Materials and methods of research.The study included 19 patients with primary a STEMI the average age was 59 6 17 years.All patients underwent PCI with reperfusion of the infarctionrelated coronary artery.The mean recanalization time was 5 (4, 6) h.Using EchoCG, final systolic volume (CSR), terminal diastolic volume (BWW), LV ejection fraction (LVEF) was msured admission, 3rd, 7th and 21st day of STEMI.The CD133þ cells level was determined by flow cytometry of venous blood samples in the same periods.There was a control group of healthy volunteers (6 males) the average age was 41 6 9 years (absolutely healthy).Results: Carrying out an individual analysis of CD 133þ cells level circulating in patients with acute myocardial infarction, they found a multidirectional, nonlinear dynamics of their content by the 3rd day of MI.Due to this dynamics, all patients were divided into 2 subgroups: the 1st subgroup consisted of the patients with initially low level and it's further increasing on the 3rd day of MI, the 2nd subgroup -patients with a decrease level to a 3-m day of illness.Intragroup and intergroup differences were revealed on different days of the disease (p <0.05).A positive correlation was established between the relative amount of CD133þ cells in patients of the 2nd subgroup on the 7th day of MI and LVEF on the 21st day (R ¼ 0.7, p ¼ 0.02).Also, a relationship was found between the low absolute amount of CD 133þ cells in the first day of acute myocardial infarction and signs of chronic heart failure (CHF) after 6 months of the disease (p <0.05).Namely: in patients of the first subgroup, 6 months after of acute MI, minimal clinical manifestations of CHF were observed.This fact confirms that a high release of CD133þ cells into the peripheral channel is associated with a favorable course and disease prognosis. Conclusion:We showed the response of marrow to acute myocardial infarction, as release of CD133þ cells into the blood.It was found that the high mobilization of these cells both on the 1st day of MI and during the whole observation period was associated with a favorable course and disease prognosis, according to improvement of clinical and echocardiographic parameters.

Key points Transforming growth‐factor‐β (TGF‐β) 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‐β 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‐β enhanced bradykinin‐induced RhoA translocation, Rho‐kinase‐dependent phosphorylation and contraction, but partially suppressed bradykinin‐induced RhoA activity (RhoA‐GTP content). TGF‐β enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF‐β 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‐β target gene, an important regulator of Rho‐kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper‐responsiveness. AbstractTransforming growth factor‐β (TGF‐β), 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‐β 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‐β 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‐β 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‐β on RhoA‐GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF‐β 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‐β 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.

The acceleration of myocardial relaxation produced by β-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca2+ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (ktr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca2+ sensitivity.

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.

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.

Adenylyl cyclase (AC) is a key signalling enzyme for many GPCRs and catalyses the conversion of ATP to cAMP which, in turn, is a crucial determinant of many biological responses. β-Adrenoceptor agonists (β2) are commonly prescribed as bronchodilators for respiratory diseases such as asthma and COPD and yet the intracellular signalling mechanisms are still not fully understood. Recently we have shown, using tracheal tissue from WT and GM mice, that, of the 10 different isoforms of AC, AC6 plays a central role in β-agonist, but not PGE2, responses (Birrell et al. BJP 2015). The aim of this study was to determine if this mechanism is apparent in translational studies utilising human airway smooth muscle cells (HASMC). We showed that primary HASMCs express AC6 at the mRNA and protein level. We developed a shRNA sequence and vector that targeted AC6 (60+% knockdown of expression). We showed that cAMP production, as measured by FRET, in response to a β2 agonist was significantly reduced in HASMCs with reduced AC6 expression, whereas responses to forskolin or EP4 receptor agonist (receptor responsible for PGE2-induced relaxation) were not altered. To confirm AC6 functional role of we employed a technique which can detect the contractile status of cultured cells, Ptychography (VL21 Phasefocus UK). Using this system we showed that ACh induced “contraction”, which is reduced by the β2 agonist in control cells, was not in the cells treated with AC6-shRNA. Responses to a PKA agonist (downstream from AC) however were comparable. We conclude that AC6 plays a critical role in relaxation of HASMCs to β2 agonists; these results further unravel the signalling pathway of this extensively prescribed class of medicine.

