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A Daugherty

Publications and source records attributed to A Daugherty.

69 records · Page 4Linked to original sources
PubMed1989

Non-invasive detection of protein metabolism in vivo by n.m.r. spectroscopy. Application of a novel 19F-containing residualizing label.

Protein residualizing labels facilitate localization of tissue sites of protein catabolism and the quantification of protein accumulation because of their prolonged intracellular retention of protein accumulation because of their prolonged intracellular retention times. Radioiodinated residualizing labels have been used to define the metabolism of a wide variety of proteins, but this has necessitated destructive analysis. Here we describe the implementation and validation of a novel 19F-containing residualizing label for protein, NN-dilactitol-3,5-bis(trifluoromethyl)benzylamine (DLBA), that permits the non-invasive assessment of protein accumulation and catabolism by n.m.r. spectroscopy in vivo. DLBA comprises a reporter molecule containing six equivalent 19F atoms. 19F is strongly n.m.r.-active, has 100% natural abundance, and is present in minimal background concentrations in soft tissues. We validated the use of DLBA as a protein-labelling compound by coupling to asialofetuin (ASF), a protein that is recognized exclusively by hepatic tissue via a saturable receptor-mediated process. Coupling of DLBA to ASF by reductive amination had no effect on the physiological receptor-mediated uptake of the protein in rat liver in vivo. The 19F-n.m.r. spectrum of DLBA exhibited a single peak that was subject to a small chemical-shift change and broadening after coupling to ASF. Pronase digestion of DLBA-ASF was performed to simulate intracellular degradation products, and resulted in a narrower set of resonances, with chemical shifts intermediate between those of uncoupled DLBA and DLBA-ASF. Intravenous administration of DLBA-ASF to rats followed by quantification of 19F in homogenates of liver tissue indicated that the half-life of residence time of degradation products from DLBA-ASF in liver was approx. 2 days. This intracellular half-life was comparable with that described for similar residualizing labels that contain radioiodide as a reporter. Similar results for the half-life of retention were obtained non-destructively and non-invasively in situ with the use of a whole-body radio-frequency antenna to acquire sequential spectra over 80 h after intravenous administration of DLBA-ASF. Quantification of these spectra demonstrated an initial accumulation of DLBA-ASF in liver followed by an expected gradual loss of 19F-labelled degradation products. The approach developed offers promise for the sequential and longitudinal characterization of metabolism of specific proteins in individual experimental animals and ultimately in human subjects.

Animals↗
PubMed1989

Probucol attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits.

1. Probucol was administered to rabbits fed a cholesterol-enriched (2% wt/wt) diet to determine potential anti-atherogenic effects in a preparation in which the disease process is due to elevated plasma concentrations of cholesterol ester-rich very low density lipoproteins (CER-VLDL). 2. Probucol was supplemented to the diet at 1% wt/wt which resulted in plasma concentrations rising steadily to 53 +/- 8 micrograms ml-1 after 14 days, with no significant changes during continued administration. Dietary consumption and body weight gains were comparable in the drug-treated and control groups during the observation period. 3. Probucol treatment did not significantly affect plasma concentrations of total cholesterol, unesterified cholesterol, triglycerides or phospholipids. 4. The concentration of CER-VLDL in plasma and its physicochemical characteristics were not significantly changed during administration of probucol. CER-VLDL from both control and probucol-treated animals was a potent stimulant of the augmentation of the intracellular incorporation of [3H]-oleate into cholesteryl-[3H]-oleate in cultured macrophages. 5. Despite the lack of effect of probucol on concentrations of plasma lipids and the cell interaction characteristics of CER-VLDL, administration of the drug markedly decreased the extent of intimal aortic surface area covered by grossly discernible atherosclerotic lesions from 55.6 +/- 11.8% to 11.6 +/- 1.9% in thoracic sections, and from 49.1 +/- 10.2% to 7.2 +/- 0.4% in abdominal sections. Furthermore, probucol treatment significantly reduced the deposition of total cholesterol in vascular tissue. 6. Probucol reduced the extent of aortic atherosclerosis produced by diet-induced hypercholesterolemia in rabbits. This reduction occurred in the absence of any significant change in the characteristics of plasma lipoproteins that were determined. These results indicate that either there is a role of oxidation in the disease process of this animal model of atherosclerosis or that probucol is acting via a presently undefined mechanism.

Animals↗
PubMed1989

Myocyte contracture, vascular resistance, and vascular permeability after global ischemia in isolated hearts from alloxan-induced diabetic rabbits.

