Bovine Pulmonary Artery Smooth Muscle Research Articles

Role of PKCζ-NADPH oxidase signaling axis in PKCα-mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells.

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C-ζ (PKC-ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC-α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC-ζ, NADPH oxidase, and PKC-α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC-ζ and NADPH oxidase attenuate AngII induced increase in PKC-α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC-α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC-ζ and NADPH oxidase activities. Overall, PKCζ-NADPH oxidase-PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII-mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.

Extended hypoxia-mediated H2 S production provides for long-term oxygen sensing.

Numerous studies have shown that H2 S serves as an acute oxygen sensor in a variety of cells. We hypothesize that H2 S also serves in extended oxygen sensing. Here, we compare the effects of extended exposure (24-48hours) to varying O2 tensions on H2 S and polysulphide metabolism in human embryonic kidney (HEK 293), human adenocarcinomic alveolar basal epithelial (A549), human colon cancer (HTC116), bovine pulmonary artery smooth muscle, human umbilical-derived mesenchymal stromal (stem) cells and porcine tracheal epithelium (PTE) using sulphur-specific fluorophores and fluorometry or confocal microscopy. All cells continuously produced H2 S in 21% O2 and H2 S production was increased at lower O2 tensions. Decreasing O2 from 21% to 10%, 5% and 1% O2 progressively increased H2 S production in HEK293 cells and this was partially inhibited by a combination of inhibitors of H2 S biosynthesis, aminooxyacetate, propargyl glycine and compound 3. Mitochondria appeared to be the source of much of this increase in HEK 293 cells. H2 S production in all other cells and PTE increased when O2 was lowered from 21% to 5% except for HTC116 cells where 1% O2 was necessary to increase H2 S, presumably reflecting the hypoxic environment in vivo. Polysulphides (H2 Sn , where n=2-7), the key signalling metabolite of H2 S also appeared to increase in many cells although this was often masked by high endogenous polysulphide concentrations. These results show that cellular H2 S is increased during extended hypoxia and they suggest this is a continuously active O2 -sensing mechanism in a variety of cells.

Role of PLD-PKCζ signaling axis in p47phox phosphorylation for activation of NADPH oxidase by angiotensin II in pulmonary artery smooth muscle cells.

We sought to determine the mechanism by which angiotensin II (ANGII) stimulates NADPH oxidase-mediated superoxide (O2 .- ) production in bovine pulmonary artery smooth muscle cells (BPASMCs). ANGII-induced increase in phospholipase D (PLD) and NADPH oxidase activities were inhibited upon pretreatment of the cells with chemical and genetic inhibitors of PLD2, but not PLD1. Immunoblot study revealed that ANGII treatment of the cells markedly increases protein kinase C-α (PKC-α), -δ, -ε, and -ζ levels in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PKC-ζ, but not PKC-α, -δ, and -ε, attenuated ANGII-induced increase in NADPH oxidase activity without a discernible change in PLD activity. Transfection of the cells with p47phox small interfering RNA inhibited ANGII-induced increase in NADPH oxidase activity without a significant change in PLD activity. Pretreatment of the cells with the chemical and genetic inhibitors of PLD2 and PKC-ζ inhibited ANGII-induced p47phox phosphorylation and subsequently translocation from cytosol to the cell membrane, and also inhibited its association with p22phox (a component of membrane-associated NADPH oxidase). Overall, PLD-PKCζ-p47phox signaling axis plays a crucial role in ANGII-induced increase in NADPH oxidase-mediated O2 .- production in the cells.

Protective role of epigallocatechin-3-gallate in NADPH oxidase-MMP2-Spm-Cer-S1P signalling axis mediated ET-1 induced pulmonary artery smooth muscle cell proliferation.