Key points We evaluated the hypothesis that an increase in the hydrogen sulphide concentration in pulmonary artery smooth muscle cells (PASMCs) causes hypoxic pulmonary vasoconstriction (HPV) by examining the effects of the sulphide donor cysteine and sulphide‐synthesis blockers on HPV in isolated rat intrapulmonary arteries (IPAs). Cysteine (1 m m ) enhanced HPV and also the contraction to prostaglandin F2α (PGF 2α ) and both effects were abolished by the cystathionine γ‐lyase (CSE) blocker propargylglycine (PAG, 1 m m ), which had little or no non‐selective effect on contraction at this concentration. Neither PAG nor the cysteine aminotransferase (CAT) antagonist aspartate affected HPV in normal physiological saline solution (PSS), or in PSS containing physiological concentrations of cysteine, cystine and glutamate, whereas dithiothreitol (DTT), proposed to enhance HPV by converting mitochondrial thiosulphate to sulphide, instead abolished HPV. PAG markedly diminished whereas DTT did not affect cysteine‐induced sulphide release from liver pieces. The results do not support the proposal that hydrogen sulphide plays a role in HPV. Abstract An increase in the H 2 S (hydrogen sulphide, hereafter sulphide) concentration in pulmonary artery smooth muscle cells (PASMCs) has been proposed to mediate hypoxic pulmonary vasoconstriction (HPV). We evaluated this hypothesis in isolated rat intrapulmonary arteries (IPAs) by examining the effects of the sulphide precursor cysteine and sulphide‐synthesis blockers on HPV and also on normoxic pulmonary vasoconstriction (NPV) stimulated by prostaglandin F2α (PGF 2α ) and by the drug LY83583, which causes contraction in IPAs by increasing cellular reactive oxygen species levels. Experiments with several blockers of cystathionine γ‐lyase (CSE), the enzyme responsible for sulphide synthesis in the vasculature, demonstrated that propargylglycine (PAG, 1 m m ) had little or no effect on the NPV caused by PGF 2α or LY83583. Conversely, other CSE antagonists tested, aminooxyacetic acid (AOAA, 100 μ m ), β‐cyanoalanine (BCA, 500 μ m ) and hydroxylamine (HA, 100 μ m ), altered the NPV to PGF 2α (BCA increased, HA inhibited) and/or LY83583 (BCA increased, AOAA and HA inhibited). Preincubating IPAs in physiological saline solution (PSS) containing 1 m m cysteine increased the amplitude of the NPV to PGF2 α by ∼50%, and had a similar effect on HPV elicited by hypoxic challenge with 0% O 2 . The enhancement of both responses by cysteine was abolished by pretreatment with 1 m m PAG. Measurements carried out with an amperometric electrode demonstrated that incubation with 1 m m cysteine under anoxic conditions (to minimize sulphide oxidation) greatly potentiated the release of sulphide from pieces of rat liver and that this release was strongly antagonized by PAG, indicating that at this concentration PAG could enter cells intact and antagonize CSE. PAG at 1 m m had no effect on HPV recorded in control PSS, or in PSS supplemented with physiological concentrations of cysteine (10 μ m ), cystine (50 μ m ) and glutamate (100 μ m ) in order to prevent the possible depletion of intracellular cysteine during experiments. Application of a combination of 1 m m cysteine and 1 m m α‐ketoglutarate to promote sulphide synthesis via the cysteine aminotransferase/mercaptopyruvate sulphurtransferase (CAT/MST) pathway caused an increase in HPV similar to that observed for cysteine. This was partially blocked by the CAT antagonist aspartate (1 m m ) and also by PAG. However, HPV was not increased by 1 m m α‐ketoglutarate alone, and HPV in the absence of α‐ketoglutarate and cysteine was not attenuated by aspartate. Pretreatment of IPAs with dithiothreitol (DTT, 1 m m ), proposed to promote the conversion of mitochondrial thiosulphate to sulphide, did not increase the release of sulphide from pieces of rat liver in either the presence or the absence of 1 m m cysteine, and virtually abolished HPV. The results provide evidence that the sulphide precursor cysteine can promote both NPV and HPV in rat IPA by generating sulphide via a PAG‐sensitive pathway, presumably CSE. However, HPV evoked under control conditions was unaffected by the blockade of CSE. Moreover, HPV was not affected by the CAT antagonist aspartate and was blocked rather than enhanced by DTT. The data therefore indicate that sulphide generated by CSE or CAT/MST or from thiosulphate is unlikely to contribute to O 2 sensing during HPV in these arteries.

