J
Mol Cell Cardiol22,
687-695
Oxygen
I. Tong
(1990)
Radical-mediated
Mak,
Injury by Propranolol
of Myocytes-protection
Jay H. Kramer, Anthony M. Freedman, and William B. Weglicki
Suzanne
Y. H. Tse
Department of Medicine, Division of Experimental Medicine, George Washington University Medical Center, 2300 Eye Street NW, Washington, DC 20037, USA (Received 22 June 1989, accepted in revised form 17 January 1990) I. T. MAK, J. H. KRAMER, A. M. FREEDMAN, S. Y. H. TSE AND W. B. WEGLICKI. Oxygen Radical-mediated Injllry of Myocytes-protection by Propranolol. 3ournnl of Molecular and Cellular Cardiology (1990) 22, 687495. The effects of propranolol and atenolol on free radical mediated injury in myocytes were examined. Freshly isolated adult canine myocytes were incubated with a superoxide generating (from dihydroxyfumarate) and Fecatalyzed free radical system. Exposure of the myocytes to free radicals for 20 min resulted in more than a 5-fold increase in thiobarbituric acid reactant (peroxide) formation and elevated levels of lactate dehydrogenase (LDH) activity released into the media compared to controls. Ultrastructurally, severe sarcolemmal damage, mitochondrial and myotibril derangements were evident. At 40 min, cellular viability (trypan blue exclusion) in the samples exposed to free radicals decreased to about one-third of controls; concomitantly, major losses in total cellular phospholipids occurred. When the cells were pretreated with 200 PM propranolol before the addition of free radicals, both peroxide formation and increased LDH release were inhibited; in agreement, complete ultrastructural preservation was observed. In addition, the subsequent losses in cellular viability and phospholipids were prevented. For comparison, the more water soluble beta-blocker, atenolol at 200 FM was shown ineffective in providing significant protection against the induced injury. The results suggest that propranolol may provide antiperoxidative protection to myocytes when elevated levels of free radicals are present. KEY WORDS: Free radicals; Viability; Phospholipids;
Isolated Propranolol
canine and
myocytes; atenolol.
Introduction Due to its amphiphilic nature, propranolol binds to non-receptor hydrophobic membrane sites and may provide membrane activity independent of its adrenergic blocking effect [4, lo]. During myocardial ischemia, free oxygen radicals have been documented to increase upon reperfusion [Z, 8, 181; and free radicalmediated lipid peroxidation has been implicated to play a major role in the pathogenesis of ischemic/reperfusion injury [S, 1.5, 201. The phospholipid-rich sarcolemma of ventricular myocytes may be a critical site of free radical reaction; we previously demonstrated that purified sarcolemmal membranes from canine myocytes are highly susceptible to free radical-mediated peroxidative attack [9]. In a recent study, by using the isolated sarPIease address George Washington 002-2828/90/060687
all
correspondence to: I. T. Mak, Ph.D., University Medical Center, 2300 Eye + 09 $03.00/O
Lipid
peroxidation;
Membrane
damage;
Ultrastructure;
colemmal membrane preparations, we have demonstrated that propranolol in micromolar concentration provided significant protection against free radical-mediated lipid peroxidation [13]. In the present study, we sought to determine whether the membrane protective effect of propranolol against free radical injury might be translated into cytoprotective effects at the myocellular level. For this purpose, freshly isolated myocytes from adult canine ventricular tissue were exposed to exogenously generated oxygen free radicals in the presence or absence of the beta-blockers, and both biochemical and morphological endpoints (of myocytic injury were monitored. The results suggest that propranolol may provide antiperoxidative protection to myocytes when excessive free radicals are present. Division of Experimental Street NW, Washington,
DC
Medicine, 20037,
0
1990
Ross USA. Academic
Hall, Press
Rm.
45t2,
Limited
688
I. Tong Mak et al. Materials
and Methods Chemicals
D,L-Propranolol, atenolol, dihydroxyfumarate (DHF), ADP, FeCls and P-thiobarbituric acid (TBA) were obtained from Sigma Chemical. Isolation of myocytes Adult canine myocytes were isolated from ventricular tissue by a modification of the enzymatic digestion procedure [19] described recently [Z,?]. The final preparation is typically 90% to 95% free of contaminating smooth muscle and non-myocytic cells. Incubation procedure The isolated myocytes (1.5 x 106/ml) were resuspended in the incubation buffer consisting of 5 mM Na-acetate, 1.2 mM MgS04, 120 mM NaCl, and 10 mM potassium phosphate, pH 7.2. Calcium was not included due to its interference with our iron-catalyzed free radical generation system. The cells were preincubated under continuous gassing with 95% oxygen, 5% carbon dioxide, with or without the selected beta-blocker (50-200 PM) for 10 min at 30°C. Oxygen free radicals were generated by the final additions of Fe3+-ADP (0.025 mM FeC13 chelated by 0.250 mM ADP) and 1.67 mM dihydroxyfumarate which generates superoxide anions and hydroxyl radicals [7, 11, 121. At various times of incubation, aliquots of the cellular suspension were retrieved for determinations of TBA-reactive substances, free and cell-bound activities of lactate dehydrogenase (LDH), trypan blue exclusion cellular contents of and phospholipids. Assays and measurements Lipid peroxidation in the cell samples were assessed as the formation of the TBA reactive substances which were determined by the calorimetric method described previously [12] and expressed as malondialdehyde (MDA) equivalents. To prevent nonspecific color formation, O.Olo/o BHT was included during the heating step, and the temperature was maintained at 80°C. Under these conditions, no interference from either propranolol or atenolol (up to 200 PM) was found. LDH
activities in both the incubation media and in the cell pellet (homogenized in 0.1 o/o TritonX-100) were determined at 30°C by measuring the oxidation rate of lactate to pyruvate which was followed spectrophotometrically at 340 nm due to NAD+/NADH conversion [I]. Viability of myocytes was monitored morphologically and by trypan blue (0.1%) exclusion. Ultrastructural studies Samples taken for electron microscopic analysis after 20 min incubation, were fixed overnight in 3% glutaraldehyde, 0.1 M phosphate buffer at pH 7.4. Subsequently, the cells were pelleted and post-fixed with 1 o/o osmium tetroxide for l-2 h. The samples were then dehydrated through a graded series of ethanol, infiltrated with propylene oxide before embedding in Epon 812. Ultrathin sections (60 to 90 nm) were counter stained with lead citrate and uranyl acetate and examined in a JEOL 1OOB transmission electron microscope. Lipid extraction and ana&ses Total myocytic lipids were extracted by the modified procedure of Bligh and Dyer as described previously [21]. Phospholipids were separated by high pressure liquid chromatography (HPLC) on a 5 p Ultrasphere Si analytical column (25 cm) under isocratic condition with a solvent system consisting of acetonitrile: methanol: HzS04, 100 : 3 : 0.1. The solvent mixture was vacuum-filtered through 0.2 p nylon filters, purged and saturated with helium gas before use. The elutions were monitored at 210 nm; different phospholipid species were identified by comparing the retention times to those of standards. Signals and areas were recorded and integrated with a Shimatzu Chromatopac C-R3A integrator. Semiquantitative changes in total cellular phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the presence of free radicals with or without beta-blockers were expressed as percentage of control samples (myocytes in buffer alone). More quantitative estimation of the phospholipid losses in the PC and PE due to free radicals were determined by inorganic phosphate assay [Ia, 211.
