Jocelyn W. Dow

A possible protective role for sulphydryl compounds in acute alcoholic liver injury

Abstract The role of various sulphydryl compounds in protective mechanisms against acute ethanol toxicity has been investigated in the mouse. Intraperitoneal administration of varying doses of ethanol (0–6 g/kg as 20 per cent, w/v solutions) produced a linear dose response reduction in hepatic reduced glutathione (GSH) levels. The time scale of this effect suggested that GSH depletion occurred as a consequence of hepatic damage rather than contributing towards it. It was also demonstrated that the sulphydryl compounds β-mercaptoethylamine-HCl, cysteine and methionine significantly increased the survival rate of mice given a lethal dose of ethanol.

Accumulation and salvage of adenosine and inosine by isolated mature cardiac myocytes

Reoxygenation of ischaemic, energy-depleted heart does not result in sufficiently rapid regeneration of normal adenine nucleotide concentrations for preservation of cardiac function and structure. Salvage of nucleoside as a mechanism for restoration of ATP in the post-ischaemic myocardium is limited by efflux of adenosine during ischaemia. Isolated cardiac myocytes have been used to establish the kinetics of uptake and salvage of adenosine and inosine, measuring the distribution of radioactive nucleoside incorporated into ATP, ADP and AMP. Maximum rates of catalysis of reactions on the salvage pathway, and of enzymes competing for substrates on the pathway, have been established in myocyte extracts. Myocytes have little capacity to salvage or catabolise inosine. Enzyme measurements indicate that salvage of adenosine should proceed at 7-8-times the rate exhibited by intact myocytes dependent upon extracellular adenosine as substrate. The data indicate that the rate of transport of adenosine is not determined by its metabolic utilization, but is the rate-limiting step in the salvage of adenosine.

Uptake of bepridil into isolated ventricular myocytes: Retention by actin

Isolated rat ventricular myocytes accumulate large amounts of bepridil intracellularly. The mode of transport is uncertain but uptake is of such a magnitude as to mask possible sarcolemmal binding sites. Bepridil is tightly bound within myocytes, there being only a minimal efflux into bepridil-free medium. Purified F-actin binds bepridil in excess of 2 moles/mole actin, sufficient to account for the uptake of bepridil by myocytes incubated in 10 microM drug. At higher bepridil concentrations the amount transported into myocytes suggests that the drug may be distributed between actin-bound and cytoplasmic pools. Bepridil may alter cardiac contractility by a direct influence on the contractile filaments.

Metabolism and salvage of adenine and hypoxanthine by myocytes isolated from mature rat heart.

Adenine and hypoxanthine can be utilised by cardiac muscle cells as substrates for the synthesis of ATP. A possible therapeutic advantage of these compounds as high-energy precursors is their lack of vasoactive properties. Myocytes isolated from mature rat heart have been used to establish in kinetic detail the capacity of the heart to incorporate adenine, hypoxanthine and ribose into cellular nucleotides. Maximum rates of catalysis by enzymes on the salvage pathways have been established. Whilst the rate of incorporation of adenine into the ATP pool appears to depend upon intracellular concentrations of adenine and phosphoribosylpyrophosphate, for hypoxanthine the pattern is more complex. Hypoxanthine is salvaged at a slow rate compared with adenine, and is incorporated into GTP and IMP as well as into adenine nucleotides. The rate of incorporation of hypoxanthine into both IMP and ATP is accelerated in myocytes incubated with ribose. However, the rate-limiting reaction appears to be that catalysed by adenylosuccinate synthetase, for the rate of ATP synthesis is not accelerated when hypoxanthine concentration is increased from 10 to 50 microM, while the rate of IMP synthesis is more than doubled. Adenine and hypoxanthine phosphoribosyl transferases are present in equal catalytic amounts, but rat cardiac myocytes have very little adenylosuccinate synthetase activity. Exogenous ribose is incorporated into adenine nucleotides in amounts equimolar with adenine or hypoxanthine.