Aims Sphingosylphosphorylcholine (SPC) elicits vasoconstriction at micromolar concentrations. At lower concentrations (<= 1 mu mol/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 amyograph were challenged with similar to 25 mu mol/L [K+] to induce a small vasoconstriction. SPC (1 mu mol/L) dramatically potentiated this constriction in all arteries by similar to 400%. The potentiation was greatly suppressed or abolished by inhibition of phospholipase C (PLC; U73122), PKC epsilon (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 PKC epsilon. SPC (1 mu mol/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.

BACKGROUND AND PURPOSEThe 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 APPROACHContraction was recorded in intact or alpha-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). [Ca2+](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 RESULTSContractile responses in intact bronchioles were inhibited by antagonists of SrcFK, FAK and Rho-kinase, while after alpha-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 Ca2+ depletion and equivalent [Ca2+](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 IMPLICATIONSSrcFK mediates Ca2+-sensitization in ASM, while SrcFK and FAK together and individually influence multiple Ca2+ 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.

Aims beta-catenin has been shown to be regulated by inducible nitric oxide synthase (NOS) in endothelial cells. We investigated here whether beta-catenin interacts with and regulates endothelial NOS (eNOS) and whether eNOS activation promotes beta-catenin signalling.Methods and results We identified beta-catenin as a novel eNOS binding protein in human umbilical vein endothelial cells (HUVECs) by mass spectroscopy and western blot analyses of beta-catenin and eNOS immunoprecipitates. This was confirmed by in situ proximity ligation assay. eNOS activity, assessed by cGMP production and eNOS phosphorylation (Ser1177), was enhanced in beta-catenin(-/-) mouse pulmonary endothelial cells (MPECs) relative to wild-type MPECs. eNOS activation (using adenosine, salbutamol, thrombin, or histamine), or application of an NO donor (spermine NONOate) or cGMP-analogue (8-bromo-cGMP) caused nuclear translocation of beta-catenin in HUVEC as shown by western blotting of nuclear extracts. Exposure to spermine NONOate, 8-bromo-cGMP, or sildenafil (a phosphodiesterase type 5 inhibitor) also increased the expression of beta-catenin-dependent transcripts, IL-8, and cyclin D1. Stimulation of wild-type MPECs with basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), spermine NONOate, 8-bromo-cGMP, or sildenafil increased tube length relative to controls in an angiogenesis assay. These responses were abrogated in beta-catenin(-/-) MPECs, with the exception of that to bFGF which is NO-independent. In C57BL/6 mice, subcutaneous VEGF-supplemented Matrigel plugs containing beta-catenin(-/-) MPECs exhibited reduced angiogenesis compared with plugs containing wildtype MPECs. Angiogenesis was not altered in bFGF-supplemented Matrigel.Conclusion These data reveal bidirectional cross-talk and regulation between the NO-cGMP and beta-catenin signalling pathways.