Antiperoxidant
Effect of Propranolol
on Myocytes
689
Results Lipid peroxidation and L DH release The employed isolation procedure yielded 65 to 85% rod-shaped myocytes which excluded trypan blue dye indicating a good preservation of sarcolemmal integrity. We have previously demonstrated that isolated sarcolemmal membranes were relatively sensitive to oxygen free radical-mediated lipid peroxidation as assessed by the TBA-MDA assay [9]. In the present study, when the ventricular myocytes were incubated with the superoxide anion generating, iron-catalyzed free radical system for 20 min, a 5-fold higher level of TBAreactive substances (or MDA equivalents) accumulated in the samples compared to the controls [Fig. 1 (a)]. Concomitantly, higher levels of LDH activities were released from cell samples exposed to free radicals for 20 min, suggesting the development of increased sarcolemmal membrane permeability [Fig. 1 (b)]. We have demonstrated that propranolol, a highly lipophilic beta-blocker, was most effective in inhibiting free radicalinduced lipid peroxidation in isolated sarcolemmal membranes [13]. When the myocytes were pretreated with 20Op~ propranolo1 before adding the free radical components, the induced TBA-reactive substances were inhibited 85%, and the level of LDH release was not statistically different from that of controls [Fig. 1 (a, b)]. Propranolol at 100 PM and 50 ,UM provided 69% and 49%, respectively, inhibition of the TBA-reactant formation. For the purpose of clarity, only data for 200 pM propranolol were presented. For comparison, when the cell samples were pretreated with the more water soluble beta-blocker atenolol (200 PM), the induced formation of the TBA-reactive substances was not inhibited [Fig. 1 (a)]; nor was the increased LDH release significantly reduced [Fig. 1 (b)]. Electron micrograph Up to 20 min of incubation, light level morphological examinations indicated no substantial changes in the percentage of rod-shaped myocytes with or without exposing to free radicals. To confirm the free radical-induced membrane damage and the effects of the twobeta-blockers, samples at 20 min of incubation
Cont.
R.
R. + Prop R. + Aten
FIGURE 1. Effects of propranolol and atenolol on free radical-mediated lipid peroxidation (a) and LDH release (b) in isolated canine myocytes. Controls were cell samples incubated in buffer alone. Experimental samples were preincubated with or without the selected beta-blocker 1200 P’M each) for 10 min before the addition of [Fe-ADP and DHFl CR.). After 20 min ofincubation. samoles were assayed for net LDH release (expressed as 0/0 total1 activity) and total TBA-reactive substance (TBA-RS) formation as described in the text. jV = 4-6 + s.D.; values for R. + Propranolol are significantly (P < 0.01) different from R. alone but not different from Controls.
were taken for electron microscopic analysi,s. A representative control cell showed normal ultrastructure [Fig. 2 (a)] which compares favorably with in vivo tissue. The intact sarcomere length was slightly reduced from 2.2 pha, found in tissue [16], to 1.9 pm, as the is lated cells were no longer held in tension. cf therwise, the myofibrillar structures clearly demonstrated banding and close packing. The mitochondria were of normal cylindrical shape with densely and orderly packed cristae. Ultrastructural changes due to free radical1 attack are represented in Figure 2(b). Majo’r damage to the sarcolemma was revealed with membrane loss prevalent. The mitochondriar, while appearing to be normal, tended to be randomly distributed compared to the regular spacing observed between myofibrils in control cells. Moreover, the myofibrils exhibited
690
I. Tong Mak et al.
Antiperoxidant
Effect
of Propranolol
on Myocytes
69
FIGURE 2. Transmission electron micrograph of isolated myocytes incubated under different conditions fox . 20 min. (a) Control. The myocyte had normal ultrastructure. X25000. (b) T wenty min exposure to free radicals. Dam .age to the sarcolemma and the a&n-Z line interactions, derangements of the mitochondria and myofibrils, were obserl red. X22000, (c) Propranolol (200 PM) and free radical exposure. Propranolol pretreatment completely preserved the normal myocytic ultrastructure. X22000; (d) Atenolol (200 PM) and free radical exposure. Atenolol pretreater Lent provided no protection against the ultrastructural alternations shown in (b). X22000.
I. Tong
692
severe derangement in banding pattern, and the 2 lines become wider and more diffuse, suggesting possible breakdown in the actin-Z line interactions. Finally, swelling and rupture of the T-tubules were observed as large vacuoles throughout the cells. Cells preincubated with propranolol or atenolol followed by free radical exposure, are shown in Figures 2(c) and 2 (d), respectively. The propranololtreated cells exhibited complete ultrastructural preservation, and appeared similar to the control cells. In contrast, the atenololtreated’myocytes showed no such protection and revealed similar damage to the untreated cells. Cellular viability Addition of the free radical components to the isolated myocytes resulted in a time dependent loss in cellular viability as assessed by trypan blue penetration (Fig. 3); by 40 min, 72% of the initial viable cells were permeable to the dye. Controls samples incubated in the buffer alone for 40 min only experienced a minor loss ( lO-l5o/o) in viability. Pretreatment of the free radical exposed cells with 200 PM propranolol effectively prevented the induced loss of viability; the resulting y. of viable cells following both 20 and 40 min incubation were not statistically different from
Time
Mak
et al.
