Jane McHowat

Cellular uncoupling induced by accumulation of long-chain acylcarnitine during ischemia.

Long-chain acylcarnitines (LCACs) increase rapidly within minutes after the onset of ischemia in vivo or hypoxia in vitro and produce a time-dependent reversible reduction in gap junctional conductance in isolated myocyte pairs. The present study was performed to assess whether LCACs contribute to cellular uncoupling in response to ischemia in isolated blood-perfused rabbit papillary muscles by use of simultaneous measurements of transmembrane action potentials, extracellular electrograms, extracellular K+, and tissue LCACs and ATP. LCACs increased threefold in response to 20 minutes of no-flow ischemia from 127 +/- 5 to 397 +/- 113 pmol/mg protein (P < .01), concomitant with the onset of cellular uncoupling, extracellular K+ accumulation, and a marked reduction in conduction velocity and action potential duration. To assess whether inhibition of the accumulation of LCACs modified the electrophysiological alterations during ischemia, muscles were pretreated with either sodium 2-(5-(4-chlorophenyl)-pentyl)-oxirane-2-carboxylate (POCA, 10 mumol/L) or oxfenicine (100 mumol/L), inhibitors of carnitine acyltransferase I. Both POCA and oxfenicine completely prevented the increase in LCACs even with 40 minutes of ischemia (138 +/- 37 and 56 +/- 4 pmol/mg protein, respectively), associated with a marked delay in the onset and progression of cellular uncoupling and ischemic contracture. Although POCA and oxfenicine did not affect either the initial early rise in extracellular K+ or the initial fall in conduction velocity, both agents markedly delayed the secondary rise in extracellular K+ as well as the secondary fall in conduction velocity, independent of the level of tissue ATP. Thus, LCACs accumulate during myocardial ischemia and contribute substantially to the initiation of cell-to-cell uncoupling. Inhibition of carnitine acyltransferase I and prevention of the increase in LCACs markedly delays cellular uncoupling and development of ischemic contracture in response to ischemia.

Neural Dysfunction and Metabolic Imbalances in Diabetic Rats: Prevention by Acetyl-L-Carnitine

The rationale for these experiments is that administration of L-carnitine and/or short-chain acylcarnitines attenuates myocardial dysfunction 1) in hearts from diabetic animals (in which L-carnitine levels are decreased); 2) induced by ischemia-reperfusion in hearts from nondiabetic animals; and 3) in nondiabetic humans with ischemic heart disease. The objective of these studies was to investigate whether imbalances in carnitine metabolism play a role in the pathogenesis of diabetic peripheral neuropathy. The major findings in rats with streptozotocin-induced diabetes of 4–6 weeks duration were that 24-h urinary carnitine excretion was increased ∼ twofold and L-carnitine levels were decreased in plasma (46%) and sciatic nerve endoneurium (31%). These changes in carnitine levels/excretion were associated with decreased caudal nerve conduction velocity (10–15%) and sciatic nerve changes in Na+-K+-ATPase activity (decreased 50%), Mg2+-ATPase (decreased 65%), 1,2-diacyl-sn-glycerol (DAG) (decreased 40%), vascular albumin permeation (increased 60%), and blood flow (increased 65%). Treatment with acetyl-L-carnitine normalized plasma and endoneurial L-carnitine levels and prevented all of these metabolic and functional changes except the increased blood flow, which was unaffected, and the reduction in DAG, which decreased another 40%. In conclusion, these observations 1) demonstrate a link between imbalances in carnitine metabolism and several metabolic and functional abnormalities associated with diabetic polyneuropathy and 2) indicate that decreased sciatic nerve endoneurial ATPase activity (ouabain-sensitive and insensitive) in this model of diabetes is associated with decreased DAG.

Dissociation between cellular K+ loss, reduction in repolarization time, and tissue ATP levels during myocardial hypoxia and ischemia.

