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Biomedical subjects

P A Insel

Publications and source records attributed to P A Insel.

At least 109 records · Page 6Linked to original sources
PubMed1990

Differential activation of protein kinase C alpha is associated with arachidonate release in Madin-Darby canine kidney cells.

The heterogeneity of the protein kinase C (PKC) gene family strongly suggests that different isoforms may have distinct functions in mediating signal transduction. However, there is very little direct evidence for this. PKC has been implicated in arachidonate (AA) release in many cell types. We sought to investigate whether bradykinin- and phorbol ester-stimulated AA release in Madin-Darby canine kidney (MDCK) cells was correlated with differential activation of PKC isoforms. Using phorbol esters to (i) activate the enzyme and (ii) to down-regulate it, we report that differential activation (translocation) of PKC alpha is associated with AA release in MDCK cells and that specific down-regulation of PKC alpha is associated with a loss of AA release in response to stimulation with dioctanoylglycerol and phorbol ester. We also demonstrate that bradykinin-stimulated AA release was associated with differential activation of PKC alpha and was inhibited in PKC alpha down-regulated cells. Thus, we conclude that the PKC alpha isoform is likely to be responsible for mediating AA release in these cells.

Animals↗
PubMed1990

ras-transformation of MDCK cells alters responses to phorbol ester without altering responses to bradykinin.

The results of studies to evaluate the hypothesis that the 21 kDa GTP-binding protein derived from the ras oncogene is involved in regulation and coupling of hormone receptors to phospholipase activity have thus far been inconsistent. We therefore examined the effect of H-ras transformation on basal, tumor-promoting phorbol ester (TPA)-stimulated, and bradykinin-mediated phospholipid hydrolysis in Madin Darby canine kidney cells (MDCK) by comparing H-ras-transformed MDCK cells (MDCK-RAS) to two non-transformed strains of MDCK cells (MDCK-D1 and MDCK-ATCC). In unstimulated MDCK-RAS, diacylglycerol (DAG), inositol phosphate accumulation, and choline phosphate release were increased while arachidonic acid and arachidonic acid metabolite (AA) release was not increased, suggesting that ras transformation increased phospholipase C activity. Protein kinase C (PK-C) activity was decreased, and specific binding of [3H]phorbol ester was reduced in MDCK-RAS relative to the non-transformed MDCK cells suggesting that elevated DAG may activate and thereby down-regulate PK-C. Consistent with this finding in MDCK-RAS, TPA-stimulated AA release and subsequent prostaglandin E2 production were decreased, while TPA-stimulated choline phosphate release was increased. Bradykinin receptor-stimulated phospholipid hydrolysis in MDCK-RAS was similar to that of non-transformed cells, suggesting that the ras-derived protein does not directly couple bradykinin receptors to phospholipases in MDCK cells. However, the ability of TPA-treatment to inhibit bradykinin-stimulated phosphoinositide hydrolysis and enhance bradykinin-stimulated AA release was attenuated in MDCK-RAS. Additionally, in MDCK-RAS the conversion of arachidonic acid to prostaglandin E2 was substantially reduced. We conclude that ras transformation of MDCK cells increases DAG levels, thereby activating and, in turn, down-regulating PK-C and certain responses to TPA. Since activation of PK-C may result in a variety of effects on signal transduction pathways, we propose that increased DAG and altered PK-C levels associated with ras transformation may account for the inconsistent effects previously observed in studies evaluating the effect of ras transformation on phospholipases and other signal transduction systems.

Animals↗
PubMed1990

Beta-adrenergic receptors and the Gs protein in myocardial ischemia and injury.