controls. Lower concentrations of propranolol (100 pM and 50 ,UM) provided intermediate levels of viability protection (Fig. 3 inset). On the other hand, pretreatment of the samples with 200 pM atenolol produced no significant protection during the entire period of incubation (Fig. 3). Phospholipid losses At the end of incubation (40 min), additional sample aliquots (1 ml cell suspensions) were processed for total cellular lipid determination, and changes in PC and PE, the two major membrane structural phospholipids, were analyzed by HPLC. Figure 4 represents the elution profiles of the lipid samples from the selected incubation conditions. As indicated, incubation of the cells wi,th free radicals resulted in large losses of both PE and PC. Reduction in PS/PI was also evident as reflected by the decreased elution peak near the retention time 6.2. The inclusion of propranolo1 (200 PM) during incubation prevented the degradation of these phospholipids. Figure 5 summarizes the changes of PE and PC, each expressed as o/0 control of the integrated PE or PC peak area within the same preparation. Free radical treatment alone induced a 57% reduction in PE, and a 32% reduction in PC signals. Since phospholipid UV absorbance is
(min)
FIGURE 3. Protective effect of propranolol against free radical-mediated loss of myocytic viability. Myocytes were incubated under the same conditions as described in Figure 1. After 20 and 40 min of incubation, the samples were assayed for cellular viability which is expressed as the percentage of initial cells (time zero) that excluded Trypan blue (0.1% w/v). n = 4-8 + SD.; values for R. + Propranolol (20 and 40 min) are significantly (P < 0.05) different from R. alone but not different from controls. Inset: Concentration-dependent protective effect of propranolol on viability; samples were fkom 40 min of incubation and values are means of 2 to 6 separate preparations.
Antiperoxidant
Effect of Propranolol
on Myocytes
693 PE
D I
PC
PE
: N
I Control
R* alone
R- + propranolol
FIGURE 4. HPLC separation of the lipid extracts from cell samples incubated with buffer alone (control), with free radicals (R.) or with free radicals plus propranolol (R. + prop.). The lipid extracts were derived from samples after 40 min of incubation under the same conditions as described in Figure 1. Chromatographic conditions: column 5 /L Ultrasphere Si analytical column; eluent solvent, acetonitrile: methanol: HzSO 4, 100:3:0.1; flow rate, 1 ml/min under isocratic condition; detector, ultraviolet absorbance
primarily reflected by the level of unsaturated fatty acids present in each species, we subsequently collected the phospholipid peaks eluted from the HPLC and quantified the changes in PE and PC by inorganic phosphate assay. The results of 334 separate determinations indicated that the mol o/o PE loss is
about 28% (72.2 f 13.3% control), and that of PC is about 24% (76.1 f 7.8% control). Both the UV absorbance and the inorganic phosphate method indicates that propranolol pretreatment prevented these losses induced by free radicals (Fig. 5). Atenolol, at 200 PM, did not prevent the free radical induced losses of PE and PC (data not shown).
Discussion
R.
R- + Prop. PE
R.
R. + Prop. PC
FIGURE 5. Protective effect ofpropranolol against free radical-mediated PE and PC losses in myocytes. Experimental conditions were as described in Figures 1 and 3. Values for PE and PC are expressed as y0 UV signal compared to the corresponding phospholipid peaks of the control samples. n = 4 f SD.; values for R. alone are significantly (P < 0.05) different from R. + propranolol which are not different from controls.
we have shown that 200 ,UM propranolol inhibited about 85% of the free radical induced TBA-reactive product formation. Due to its sensitivity and simplicity, the TBA method is the most common assay used to determine membrane lipid peroxidation in vitro. However due to the potential non-specificity of the TBA reaction, the assay can only be used as a qualitative indicator of lipid peroxidation [3]. Nevertheless, the samples exposed to the free radicals did accumulate substantial amounts of TBA reactive substances suggestive of peroxidative degradation of unsaturated phospholipids. The observed major losses in PC and PE measured by HPLC and inorganic phosphate serves to confirm this occurrence. The greater losses in PE compared to PC is consistent with the higher level of polyunsaturated fatty acids present in PE which renders
694
I. Tong l&k
it more susceptible to free radical attack. However, the present data do not rule out the participation of phospholipase activation; the phospholipid losses could result in part from phospholipase-mediated lipolytic reactions. With highly purified hepatic lysosomes, we have demonstrated that peroxidized phospholipids might be more susceptible to lipase degradation [,?I]. It remains to be determined whether a similar process would have occurred in our cellular system. Since free radicals were generated in the extracellular media, presumably the initial membrane damage occurred at the sarcolemmal site which then led to the loss of permeability and cell death. This is supported by the observed increased release of LDH and loss of the permeability barrier to trypan blue during free radical exposure. Ultrastructural analysis by electron microscopy revealed not only that the sarcolemmal membrane was damaged, but that both mitochondrial and myofibril arrangements were disrupted. Propranolol, but not atenolol, pretreatment effectively reduced the induced peroxide formation and the associated LDH release. Subsequently, losses in cellular viability and phospholipids were prevented. In confirmation of these biochemical measurements, propranolol pretreatment completely preserved the ultrastructure of the cell. We have previously shown that several beta-blockers possess various degrees of antiperoxidative effects on isolated sarcolemmal membranes; propranolol, being highly lipophilic, was the most potent agent [13]. The present study demonstrated that propranolol at 200 FM effectively protected the isolated myocytes against oxygen radical-mediated injury. For comparison, atenolol, which was shown to exhibit only a modest antiperoxidative effect on the membrane model, provided little protection against the cellular peroxidative injury. Separate experiments were performed which indicated that propranolol at 200 PM did not affect the rate of DHF autooxidation or superoxide anion generation in our system [I.!?]. In a more recent study, the anti-radical effects of propranolol has been further studied by ESR spin trapping technique [II]. It was found that propranolol (500 PM) only had a minor scavenging effect (less than 20%) on the hydroxyl radicals generated
et al.
