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Biomedical subjects

R A Kelly

Publications and source records attributed to R A Kelly.

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

Cardiac glycosides and congestive heart failure.

Although the cardiac glycosides are universally acknowledged to be important agents in the drug therapy of advanced congestive heart failure (CHF), their role in the treatment of more moderate CHF, particularly in patients in sinus rhythm, remains controversial. Over the past decade, several randomized clinical trials have been undertaken to help clarify the appropriate use of the cardiac glycosides in these patients. Although the data are not conclusive, the available evidence indicates that digoxin is efficacious and relatively safe in patients with CHF whether given alone or in combination with vasodilators. Ongoing myocardial ischemia, hypokalemia and reduced drug clearance due to renal disease or drug interactions remain the clinical parameters most closely associated with digitalis toxicity. However, the recent introduction and widespread availability of a safe and rapidly effective antidote to digitalis preparations--Fab fragments of antidigoxin antibodies--offers the clinician a greater margin of safety in the use of the cardiac glycosides than has been available in the past.

Digitalis Glycosides↗
PubMed1990

Effects of acute and chronic uremia on active cation transport in rat myocardium.

As abnormalities of active cation transport could contribute to the genesis of uremic cardiomyopathy, we investigated myocardial sodium pump function in rats with acute renal failure (ARF) and with a model of experimental chronic renal failure (CRF) that has metabolic similarities to advanced chronic uremia in humans. CRF rats were hypertensive and had left ventricular hypertrophy (33% higher heart:body weight ratio; P less than 0.01) at four weeks compared to pair-fed sham-operated rats. Importantly, both ouabain- and furosemide-sensitive 86Rb uptake rates were unchanged in left ventricular myocardial slices from CRF, and the intracellular sodium concentration was not different from that of control rats even though skeletal muscle sodium was increased, as we found previously (J Clin Invest 81:1197, 1988). Insulin-stimulated, ouabain-sensitive 86Rb influx was also preserved. There also were no abnormalities in myocardium cation transport in rats with ARF. However, [3H]ouabain binding was decreased 45% in CRF rats (P less than 0.01); it was unchanged in acute uremia. Decreased ouabain binding in chronic uremia was due entirely to fewer low affinity [3H]ouabain binding sites (the binding affinity for ouabain was unaffected). We conclude that in chronic, (but not acute) renal failure, sodium pump number is reduced in myocardium but intracellular sodium is unchanged and active cation flux rates are maintained. These results emphasize that in rats with chronic uremia, intracellular sodium homeostasis is preserved in myocardium, despite the presence of marked abnormalities of active cation transport in skeletal muscle that are characteristic of chronic uremia.

Acute Kidney Injury↗
PubMed1990

In situ calibration of fura-2 and BCECF fluorescence in adult rat ventricular myocytes.

Quantitation of Ca+ and H+ activities within cells using presently available fluorescent probes is optimal when the fluorescence signal is calibrated in situ after each experiment. Fura-2 and 2',7'-bis(2-carboxy-ethyl)-5,6-carboxyfluoroscein (BCECF) are difficult to calibrate in freshly dissociated adult cardiac myocytes because calibration procedures produce cellular hypercontracture. In situ calibration was accomplished in rat ventricular cells by saturating fura-2 with La3+, an agent known to produce myocardial relaxation. Since fura-2 has different spectral properties when complexed with La3+ than with Ca2+, scaling factors were defined in vitro and then verified by experiments in cultured neonatal myocytes. In adult rat myocytes using the La3+ method, intracellular Ca2+ concentration ([Ca2+]i) was 131 +/- 47 nM (n = 14) in quiescent cells; diastolic [Ca2+]i and systolic [Ca2+]i in myocytes stimulated at 1 Hz were 140 +/- 56 and 1,088 +/- 211 nM (n = 5), respectively. BCECF fluorescence was calibrated in situ by a method that prevented cellular hypercontracture and reported a pH value of 7.10 +/- 0.10 in cells stimulated at 1.5 Hz. An additional advantage of both methods is that the buffers employed prevented large changes in the redox state of intracellular pyridine nucleotides, thus preventing a change in cellular autofluorescence during the calibration procedure.

Aging↗
PubMed1990

Efficacy and safety of perhexiline maleate in refractory angina. A double-blind placebo-controlled clinical trial of a novel antianginal agent.

