Grape seed procyanidin suppresses inflammation in cigarette smoke-exposed pulmonary arterial hypertension rats by the PPAR-γ/COX-2 pathway.

BACKGROUND AND AIM: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling, which is mainly caused by inflammation. Inhibiting inflammation can relieve PAH. Grape seed procyanidin (GSP) possesses remarkable anti-inflammatory property and vascular protective function. In this experiment, we verified the anti-inflammatory property of GSP in cigarette smoke-exposed PAH rats and revealed its molecular mechanism. METHODS AND RESULTS: In vivo, 45 Sprague Dawley (SD) rats were divided into 5 groups randomly, treated with normoxia/cigarette smoke (CS)/GSP + CS/CS + solvent/GSP. After GSP + CS administration, a decrease in mPAP, PVR, RVHI, WT%, and WA% was detected in the rats as compared to those treated with CS. In vitro, the proliferation of pulmonary arterial smooth muscle cells (PASMCs) caused by cigarette smoke extract (CSE) was effectively attenuated with GSP + CSE administration. Furthermore, GSP significantly increased the expression of peroxisome proliferator-activated receptor γ (PPAR-γ) together with the lowered expression level of cyclooxygenase 2 (COX-2) in PASMCs co-incubated with CSE. CONCLUSION: These findings indicate that GSP ameliorates inflammation by the PPAR-γ/COX-2 pathway and finally inhibits the proliferation of PASMCs, which leads to pulmonary vascular remodeling.

Effects of toceranib compared with sorafenib on monocrotaline-induced pulmonary arterial hypertension and cardiopulmonary remodeling in rats.

Sorafenib reverses pulmonary arterial hypertension (PAH) and cardiopulmonary remodeling (CPR), but the effects of toceranib are unknown. This study investigated anti-remodeling effects and determined optimal doses of toceranib and sorafenib on monocrotaline (MCT)-induced PAH and CPR in rats. MCT-treated rats were orally treated with a 14-day course of sorafenib (10, 30, or 100 mg/kg), toceranib (1, 3, or 10 mg/kg), or water. Both sorafenib and toceranib significantly reversed the right ventricular (RV) hypertrophy at 10 mg/kg, but only sorafenib significantly improved the RV systolic and mean pressures. Sorafenib significantly normalized the B-type natriuretic peptide mRNA level of the RV and increased the non-muscularized pulmonary artery percentage. However, these effects were only observed at the highest toceranib dose, and neither toceranib dose reduced the fully muscularized pulmonary artery percentage. Further, the inhibition on vascular endothelial growth factor (VEGF) signaling was stronger in sorafenib than in toceranib. Besides the stronger inhibition on mitogen-activated protein kinase signaling, the greater reversal ability of sorafenib may be also due to the simultaneous blockade on the C-X-C chemokine receptor type 4 and autophagy induction. Toceranib insignificantly reversed CPR, and a high-dose therapy did not improve the RV hemodynamic outcomes. Sorafenib significantly reversed CPR, and a low-dose sorafenib therapy may be a suitable therapeutic agent for PAH.

Tetrahydrobiopterin oral therapy recouples eNOS and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs.

We previously showed that newborn piglets who develop pulmonary hypertension during exposure to chronic hypoxia have diminished pulmonary vascular nitric oxide (NO) production and evidence of endothelial NO synthase (eNOS) uncoupling (Fike CD, Dikalova A, Kaplowitz MR, Cunningham G, Summar M, Aschner JL. Am J Respir Cell Mol Biol 53: 255-264, 2015). Tetrahydrobiopterin (BH4) is a cofactor that promotes eNOS coupling. Current clinical strategies typically invoke initiating treatment after the diagnosis of pulmonary hypertension, rather than prophylactically. The major purpose of this study was to determine whether starting treatment with an oral BH4 compound, sapropterin dihydrochloride (sapropterin), after the onset of pulmonary hypertension would recouple eNOS in the pulmonary vasculature and ameliorate disease progression in chronically hypoxic piglets. Normoxic (control) and hypoxic piglets were studied. Some hypoxic piglets received oral sapropterin starting on day 3 of hypoxia and continued throughout an additional 7 days of hypoxic exposure. Catheters were placed for hemodynamic measurements, and pulmonary arteries were dissected to assess eNOS dimer-to-monomer ratios (a measure of eNOS coupling), NO production, and superoxide (O2·-) generation. Although higher than in normoxic controls, pulmonary vascular resistance was lower in sapropterin-treated hypoxic piglets than in untreated hypoxic piglets. Consistent with eNOS recoupling, eNOS dimer-to-monomer ratios and NO production were greater and O2·- generation was less in pulmonary arteries from sapropterin-treated than untreated hypoxic animals. When started after disease onset, oral sapropterin treatment inhibits chronic hypoxia-induced pulmonary hypertension at least in part by recoupling eNOS in the pulmonary vasculature of newborn piglets. Rescue treatment with sapropterin may be an effective strategy to inhibit further development of pulmonary hypertension in newborn infants suffering from chronic cardiopulmonary conditions associated with episodes of prolonged hypoxia.