in the aqueous phase of the system. However, pretreatment of the sarcolemmal membranes with propranolol (up to 100 ,UM) effectively blocked the free radical-induced carboncentered radicals generated from the membrane phospholipids. In addition, D- and Lpropranolol were found equally effective. The combined data suggest that, rather than betablockade, it is the lipophilic interaction between propranolol and the cell membrane that interrupts the free radical chain reaction and thereby provide its anti-radical protection. Atenolol is about 3 orders of magnitude less soluble in lipid than propranolol [5J. Presumably, the atenolol-membrane interaction would be substantially less and thus, provide only minimal protection against free radical injury of the cell. In unpublished data, when the myocytes were pretreated with lidocaine (200 PM), which is a well-known local anesthetic and is relatively lipophilic, no protection against either the induced lipid peroxidation or loss of viability was observed. This observation suggests that the protective effects of propranolol depend on its “true” antioxidant activity rather than on its lipid solubility alone. In conclusion, the study has demonstrated that the membrane anti-peroxidative properties of propranolol could be translated into cytoprotective effects at the myocellular level. However, the clinical relevance of these findings should be interpreted with caution, since the concentrations of drug required ( > 50 PM) to mediate the protective effects were notably higher than the therapeutic concentrations [5J. Nevertheless, long term administration of propranolol may produce much higher accumulation in myocardial tissue [17,23]. In a study using Purkinje fibers [17J, a 40-fold accumulation of propranolol was observed at equilibrium which required 180 min of incubation. It was also demonstrated that platelets could accumulate propranolol lo-30-fold over plasma concentrations [,!?.?I. These findings suggest that longer time of incubation may lead to higher membrane concentration, though the process might depend not only on the lipophilicity of the drug, but aiso on other cellular uptake mechanisms, Thus the combined information and our experimental results suggest the possibility that propranolol may mediate a similar myocellular protective
Antiperoxidant
Effect of ProPranolol
on Myoeytes
695
effect in vivo, especially when excessive free from the American Heart Association, radicals are present during ischemia- Nation’s Capitol AEiliate, and by NIH grants POl-HL38079 and ROl-HL36418. The reperfusion episodes. authors wish to thank Lisa Kopyta and Tony Hursey for providing excellent technical asAcknowledgements sistance,and Leila Binder for performing part This work was supported by a Grant-in-Aid of the lipid analysis.
References 1
E., DORFMAN, L. E., WACKIER, W. E. C. Serum lactic dehydrogenase activity: an analytical assessment IDA assays. Clin Chem 9,391-399 (1963). 2 BAKER, J. E., FELIX, C. C., OLINGER, G. N., KALYANABAMAN, B. Myocardial ischemia and reperlusion: direct evidence for free radical generation by electron spin resonance spectroscopy. Proc Natl Acad Sci 85, 2786-2789 (1988). 3 BIRD, R. P., DRAPER, H. H. Comparative studies on different methods of malonaldehyde determination. Met.h Enzymol105,299-305 (1984). 4 CONOLLY, M. E., KERSTING, F., DELLERY, C. T. The clinical pharmacology of beta-adrenoceptor-blocking drugs. Progr Cardiovasc Dis 19,203-234 ( 1972). 5 CRUICKSHANK, J. M., The clinical importance of cardioselectivity and lipophilicity in beta blockers. Am Heart J llMt, 160-178 (1980). 6 FLAHERTY, J. T., WEISFELDT, M. L. Reperfusion injury. Free Radical Biol Med 5,409-419 (1988). 7 GOS~IN, S. A., FRIDOVICH, I. The role of superoxide radical in a nonenzymatic hydroxylation. Arch Biochem Biophys 153,778783 (1977). 8 KRAMER, J. H., ARROYO, C. M., DICKENS, B. F., WEGLICKI, W. B. Spin trapping evidence that graded myocardial ischemia alters post-ischemic superoxide production. Free Radical Biol Med 3, 153-159 (1987). 9 KRAMER, J. H., MAK, I. T., WEGLICKI, W. B. Differential sensitivity of sarcolemmal and microsomal enzymes 1.0 inhibition by free radical-induced lipid peroxidation. Circ Res 55, 120-124 (1984). 10 LEFKOWITZ, R. J. Direct binding studies of adrenergic receptors: biochemical, physiological and clinical implications. Ann Intern Med 91,45&458 (1976). 11 MAK, I. T., ARROYO, C. M., WEGLICKI, W: B. Inhibition ofsarcolemmal carbon-centered free radical formation by propranolol. Circ Res 65, 1151-l 156 (1989). 12 MAK, I. T., MISRA, H. P., WEGLICIU, W. B. Temporal relationship of free radical-induced lipid peroxidation and loss of latent enzyme activity in highly enriched hepatic lysosomes. J Biol Chem 258, 13733-13737 (1983). 13 MAX, I. T., WEGLICKI, W. Protection by beta-blocking agents against free radical-mediated sarcolemmal lipid peroxidation. Circ Res 63, 262-266 (1988). 14 MARINETTI, G. V. Chromatographic separation, identification and analysis ofphospholipids. J Lipid RES 3, l-l 1 (1962). 15 MEERSON, F. Z., KAGAN, V. E., KOZLOV, Y. P., BELKINA, L. M., ARKHIPENKO, Y. V. The role oflipid peroxidation in pathogenesis of ischemic damage and antioxidant protection of the heart. Basic Res Cardiol77,465-48 1 ( 1982). 16 PIPER, H. M., PROBST, I., SCHWARTZ, P. SPAHR, R., SPIECKERMANN, P. G. The adult heart cell maintained in culture. In: The Heart Cell in C&are, Pinson, A. (Ed.) Vol. III. pp. 49-75, CRC Press, Inc., Boca Raton, Fl. (1987). ! 7 PRUETT, J. K., WALLE, T., WALLE, U. K. Propranolol effects on membrane repolarization tissue content and the influence of exposure time. J Pharmacol Exp Ther 215,539543 ( 1980). 18 RAO, P. S., COHEN, M. V., MUELLER, H. S. Production of free radicals and lipid peroxides in early experimental myocardial ischemia. J Mol Cell Cardiol 15, 7 13-7 16 ( 1983). 19 SPANIER, A. M., WEGLICKI, W. B. Calcium-tolerant adult canine myocytes: preparation and response to anoxia/acidosis. Am J Physiol243, H44&455 (1982). 20 WEGLICKI, W. B., ARROYO, C. M., KRAMER, J. H., MAK, I. T., LEIBOFF, R. H., MERGNER, G. W., DICKENS, B. F. Applications of spin trapping/ESR techniques in models of cardiovascular injury. In: Oxy-Radicals in Molwukzr Biology and Pathology Cerutti, P. A., Fridovich, I. and McCord, J. H. (Eds) pp. 357-364; Alan R. Liss Inc., N.Y. (1988). 21 WEGLICKI, W. B., I~CKENS, B. F., MAK, I. T. Enhanced lysosomal phospholipid degradation and lysophospholipid production due to free radicals. Biochem Biophys Res Commun 124, 229-235 (1984). 22 WEGLICKI, W. B., KRAMER, J. H., MAK, 1. T., DICKENS, B. F., PHILLIPS, T. M. Biochemistry of sarcolemma of isolated cardiocytes. In: Isolated Adult Cmdiocytes Piper, H. and Isenberg, G. (Eds) Vol I. pp. 145-161, CRC Press, Inc., Boca Raton, Fl. (1988). 23. WEKSLER, B. B., GILLICK, M., PINK, J. Effect of propranolol on platelet function. Blood 49, 183-196 (1977). AMADOR,
current
Mol Cell Cardiol22,
687-695
Oxygen
I. Tong
(1990)
Radical-mediated
Mak,
Injury by Propranolol
of Myocytes-protection
Jay H. Kramer, Anthony M. Freedman, and William B. Weglicki
Suzanne
Y. H. Tse