Despite large gains in the medical and surgical treatment of angina pectoris in the past two decades, many patients are refractory to conventional medical therapy and are unsuitable for a first or, more commonly, repeat coronary revascularization procedure. We evaluated the efficacy of perhexiline maleate, a drug with an antianginal mechanism of action in humans that is as yet unknown, by using a randomized double-blind placebo-controlled crossover design in 17 patients with refractory angina who continued to receive maximal antianginal therapy, typically including nitrates, a beta-blocker, and a calcium channel antagonist. In view of perhexiline's potential for hepatic and neurological toxicity, plasma drug levels were monitored and maintained in the 150-600 ng/ml range. Sixty-three percent of patients were judged perhexiline responders by objective exercise testing criteria, as compared with 18% of patients on placebo (p less than 0.05). By blinded review of subjective measures of anginal frequency and severity, 65% of patients noted an improvement while on perhexiline, whereas no patient identified the placebo phase with improvement. Side effects observed in 29% of patients were minor and related to transient elevations of blood levels of more than 600 ng/ml; no patient suffered hemodynamic or cardiac conduction abnormalities attributable to perhexiline. With attention to the pharmacokinetics of perhexiline's elimination in individual patients, this novel antianginal agent seems to be safe and effective and deserves further evaluation in patients already receiving maximal antianginal therapy who are not candidates for revascularization procedures.

Aged↗
PubMed1990

Endothelin enhances the contractile responsiveness of adult rat ventricular myocytes to calcium by a pertussis toxin-sensitive pathway.

It has long been assumed that the primary influences regulating cardiac contractility are the extent of mechanical loading of muscle fibers and the activity of the autonomic nervous system. However, the vasoactive peptide endothelin, initially found in vascular endothelium, is among the most potent positively inotropic agents yet described in mammalian myocardium. In isolated adult rat ventricular cells, endothelin's action was slow in onset but very long lasting with an EC50 of 50 pM that approximates the reported KD of the peptide for its receptor in rat heart. When the calcium activity of the buffer superfusing isolated single fura-2-loaded myocytes paced at 1.5 Hz was varied from 0.1 to 0.9 mM [Ca2+]o, 100 pM endothelin increased contractile amplitude with no significant change in diastolic or systolic [Ca2+]i, thus appearing to sensitize the myofilaments to intracellular calcium. Pertussis toxin, or prior exposure to a beta-adrenergic agonist, reduced or abolished the increase in myocyte contractility induced by endothelin. This novel and potent pharmacologic action of endothelin points to the potential importance of local, paracrine factors, perhaps derived from microvascular endothelium or endocardium, in the control of the contractile function of the heart.

Animals↗
PubMed1989

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.

Animals↗
PubMed1989

Hemodialysis and red cell cation transport in uremia: role of membrane free fatty acids.

Active and facilitated cation transport in erythrocytes of uremic patients may be improved acutely by hemodialysis, although the mechanisms remain unknown. As nonesterified fatty acids (NEFA) can affect Na+ pump activity in vitro, changes in plasma and red cell membrane NEFA content following a single hemodialysis procedure were examined and compared with acute changes in erythrocyte cation flux rates in 34 hemodialysis patients. In nonsodium-loaded cells, small changes in Na+ pump flux with dialysis did correlate with changes in intracellular Na+ content (r = 0.59; N = 17; P less than 0.01). On average, neither maximal Na+ pump activity nor Na+/Li+ counter-transport flux improved with dialysis, but Na+/K+/Cl- cotransport rates rose 25% post-dialysis (P less than 0.02). Plasma NEFA levels rose 87% following hemodialysis but erythrocyte membrane NEFA content declined by 23% (P less than 0.001). Importantly, 24 of the 34 subjects studied had a decrease in erythrocyte membrane NEFA content of greater than 10%, and in these patients, the fall in membrane NEFA correlated with an increase in ouabain-sensitive Na+ efflux (r = 0.564; P less than 0.01). The effects of hemodialysis on both erythrocyte NEFA content and Na+ pump flux could be reproduced by incubating pre-dialysis cells in fatty acid-free albumin. We conclude that acute changes in membrane NEFA may modulate active cation transport in uremic erythrocytes.

Aged↗
PubMed1989

The search for the endogenous digitalis: an alternative hypothesis.

