Heart Sarcolemmal Research Articles

Alterations of sarcolemmal phospholipase D and phosphatidate phosphohydrolase in congestive heart failure

Phospholipase D 2 (PLD2) is the major PLD isozyme associated with the cardiac sarcolemmal (SL) membrane. Hydrolysis of SL phosphatidylcholine (PC) by PLD2 produces phosphatidic acid (PA), which is then converted to 1,2 diacylglycerol (DAG) by the action of phosphatidate phosphohydrolase type 2 (PAP2). In view of the role of both PA and DAG in the regulation of Ca 2+ movements and the association of abnormal Ca 2+ homeostasis with congestive heart failure (CHF), we examined the status of both PLD2 and PAP2 in SL membranes in the infarcted heart upon occluding the left coronary artery in rats for 1, 2, 4, 8 and 16 weeks. A time-dependent increase in both SL PLD2 and PAP2 activities was observed in the non-infarcted left ventricular tissue following myocardial infarction (MI); however, the increase in PAP2 activity was greater than that in PLD2 activity. Furthermore, the contents of both PA and PC were reduced, whereas that of DAG was increased in the failing heart SL membrane. Treatment of the CHF animals with imidapril, an angiotensin-converting enzyme (ACE) inhibitor, attenuated the observed changes in heart function, SL PLD2 and PAP2 activities, as well as SL PA, PC and DAG contents. The results suggest that heart failure is associated with increased activities of both PLD2 and PAP2 in the SL membrane and the beneficial effect of imidapril on heart function may be due to its ability to prevent these changes in the phospholipid signaling molecules in the cardiac SL membrane.

Ca2+-antagonists inhibit the N-methyltransferase-dependent synthesis of phosphatidylcholine in the heart.

Evidence indicates that, in addition to the L-type Ca2+ channel blockade, Ca2+-antagonists target other functions including the Ca2+-pumps. This study was conducted to test the possibility that the reported inhibition of heart sarcolemmal (SL) and sarcoplasmic reticular (SR) Ca2+-pumps by verapamil and diltiazem could be due to drug-induced depression of phosphatidylethanolamine (PE) N-methylation which modulates these Ca2+-transport systems. Three catalytic sites individually responsible for the synthesis of PE monomethyl (site I), dimethyl (site II) and trimethyl (phosphatidylcholine (PC), site III) derivates were examined in SL and SR membranes by employing different concentrations of S-adenosyl-L-methionine (AdoMet). Total methyl group incorporation into SL PE, in vitro, was significantly depressed by 10(-6)-10(-3) M verapamil or diltiazem at site III. The catalytic activity of site I was inhibited by 10(-3) M verapamil only, whereas the site II activity was not affected by these drugs. The inhibition induced by verapamil or diltiazem (10(-5) M) was associated with a depression of the Vmax value without any change in the apparent affinity for AdoMet. Both drugs decreased the SR as well as mitochondrial PE N-methylation at site III. A selective depression of site III activity was also observed in SL isolated from hearts of rats treated with verapamil in vivo. Furthermore, administration of [3H-methyl]-methionine following the treatment of animals with verapamil, reduced the synthesis of PC by N-methyltransferase. Verapamil also depressed the N-methylation-dependent positive inotropic effect induced by methionine in the isolated Langendorff heart. Both agents depressed the SL Ca2+-pump and although diltiazem also inhibited the SR Ca2+-pump, verapamil exerted a stimulatory effect. In addition, verapamil decreased SR Ca2+-release. These results suggest that verapamil and diltiazem alter the cardiac PE N-methyltransferase system. This action is apparently additional to the drugs' effect on L-type Ca2+ channels and may serve as a biochemical mechanism for the drugs' inhibition of the cardiac Ca2+-pumps and altered cardiac function.

Changes in sarcolemmal PLC isoenzymes in postinfarct congestive heart failure: partial correction by imidapril.