Department of Medicine, Division of Experimental Medicine, George Washington University Medical Center, 2300 Eye Street NW, Washington, DC 20037, USA (Received 22 June 1989, accepted in revised form 17 January 1990) I. T. MAK, J. H. KRAMER, A. M. FREEDMAN, S. Y. H. TSE AND W. B. WEGLICKI. Oxygen Radical-mediated Injllry of Myocytes-protection by Propranolol. 3ournnl of Molecular and Cellular Cardiology (1990) 22, 687495. The effects of propranolol and atenolol on free radical mediated injury in myocytes were examined. Freshly isolated adult canine myocytes were incubated with a superoxide generating (from dihydroxyfumarate) and Fecatalyzed free radical system. Exposure of the myocytes to free radicals for 20 min resulted in more than a 5-fold increase in thiobarbituric acid reactant (peroxide) formation and elevated levels of lactate dehydrogenase (LDH) activity released into the media compared to controls. Ultrastructurally, severe sarcolemmal damage, mitochondrial and myotibril derangements were evident. At 40 min, cellular viability (trypan blue exclusion) in the samples exposed to free radicals decreased to about one-third of controls; concomitantly, major losses in total cellular phospholipids occurred. When the cells were pretreated with 200 PM propranolol before the addition of free radicals, both peroxide formation and increased LDH release were inhibited; in agreement, complete ultrastructural preservation was observed. In addition, the subsequent losses in cellular viability and phospholipids were prevented. For comparison, the more water soluble beta-blocker, atenolol at 200 FM was shown ineffective in providing significant protection against the induced injury. The results suggest that propranolol may provide antiperoxidative protection to myocytes when elevated levels of free radicals are present. KEY WORDS: Free radicals; Viability; Phospholipids;
Isolated Propranolol
canine and
myocytes; atenolol.
Introduction Due to its amphiphilic nature, propranolol binds to non-receptor hydrophobic membrane sites and may provide membrane activity independent of its adrenergic blocking effect [4, lo]. During myocardial ischemia, free oxygen radicals have been documented to increase upon reperfusion [Z, 8, 181; and free radicalmediated lipid peroxidation has been implicated to play a major role in the pathogenesis of ischemic/reperfusion injury [S, 1.5, 201. The phospholipid-rich sarcolemma of ventricular myocytes may be a critical site of free radical reaction; we previously demonstrated that purified sarcolemmal membranes from canine myocytes are highly susceptible to free radical-mediated peroxidative attack [9]. In a recent study, by using the isolated sarPIease address George Washington 002-2828/90/060687
all
correspondence to: I. T. Mak, Ph.D., University Medical Center, 2300 Eye + 09 $03.00/O
Lipid
peroxidation;
Membrane
damage;
Ultrastructure;
colemmal membrane preparations, we have demonstrated that propranolol in micromolar concentration provided significant protection against free radical-mediated lipid peroxidation [13]. In the present study, we sought to determine whether the membrane protective effect of propranolol against free radical injury might be translated into cytoprotective effects at the myocellular level. For this purpose, freshly isolated myocytes from adult canine ventricular tissue were exposed to exogenously generated oxygen free radicals in the presence or absence of the beta-blockers, and both biochemical and morphological endpoints (of myocytic injury were monitored. The results suggest that propranolol may provide antiperoxidative protection to myocytes when excessive free radicals are present. Division of Experimental Street NW, Washington,
DC
Medicine, 20037,
0
1990
Ross USA. Academic
Hall, Press
Rm.
45t2,
Limited
688
I. Tong Mak et al. Materials
and Methods Chemicals
D,L-Propranolol, atenolol, dihydroxyfumarate (DHF), ADP, FeCls and P-thiobarbituric acid (TBA) were obtained from Sigma Chemical. Isolation of myocytes Adult canine myocytes were isolated from ventricular tissue by a modification of the enzymatic digestion procedure [19] described recently [Z,?]. The final preparation is typically 90% to 95% free of contaminating smooth muscle and non-myocytic cells. Incubation procedure The isolated myocytes (1.5 x 106/ml) were resuspended in the incubation buffer consisting of 5 mM Na-acetate, 1.2 mM MgS04, 120 mM NaCl, and 10 mM potassium phosphate, pH 7.2. Calcium was not included due to its interference with our iron-catalyzed free radical generation system. The cells were preincubated under continuous gassing with 95% oxygen, 5% carbon dioxide, with or without the selected beta-blocker (50-200 PM) for 10 min at 30°C. Oxygen free radicals were generated by the final additions of Fe3+-ADP (0.025 mM FeC13 chelated by 0.250 mM ADP) and 1.67 mM dihydroxyfumarate which generates superoxide anions and hydroxyl radicals [7, 11, 121. At various times of incubation, aliquots of the cellular suspension were retrieved for determinations of TBA-reactive substances, free and cell-bound activities of lactate dehydrogenase (LDH), trypan blue exclusion cellular contents of and phospholipids. Assays and measurements Lipid peroxidation in the cell samples were assessed as the formation of the TBA reactive substances which were determined by the calorimetric method described previously [12] and expressed as malondialdehyde (MDA) equivalents. To prevent nonspecific color formation, O.Olo/o BHT was included during the heating step, and the temperature was maintained at 80°C. Under these conditions, no interference from either propranolol or atenolol (up to 200 PM) was found. LDH
activities in both the incubation media and in the cell pellet (homogenized in 0.1 o/o TritonX-100) were determined at 30°C by measuring the oxidation rate of lactate to pyruvate which was followed spectrophotometrically at 340 nm due to NAD+/NADH conversion [I]. Viability of myocytes was monitored morphologically and by trypan blue (0.1%) exclusion. Ultrastructural studies Samples taken for electron microscopic analysis after 20 min incubation, were fixed overnight in 3% glutaraldehyde, 0.1 M phosphate buffer at pH 7.4. Subsequently, the cells were pelleted and post-fixed with 1 o/o osmium tetroxide for l-2 h. The samples were then dehydrated through a graded series of ethanol, infiltrated with propylene oxide before embedding in Epon 812. Ultrathin sections (60 to 90 nm) were counter stained with lead citrate and uranyl acetate and examined in a JEOL 1OOB transmission electron microscope. Lipid extraction and ana&ses Total myocytic lipids were extracted by the modified procedure of Bligh and Dyer as described previously [21]. Phospholipids were separated by high pressure liquid chromatography (HPLC) on a 5 p Ultrasphere Si analytical column (25 cm) under isocratic condition with a solvent system consisting of acetonitrile: methanol: HzS04, 100 : 3 : 0.1. The solvent mixture was vacuum-filtered through 0.2 p nylon filters, purged and saturated with helium gas before use. The elutions were monitored at 210 nm; different phospholipid species were identified by comparing the retention times to those of standards. Signals and areas were recorded and integrated with a Shimatzu Chromatopac C-R3A integrator. Semiquantitative changes in total cellular phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the presence of free radicals with or without beta-blockers were expressed as percentage of control samples (myocytes in buffer alone). More quantitative estimation of the phospholipid losses in the PC and PE due to free radicals were determined by inorganic phosphate assay [Ia, 211.
