PubMed Health⌕ Search

Biomedical subjects

L L Brunton

Publications and source records attributed to L L Brunton.

At least 73 records · Page 4Linked to original sources
PubMed1982

Functional compartments in cyclic nucleotide action.

Cyclic AMP-dependent protein kinase (cAMP-PK) is a ubiquitous enzyme that, when activated by cAMP, is capable of phosphorylating a variety of intracellular proteins. The central postulate of cAMP-mediated hormone action is that hormones regulate intracellular cAMP concentration and cAMP-PK mediates the effects of this second messenger. Although this postulate accurately describes cAMP action in certain systems, it does not adequately provide for recent observations of the accumulation of cAMP and the activation of protein kinase without the anticipated effects on protein kinase's substrates. Both biochemical and cytochemical technics provide evidence that hormonally-specific regulation of cAMP action occurs and is important. Our thesis is that hormonal regulation of metabolic events via cAMP is localized intracellular phenomenon. We propose that occupation of some cell-surface hormone receptors leads to cAMP accumulation and the activation of protein kinase in subcellular compartments, with the consequent phosphorylation of specific, rather than all, substrates of protein kinase. circumstances potentially contributing to this specificity include: (a) physical and kinetic compartmentation of hormone-receptor-adenylate cyclase complexes non-randomly within the cell membrane; and, (b) a fixed spatial relationship of hormonally activated adenylate cyclase and specific intracellular regions by the participation of cytoskeletal proteins.

Alprostadil↗
PubMed1981

Cautions on the use of the heat stable inhibitor of protein kinase: studies with S49 lymphoma cells.

When crude preparations of protein kinase inhibitor (PKI) are added to homogenates of S49 lymphoma cells, protein kinase activity increases rather than decreases. This is not an increase in the activity of cyclic AMP-dependent protein kinase, which is completely inhibited by PKI, nor an increase in either cyclic GMP-dependent or Ca ++-dependent kinases. The increased kinase activity is not due to PKI itself but to one or more protein contaminants which serve as substrates for an S49 cell kinase which is cyclic nucleotide- and Ca ++ -independent. These contaminating substrates can be readily separated from PKI by DEAE cellulose chromatography. Although the crude PKI preparations sold by the major biochemical supply houses may be satisfactory for some purposes, we suggest that crude PKI be further purified when used to assess kinase activity in systems as complex as homogenates of whole cells. Otherwise, the presence of contaminating proteins in crude preparations of PKI can lead to erroneous conclusions about the type and quantity of protein kinase in the cell.

Animals↗
PubMed1980

Export of cyclic AMP by mammalian reticulocytes.

Suspensions of reticulocyte-enriched red cells produce and extrude cyclic AMP in proportion to their reticulocyte content. When reticulocytes are treated with the beta-adrenergic agonist isoproterenol, up to 3.5 pmoles of cyclic AMP/min/mg protein appear in the extracellular medium. Extruded cyclic AMP can occur against apparent concentration gradients and is inhibited by agents (iodoacetate, dinitrophenol) that deplete cellular ATP, as well as by probenecid and prostaglandin A1. Extrusion of cyclic AMP depends on the availability of intracellular cyclic AMP but is not obligatorily coupled to adenylate cyclase activity: extrusion continues following termination of a pulse of stimulation and exhibits a temperature dependence that differs from that for cyclic AMP production in response to isoproterenol. Erythropoietin affects neither production nor extrusion of cyclic AMP by reticulocytes. In whole blood, cyclic AMP extruded by reticulocytes may be a significant source of plasma cyclic nucleotide.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1979

Depressed cyclic AMP levels in airways smooth muscle from asthmatic dogs.

We tested mongrel dogs by intradermal injection and tracheobronchial aerosol challenge with Ascaris suum antigen extract. All dogs were skin-test positive but could be segregated, on the basis of airways resistance measurements, into "asthmatic" (bronchoreactive) and "nonasthmatic" (nonbronchoreactive) groups. By using tracheal rings from these dogs, we measured the abilities of the beta-adrenergic agonist, isoproterenol, to relax tracheal smooth muscle contracted by methacholine and to cause cyclic AMP (cAMP) accumulation in the presence and absence of methacholine. The magnitude of relaxation induced by any concentration of isoproterenol was always less in the smooth muscle from "asthmatic" dogs. In the same tissues the concentrations of cAMP after in vitro equilibration, but prior to isoproterenol addition, were significantly less in the "asthmatic" than "nonasthmatic" samples. The accumulation of cAMP due to isoproterenol was similar in both groups for every dose of isoproterenol so that the initial difference between groups in cAMP concentration was maintained in an additive fashion over the entire dose-response curve. Total protein content of trachealis muscles from both groups of dogs was the same. We conclude that beta-adrenergically sensitive adenylate cyclase is not impaired in tracheal smooth muscle from "asthmatic" dogs; rather, the basal concentration of cAMP is depressed in "asthmatic" airway smooth muscle, and this difference is maintained throughout the isoproterenol dose-response curve. The depressed intracellular cAMP concentrations may be related to the decreased relaxation induced by isoproterenol in the "asthmatic" tracheal smooth muscle.

