Expression and muscarinic receptor coupling of Lyn kinase in cultured human airway smooth muscle cells.

Src family tyrosine kinases are signaling intermediates in a diverse array of cellular events including cell differentiation, motility, proliferation, and survival. In nonairway smooth muscle cells, muscarinic receptors directly interact with Src family tyrosine kinases. As little is known about the expression and signaling of these Src family tyrosine kinases in human airway smooth muscle cells, we determined the expression of Src family members and characterized the muscarinic receptor-mediated activation of Lyn kinase in these cells.

Lipopolysaccharide-induced sensitization of adenylyl cyclase activity in murine macrophages.

LPS is known to modulate macrophage responses during sepsis, including cytokine release, phagocytosis, and proliferation. Although agents that elevate cAMP reverse LPS-induced macrophage functions, whether LPS itself modulates cAMP and whether LPS-induced decreases in proliferation are modulated via a cAMP-dependent pathway are not known. Murine macrophages (RAW264.

Isoproterenol induces actin depolymerization in human airway smooth muscle cells via activation of an Src kinase and GS.

In a previous study, we showed that isoproterenol induced actin depolymerization in human airway smooth muscle cells by both protein kinase A (PKA)-dependent and -independent signaling pathways. We now investigate the signaling pathway of PKA-independent actin depolymerization induced by isoproterenol in these cells. Cells were briefly exposed to isoproterenol or PGE(1) in the presence and absence of specific inhibitors of Src-family tyrosine kinases, phosphatidylinositol-3-kinase (PI3 kinase), or MAP kinase, and actin depolymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I.

A mechanism for rapacuronium-induced bronchospasm: M2 muscarinic receptor antagonism.

Background: A safe and effective ultra-short-acting nondepolarizing neuromuscular blocking agent is required to block nicotinic receptors to facilitate intubation. Rapacuronium, which sought to fulfill these criteria, was withdrawn from clinical use due to a high incidence of bronchospasm resulting in death. Understanding the mechanism by which rapacuronium induces fatal bronchospasm is imperative so that newly synthesized neuromuscular blocking agents that share this mechanism will not be introduced clinically.

Tumor necrosis factor alpha stimulates adenylyl cyclase activity in human myometrial cells.

Cytokines such as tumor necrosis factor alpha (TNFalpha) have been implicated in amniotic fluid infections and preterm and term labor. The underlying mechanisms are incompletely understood. In some smooth muscle cells, TNFalpha affects function of the beta-adrenergic/adenylyl cyclase pathway.

Despite in vitro increase in cyclic guanosine monophosphate concentrations, intracarotid nitroprusside fails to augment cerebral blood flow of healthy baboons.

Background: During cerebral angiography, intracarotid infusion of sodium nitroprusside (SNP), an endothelium-independent nitric oxide donor, fails to increase cerebral blood flow (CBF) of human subjects. A confounding effect of intracranial pathology or that of radiocontrast could not be ruled out in these experiments. The authors hypothesized that, if nitric oxide was a significant regulator of CBF of primates, then intracarotid SNP will augment CBF of baboons.

Asthma, allergy, and airway hyperresponsiveness are not linked to the beta(2)-adrenoceptor gene.

Study Objectives: To exclude genetic linkage between the beta(2)-adrenoceptor gene and asthma, allergy, and methacholine airway hyperresponsiveness.

Design: The current study used six distinct intragene markers within the beta(2)-adrenoceptor gene, and evaluated genetic linkage between the beta(2)-adrenoceptor and asthma, allergy, or methacholine airway hyperresponsiveness in eight multiplex families.

Patients: Forty-nine members of eight multiplex families with a high incidence of asthma.

Actin depolymerization via the beta-adrenoceptor in airway smooth muscle cells: a novel PKA-independent pathway.

Actin is a major functional and structural cytoskeletal protein that mediates such diverse processes as motility, cytokinesis, contraction, and control of cell shape and polarity. While many extracellular signals are known to mediate actin filament polymerization, considerably less is known about signals that mediate depolymerization of the actin cytoskeleton. Human airway smooth muscle cells were briefly exposed to isoproterenol, forskolin, or the cAMP-dependent protein kinase A (PKA) agonist stimulatory diastereoisomer of adenosine 3',5'-cyclic monophosphate (Sp-cAMPS).

Inhibition of geranylgeranylation blocks agonist-induced actin reorganization in human airway smooth muscle cells.

To determine whether RhoA isoprenylation (geranylgeranylation) is required for agonist-induced actin cytoskeleton reorganization (measured by an increase in the filamentous F- to monomeric G-actin ratio), human airway smooth muscle cells were treated for 72 h with inhibitors of geranylgeranyltransferase I. Geranylgeranyltransferase inhibitor (GGTI)-2147 or -286 pretreatment completely blocked the increase in the F- to G-actin fluorescence ratio when cells were stimulated with lysophosphatidic acid (LPA), endothelin, or carbachol. In contrast, LPA or endothelin induced actin cytoskeletal reorganization in cells treated with farnesyltransferase inhibitor (FTI)-277 to inactivate Ras.

