LPP expression during in vitro smooth muscle differentiation and stent-induced vascular injury.

Lipoma preferred partner (LPP) has been identified as a protein highly expressed in smooth muscle (SM) tissues. The aim of the present study was to determine mechanisms that regulate LPP expression in an in vitro model of SM cell (SMC) differentiation and in stent-induced pig coronary vessel injury. All trans-retinoic acid treatment of A404 cells induced a strong increase in LPP, as well as SM alpha-actin, SM myosin heavy chain, and smoothelin mRNA levels, in a Rho kinase (ROK)-dependent manner. Adenovirus mediated overexpression of myocardin in A404 cells significantly increased LPP mRNA expression. Interestingly, inactivation of RhoA with C3-exoenzyme or treatment with ROK inhibitors strongly inhibited myocardin mRNA expression in retinoic acid-treated A404 cells or human iliac vein SMCs. LPP silencing with short interfering RNA significantly decreased SMC migration. LPP expression was also markedly decreased in focal adhesion kinase (FAK)-null cells known to have impaired migration but rescued with inducible expression of FAK. LPP expression in FAK-null fibroblasts enhanced cell spreading. In stented pig coronary vessels, LPP was expressed in the neointima of cells lacking smoothelin and showed expression patterns identical to those of SM alpha-actin. In conclusion, LPP appears to be a myocardin-, RhoA/ROK-dependent SMC differentiation marker that plays a role in regulating SMC migration.

Estradiol modulates protein kinase C activity in the rat pituitary in vivo and in vitro.

17 beta-Estradiol (E2) alters different functions of pituitary cells, including cell sensitivity to several neurohormones such as LHRH, TRH, somatostatin, or dopamine, presumably by affecting receptor coupling mechanisms. Attempting to pinpoint the membrane processes underlying this modulation, we studied the effect of E2 on pituitary kinase-C (PKC) activity, a major signal transduction enzyme. The distribution of calcium- and phospholipid-dependent partially purified PKC (chromatography on DEAE-52 cellulose columns) was evaluated in membrane and cytosol fractions from anterior pituitaries of ovariectomized (OVX) or OVX plus E2-treated rats. E2 administration by implants to OVX animals increased significantly both soluble and particulate enzyme activity. The effect increased progressively from 24 h to 5 days after E2 treatment. Administration of 17 alpha-estradiol, an inactive stereoisomer of E2, was ineffective, pointing to stereospecific interaction. Total destruction of neural connections to the pituitary (complete hypothalamic lesions) did not modify the enzyme response to E2 administration, indicating a direct effect of the steroid on pituitary PKC activity. A direct E2 (10(-9) M) effect was confirmed in primary mixed cultures of pituitary cells; it was time dependent (15-96 h) and specific, and reflects a genomic E2 action. E2 treatment for shorter times had no effect on the enzyme levels or the membrane redistribution of PKC activity. In contrast, under the same experimental conditions phorbol esters (12-O-tertadecanoyl-phorbol-13-acetate (TPA] induced a rapid and sustained translocation of the enzyme. PKC activity was found in all pituitary cell types, with maximal activity in fractions of gonadotropes and thyrotropes, as evaluated in cultures enriched in certain types of pituitary cells separated by means of unit gravity gradient sedimentation. E2 treatment (10(-9) M; 72 h) significantly increased both soluble and particulate enzyme levels in all cell types. In addition, administration of E2 (10(-9) M; 72 h) to cell cultures strongly increased the TPA-evoked LH and PRL release. These results indicate that E2-induced changes in pituitary function include selective effects of the steroid on PKC activity involved at different levels in the coupling mechanisms.

Phosphorylation of telokin by cyclic nucleotide kinases and the identification of in vivo phosphorylation sites in smooth muscle.