Is arginase a potential drug target in tobacco-induced pulmonary endothelial dysfunction?

BACKGROUND: Tobacco-induced pulmonary vascular disease is partly driven by endothelial dysfunction. The bioavailability of the potent vasodilator nitric oxide (NO) depends on competition between NO synthase-3 (NOS3) and arginases for their common substrate (L-arginine). We tested the hypothesis whereby tobacco smoking impairs pulmonary endothelial function via upregulation of the arginase pathway. METHODS: Endothelium-dependent vasodilation in response to acetylcholine (Ach) was compared ex vivo for pulmonary vascular rings from 29 smokers and 10 never-smokers. The results were expressed as a percentage of the contraction with phenylephrine. We tested the effects of L-arginine supplementation, arginase inhibition (by N(omega)-hydroxy-nor-l-arginine, NorNOHA) and NOS3 induction (by genistein) on vasodilation. Protein levels of NOS3 and arginases I and II in the pulmonary arteries were quantified by Western blotting. RESULTS: Overall, vasodilation was impaired in smokers (relative to controls; p < 0.01). Eleven of the 29 smokers (the ED(+) subgroup) displayed endothelial dysfunction (defined as the absence of a relaxant response to Ach), whereas 18 (the ED(-) subgroup) had normal vasodilation. The mean responses to 10(-4) M Ach were -23 ± 10% and 31 ± 4% in the ED(+) and ED(-) subgroups, respectively (p < 0.01). Supplementation with L- arginine improved endothelial function in the ED(+) subgroup (-4 ± 10% vs. -32 ± 10% in the presence and absence of L- arginine, respectively; p = 0.006), as did arginase inhibition (18 ± 9% vs. -1 ± 9%, respectively; p = 0.0002). Arginase I protein was overexpressed in ED(+) samples, whereas ED(+) and ED(-) samples did not differ significantly in terms of NOS3 expression. Treatment with genistein did not significantly improve endothelial function in ED(+) samples. CONCLUSION: Overexpression and elevated activity of arginase I are involved in tobacco-induced pulmonary endothelial dysfunction.

Exposure to supplemental oxygen downregulates antioxidant enzymes and increases pulmonary arterial contractility in premature lambs.

BACKGROUND: The optimal oxygen concentration for the resuscitation of premature infants remains controversial. OBJECTIVES: We studied the effects of 21 versus 100% oxygen at initial resuscitation and also the effects of 24-hour exposure to 100% oxygen on arterial blood gases, oxidant lung injury, activities of lung antioxidant enzymes (AOEs) and isolated pulmonary artery (PA) contractility in preterm newborn lambs. METHODS: Preterm lambs at 128 days' gestation (term = 145 days) were delivered and ventilated with 21 (RAR; n = 5) or 100% oxygen (OXR; n = 5) for the first 30 min of life. Subsequently, FiO2 was adjusted to maintain an arterial PO2 (PaO2) between 45 and 70 mm Hg for 24 h. A third group of lambs was mechanically ventilated with 100% oxygen for 24 h (OX24; n = 5). RESULTS: Oxidized glutathione levels in whole blood correlated highly with PaO2. Reduced to oxidized glutathione ratio was significantly different between the groups, the ratio increasing with decreasing oxygen exposure. The OX24 group had significantly higher activities of lipid hydroperoxide and myeloperoxidase and significantly lower activities of superoxide dismutase, catalase and glutathione peroxidase in the lung at 24 h. Activities of AOEs correlated inversely with alveolar PO2. PA contractility to norepinephrine and KCl was greater with increasing oxygen exposure. Pretreatment with superoxide dismutase and catalase significantly reduced PA contractility in the OXR and OX24 groups, but not in the RAR group. CONCLUSIONS: We conclude that ventilated premature lambs are unable to appropriately increase AOE activity in response to hyperoxia and that increasing exposure to oxygen aggravates systemic oxidant stress, oxidant lung injury and pulmonary arterial contractility in these lambs.