We have examined the changes in quantity and activity of cardiac sarcolemmal (SL) phosphoinositide-phospholipase C (PLC)-beta(1), -gamma(1), and -delta(1) in a model of congestive heart failure (CHF) secondary to large transmural myocardial infarction (MI). We also instituted a late in vivo monotherapy with imidapril, an ANG-converting enzyme (ACE) inhibitor, to test the hypothesis that its therapeutic action is associated with the functional correction of PLC isoenzymes. SL membranes were purified from the surviving left ventricle of rats in a moderate stage of CHF at 8 wk after occlusion of the left anterior descending coronary artery. SL PLC isoenzymes were examined in terms of protein mass and hydrolytic activity. CHF resulted in a striking reduction (to 6-17% of controls) of the mass and activity of gamma(1)- and delta(1)-isoforms in combination with a significant increase of both PLC beta(1) parameters. In vivo treatment with imidapril (1 mg/kg body wt, daily, initiated 4 wk after coronary occlusion) improved the contractile function and induced a partial correction of PLCs. The mass of SL phosphatidylinositol 4,5-bisphosphate and the activities of the enzymes responsible for its synthesis were significantly reduced in post-MI CHF and partially corrected by imidapril. The results indicate that profound changes in the profile of heart SL PLC-beta(1), -gamma(1), and -delta(1) occur in CHF, which could alter the complex second messenger responses of these isoforms, whereas their partial correction by imidapril may be related to the mechanism of action of this ACE inhibitor.

Phospholipase A2-Mediated Activation of Phospholipase D in Rat Heart Sarcolemma

The effect of phospholipase A2(PLA2)-dependent release of unsaturated fatty acids (FA) on phospholipase D (PLD) function was examined in purified sarcolemmal (SL) membranes isolated from rat heart. PLD hydrolytic activity was determined by measuring either [14C] phosphatidic acid formation from exogenous [14C] phosphatidylcholine (PtdCho) or [3H] choline release from prelabelled SL Ptd[3H]choline. SL membranes with endogenous [3H] PtdCho that were prelabelled with [3H] myristic acid were used for testing PLD transphosphatidylation activity. Exogenouscis-unsaturated FA, arachidonate and oleate, significantly enhanced the [3H] choline formation at 50 and 100μm, respectively; their effect was maximal at 250μmand declined at higher concentrations. Use of melittin (which stimulates membrane-bound PLA2, thus releasing FA) or exogenous PLA2reproduced the stimulatory effect of added arachidonate and oleate. Under melittin, PLA2-dependent FA release was strongly correlated (r=0.99) to the PLD-dependent phosphatidic acid formation. Arachidonate- or melittin-enhanced PLD transphosphatidylation activity confirmed the augmented catalytic rate of PLD by these agents. Melittin-evoked PLD activation was completely blocked by 1μmE-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a selective inhibitor of Ca2+-independentvCa2+-dependent PLA2, thus indicating that PLD stimulation under melittin occurred via PLA2. Activity measurement and Western blotting studies revealed the presence of a Ca2+-independent, high molecular weight (110 kDa) PLA2in the SL membrane, and its immunoprecipitation by monoclonal antibodies significantly reduced the melittin-related PLD stimulation. These results suggest that Ca2+-independent PLA2and subsequent endogenous mobilization ofsn-2 unsaturated FA modulate PLD activity in heart SL membranes. This event may occur in physiological conditions via hormonal stimulation of membranal PLA2as well as in heart diseases characterized by PLA2pathological dysfunction.

Biophysical characterization of rat cardiac Ca2+/Mg2+ ecto-ATPase (myoglein).

Sarcolemmal Ca2+/Mg2+ ecto-ATPase (Myoglein; MW 180 kD) is a membrane bound enzyme which requires a millimolar concentration of either Ca2+ or Mg2+ for maximal hydrolysis of ATP. The isoelectric point (pI) of the cardiac ecto-ATPase was 5.7. The purified Ca2+/Mg2+ ecto-ATPase from the rat heart sarcolemmal appeared as a single band with MW approximately 90 kD in the SDS-PAGE. In order to understand the nature of this enzyme, the 90 kD band in the SDS-PAGE was electroeluted; the analysis of the eluate showed 2 prominent bands with MW approximately 90 and 85 kD. The presence of 2 bands was further confirmed by gradient gel (10-20%) electrophoresis in 0.375 M Tris-HCl buffer, pH 8.8. Analysis of the purified Ca2+/Mg2+ ecto-ATPase as well as the electroeluted protein in a non-equilibrium linear two dimensional electrophoresis (Ampholyte pI 3.0-10.0) also showed two distinct bands. Mass spectroscopic analysis of the enzyme using different matrix combinations revealed the presence of multi-components indicating microheterogeneity in the protein structure. Treatment of the ecto-ATPase with DL-dithiothreitol did not alter the pattern of mass spectroscopic analysis and this indicated that the microheterogeneity may be due to some posttranslational modifications. It is concluded that rat cardiac Ca2+/Mg2+ ecto-ATPase is an acidic protein having two subunits. Furthermore, the enzyme shows microheterogeneity in its molecular structure.