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Results Lipid peroxidation and L DH release The employed isolation procedure yielded 65 to 85% rod-shaped myocytes which excluded trypan blue dye indicating a good preservation of sarcolemmal integrity. We have previously demonstrated that isolated sarcolemmal membranes were relatively sensitive to oxygen free radical-mediated lipid peroxidation as assessed by the TBA-MDA assay [9]. In the present study, when the ventricular myocytes were incubated with the superoxide anion generating, iron-catalyzed free radical system for 20 min, a 5-fold higher level of TBAreactive substances (or MDA equivalents) accumulated in the samples compared to the controls [Fig. 1 (a)]. Concomitantly, higher levels of LDH activities were released from cell samples exposed to free radicals for 20 min, suggesting the development of increased sarcolemmal membrane permeability [Fig. 1 (b)]. We have demonstrated that propranolol, a highly lipophilic beta-blocker, was most effective in inhibiting free radicalinduced lipid peroxidation in isolated sarcolemmal membranes [13]. When the myocytes were pretreated with 20Op~ propranolo1 before adding the free radical components, the induced TBA-reactive substances were inhibited 85%, and the level of LDH release was not statistically different from that of controls [Fig. 1 (a, b)]. Propranolol at 100 PM and 50 ,UM provided 69% and 49%, respectively, inhibition of the TBA-reactant formation. For the purpose of clarity, only data for 200 pM propranolol were presented. For comparison, when the cell samples were pretreated with the more water soluble beta-blocker atenolol (200 PM), the induced formation of the TBA-reactive substances was not inhibited [Fig. 1 (a)]; nor was the increased LDH release significantly reduced [Fig. 1 (b)]. Electron micrograph Up to 20 min of incubation, light level morphological examinations indicated no substantial changes in the percentage of rod-shaped myocytes with or without exposing to free radicals. To confirm the free radical-induced membrane damage and the effects of the twobeta-blockers, samples at 20 min of incubation
Cont.
R.
R. + Prop R. + Aten
FIGURE 1. Effects of propranolol and atenolol on free radical-mediated lipid peroxidation (a) and LDH release (b) in isolated canine myocytes. Controls were cell samples incubated in buffer alone. Experimental samples were preincubated with or without the selected beta-blocker 1200 P’M each) for 10 min before the addition of [Fe-ADP and DHFl CR.). After 20 min ofincubation. samoles were assayed for net LDH release (expressed as 0/0 total1 activity) and total TBA-reactive substance (TBA-RS) formation as described in the text. jV = 4-6 + s.D.; values for R. + Propranolol are significantly (P < 0.01) different from R. alone but not different from Controls.
were taken for electron microscopic analysi,s. A representative control cell showed normal ultrastructure [Fig. 2 (a)] which compares favorably with in vivo tissue. The intact sarcomere length was slightly reduced from 2.2 pha, found in tissue [16], to 1.9 pm, as the is lated cells were no longer held in tension. cf therwise, the myofibrillar structures clearly demonstrated banding and close packing. The mitochondria were of normal cylindrical shape with densely and orderly packed cristae. Ultrastructural changes due to free radical1 attack are represented in Figure 2(b). Majo’r damage to the sarcolemma was revealed with membrane loss prevalent. The mitochondriar, while appearing to be normal, tended to be randomly distributed compared to the regular spacing observed between myofibrils in control cells. Moreover, the myofibrils exhibited
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FIGURE 2. Transmission electron micrograph of isolated myocytes incubated under different conditions fox . 20 min. (a) Control. The myocyte had normal ultrastructure. X25000. (b) T wenty min exposure to free radicals. Dam .age to the sarcolemma and the a&n-Z line interactions, derangements of the mitochondria and myofibrils, were obserl red. X22000, (c) Propranolol (200 PM) and free radical exposure. Propranolol pretreatment completely preserved the normal myocytic ultrastructure. X22000; (d) Atenolol (200 PM) and free radical exposure. Atenolol pretreater Lent provided no protection against the ultrastructural alternations shown in (b). X22000.
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severe derangement in banding pattern, and the 2 lines become wider and more diffuse, suggesting possible breakdown in the actin-Z line interactions. Finally, swelling and rupture of the T-tubules were observed as large vacuoles throughout the cells. Cells preincubated with propranolol or atenolol followed by free radical exposure, are shown in Figures 2(c) and 2 (d), respectively. The propranololtreated cells exhibited complete ultrastructural preservation, and appeared similar to the control cells. In contrast, the atenololtreated’myocytes showed no such protection and revealed similar damage to the untreated cells. Cellular viability Addition of the free radical components to the isolated myocytes resulted in a time dependent loss in cellular viability as assessed by trypan blue penetration (Fig. 3); by 40 min, 72% of the initial viable cells were permeable to the dye. Controls samples incubated in the buffer alone for 40 min only experienced a minor loss ( lO-l5o/o) in viability. Pretreatment of the free radical exposed cells with 200 PM propranolol effectively prevented the induced loss of viability; the resulting y. of viable cells following both 20 and 40 min incubation were not statistically different from
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controls. Lower concentrations of propranolol (100 pM and 50 ,UM) provided intermediate levels of viability protection (Fig. 3 inset). On the other hand, pretreatment of the samples with 200 pM atenolol produced no significant protection during the entire period of incubation (Fig. 3). Phospholipid losses At the end of incubation (40 min), additional sample aliquots (1 ml cell suspensions) were processed for total cellular lipid determination, and changes in PC and PE, the two major membrane structural phospholipids, were analyzed by HPLC. Figure 4 represents the elution profiles of the lipid samples from the selected incubation conditions. As indicated, incubation of the cells wi,th free radicals resulted in large losses of both PE and PC. Reduction in PS/PI was also evident as reflected by the decreased elution peak near the retention time 6.2. The inclusion of propranolo1 (200 PM) during incubation prevented the degradation of these phospholipids. Figure 5 summarizes the changes of PE and PC, each expressed as o/0 control of the integrated PE or PC peak area within the same preparation. Free radical treatment alone induced a 57% reduction in PE, and a 32% reduction in PC signals. Since phospholipid UV absorbance is
(min)
FIGURE 3. Protective effect of propranolol against free radical-mediated loss of myocytic viability. Myocytes were incubated under the same conditions as described in Figure 1. After 20 and 40 min of incubation, the samples were assayed for cellular viability which is expressed as the percentage of initial cells (time zero) that excluded Trypan blue (0.1% w/v). n = 4-8 + SD.; values for R. + Propranolol (20 and 40 min) are significantly (P < 0.05) different from R. alone but not different from controls. Inset: Concentration-dependent protective effect of propranolol on viability; samples were fkom 40 min of incubation and values are means of 2 to 6 separate preparations.