Involvement of RhoA/Rho kinase signaling in protection against monocrotaline-induced pulmonary hypertension in pneumonectomized rats by dehydroepiandrosterone.

RhoA/Rho kinase (ROCK) signaling plays a key role in the pathogenesis of experimental pulmonary hypertension (PH). Dehydroepiandrosterone (DHEA), a naturally occurring steroid hormone, effectively inhibits chronic hypoxic PH, but the responsible mechanisms are unclear. This study tested whether DHEA was also effective in treating monocrotaline (MCT)-induced PH in left pneumonectomized rats and whether inhibition of RhoA/ROCK signaling was involved in the protective effect of DHEA. Three weeks after MCT injection, pneumonectomized rats developed PH with severe vascular remodeling, including occlusive neointimal lesions in pulmonary arterioles. In lungs from these animals, we detected cleaved (constitutively active) ROCK I as well as increases in activities of RhoA and ROCK and increases in ROCK II protein expression. Chronic DHEA treatment (1%, by food for 3 wk) markedly inhibited the MCT-induced PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 33+/-5 and 16+/-1 mmHg, respectively) and severe pulmonary vascular remodeling in pneumonectomized rats. The MCT-induced changes in RhoA/ROCK-related protein expression were nearly normalized by DHEA. A 3-wk DHEA treatment (1%) started 3 wk after MCT injection completely inhibited the progression of PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 47+/-3 and 30+/-3 mmHg, respectively), and this treatment also resulted in 100% survival in contrast to 30% in DHEA-untreated rats. These results suggest that inhibition of RhoA/ROCK signaling, including the cleavage and constitutive activation of ROCK I, is an important component of the impressive protection of DHEA against MCT-induced PH in pneumonectomized rats.

Differential effects of chronic hypoxia and intermittent hypocapnic and eucapnic hypoxia on pulmonary vasoreactivity.

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension (PH) and right heart failure, similar to chronic sustained hypoxia (CH). Supplemental CO(2), however, attenuates hypoxic PH. We therefore hypothesized that, similar to CH, IH elicits PH and associated increases in arterial endothelial nitric oxide synthase (eNOS) expression, ionomycin-dependent vasodilation, and receptor-mediated pulmonary vasoconstriction. We further hypothesized that supplemental CO(2) inhibits these responses to IH. To test these hypotheses, we measured eNOS expression by Western blot in intrapulmonary arteries from CH (2 wk, 0.5 atm), hypocapnic IH (H-IH) (3 min cycles of 5% O(2)/air flush, 7 h/day, 2 wk), and eucapnic IH (E-IH) (3 min cycles of 5% O(2), 5% CO(2)/air flush, 7 h/day, 2 wk) rats and their respective controls. Furthermore, vasodilatory responses to the calcium ionophore ionomycin and vasoconstrictor responses to the thromboxane mimetic U-46619 were measured in isolated saline-perfused lungs from each group. Hematocrit, arterial wall thickness, and right ventricle-to-total ventricle weight ratios were additionally assessed as indexes of polycythemia, arterial remodeling, and PH, respectively. Consistent with our hypotheses, E-IH resulted in attenuated polycythemia, arterial remodeling, RV hypertrophy, and eNOS upregulation compared with H-IH. However, in contrast to CH, neither H-IH nor E-IH increased ionomycin-dependent vasodilation. Furthermore, H-IH and E-IH similarly augmented U-46619-induced pulmonary vasoconstriction but to a lesser degree than CH. We conclude that maintenance of eucapnia decreases IH-induced PH and upregulation of arterial eNOS. In contrast, increases in pulmonary vasoconstrictor reactivity following H-IH are unaltered by exposure to supplemental CO(2).

Prolonged relaxation consistent with persistent soluble guanylyl cyclase activation in canine pulmonary artery following brief treatment with nitric oxide donors.