Modification of heart sarcolemmal phosphoinositide pathway by lysophosphatidylcholine

Although lysophosphatidylcholine (lyso-PtdCho) accumulates in the sarcolemmal (SL) membrane and alters its function during myocardial ischemia and diabetic cardiomyopathy, the effects of lyso-PtdCho on SL signalling processes have not yet been investigated. The present study was carried out to examine the actions of lyso-PtdCho on the rat heart SL membrane enzymes involved in the phosphoinositide pathway. Different lyso-PtdCho species (10 to 200 μM) inhibited the activities of both phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase in the SL membrane in a concentration-dependent manner. The inhibitory potency of lyso-PtdCho compounds for phosphatidylinositol kinase was lyso-PtdCho plasmalogen >1-oleoyl-lyso-PtdCho >1-stearoyl-lyso-PtdCho >1-palmitoyl-lyso-PtdCho, and that for phosphatidylinositol-4-phosphate kinase was lyso-PtdCho plasmalogen >1-oleoyl-lyso-PtdCho >1-palmitoyl-lyso-PtdCho >1-stearoyl-lyso-PtdCho. The inhibitory effect of lyso-PtdCho on phosphatidylinositol-4-phosphate kinase was greater than that on phosphatidylinositol kinase. Lyso-PtdCho structural analogues, such as phosphatidylcholine, lysophosphatidic acid, lysophosphatidylethanolamine, L-α-glycerophosphate, oleate and phosphorylcholine, did not affect the phosphoinositide kinases, suggesting that the intact structure of lyso-PtdCho was required for the inhibition of the kinases. The detrimental action of lyso-PtdCho on PtdIns kinase was potentiated by acidosis. Unlike Ca 2+, ATP (0.1 and 4 mM) increased lyso-PtdCho-induced deactivation of the kinases. Both enzyme activities were found to be depressed in the ischemic-reperfused or diabetic hearts. None of the tested lyso-PtdCho species altered phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2) hydrolysis by SL phospholipase C. These results indicate that accumulation of lyso-PtdCho in the SL membrane under pathological conditions may diminish the availability of the PtdIns(4,5)P 2 substrate for the production of second messengers by receptor-linked phospholipase C.

Abnormal synthesis of n-methylated phospholipids during calcium paradox of the heart

Phosphatidylethanolamine (PtdEtn) N-methyltransferase activity that synthesizes phosphatidylcholine (PtdCho) via formation of methylated intermediates (phosphatidyl-N-monomethylethanolamine, PtdEtnMe and phosphatidyl-N,N-dimethylethanolamine, PtdEtnMe2) was comparatively studied in rat heart sarcolemmal (SL), sarcoplasmic reticular (SR) and mitochondrial fractions during Ca2+ paradox. Perfusion (5 min) with Ca(2+)-free medium followed by reperfusion (5 min) with Ca(2+)-containing medium produced a marked rise in resting tension without any recovery of contractile force. Methyltransferase catalytic sites I, II and III which synthesize PtdEtnMe, PtdEtnMe2 and PtdCho, respectively, were assayed by measuring the [3H] methyl group incorporation from 0.055, 10 and 150 microM S-adenosyl-L-[3H-methyl] methionine into membrane PtdEtn molecules. Five minutes of perfusion with Ca(2+)-free medium did not affect either SL or SR N-methyltransferase systems. Ca(2+)-readmission for 1 to 5 min induced a selective, time-dependent depression of SL site II and SR site I methyltransferase activities. Individual N-methylated phospholipids specifically formed at the two sites reflected these changes. The above abnormalities were differently influenced by the duration (1-5 min) of Ca(2+)-free perfusion and were characterized by different kinetic alterations. The mitochondrial methylation system was not affected under Ca2+ paradox. The results suggest that reduced synthesis of SL N-methylated phospholipids may contribute to the contractile dysfunction observed in Ca2+ paradox.