Coronary vascular hemodynamics, albumin permeation, and myocyte contractility were assessed in isolated hearts from 6-mo alloxan-induced diabetic (ALX-D) rabbits during 3 h of reperfusion after 40 min of global no-flow ischemia. Residue-detection data, generated during the single passage of a bolus of 125I-labeled bovine serum albumin (125I-BSA) through the coronary vasculature, were used to estimate indices of vascular function, including the mean transit time of 125I-BSA, the fractional rate of intravascular clearance of 125I-BSA, and 125I-BSA permeation of coronary vessels. During reflow after ischemia in hearts from control rabbits, vascular resistance increased approximately three times that at baseline, left ventricular end-diastolic pressure (LVEDP) increased 8-10 times, and maximum +dP/dt recovered 0.4 times baseline, whereas the fractional rate of washout of intravascular 125I-BSA decreased to less than one-half of baseline values (was prolonged 2-fold), and albumin permeation and mean-transit time were increased 3 and 5 times baseline, respectively. In hearts from diabetic rabbits, vascular resistance was similar to the control group before ischemia but increased only one-third as much during reflow after ischemia. Increases in LVEDP during reflow were approximately 50% lower than controls, and +dP/dt recovered approximately 2.5 times more than in control hearts. 125I-BSA permeation in diabetics was similar to controls before ischemia, but during reflow increased 6 times (approximately 2 times controls). Washout of intravascular 125I-BSA was prolonged approximately 20% versus baseline during 3 h of reflow in hearts from diabetic rabbits. Thus, ALX-D in the rabbit delayed ischemia-reperfusion injury to myocytes and vascular smooth muscle cells while increasing vascular albumin permeation.

Animals↗
PubMed1988

Dependence of metabolic and structural heterogeneity of cholesterol ester-rich very low density lipoproteins on the duration of cholesterol feeding in rabbits.

Cholesterol ester-rich (CER) VLDL accumulate rapidly in the plasma of rabbits fed cholesterol-enriched diets. However, the major loci of enhanced synthesis of subfractions of CER-VLDL, their interaction with macrophages, and their relative contribution to atherogenesis have not yet been elucidated. To determine whether anabolism is hepatic or intestinal, subfractions of CER-VLDL were characterized at selected intervals from day 0 to 60 of cholesterol feeding. Rate zonal ultracentrifugation of CER-VLDL from rabbits fed cholesterol for 4 and 60 d demonstrated an early increase of the proportion of cholesterol carried in the intestinally-derived fraction (designated as Fx-I) of VLDL compared with that in hepatically-derived particles (Fx-H). Quantification by size exclusion HPLC also demonstrated that Fx-I was a prominent CER-VLDL component at day 4, while Fx-H particles became increasingly prominent with further cholesterol feeding. At both 4 and 60 d Fx-I stimulated cholesterol esterification and intracellular cholesterol content in macrophages more than the corresponding Fx-H did. In fact, Fx-H harvested at 4 d produced no cholesterol ester deposition. In contrast, Fx-H harvested at 60 d markedly stimulated cholesterol esterification and intracellular cholesterol content. Thus, both compositional and metabolic characteristics of CER-VLDL changed as a function of the duration cholesterol feeding.

Animals↗
PubMed1987

Inhibition of cholesteryl ester deposition in macrophages by calcium entry blockers: an effect dissociable from calcium entry blockade.

The effects of calcium entry blockers on stimulated cholesteryl [3H]-oleate deposition in cultured macrophages were characterized in order to elucidate mechanisms underlying possible antiatherosclerotic effects. Stimulation of intracellular cholesteryl [3H]-oleate deposition was initiated by incubation of macrophages with beta-very low density lipoproteins (beta-VLDL). Nifedipine (Class I) markedly reduced cholesteryl [3H]-oleate deposition at all concentrations tested. However, Bay K 8644, a dihydropyridine which is known to stimulate calcium entry, also reduced cholesteryl [3H]-oleate deposition with a similar potency to nifedipine. The effects of three Class II calcium entry blockers were evaluated: verapamil, methoxyverapamil, and diltiazem. Verapamil inhibited cholesteryl [3H]-oleate deposition in a concentration-dependent manner. Similarly, methoxyverapamil reduced cholesteryl [3H]-oleate deposition in a concentration-dependent manner although the reduction was not as great as that produced by verapamil. In contrast, diltiazem at any concentration tested did not inhibit cholesteryl [3H]-oleate deposition. Flunarizine (a Class III calcium entry blocker) produced a modest stimulation of cholesteryl [3H]-oleate deposition at the lowest concentration used (10(-7)M) but marked depression at the highest concentration (10(-5)M). The results indicate calcium entry blockers may exert protective effects on the development of atherosclerosis in animal models of diet-induced hyperlipidaemia by inhibiting intracellular cholesteryl ester deposition, but this effect may not be related to their calcium entry-blocking effects.