The mechanisms underlying the marked increase in [K+]o in response to ischemia are not fully understood. Accordingly, the present study was performed to assess the contribution of ATP-regulated K+ channels by using simultaneous measurements of cellular K+ efflux, [K+]o, transmembrane action potentials, and tissue ATP, ADP, phosphocreatine, and creatine content in a unique isolated, blood-perfused papillary muscle preparation during hypoxia compared with ischemia. During 15 minutes of hypoxic perfusion (PO2, 6.1 +/- 0.9 mm Hg) with normal [K+]o of 4.1 +/- 0.1 mM, action potential duration (APD) was not altered even though tissue ATP levels decreased markedly from 33.5 +/- 1.8 to 14.7 +/- 2.0 nmol.mg protein-1 (p < 0.01). Net cellular K+ efflux, based on measured differences of [K+] between the venous effluent and the perfusate, was 13.23 +/- 0.79 mumol.g wet wt-1 during hypoxia. In contrast, after 15 minutes of zero-flow ischemia, APD at 80% of repolarization (APD80) decreased by 47% from 171 +/- 5 to 92 +/- 5 msec (p < 0.01), but integrated net cellular K+ efflux over 15 minutes of ischemia was 8.4-fold less (1.57 +/- 0.13 mumol.g wet wt-1) than during hypoxia. Tissue ATP levels, however, decreased by only 35.2% to 21.7 +/- 2.1 nmol.mg protein-1, which was significantly less than that induced by 15 minutes of hypoxia. Perfusion with hypoxic blood containing high [K+]o of 10.3 +/- 0.3 mM resulted in APD shortening similar to that observed during ischemia. Cellular K+ loss, however, was inhibited markedly by high [K+]o perfusion (only 4.51 +/- 0.28 mumol.g wet wt-1). Pretreatment with glibenclamide (5 microM), a drug that has been reported to inhibit ATP-regulated K+ channels and accelerate glycolysis in normoxic tissue, partially inhibited cellular K+ efflux during hypoxic perfusion with normal [K+]o (7.35 +/- 0.71 versus 13.23 +/- 0.79 mumol.g wet wt-1, p < 0.01) but had no significant influence on repolarization time or tissue ATP levels. Although glibenclamide partially prevented action potential shortening induced by hypoxic perfusion in the presence of elevated [K+]o, the proportion of cellular K+ efflux reduced by glibenclamide was less (23%) than that observed with glibenclamide in hypoxic perfusion with normal [K+]o (44%).(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of gap junctional conductance by long-chain acylcarnitines and their preferential accumulation in junctional sarcolemma during hypoxia.

Electrophysiological and biochemical sequelae of myocardial ischemia occur within minutes of the onset of myocardial ischemia in vivo. Both conduction delay and conduction block occur rapidly within the same time interval as the accumulation of long-chain acylcarnitines. In the present study, double whole-cell voltage-clamp procedures were used to assess the influence of long-chain acylcarnitines on gap junctional conductance in isolated pairs of canine ventricular myocytes. Long-chain acylcarnitine (5 microM) decreased gap junctional conductance from 153 to 48 nS in a time-dependent and reversible manner. Although the amplitude of junctional current was reduced by 68%, the current continued to demonstrate a linear current-voltage relation. The extent of endogenous accumulation of long-chain acylcarnitines in junctional regions of the sarcolemma was assessed in isolated myocytes in which endogenous free, short-chain, and long-chain acylcarnitine pools had been equilibrated with [3H]carnitine. Under normoxic conditions, long-chain acylcarnitines were not detectable in junctional sarcolemma of myocytes as assessed using electron microscopic autoradiography. Exposure of myocytes to hypoxia (PO2, < 15 mm Hg) for 10 minutes resulted in the preferential accumulation of endogenous long-chain acylcarnitines in junctional sarcolemma (173 +/- 5 x 10(5) molecules/microns 3), a concentration that was sevenfold greater than that found in nonjunctional sarcolemma. Therefore, endogenous long-chain acylcarnitines accumulate preferentially in junctional regions of the sarcolemma during short intervals of hypoxia. Exogenously supplied long-chain acylcarnitines can markedly decrease cellular coupling in a reversible manner, suggesting that this amphiphile may contribute to the marked slowing in conduction velocity in the ischemic heart in vivo, not only by suppressing the rapid Na+ inward current directly, as has been shown previously, but also by decreasing cellular coupling.

Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes

Accelerated phospholipid catabolism occurs early after the onset of myocardial ischemia and is likely to be mediated by the activation of one or more phospholipases in ischemic tissue. We hypothesized that hypoxia increases phospholipase A2 (PLA2) activity in isolated ventricular myocytes, resulting in increased lysophospholipid and arachidonic acid production, contributing to arrhythmogenesis in ischemic heart disease. The majority of ventricular myocyte arachidonic acid was found in plasmalogen phospholipids. Hypoxia increased membrane-associated, Ca2+-independent, plasmalogen-selective PLA2 activity, resulting in increased arachidonic acid release and lysoplasmenylcholine production. Pretreatment with the specific Ca2+-independent PLA2 inhibitor bromoenol lactone blocked hypoxia-induced increases in PLA2 activity, arachidonic acid release, and lysoplasmenylcholine production. Lysoplasmenylcholine produced action potential derangements, including shortening of action potential duration, and induced early and delayed afterdepolarizations in normoxic myocytes. The electrophysiological alterations induced by lysoplasmenylcholine would likely contribute to the initiation of arrhythmogenesis in the ischemic heart.Accelerated phospholipid catabolism occurs early after the onset of myocardial ischemia and is likely to be mediated by the activation of one or more phospholipases in ischemic tissue. We hypothesized that hypoxia increases phospholipase A2(PLA2) activity in isolated ventricular myocytes, resulting in increased lysophospholipid and arachidonic acid production, contributing to arrhythmogenesis in ischemic heart disease. The majority of ventricular myocyte arachidonic acid was found in plasmalogen phospholipids. Hypoxia increased membrane-associated, Ca2+-independent, plasmalogen-selective PLA2activity, resulting in increased arachidonic acid release and lysoplasmenylcholine production. Pretreatment with the specific Ca2+-independent PLA2 inhibitor bromoenol lactone blocked hypoxia-induced increases in PLA2 activity, arachidonic acid release, and lysoplasmenylcholine production. Lysoplasmenylcholine produced action potential derangements, including shortening of action potential duration, and induced early and delayed afterdepolarizations in normoxic myocytes. The electrophysiological alterations induced by lysoplasmenylcholine would likely contribute to the initiation of arrhythmogenesis in the ischemic heart.

Genetic and pharmacologic evidence that calcium-independent phospholipase A2β regulates virus-induced inducible nitric-oxide synthase expression by macrophages

Recent evidence supports a regulatory role for the calcium-independent phospholipase A2 (iPLA2) in the antiviral response of inducible nitric-oxide synthase (iNOS) expression by macrophages. Because two mammalian isoforms of iPLA2 (iPLA2β and iPLA2 γ) have been cloned and characterized, the aim of this study was to identify the specific isoform(s) in macrophages that regulates the expression of iNOS in response to virus infection. Bromoenol lactone (BEL), a suicide substrate inhibitor of iPLA2, inhibits the activity of both isoforms at low micromolar concentrations. However, the R- and S-enantiomers of BEL display ∼10-fold greater potency for inhibition of the enzymatic activity of iPLA2γ and iPLA2β, respectively. In this study, we show that the iPLA2β-selective (S)-BEL inhibits encephalomyocarditis virus (EMCV)-induced iNOS expression, nitric oxide production, and iPLA2 enzymatic activity in macrophages in a concentration-related manner that closely resembles the inhibitory properties of racemic BEL. cAMP response element-binding protein (CREB) is one downstream target of iPLA2 that is required for the transcriptional activation of iNOS in response to virus infection, and consistent with the effects of BEL enantiomers on iNOS expression, (S)-BEL more effectively inhibits EMCV-induced CREB phosphorylation than (R)-BEL in macrophages. Using macrophages isolated from iPLA2β-null mice, virus infection fails to stimulate iNOS mRNA accumulation and protein expression, thus providing genetic evidence that iPLA2β is required for EMCV-induced iNOS expression. These findings provide evidence for a signaling role for iPLA2β in virus-induced iNOS expression by macrophages.

Gradient elution reversed-phase chromatographic isolation of individual glycerophospholipid molecular species.

We describe a gradient elution reversed-phase high-performance liquid chromatographic approach for isolation of individual glycerophospholipid molecular species which greatly improves resolution and reduces run time compared to isocratic techniques. Separations were optimized and elution order and retention time data established by synthesizing 37 different homogeneous phospholipids comprising the major alkylacyl, diacyl and plasmalogen molecular species in samples derived from mammalian sources. Empirical equations which predict the elution order of individual species were derived. The method was validated with the use of complex mixtures of choline and ethanolamine glycerophospholipid species from isolated rabbit cardiomyocytes and porcine endothelial cells.