Characterization of adenylyl cyclase in heart sarcolemma in the absence or presence of alamethicin.

Alamethicin is commonly used as an agent for unmasking the latent enzyme activities in vesicular membrane preparations; however, relatively little is known about the effect of this agent on the characteristics of adenylyl cyclase in heart sarcolemma. By employing rat heart sarcolemmal preparation, we observed 5 to 6 fold increase in adenylyl cyclase activity upon treatment with alamethicin. Kinetic experiments using various concentrations of MgATP revealed that the increase in adenylyl cyclase activity in alamethicin treated membranes was associated with an increase in Vmax as well as affinity of the substrate for the enzyme. Dose-responses of the control and alamethicin-treated preparations to various activators of adenylyl cyclase revealed that the sensitivity of the enzyme to forskolin, NaF and GppNHp, was markedly increased upon treating sarcolemma with alamethicin. The activation of adenylyl cyclase by forskolin was also enhanced by increasing the concentration of alamethicin in the incubation medium. Furthermore, there was a greater increase in adenylyl cyclase activity with different concentrations of Mn2+ in the presence of alamethicin. These results suggest that alamethicin treatment alters the characteristics of adenylyl cyclase in addition to unmasking the enzyme activity in the purified sarcolemmal vesicular preparation.

Nitrendipine modulates the sodium binding site of cardiac (Na, K)-ATPase

The present study deals with the effect of calcium entry blocking agent, nitrendipine, on the rat heart sarcolemmal (Na, K)-ATPase. Nitrendipine ( 1μ mol 1) exerted a stimulatory effect on (Na, K)-ATPase activity. Kinetic analysis of enzyme activation by increasing concentrations of sodium in the presence of nitrendipine revealed a 67% increase in Vmax value and a decrease of the apparent Ka value to 33% of that in control. The observed influence on (Na, K)-ATPase may contribute indirectly to the calcium antagonistic effect of nitrendipine.

Effects of some L-carnitine derivatives on heart membrane ATPases

In order to understand the role of carnitine metabolites in the genesis of cellular dysfunction and damage due to myocardial ischemia, the effects of 1-100 microM L-carnitine, acetylcarnitine, propionylcarnitine, and palmitoylcarnitine were investigated on rat heart sarcolemmal, sarcoplasmic reticular, and mitochondrial ATPase activities. Palmitoylcarnitine, unlike acetylcarnitine, propionylcarnitine and carnitine, produced marked inhibitory actions on sarcolemmal Na,K-ATPase and Ca2(+)-stimulated ATPase, as well as sarcoplasmic reticular Ca2(+)-stimulated ATPase activities; Na,K-ATPase was most sensitive. Although palmitoylcarnitine, unlike carnitine or its short-chain fatty-acid derivatives, also depressed sarcolemmal Ca2+ ATPase or Mg2+ ATPase, sarcoplasmic reticular Mg2+ ATPase, and mitochondrial Mg2+ ATPase, mitochondria were less sensitive in comparison to other organelles. Myofibrillar Ca2(+)-stimulated ATPase was slightly inhibited by very high concentrations of palmitoylcarnitine only. It is suggested that the observed depression of the sarcolemmal Na(+)-pump system by low concentrations of long-chain acyl derivatives of carnitine may contribute towards the pathogenesis of arrhythmias due to myocardial ischemia. Furthermore, the inhibition of Ca2(+)-pump mechanisms in the sarcolemmal and sarcoplasmic reticular membranes by relatively high concentrations of palmitoylcarnitine may result in the occurrence of intracellular Ca2+ overload and subsequent cell damage, as well as cardiac dysfunction due to myocardial ischemia.

