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C W Emala

Publications and source records attributed to C W Emala.

35 records · Page 2Linked to original sources
PubMed1998

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 3.6 +/- 0.4-fold (mean +/- SE; n = 5 experiments). Preincubation with pertussis toxin, Clostridium C3 exoenzyme, or tyrosine kinase inhibitors reduced the carbachol-induced increase in stress fiber formation and significantly decreased the F- to G-actin ratio, whereas a mitogen-activated protein kinase inhibitor, a phosphatidylinositol 3-kinase inhibitor, and a protein kinase C inhibitor were without effect. This study demonstrates that in cultured human airway smooth muscle cells, muscarinic-receptor activation induces stress fiber formation via a pathway involving a pertussis-sensitive G protein, Rho proteins, and tyrosine phosphorylation.

ADP Ribose Transferases↗
PubMed1998

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. F- to G-actin ratios were measured with dual-fluorescence labeling after 5 min of carbachol exposure, which is known to increase the F- to G-actin ratio. Cells in parallel wells were harvested for immunoblot analysis of G protein alpha-subunit expression. Oligonucleotide antisense treatment decreased protein expression of the respective G protein alpha-subunit. Antisense depletion of the Galphai-2 protein but not of Galphai-3 or Gqalpha protein blocked the carbachol-induced increase in the F- to G-actin ratio. These results show that the Galphai-2 protein couples muscarinic receptors to stress fiber formation in ASM.

Carbachol↗
PubMed1997

Qualitative immunoblot analysis of PKC isoforms expressed in airway smooth muscle.

Protein kinase C (PKC) was originally identified as a single serine/ threonine protein kinase with calcium- and phospholipid-dependent activity, but more recently PKC has been found to consist of a family of multiple isoenzymes with different biochemical characteristics, substrates, and cofactor requirements. PKC is particularly important in regulating airway smooth muscle (ASM) tone. Although a previous investigation has demonstrated PKC-beta, -delta, -epsilon, -theta and -zeta in canine trachealis muscle, additional PKC isoforms have not been characterized in ASM. Therefore, immunoblot analysis using nine isotype-specific antibodies was used to further characterize the expression of PKC isoforms in porcine ASM. In addition to the previously described beta-, delta-, epsilon-, and zeta-isoforms in ASM, the calcium-dependent alpha-isoform, and the calcium- and diacylglycerol-independent isoforms iota/lambda and mu were identified. This study demonstrates multiple PKC isoforms in porcine ASM that can participate in intracellular signaling pathways in this tissue.

Animals↗
PubMed1997

TNF-alpha inhibits isoproterenol-stimulated adenylyl cyclase activity in cultured airway smooth muscle cells.

Inflammation, increased cytokine production, and decreased responsiveness of airway smooth muscle (ASM) to beta-adrenergic agonists are characteristics of asthma. We questioned whether the cytokine tumor necrosis factor-alpha (TNF-alpha) directly impaired beta-adrenergic signal transduction in cultured canine ASM cells. Confluent ASM cells exposed to TNF-alpha (0.1-10 ng/ml) for 72 h showed lower maximal levels of adenylyl cyclase activity in response to isoproterenol (10 ng/ml; 14 +/- 4.3 vs. 7.5 +/- 1.3 pmol adenosine 3',5'-cyclic monophosphate x well(-1) x 20 min(-1), control vs. treated, respectively), despite no changes in beta-adrenergic receptor numbers (maximum number of binding sites = 4.8 +/- 0.72 vs. 4.5 +/- 0.81 fmol/mg protein, control vs. treated, respectively). Adenylyl cyclase activities in response to prostaglandin E1, NaF, or forskolin were not different in treated and untreated cells. These results demonstrate that a cytokine known to be increased during exacerbation of asthmatic symptoms directly impairs beta-adrenergic function in ASM cells and suggests a mechanism by which inflammation impairs beta-adrenergic receptor signal transduction in asthma.

Adenosine Diphosphate Ribose↗
PubMed1997

Glucocorticoid treatment decreases muscarinic receptor expression in canine airway smooth muscle.