The signalling pathway involving MMP-2 and sphingosine-1-phosphate (S1P) in endothelin-1 (ET-1) induced pulmonary artery smooth muscle cell (PASMC) proliferation is not clearly known. We, therefore, investigated the role of NADPH oxidase derived O2.--mediated modulation of MMP2-sphingomyeline-ceramide-S1P signalling axis in ET-1 induced increase in proliferation of PASMCs. Additionally, protective role of the tea cathechin, epigallocatechin-3-gallate (EGCG), if any, in this scenario has also been explored. ET-1 markedly increased NADPH oxidase and MMP-2 activities and proliferation of bovine pulmonary artery smooth muscle cells (BPASMCs). ET-1 also caused significant increase in sphingomyelinase (SMase) activity, ERK1/2 and sphingosine kinase (SPHK) phosphorylations, and S1P level in the cells. EGCG inhibited ET-1 induced increase in SMase activity, ERK1/2 and SPHK phosphorylations, S1P level and the SMC proliferation. EGCG also attenuated ET-1 induced activation of MMP-2 by inhibiting NADPH oxidase activity upon inhibiting the association of the NADPH oxidase components, p47phox and p67phox in the cell membrane. Molecular docking study revealed a marked binding affinity of p47phox with the galloyl group of EGCG. Overall, our study suggest that ET-1 induced proliferation of the PASMCs occurs via NADPH oxidase-MMP2- Spm- Cer-S1P signalling axis, and EGCG attenuates ET-1 induced increase in proliferation of the cells by inhibiting NADPH oxidase activity.

Effects of Hypoxia on intracellular H2S and Polysulfides: Implications in O2 Sensing

The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic responses is a contentious issue. Vascular oxygen sensing is generally attributed to hypoxic generation of reactive oxygen species (ROS). However, we recently found that methods commonly employed to measure ROS cannot distinguish between ROS and reactive sulfur species (RSS) and that RSS and ROS have similar effects on intracellular redox status. Our previous studies suggest that metabolism of hydrogen sulfide (H2S) is the O2 sensor, as H2S mimics hypoxia in numerous systems and H2S metabolism is inversely coupled to O2 availability at physiological levels. In the present study we measure intracellular production of H2S and H2Sn using 7‐Azido‐4‐Methylcoumarin (AzMC) and 3,6‐DI(O‐Thiosalicyl) (SSP4), respectively in HEK‐293 cells and bovine pulmonary artery smooth muscle cells (BPASMC) under both normoxic (21% O2) and hypoxic (0.5% O2) conditions over a 48 hr period. In addition, we attempted to augment H2S and H2Sn production via the known intracellular pathways through addition of substrates (to increase production) and inhibitors (to decrease production). We found that hypoxia increased AzMC fluorescence by 50%, but did not affect SSP4 fluorescence. In normoxia, AzMC fluorescence did not change, whereas SSP4 fluorescence increased two‐fold. These results suggest that hypoxia increases intracellular H2S concentration and concomitantly prevents further the increase in H2Sn concentration. Intracellular H2S and H2Sn was not affected by either cysteine or cystine (10 and 100 uM). Both propargylglycine (10 mM), an inhibitor of cystathionine ã‐lyase and aspartate (1 mM) an inhibitor of cysteine aminotransferase reduced hypoxia stimulated AzMC fluorescence in HEK‐293 cells. Aminooxyacetate, an inhibitor of cystathionione â‐synthase, greatly increased AzMC fluorescence in HEK‐293 cells in normoxia and hypoxia. These are the first measurements of oxygen‐dependent changes in intracellular H2S and H2Sn in living cells and they support our hypothesis that RSS are directly coupled to O2 sensing.Support or Funding InformationNSF Grant IOS‐144‐6310 (KRO), NSF GRFP DGE‐131‐3583 (ERD)

Cross-talk between NADPH oxidase-PKCα-p(38)MAPK and NF-κB-MT1MMP in activating proMMP-2 by ET-1 in pulmonary artery smooth muscle cells.