Animals↗
PubMed1979

Hormonally specific expression of cardiac protein kinase activity.

The relationship between the effects of isoproterenol and prostaglandin E(1) (PGE(1)) on contractile state, cyclic AMP accumulation, and the activation states of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37), phosphorylase kinase, glycogen synthase, and glycogen phosphorylase have been studied in the isolated perfused rat heart. Perfusion of hearts with isoproterenol (10 or 80 nM) caused enhancement of left ventricular dP/dt (P, pressure), increased intracellular cyclic AMP, increased the activation states of protein kinase, phosphorylase kinase, glycogen phosphorylase, and conversion of glycogen synthase to a less active form. PGE(1) (2 or 30 muM) increased cyclic AMP accumulation and activated protein kinase, but caused no detectable changes in dP/dt or the activation states of the protein kinase substrates involved in glycogen metabolism. Perfusion of hearts with either 10 nM isoproterenol or 30 muM PGE(1) produced comparable increases in cyclic AMP accumulation and protein kinase activity. Exposure of hearts to a combination of these agents caused additive effects on cyclic AMP content and protein kinase activity. However, values for phosphorylase kinase, glycogen phosphorylase, glycogen synthase, and dP/dt did not differ from those observed in the presence of 10 nM isoproterenol alone. The failure of PGE(1) to stimulate phosphorylation of protein kinase substrates was not due to an increase in phosphorylase phosphatase activity. We conclude that an increase in intracellular cyclic AMP and the subsequent activation of protein kinase are insufficient to change either the activities of phosphorylase kinase, glycogen phosphorylase, and glycogen synthase or the inotropic state of heart muscle.

Animals↗
PubMed1977

Expression of genes for metabolism of cyclic adenosine 3':5'-monophosphate in somatic cells. beta-Adrenergic and PGE1 receptors in parental and hybrid cells.

Using the ligands [125I]iodohydroxybenzylpindolol and [3H]prostaglandin E1 ([3H]PGE1), we have studied the relationship of receptors for beta-adrenergic agents and for PGE1 to adenylate cyclase in membranes of parental, hybrid, and variant mammalian cell lines. Fusion of parental clones responsive to beta-adrenergic agonists (beta+) with unresponsive clones (beta-) produced hybrid clones with a greatly diminished beta-adrenergic response; beta+ X beta leads to beta-. Binding studies with [125I]iodohydroxybenzylpindolol showed a decreased concentration of beta receptors in six such hybrid clones. Thus, paucity of beta-adrenergic receptors is probably a sufficient, albeit not necessarily complete, explanation for the decreased beta-adrenergic responsiveness of the hybrid clones. When a clone with beta receptor but without apparent adenylate cyclase activity (HC-1) was hybridized with a beta- clone that has adenylate cyclase (B82), a responsive hybrid clone was obtained. In nine cell hybrids produced by the fusion of clones responsive (PGE1+) and unresponsive (PGE1-) to PGE1, high affinity binding sites for [3H]PGE1 were expressed in the same manner as was PGE1-sensitive adenylate cyclase: PGE1+ X PGE1 leads to PGE1+. The chemical specificities and affinities of the parental receptors and responsive adenylate cyclases were faithfully reproduced in the hybrid clones. Activation by PGE1 was proportional to the occupation of the high affinity receptors. In a wild type lymphoma clone (24.3.2), the concentration dependences for binding of [3H]PGE1 and for activation of adenyalte cyclase by PGE1 were identical. In a variant lymphoma clone (94.15.1) lacking adenylate cyclase activity, no high affinity receptors for PGE1 were detected, whereas beta-adrenergic receptors have been demonstrated in this variant clone (Insel, P.A., Maguire, M.E., Gilman, A.G., Coffino, P., Bourne, H., and Melmon, K. (1976) Mol. Pharmacol. 12, 1062-1069). Hybrid cells formed by the fusion of 94.15.1 with cell line RAG (PGE1-) responded to PGE1. Clone 94.15.1 may have receptors for PGE1 of reduced affinity or in low concentration. Alternatively, RAG and 94.15.1 may have complementary genetic defects such that the RAG X 94.15.1 hybrid cells express a hormonally responsive receptor-adenylate cyclase system.

Adenylyl Cyclases↗
PubMed1977

Characterization of the Chinese hamster ovary cell assay for the enterotoxins of Vibrio cholerae and Escherichia coli and for specific antisera, and toxoid.