The purpose of this study was to explore alterations in the life cycle of adrenergic receptors and the Gs protein in the heart of ischemic animals. In initial experiments left anterior descending coronary artery occlusion was performed in guinea pigs. Sarcolemmal (SL) and light vesicle (LV) (presumably intracellular) fractions were prepared. Both fractions contained a substantial number of beta-adrenergic receptors and alpha 1-adrenergic receptors: the relative proportion of beta-adrenergic receptors in LV/SL was greater than for alpha 1-adrenergic receptors. Myocardial ischemia produced a rapid externalization of beta-adrenergic receptors from LV to SL. alpha 1-adrenergic receptors also increased in SL but without an apparent externalization from LV. Pretreatment of animals with either the non-selective beta-antagonist propranolol or the beta 1-selective antagonist atenolol increased the number of SL beta-receptors and blunted the ischemia-induced increase in SL beta-adrenergic receptors. Treatment with the partial agonist pindolol did not cause up-regulation of beta-receptors, and did not block ischemia-induced externalization. In the second part of this study, we have begun to examine post-receptor events in a rat model of myocardial ischemia. Ligation of the distal left main coronary artery in the rat led to an increase in SL beta-receptors. As G proteins play a pivotal role in transducing receptor occupancy to activation of effector molecules, we measured levels of Gs which stimulates adenylate cyclase activity, using an ELISA technique. In rat SL the amount of alpha s markedly decreased within 15 min of coronary occlusion. There was no transfer of Gs activity to the light vesicle fraction. These studies indicate the dynamic nature of adrenergic receptors and the alpha s protein in the sarcolemma in myocardial ischemia. Changes in adrenergic receptor number and in G protein expression may contribute to the altered pathophysiology of the ischemic heart.

Animals↗
PubMed1990

Asthma: a disorder of adrenergic receptors?

For the past 20 years, the hypothesis that bronchial asthma represents an imbalance between deficient activity of the beta-adrenergic receptor pathway and/or enhanced activity of alpha-adrenergic/muscarinic cholinergic pathways in the regulation of airway smooth muscle has been a popular idea. Although some evidence supports this hypothesis, direct proof of its validity has been elusive. Concepts regarding the pathobiology of asthma have shifted from a focus on changes in smooth muscle per se to the prominent inflammatory nature of this disorder. We review some of the evolving information regarding the pathogenesis of this disorder and discuss how altered neurohormonal regulation of key inflammatory cells may contribute to the asthmatic condition.

Asthma↗
PubMed1990

The beta-adrenoceptor antagonist carteolol and its metabolite 8-hydroxycarteolol have different intrinsic sympathomimetic activities.

We have tested the effects of carteolol and 8-hydroxycarteolol on cAMP generation in S49 lymphoma and BC3H1 smooth muscle-like cells. Carteolol was a high affinity beta-adrenoceptor antagonist in both systems but did not stimulate cAMP accumulation. The metabolite 8-hydroxycarteolol also was a high affinity antagonist. In contrast to its parent compound, however, it possessed an agonistic component which was considerably stronger than that of other beta-adrenoceptor blockers with intrinsic sympathomimetic activity, e.g. dichloroisoprenaline, pindolol, or celiprolol. These results suggest that metabolites can possess different pharmacodynamic properties in terms of beta-adrenoceptor interaction relative to parent compounds.

Carteolol↗
PubMed1990

G-protein linked polyphosphoinositide phospholipase C activity in cell membranes from clonally derived and ras-transformed Madin-Darby canine kidney cells.

Membranes prepared from clone D1 of Madin-Darby canine kidney (MDCK) cells contain activity that can be attributed to Gp, a guanine nucleotide binding protein linked to phosphatidylinositol 4,5-bisphosphate dependent phospholipase C. Polyphosphoinositides are produced by addition of GTP, nonhydrolyzable GTP analogs, or fluoroaluminate. This production is inhibited by guanosine 5'-(beta-thiodiphosphate). While Ca2+ at 1 microM or more can generate high yields of inositol phosphates, guanine nucleotide activation of Gp can potentiate this Ca2(+)-dependent yield at resting levels of the cation. Membranes from cells expressing large amounts of ras-p21 exhibit small differences in guanine nucleotide induced polyphosphoinositide quantities. The greatest difference between normal and ras membranes was seen with AlF4- incubation. Of the three inositol phosphates measured, only the inositol bisphosphate yield was greatly increased in ras membranes compared with membranes from both parental and the D-1 clone of MDCK cells. From these data, we conclude that the presence of ras-p21 may affect production of polyphosphoinositides in MDCK cell membranes by some means other than direct participation in phospholipase C activation.