The universal presence of a binding site for cardiac glycosides on Na+-K+-ATPase has engendered speculation as to whether it also serves as a receptor for an endogenous digitalis-like hormone or autacoid. If such a hormone were to exist, it could play a role in sodium homeostasis and in the pathophysiology of primary hypertension and uremia. However, we believe that this hypothesis rests on unproven assumptions. Although typical of many toxins and drugs, binding to a single protein that acts as both its receptor and effector mechanism at the cell membrane, thereby directly affecting transmembrane ion flux, would be unusual for a hormone or autacoid. As an alternative hypothesis for the evolutionary conservation of the cardiac glycoside binding site, we suggest that its endogenous ligand may exist within the cell. After cotranslational insertion of the alpha- and beta-subunits into the membrane of the rough endoplasmic reticulum, Na+-K+-ATPase, like most integral membrane proteins, 1) must be targeted through a complex network of intracellular organelles to the correct plasmalemmal domain, 2) must be monitored for appropriate protein conformation and subunit assembly, and perhaps 3) could have its catalytic function regulated before insertion in the cell membrane. Because the lumina of the endoplasmic reticulum, Golgi, and other organelles and vesicles are topologically equivalent to the outside of the cell, all three functions could be subserved by an intraorganellar ligand for the cardiac glycoside binding site.

Animals↗
PubMed1988

Abnormal cation transport in uremia. Mechanisms in adipocytes and skeletal muscle from uremic rats.

The cause of the abnormal active cation transport in erythrocytes of some uremic patients is unknown. In isolated adipocytes and skeletal muscle from chronically uremic chronic renal failure rats, basal sodium pump activity was decreased by 36 and 30%, and intracellular sodium was increased by 90 and 50%, respectively, compared with pair-fed control rats; insulin-stimulated sodium pump activity was preserved in both tissues. Lower basal NaK-ATPase activity in adipocytes was due to a proportionate decline in [3H]ouabain binding, while in muscle, [3H]ouabain binding was not changed, indicating that the NaK-ATPase turnover rate was decreased. Normal muscle, but not normal adipocytes, acquired defective Na pump activity when incubated in uremic sera. Thus, the mechanism for defective active cation transport in CRF is multifactorial and tissue specific. Sodium-dependent amino acid transport in adipocytes closely paralleled diminished Na pump activity (r = 0.91), indicating the importance of this defect to abnormal cellular metabolism in uremia.

Adipose Tissue↗
PubMed1987

High-performance liquid chromatographic separation of femtomolar quantities of endogenous carboxylic acids, including arachidonic acid metabolites, as 4-bromomethyl-7-acetoxycoumarin derivatives.

A major limitation of high-performance liquid chromatographic techniques for measuring biologically active eicosanoids has been the inadequate sensitivity of most on-line detection systems. In addition, the availability of a technique suitable for measuring small quantities of non-esterified fatty acids (NEFAs) in plasma would allow longitudinal studies of plasma levels of these lipids in small animals. To improve the sensitivity of detection, the compounds with acyl groups containing carboxylic acids were derivatized with the highly fluorescent compound, 4-bromomethyl-7-acetoxycoumarin. All classes of NEFA and arachidonic acid metabolites, including the cyclooxygenase and lipoxygenase products, and hydroxy acid compounds could be derivatized with this reagent. The derivatized metabolites were separated with a reversed-phase high-performance liquid chromatographic system using a radial compression column and a gradient elution technique. Reproducible measurements of plasma NEFAs from as little as 5 microliters of plasma, and femtomolar concentrations of eicosanoids, could be detected using an on-line fluorescent spectrometer. This improvement in sensitivity should permit the quantification of all eicosanoids, including the leukotrienes, in biologic fluids and the longitudinal measurement of changes in plasma NEFA levels in small animals.

Animals↗
PubMed1987

Na+, K+, and BP homeostasis in man during furosemide: effects of prazosin and captopril.

Furosemide increases sodium (Na+) and potassium (K+) excretion but if dietary salt is provided, a compensatory reduction in Na+ and K+ excretion follows which restores neutral balances within 18 to 24 hours. This compensation is not interrupted by blockade of the renin-angiotensin-aldosterone system (RAA) alone with captopril. Since plasma norepinephrine concentration increases after furosemide and alpha 1 adrenoreceptors can mediate enhanced Na+ reabsorption, we administered prazosin (2 mg 6 hr-1) to six normal volunteers consuming a daily intake of 270 mmol of Na+ and 75 mmol of K+, and added captopril (25 mg 6 hr-1) for an additional day to block the RAA system concurrently. Furosemide (40 mg day-1) was given for the last four days. Prazosin given alone before the diuretic reduced (P less than 0.05) BP and plasma angiotensin II (AII) concentration and increased body weight and heart rate. However, when given with furosemide, neither prazosin nor prazosin with captopril modified the short-term natriuretic or kaliuretic responses to furosemide, or the ensuing compensatory reductions in Na+ and K+ excretion. Accordingly, cumulative balances for Na+ and K+ remained neutral over four days of diuretic administration. Neither drug altered the renal responsiveness to the diuretic which was assessed from the relationship between renal Na+ and K+ excretion and diuretic elimination. Although the BP was maintained when furosemide was given alone, when given with prazosin and captopril, the mean BP fell by 13 +/- 5 mm Hg (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Acidosis, not azotemia, stimulates branched-chain, amino acid catabolism in uremic rats.