Treatment of bovine pulmonary artery smooth muscle cells with endothelin-1 (ET-1) caused an increase in the expression and activation of proMMP-2 in the cells. The present study was undertaken to determine the underlying mechanisms involved in this scenario. We demonstrated that (i) pretreatment with NADPH oxidase inhibitor, apocynin; PKC-α inhibitor, Go6976; p(38)MAPK inhibitor SB203580 and NF-κB inhibitor, Bay11-7082 inhibited the expression and activation of proMMP-2 induced by ET-1; (ii) ET-1 treatment to the cells stimulated NADPH oxidase and PKCα activity, p(38)MAPK phosphorylation as well as NF-κB activation by translocation of NF-κBp65 subunit from cytosol to the nucleus, and subsequently by increasing its DNA-binding activity; (iii) ET-1 increases MT1-MMP expression, which was inhibited upon pretreatment with apocynin, Go6976, SB293580, and Bay 11-7082; (iv) ET-1 treatment to the cells downregulated TIMP-2 level. Although apocynin and Go6976 pretreatment reversed ET-1 effect on TIMP-2 level, yet pretreatment of the cells with SB203580 and Bay 11-7082 did not show any discernible change in TIMP-2 level by ET-1. Overall, our results suggest that ET-1-induced activation of proMMP-2 is mediated via cross-talk between NADPH oxidase-PKCα-p(38)MAPK and NFκB-MT1MMP signaling pathways along with a marked decrease in TIMP-2 expression in the cells.

Cross-talk between p(38)MAPK and G iα in regulating cPLA 2 activity by ET-1 in pulmonary smooth muscle cells.

Endothelin-1 (ET-1) is known as the most potent vasoconstrictor yet described. Infusion of ET-1 into isolated rabbit lung has been shown to cause pulmonary vasoconstriction with the involvement of arachidonic acid metabolites. Given the potency of arachidonic acid metabolites, the activity of phospholipase A2 must be tightly regulated. Herein, we determined the mechanisms by which ET-1 stimulates cPLA2 activity during ET-1 stimulation of bovine pulmonary artery smooth muscle cells. We demonstrated that (i) treatment of bovine pulmonary artery smooth muscle cells with ET-1 stimulates cPLA2 activity in the cell membrane; (ii) ET-1 caused increase in O 2 (·-) production occurs via NADPH oxidase-dependent mechanism; (iii) ET-1-stimulated NADPH oxidase activity is markedly prevented upon pretreatment with PKC-ζ inhibitor, indicating that PKC-ζ plays a prominent role in this scenario; (iv) ET-1-induced NADPH oxidase-derived O 2 (·-) stimulates an aprotinin sensitive protease activity due to prominent increase in [Ca(2+)]i; (v) the aprotinin sensitive protease plays a pivotal role in activating PKC-α, which in turn phosphorylates p(38)MAPK and subsequently Giα leading to the activation of cPLA2. Taken together, we suggest that cross-talk between p(38)MAPK and Giα with the involvement of PKC-ζ, NADPH oxidase-derived O 2 (·-) , [Ca(2+)]i, aprotinin-sensitive protease and PKC-α play a pivotal role for full activation of cPLA2 during ET-1 stimulation of pulmonary artery smooth muscle cells.

Identification, purification and partial characterization of low molecular weight protein inhibitor of Na⁺/K⁺-ATPase from pulmonary artery smooth muscle cells.

We have identified a novel endogenous low mol wt. (15.6 kDa) protein inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of bovine pulmonary artery smooth muscle cells. The inhibitor showed different affinities toward the α₂β₁ and α₁β₁ isozymes of Na(+)/K(+)-ATPase, where α₂ is more sensitive than α₁. The inhibitor interacted reversibly to the E1 site of the enzyme and blocked the phosphorylated intermediate formation. Circular dichroism study suggests that the inhibitor causes an alteration in the confirmation of the enzyme.

Role of PKC-ζ in NADPH oxidase–PKCα–Giα axis dependent inhibition of β-adrenergic response by U46619 in pulmonary artery smooth muscle cells

