William B. Weglicki

Enhanced lysosomal phospholipid degradation and lysophospholipid production due to free radicals.

To pursue the hypothesis that peroxidized lipids may become preferred substrates for endogenous phospholipases, we injured hepatic lysosomes by adding an exogenous free radical generating system [dihydroxyfumurate + Fe3+-ADP]; this system rapidly lysed hepatic lysosomes at pH 6.0, with maximal changes at 30 min. The production of malondialdehyde [MDA] plateaued rapidly. At 20 min the degradation of phosphatidylethanolamine [PE] was greater than phosphatidylcholine [PC]: 52% and 17%, respectively. Sphingomyelin and neutral lipids did not decrease. Most interesting was the significant increase of lysoPC [329%; p less than 0.05] at 10 min and [381%; p less than 0.01] after 20 min of incubation; lysoPE production became significant [766%; p less than 0.05] at 20 min. This enhanced production of lysoPC and lysoPE suggests a new mechanism to increase the production of amphiphilic lipids during ischemia, that is active at moderately acid pH without added calcium.

Biochemical mechanisms for the inotropic effect of the cardiotonic drug milrinone.

Milrinone is a new inotropic agent for the treatment of refractory congestive heart failure. Our understanding of the mechanisms(s) of action of this synthetic cardiotonic drug is incomplete. We examined the effects of milrinone and the parent compound amrinone on sarcoplasmic reticulum function (45Ca-uptake and Ca-ATPase); radioligand binding to adenosine, beta-adrenergic, and cholinergic muscarinic receptors; cyclic AMP accumulation; and inhibition of various forms of cyclic AMP phosphodiesterases. Comparisons were made to observe how these effects correlate with the inotropic response of heart. Milrinone was shown to be a potent phosphodiesterase inhibitor that was 40 times more potent than amrinone and 10 times more potent at inhibiting the high-affinity (Km = 0.23 microM) form (Ki = 22 microM) than the low-affinity (Km = 140 microM) form (Ki = 225 microM) of cyclic AMP phosphodiesterase in heart. The potency of milrinone as a phosphodiesterase inhibitor was the same in the presence and absence of calcium. Concentrations of milrinone that increased cyclic AMP accumulation also produced positive inotropy. A comparison of milrinone with amrinone and methylxanthines revealed the order of potency to be isobutylmethylxanthine greater than milrinone greater than theophylline greater than caffeine greater than amrinone. Milrinone and amrinone had no effect on 45Ca-uptake or Ca-ATPase activity in myocyte sarcoplasmic reticulum. However, milrinone did bind weakly to adenosine receptors (KD = 466 microM) but not to cholinergic muscarinic or beta-adrenergic receptors. Also, in combination with isoproterenol high concentrations of milrinone blocked the negative inotropic response to the adenosine agonist phenylisopropyladenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of cyclic AMP-dependent protein kinase activity by the cardiotonic drugs amrinone and milrinone

Amrinone and milrinone are new cardiotonic drugs that have potent inotropic and vasodilatory properties. The mechanism of action of these agents is controversial, but the positive inotropic component is thought to be due to the inhibition of phosphodiesterase. Because amrinone and milrinone have been shown to be involved primarily in cyclic AMP-mediated processes, we examined the effect of these agents on cyclic AMP-dependent protein kinase. The results indicate that amrinone and milrinone inhibit cyclic AMP-dependent protein kinase activity by competing with ATP but not cyclic AMP binding sites. Dissociation constants (Ki) of amrinone and milrinone for ATP binding sites on protein kinase were calculated to be 100-300 microM and 842 microM, respectively. The phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) had no effect on protein kinase activity. Amrinone and milrinone inhibited the catalytic subunit of protein kinase to the same degree as the holo-enzyme by competitively inhibiting the binding of ATP. Amrinone and milrinone had no effect on phospholipid-sensitive, calcium-dependent protein kinase indicating that there may be differences in the ATP binding sites on these two protein kinases. Inhibition of cyclic AMP-dependent protein kinase by amrinone and milrinone occurs at concentrations higher than those used clinically. However, because amrinone and milrinone are lipophilic drugs, they may be useful tools for the investigation of protein kinase mediated reactions.

Abnormal electrical activity induced by H2O2 in isolated canine myocytes.