Recent work has indicated that prolonged treatment with nitric oxide (NO) donors results in tissue storage of NO as S-nitrosothiols and N-nitrosamines. The possibility thus exists that NO treatment may result in the development of tissue stores of NO with functionally significant effects following removal of the original NO source. In these studies, the effects of 10 min treatment with two chemically distinct NO sources, S-nitrosoglutathione (GSNO) and (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NO) were determined in canine pulmonary artery using a superfusion system that permitted continuous isometric force recording during addition and removal of the NO donors. Relaxation that persisted for up to 1 h after removal of the NO source, was demonstrated for both NO sources, but at lower concentrations relative to the relaxant EC(50) for GSNO versus DEA-NO. Persistent relaxation with both NO sources was fully reversed by both the sGC inhibitor, ODQ, and an inhibitor of cGMP-dependent protein kinase, Rp-8-Br-PET-cGMPS, indicating that persistent relaxation was consistent with persistent activation of the sGC-cGMP signaling pathway. In separate measurements, a GSNO-induced persistent increase in both tissue cGMP ([cGMP](i)) and relaxation were fully reversed by both ODQ and the thiol reducing agent dithiothreitol (DTT). The results indicate that vascular smooth muscle is capable of converting short-lived NO responses following short term exposure to NO donors by a mechanism consistent with prolonged sGC activation, resulting in persistent relaxation. Reversal of this cGMP-dependent process with DTT suggests that it occurs via mechanisms that are thiol redox sensitive.

Changes in pulmonary arteriole protein kinase calpha expression associated with supplemental L-arginine in broilers during cool temperature exposure.

The present study was conducted to examine the effect of supplemental L-arginine on pulmonary arteriole protein kinase Calpha (PKCalpha) expression in broilers exposed to cool temperature, to investigate further the molecular mechanisms of supplemental L-arginine on modulating pulmonary vascular functions in hypertensive broilers. Broilers were subjected to sub-thermoneutral (cool) temperature to induce pulmonary hypertension syndrome (PHS), and an additional 10 g/kg L-arginine was added to the basal diet to evaluate the effects of supplemental L-arginine on PHS mortality, plasma nitric oxide (NO) production and pulmonary arterioles PKCalpha expression. Supplemental L-arginine reduced PHS mortality but did not affect right/total ventricle (RV/TV) ratios in clinically healthy birds. Birds fed additional L-arginine had increased plasma NO and decreased PKCalpha protein expression in pulmonary arterioles; NO production was negatively correlated with PKCalpha expression. These results demonstrated that supplemental L-arginine diminished PKCalpha expression in birds exposed to cool temperature. It is suggested that NO-induced loss of PKCalpha expression might be partially responsible for its effects on dilating pulmonary vasculature and inhibiting pulmonary vascular remodelling in vivo.

Alkaline stress-induced apoptosis in human pulmonary artery endothelial cells.

The effect of alkaline stress, or an increase in extracellular pH (pHext), on cell viability is poorly defined. Human pulmonary artery endothelial cells (HPAEC) were subjected to alkaline stress using different methods of increasing pHext. Viability and mode of cell death following alkaline stress were determined by assessing nuclear morphology, ultrastructural features, and caspase-3 activity. Incubation of monolayers in media set to different pHext values (7.4-8.4) for 24-h induced morphological changes suggesting apoptosis (35-45% apoptotic cells) following severe alkaline stress. The magnitude of apoptosis was related to the severity of alkaline stress. These findings were confirmed with an assessment of ultrastructural changes and caspase-3 activation. While there was no difference in the intracellular calcium level ([Ca(2+)](i)) in monolayers set to pHext 7.4 versus 8.4 following the first hour of alkaline stress, blockade of calcium uptake with the chelator, EGTA, potentiated the magnitude of apoptosis under these conditions. Potentiation of apoptosis was reduced by calcium supplementation of the media. Finally, alkaline stress was associated with an increase in intracellular pH. This is the first report of apoptosis following alkaline stress in endothelial cells in the absence of other cell death stimuli.

Role of an aprotinin-sensitive protease in protein kinase Calpha-mediated activation of cytosolic phospholipase A2 by calcium ionophore (A23187) in pulmonary endothelium.

Treatment of bovine pulmonary artery endothelial cells with the calcium ionophore, A23187, stimulates the cell membrane associated protease activity, phospholipase A2 (PLA2) activity, and arachidonic acid (AA) release from the cells. Pretreatment of the cells with arachidonyl-trifluomethylketone (AACOCF3), a cPLA2 inhibitor, but not bromoenollactone (BEL), a iPLA2 inhibitor, prevents A23187 stimulated PLA2 activity and AA release without producing an appreciable alteration of the protease activity. Pretreatment of the cells with aprotinin, an ambient protease inhibitor, prevents the increase in the protease activity and cPLA2 activity in the membrane and AA release from the cells caused by both low and high doses of A23187, and also inhibits protein kinase C (PKC) activity caused by high doses of A23187. Immunoblot study of the endothelial cell membrane isolated from A23187 (10 microM)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. Immunoblot study of the endothelial membrane with polyclonal cPLA2 antibody revealed that treatment of the cells with A23187 dose-dependently increases cPLA2 immunoreactive protein profile in the membrane. It therefore appears from the present study that treatment of the cells with a low dose of A23187 (1 microM) causes a small increase in an aprotinin-sensitive protease activity and that stimulates cPLA2 activity in the cell membrane without an involvement of PKC. By contrast, treatment of the cells with a high dose of 10 microM of A23187 causes optimum increase in the protease activity and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA2 activity in the cell membrane. Although pretreatment of the cells with pertussis toxin caused ADP ribosylation of a 41 kDa protein in the cell membrane, it did not inhibit the cPLA2 activity and AA release caused by both low and high doses of A23187.