Recent Insights Pertaining to Sarcolemmal Phospholipid Alterations Underlying Arrhythmogenesis in the Ischemic Heart

Sarcolemmal Phospholipid Alterations and Arrhythmogenesis. Myocardial ischemia in vivo is associated with dramatic electrophysiologic alterations that occur within minutes of cessation of coronary flow and are rapidly reversible with reperfusion. This suggests that subtle and reversible biochemical alterations within or near the sarcolemma may contribute to the electrophysiologic derangements. Our studies have concentrated on two amphipathic metabolites, long‐chain acylcarnitines and lysophosphatidylcholine (LPC). which have been shown to increase rapidly in ischemic tissue in vivo and to elicit electrophysiologic derangements in normoxic tissue in vitro. Incorporation of these amphiphiles into the sarcolemma at concentrations of 1 to 2 mole %, elicits profound electrophysiologic derangements analogous to those observed in ischemic myocardium in vivo. The pathophysiological effects of the accumulation of these amphiphites are thought to be mediated by alterations in the biophysical properties of the Sarcolemmal membrane, although there is a possibility of a direct effect upon ion channels. Inhibition of carnitine acyltransferase I (CAT‐I) in the ischemic cat heart was found to prevent the increase in long‐chain acylcarnitines and LPC and to significantly reduce the incidence of malignant arrhythmias including ventricular tachycardia and fibrillation. This review focuses on the electrophysiologic derangements that are observed during early ischemia and presents data supporting the concept that accumulation of these amphiphiles within the sarcolemma contributes to these changes. The potential contribution of these amphiphiles to the increases in extracellular potassium and intracellular calcium are examined. Finally, recent data pertaining to the accumulation of long‐chain acylcarnitines on cell‐to‐cell uncoupling are presented. In addition to the events reviewed here, there are many other alterations that occur during early myocardial ischemia, but the results from multiple studies over the past two decades indicate that the accumulation of these amphiphiles contributes importantly to arrhythmogenesis and that development of specific inhibitors of CAT‐I or phospholipase A may be a promising therapeutic strategy to attenuate the incidence of lethal arrhythmias associated with ischemic heart disease in man.

Thrombin activates a membrane-associated calcium-independent PLA2 in ventricular myocytes

Activation of phospholipase A2(PLA2) and accumulation of lysophosphatidylcholine contribute importantly to arrhythmogenesis during acute myocardial ischemia. We examined thrombin stimulation of PLA2 activity in isolated ventricular myocytes. Basal and thrombin-stimulated cardiac myocyte PLA2 activity demonstrated a distinct preference for sn-1 ether-linked phospholipids with arachidonate esterified at the sn-2 position. The majority of PLA2 activity was calcium independent and membrane associated. Thrombin stimulation of membrane-associated PLA2 occurs in a time- and concentration-dependent fashion. An increase in PLA2 activity was also observed using the synthetic peptide SFLLRNPNDKYEPF (the tethered ligand generated by thrombin cleavage of its receptor). Bromoenol lactone, a selective inhibitor of calcium-independent PLA2, completely blocked thrombin-stimulated increases in PLA2 activity and arachidonic acid release. No significant inhibition of thrombin-induced PLA2 was observed following pretreatment with mepacrine or dibucaine. These data confirm the presence of high-affinity thrombin receptors on isolated cardiac myocytes and demonstrate the specific activation of a unique membrane-associated, calcium-independent PLA2 following thrombin receptor ligation.

Calcium-independent phospholipase A2 in isolated rabbit ventricular myocytes

We characterized phospholipase A2 (PLA2) activity in isolated rabbit ventricular myocytes with respect to subcellular distribution, substrate specificity, and Ca2+ dependency. Membrane-associated PLA2 was found to be an order of magnitude greater than cytosolic PLA2. Ventricular myocyte PLA2 activity was enhanced following protease-activated receptor stimulation with thrombin and was found to be largely Ca2+-independent and selective for phospholipid substrates containing arachidonic acid at the sn-2 position. Immunoblot analysis using an antibody to cytosolic Ca2+-independent PLA2 from Chinese hamster ovary cells recognized a membrane-associated protein with a molecular mass of approximately 80 kDa; however, differences in pH optima, response to inhibitors, and substrate selectivity of membrane-associated and cytosolic PLA2 activity suggest the presence of multiple Ca2+-independent PLA2. Pretreatment with bromoenol lactone, a specific inhibitor of Ca2+-independent PLA2, significantly attenuated membrane-associated and cytosolic PLA2 in unstimulated and thrombin-stimulated myocytes. Pretreatment with methyl arachidonyl fluorophosphonate, mepacrine, or dibucaine had no significant effect on PLA2 activity under all conditions tested. Ventricular myocyte PLA2 activity was significantly inhibited by ATP, GTP, and their nonhydrolyzable analogs and was regulated by protein kinase C activity. These studies demonstrate the presence of one or more unique membrane-associated Ca2+-independent PLA2 in isolated ventricular myocytes that exhibit a preference for phospholipids with arachidonate at the sn-2 position and that are activated by thrombin stimulation.