Treatment of bovine pulmonary artery smooth muscle cells (BPASMCs) with U46619 attenuated isoproterenol caused stimulation of adenyl cyclase activity and cAMP production. Pretreatment with SQ29548 (Tp receptor antagonist), apocynin (NADPH oxidase inhibitor) and Go6976 (PKC-α inhibitor) eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity. Pretreatment with SQ29548 and apocynin prevented U46619 induced increase in NADPH oxidase activity, PKC-α activity and Giα phosphorylation. However, pretreatment with CZI, a PKC-ζ inhibitor, markedly, but not completely, inhibited U46619 induced increase in NADPH oxidase activity, PKC-α activity, Giα phosphorylation and also significantly eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976 inhibited U46619 induced increase in Giα phosphorylation, but not PKC-ζ activity and NADPH oxidase activity. Pretreatment with pertussis toxin eliminated U46619 caused attenuation of isoproterenol stimulated adenyl cyclase activity without any discernible change in PKC-ζ, NADPH oxidase and PKC-α activities. Transfection of the cells with Tp, PKC-ζ and PKC-α siRNA duplexes corroborate the findings observed with their respective pharmacological inhibitors on the responses produced by U46619. Taken together, we suggest involvement of PKC-ζ in U46619 caused attenuation of isoproterenol stimulated β-adrenergic response, which is regulated by NADPH oxidase–PKCα–Giα axis in pulmonary artery smooth muscle cells.

Activation of proMMP-2 by U46619 occurs via involvement of p38MAPK-NFκB-MT1MMP signaling pathway in pulmonary artery smooth muscle cells

We investigated the mechanism by which TxA2 mimetic, U46619, activates proMMP-2 in bovine pulmonary artery smooth muscle cells. Our study showed that treatment of the cells with U46619 caused an increase in the expression and subsequently activation of proMMP-2 in the cells. Pretreatment with p(38)MAPK inhibitor, SB203580; and NF-κB inhibitor, Bay11-7082 inhibited the expression and activation of proMMP-2 induced by U46619. U46619 also induced increase in MT1-MMP expression, which was inhibited upon pretreatment with SB203580 and Bay11-7082. U46619 treatment to the cells stimulated p(38)MAPK activity as well as NF-κB activation by IκB-α phosphorylation, translocation of NF-κBp65 subunit from cytosol to nucleus and subsequently, by increasing its DNA-binding activity. Induction of NF-κB activation seems to be mediated through IKK, as transfection of cells with either IKKα or IKKβ siRNA prevented U46619-induced phosphorylation of IκB-α and NF-κBp65 DNA-binding activity. U46619 treatment to the cells also downregulated the TIMP-2 level. Pretreatment of the cells with SB203580 and Bay11-7082 did not show any discernible change in TIMP-2 level by U46619. Overall, U46619-induced activation of proMMP-2 is mediated via involvement of p(38)MAPK-NFκB-MT1MMP signaling pathway with concomitant downregulation of TIMP-2 expression in bovine pulmonary artery smooth muscle cells.

Role of PKC- in NF- B-MT1-MMP-mediated activation of proMMP-2 by TNF- in pulmonary artery smooth muscle cells

We sought to evaluate the mechanism(s) associated with pro matrix metalloprotease 2 (proMMP-2) activation in bovine pulmonary artery smooth muscle cells. Preincubation of cells with anti-TNFR1 antibody prevented tumour necrosis factor-α (TNF-α)-induced proMMP-2 activation and increase in membrane type 1 matrix metalloprotease (MT1-MMP) expression as well as inhibition of tissue inhibitor of metalloproteinase 2 (TIMP-2) expression, indicating the role of TNFR1 receptor during TNF-α stimulation. Anti-MT1-MMP antibody abrogated proMMP-2 activation by TNF-α-stimulated cell membrane, suggesting the involvement of MT1-MMP in proMMP-2 activation. Induction of MT1-MMP expression in response to TNF-α occurs via activation of nuclear factor (NF)-κB on inhibitory κB kinase (IKK) activation and subsequently phosphorylation/degradation of IκB-α. Inhibition of protein kinase C (PKC)-α activity by Go6976 and PKC-α siRNA prevented TNF-α-induced IKK activity, IκB-α phosphorylation/degradation and NF-κB activation. Inhibition of PKC-α activity also prevented TNF-α-induced MT1-MMP expression and proMMP-2 activation as well as down regulation of TIMP-2 expression. Inhibition of IκB-α phosphorylation by PS-1145, an IKK selective inhibitor, prevented TNF-α-induced increase in MT1-MMP expression and proMMP-2 activation, which although did not alter inhibition of TIMP-2 expression. Overall, we unravelled a hitherto unknown mechanism of the involvement of PKC-α in proMMP-2 activation and inhibition of TIMP-2 expression by NF-κB-MT1-MMP-dependent and -independent pathway, respectively, during TNF-α stimulation in pulmonary artery smooth muscle cells.