Animals↗
PubMed1990

Beta-adrenoceptor metabolism in wild-type, Gs, and protein kinase A-variant S49 cells.

To determine the role of the stimulatory guanine nucleotide-binding protein, Gs, and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase in the basal metabolism of beta-adrenergic receptors in S49 lymphoma cells, we measured the return of receptor number and function after irreversible blockade of receptors. After inactivation of receptors with the irreversible ligand N8-(bromoacetyl)-N'-[3-(4-indolyoxy)-2-hydroxypropyl]-(2)-1,8-diam ino-p- methane (BIM), beta-adrenergic receptors (defined as [125I]iodocyanopindolol binding sites) reappeared in a biphasic manner, the faster phase having a half-time (t 1/2) of 3-8 h (approximately 50% of the sites) and the slower phase greater than 40 h. Although the slow phase is not readily explained, recovery of binding sites during the first 10 h matched recovery of receptor function after BIM treatment (as measured by stimulation of cAMP accumulation) and recovery of receptor sites after downregulation induced by the agonist isoproterenol. Thus quantifying receptor recovery during the first 10 h after BIM treatment appears to be a reasonable method for examining basal receptor metabolism in S49 cells. Measured in this way, metabolism of beta-adrenergic receptors is very similar in wild-type S49 and the following variant clones: cyc- (absent Gs alpha), UNC and H21a (defective Gs alpha), and kin- (lacking cAMP-dependent protein kinase activity). Although previous data have demonstrated that agonist-promoted downregulation of beta-adrenergic receptors requires functional receptor-Gs coupling, the current data suggest that neither Gs nor cAMP-dependent protein kinase activity plays an important role in the regulation of basal metabolism of beta-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMed1990

Elevated extracellular K+ enhances arachidonic acid release in MDCK-D1 cells.

Depolarization can alter the expression of membrane receptors and change certain receptor-mediated events, but previous studies have not assessed the impact of depolarization on generation of arachidonic acid and its metabolites (AA) in nonexcitable tissues. We assessed AA generation in Madin-Darby canine kidney (MDCK) cells grown for 3 days in increased extracellular [K+], which is known to acutely depolarize these cells. Growth under these conditions resulted in decreases in the number of alpha 1-adrenergic receptors (alpha 1 AR), a small decrease in receptor-mediated phosphoinositide hydrolysis, but increases in alpha 1 AR-mediated prostaglandin E2 formation and AA release. Calcium ionophore (A23187)-, melittin-, and bradykinin-stimulated AA release were also enhanced. The reduction in alpha 1 AR number and increased AA release were substantially reduced or eliminated when K(+)-treated cells were grown in the absence of extracellular calcium. The results provide evidence that hormone-stimulated AA release and prostaglandin production can be enhanced by chronic exposure to elevated extracellular K+ concentration, perhaps as a consequence of a depolarization-induced enhancement in phospholipase A2 activity. The results provide evidence for the parallel and independent regulation of the pathways for receptor-mediated phosphoinositide hydrolysis (phospholipase C activation) and AA release (phospholipase A2 activation) in MDCK cells.

Animals↗
PubMed1990

Pertussis toxin treatment of whole blood. A novel approach to assess G protein function in congestive heart failure.

This study was designed to assess G protein function in mononuclear leukocytes (MNL) of patients with congestive heart failure (CHF). MNL membranes were ADP-ribosylated in vitro in the presence of pertussis or cholera toxin. The amount of pertussis toxin substrates did not differ significantly between CHF patients (6,100 +/- 224 fmol/mg, n = 23) and age-matched healthy control subjects (5,812 +/- 972 fmol/mg protein, n = 19). Among the CHF patients, no differences were observed between those with idiopathic and ischemic CHF. The amount of cholera toxin substrates also did not differ significantly between CHF patients (7,522 +/- 1,405 fmol/mg protein, n = 11) and control subjects (5,654 +/- 707 fmol/mg protein, n = 14). Moreover, basal and isoproterenol- and prostaglandin E1-stimulated cyclic AMP (cAMP) accumulation in MNL was similar in control subjects and patients. To detect more subtle alterations of the cAMP-generating system, we incubated anticoagulated blood with 250-400 ng/ml pertussis toxin for 4 hours at 37 degrees C. This treatment completely ADP-ribosylated the MNL pertussis toxin substrates. Incubation with pertussis toxin did not change basal or prostaglandin E1-stimulated cAMP generation in MNL of control subjects, but it significantly enhanced stimulated generation (443 +/- 44 vs. 643 +/- 93 pmol/10(7) cells, p less than 0.025) in MNL of CHF patients. This enhancement was most pronounced in the most severely ill patients (New York Heart Association class IV) and correlated with plasma norepinephrine levels, another marker of CHF severity (r = 0.798, n = 11, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1990