The Ca(2+)-independent acceleration of dephosphorylation of the regulatory light chain of smooth muscle myosin and relaxation of smooth muscle by telokin are enhanced by cyclic nucleotide-activated protein kinase(s) [Wu et al. (1998) J. Biol. Chem. 273, 11362-113691. The purpose of this study was to determine the in vivo site(s) and in vitro rates of telokin phosphorylation and to evaluate the possible effects of sequential phosphorylation by different kinases. The in vivo site(s) of phosphorylation of telokin were determined in rabbit smooth muscles of longitudinal ileum and portal vein. Following stimulation of ileum with forskolin (20 microM) the serine at position 13 was the only amino acid to exhibit increased phosphorylation. Rabbit portal vein telokin was phosphorylated on both Ser-13 and -19 as a result of forskolin and GTPgammaS stimulation in vivo. Point mutation of Ser-13 (to Ala or Asp) abolished in vitro phosphorylation by cyclic nucleotide-dependent protein kinases.

M1 and M3 muscarinic receptors in human pulmonary arteries.

1. Acetylcholine (ACh) and the M1 agonists (McN-A-343 or PD142505) relaxed human isolated pulmonary arteries which were pre-contracted with noradrenaline (10 microM). In preparations where the endothelium had been removed ACh induced a contractile response whereas the M1 agonists (McN-A-343 or PD142505) had no effect. 2. ACh- and McN-A-343-induced relaxations were abolished after treatment of endothelium-intact preparations with the drug combination NG-nitro-L-arginine (L-NOARG: 0.1 mM) and indomethacin (1.7 microM). 3. The affinity (pKB value) for pirenzepine was higher in human pulmonary arteries when tissues were relaxed with McN-A-343 as compared with ACh (pKB values, 7.71 +/- 0.30 (n = 4) and 6.68 +/- 0.15 (n = 8), respectively). In addition, the affinity for pFHHSiD against McN-A-343- and ACh-induced relaxations was 6.86 +/- 0.13 (n = 3) and 7.35 +/- 0.11 (n = 9) respectively. 4. The low affinities for methoctramine in human isolated pulmonary arteries with the endothelium either intact or removed, suggested the lack of involvement of M2 and M4 receptors in the Ach responses. 5. Phenoxybenzamine (3 microM: 30 min) abolished both ACh contraction and relaxation in human pulmonary artery. The ACh contraction was present when the phenoxybenzamine treatment was preceded by incubation with pFHHSiD (2 microM) but not with pirenzepine (1 microM). In addition, the ACh relaxation was present when preparations were treated with either pFHHSiD (2 microM) or pirenzepine (1 microM), before exposure to phenoxybenzamine. 6. These results in human isolated pulmonary arteries support the notion that only M3 receptors, on smooth muscle, mediate the ACh-induced contraction whereas M3 and M1 receptors are involved in the endothelium-dependent ACh-induced relaxation.

Effects of beta 2-adrenoceptor agonists on anti-IgE-induced contraction and smooth muscle reactivity in human airways.

1. The beta 2-adrenoceptor agonists, salbutamol, salmeterol and RP 58802 relaxed basal tone of human isolated bronchial smooth muscle. Salmeterol- and RP 58802-induced relaxations persisted for more than 4 h when the medium was constantly renewed after treatment. 2. Salbutamol, salmeterol and RP 58802 reversed histamine-induced contractions in human airways (pD2 values: 6.15 +/- 0.21, 6.00 +/- 0.19 and 6.56 +/- 0.12, respectively). 3. Anti-IgE-induced contractions were significantly inhibited immediately after pretreatment of preparations with beta 2-adrenoceptor agonists (10 microM). However, when tissues were treated with beta 2-agonists and then washed for a period of 4 h, salmeterol was the only agonist which significantly inhibited the anti-IgE response. 4. Histamine response curves were shifted to the right immediately after pretreatment of tissues with the beta 2-adrenoceptor agonists (10 microM; 20 min), but maximal contractions were not affected. After a 4 h washing period, the histamine curves were not significantly different from controls. Concentration-effect curves to acetylcholine (ACh) or leukotriene C4 (LTC4) were not significantly modified after beta 2-agonist pretreatment. 5. These results suggest that beta 2-adrenoceptor agonists may prevent anti-IgE-induced contraction by inhibition of mediator release rather than alterations of those mechanisms involved in airway smooth muscle contraction.