Antiremodeling effects of iloprost and the dual-selective phosphodiesterase 3/4 inhibitor tolafentrine in chronic experimental pulmonary hypertension.

Severe pulmonary hypertension is a disabling disease with high mortality. We investigated acute and chronic effects of iloprost, a long-acting prostacyclin analogue, and the dual-selective phosphodiesterase 3/4 inhibitor tolafentrine in monocrotaline-induced pulmonary hypertension in rats. Twenty-eight and 42 days after administration of the alkaloid, right ventricular systolic pressure increased from 25.8+/-2.0 to 62.9+/-3.4 and 70.5+/-7.4 mm Hg, with concomitant decline in cardiac index, central venous oxygen saturation, and arterial oxygenation. Marked right heart hypertrophy was demonstrated by the strongly elevated ratio of right ventricle/left ventricle plus septum weight, and massive thickening of the precapillary artery smooth muscle layer was shown histologically. Western blot analysis demonstrated increased levels of matrix metalloproteinases (MMPs) -2 and -9 and increased gelatinolytic activities in isolated pulmonary arteries. In these animals, both intravenous iloprost and tolafentrine displayed characteristic features of pulmonary vasodilators. When chronically infused from days 14 to 28, both agents significantly attenuated all monocrotaline-induced hemodynamic and gas exchange abnormalities as well as right heart hypertrophy. Full normalization of all variables including right ventricle size was achieved on combined administration of both agents during this period. This was also true for MMP-2 and MMP-9 expression and activity. Moreover, when iloprost plus tolafentrine was used for late therapeutic intervention, with infusion from days 28 to 42 after full establishment of severe pulmonary hypertension and cor pulmonale, hemodynamic, gas exchange, and cardiac and pulmonary vascular remodeling changes were significantly reversed. We conclude that the combined administration of iloprost and a dual-selective phosphodiesterase 3/4 inhibitor prevents and reverses the development of pulmonary hypertension and cor pulmonale in response to monocrotaline in rats. This regimen may therefore offer a possible antiremodeling therapy in severe pulmonary hypertension.

[Regulation of the expression of pulmonary arterial collagen by protein kinase C and breviscapine in chronic hypoxic rats].

OBJECTIVE: To investigate the effects of protein kinase C and breviscapine on the expression of pulmonary arterial collagen in chronic hypoxic rats. METHODS: Thirty-six rats were randomly divided into three groups: control group (A), hypoxic group(B),hypoxic + breviscapine(bre.)group (C). The ultrastructure of pulmonary arterioles was observed by electron microscope; the PKC activities of lung tissues were measured by radioactivity; the expression of PKC and collagen I and III in arterioles was observed by immunohistochemistry; the expression of procollagen I and III mRNA in arterioles was observed by in situ hybridization. The averages of integral light density( A )of PKC, collagen I and III and procollagen I and III mRNA in pulmonary arterioles were detected by image analysor and their relative contents were calculated. RESULTS: (1) The mean pulmonary arterial pressure (mPAP) and the weight ratio of RV to LV + S in group B were higher than those in group A(P < 0.01); the mPAP and the weight ratio of RV to LV + S in group C were lower than those in group B (P < 0.01). (2)Electron microscopy showed breviscapine could inhibit the deposition of collagenous fibers in pulmonary arterioles induced by hypoxia.(3) The total,cytosolic and particulate fractions of PKC activity and the ratio of particulate fraction to total PKC activity in group B were higher than those in group A(P < 0.01); the total,cytosolic and particulate fractions of PKC activity and the ratio of particulate fraction to total PKC activity in group C were lower than those in group B (P < 0.05). (4) The A values of PKC, collagen I and procollagen I mRNA in pulmonary arterioles were higher in group B than in group A(P < 0.01),and the A values of PKC, collagen I and procollagen I mRNA in pulmonary arterioles were lower in group C than in group B(P < 0.01); the differences in the A values of collagen III and procollagen III mRNA in pulmonary arterioles were not significant among the three groups (P > 0.05).(5)There were good correlations between the PKC activity of the lung tissues and the A values of collagen I and procollagen I mRNA in pulmonary arterioles(P < 0.05),and between the A values of PKC in pulmonary arterioles and those of collagen I and procollagen I mRNA in pulmonary arterioles(P < 0.01). CONCLUSIONS: The PKC signal pathway regulates the expression of pulmonary arterial collagen in chronic hypoxic rats which may play an important role in the pathogenesis of pulmonary hypertension and structural remodeling of pulmonary arterials breviscapine can lower hypoxic pulmonary hypertension by inhibiting the effect of PKC and decreasing the expression of pulmonary arterial collagen.

