Hiroko Hashizume

Propofol Improves Functional and Metabolic Recovery in Ischemic Reperfused Isolated Rat Hearts

Propofol attenuates mechanical dysfunction, metabolic derangement, and lipid peroxidation by exogenous administration of H2 O2 in the Langendorff rat heart.In this study, we examined the effects of propofol on mechanical and metabolic changes, as well as on lipid peroxidation induced by ischemia-reperfusion, in isolated, working rat hearts. Rat hearts (in control-modified Krebs-Henseleit bicarbonate buffer) were treated with two doses (25 micro M and 50 micro M) of propofol in an intralipid vehicle. In the first protocol, propofol was administered during the preischemic and reperfusion period, whereas in the second, it was only administered during the reperfusion period. Ischemia (15 min) decreased peak aortic pressure (PAOP), heart rate (HR), rate-pressure product (RPP), coronary flow (CF), and tissue concentrations of adenosine triphosphate (ATP) and creatine phosphate. After postischemic reperfusion (20 min), the CF and tissue concentration of ATP recovered incompletely; however, PAOP, HR, and RPP did not. Ischemia-reperfusion also increased the tissue concentration of malondialdehyde (MDA). In both protocols, both doses of propofol enhanced recovery of PAOP, HR, RPP, CF, and tissue concentration of ATP during reperfusion, and inhibited the tissue accumulation of MDA. These results indicate that propofol improves recovery of mechanical function and the energy state in ischemic reperfused isolated rat hearts, and the mechanism may involve the reduction of lipid peroxidation during postischemic reperfusion. Implications: We evaluated the possible cardioprotective effects of propofol in isolated, working rat hearts subjected to 15-min ischemia, followed by 20-min reperfusion. We observed that propofol attenuated mechanical dysfunction, metabolic derangement, and lipid peroxidation during reperfusion. This latter finding seems to be one mechanism for cardioprotective effects of propofol. (Anesth Analg 1998;86:252-8)

Effects of antiischemic drugs on veratridine-induced hypercontracture in rat cardiac myocytes

The effects of different groups of substances (beta-adrenoceptor antagonists, Ca2+ channel blockers and vasodilators) which are known to have antiischemic properties were studied on veratridine-induced hypercontracture. Veratridine increases Na+ influx by slowing the inactivation process of the Na+ channel, thereby inducing a continuously increased Na+ entry in depolarized cells. Veratridine (6.3 x 10(-6) M) produced a change in cell shape from rod-shape to round, resulting from hypercontracture of cells. Before treatment with veratridine the proportion of rod-shaped cells was 70% and fell to 0% 5 min after the treatment with veratridine. dl-Propranolol, d-propranolol, l-penbutolol, d-penbutolol, nisoldipine, and dilazep all inhibited veratridine-induced hypercontracture dose dependently. In contrast, acebutolol, atenolol, timolol, nifedipine, diltiazem, and nitroglycerin did not inhibit the rounding of cells. Concomitantly with the rounding of cells, the [Ca2+]i was increased by veratridine. dl-Propranolol, d-propranolol and dilazep prevented the increase of [Ca2+]i induced by veratridine, whereas timolol and nitroglycerin did not. These results show that dl-propranolol, d-propranolol, l-penbutolol, d-penbutolol, nisoldipine, and dilazep possess Na+ channel blocking actions on the veratridine-modified Na+ channel, thereby preventing excessive Na+ influx and secondary Ca2+ overload.

Ranolazine Attenuates Palmitoyl-l-carnitine-induced Mechanical and Metabolic Derangement in the Isolated, Perfused Rat Heart

The effect of ranolazine, a novel anti‐ischaemic drug that stimulates the activity of pyruvate dehydrogenase, on palmitoyl‐l‐carnitine‐induced mechanical dysfunction and metabolic derangement in isolated perfused rat hearts has been studied and compared with the effect of dichloroacetate, an activator of pyruvate dehydrogenase. Rat hearts paced electrically were perfused aerobically at constant flow by the Langendorff technique. Palmitoyl‐l‐carnitine (4 μm) increased left ventricular end‐diastolic pressure and reduced left ventricular developed pressure (i.e. induced mechanical dysfunction); it also reduced tissue levels of adenosine triphosphate and increased tissue levels of adenosine monophosphate (i.e. induced metabolic derangement). These functional and metabolic alterations induced by palmitoyl‐l‐carnitine were attenuated by ranolazine (5, 10, and 20 μm) in a concentration‐dependent manner. In contrast, dichloroacetate (1 and 10 mm) did not attenuate palmitoyl‐l‐carnitine‐induced mechanical and metabolic derangement. In the normal (palmitoyl‐l‐carnitine‐untreated) heart, however, ranolazine did not modify mechanical function and energy metabolism.