Degradation of acetylcholine in human airways: role of butyrylcholinesterase.

1. Neostigmine and BW284C51 induced concentration-dependent contractions in human isolated bronchial preparations whereas tetraisopropylpyrophosphoramide (iso-OMPA) was inactive on airway resting tone. 2. Neostigmine (0.1 microM) or iso-OMPA (100 microM) increased acetylcholine sensitivity in human isolated bronchial preparations but did not alter methacholine or carbachol concentration-effect curves. 3. In the presence of iso-OMPA (10 microM) the bronchial rings were more sensitive to neostigmine. The pD2 values were, control: 6.05 +/- 0.15 and treated: 6.91 +/- 0.14. 4. Neostigmine or iso-OMPA retarded the degradation of acetylcholine when this substrate was exogenously added to human isolated airways. A marked reduction of acetylcholine degradation was observed in the presence of both inhibitors. Exogenous butyrylcholine degradation was prevented by iso-OMPA (10 microM) but not by neostigmine (0.1 microM). 5. These results suggest the presence of butyrylcholinesterase activity in human bronchial muscle and this enzyme may co-regulate the degradation of acetylcholine in this tissue.

Leukotriene receptors on human pulmonary vascular endothelium.

1. Cysteinyl-leukotrienes cause contractions and/or relaxations of human isolated pulmonary vascular preparations. Although, the localization and nature of the receptors through which these effects are mediated have not been fully characterized, some effects are indirect and not mediated via the well-described LT1 receptor. 2. In human pulmonary veins (HPV) with an intact endothelium, leukotriene D4 (LTD4) induced contraction above basal tone. This response was observed at lower concentrations of LTD4 in the presence of nitric oxide synthase inhibitor N omega-nitro-L-arginine (L-NOARG). Contractions (in the absence and presence of L-NOARG) were partially blocked by the LT1 antagonists (MK 571 and ICI 198615). 3. LTD4 relaxed HPV previously contracted with noradrenaline. This relaxation was potentiated by LT1 antagonists, but was abolished by removal of the endothelium. LTD4 also relaxed human pulmonary arteries (HPA) precontracted with noradrenaline but this effect was not modified by LT1 antagonists. 4. The results suggest that contraction of endothelium-intact HPV by LTD4 is partially mediated via LT1 receptors. Further, in endothelium-intact HPV, this contraction was opposed by a relaxation induced by LTD4, dependent on the release of nitric oxide, which was mediated, at least in part, via a non-LT1 receptor. In addition, LTD4 relaxation on contracted HPA was not mediated by LT1 receptors. 5. The mechanical effects of LTD4 on human pulmonary vasculature are complex and involve both direct and indirect mechanisms mediated via at least two types of cysteinyl-leukotriene receptors.

Antagonism of leukotriene responses in human airways by BAY x7195.

Contractions induced by leukotriene and anti-IgE (sheep antiserum to human IgE) were antagonized by pretreatment of human airways with the cysteinyl leukotriene receptor antagonist BAY x7195 ((4S)-[4-carboxyphenylthio]-7-[4-(4-phenoxybutoxy)-phenyl]-h ept-5-(z)- enoic acid). However, this receptor antagonist did not inhibit either leukotriene D4- or leukotriene C4-induced contractions in human pulmonary veins. The pA2 value for BAY x7195 in human airways against leukotriene D4 was 7.83 +/- 0.16 with a slope of 1.07 +/- 0.15 (means +/- S.E.M; n = 11). The IC50 value for BAY x7195 in human airways contracted with anti-IgE was 0.31 +/- 0.08 microM (n = 11). These results were comparable to those obtained with ICI 204,219 (4-(5-cyclopentyl-oxycarbonylamino-1-methylindol-3-ylmeth yl)-3-methoxy-N-otolyl - sulfonylbenzamide). These data demonstrate that BAY x7195 is a potent selective leukotriene receptor antagonist which may block allergic reactions in the lung.