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) exerts cardioprotective effects. eNOS binds to a number of proteins that regulate its function. Using mass spectroscopy to study eNOS immunoprecipitated from human umbilical vein endothelial cells (HUVEC), we identified β-catenin as a novel binding partner of eNOS. This previously unrecognised interaction was confirmed by western blot analyses of both eNOS and β-catenin immunoprecipitates. Further, eNOS activation (using adenosine, salbutamol, histamine or thrombin), application of an NO donor (spermine NONOate) or elevation of cGMP (using sildenafil or 8-bromo-cGMP) all increased nuclear translocation of β-catenin. Nuclear β-catenin activates T cell factor (TCF)/lymphoid enhancing factor (LEF) transcription factors. Application of spermine NONOate or elevation of cGMP increased β-catenin transcriptional activity, as assessed using a luciferase reporter assay in HUVEC transfected with TCF/LEF reporter plasmids. The role of β-catenin in regulating NO-mediated angiogenesis was assessed in wild type and β-catenin-/- mouse pulmonary endothelial cells (MPECs) using an in vitro Matrigel assay. Stimulation with vascular endothelial growth factor (VEGF; NO dependent), spermine NONOate or cGMP elevation increased tube length compared to untreated controls in wild type but not β-catenin-/- MPECs, although both exhibited similar responses to basic fibroblast growth factor (NO independent). Similarly, in C57BL/6 mice, subcutaneous Matrigel plugs containing VEGF and β-catenin siRNA contained fewer endothelial cells compared to plugs containing scrambled siRNA. We conclude that activation of NO-cGMP signalling induces nuclear translocation of β-catenin, which promotes angiogenesis. Whether this contributes to other physiological processes involving NO-mediated transcription remains to be determined.

Asthma and chronic obstructive pulmonary disease (COPD) are highly prevalent respiratory diseases characterized by airway inflammation, airway obstruction and airway hyperresponsiveness. Whilst current therapies, such as β-agonists and glucocorticoids, may be effective at reducing symptoms, they do not reduce disease progression. Thus, there is a need to identify new therapeutic targets. In this review, we summarize the potential of novel targets or tools, including anti-inflammatories, phosphodiesterase inhibitors, kinase inhibitors, transient receptor potential channels, vitamin D and protease inhibitors, for the treatment of asthma and COPD.

To determine the role of gap junctions (GJs) in hypoxic pulmonary vasoconstriction (HPV).Studies were performed in rat isolated intrapulmonary arteries (IPAs) mounted on a myograph and in anaesthetized rats. Hypoxia induced a biphasic HPV response in IPAs preconstricted with prostaglandin F-2 (PGF(2), 3 M) or 20 mM K. The GJ inhibitors 18-glycyrrhetinic acid (18-GA, 30 M), heptanol (3.5 mM), or 2-aminoethoxydiphenyl borate (2-APB) (75 M) had little effect on the transient Phase 1 of HPV, but abolished the sustained Phase 2 which is associated with Ca-2 sensitization. The voltage-dependent Ca-2 channel blocker diltiazem (10 M) had no effect on HPV, and did not alter the inhibitory action of 18-GA. Sustained HPV is enhanced by high glucose (15 mM) via potentiation of Ca-2 sensitization, in the presence of high glucose 18-GA still abolished sustained HPV. Simultaneous measurement of tension and intracellular Ca-2 using Fura PE-3 demonstrated that whilst 18-GA abolished tension development during sustained HPV, it did not affect the elevation of intracellular Ca-2. Consistent with this, 18-GA abolished hypoxia-induced phosphorylation of the Rho kinase target MYPT-1. In anaesthetized rats hypoxia caused a biphasic increase in systolic right ventricular pressure. Treatment with oral 18-GA (25 mg/kg) abolished the sustained component of the hypoxic pressor response.These results imply that GJs are critically involved in the signalling pathways leading to Rho kinase-dependent Ca-2 sensitization during sustained HPV, but not elevation of intracellular Ca-2, and may explain the dependence of the former on an intact endothelium.