Expression of 5-lipoxygenase in pulmonary artery endothelial cells.

Increased expression of 5-lipoxygenase (5LO) in pulmonary artery endothelial cells (PAECs) has been observed in disease states such as pulmonary hypertension and allergen challenge. To understand the function of endothelial 5LO, we examined the expression of this enzyme in normally cultured human PAECs and its characteristics when overexpressed. A small amount of 5LO message and protein was detected by reverse-transcriptase-mediated PCR (RT-PCR) and Western blotting in PAECs. Sequencing of the RT-PCR products that overlapped the entire coding region of 5LO mRNA indicated that the sequence of PAEC 5LO was identical with that of leucocyte 5LO. Incubation of the PAECs with A23187 and arachidonic acid led to a small production of 5-hydroxyeicosatetraenoic acid (5-HETE) (46-98 pmol/4x10(6) cells) but no leukotrienes. Overexpression of 5LO in PAECs by adenovirus-mediated gene transfer revealed that the enzyme was localized in the nucleus. Incubation of the transduced cells with A23187 (5 microM) caused the production of both 5LO products and downstream leukotrienes. The proportions of the produced leukotriene A(4) (LTA(4)) hydrolates (sum of 6-trans-LTB(4) and 12-epi-6-trans-LTB(4)), LTB(4) and cysteinyl leukotriene were approx. 17:14:10. cGMP production in the 5LO-transduced PAECs was decreased by 33+/-14% on stimulation with A23187. These results show that cultured PAECs express a minimal amount of 5LO, which can generate some 5-HETE, but not leukotrienes. However, increased expression of 5LO in PAECs can lead to the production of all downstream leukotrienes, which could potentially cause endothelial dysfunction in the pulmonary vasculature.

Molecular cloning, characterization and expression of a nitric oxide synthase from porcine pulmonary artery endothelial cells.

The lack of sequence information and clones of porcine pulmonary artery endothelial cell (PAEC) constitutive nitric oxide synthase (ecNOS) cDNA limits comparative analysis between porcine and human PAEC. Therefore, we cloned, characterized and expressed the ecNOS cDNA from porcine PAEC. Two oligonucleotide primers were designed based on the published human ecNOS cDNA sequence and used to clone porcine PAEC ecNOS using 5' and 3' rapid amplification of cDNA ends reverse transcriptase polymerase chain reaction technique. A full-length ecNOS cDNA was cloned and sequenced, representing a protein of 1205 amino acids with a molecular mass of 134 kDa. A mammalian expression vector (pcDNA3) containing this cDNA was transfected into COS-7 cells, and ecNOS activity was detected by monitoring the formation of [3H]-citrulline from [3H]-L-arginine. Expression of ecNOS activity was predominantly associated (> 90%) with the total membrane fraction of these transfected cells. The deduced amino acid sequence of porcine ecNOS cDNA, containing binding sites for NADPH, flavin adenine dinucleotide and bound flavin mononucleotide, shows 94% identity to human ecNOS. The molecular weight of porcine ecNOS mRNA was estimated to be 4.7 kb by Northern blot analysis, similar to human ecNOS mRNA. This suggests that porcine ecNOS is similar to human ecNOS in deduced amino acid sequence and structure.

Studies of myofibrillar ATPase in ragweed-sensitized canine pulmonary smooth muscle.

The maximal shortening velocities of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized dogs were significantly higher than those of muscles from their littermate controls. Myofibrils of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized and control dogs were obtained with use of Triton X-100 homogenizing solution. The myofibrillar adenosinetriphosphatase (ATPase) activities of the sensitized tissues were significantly higher (P less than 0.05) than those of their respective controls.