Animals↗
PubMed1987

Increased ischemia-reperfusion injury to the heart associated with short-term, diet-induced hypercholesterolemia in rabbits.

The effects of increased dietary cholesterol content on coronary vascular hemodynamics and endothelial cell transport function were assessed in isolated rabbit hearts during 3.5 hours of reperfusion after 30 minutes of global, no-flow ischemia. In control hearts from rabbits fed normal chow, perfusion pressure, left ventricular end-diastolic pressure, maximum +dP/dt, and the rate of intravascular clearance of radiolabelled albumin remained constant during 5 hours of continuous perfusion, while the mean transit time of radiolabelled albumin increased 1.6 X baseline. In ischemic hearts from rabbits fed normal chow, perfusion pressure increased 59% during reperfusion while left ventricular end-diastolic pressure and maximum +dP/dt returned toward control levels. The rate of intravascular clearance of radiolabelled albumin decreased 36%, and the mean transit time of albumin increased approximately 3 X baseline. Ischemia-reperfusion injury to the cardiac vasculature and musculature was markedly increased in hearts of rabbits fed chow supplemented with 2% cholesterol for 2-3 weeks compared to rabbits fed the same diet for a longer duration (5-16 weeks) or rabbits fed normal chow. Prior to ischemia, permeation of the coronary vasculature by albumin was increased twofold in rabbits fed cholesterol for 2-3 weeks while myocyte contractile function was normal relative to chow-fed controls or the group fed cholesterol for 5-16 weeks. These effects of acute cholesterol feeding precede occlusive atherosclerotic coronary artery disease and occur at plasma cholesterol concentrations one third of those in rabbits fed cholesterol for the longer duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1986

The role of catecholamines in the production of ischaemia-induced ventricular arrhythmias in the rat in vivo and in vitro.

The role of catecholamines in the production of ischaemia-induced ventricular arrhythmias in vivo and in vitro was studied using coronary artery ligation in the rat. Increases in plasma catecholamine concentrations during coronary artery ligation in pentobarbitone-anaesthetized animals were prevented by either acute adrenalectomy or chronic adrenal demedullation, but these procedures did not protect against the occurrence of ventricular arrhythmias. Thus plasma catecholamines were not obligatory mediators of arrhythmogenesis. Three protocols were used in vitro to evaluate the possible influence of intramyocardial release of noradrenaline, produced by the local conditions of ischaemia, on the production of ventricular arrhythmias. During coronary artery ligation in isolated perfused hearts, no enhanced output of 3H could be detected from [3H]-noradrenaline loaded hearts, even in the presence of inhibitors of catecholamine uptake processes, although washout of lactate from ischaemic regions was readily demonstrable. Both optical isomers of propranolol were equally effective in reducing the incidence of arrhythmias, implying a non-specific effect, since the (+)-isomer possesses considerably less beta-adrenoceptor blocking activity. The equipotency of optical isomers of propranolol combined with a lack of effect of atenolol suggested that arrhythmia production was not a consequence of beta-adrenoceptor stimulation. The alpha-adrenoceptor blockers phentolamine and prazosin, both exerted antiarrhythmic actions of similar potency, but phenoxybenzamine and trimazosin had no significant effects. An evaluation of the pharmacological properties of the alpha-adrenoceptor blockers showed that those drugs which had demonstrable local anaesthetic properties also exerted significant antiarrhythmic effects. No relationship was found between potency of alpha-adrenoceptor blockade and antiarrhythmic efficacy. The overall conclusion from these multifaceted approaches was that catecholamines were not necessary mediators of the early phase of ventricular arrhythmias in the rat.

Adrenal Medulla↗
PubMed1986

Effect of fatty acid on performance and lipid content of hearts from diabetic rabbits.

