Inhibition of long-chain acylcarnitine accumulation during coronary artery occlusion does not alter infarct size in dogs.

We tested whether inhibition of carnitine acyl-transferase-1 (CAT-1) during coronary artery occlusion can limit infarct size (IS) by suppressing accumulation of long-chain acylcarnitines (LCAs), potentially cytotoxic intermediates of fatty acid metabolism. The CAT-1 inhibitor 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA) was administered to dogs before 90-min occlusion and 4-h reperfusion of the left anterior descending or left circumflex coronary artery (LAD, LCX). Dogs in the LAD occlusion series received 7.5 (n = 5) or 15 (n = 2) mg/kg POCA intravenously (i.v.); dogs in the LCX occlusion series received 15 mg/kg i.v. (n = 7); an equal number were treated with drug vehicle. Biopsies were obtained for determination of myocardial LCAs. The region at risk and IS were delineated by dye injection and tetrazolium staining. In vehicle-treated dogs, myocardial LCAs (in picomoles per milligram of wet weight +/- SEM) increased from 11 +/- 3 to a peak of 75 +/- 24 during LAD occlusion and from 32 +/- 10 to 192 +/- 55 during LCX occlusion. In POCA-treated dogs LCAs increased from 12 +/- 2 to only 33 +/- 13 pmol/mg wet weight during LAD occlusion (p < 0.05 vs. vehicle) and did not increase significantly during LCX occlusion; 22 +/- 8 to 27 +/- 5 pmol/mg wet weight (p < 0.005 vs. vehicle). LCX occlusion resulted in larger areas at risk and larger infarcts (as a percentage of left ventricle) than did LAD occlusion. IS as a percentage of the region at risk did not differ significantly among the experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Reperfusion-induced accumulation of long-chain acylcarnitines in previously ischemic myocardium.

Long-chain acylcarnitines (LCA) have been shown to accumulate during myocardial ischemia and to contribute to malignant derangements characteristic of ischemia. We detail the time course of the increase in LCA levels during both ischemia and reperfusion. Evidence indicates an additional specific reperfusion-induced increase in LCA that peaks at 2 min and decreases to basal levels by 30 min. This increase in LCA during reperfusion is observed after 2-, 10-, or 20-min ischemia and is inhibited by the presence of the carnitine palmitoyl transferase 1 (CPT1) inhibitor phenyloxirane carboxylic acid (POCA). A role for increased LCA in mediating "reperfusion damage" is not indicated, however, because POCA did not attenuate either the incidence of ventricular fibrillation (VF) during early reperfusion or the survival rate of rats undergoing 24-h reperfusion after 10-min occlusion.

Mechanisms contributing to the arrhythmogenic influences of alpha 1-adrenergic stimulation in the ischemic heart.

The majority of deaths associated with ischemic heart disease occur suddenly because of disturbances in cardiac rhythm culminating in ventricular fibrillation. Past research has focused on elucidating the biochemical membrane mechanisms responsible for the adverse electrophysiologic alterations in the ischemic heart, with major emphasis on the influence of adrenergic neural factors. It has been demonstrated that both alpha 1-and beta-adrenergic mechanisms contribute to arrhythmogenesis in the ischemic heart. In the normal heart, alpha 1-adrenergic input has very little effect on electrophysiologic indices. However, during early ischemia and reperfusion, enhanced alpha 1-adrenergic responsivity associated with a twofold reversible increase in alpha 1-adrenergic receptors in vivo has been demonstrated. Likewise, in a variety of species, alpha 1-adrenergic inhibition with prazosin markedly decreases the incidence of malignant ventricular arrhythmias associated with either myocardial ischemia or subsequent reperfusion. One major manifestation of alpha 1-adrenergic receptor activation during reperfusion of ischemic myocardium is an increase in intracellular calcium ion (Ca2+). It has been demonstrated that reperfusion of ischemic myocardium increases intracellular Ca2+ in reversibly injured tissue, and that the gain in intracellular Ca2+ is prevented by alpha 1-adrenergic inhibition with hydroxyphenylethyl aminomethyl tetralone, even when administered just prior to reperfusion. Subsequently, it was demonstrated that the alpha 1-adrenergic-induced increase in mitochondrial Ca2+ contributes to the decline in mitochondrial function. These findings suggest that even single-dose intervention with alpha 1-adrenergic inhibitors may improve markedly the functional recovery and extent of ultimate necrosis in humans after coronary thrombolysis. To investigate the mechanisms responsible for the increase in alpha 1-adrenergic receptors during ischemia, we used isolated adult canine ventricular myocytes exposed to hypoxia. Thirty minutes of hypoxia at 25 degrees C or 10 minutes of hypoxia at 37 degrees C resulted in a threefold reversible increase in the density of surface alpha 1-adrenergic receptors and a threefold increase in the cellular content of long-chain acylcarnitines. Inhibition of carnitine acyltransferase I abolished not only the accumulation of long-chain acylcarnitines during hypoxia but also the increase in alpha 1-adrenergic receptors. Exposure of normoxic myocytes to exogenous long-chain acylcarnitines (1 mumol/liter) for 10 minutes also increased alpha 1-adrenergic receptor number. These findings indicate that the sarcolemmal accumulation of long-cha

Enhanced inositol trisphosphate response to alpha 1-adrenergic stimulation in cardiac myocytes exposed to hypoxia.

