessel, we studied second and third generation middle cerebral arteries (MCA; approximately 75-microm internal diameter before mounting). MCA were isolated from normoxic (inspired oxygen = 125 Torr) and hypoxic (8 wk at 3,960 m; inspired oxygen = 90 Torr) guinea pigs, and their vasoconstrictor responses were determined to the thromboxane mimetic U-46619 by using dual-pipette video microscopy. Arteries from hypoxic animals had greater contractile sensitivity to U-46619 compared with those of the normoxic animals (-log EC50 = 7.86 +/- 0.11 vs. 7.62 +/- 0.06, respectively, P < 0.05). Addition of the nitric oxide (NO) inhibitor nitro-L-arginine (200 microM) to the vessel bath eliminated the differences in contractile sensitivity between the MCA from the normoxic and chronically hypoxic groups. Supplementation with L-arginine in the drinking water sufficient to raise plasma L-arginine levels 41% reduced MCA contractile sensitivity to U-46619 in the normoxic group (-log EC50 = 7.22 +/- 0.31, P < 0.05 compared with the nonsupplemented normoxic group) but not in the chronically hypoxic group. These results show that chronic hypoxia increases the sensitivity of the MCA to the vasoconstrictor U-46619, likely because of a reduction in NO production and/or activity.     

RhoA/Rho kinase and nitric oxide modulate the agonist-induced pulmonary artery diameter response time

We studied the amplitude and response time (RT; time to 50% of maximal response) of pulmonary vasoreactivity and investigated whether the characteristics of pulmonary vasoreactivity could be modulated by endothelium removal, nitric oxide (NO) synthase inhibition [N(G)-nitro-L-arginine (L-NNA)], RhoA activation [lysophosphatidic acid (LPA)] and Rho kinase inhibition (Y-27632). Slow acetylcholine-induced pulmonary vasodilation (262 +/- 5 s) was not due to the RT of endothelial NO release (45-55 s) and was always longer than RT in renal arteries (15 +/- 4 s). The rate-determining step is located in the smooth muscle cells. This was confirmed by the existing differences between the RT of the NO solution and KCl-induced renal and pulmonary vasoreactivity in endothelium-denuded arteries. We found that the pulmonary contractile amplitude increases and the RT decreases by L-NNA or LPA. In contrast, Y-27632 reduced the contractile amplitude and increased the RT in pulmonary arteries. These phenomena were dependent on the contractile stimulus (phenylephrine or KCl). In conclusion, slow pulmonary vasoreactivity is a smooth muscle cell characteristic that can be enhanced by RhoA and NO or endothelium removal. These effects were counteracted by Rho kinase inhibition. We show a role for RhoA/Rho kinase and NO in the modulation of pulmonary vascular reactivity.     

Effects of thromboxane analog U46619 on endothelial damaged canine coronary arteries in vivo

We studied the effects of the thromboxane analog, U46619, infused into the left anterior descending (LAD) artery of intact dogs before and after producing endothelial denudation of the mid portion of the LAD. Proximal artery cross-sectional area (CSA) decreased by 47% with 0.1 microgram/min infusion of U46619 with intact and denuded endothelium, while resting CSA reduced spontaneously following denudation. Coronary resistance vessels demonstrated a marked constrictor response to U46619 with a rise in resistance and a fall in flow and myocardial O2 consumption. U46619 produces significant narrowing of proximal epicardial coronary arteries as well as resistance coronary vessels. This effect could cause ischemia in patients with moderate coronary atherosclerosis.