Oxytocin and lysophosphatidic acid induce stress fiber formation in human myometrial cells via a pathway involving Rho-kinase.

The actin cytoskeleton is important for stress fiber formation and contributes to the initiation and maintenance of smooth muscle contraction. To determine if oxytocin and lysophosphatidic acid (LPA) induce stress fiber formation, cultured human myometrial cells were exposed to oxytocin (10(-5) M) or LPA (10(-6) M), and filamentous (F) and globular (G) actin pools were stained with fluorescein isothiocyanate-phalloidin and Texas red DNase I, respectively. The F- to G-actin fluorescent-staining ratio was measured by fluorescence microscopy.

Decreased adenylyl cyclase protein and function in airway smooth muscle by chronic carbachol pretreatment.

Cellular levels of cAMP are an important determinant of airway smooth muscle tone. We have previously shown that chronic (18 h) but not acute (30 min or 2 h) pretreatment with the muscarinic receptor agonist carbachol resulted in decreased adenylyl cyclase activity in response to GTP, isoproterenol, or forskolin via a pathway blocked by the protein kinase C inhibitor staurosporine. The present study was designed to determine if carbachol-induced decreases in adenylyl cyclase activity were due to regulatory events at the level of either G(s)alpha or adenylyl cyclase.

TNF-alpha increases transcription of Galpha(i-2) in human airway smooth muscle cells.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that has an important role in the regulation of airway smooth muscle tone and reactivity. We have shown previously that TNF-alpha upregulates the expression of Galpha(i-2) protein without significantly increasing G(s)alpha protein and enhances adenylyl cyclase inhibition by carbachol in cultured human airway smooth muscle cells (Hotta K, Emala CW, and Hirshman CA. Am J Physiol Lung Cell Mol Physiol 276: L405-L411, 1999).

Cellular signaling by the potent bronchoconstrictor endothelin-1 in airway smooth muscle.

Objective: The objective of this study was to determine whether the potent bronchoconstrictor endothelin-1 was coupled to the activation of the inositol phosphate and/or inhibition of the cyclic adenine monophosphate second messenger pathways in porcine airway smooth muscle.

Design: Prospective, controlled, in vitro, nonblinded study.

Setting: University biochemical and molecular biological research laboratory.

Myometrial adenylyl cyclase protein decreases on the last day of pregnancy in the rat.

To determine whether gestation-related changes in responsiveness of the rat uterus to beta-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12-14) and then late (Days 18-20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the beta-adrenergic receptor (isoproterenol, 10(-4) M), the G protein (GTP, 10(-5) M), or the adenylyl cyclase enzyme (MnCl(2), 20 mM), was similarly altered during gestation.

Actin reorganization in airway smooth muscle cells involves Gq and Gi-2 activation of Rho.

Extracellular stimuli induce cytoskeleton reorganization (stress-fiber formation) in cells and Ca2+ sensitization in intact smooth muscle preparations by activating signaling pathways that involve Rho proteins, a subfamily of the Ras superfamily of monomeric G proteins. In airway smooth muscle, the agonists responsible for cytoskeletal reorganization via actin polymerization are poorly understood. Carbachol-, lysophosphatidic acid (LPA)-, and endothelin-1-induced increases in filamentous actin staining are indicative of actin reorganization (filamentous-to-globular actin ratios of 2.

Cholinesterase activity is similar in C3H/HeJ and A/J mice and does not account for their different bronchoconstrictor responsiveness.

Significant interstrain variation in airway responsiveness to acetylcholine (ACh) exists in inbred mouse strains. We hypothesized that part of the variation may be due to between-strain differences in cholinesterase activity. We asked if administration of neostigmine (an acetylcholinesterase inhibitor) and/or succinylcholine (an agent which competes for and inhibits butyrylcholinesterase) altered ACh responsiveness in hyporesponsive C3H/HeJ and hyperresponsive A/J mouse strains.

Gialpha but not gqalpha is linked to activation of p21(ras) in human airway smooth muscle cells.

Airway smooth muscle hypertrophy contributes to the narrowing of asthmatic airways. Activation of the mitogen-activated protein kinases is an important event in mediating cell proliferation. Because the monomeric G protein p21(ras) is an important intermediate leading to activation of mitogen-activated protein kinases, we questioned which heterotrimeric G protein-coupled receptors were linked to the activation of p21(ras) in cultured human airway smooth muscle and which of the heterotrimeric G protein subunits (alpha or betagamma) transmitted the activation signal.