The morphologic change in Chinese hamster ovary (CHO) cells that is associated with increased intracellular concentrations of adenosine 3':5'-cyclic phosphate (cyclic AMP) provided a highly sensitive assay for the heat-liable enterotoxins of Vibrio cholerae and Escherichia coli. The similarity between the responses of CHO cells and those of intestinal mucosa to enterotoxins was demonstrated by the inhibition of these effects by the ganglioside galactosyl-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide (GM1), as well as by specific antisera. The antisera against either enterotoxin were more effective in inhibiting the effects of homologous toxin than those of heterologous toxin, but cross-neutralization also occurred in both cases. When choleragenoid, the natural toxoid of cholera toxin, was incubated with the CHO cells, the subsequent effects of the toxin on the morphology of the cells were prevented as were the effects of the toxin on intestinal secretion. However, choleragenoid did not prevent the effects of E. coli toxin. The similarity between the action of enterotoxins on CHO cell morphology and on secretion by the small intestine suggests that there are similar binding sites in these tissues. The potential usefulness of the CHO cell assay in quantitative measurement of antitoxic immunity is thus demonstrated.

Animals↗
PubMed1976

Binding of (3H)prostaglandin E1 to putative receptors linked to adenylate cyclase of cultured cell clones.

A method for assessing the binding of 3H-labeled prostaglandin E1 ([3H]PGE1) to cell membranes has been developed and used to study the interaction of [3H]PGE1 with membranes from cultured mammalian cells. Receptor sites were identified by correlation of the potency of a series of compounds to compete for [3H]PGE1 binding sites and to stimulate adenylate cyclase activity, by correlation of rates of binding and change in enzyme activity, and by the correspondence of [3H]PGE1-binding activity with the presence or absence of PGE1-sensitive adenylate cyclase in several clones. In clone B82, a murine L-cell, [3H]PGE1 binds with an activation energy of 14 kcal/mol to a class of sites with an affinity of 0.5 X 10(8) M-1 and a capacity of 150 fmol/mg of protein. Concentration dependence of adenylate cyclase activation by PGE1 (KD =30 nM) and kinetic analysis of [3H]PGE1 binding (k1 = 4 X 10(6) liters/mol/min, k-1 0.15/min) verify this affinity. Concentration dependence and specificity of binding and activation of adenylate cyclases in neuroblastoma clone N4TG1 and N18TG2 substantiate the method. In several clones that lack PGE1-responsive adenylate cyclase, no specific [3H]PGE1 binding is detectable.

Adenylyl Cyclases↗
PubMed1974

Cyclic adenosine monophosphate and alteration of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for the enterotoxins of Vibrio cholerae and Escherichia coli.

The major limitation to our understanding of the clinical importance of enterotoxigenic Escherichia coli in diarrheal illness has been the lack of a simple rapid assay for the enterotoxin produced by certain E. coli. On the basis of the activation of adenylate cyclase by heat-labile enterotoxin of E. coli (LT) and by cholera toxin (CT) in intestinal and other tissues, cultured Chinese hamster ovary (CHO) cells with known morphological responses to dibutyryl cyclic adenosine 5'-monophosphate (AMP) were exposed to these enterotoxins. Crude culture filtrates of LT-producing E. coli and CT stimulated cyclic AMP accumulation and cell elongation in CHO cells. The similarity of time course, concentration dependence, and potentiation by phosphodiesterase inhibitors suggested cyclic AMP mediation of the morphological change. Heat inactivated CT and LT in this system. Choleragenoid inhibited CT; antiserum against CT inhibited both enterotoxin effects. In contrast to culture filtrates of 16 strains of E. coli known to produce LT, culture filtrates from 13 E. coli that do not produce LT did not alter CHO cell morphology. The morphological change is a simple, specific assay for these enterotoxins and detect 3 x 10(-17) mol of CT or a 1:250 dilution of crude culture filtrate of LT-producing E. coli 334.

Animals↗
PubMedDate not supplied

Post-receptor modulation of the effects of cyclic AMP in isolated cardiac myocytes.

The actions of cyclic AMP are subject to several levels of post-receptor modulation in cardiac tissue. Isoproterenol and prostaglandin E1 both stimulate cAMP accumulation, but only isoproterenol causes activation of particulate cAMP-dependent protein kinase, leading to activation of phosphorylase kinase and glycogen phosphorylase, and inhibition of glycogen synthase. Through the use of isolated, adult ventricular myocytes, we have determined that the hormone-specific activation of glycogen phosphorylase is due to subcellular compartmentation of cAMP. There is some evidence that cyclic nucleotide phosphodiesterases, whose activity is stimulated by alpha 1-adrenergic agonists in isolated myocytes, may have a role in compartmentation. Phosphoinositide hydrolysis is stimulated by alpha 1 and muscarinic agonists, presumably leading to activation of protein kinase C, which in turn has multiple effects on hormone-sensitive adenylate cyclase.

Alprostadil↗