Cholinesterase inhibition by vecuronium and pancuronium in human airways.

Vecuronium (100 microM) but not pancuronium (100 microM) increased the sensitivity of human isolated bronchial preparations (HBP) to exogenous acetylcholine (ACh). The pD2 values obtained from concentration-dependent contractions were: control, 4.59 +/- 0.29 vecuronium, 5.86 +/- 0.31. Vecuronium or pancuronium (100 microM) significantly decreased (50%) the neostigmine contractions in HBP. In addition, vecuronium was more potent than pancuronium in preventing exogenous ACh degradation. These results suggest that vecuronium and pancuronium may have physiological effects in human airways by inhibiting both the tissue cholinesterases and muscarinic (M3) receptors.

Leukotriene synthesis inhibition and anti-ige challenge of human lung parenchyma.

The leukotriene (LT) synthesis inhibitors BAY x1005 and MK-886 were evaluated in human lung parenchyma challenged with an anti-IgE. The anti-IgE-induced LTE4 release was time- and dose-dependent. Treatment of the parenchyma with indomethacin (3 microM) prior to anti-IgE challenge inhibited the 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) release and enhanced (36%) the quantities of LTE4 detected during IgE-stimulations. BAY x1005 and MK-886 were assessed in the presence of indomethacin (3 microM) and the IC50 values for both inhibitors were similar (0.13 microM). BAY x1005 (1 microM) produced the same percent of inhibition of anti-IgE-induced LTE4 release in the presence or absence of indomethacin. BAY x1005 (1 microM) did not alter the 6-keto PGF1 alpha release during anti-IgE challenge. The results indicate that BAY x1005 and MK-886 are potent inhibitors of LT synthesis when human lung parenchyma were stimulated by an anti-IgE.

Antigen stimulation of human pulmonary smooth muscle: an in vitro model of inflammation.

Human airways in vitro contract when stimulated by anti-IgE, whereas human pulmonary vessels relax. Leukotriene D4 (LTD4) induced a contractile response in the airways, while in pulmonary vessels both contractions and relaxations were observed. The LTD4 contractions in airways were blocked by cysLT1 receptor antagonists (MK 571, ICI 198615, and BAY x7195). In contrast none of the compounds affected the LTD4 contractions of pulmonary veins. These results suggest that the leukotrienes which are released during antigen challenge of airways and pulmonary vessels may be acting at distinct receptors in the human lung.

Inhibition of p42 and p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery.

Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon reverses the inhibition. The caldesmon kinase is believed to be mitogen-activated protein (MAP) kinase. MAP kinases are activated during vascular stimulation, but a cause-and-effect relationship between kinase activity and contraction has not been established. We examined the role of MAP kinase in contraction using PD-098059, an inhibitor of MAP kinase kinase (MEK). MAP kinase activity was assessed using an anti-active MAP kinase antibody and direct measurement of MAP kinase catalyzed phosphorylation of myelin basic protein, MBP-(95-98). MAP kinase phosphorylation, stimulated by histamine (50 microM) or phorbol 12,13-dibutyrate (PDBu, 0.1 microM), was inhibited by PD-098059 (100 microM). PD-098059 did not alter the sensitivity or the maximal level of force in smooth muscle stimulated by histamine or PDBu, nor did PD-098059 affect contraction of beta-escin-permeabilized tissue. Our data suggest that p44 and p42 MAP kinases are not involved in regulation of vascular smooth muscle contraction. These results do not, however, preclude a role for other isoforms of the MAP kinase family.

Response to anti-human IgE in human pulmonary arteries. Regulation by endothelium.