Myocardial ischemia elicits an enhanced responsivity to alpha 1-adrenergic stimulation and a reversible increase in alpha 1-adrenergic receptor number. In adult cardiac myocytes, alpha 1-adrenergic receptor number increases two- to threefold after 10 min of hypoxia, an increase similar to that seen during ischemia in vivo. To determine whether this increase in alpha 1-adrenergic receptor number leads to an enhanced synthesis of inositol trisphosphate, the intracellular second messenger for the alpha 1-adrenergic receptor, the mass of inositol trisphosphate was quantified by a novel procedure developed in our laboratory that circumvents problems associated with using labeled precursors. The peak increases in inositol trisphosphate levels of three- to fourfold were measured after 30 s of norepinephrine stimulation and exhibited a 50% effective concentration (EC50) of 7.9 x 10(-8) M. Hypoxia produced a marked leftward shift in the dose-response curve for the production of inositol trisphosphate in response to norepinephrine stimulation (EC50 = 1.2 x 10(-8) M). Hypoxia also induced a 100-fold reduction in the concentration of norepinephrine required to elicit a threshold increase in inositol trisphosphate (10(-9) M), compared with control normoxic myocytes (10(-7) M). Thus, hypoxia, which increases alpha 1-adrenergic receptor density, also leads to an enhanced production of inositol trisphosphate and could account for the enhanced alpha 1-adrenergic responsivity in the ischemic heart in vivo, which is known to facilitate arrhythmogenesis.

Anion exchange chromatographic separation of inositol phosphates and their quantification by gas chromatography.

The direct measurement of mass of inositol trisphosphate from biologic samples is described. Separation of inositol monophosphate, bisphosphate, trisphosphate, and inositol tetrakisphosphate was achieved using anion exchange chromatography with a sodium sulfate gradient. In addition, separation of the isomers of each inositol phosphate was performed using HPLC procedures. The individual inositol phosphate fractions were subsequently dephosphorylated and desalted. The myo-inositol from each fraction was then derivatized to the hexatrimethylsilyl derivative and the myo-inositol derivatives were quantified by a novel gas chromatographic analysis using the hexatrimethylsilyl derivative of chiro-inositol as an internal concentration reference. This method is a reproducible and relatively rapid procedure for the direct quantification of inositol phosphate mass which overcomes many of the problems associated with the use of radiolabeled precursors. The method is a significant improvement over existing procedures for the quantitative determination of the mass of inositol phosphate by virtue of improved recovery, sensitivity, and technical simplicity. The applicability of this method is illustrated by the quantitative determination of inositol trisphosphate in response to norepinephrine stimulation of adult canine myocytes and cerebral cortical brain slices and by measurement of the isomers of inositol trisphosphate in isolated myocytes.

Transient accumulation of inositol (1,3,4,5)-tetrakisphosphate in response to alpha 1-adrenergic stimulation in adult cardiac myocytes.

To investigate the response to catecholamine stimulation of adult cardiac myocytes and the metabolism of inositol (1,4,5)-trisphosphate (1,4,5-IP3) and inositol (1,3,4,5)-tetrakisphosphate (IP4), we have employed a procedure developed in our laboratory to directly measure the mass of inositol phosphates after separation of individual isomers of inositol phosphates by high performance liquid chromatography. Control, unstimulated myocytes, contained low levels of inositol (1,4)-bisphosphate (1,4-IP2), inositol (1,3)-bisphosphate (1,3-IP2), inositol (3,4)-bisphosphate (3,4-IP2), inositol (1,3,4)-trisphosphate (1,3,4-IP3), 1,4,5-IP3 and IP4. Stimulation with norepinephrine for 30 seconds produced peak 1,4,5-IP3 and IP4 levels which rapidly returned to basal values by 60 seconds of norepinephrine stimulation. 1,4-IP2, 1,3-IP2 and 1,3,4-IP3 were increased markedly but only after stimulation with norepinephrine for 60 seconds. These results indicate a rapid yet transient increase in 1,4,5-IP3 and IP4 in response to norepinephrine stimulation and are the first quantitative measurements of the isomers of inositol phosphates in cardiac tissue.