Effects of β-adrenoceptor antagonists on Ca2+-overload induced by lysophosphatidylcholine in rat isolated cardiomyocytes

1 The effects of β‐adrenoceptor antagonists including (−)− and (+)‐propranolol, (−)− and (+)‐penbutolol, timolol, pindolol, atenolol, acebutolol and practolol on the Ca2+‐overload induced by lysophosphatidylcholine (LPC) were examined in isolated cardiomyocytes of the rat. 2 Fura‐2 was used for measurement of the intracellular calcium concentration ([Ca2+]i). LPC (15 μm) produced a rapid increase in [Ca2+]i from 72±5 to 3042±431 nM which coincided with a decrease in the percentage of rod‐shaped cells from 69±2 to 5±2%. 3 Preincubation with (−)−propranolol (20 μm), (+)‐propranolol (50 μm), or (−)− or (+)‐penbutolol (20 μm), the lipophilicity of which is higher than other β‐adrenoceptor antagonists, significantly inhibited both the increase in [Ca2+]i and the cell‐shape change induced by 15 μm LPC. The inhibitory effects of the four drugs on the LPC‐induced increase in [Ca2+]i and cell‐shape change were concentration‐dependent. The IC50s of (−)−propranolol, (+)‐propranolol, (−)− and (+)‐penbutolol for the increase in [Ca2+]i were 1.28, 10.50, 0.67 and 0.76 μm, respectively. 4 Pretreatment with pindolol, timolol, acebutolol, practolol, atenolol or lignocaine did not inhibit the increase in [Ca2+]i and the morphological change induced by LPC. 5 LPC markedly increased the release of creatine phosphokinase from 9±1 to 45±2% which could be significantly reduced by (−)− or (+)‐propranolol but not by acebutolol or timolol. 6 The protective effects of (−)− and (+)‐propranolol, (−)− and (+)‐penbutolol against the Ca2+‐overload induced by LPC were not associated with the β‐adrenoceptor antagonistic action, but probably with an unknown action which is related to the preservation of membrane integrity. Further studies are necessary to clarify the exact mechanisms of the protective action of these β‐adrenoceptor antagonists against the Ca2+‐overload induced by LPC.

Accumulation of nonesterified fatty acids in the dog myocardium during coronary artery occlusion determined by a method using 9-anthryldiazomethane.

SummaryThe levels of nonesterified fatty acids (NEFA) in the myocardium during ischemia were determined by a simple method, which requires neither previous separation with thin-layer chromatography nor heating. After being extracted with Folchs solution, NEFA were subjected to fluorescent labeling with 9-anthryldiazomethane at room temperature, separation with high-pressure liquid chromatography, and then detection by a flow-through fluorometer. Calibration and validation studies revealed that this method was satisfactory. The left anterior descending coronary artery was completely occluded for 90 min in dogs anesthetized with pentobarbital. In the nonischemic myocardium, the levels of lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic, and arachidonic acids were 5.31, 15.85, 47.06, 2.59, 23.92, 37.90, 38.69, and 3.99 nmol/g wet tissue, respectively, and those in the ischemic myocardium 5.67, 22.16, 75.94, 6.65, 42.67, 61.75, 70.06, and 13.39 nmol/g wet tissue, respectively, the total NEFA in the former being 175.3 and that in the latter 298.3 nmol/g wet tissue. The increase in myocardial NEFA during ischemia was significant except for lauric and myristic acids, and the increase in arachidonic acid was the greatest. The ratio of the level of arachidonic acid in the ischemic myocardium to that in the nonischemic myocardium was 335.6%.

Differential effects of Ca2+ channel blockers on Ca2+ overload induced by lysophosphatidylcholine in cardiomyocytes.

The effects of Ca2+ channel blockers (verapamil, diltiazem, nicardipine, bepridil and flunarizine) on Ca2+ overload induced by lysophosphatidylcholine were examined in rat isolated cardiomyocytes. Addition of lysophosphatidylcholine (15 microM) produced Ca2+ overload as evidenced by a marked increase in the concentration of intracellular Ca2+ and hypercontracture of cells. Verapamil, flunarizine and bepridil concentration dependently inhibited the lysophosphatidylcholine-induced Ca2+ overload, whereas diltiazem and nicardipine did not. Lysophosphatidylcholine increased the release of creatine kinase, which was significantly attenuated by verapamil, flunarizine or bepridil (5 microM for each), but not by diltiazem or nicardipine (20 microM for each). Verapamil, flunarizine, bepridil (which attenuated the lysophosphatidylcholine-induced Ca2+ overload) and nicardipine (which did not) inhibited the veratridine-induced increase in the concentration of intracellular Na+ (indicated by the increase in fluorescence ratio of Na(+)-binding benzofuran isophthalate) and cell contracture, whereas diltiazem did not. These results suggest that verapamil, bepridil and flunarizine attenuate the Ca2+ overload induced by lysophosphatidylcholine, and that the Ca2+ channel blocking action of these drugs does not contribute substantially to this effect. The Na+ channel inhibition together with high lipophilicity of these drugs may be important for the attenuation of the lysophosphatidylcholine-induced Ca2+ overload.