Initial reports concerning anaphylactic reactions in the lung have demonstrated that histamine is released, and this mediator may be responsible for the severe hypotension observed in vivo in sensitized animals. However, those mechanisms involved in the antigen-vascular interactions have not been elucidated. Human isolated pulmonary arterial preparations relaxed when challenged with anti-human IgE (a-IgE). This response was associated with a release of histamine and PGI2. Both the relaxation and the release of PGI2 were attenuated by removal of the endothelium or by prior treatment of the tissues with chlorpheniramine. Indomethacin also significantly reduced the relaxations produced by a-IgE. In addition, L-NOARG in the presence of indomethacin blocked the a-IgE-dependent relaxation. Stimulation of these tissues with histamine also induced relaxations which were endothelium dependent and blocked by chlorpheniramine and L-NOARG in the presence of indomethacin. These results suggest that the relaxations via products of the cyclooxygenase and NO pathways were mediated by histamine release which stimulated the endothelium.

Anti-IgE Response in Human Airways: Relative Contribution of Inflammatory Mediators.

Heman airway preparations at resting tone were relaxed with either the leukotriene synthesis inhibitor BAY x1005 (3 muM), chlorpheniramine (1 muM) or the thromboxane receptor antagonist BAY u3405 (0.1 muM). The response to anti-IgE (1:1000) was 58 +/- 8% of acetylcholine pre-contraction (2.19 +/- 0.28 g). Indomethacin (3 muM) enhanced the anti-IgE-induced contraction by 28%. The anti-IgE maximal response was not modified by either chlorpheniramine, BAY x1005 or BAY u3405. When the tissues were treated with either BAY xl005/indomethacin or BAY x1005/chlorpheniramine, the anti-IgE-induced contraction was reduced. In addition, in presence of BAY xl005/indomethacin/chlorpheniramine the response was completely blocked. These results suggest that mediatots released during anti-IgE challenge cause airway contraction which may mask the evaluation of the leukotriene component.

Histamine receptors on human isolated pulmonary arterial muscle preparations: effects of endothelial cell removal and nitric oxide inhibitors.

Human isolated intact pulmonary arterial muscle ring preparations which were precontracted with serotonin (10 microM) relaxed when stimulated with low concentrations of histamine, 2-[2-thiazolyl]ethylamine or 2-[pyridyl]ethylamine (pD2 values: 8.66 +/- 0.22, 7.10 +/- 0.06 and 6.20 +/- 0.26, respectively) or contracted at higher concentrations of these agonists. This relaxant response was obliterated in endothelial denuded tissues. Chlorpheniramine (H1-antagonist; 0.25 and 2.5 microM) induced a small contractile response in the tissues at resting tone (0.08 +/- 0.03 g and 0.10 +/- 0.10 g, respectively). Chlorpheniramine also shifted the histamine relaxation curves to the right (pD2 values: control, 8.85 +/- 0.31; 0.25 microM, 6.90 +/- 0.41; and 2.5 microM, 5.58 +/- 0.30; N = 6). Dimaprit (H2-agonist) induced a small relaxation (20%) in both intact and denuded tissues. Treatment of the tissues with cimetidine (H2-antagonist; 50 microM), burimamide (H2/H3-antagonist; 10 microM) and impromidine (H2-agonist/H3-antagonist; 1 microM) did not alter histamine-induced relaxation or contraction. Indomethacin (1.7 microM) caused a small contraction in these tissues and significantly reduced the histamine relaxation. The nitric oxide inhibitors (L-NG-monoethyl-L-arginine, 30 and 300 microM; or L-NG-nitroarginine, 30 and 300 microM) induced a slight and variable contraction in the preparations. However, these inhibitors, only in the presence of indomethacin, inhibited the relaxant effects of histamine and potentiated the contractions induced by this amine. These data suggest that a dual endogenous vasodilatory mechanism is present in human isolated pulmonary arterial muscle preparations and that products of the cyclooxygenase and endothelium-derived relaxing factor-nitric oxide pathway may interact to regulate histamine stimulation of H1-receptors.

Leukotriene D4 contractions in human airways are blocked by SK&F 96365, an inhibitor of receptor-mediated calcium entry.