Free oxygen radicals may participate in a variety of pathological cardiac conditions which are associated with an increased incidence of arrhythmias. For example, adriamycin, an anti-cancer agent that undergoes redox-cycling and produces radicals, causes arrhythmias that restrict its therapeutic usefulness.1 The arrhythmias that accompany reperfusion of ischemic hearts are reduced by the presence of allopurinol2 or free radical scavengers3,4 and enhanced by Fe-ADP4. Such reports imply that radicals have a direct effect on the electrical activity of myocardial cells. Yet this indirect evidence is weak because it relies on the production of radicals during pathological conditions which simultaneously produce hypoxia, acidosis, hyperkalemia or other biochemical changes. Previously, we have described a distinct sequence of electrophysiological changes that occur in the presence of free-radical-generating systems consisting of 3 mM dyhydroxyfumaric acid or xanthine:xanthine oxidase.5,6 The changes induced by exogenous generating systems follow a reproducible pattern. They are not readily reversed by removal of the generating system but can be partially prevented by the addition of the free radical scavengers superoxide dismutase and catalase.6 The generating systems used previously should produce a spectrum of free radicals in our superfusion solutions, but they also require the addition of one or more compounds to the superfusate.

Characteristics of multiple forms of the acidic triacylglycerol lipase(s) of canine cardiac myocytes

Acidic lipase activity was extracted by digitonin treatment from particulate fractions prepared from isolated adult canine myocytes. Both methylumbelliferyloleate (MUO) and trioleoylglycerol were hydrolyzed with an apparent Km of 13 and 135 microM, respectively. The primary products of trioleoylglycerol lipolysis were oleic acid and 1,2-dioleoylglycerol. Hydrolysis of either MUO or triacylglycerol was stimulated in vitro by the addition of cardiolipin or Triton X-100. Triton X-100 alone was sufficient for maximal stimulation of MUO hydrolysis, but cardiolipin further stimulated triacylglycerol lipolysis in the presence of an optimal concentration of Triton X-100. Cardiolipin increased the Vmax without altering the Km for trioleoylglycerol. Upon gel filtration chromatography the 4-methylumbelliferyloleate and triacylglycerol lipase activities eluted in regions consistent with molecular weights of approx. 47 000 and 55 000, respectively. Chromatofocusing revealed predominantly one form of acidic 4-methylumbelliferyloleate hydrolase (pI approx. 6.3), whereas acidic triacylglycerol lipase activity eluted continuously in the pH gradient from 7.2 to 4.3 with no clearly predominant peak of activity. Two forms of both 4-methylumbelliferyloleate and triacylglycerol lipase were eluted from columns of carboxymethyl Bio-Gel at pH 5.7; one form of each lipase activity was not bound and another form of each lipase was eluted with 50-60 mM KCl. The non-bound forms of each lipase were indistinguishable from their respective carboxymethyl-bound forms on the basis of pH dependency or kinetically (similar Km). The non-bound and carboxymethyl-bound peaks of lipolytic activity differed in the ratios of 4-methylumbelliferyloleate hydrolase to triacylglycerol lipase activity. The results suggest that the cardiac myocyte contains multiple forms of acidic lipase, and that the catalytic units primarily responsible for the hydrolysis of methylumbelliferyl esters and triacylglycerols may not be identical.

Role of Tissue and Circulating Substance P in Cardiovascular Injury Associated with Mg-Deficiency

Hypomagnesemia is a common electrolyte deficiency found among hospitalized patients and is particularly prevalent in selected patient populations, such as alcoholics, diabetics, and those receiving diuretics and other magnesium-wasting drugs. Clinical complications as a result of magnesium deficiency were documented in a recent prospective study in which hypomagnesemia, which was present at the time of admission of critically ill patients, was associated with a statistically significant higher mortality rate [1]. Magnesium deficiency has also been associated with adverse cardiovascular conditions, such as sudden death, ventricular and atrial arrhythmias, coronary spasm, and cardiomyopathies. In one study of patients with heart disease, 45% of patients with myocardial infarction were reported to be hypomagnesemic [2].

Perturbations of sarcolemmal and microsomal enzymes by amphiphilic lipids and drugs

Sarcolemmal (SL) and microsomal (MC) membranes were prepared from adult canine cardiocytes. SL Na+, K+-ATPase (2.35 mumole/min per mg) was enriched 117-fold over the homogenate and MC rotenone-insensitive NADH cytochrome c reductase (RINCR) was enriched 41-fold. Preincubation of SL with 50 microM arachidonyl-CoA (20:4 CoA) stimulated Na+, K+-ATPase almost 2-fold; 250 microM 20:4 CoA inhibited the enzyme by 85%. However, RINCR was inhibited 80% by only 0.2 microM 20:4 CoA. Thus, each of these myocardial lipid-dependent enzymes showed a different sensitivity to perturbation by lipid amphiphiles. In further experiments, SL preincubated with 50 microM 20:4 CoA + 2.5 mM propranolol (which had no effect alone) exhibited a synergistic inhibition of the Na+, K+-ATPase: The enzymatic activity declined 8.5-fold when compared to sarcolemma treated with 50 microM 20:4 CoA alone. Thus, the presence of lipid amphiphiles may result in greater inhibition of the Na+, K+-ATPase when propranolol is present in the membrane.