TNF-alpha upregulates Gialpha and Gqalpha protein expression and function in human airway smooth muscle cells.

Chronic inflammation is a characteristic feature of asthma. Multiple inflammatory mediators are released within the asthmatic lung, some of which may have detrimental effects on signal transduction pathways in airway smooth muscle. The effects of tumor necrosis factor (TNF)-alpha on the expression and function of muscarinic receptors and guanine nucleotide-binding protein (G protein) alpha-subunits were examined in human airway smooth muscle cells.

Role of M2 muscarinic receptors in airway smooth muscle contraction.

Airway smooth muscle expresses both M2 and M3 muscarinic receptors with the majority of the receptors of the M2 subtype. Activation of M3 receptors, which couple to Gq, initiates contraction of airway smooth muscle while activation of M2 receptors, which couple to Gi, inhibits beta-adrenergic mediated relaxation. Increased sensitivity to intracellular Ca2+ is an important mechanism for agonist-induced contraction of airway smooth muscle but the signal transduction pathways involved are uncertain.

Hypocapnia-induced contraction of porcine airway smooth muscle.

Hypocapnia constricts peripheral airways in vivo. This study investigated the role of airway smooth muscle in this phenomenon and the mechanism of hypocapnia-induced contraction in vitro. Isometric tension, intracellular pH (pHi) and intracellular free calcium concentration ([Ca2+]i) were measured in porcine airway smooth muscles suspended in organ baths in the presence of 5% or 0% CO2.

Galphai-2 is required for carbachol-induced stress fiber formation in human airway smooth muscle cells.

To determine which heterotrimeric G protein couples muscarinic receptors to stress fiber formation [measured by an increase in the filamentous (F)- to monomeric (G)-actin ratio] in human airway smooth muscle (ASM) cells, cultured human ASM cells expressing the M2 muscarinic receptor were grown to confluence. Cells were exposed for 6 days to 10 microM antisense oligonucleotides designed to specifically bind to the mRNA encoding Galphai-2, Galphai-3, or Gqalpha. A randomly scrambled oligonucleotide served as a control.

Chronic oxytocin pretreatment inhibits adenylyl cyclase activity in cultured rat myometrial cells.

To determine whether chronic oxytocin pretreatment inhibits adenylyl cyclase, we compared adenylyl cyclase activity in membranes prepared from cultured, immortalized rat myometrial cells that were untreated or pretreated for 24 h with oxytocin. Chronic oxytocin pretreatment (1 x 10(-5) M for 24 h) attenuated basal, guanosine triphosphate (1 x 10(-5) M)-, isoproterenol (1 x 10(-4) M)-, forskolin (1 x 10(-5) M)-, MnCl2 (20 mM)- or NaF (1 x 10(-2) M)-stimulated adenylyl cyclase activity by 27 +/- 5% to 39 +/- 11% (n = 6, p < 0.05).

Role of G proteins in agonist-induced Ca2+ sensitization of tracheal smooth muscle.

Increased sensitivity to intracellular Ca2+ concentration ([Ca2+]) is an important mechanism for agonist-induced contraction of airway smooth muscle, but the signal transduction pathways involved are uncertain. We studied Ca2+ sensitization with acetylcholine (ACh) and endothelin (ET)-1 in porcine tracheal smooth muscle by measuring contractions at a constant [Ca2+] in strips permeabilized with alpha-toxin or beta-escin. The peptide inhibitor G protein antagonist 2A (GP Ant-2A), which has selectivity for Gq over Gi, inhibited contractile responses to ET-1, ACh, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), but the proportional inhibition of ACh responses was less than that of ET-1.

Adenylyl cyclase messenger ribonucleic acid in myometrium: splice variant of type IV.

Regulation of uterine tone during pregnancy and parturition involves G protein-coupled receptors that modulate cellular levels of cAMP. Cyclic AMP is synthesized by a family of at least nine adenylyl cyclase enzymes that have unique tissue distributions, basal activities, and modes of regulation. Little is known regarding the expression of adenylyl cyclase isoforms in myometrium.

Carbachol-induced actin reorganization involves Gi activation of Rho in human airway smooth muscle cells.

To determine whether M2 muscarinic receptors are linked to the monomeric G protein Rho, we studied the effect of carbachol on actin reorganization (stress fiber formation) in cultured human airway smooth muscle cells that expressed mainly M2 muscarinic receptors by dual-fluorescence labeling of filamentous (F) and monomeric (G) actin. F-actin was labeled with FITC-labeled phalloidin, and G-actin was labeled with Texas Red-labeled DNase I. Carbachol stimulation induced stress fiber formation (increased F-actin staining) in the cells and increased the F- to G-actin ratio 3.