PALMITOYL-L-CARNITINE MODIFIES THE MYOCARDIAL LEVELS OF HIGH-ENERGY PHOSPHATES AND FREE FATTY ACIDS

Long-chain acylcarnitines, such as palmitoyl-L-carnitine (PALCAR), are known to accumulate in the myocardium during ischemia. We examined whether exogenous PALCAR modifies the myocardial levels of high-energy phosphates (HEP) and free fatty acids (FFA) in the heart, and whetherd-cis-diltiazem andl-cis-diltiazem, an optical isomer having less potent Ca2+ channel blocking action thand-cis-diltiazem, attenuate the PALCAR-induced myocardial changes. Rat hearts were perfused aerobically at a constant flow according to the Langendorffs technique, while being paced electrically. PALCAR (5 μM) decreased the tissue levels of adenosine triphosphate and creatine phosphate and increased the tissue level of adenosine monophosphate, and produced mechanical dysfunction. In addition, PALCAR (5 μM) increased markedly the tissue levels of FFA, especially those of arachidonic and palmitoleic acids, and the release of creatine kinase (CK) from the myocardium. These alterations in the myocardial levels of HEP and FFA induced by PALCAR were significantly attenuated byd-cis-diltiazem (15 μM) orl-cis-diltiazem (15 μM). Both drugs also attenuated the PALCAR-induced CK release. The present study demonstrates that PALCAR modifies the tissue levels of HEP and FFA in the heart and that bothd-cis andl-cis-diltiazem protect the myocardium against the PALCAR-induced changes through mechanisms other that Ca2+ channel blocking action.

Effects of CP-060S, a novel Ca2+ channel blocker, on oxidative stress in cultured cardiac myocytes

The effect of (-)-(S)-2-[3,5-bis(1, 1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[N-methyl-N-[2-(3, 4-methylenedioxyphenoxy)ethyl]amino]propyl]-1,3-thiazolidin- 4-one hydrogen fumarate (CP-060S), a novel Ca(2+) channel blocker, on hydrogen peroxide (H(2)O(2))-induced cytotoxicity was studied in cultured rat cardiac myocytes. The CP-060S effect was compared with that of CP-060R, an optical isomer of CP-060S with a less potent Ca(2+) channel blocking action than CP-060S. H(2)O(2) increased the release of lactate dehydrogenase from cardiac myocytes and decreased the formation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) formazan in cardiac myocytes (i.e., cytotoxic action). Both CP-060S (1 microM) and CP-060R (1 microM) attenuated to a similar extent the foregoing alterations induced by H(2)O(2). On the other hand, 1,3-dimethyl-2-thiourea (10 mM), a scavenger of both H(2)O(2) and hydroxyl radical, also attenuated the H(2)O(2)-induced cytotoxicity whereas diltiazem (10 microM) did not. In an experiment using electron spin resonance (ESR) with 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), a spin-trapping agent, both CP-060S and CP-060R decreased the intensity of DMPO-hydroxyl radical signal concentration dependently. These results suggest that CP-060S protects cardiac myocytes from oxidative stress through its radical scavenging action.

Propranolol inhibits accumulation of non-esterified fatty acids in the ischemic dog heart

The effects of propranolol and d-propranolol on the accumulation of non-esterified fatty acids (NEFA) induced by complete occlusion of the left anterior descending coronary artery (LAD) for 90 min, were investigated in dogs anesthetized with pentobarbital. The myocardial levels of eight NEFA (lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic and arachidonic acids) were determined after fluorescent labelling of the NEFA with 9-anthryldiazomethane. LAD occlusion increased the myocardial levels of NEFA, especially those of arachidonic and palmitoleic acids; arachidonic acid increased by 3.5 times and palmitoleic acid by 2.9 times. Pretreatment with propranolol (1 mg/kg) inhibited almost completely the accumulation of NEFA induced by occlusion of the LAD, whereas pretreatment with d-propranolol (1 mg/kg) did not inhibit the accumulation of NEFA, although it tended to inhibit the accumulation of palmitoleic, stearic and arachidonic acids. These results suggest that the inhibitory action of propranolol on the accumulation of NEFA in the myocardium during ischemia is mainly due to its antagonistic effect at beta-adrenoceptors and probably partly due to its effect as a local anesthetic.

A new approach to the development of anti-ischemic drugs: Protective drugs against cell injury induced by lysophosphatidylcholine

Recent studies have revealed that lysophosphatidylcholine (LPC) produces mechanical and metabolic derangements in perfused working rat hearts and Ca2+-overload in isolated cardiac myocytes. Thus, LPC possesses an ischemia-like effect on the heart. Therefore, a drug that possesses an anti-LPC action would protect or improve ischemia/reperfusion damage. We examined the effects of various anti-ischemic drugs on the Ca2+ overload induced by LPC. Our data suggest that a drug with high lipophilicity possesses a protective effect on cell injury induced by LPC, probably because of preservation of membrane integrity.