Peripheral adrenergic receptors in hypertension.

Increased sympathoadrenal activity appears to play an important role in the development or maintenance of elevated blood pressure in hypertensive patients and various animal models of hypertension. Alterations of adrenergic receptor number or responsiveness might contribute to this increased activity. We therefore reviewed the data on adrenergic receptor alterations in hypertension with special emphasis on several key cardiovascular tissues (i.e., heart, vascular smooth muscle, and kidney) and on lymphocytes and platelets as human tissues available for such studies. The data suggest that the number of alpha-adrenergic receptors in hypertension is regulated by catecholamines, dietary salt intake, and genetic factors. Increases in renal alpha-adrenergic receptor number may be etiologic in genetic forms of essential hypertension. beta-Adrenergic receptor alterations in states of elevated blood pressure do not appear to be specific for genetic hypertension. Desensitization of beta-adrenergic receptor function in hypertensive animals and patients contrasts with reports of decreased, unchanged, and increased beta-adrenergic receptor number, suggesting that signal transduction of beta-adrenergic (and possibly other) receptors that stimulate adenylyl cyclase is disturbed in hypertension. The mechanisms of such heterologous desensitization in states of elevated blood pressure remain to be elucidated.

Animals↗
PubMed1990

Adrenergic control of circulating lymphocyte subpopulations. Effects of congestive heart failure, dynamic exercise, and terbutaline treatment.

The current studies were undertaken to explore the relationship between enhanced sympathetic nervous activity and lymphocyte subset distribution in three settings: congestive heart failure, dynamic exercise, and beta-adrenergic agonist treatment. We compared the number and subset distribution of circulating lymphocytes in 36 patients with congestive heart failure and 31 age-matched control subjects. The number of circulating lymphocytes was lower in heart failure than in control. This was due to a reduction in Tsuppressor/cytotoxic and natural killer cells without significant alteration of Thelper cells. The extent of the alteration was similar in patients with idiopathic and ischemic heart failure, but the reduction was more pronounced in patients with New York Heart Association class III-IV than in class I-II. The plasma catecholamine elevation in heart failure was also independent of etiology but more pronounced in the more severely ill patients. We also assessed lymphocyte subsets after acute stimulation of sympathetic activity by dynamic exercise and after treatment with the beta-adrenergic agonist terbutaline. Dynamic exercise until exhaustion increased the number of circulating lymphocytes in healthy controls and heart failure patients in a subset-selective manner. By contrast, a 7-d treatment with terbutaline caused a reduction in the circulating number of lymphocytes in some subsets that was identical to that seen in heart failure patients. We conclude that prolonged sympathetic activity reduces the number of circulating lymphocytes by a beta-adrenergic mechanism. Such alterations might be involved in the pathophysiology of heart failure and other disease states involving increased activity of the sympathetic nervous system.

Adult↗
PubMed1990

Amplification of cyclic AMP generation reveals agonistic effects of certain beta-adrenergic antagonists.