Immunological cross-reactivity between Streptococcus mutans and human heart tissue examined by cross-immunization experiments

Hyperimmunization of rabbits with Streptococcus mutans or other related cariogenic streptococci sometimes induces serum antibodies that react with human heart muscle. To determine whether antigen I/II (AgI/II), a major surface protein antigen present in most human isolates of these organisms, was responsible for inducing cross-reactive antibodies, we tested it for antigenic similarity to heart components, exploiting the ability of immune systems to mount anamnestic responses to antigens previously encountered. Mice immunized with a strain of Streptococcus pyogenes type M6, known to be heart cross-reactive, or with intact S. mutans cells developed antibodies that could be detected on a human heart sarcolemmal preparation. However, mice immunized with AgI/II and boosted with sarcolemma were unable to develop significant antisarcolemmal antibodies attributable to prior sensitization by AgI/II. Similarly, AgI/II was unable to recall antisarcolemmal responses in mice previously immunized with sarcolemma. Nevertheless, strong immunoglobulin G antibody responses to AgI/II were detected at the single-cell level in spleens and as circulating antibodies in all mice immunized with AgI/II or AgI/II-bearing S. mutans. We conclude that the ability of S. mutans to induce heart-reactive antibodies is not due to antigenic similarity between AgI/II and components of human heart but may be caused by other cross-reactive antigens in the bacterial cells or by nonspecific stimulation of the immune system.

Inhibition of cardiac phosphatidylethanolamine N-methylation by oxygen free radicals

This study was undertaken to examine the effects of oxygen free radicals on phosphatidylethanolamine (PE) N-methylation in rat heart sarcolemmal (SL) and sarcoplasmic reticular (SR) membranes. Three catalytic sites involved in the sequential methyl transfer reaction were studied by assaying the incorporation of radiolabeled methyl groups from S- adenosyl- l-methionine (0.055, 10, and 150 μM) into SL or SR PE molecules under optimal conditions. In the presence of xanthine + xanthine oxidase (superoxide anion radicals generating system), PE N-methylation was inhibited at site I and III in the heavy SL fraction isolated by the hypotonic shock-LiBr treatment method. In the light SL fraction isolated by sucrose-density gradient, a significant inhibition of PE N-methylation was seen at all three sites. These inhibitory effects of xanthine + xanthine oxidase on PE N-methylation were prevented by the addition of superoxide dismutase. Hydrogen peroxide showed a significant inhibition of PE N-methylation at site I in the heavy SL fraction, and at site I and II in the light SL fraction. Catalase blocked the inhibitory effects of hydrogen peroxide. The effects of both xanthine + xanthine oxidase and hydrogen peroxide on the SR membranes were similar to those seen for the heavy SL fraction. These results suggest that, in addition to lipid peroxidation, the oxygen free radicals may affect the function of cardiac membranes by decreasing the phospholipid N-methylation activity.

Stimulation of phospholipid N-methylation by isoproterenol in rat hearts.

Phosphatidylethanolamine (PtdEtn) N-methyltransferase activities were studied in rat heart sarcolemmal and sarcoplasmic reticular fractions after a single intraperitoneal injection of isoproterenol (0.5-5.0 mg/kg). Three active sites (I, II, and III) for PtdEtn N-methylation were assayed by measurement of [3H]methyl group incorporation from 0.055, 10, and 150 microM S-adenosyl-L-[methyl-3H]methionine into membrane PtdEtn molecules. Total methylation activity for catalytic site I of both sarcolemma and sarcoplasmic reticulum was stimulated within 2 minutes by isoproterenol in a dose-dependent manner. Although the increased methyltransferase activity in sarcoplasmic reticulum was normalized at 10 minutes, the enzyme activity in sarcolemma was normalized at 5 minutes but was again increased at 10-30 minutes after isoproterenol injection. No changes in response to isoproterenol were seen for site II and III N-methylation activities in either membrane. Individual N-methylated phospholipids (phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine), which specifically formed at each site, showed similar behavior. Pretreatment of the animals with a beta-blocking drug, atenolol, for 2 days prevented the isoproterenol-induced changes in hemodynamic parameters and sarcolemmal methylation without affecting the enhanced methylation activities in sarcoplasmic reticulum. In vitro addition of cyclic AMP-dependent protein kinase (catalytic subunit) plus Mg-ATP enhanced methyltransferase activities in sarcolemma and sarcoplasmic reticulum from control hearts by 2.7- and 2.3-fold, respectively; however, under the same in vitro conditions, only about 20% activation was seen in both subcellular membranes isolated from the heart of isoproterenol-injected animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of membrane methylation on heart sarcolemmal 5′-nucleotidase activity: G. Paolillo, S. Majumder, C. Ou, V. Panagia. Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Department of Anatomy, University of Manitoba, Winnipeg, Canada