In human bronchial muscle preparations, nifedipine (3 microM) significantly inhibited the histamine, ACh and KCl contractions. However, the dihydropyridine did not modify the contractile responses induced by either leukotriene D4 (LTD4) or anti-human IgE (a-IgE). In human airways, SK&F 96365 (30 microM and 100 microM) markedly reduced the KCl and, at the higher concentration, LTD4 maximal contractions. In addition, when preparations were treated with nifedipine (3 microM), SK&F 96365 (100 microM) significantly blocked responses to both LTD4 and a-IgE. The calcium chelating agent ethylene glycol-bis (beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (4 mM) also inhibited the a-IgE-induced contractions. These data demonstrate that the nifedipine-resistant component of the LTD4 and a-IgE contractions was inhibited by SK&F 96365 and suggest that the cysteinyl-leukotriene receptor in human airways may be intimately linked with a receptor-operated calcium-entry mechanism.

Sodium hydrosulfite contractions of smooth muscle are calcium and myosin phosphorylation independent.

In an effort to further understand the processes underlying hypoxic pulmonary vasoconstriction, we examined the mechanism by which sodium hydrosulfite (Na2S2O4), a potent reducing agent and oxygen scavenger, induces smooth muscle contraction. In rat pulmonary arterial strips, sodium hydrosulfite (10 mM) induced contractions that were 65.9 +/- 12.8% of the response to 60 mM KCl (n = 9 segments). Contractions were not inhibited by nisoldipine (5 microM) or by repeated stimulation with caffeine (10 mM), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (10 microM), or cyclopiazonic acid (10 microM), all of which eliminated responses to contractile agonists. Maximum force generation after exposure to sodium hydrosulfite was 0.123 +/- 0.013 mN in the presence of 1.8 mM calcium and 0.127 +/- 0.015 mN in the absence of calcium. Sodium hydrosulfite contractions in pulmonary arterial segments were not due to the generation of H2O2 and occurred in the presence of chelerythrine (10 microM), which blocked phorbol ester contractions, and solution hyperoxygenation. Similar contractile responses were obtained in rat aortic and tracheal smooth muscles. Finally, contractions occurred in the complete absence of an increase in myosin light chain phosphorylation. Therefore sodium hydrosulfite-induced smooth muscle contraction is not specific to pulmonary arterial smooth muscle, is independent of calcium and myosin light chain phosphorylation, and is not mediated by either hypoxia or protein kinase C.

Regulation by GDI of RhoA/Rho-kinase-induced Ca2+ sensitization of smooth muscle myosin II.

We characterized the role of guanine nucleotide dissociation inhibitor (GDI) in RhoA/Rho-kinase-mediated Ca2+ sensitization of smooth muscle. Endogenous contents (approximately 2-4 microM) of RhoA and RhoGDI were near stoichiometric, whereas a supraphysiological GDI concentration was required to relax Ca2+ sensitization of force by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). GDI also inhibited Ca2+ sensitization by GTP. G14V RhoA, by alpha-adrenergic and muscarinic agonists, and extracted RhoA from membranes. GTPgammaS translocated Rho-kinase to a Triton X-114-extractable membrane fraction. GTP. G14V RhoA complexed with GDI also induced Ca2+ sensitization, probably through in vivo dissociation of GTP. RhoA from the complex, because it was reversed by addition of excess GDI. GDI did not inhibit Ca2+ sensitization by phorbol ester. Constitutively active Cdc42 and Rac1 inhibited Ca2+ sensitization by GTP. G14V RhoA. We conclude that 1) the most likely in vivo function of GDI is to prevent perpetual "recycling" of GDP. RhoA to GTP. RhoA; 2) nucleotide exchange (GTP for GDP) on complexed GDP. RhoA/GDI can precede translocation of RhoA to the membrane; 3) activation of Rho-kinase exposes a hydrophobic domain; and 4) Cdc42 and Rac1 can inhibit Ca2+ sensitization by activated GTP. RhoA.