Role of PKCα–p38MAPK–Giα axis in NADPH oxidase derived [formula omitted]-mediated activation of cPLA2 under U46619 stimulation in pulmonary artery smooth muscle cells

We have recently reported that treatment of bovine pulmonary artery smooth muscle cells with the thromboxane A2 mimetic, U46619 stimulated NADPH oxidase derived O2·- level, which subsequently caused marked increase in [Ca2+]i[17]. Herein, we demonstrated that O2·--mediated increase in [Ca2+]i stimulates an aprotinin sensitive proteinase activity, which proteolytically activates PKC-α under U46619 treatment to the cells. The activated PKC-α then phosphorylates p38MAPK and that subsequently caused Giα phosphorylation leading to stimulation of cPLA2 activity in the cell membrane.

Heterotypic cell pair co-culturing on patterned microarrays.

We present a pair-wise co-culturing technique that creates large numbers of heterotypic cell pairs in patterned arrays. Lithographic patterning produces arrays with thousands of traps, each designed to accommodate only two cells and confine them at these sites for co-culturing. Two variants are introduced: a random seeding method that sediments a mixture of two cell types onto the array, and an approach that incorporates ferromagnetic thin films into the arrays and attracts cells that have been attached to ferromagnetic nanowires to the array sites through dipole interactions. The array technique includes the utilization of custom image analysis software that extracts data from multi-channel fluorescence images and records information about the cells in every trap, enabling the acquisition of accurate, high-statistics data. The applicability of the technique was demonstrated in experiments examining proliferation rates in pairs of bovine pulmonary artery endothelial and smooth muscle cells. Results demonstrated that heterotypic interactions favored smooth muscle cell proliferation while disfavoring endothelial cell proliferation. This is one example of a variety of cell-cell interactions that could be probed with this method.

Anti-proliferative effects of O-acyl-low-molecular-weight heparin derivatives on bovine pulmonary artery smooth muscle cells.

Heparin (HP) inhibits the growth of several cell types in vitro including bovine pulmonary artery (BPA) smooth muscle cells (SMCs). In initial studies we discovered that an O-hexanoylated low-molecular-weight (LMW) HP derivative having acyl groups with 6-carbon chain length was more potent inhibitor of BPA-SMCs than the starting HP. We prepared several O-acylated LMWHP derivatives having 4-, 6-, 8-, 10-, 12-, and 18- carbon acyl chain lengths to determine the optimal acyl chain length for maximum anti-proliferative properties of BPA-SMCs. The starting LMWHP was prepared from unfractionated HP by sodium periodate treatment followed by sodium borohydride reduction. The tri-n-butylammonium salt of this LMWHP was O-acylated with butanoic, hexanoic, octanoic, decanoic, dodecanoic, and stearyl anhydrides separately to give respective O-acylated LMWHP derivatives. Gradient polyacrylamide gel electrophoresis (PAGE) was used to examine the average molecular weights of those O-acylated LMWHP derivatives. NMR analysis indicated the presence of one O-acyl group per disaccharide residue. Measurement of the inhibition of BPA-SMCS as a function of O-acyl chain length shows two optima, at a carbon chain length of 6 (O-hexanoylated LMWHP) and at a carbon chain length 12-18 (O-dodecanoyl and O-stearyl LMWHPs). A solution competition SPR study was performed to test the ability of different O-acylated LMWHP derivatives to inhibit fibroblast growth factor (FGF) 1 and FGF2 binding to surface-immobilized heparin. All the LMWHP derivatives bound to FGF1 and FGF2 but each exhibited slightly different binding affinity.