Some beta-adrenergic receptor (beta AR) antagonists, in addition to blocking receptor-mediated responses, possess agonistic properties or intrinsic sympathomimetic activity (ISA). In this study we describe several techniques for amplification of cAMP levels as a measure of agonistic activity, and we apply these techniques to the study of beta AR antagonists with ISA. We show that 1) a variety of beta AR antagonists with ISA, including alprenolol and cyanopindolol, enhance cyclic AMP accumulation in S49 lymphoma cells if cells are also incubated with the diterpene forskolin; 2) beta AR blockers with ISA stimulate cAMP accumulation in the presence of a water-soluble analog of forskolin but not in the presence of 9,11-dideoxyforskolin (which does not activate adenylyl cyclase); 3) the potentiation by forskolin is not unique to S49 cells but is also observed in BC3H1 smooth muscle-derived cells; 4) stimulation of cAMP accumulation by beta-blockers with ISA occurs in S49 cells in three additional settings that do not involve the use of forskolin, after pretreatment with pertussis toxin to inactivate the inhibitory guanine nucleotide binding protein, after pretreatment with [D-Trp8]-somatostatin to sensitize adenylyl cyclase, and using a radioimmunoassay to quantitate levels of cellular cAMP. We conclude that beta AR antagonists with ISA can weakly stimulate intracellular cAMP accumulation, but this stimulation is not easily detected. Elevation of cAMP levels may account for the agonistic effects of these drugs or, at least provides a measure of stimulatory guanine nucleotide-binding protein activation by these compounds.

Adenylate Cyclase Toxin↗
PubMed1990

Nonadrenergic [3H]idazoxan binding sites are physically distinct from alpha 2-adrenergic receptors.

We have recently demonstrated that the alpha 2-adrenergic radioligand [3H]idazoxan also labels additional sites that do not recognize catecholamines but bind with high affinity several chemically distinct drugs previously assumed to be highly selective for alpha 2-adrenergic receptors [Mol. Pharmacol. 35:324-330 (1989)]. We now have used three approaches to distinguish the nonadrenergic [3H]idazoxan sites from alpha 2-adrenergic receptors. (a) No nonadrenergic [3H]idazoxan binding sites were found in COS-7 cells transfected with the genes for the two known alpha 2-adrenergic receptor subtypes. (b) The ratio of alpha 2-adrenergic and nonadrenergic [3H]idazoxan sites in human platelet membranes varied considerably between various donors. (c) Highly purified platelet plasma membranes were enriched for alpha 2-adrenergic receptors but did not contain any nonadrenergic [3H]idazoxan binding sites. We conclude that the nonadrenergic [3H]idazoxan binding sites are not co-expressed with alpha 2-adrenergic receptors and at least in human platelets may be located in an intracellular compartment.

Animals↗
PubMed1989

Structure and function of alpha-adrenergic receptors.

Alpha 1- and alpha 2-adrenergic receptors are the initial recognition sites on a wide variety of catecholamine-responsive target cells. This article addresses several major questions related to subtypes, structure, signal transduction mechanisms, and regulation of alpha 1- and alpha 2-adrenergic receptors. The application of biochemical and cell and molecular biologic techniques has provided many new insights regarding alpha-adrenergic receptors. Two (and perhaps three) distinct alpha 2-adrenergic receptor subtypes have been identified, and subtypes may exist for alpha 1-adrenergic receptors as well. These multiple subtypes imply much greater diversity among alpha-adrenergic receptors than among beta-adrenergic receptors. Alpha-adrenergic receptors are membrane glycoproteins with several common structural features (including seven membrane-spanning domains with extracellular amino terminus and intracellular carboxyl terminus) that are shared with other types of membrane receptors linked to guanine nucleotide-binding regulatory (G) proteins. These G proteins appear to link alpha-adrenergic receptors to multiple effector systems, including enzymes such as adenylate cyclase and phospholipases, and ion channels. The receptors themselves are dynamic entities, the number of which is regulated as a consequence of a poorly understood life cycle. Although unproven, it seems likely that several important clinical disorders represent alterations in alpha-adrenergic receptors themselves or in the G proteins or effector systems to which these receptors couple. New tools for studying receptor structure and function should help clarify the numerous, inadequately understood issues regarding alpha-adrenergic receptors and their possible alteration in disease states.

Affinity Labels↗
PubMed1989

Quantitation of a guanine nucleotide binding regulatory protein by an enzyme-linked immunosorbent competition assay.