To investigate branched-chain, amino acid metabolism (BCAA) in muscle in chronic renal failure (CRF), we studied rats with moderately severe uremia (PUN 110 approximately mg/dl) and spontaneous metabolic acidosis (bicarbonate, 19 +/- 1 mEq/liter). Plasma BCAA levels in CRF compared to pair-fed control rats were approximately 15% lower and muscle valine was 93 microM lower (P less than 0.05). BCAA metabolism was measured in incubated epitrochlearis muscles using L-[1-14C]valine or L-[1-14C]leucine in the presence and absence of insulin. BCAA decarboxylation was increased (P less than 0.05) and insulin-stimulated BCAA incorporation into protein was blunted (P less than 0.05) by CRF. Since we have found that metabolic acidosis, by itself, stimulates muscle branched-chain, ketoacid dehydrogenase activity, another group of CRF and control rats was given NaHCO3 which corrected the acidosis, but not the azotemia. BCAA decarboxylation in muscle was reduced in CRF rats given NaHCO3, and this was reflected in increased plasma and muscle BCAA concentrations. We conclude that in CRF, chronic metabolic acidosis stimulates BCAA decarboxylation in skeletal muscle and this could contribute to the reduced intra- and extracellular concentrations of BCAA. Correction of acidosis should be a goal of therapy in CRF, especially when dietary regimens restrict intake of BCAA.

Acidosis↗
PubMed1987

Branched-chain amino acid metabolism in rat muscle: abnormal regulation in acidosis.

Branched-chain amino acid (BCAA) metabolism is frequently abnormal in pathological conditions accompanied by chronic metabolic acidosis. To study how metabolic acidosis affects BCAA metabolism in muscle, rats were gavage fed a 14% protein diet with or without 4 mmol NH4Cl X 100 g body wt-1 X day-1. Epitrochlearis muscles were incubated with L-[1-14C]-valine and L-[1-14C]leucine, and rates of decarboxylation, net transamination, and incorporation into muscle protein were measured. Plasma and muscle BCAA levels were lower (P less than 0.05) in acidotic rats. Rates of valine and leucine decarboxylation and net transamination were higher (P less than 0.05) in muscles from acidotic rats; these differences were associated with a 79% increase in the total activity of branched-chain alpha-keto acid dehydrogenase and a 146% increase in the activated form of the enzyme. We conclude that acidosis affects the regulation of BCAA metabolism by enhancing flux through the transaminase and by directly stimulating oxidative catabolism through activation of branched-chain alpha-keto acid dehydrogenase.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1987

Plasma nonesterified fatty acids in the Dahl rat. Response to salt loading.

The link between dietary salt intake and the development of hypertension in the salt-sensitive Dahl strain of rats remains elusive. There is evidence that Dahl salt-sensitive rats (DS) produce less vasodilator and natriuretic prostaglandins in response to salt loading than do control salt-resistant rats (DR), although the reason for this blunted response is unknown. We examined the effects of chronic dietary salt loading on the plasma levels of nonesterified fatty acids in DS and DR. Animals were fed the same chow containing either 0.4% or 4% NaCl (wt/wt). At 12 weeks, 75 microliters of tail capillary blood was obtained from restrained, conscious rats, and principal nonesterified fatty acids were measured by high performance liquid chromatography. Total nonesterified fatty acids rose in the 15 DR on high salt diets compared with values in 11 rats eating low salt (0.57 +/- 0.05 vs 0.35 +/- 0.01 mM; p less than 0.001). The greatest changes occurred in levels of arachidonic acid (+287%) and in the arachidonic precursors, linoleic (+89%) and linolenic (+107%) acids. In marked contrast, there was no change in levels of plasma nonesterified fatty acids in DS fed 4% NaCl compared with DS fed 0.4% NaCl. These observations suggest that defective production of natriuretic and vasodilator prostaglandins by DS may be due in part to an inability to produce or release eicosanoid precursors from phospholipid stores in response to dietary salt.