Threshold of peroxynitrite cytotoxicity in bovine pulmonary artery endothelial and smooth muscle cells

Peroxynitrite is widely reported as highly cytotoxic; yet recent evidence indicates that at certain concentrations, it can induce pulmonary cell hyper-proliferation and tissue remodelling. This study aimed to establish the threshold concentration of peroxynitrite to induce functional impairment of bovine pulmonary artery endothelial (PAEC) and smooth muscle cells (PASMC). PAEC or PASMC were exposed to solution of peroxynitrite or 3-morpholinosydnonimine (SIN-1). Twenty-four hour cell viability, DNA synthesis, and protein biochemistry were assessed by trypan blue dye exclusion, [3H] thymidine incorporation and western blot analysis, respectively. Threshold concentration of peroxynitrite to significantly impair viability of PAEC and PASMC was 2μM peroxynitrite. In PASMC and PAEC, low concentrations of peroxynitrite (2nM–0.2μM) increased cell proliferation and did not activate p38 MAP kinase. The decrease in DNA synthesis and cell viability caused by 2μM peroxynitrite was associated with caspase-3 cleavage but not p38 activation. Also, 2–20μM peroxynitrite significantly activated poly ADP ribose polymerase and stress activated kinase JNK in PAEC. However, the higher concentration of 20μM peroxynitrite did cause a threefold increase in p38 activation. In conclusion, the threshold for the cytotoxic effects of peroxynitrite was 2μM; which caused apoptotic cell death independent of p38 MAP kinase activation in pulmonary artery cells.

Role of TGF-β1 and TNF-α in IL-1β mediated activation of proMMP-9 in pulmonary artery smooth muscle cells: Involvement of an aprotinin sensitive protease

We investigated the role of TGF-β1 and TNF-α in mediating the effect of IL-1β in activating proMMP-9 and proMMP-2, and the involvement of an aprotinin sensitive protease in this scenario in bovine pulmonary artery smooth muscle cells. IL-1β induces TGF-β1 mediated stimulation of 92 kDa proMMP-9 and 72 kDa proMMP-2 mRNA and protein expression; whereas, the elevated level of TNF-α promotes activation of proMMP-9 and proMMP-2. Interestingly, TNF-α induced activation of proMMP-9 appeared to be mediated via a 43 kDa aprotinin sensitive protease. TNF-α inhibited aprotinin and TIMP-1 mRNA and protein expression, which apparently facilitated the proteolytic conversion of proMMP-9 to MMP-9 with the involvement of the aprotinin sensitive protease. The aprotinin sensitive protease did not activate proMMP-2 under IL-1β stimulation, albeit a marked inhibition of TIMP-2 mRNA and protein expression were elicited by TNF-α. Thus, IL-1β induced stimulation of the two progelatinases occurs via different mechanisms.

Peroxynitrite stimulates pulmonary artery endothelial and smooth muscle cell proliferation: Involvement of ERK and PKC

Background There is evidence that peroxynitrite is generated in pulmonary hypertension and we have therefore investigated whether peroxynitrite can cause proliferation of pulmonary artery cells. Methods Bovine pulmonary artery endothelial (PAEC) and smooth muscle cells (PASMC) were exposed to peroxynitrite solution or to the peroxynitrite generating compound, 3-morpholinosydnonimine (SIN-1). Vascular cell proliferation was determined by cell count and 3H-thymidine incorporation. Protein biochemistry was by western blot analysis. Results Transient exposure to peroxynitrite stimulated the proliferation of PASMC (peroxynitrite 0.2 nM–2 μM) and PAEC (peroxynitrite 0.2 μM). Peroxynitrite 0.2 μM stimulated DNA synthesis in PASMC cell by 200 ± 22% and in PAEC by 137 ± 4%. DNA synthesis in PAEC and PASMC was also stimulated by the peroxynitrite generator SIN-1 2 μM. Cell proliferation was accompanied by activation of ERK, which peaked at 15 min and remained elevated for 12 h in PASMC. However peroxynitrite at the concentrations used in this study did not activate the stress pathways p38 mitogen activated protein kinase (MAPK) or Jun N-terminal kinase (JNK). Peroxynitrite-induced proliferation and ERK phosphorylation in PASMC were abolished by the peroxynitrite scavenger ebselen 5 μM. Peroxynitrite-induced proliferation and extracellular signal-regulated kinase (ERK) phosphorylation in PASMC was prevented by selective inhibitors of MAP kinase kinase (MEK) (U0126 5 μM, PD98059 50 μM), Raf-1 (Raf-1 kinase inhibitor 10 μM), Ras (FPT II and FPT III 10 μM) and protein kinase C (PKC) (GF109203X 10 μM). Inhibition of EGF or PDGF receptor signaling using AG-1296, AG-1478 or imatinib prevented peroxynitrite-induced cell proliferation and ERK phosphorylation in PASMC. Conclusion Peroxynitrite can stimulate proliferation of pulmonary artery cells, involving ERK, PKC and EGF or PDGF receptors.