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693 PE
D I
PC
PE
: N
I Control
R* alone
R- + propranolol
FIGURE 4. HPLC separation of the lipid extracts from cell samples incubated with buffer alone (control), with free radicals (R.) or with free radicals plus propranolol (R. + prop.). The lipid extracts were derived from samples after 40 min of incubation under the same conditions as described in Figure 1. Chromatographic conditions: column 5 /L Ultrasphere Si analytical column; eluent solvent, acetonitrile: methanol: HzSO 4, 100:3:0.1; flow rate, 1 ml/min under isocratic condition; detector, ultraviolet absorbance
primarily reflected by the level of unsaturated fatty acids present in each species, we subsequently collected the phospholipid peaks eluted from the HPLC and quantified the changes in PE and PC by inorganic phosphate assay. The results of 334 separate determinations indicated that the mol o/o PE loss is
about 28% (72.2 f 13.3% control), and that of PC is about 24% (76.1 f 7.8% control). Both the UV absorbance and the inorganic phosphate method indicates that propranolol pretreatment prevented these losses induced by free radicals (Fig. 5). Atenolol, at 200 PM, did not prevent the free radical induced losses of PE and PC (data not shown).
Discussion
R.
R- + Prop. PE
R.
R. + Prop. PC
FIGURE 5. Protective effect ofpropranolol against free radical-mediated PE and PC losses in myocytes. Experimental conditions were as described in Figures 1 and 3. Values for PE and PC are expressed as y0 UV signal compared to the corresponding phospholipid peaks of the control samples. n = 4 f SD.; values for R. alone are significantly (P < 0.05) different from R. + propranolol which are not different from controls.
we have shown that 200 ,UM propranolol inhibited about 85% of the free radical induced TBA-reactive product formation. Due to its sensitivity and simplicity, the TBA method is the most common assay used to determine membrane lipid peroxidation in vitro. However due to the potential non-specificity of the TBA reaction, the assay can only be used as a qualitative indicator of lipid peroxidation [3]. Nevertheless, the samples exposed to the free radicals did accumulate substantial amounts of TBA reactive substances suggestive of peroxidative degradation of unsaturated phospholipids. The observed major losses in PC and PE measured by HPLC and inorganic phosphate serves to confirm this occurrence. The greater losses in PE compared to PC is consistent with the higher level of polyunsaturated fatty acids present in PE which renders
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it more susceptible to free radical attack. However, the present data do not rule out the participation of phospholipase activation; the phospholipid losses could result in part from phospholipase-mediated lipolytic reactions. With highly purified hepatic lysosomes, we have demonstrated that peroxidized phospholipids might be more susceptible to lipase degradation [,?I]. It remains to be determined whether a similar process would have occurred in our cellular system. Since free radicals were generated in the extracellular media, presumably the initial membrane damage occurred at the sarcolemmal site which then led to the loss of permeability and cell death. This is supported by the observed increased release of LDH and loss of the permeability barrier to trypan blue during free radical exposure. Ultrastructural analysis by electron microscopy revealed not only that the sarcolemmal membrane was damaged, but that both mitochondrial and myofibril arrangements were disrupted. Propranolol, but not atenolol, pretreatment effectively reduced the induced peroxide formation and the associated LDH release. Subsequently, losses in cellular viability and phospholipids were prevented. In confirmation of these biochemical measurements, propranolol pretreatment completely preserved the ultrastructure of the cell. We have previously shown that several beta-blockers possess various degrees of antiperoxidative effects on isolated sarcolemmal membranes; propranolol, being highly lipophilic, was the most potent agent [13]. The present study demonstrated that propranolol at 200 FM effectively protected the isolated myocytes against oxygen radical-mediated injury. For comparison, atenolol, which was shown to exhibit only a modest antiperoxidative effect on the membrane model, provided little protection against the cellular peroxidative injury. Separate experiments were performed which indicated that propranolol at 200 PM did not affect the rate of DHF autooxidation or superoxide anion generation in our system [I.!?]. In a more recent study, the anti-radical effects of propranolol has been further studied by ESR spin trapping technique [II]. It was found that propranolol (500 PM) only had a minor scavenging effect (less than 20%) on the hydroxyl radicals generated
et al.