Animals↗
PubMed1987

Endogenous cardiac glycosidelike compounds.

The possibility that endogenous inhibitors of the sodium pump exist and bind to the cardiac glycoside binding site on Na+,K+-adenosine triphosphatase (ATPase) has been a source of much controversy. Although numerous hormones and inorganic ions that modulate Na+,K+-ATPase activity have been described, most of these affect the sodium pump indirectly by varying the intracellular sodium concentration or by increasing the number of enzyme units. None of these endogenous compounds has been shown conclusively to modulate sodium pump activity by binding to the cardiac glycoside binding site on Na+,K+-ATPase. However, the near-universal presence of three high-affinity binding sites on the alpha-subunit of the enzyme has engendered much speculation that endogenous ligands for these receptors must exist. In addition, none of the hormones known to indirectly affect sodium pump activity in vivo has been shown to modulate Na+,K+-ATPase activity in response to extracellular volume expansion or to play a role in the pathogenesis of hypertension or chronic renal failure, conditions in which a circulating inhibitor of Na+,K+-ATPase has been implicated. This report presents a condensed history of the search for endogenous inhibitors of Na+,K+-ATPase and describes recent advances in the field. Despite progress in identifying and characterizing compounds that could affect Na+,K+-ATPase activity in vivo, definitive proof for the existence of endogenous ligands for the cardiac glycoside binding site remains elusive.

Animals↗
PubMed1987

Mechanisms for defects in muscle protein metabolism in rats with chronic uremia. Influence of metabolic acidosis.

Chronic renal failure (CRF) is associated with metabolic acidosis and abnormal muscle protein metabolism. As we have shown that acidosis by itself stimulates muscle protein degradation by a glucocorticoid-dependent mechanism, we assessed the contribution of acidosis to changes in muscle protein turnover in CRF. A stable model of uremia was achieved in partially nephrectomized rats (plasma urea nitrogen, 100-120 mg/dl, blood bicarbonate less than 21 meq/liter). CRF rats excreted 22% more nitrogen than pair-fed controls (P less than 0.005), so muscle protein synthesis and degradation were measured in perfused hindquarters. CRF rats had a 90% increase in net protein degradation (P less than 0.001); this was corrected by dietary bicarbonate. Correction of acidosis did not reduce the elevated corticosterone excretion rate of CRF rats, nor did it improve a second defect in muscle protein turnover, a 34% lower rate of insulin-stimulated protein synthesis. Thus, abnormal nitrogen production in CRF is due to accelerated muscle proteolysis caused by acidosis and an acidosis-independent inhibition of insulin-stimulated muscle protein synthesis.

Acidosis↗
PubMed1986

Identification of NaK-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids.

Elevated plasma levels of factors with cardiac glycoside-like activity have been implicated in the response to volume expansion in animals and in the pathogenesis of certain human diseases. We recently described four fractions (IR1, EI1, EI2, EI3) from normal human plasma that inhibit NaK-ATPase, displace ouabain from the enzyme, and exhibit digoxin-like immunoreactivity (Kelly, R. A., O'Hara, D. S., Canessa, M. L., Mitch, W. E., and Smith, T. W. (1985) J. Biol. Chem. 260, 11396-11405). In this report, we identify the active component of these plasma fractions as long-chain nonesterified fatty acids (NEFA) and lysophospholipids. These lipids were present in fractions EI1, EI2, and EI3 in quantities sufficient to account for all of the NaK-ATPase inhibitory activity. The digoxin-like immunoreactivity in fraction IR1 could be attributed to hydrocortisone and other endogenous steroids. To explore the nature of the lipid-NaK-ATPase interactions, we examined the effects of various ATP or sodium concentrations on the NaK-ATPase activity measured in the presence of NEFA. Varying sodium did not affect the inhibition of NaK-ATPase by linoleic acid. At less than 0.15 mM ATP, linoleic acid stimulated NaK-ATPase, but at higher ATP concentrations, the enzyme was progressively inhibited. In summary, NEFA and lysophospholipids, at levels similar to those occurring in human plasma, may account for all of the NaK-ATPase inhibitory activity observed in human plasma fractions. These lipids probably do not directly regulate NaK-ATPase in vivo under normal physiologic conditions, but may alter the sodium pump in disease states characterized by abnormalities in lipid metabolism or plasma protein binding.

Animals↗