We have developed a new method using a competitive enzyme-linked immunosorbent assay, ELISA, for the determination of levels of the stimulatory guanine nucleotide binding protein, Gs, in membrane extracts. The method is based on the use of antipeptide antibodies generated in rabbits directed against amino acids 28-42 in the alpha-subunit, alpha s, of Gs. The peptide is utilized as the stationary phase in the ELISA and anti-alpha s antibody bound to the microtiter plate is assessed by a peroxidase-coupled anti-rabbit immunoglobulin G antibody that yields detectable color development at 490 nm. Gs purified from rabbit liver is utilized as the standard to assess the ability of Gs present in cholate extracts of membrane samples to compete with bound peptides for primary antibody. This assay provides a direct means to quantify changes in levels of native Gs in membranes and cells.

Animals↗
PubMed1989

Stimulation of beta-adrenergic receptors of S49 lymphoma cells redistributes the alpha subunit of the stimulatory G protein between cytosol and membranes.

The stimulatory guanine nucleotide-binding protein (Gs), which links cell-surface receptors to second-messenger effector systems, is assumed to be confined to plasma membranes. In the current studies we tested whether Gs redistributes within cells by treating S49 lymphoma cells with the beta-adrenergic agonist isoproterenol, then separating cytosol and crude membrane fractions (defined as pellet and supernatant, respectively, after centrifugation for 1 hr at 150,000 x g), and assaying fractions for the alpha subunit of Gs (alpha s) using a competitive ELISA and reconstitution techniques. Under basal conditions, a small (10%) pool of alpha s was identified in supernatant fractions of S49 cells. The size of this pool decreased in the first 15 min after agonist treatment of cells. This decrease was blocked by a beta-adrenergic receptor antagonist and did not occur in an S49 variant, UNC, which lacks functional interaction between receptors and Gs. The size of the alpha s pool in supernatant fractions increased to almost 50% of total cellular alpha s during a 1-hr incubation of cells with isoproterenol. Before isoproterenol treatment only the competitive ELISA was sensitive enough to detect cytosolic alpha s, whereas at later time points (greater than or equal to 30 min) the presence of alpha s in the cytosol was confirmed by both immunoblotting and by reconstitution of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in Gs-deficient membranes derived from cyc-S49 cells. In contrast to membrane alpha s, cytosolic alpha s did not require activation (e.g., by AlF4-) in the reconstitution assay to stimulate adenylyl cyclase. Use of an antibody that selectively recognizes monomeric dissociated alpha s, but not heterotrimeric alpha s, indicated that cytosolic alpha s is monomeric. These data indicate that alpha s is not exclusively localized to the plasma membrane and that agonist treatment redistributes this protein within target cells.

Adenylyl Cyclases↗
PubMed1989

Renal alpha-adrenergic receptor alterations: a cause of essential hypertension?

Based on a review of literature in various fields of research related to hypertension, we develop a new working hypothesis on the pathophysiology of genetically determined increases in blood pressure. According to our hypothesis, the primary defect is located in the kidneys. Renal alpha-adrenergic receptor density is increased in the early stages of the disease, before increases in blood pressure occur. Most renal alpha-adrenergic receptors are located in the proximal tubules and enhance Na+ reabsorption. A genetically determined increase of alpha 1- or alpha 2- or of both alpha-adrenergic receptor subtypes would impair Na+ excretion and, together with increased Na+ intake, would lead to positive Na+ balance. Subsequently, various mechanisms would be activated to restore a neutral Na+ balance, including the secretion of a natriuretic factor that inhibits Na+/K+-ATPase. Inhibition of Na+/K+-ATPase in extrarenal tissues would increase the intracellular concentration of Na+ and, via Na+/Ca2+ exchange, of Ca2+. Elevated intracellular Ca2+ would enhance vascular smooth muscle contractility and neuronal transmitter release, thereby leading to vasoconstriction and to increases in blood pressure. We thus hypothesize that hypertension is a homeostatic response designed to protect blood volume from a genetically determined renal alpha-adrenergic receptor-mediated increase in Na+ retention.

Humans↗