in the aqueous phase of the system. However, pretreatment of the sarcolemmal membranes with propranolol (up to 100 ,UM) effectively blocked the free radical-induced carboncentered radicals generated from the membrane phospholipids. In addition, D- and Lpropranolol were found equally effective. The combined data suggest that, rather than betablockade, it is the lipophilic interaction between propranolol and the cell membrane that interrupts the free radical chain reaction and thereby provide its anti-radical protection. Atenolol is about 3 orders of magnitude less soluble in lipid than propranolol [5J. Presumably, the atenolol-membrane interaction would be substantially less and thus, provide only minimal protection against free radical injury of the cell. In unpublished data, when the myocytes were pretreated with lidocaine (200 PM), which is a well-known local anesthetic and is relatively lipophilic, no protection against either the induced lipid peroxidation or loss of viability was observed. This observation suggests that the protective effects of propranolol depend on its “true” antioxidant activity rather than on its lipid solubility alone. In conclusion, the study has demonstrated that the membrane anti-peroxidative properties of propranolol could be translated into cytoprotective effects at the myocellular level. However, the clinical relevance of these findings should be interpreted with caution, since the concentrations of drug required ( > 50 PM) to mediate the protective effects were notably higher than the therapeutic concentrations [5J. Nevertheless, long term administration of propranolol may produce much higher accumulation in myocardial tissue [17,23]. In a study using Purkinje fibers [17J, a 40-fold accumulation of propranolol was observed at equilibrium which required 180 min of incubation. It was also demonstrated that platelets could accumulate propranolol lo-30-fold over plasma concentrations [,!?.?I. These findings suggest that longer time of incubation may lead to higher membrane concentration, though the process might depend not only on the lipophilicity of the drug, but aiso on other cellular uptake mechanisms, Thus the combined information and our experimental results suggest the possibility that propranolol may mediate a similar myocellular protective
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effect in vivo, especially when excessive free from the American Heart Association, radicals are present during ischemia- Nation’s Capitol AEiliate, and by NIH grants POl-HL38079 and ROl-HL36418. The reperfusion episodes. authors wish to thank Lisa Kopyta and Tony Hursey for providing excellent technical asAcknowledgements sistance,and Leila Binder for performing part This work was supported by a Grant-in-Aid of the lipid analysis.
References 1
E., DORFMAN, L. E., WACKIER, W. E. C. Serum lactic dehydrogenase activity: an analytical assessment IDA assays. Clin Chem 9,391-399 (1963). 2 BAKER, J. E., FELIX, C. C., OLINGER, G. N., KALYANABAMAN, B. Myocardial ischemia and reperlusion: direct evidence for free radical generation by electron spin resonance spectroscopy. Proc Natl Acad Sci 85, 2786-2789 (1988). 3 BIRD, R. P., DRAPER, H. H. Comparative studies on different methods of malonaldehyde determination. Met.h Enzymol105,299-305 (1984). 4 CONOLLY, M. E., KERSTING, F., DELLERY, C. T. The clinical pharmacology of beta-adrenoceptor-blocking drugs. Progr Cardiovasc Dis 19,203-234 ( 1972). 5 CRUICKSHANK, J. M., The clinical importance of cardioselectivity and lipophilicity in beta blockers. Am Heart J llMt, 160-178 (1980). 6 FLAHERTY, J. T., WEISFELDT, M. L. Reperfusion injury. Free Radical Biol Med 5,409-419 (1988). 7 GOS~IN, S. A., FRIDOVICH, I. The role of superoxide radical in a nonenzymatic hydroxylation. Arch Biochem Biophys 153,778783 (1977). 8 KRAMER, J. H., ARROYO, C. M., DICKENS, B. F., WEGLICKI, W. B. Spin trapping evidence that graded myocardial ischemia alters post-ischemic superoxide production. Free Radical Biol Med 3, 153-159 (1987). 9 KRAMER, J. H., MAK, I. T., WEGLICKI, W. B. Differential sensitivity of sarcolemmal and microsomal enzymes 1.0 inhibition by free radical-induced lipid peroxidation. Circ Res 55, 120-124 (1984). 10 LEFKOWITZ, R. J. Direct binding studies of adrenergic receptors: biochemical, physiological and clinical implications. Ann Intern Med 91,45&458 (1976). 11 MAK, I. T., ARROYO, C. M., WEGLICKI, W: B. Inhibition ofsarcolemmal carbon-centered free radical formation by propranolol. Circ Res 65, 1151-l 156 (1989). 12 MAK, I. T., MISRA, H. P., WEGLICIU, W. B. Temporal relationship of free radical-induced lipid peroxidation and loss of latent enzyme activity in highly enriched hepatic lysosomes. J Biol Chem 258, 13733-13737 (1983). 13 MAX, I. T., WEGLICKI, W. Protection by beta-blocking agents against free radical-mediated sarcolemmal lipid peroxidation. Circ Res 63, 262-266 (1988). 14 MARINETTI, G. V. Chromatographic separation, identification and analysis ofphospholipids. J Lipid RES 3, l-l 1 (1962). 15 MEERSON, F. Z., KAGAN, V. E., KOZLOV, Y. P., BELKINA, L. M., ARKHIPENKO, Y. V. The role oflipid peroxidation in pathogenesis of ischemic damage and antioxidant protection of the heart. Basic Res Cardiol77,465-48 1 ( 1982). 16 PIPER, H. M., PROBST, I., SCHWARTZ, P. SPAHR, R., SPIECKERMANN, P. G. The adult heart cell maintained in culture. In: The Heart Cell in C&are, Pinson, A. (Ed.) Vol. III. pp. 49-75, CRC Press, Inc., Boca Raton, Fl. (1987). ! 7 PRUETT, J. K., WALLE, T., WALLE, U. K. Propranolol effects on membrane repolarization tissue content and the influence of exposure time. J Pharmacol Exp Ther 215,539543 ( 1980). 18 RAO, P. S., COHEN, M. V., MUELLER, H. S. Production of free radicals and lipid peroxides in early experimental myocardial ischemia. J Mol Cell Cardiol 15, 7 13-7 16 ( 1983). 19 SPANIER, A. M., WEGLICKI, W. B. Calcium-tolerant adult canine myocytes: preparation and response to anoxia/acidosis. Am J Physiol243, H44&455 (1982). 20 WEGLICKI, W. B., ARROYO, C. M., KRAMER, J. H., MAK, I. T., LEIBOFF, R. H., MERGNER, G. W., DICKENS, B. F. Applications of spin trapping/ESR techniques in models of cardiovascular injury. In: Oxy-Radicals in Molwukzr Biology and Pathology Cerutti, P. A., Fridovich, I. and McCord, J. H. (Eds) pp. 357-364; Alan R. Liss Inc., N.Y. (1988). 21 WEGLICKI, W. B., I~CKENS, B. F., MAK, I. T. Enhanced lysosomal phospholipid degradation and lysophospholipid production due to free radicals. Biochem Biophys Res Commun 124, 229-235 (1984). 22 WEGLICKI, W. B., KRAMER, J. H., MAK, 1. T., DICKENS, B. F., PHILLIPS, T. M. Biochemistry of sarcolemma of isolated cardiocytes. In: Isolated Adult Cmdiocytes Piper, H. and Isenberg, G. (Eds) Vol I. pp. 145-161, CRC Press, Inc., Boca Raton, Fl. (1988). 23. WEKSLER, B. B., GILLICK, M., PINK, J. Effect of propranolol on platelet function. Blood 49, 183-196 (1977). AMADOR,
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