Daunorubicin-induced myocardial failure: Reversal by digoxin of an inability to use ATP for contraction

Abstract Daunorubicin (DNR) is an anti-cancer drug limited by fatal cardiac complications in which rhythm disturbances precede ventricular failure. We have used isolated working rat hearts to study the mechanism of myocardial DNR-induced work failure. DNR perfused at 19 to 38 μmol/l severely disturbed the aortic pressure profile and produced complete contractile failure within 90 min. It rapidly accumulates in isolated rat hearts to 2 μmol/g dry weight. Cardiac mechanical activity is not restored after removal of DNR from the perfusate. Nor is bound DNR eluted by DNR-free perfusion. DNR produces irreversible cardiac arrest with paradoxical conservation of ATP and creatine phosphate. Thus, DNR-induced work failure is associated with an inability to use ATP. Routes of ATP production appear functional. Normal production of lactate during perfusion indicates conservation of oxidative metabolism. At the end of the experimental period DNR-inhibited cells contain more lactate than controls. Myocardial fluid distribution is disturbed only at high perfusion concentrations of DNR. Total protection against DNR-induced myocardial work failure is provided by prior digitalization of the donor rats.

Amino acid transport and protein synthesis in energetically-stable calcium-tolerant isolated cardiac myocytes

Myocytes isolated by enzymic dispersion from adult rat ventricular tissue are shown to be energetically stable in the presence of 0.5 mM CaCl2. ATP and ADP content and rates of lactate production are comparable with those of intact myocardial tissue and consistent with these cells being tightly coupled. Addition of 2,4-dinitrophenol precipitates rapid changes in adenine nucleotide concentrations and a 10-fold increase in lactate production. Cardiac myocytes selectively transport neutral amino acids of the A and L classes. Transport of the amino acid analogue alpha-aminoisobutyric acid is an active, temperature-dependent and insulin-sensitive process. The apparent Km for alpha-aminoisobutyric acid transport is similar to that determined for embryonic cardiac cells. Mature myocytes incorporate labelled amino acids into cytoplasmic proteins with molecular weights ranging from 10 000 to 150 000. Newly synthesised protein is metabolically stable. The data establishes calcium-tolerant myocytes as an experimental system offering many advantages over whole hearts for short- and long-term studies of protein synthesis and catabolism.

Adenine nucleotide synthesis de novo in mature rat cardiac myocytes

Inadequate oxygenation of cardiac muscle leads to rapid loss of high energy compounds essential for contractile function. ATP can be regenerated by synthesis de novo, a route operating at a relatively slow rate in the heart. Myocytes isolated from mature rat heart have been used to measure the rate of ATP synthesis de novo from both [14C]glycine and [14C]ribose. Incorporation of glycine into ATP is accelerated 10-fold in the presence of 1 mM ribose. Myocytes also accumulate both precursors into IMP and four other metabolites on the de novo synthesis pathway. These metabolites represent 80% of the glycine entering the pathway. The potential of de novo synthesis for restoration of adenine nucleotides appears to be limited by the rates of early reactions, adenylosuccinate synthetase being only one of the enzymes operating at a sufficiently slow rate to make this pathway an inherently weak route for the restoration of normal energy status in post-ischemic myocardium. Interventions are being sought to alleviate these apparent metabolic delays.

Characterization of digoxin binding and daunorubicin uptake by isolated mature rat cardiac myocytes

Myocytes isolated from ventricular muscle of mature rat heart have been used for characterization of digoxin binding and to establish whether a relationship exists between digoxin binding and uptake of daunorubicin. High- and low-affinity digoxin binding sites have been identified; respectively, 0.9 +/- 0.1 X 10(7) sites/myocyte, Kd 70-77 nM and 7 +/- 2 X 10(7) sites/myocyte, Kd 1.4-1.7 microM. Myocytes accumulate daunorubicin to an intracellular concentration 30-40 times that in the medium. We find no evidence that saturation of digoxin binding sites alters daunorubicin uptake or that daunorubicin influences binding of digoxin. Alteration of sarcolemmal membrane properties is demonstrated by inhibition of amino acid transport reflected in protein synthesis rates. Calmodulin activation of phosphodiesterase appears insensitive to daunorubicin.

Features of cardiac myocytes in culture: characterisation of the failing cell

Current problems in cardiac surgery have led us to study events at the level of the single cardiac cell. At present those patients with the best functional hearts are selected for surgery. The range of patients could usefully be extended either if we could protect a high risk system, where spontaneous myocardial work is unlikely to be resumed after surgery, or if we knew how to prepare the poorer prognosis patients biochemically for successful surgery. Secondly, increasing use of successful organ grafts requires the longest maintenance interval between excision and implant of the donor heart. Optimum conditions for organ graft survival have yet to be established.