Long-chain acylcarnitines mediate the hypoxia-induced increase in alpha 1-adrenergic receptors on adult canine myocytes.

To elucidate the mechanisms responsible for the increase in alpha 1-adrenergic receptors during ischemia in vivo, we developed a procedure for measuring alpha 1-adrenergic receptors in isolated, calcium-tolerant adult canine myocytes. Specific [3H]prazosin binding was rapid, saturable, reversible, and demonstrated the expected order of potency and stereospecificity for the alpha 1-adrenergic receptor. Myocytes exposed to 30 minutes of hypoxia at 25 degrees C or only 10 minutes at 37 degrees C exhibited a twofold to threefold increase in the number of alpha 1-adrenergic receptors with no significant change in receptor affinity. This hypoxia-induced increase in receptor number was reversible by 10 minutes of reoxygenation at 37 degrees C. In contrast, more prolonged hypoxia of 80 minutes or hypotonic shock actually decreased receptor number below normoxic, control values. The concentration of long-chain acylcarnitines in myocytes also increased threefold on exposure to 30 minutes of hypoxia. Sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA, 10 microM), a potent inhibitor of carnitine acyltransferase I, not only abolished the accumulation of long-chain acylcarnitines but also the increase in alpha 1-adrenergic receptor number induced by 30 minutes of hypoxia. Likewise, incubation of normoxic cells with exogenous palmitoyl carnitine (1 microM) for 10 minutes also increased alpha 1-adrenergic receptor number in the presence or absence of POCA. Thus, hypoxia results in an increase in alpha 1-adrenergic receptors associated with an increase in endogenous long-chain acylcarnitines. Furthermore, inhibition of carnitine acyltransferase I prevents not only the sarcolemmal accumulation of long-chain acylcarnitines but also the exposure of the alpha 1-adrenergic receptor, indicating that accumulation of endogenous long-chain acylcarnitines is critical to the hypoxia-induced increase in alpha 1-adrenergic receptors on adult myocytes.

The effect of coronary artery occlusion and reperfusion on the activities of triglyceride lipase and glycerol 3-phosphate acyl transferase in the isolated perfused rat heart.

Glycerol 3-phosphate acyltransferase (GPAT) and Triglyceride lipase (TGL) were measured in homogenates from non-ischaemic and ischaemic tissue from the isolated perfused rat heart. Ischaemia was produced by occlusion of the left descending coronary artery for 10 min. Compared to activities measured in tissue from normally perfused hearts, GPAT activity measured in tissue from the ischaemic area was considerably reduced. TGL activity in the ischaemic area was markedly increased compared to activity measured in normally perfused hearts. No change was seen in GPAT or TGL activity measured in tissue from the non-ischaemic area. The change in activities produced by ischaemia were prevented by pre-perfusion with the cardio-selective beta-antagonist Atenolol. Reperfusion of the ischaemic area resulted in TGL activity returning to the value measured in tissue from normally perfused hearts. However, GPAT activity, after 1 min of reperfusion, fell to a value lower than after 10 min ischaemia. The reperfusion-induced fall in GPAT activity was prevented by pre-perfusion with the alpha 1 antagonist Doxasozin. Pre-perfusion of the alpha 2 antagonist Yohimbine resulted in a prolongation of the increased TGL activity in the ischaemic area during reperfusion. All changes in enzyme activities were prevented by injection of 6 OH-dopamine 24 h before hearts were removed. These changes in enzyme activities show that during ischaemia there is an increased beta-adrenergic drive. On reperfusion the beta-adrenergic drive is removed but an alpha 1 adrenergic drive becomes apparent.

The effect of adrenergic agents on the activities of glycerol 3-phosphate acyltransferase and triglyceride lipase in the isolated perfused rat heart.

Glycerol 3-phosphate acyltransferase (GPAT) activity and triglyceride lipase (TGL) activity were measured in homogenates from hearts perfused with adrenergic agonists and antagonists. Perfusion with adrenalin or the beta-agonist isoprenaline produced an increase in TGL activity and a fall in GPAT activity. These changes could be imitated by incubation of heart homogenates with cAMP-dependent protein kinase. The alpha 2-agonist clondine produced the opposite effect, thus it increased GPAT activity and decreased TGL activity. Methoxamine, an alpha 1-agonist, had no effect on TGL activity but reduced GPAT activity. Continuous perfusion of the beta-antagonist atenolol reduced TGL activity to half that found in controls but also reduced GPAT activity. No change was seen on continuous perfusion of alpha 1- or alpha 2-antagonists. Changes in GPAT activity were localized mainly in the microsomal enzyme. These changes are consistent with both enzymes being regulated via a cyclic-AMP dependent protein kinase system and via alpha-adrenergic mechanisms.