The relationship of external work to exogenous substrate supply and whole heart lipid content was studied in hearts isolated from 27 normal and 24 alloxan-diabetic rabbits. In addition, we evaluated hearts from seven rabbits after 4 days of untreated diabetes followed by 10 days of insulin therapy. Hearts were retrogradely perfused with erythrocyte-enriched modified Krebs-Henseleit media in which glucose, palmitate, and insulin were present in concentrations simulating diabetic plasma (13 mM, 0.4 mM, and 15 microU/ml, respectively). Diabetes was associated with a greater than 40% reduction in peak left ventricular systolic pressure, dP/dtmax, and left ventricular pressure-time index (P less than 0.01 for each). Perfusion of hearts from six diabetic animals with low concentrations of exogenous fatty acid (0.06 mM) normalized left ventricular (LV) performance. Insulin therapy also completely normalized LV performance. This improvement was associated with lower heart fatty acid and triacylglycerol content. These studies suggest that the myocardial dysfunction in hearts from rabbits with alloxan-induced diabetes of 2-wk duration may be, in part, due to enhanced sensitivity to deleterious effects of high exogenous levels of fatty acid.

Alloxan↗
PubMed1986

Metabolism of very low density lipoproteins after cessation of cholesterol feeding in rabbits. A factor potentially contributing to the slow regression of atheromatous plaques.

Aortic atheromatous plaques regress slowly in cholesterol-fed rabbits that have been returned to normal laboratory diet. To delineate metabolic factors potentially responsible for persistence of atherosclerosis under these conditions, the physical, chemical, and metabolic characteristics were determined for lipoproteins of d less than 1.006 g/ml; such lipoproteins are thought to be the major determinant of progression of atherosclerotic lesions in cholesterol-fed rabbits. At the time of return to a normal laboratory diet regimen after 3 mo of feeding with cholesterol-enriched laboratory diet, plasma cholesterol concentrations were 2,275 +/- 252 mg/dl, mostly attributable to cholesteryl ester-rich very low density lipoproteins (VLDL). On the hypercholesterolemic diet, fractional catabolic rates of plasma clearance of 125I-labeled VLDL were reduced (0.011 +/- 0.002 vs. 0.151 +/- 0.015 h-1), but the total VLDL catabolic rate was increased considerably (17.1 +/- 2.2 vs. less than 1.2 +/- 0.4 mg of protein/kg X d), because of the expansion of the endogenous pool of cholesteryl ester-rich VLDL. The total catabolic rate of VLDL was maintained above estimated control values (5.8 +/- 0.7 mg protein/kg X d) even 10 wk after return of the rabbits to a normal chow regimen, an effect attributable to continued high rates of cholesteryl ester-rich VLDL synthesis in liver. Accumulation of cholesteryl ester-rich VLDL into aortic tissue persisted at a high rate. Thus the persistence of aortic atheromatous lesions after cessation of cholesterol feeding was attributable in part to continued high rates of hepatic production of cholesteryl ester-rich VLDL and its persistent delivery into the aortic wall.

Animals↗
PubMed1985

Loci of catabolism of beta-very low density lipoprotein in vivo delineated with a residualizing label, 125I-dilactitol tyramine.

beta-Very low density lipoprotein (beta-VLDL) may be a major atherogenic lipoprotein, and knowledge of the sites of its catabolism should facilitate elucidation of mechanisms important in the regulation of its plasma concentrations. In this study, catabolic sites of beta-VLDL have been delineated in normolipidemic rabbits with a novel, radioiodinated, residualizing label, 125I-dilactitol tyramine (125I-DLT). Comparative studies of beta-VLDL and low density lipoprotein catabolism were performed with 125I-DLT conjugated to each lipoprotein and with lipoproteins iodine-labeled conventionally. Conjugation did not alter size distributions or charge characteristics of lipoprotein particles. The overall processing (binding and degradation) of lipoproteins by cultured rabbit skin fibroblasts was not influenced by 125I-DLT derivatization, suggesting that attachment of the label did not influence cell receptor-lipoprotein interactions. Furthermore, although degradation products of 125I-lipoproteins leaked out of the cells and into the medium, the degradation products of 125I-DLT lipoproteins were retained by the cells. The principal catabolic site of beta-VLDL in normolipidemic rabbits was found to be the liver with 54 +/- 4% of injected 125I retained in this organ 24 h after injection of 125I-DLT-beta-VLDL. When catabolism was normalized to tissue weight, the liver and adrenals were found to be approximately equally active in the metabolism of beta-VLDL. In agreement with results of other studies with residualizing labels, the principal organ of catabolism of 125I-DLT-LDL in vivo was the liver. The adrenals were the most highly catabolizing organ when results were normalized for tissue weight. The quantitative differences observed in the tissue distributions of injected 125I-DLT-beta-VLDL and 125I-DLT-low density lipoprotein suggested that a significant proportion of beta-VLDL is removed by tissues before conversion to low density lipoprotein.

Animals↗
PubMed1985

Carbachol and dibutyryl cyclic GMP on the vulnerability to ventricular fibrillation in rat isolated hearts.