Corticosteroids upregulate the beta-adrenergic pathway, but little is known about corticosteroid regulation of muscarinic pathways. Basenji-greyhound (BG) dogs treated for 3 days with methylprednisolone (MPS) but not deoxycorticosterone (DOC) had decreased numbers of muscarinic receptors in airway smooth muscle homogenates as determined by radioligand binding with 1-[3H]quinuclidinyl benzilate (vehicle control, 578 +/- 53 fmol/mg protein; MPS, 290 +/- 22 fmol/mg protein; DOC, 565 +/- 141 fmol/mg protein). Competition radioligand binding with the M2-selective antagonist tripitramine showed a decrease in both the M2 and M3 muscarinic receptors with no changes in receptor affinities (M2: vehicle control, 478 +/- 41 fmol/mg protein; MPS, 265 +/- 20 fmol/mg protein, M3: vehicle control, 89 +/- 13 fmol/mg protein; MPS, 25 +/- 16 fmol/mg protein). In vitro treatment of airway smooth muscle from control BG dogs with MPS had no effect on muscarinic receptor number, despite increased expression of beta-adrenergic receptors. Thus glucocorticoids indirectly decrease the expression of M2 and M3 muscarinic receptors in airway smooth muscle, which, in part, may account for their beneficial effects in the treatment of asthma.

Animals↗
PubMed1997

Chronic carbachol pretreatment decreases adenylyl cyclase activity in airway smooth muscle.

In airway smooth muscle, the regulation of adenylyl cyclase, the enzyme that synthesizes adenosine 3',5'-cyclic monophosphate, is under dual regulation by G protein-coupled receptors. It is unknown if chronic activation of muscarinic receptors in airway smooth muscle alters the stimulatory adenylyl cyclase cascade to decrease airway relaxation. Bovine airway smooth muscle pretreated with carbachol for 18 h, but not for 30 min or 2 h, showed decreased adenylyl cyclase activity under basal conditions and in response to isoproterenol, prostaglandin E1, GTP, and forskolin. The quantity of beta-adrenergic receptors or of Gi alpha proteins was unaffected by carbachol pretreatment. The effect of carbachol pretreatment was blocked by the inclusion of atropine or the protein kinase C (PKC) inhibitor staurosporine. These results suggest that chronic but not acute agonist pretreatment of muscarinic receptors decreases in adenylyl cyclase stimulation at a site distal to receptors and that this effect is mediated by the chronic activation of PKC via the M3 muscarinic receptor.

Adenylyl Cyclases↗
PubMed1996

Impaired activation of adenylyl cyclase in lung of the Basenji-greyhound model of airway hyperresponsiveness: decreased numbers of high affinity beta-adrenoceptors.

1. To evaluate mechanisms involved in the impaired beta-adrenoceptor stimulation of adenylyl cyclase in tissues from the Basenji-greyhound (BG) dog model of airway hyperresponsiveness, we compared agonist and antagonist binding affinity of beta-adrenoceptors, beta-adrenoceptor subtypes, percentage of beta-adrenoceptors sequestered, and coupling of the beta-adrenoceptor to Gs alpha in lung membranes from BG and control mongrel dogs. We found that lung membranes from the BG dog had higher total numbers of beta-adrenoceptors with a greater percentage of receptors of the beta 2 subtype as compared to mongrel lung membranes. 2. Agonist and antagonist binding affinity and the percentage of beta-adrenoceptors sequestered were not different in BG and mongrel dog lung membranes. However, the percentage of beta-adrenoceptors in the high affinity state for agonist was decreased in BG lung membranes suggesting an uncoupling of the receptor from Gs alpha. 3. Impaired coupling between the beta-adrenoceptor and G protein documented by the decreased numbers of beta-adrenoceptors in the high affinity state in BG lung membranes, is a plausible explanation for the reduced stimulation of adenylyl cyclase and the resultant reduction in airway smooth muscle relaxation in this model.

Adenylyl Cyclases↗
PubMed1995

Reduced adenylyl cyclase activation with no decrease in beta-adrenergic receptors in basenji greyhound leukocytes: relevance to beta-adrenergic responses in airway smooth muscle.

Mononuclear leukocytes (MNLs) have been used as a model of beta-adrenergic responsiveness of airway smooth muscle, but the relevance of this model remains controversial. The basenji greyhound (BG) dog model of airway hyperresponsiveness shares some features with human asthma, and airway smooth muscle shows a selective impairment in isoproterenol-stimulated adenylyl cyclase activity. In this study, MNL membranes were obtained from these same dogs, and the beta-adrenergic receptor-adenylyl cyclase cascade function was compared with that in airway smooth muscle. beta-Adrenergic receptor numbers and affinities for iodine 125-cyanopindolol were similar in the two dog groups (receptor numbers [Bmax] = 441 +/- 101 and 447 +/- 61 fmol/mg protein and dissociation constant [Kd] = 269 +/- 44 and 312 +/- 60 pmol/L for mongrel and BG MNLs, respectively). Quantities of the Gs alpha protein were not different in the membranes as determined by immunoblotting. Stimulation of adenylyl cyclase by isoproterenol (100 mumol/L) was impaired in MNL membranes of BG membranes (22% +/- 4% increase over guanosine triphosphate [10 mumol/L]) compared with mongrel membranes (47% +/- 8.6% increase over guanosine triphosphate [10 mumol/L], p < 0.05). Stimulation of adenylyl cyclase by prostaglandin E1 (10 mumol/L), NaF (10 mmol/L), or forskolin (10 mumol/L) did not differ in membranes from the two groups. No difference was found in the lymphocyte subsets in the two groups as determined by flow cytometry. These findings are qualitatively similar to studies of trachealis muscle membranes from these same dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1995

Expression of muscarinic receptor subtypes and M2-muscarinic inhibition of adenylyl cyclase in lung.

