A sensitive radioenzymatic assay for catechol drugs

Durrett, L R; Ziegler, Michael G.

doi:10.1002/jnr.490050613

Cited by 96 publications

(25 citation statements)

References 19 publications

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“…Blood samples were obtained from animals in the basal state 2 wk after initial thoracotomy, and again just before killing, after CHF was present. Levels of epinephrine and norepinephrine in plasma and myocardium were determined using a sensitive radioenzymatic assay previously described (30), and data are expressed as catecholamine per milligram wet weight of tissue or per milliliter plasma.…”

Section: Methodsmentioning

confidence: 99%

Downregulation of cardiac guanosine 5'-triphosphate-binding proteins in right atrium and left ventricle in pacing-induced congestive heart failure.

Urasawa¹,

Helmer²,

Hammond³

1993

J. Clin. Invest.

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The extent to which congestive heart failure (CHF) is dependent upon increased levels of the cardiac inhibitory GTP-binding protein (Gi), and the impact of CHF on the cardiac stimulatory GTP-binding protein (Gs) and mechanisms by which Gs may change remain unexplored. We have addressed these unsettled issues using pacing-induced CHF in pigs to examine physiological, biochemical, and molecular features of the right atrium (RA) and left ventricle (LV). CHF was associated with an 85±20% decrease in LV segment shortening (P < 0.001) and a 3.5-fold increase (P = 0.006) in the ED5, for isoproterenol-stimulated heart rate responsiveness. Myocardialfg-adrenergic receptor number was decreased 54% in RA (P = 0.004) and 57% in LV (P < 0.001), and multiple measures of adenylyl cyclase activity were depressed 49±8% in RA (P < 0.005), and 44±9% in LV (P < 0.001). Quantitative immunoblotting established that Gi and Gs were decreased in RA (Gi: 59% reduction; P < 0.0001; Gs: 28% reduction; P < 0.007) and LV (Gi: 35% reduction; P < 0.008; Gs: 28% reduction; P < 0.01 ) after onset of CHF. Reduced levels of Gi and Gs were confirmed by ADP ribosylation studies, and diminished function of Gs was established in reconstitution studies. Steady state levels for Gsa mRNA were increased in RA and unchanged in LV, and significantly more GSa was found in the supernatant (presumably cytosolic) fraction in RA and LV membrane homogenates after CHF, suggesting that increased Gs degradation, rather than decreased Gs synthesis, is the mechanism by which Gs is downregulated. We conclude that cardiac Gi content poorly predicts adrenergic responsiveness or contractile function, that decreased Gs is caused by increased degradation rather than decreased synthesis, and that alterations in fI-adrenergic receptors, adenylyl cyclase, and GTP-binding proteins are uniform in RA and LV in this model of congestive heart failure. (J. Clin. Invest. 1993. 91:939-949.)

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Section: Methodsmentioning

confidence: 99%

Downregulation of cardiac guanosine 5'-triphosphate-binding proteins in right atrium and left ventricle in pacing-induced congestive heart failure.

Urasawa¹,

Helmer²,

Hammond³

1993

J. Clin. Invest.

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“…All patients were receiving digitalis, diuretics, and at least one vasodilator (nitrates in 12, hydralazine in two, and angiotensin converting enzyme inhibitors in seven patients). All cardiac medications were withheld for [12][13][14][15][16][17][18][19][20][21][22][23][24] hours before the study. Eight healthy men (age range, 32-76 years; mean, 61±5 years) who had no history of cardiac impairment served as control subjects.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 1990

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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 El-stimulated cyclic AMP (cAMP) accumulation in MNL was similar in control subjects and patients. To detect more subtle alterations of the cAMPgenerating system, we incubated anticoagulated blood with 250-400 ng/ml pertussis toxin for 4 hours at 370 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/107 cells, p<0.025) in MNL of CHF patients. This enhancement was most pronounced in the most severely ill patients (New York Heart Association class lY) and correlated with plasma norepinephrine levels, another marker of CHF severity (r=0.798, n = 11, p <0.01). Isoproterenol-stimulated cAMP generation, which was much weaker than that produced by prostaglandin E1, was enhanced only in patients in class IV. We conclude that despite apparently unaltered amounts of subunits identifiable by ADP ribosylation with pertussis toxin, MNL of CHF patients appear to have enhanced functional activity (i.e., inhibition of cAMP formation) by these substrates. Thus, incubation of MNL with pertussis toxin reveals a tonic inhibition of cAMP generation in cells of CHF patients. The results suggest that incubation of whole blood with pertussis toxin and subsequent assessment of cAMP accumulation in MNL provide a useful way to examine G protein function in patients with cardiovascular disease. (Circulation 1990;81:1198-1204 I notropic responsiveness is markedly reduced in the hearts of experimental animals and patients suffering from congestive heart failure (CHF). A reduced density of receptors for catecholamines (,3-adrenergic)

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“…For short timecourse experiments (< 120 min) male Hartley guinea pigs (400-500 g, 4-6 wk, Simonsen Co.) received either (-)-isoproterenol (0.05-0.2 mg/kg in 10 mM HCl) or (13). The heart was removed and perfused with ice-cold 50 mM Tris-HCI (pH 7.4), and the left ventricle was trimmed and frozen at -70'C.…”

Section: Methodsmentioning

confidence: 99%

In vivo regulation of beta-adrenergic receptors on mononuclear leukocytes and heart. Assessment of receptor compartmentation after agonist infusion and acute aortic constriction in guinea pigs.

Maisel¹,

Carter²,

Insel³

et al. 1988

J. Clin. Invest.

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In animals injected with a bolus of isoproterenol, fi-adrenergic receptors in both mononuclear leukocytes (MNL) and heart were sequestered away from the cell surface, and the time course (0-120 min) and dose-response patterns were similar in the two tissues. In guinea pigs given a constant infusion of isoproterenol, 0.15 mg/(kg * h), down-regulation of total receptor number occurred more quickly and to a greater extent in the MNL than in the heart.We also compared receptor sequestration after aortic constriction-induced acute heart failure. Negligible sequestration (9%) of j-adrenergic receptors occurred in the MNL of animals treated in this manner, whereas the number of receptors in the sarcolemmal fraction decreased 61%. This selective sequestration of cardiac receptors may result from the action of high concentrations of norepinephrine (which is selective for ,81 over 2 receptors) present at sympathetic nerve-cardiac cell synapses.We conclude that although receptor redistribution occurs similarly in MNL and heart in response to a circulating nonselective agonist, ,-adrenergic receptor redistribution may occur selectively in the heart in response to such stimuli as aortic constriction-induced acute heart failure that activate the sympathetic nervous system.

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A sensitive radioenzymatic assay for catechol drugs

Cited by 96 publications

References 19 publications

Downregulation of cardiac guanosine 5'-triphosphate-binding proteins in right atrium and left ventricle in pacing-induced congestive heart failure.

Downregulation of cardiac guanosine 5'-triphosphate-binding proteins in right atrium and left ventricle in pacing-induced congestive heart failure.

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

In vivo regulation of beta-adrenergic receptors on mononuclear leukocytes and heart. Assessment of receptor compartmentation after agonist infusion and acute aortic constriction in guinea pigs.

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