Inhibitory effect of membrane methylation on heart sarcolemmal 5′-nucleotidase activity: G. Paolillo, S. Majumder, C. Ou, V. Panagia. Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Department of Anatomy, University of Manitoba, Winnipeg, Canada

Ihproved information about the composition of the active site of heart sarcolemmal (Na ++K +)-ATPase

Ihproved information about the composition of the active site of heart sarcolemmal (Na ++K +)-ATPase

The effects of myocardial ischemia and nisoldipine pretreatment on the asymmetric distribution of phosphatidylethanolamine in a canine heart sarcolemmal preparation.

We examined the distribution of phosphatidylethanolamine (PE) in the membrane bilayer of sarcolemmal preparation isolated from the ischemic and nonischemic areas of dog ventricles. The membrane preparation, isolated by the Reeves and Sutko's method, was purified ninefold over homogenates as judged from the results of measurements of (Na+K+)-ATPase and K+-p-nitrophenylphosphatase activities, sialic acid, and cholesterol. Sealed vesicles were comprised of 60% inside-out-oriented and 40% rightside-out-oriented vesicles; 30% of the total were unsealed vesicles. The results obtained from the incubation of the membrane preparation with 2,4,6-trinitrobenzenesulfonic acid (TNBS) and cycloheptaamylose-fluorescamine complex, both of which served as nonpermeable chemical probes, indicated that 80% of the total PE was accessible from the outside. By contrast, it was possible to label up to 98% of the PE by using a permeable probe, 1-fluoro-2,4-dinitrobenzene. These results suggest that PE is predominantly localized in the cytosolic side of the sarcolemmal membrane bilayer in the dog heart. Ischemic lesion was produced in the dog heart by the occlusion of a branch of the left anterior descending coronary artery for 1.5 hr followed by 3 hr of reflow. The concentrations of both total phospholipid and phosphatidylcholine and PE in the sarcolemmal fraction prepared from the ischemic area of the myocardium were significantly decreased as compared to those from the nonischemic area. The magnitude of labeling sarcolemmal PE by TNBS was reduced in the preparation from the ischemic area as compared to that from the nonischemic area. This difference was abolished when the dog received nisoldipine (an iv injection of 5 micrograms/kg twice) or chlorpromazine (infusion at a rate of 10 micrograms/kg X min plus an iv injection of 400 micrograms/kg twice). These results suggest that ischemia decreased primarily the membrane PE existing at the cytosolic side of the sarcolemmal membrane and that pharmacological intervention can prevent the change in membrane lipids induced by ischemia.

Lack of cross-reactivity of antibodies to ribosomal preparations from Streptococcus mutans with human heart and kidney antigens.

Previous studies have suggested that sera from animals immunized with whole Streptococcus mutans cells may cross-react with human and monkey heart sarcolemmal tissues. In the present study, sera and saliva from rats and rabbits immunized peripherally with ribosomal preparations from S. mutans 6715 (serotype g) or GS-5 (serotype c) were examined for their ability to react with normal human heart sarcolemmal and kidney glomerular tissues by using enzyme-linked immunosorbent and immunofluorescence assays. The results showed that antibodies to serotype g and c ribosomal preparations do not react with either the human heart or renal antigens. Sera from mice immunized with human heart tissue and from a patient with a high anti-streptolysin O titer reacted strongly with human heart sarcolemmal and kidney glomerular tissues. These data indicated that ribosomal preparations from S. mutans lack the putative human heart cross-reactive determinant and suggest that the use of an S. mutans ribosomal vaccine against dental caries may not be pathogenic to human heart or renal tissues.