The hypothesis that elevation of intracellular guanosine 3':5' cyclic monophosphate (cyclic GMP) concentrations may increase electrical stability of the myocardium was examined by determination of ventricular fibrillation thresholds (VFT) on isolated perfused hearts of the rat. Hearts were paced to circumvent any complicating effects of bradycardia. Using this system, carbachol produced a concentration-related reduction in VFT. The reduction in VFT produced by carbachol was not significantly modified by a high concentration of atenolol (10(-5)M), indicating that the increased vulnerability to ventricular fibrillation was not an indirect consequence of catecholamine release from intramyocardial stores. Atropine (10(-6)M) blocked the carbachol-induced reduction in VFT. At the concentrations of carbachol used to reduce VFT, myocardial cyclic GMP concentrations were also elevated. The dibutyryl analogue of cyclic GMP (10(-4)M) mimicked the effect of carbachol in reducing VFT. Carbachol potentiated the adrenaline (3 X 10(-7)M)-induced reduction in VFT.

Animals↗
PubMed1985

Aortic accumulation and plasma clearance of beta-VLDL and HDL: effects of diet-induced hypercholesterolemia in rabbits.

To assess the role of beta-VLDL in diet-induced atherogenesis, the in vivo metabolism and aortic accumulation of 125I-labeled beta-VLDL were investigated in cholesterol-fed rabbits and chow-fed controls. 125I-labeled HDL and 125I-labeled albumin were studied for comparison. The fractional catabolic rate of 125I-labeled beta-VLDL was reduced in cholesterol-fed rabbits (0.011 vs 0.139 hr-1), but due to the high endogenous pool, the total beta-VLDL flux was very high (13.1 vs less than 1.1 mg/kg per 24 hr). These results suggest that elevated levels of beta-VLDL during cholesterol feeding were due to an enhanced rate of synthesis, a finding confirmed in hypercholesterolemic rabbits subjected to plasmapheresis. Following acute reduction of plasma cholesterol by plasmapheresis, the quantitative increases in beta-VLDL cholesterol concentrations (210 to 364 mg/dl) over the subsequent 24 hr were in agreement with the rise calculated from the plasma clearance kinetics of 125I-labeled beta-VLDL (378 mg/dl per 24 hr). Aortic accumulation of beta-VLDL in hypercholesterolemic rabbits was increased greater than 15-fold over controls. Accumulation was predominantly in the intimal atheromatous lesions. The fractional catabolic rate of 125I-labeled HDL was increased during cholesterol feeding (0.037 vs 0.021 hr-1). A decreased rate of synthesis appeared to be responsible for the markedly depleted plasma HDL. HDL accumulation within the aorta was attenuated greater than 9-fold in cholesterol-fed rabbits compared to those fed normal chow. Plasma kinetics and aortic accumulation of 125I-labeled albumin were similar in hypercholesterolemic and control rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Isolation of low density lipoprotein from atherosclerotic vascular tissue of Watanabe heritable hyperlipidemic rabbits.

Atherogenic properties of low density lipoproteins (LDL) in vivo may reflect modification of lipoproteins associated with endothelial translocation and exposure to extracellular matrix and interstitial fluid. To examine whether modifications of LDL occur in vivo, lipoproteins were isolated from plasma and vascular tissue of Watanabe heritable hyperlipidemic (WHHL) rabbits. LDL was extracted from vascular tissue (LDL-V) by homogenization in the presence of a sodium carbonate buffer. Control experiments demonstrated that modifications did not occur under the preparative conditions used to release LDL from tissue. LDL-V contained less esterified cholesterol, but more cholesterol esters, than did LDL from plasma (LDL-P). The diameters of both LDL-V and LDL-P followed gaussian distributions, but LDL-V particles were smaller (20.3 +/- 0.1 and 26.3 +/- 0.1 nm). Mild lipid peroxidation was evident in LDL-V. The sphingomyelin content was increased in LDL-V, with less phosphatidylcholine than in LDL-P. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that apolipoprotein B was depleted in LDL-V, but Western blot analyses identified lower molecular weight proteins antigenically related to apolipoprotein B. LDL-V markedly stimulated cholesterol esterification in mouse peritoneal macrophages and also in rabbit alveolar macrophages, a cell type that did not respond to acetylated LDL. LDL-V was not recognized by cultured rabbit skin fibroblasts. Thus, LDL isolated from atherosclerotic vascular tissue in vivo was modified in a fashion that could confer atherogenic properties reflected by augmentation of cholesterol esterification in macrophages in vitro.

Animals↗