M-Calpain-mediated cleavage of Na+/Ca2+ exchanger-1 in caveolae vesicles isolated from pulmonary artery smooth muscle

Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na(+)/Ca(2+) exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 microM) in presence of CaCl(2) (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca(2+) chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca(2+) overload, which could arise due to inhibition of Ca(2+) efflux by the forward-mode NCX and that could lead to sustained Ca(2+) overload in the smooth muscle leading to pulmonary hypertension.

Effect of carboxyl-reduced heparin on the growth inhibition of bovine pulmonary artery smooth muscle cells

Heparin (HP) inhibits the proliferation of bovine pulmonary artery smooth muscle cells (BPASMC’s), among other cell types in vitro. In order to develop a potential therapeutic agent to reverse vascular remodeling, we are involved in deciphering the relationship between the native HP structure and its antiproliferative potency. We have previously reported the influence of the molecular size and the effects of various O-sulfo and N-acetyl groups of HP on growth-inhibitory activity. In this study, to understand the influence of carboxyl groups in the HP structure required for endogenous activity, a chemically modified derivative of native HP was prepared by converting the carboxyl groups of hexuronic acid residues in HP to primary hydroxyl groups. This modification procedure involves the treatment of HP with N-(3-dimethylaminopropyl)- N-ethylcarbodiimide followed by reduction with NaBH 4 to yield carboxyl-reduced heparin (CR-HP). When compared to the antiproliferative potency of native HP on cultured BPASMC’s at three dose levels (1, 10, and 100 μg/mL), the CR-HP showed significantly less potency at all the doses. These results suggest that hexuronic acid residues in both major and variable sequences in HP are essential for the antiproliferative properties of native HP.

JNK regulates serotonin-mediated proliferation and migration of pulmonary artery smooth muscle cells

JNK is a member of the MAPK family and has essential roles in inflammation and cell differentiation and apoptosis. In recent years, there have been accumulating data indicating a novel role for JNK in cell growth and migration. In this report, we demonstrate that JNK activity is necessary for serotonin (5-HT)-induced proliferation and migration of bovine pulmonary artery smooth muscle cells (PASMCs). Stimulation with 5-HT was found to lead to activation of JNK with a maximal activation at 10 min. Inhibition of JNK with its specific inhibitor, SP-600125, or its dominant-negative form, DN-JNK, significantly reduced 5-HT-stimulated [(3)H]thymidine incorporation and cyclin D1 expression. A similar inhibitory effect on SMC migration produced by 5-HT, as detected by a wound healing assay, was observed with inhibition of JNK. Furthermore, inhibition of 5-HT receptors (1B) and (2A), but not inhibition of the 5-HT transporter, blocked 5-HT-induced JNK activation. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY-294002 and wortmannin had little or no effect on 5-HT-induced JNK phosphorylation, but JNK inhibitor SP-600125 and DN-JNK blocked 5-HT-stimulated phosphorylation of Akt and its downstream effectors, p70S6K1 and S6, indicating that Akt is a downstream effector of JNK. Activation of Akt by 5-HT was blocked only minimally, if at all, by inhibitors of ERK and p38 MAPK, indicating a uniqueness of JNK MAPK in this activation of Akt. Coimmunoprecipitation showed binding of Akt to JNK, further supporting the interaction of JNK and Akt. Thus JNK is a critical molecule in 5-HT-induced PASMC proliferation and migration and may act at an important point for cross talk of the MAPK and PI3K pathways. Its activation by 5-HT is initiated through 5-HT (1B) and (2A) receptors, and its stimulation of SMC proliferation and migration occurs through the Akt pathway.