Solubilization of rat heart sarcolemma 5′-nucleotidase by phosphatidylinositol-specific phospholipase C

The influence of a phosphatidylinositol-specific phospholipase C treatment on rat heart sarcolemmal 5′-nucleotidase was investigated. Upon complete hydrolysis of all phosphatidylinositol in the sarcolemma, 75% of 5′-nucleotidase activity was found in the solubilized form. The insolubilized enzyme after this treatment has the same K m for AMP as the untreated, sarcolemmal-bound enzyme (0.04 mM), whereas the solubilized enzyme has a 40-fold increase in K m for AMP (0.16 mM). Other sarcolemmal-bound enzymes were not affected by the same treatment. Hence, the specific involvement of phosphatidylinositol in the binding of 5′-nucleotidase to the sarcolemma of the rat heart is clearly demonstrated.

Phosphorylation of low molecular weight proteins in purified preparations of rat heart sarcolemma and sarcoplasmic reticulum

A rat heart sarcolemmal preparation could be obtained in which both 5′-nucleotidase and adenylate cyclase were enriched approx. 9-fold by subjecting a homogenate to a discontinuous sucrose gradient, without the use of a high salt extraction. After incubation of this fraction with Mg[γ- 32P]ATP, the majority of 32P incorporated was present in 24000- and 9000-dalton protein components. Only when a heart cytosol fraction or a purified cyclic AMP-dependent protein kinase was added, was enhancement of 32P-incorporation found by addition of cyclic AMP. The 9000- and 24 000-dalton proteins appeared to be interconvertible. The degree of conversion could be affected by changing the temperature during solubilization of the membranes in SDS prior, to electrophoresis. This suggested that the 24 000-dalton protein does not correspond to phospholamban, first identified by others in canine heart sarcoplasmic reticulum. Moreover, it could be excluded that the 24000-dalton protein was derived from contaminating myofibrillar troponin I. When the sarcolemmal fraction was preincubated with Ca 2+, Mg 2+, ATP and oxalate, contaminating sarcoplasmic reticulum vesicles, loaded with calcium oxalate, settled to a greater density in the sucrose gradient. Membrane constituents other than those with enzymatic activity were monitored to confirm the separation between sarcolemmal and sarcoplasmic reticulum membranes: Coomassie blue staining material, sialic acid, cholesterol and phospholipid. The 24 000- and 9000-dalton proteins were equally distributed among the sarcolemmal and sarcoplasmic reticulum fractions present in the sucrose gradient. However, the rate of 32P-incorporation in the presence of heart cytosol fraction was much slower in the sarcoplasmic reticulum than in the sarcolemmal fraction.

Changes in subcellular membranes in rat heart perfused with diethyl ether

Alterations in contractile force as well as heart sarcolemmal, microsomal, and mitochondrial activities were investigated by perfusing rat hearts with different concentrations (0.5–1.5% (v/v)) of ether for 2 min. Contractile force of the perfused heart was depressed by 0.5–1.0% ether and completely abolished by 1.25–1.5% ether. The contractile force recovered fully upon reperfusion when the hearts were preperfused with 0.5–0.75% ether but the recovery was partial in hearts preperfused with 1.0–1.25% ether. The hearts preperfused with 1.5% ether did not recover their ability to generate contractile force upon reperfusion. The Na+–K+-ATPase activity of sarcolemma obtained from hearts perfused with 0.75–1.0% ether was higher than the control value. The sarcolemmal Na+–K+-ATPase, Mg2+-ATPase, Ca2+-ATPase, calcium binding, and adenylate cyclase activities were decreased in hearts perfused with 1.5% ether and these activities were further decreased upon reperfusion. The microsomal calcium binding and uptake activities were depressed in hearts perfused with 1.0–1.5% ether and these changes were irreversible. The mitochondrial calcium binding and uptake activities as well as oxidative phosphorylation activity were also depressed in hearts perfused with 1.0–1.5% ether; however, irreversible damage was seen in hearts perfused with 1.5% ether. Electron microscopic examination showed occasional vacuolization of mitochondria in hearts perfused with 1% ether and swelling of sarcoplasmic reticulum and mitochondria as well as damage to myofilaments were seen in hearts perfused with 1.5% ether. These results suggest the involvement of alterations in subcellular membrane system in the cardiotoxic actions of ether.