The relative distribution and absolute quantities of muscarinic receptor subtypes m1, m2, m3, and m4 were determined in membranes of canine trachealis muscle, bronchi, and lung parenchyma by immuno-precipitation with receptor subtype-specific rabbit polyclonal antisera. Additionally, the functional coupling of muscarinic receptors to the inhibition of adenylyl cyclase was related to the presence of m2-muscarinic receptors in each region. Immunoprecipitation identified more total muscarinic receptors in trachealis muscle than in bronchi or lung. m2-Muscarinic receptor predominated in tracheal muscle (372 +/- 85 fmol/mg protein) with fewer m3 receptors (48 +/- 5 fmol/mg protein). Bronchi contained 6.6 +/- 2.0 and 9.2 +/- 1.8 fmol/mg protein of m2 and m3 receptors, respectively. Lung parenchyma contained 13.9 +/- 3.9 fmol/mg protein of m3 receptors. Adenylyl cyclase activity increased in response to guanosine triphosphate and isoproterenol in membranes from all three lung regions, but muscarinic-mediated inhibition of adenylyl cyclase occurred only in trachealis membranes. These studies provide the first quantitative assessment of muscarinic receptor subtypes in different regions of the lung and relate the ability to measure muscarinic inhibition of adenylyl cyclase to the presence of m2 receptors.

Acetylcholine↗
PubMed1995

Basenji-greyhound dog: increased m2 muscarinic receptor expression in trachealis muscle.

Airway smooth muscle from asthmatic humans and from the Basenji-greyhound dog (BG) dog is hyporesponsive to beta-adrenergic agonist stimulation. Because adenylyl cyclase is under dual regulation in airway smooth muscle, we compared muscarinic receptor-coupled inhibition of adenylyl cyclase in airway smooth muscle from BG and mongrel dogs. Inhibition of forskolin-stimulated adenylyl cyclase activity by the muscarinic M2 agonist oxotremorine was greater in airway smooth muscle membranes from BG compared with mongrel controls. Quantitative immunoprecipitation studies showed increased numbers of m2 but not m3 muscarinic receptors in the BG airway smooth muscle. The enhanced ability of muscarinic agonists to inhibit adenylyl cyclase in BG airway smooth muscle may be due to the greater numbers of muscarinic m2 receptors, which may account in part for impaired airway smooth muscle relaxation in the BG model of airway hyperresponsiveness.

Adenylyl Cyclase Inhibitors↗
PubMed1994

G protein subunits in lung cells.

Many hormones and neurotransmitters bind to membrane-bound receptors that are coupled to signal generating enzymes or ion channels via signal transducing GTP-binding proteins termed G proteins. Although receptors and second messengers have been extensively studied in cells of the respiratory system, the G proteins responsible for the coupling of these proteins have not been well-characterized. Therefore, we used immunoblot analysis to determine expression of G protein alpha and beta subunits in membranes prepared from cells and tissues of the respiratory system, including cultured canine tracheal epithelium, cleanly dissected canine tracheal smooth muscle, canine large conducting airways, and canine and human lung parenchyma. The two isoforms of Gs alpha (45 and 52 kDa) were present in all tissues, with a predominant expression of the 45 kDa isoform. Plasma membranes prepared from canine tracheal epithelium and muscle, and human lung parenchyma, contained greater amounts of Gs alpha than membranes prepared from canine bronchus and lung. Relative levels of immunoreactive G(i) alpha(2), G(i) alpha(3), Gq/G11 alpha, beta 1 and beta 2 were similar in all of the tissues studied. By contrast, G(o) alpha was absent in cultured tracheal epithelium, and tracheal smooth muscle expressed greater amounts of G(i) alpha(2) compared to G(i) alpha(3). Specificity of G protein expression can provide one regulatory mechanism for functional biochemical pathways within cells. The demonstration of specific G protein subunits is the first step in the molecular characterization of the regulation of these pathways, both in normal tissues and in disease states.

Amino Acid Sequence↗
PubMed1994

Signal-transducing G proteins: basic and clinical implications.

The pivotal role that G proteins play in transmembrane signal transduction is highlighted by the rapidly expanding list of receptors and effector molecules that are coupled through G proteins. G proteins are poised to allow discrimination and diversification of cellular signals into the cytosolic milieu. The utilization of an evolutionarily conserved "GTPase clock" by G proteins, offers insight into the fundamental role these proteins play in biology. Knowledge of the implication of altered expression or function of G proteins in human disease is now emerging. It is not surprising that deficiency or expression of altered forms of these important proteins can lead to global or restricted metabolic disturbances, depending upon the distribution and role of the G protein. Human disorders, including heart failure, alcoholism, endocrine abnormalities, and neoplasia, are now recognized as due in part to altered expression or function of G proteins.

Amino Acid Sequence↗
PubMed1994

Characterization of GTP-binding proteins coupled to inhibition of adenylyl cyclase in guinea pig tracheal epithelial cells.

Many important airway epithelial cell functions are regulated by intracellular cAMP. Adenylyl cyclase, the enzyme that synthesizes cAMP, is under dual regulation in many cells, but muscarinic agonists have not been shown to inhibit adenylyl cyclase in human and dog epithelial cells, despite the presence of muscarinic receptors. We question whether the lack of inhibition was related to the absence of a component of the inhibitory pathway or a lack of coupling between the components. The GTP-binding regulatory proteins (G proteins) that regulate adenylyl cyclase activity in airway epithelium have not been well characterized. We used primary cultures of guinea pig tracheal epithelial cells as a model system and identified the G proteins that modulate adenylyl cyclase activity. Immunoblot analysis demonstrated the presence of alpha subunits corresponding to stimulatory (Gs alpha) and inhibitory [Gi alpha (2) and Gi alpha (3)] G proteins as well as beta chains. These G proteins were functionally coupled to stimulation and inhibition of adenylyl cyclase in epithelial membrane preparations. Pertussis toxin-catalyzed [32P]ADP-ribosylation of Gi alpha was significantly reduced by 100 microM GTP gamma S (78.4 +/- 3.6% of control), by 100 mM NaF (41.9 +/- 9.1% of control), and by carbachol (100 microM) (29.2 +/- 9.0% of control). Atropine (10 microM) inhibited the carbachol effect by greater than 90%, suggesting that the muscarinic receptors were functionally coupled to Gi proteins. beta-Adrenergic agonists increased adenylyl cyclase activity, but muscarinic agonists failed to inhibit this enzyme. In summary, guinea pig tracheal epithelial membranes contain muscarinic receptors, Gi alpha (2) and adenylyl cyclase, which are appropriately coupled.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1983

Invited review: bacterial flagellar sheaths: structures in search of a function.

Although bacterial flagellar sheaths were observed over 30 years ago, they may still be characterized as structures in search of a function. In addition to true sheaths, bacterial flagella may possess other adornments that cause an increase in the organelle's cross-sectional diameter. These "complex flagella" are sharply differentiated from sheathed flagella. Immunological and chemical distinctions have been found between flagellar sheaths, flagellar cores, and LPS layers inferred to be the sheath sensu stricto. Although complex flagella may serve as specific receptors for flagellotropic phages or in allowing for more efficient swimming in viscous environments, similar functions have not yet been attributed to true sheaths. It is postulated that flagellar sheaths may allow for specific interaction between a bacterium and a surface. In addition, there is a problem as to the relationship between a rapidly rotating flagellum and the sheath.

Antigens, Bacterial↗
PubMedDate not supplied

Expression of adenylyl cyclase V/VI mRNA and protein is upregulated in cyanotic infant human myocardium.

We have previously demonstrated that both basal and isoproterenol-stimulated activities of myocardial adenylyl cyclase were greater in cyanotic patients with tetralogy of Fallet (TOF) than those in acyanotic patients. However, it was not determined whether increased enzyme activity was related to a similar increase in adenylyl cyclase protein and mRNA expression. In the current study, we examined the mRNA and protein expression of cardiac adenylyl cyclase, types V and VI, in cyanotic and acyanotic patients with TOF. Ribonuclease protection assays and immunoblotting were performed on myocardial specimens obtained from cyanotic patients with TOF and acyanotic patients with TOF or ventricular septal defect. We demonstrated that in both cyanotic and acyanotic patients, there was more type V adenylyl cyclase mRNA than type VI. Types V and VI cardiac adenylyl cyclase mRNA were significantly increased in myocardium of the cyanotic group compared to the acyanotic group. Protein expression of both V and VI adenylyl cyclases was correspondingly upregulated in cyanotic patients compared to acyanotic patients. Our results indicate that gene and protein expression of cardiac adenylyl cyclases, types V and VI, is increased in the cyanotic myocardium. These results suggest that chronic hypoxemia may regulate the expression of adenylyl cyclase enzymes.

Adenylyl Cyclases↗