Functional coupling of angiotensin II type 1 receptor with insulin resistance of energy substrate uptakes in immortalized cardiomyocytes (HL-1 cells).

BACKGROUND AND PURPOSE: Increased angiotensin II levels and insulin resistance coexist at the early stages of cardiomyopathies. To determine whether angiotensin II increases insulin resistance in cardiomyocytes, we studied the effect of angiotensin II on basal and insulin-stimulated transport rate of energy substrates in immortalized cardiomyocytes (HL-1 cells). EXPERIMENTAL APPROACH: Glucose and palmitic acid uptakes were measured using [(3)H]2-deoxy-D-glucose and [(14)C]palmitic acid, respectively, in cells exposed or not exposed to angiotensin II (100 nM), angiotensin II plus irbesartan or PD123319, type 1 and 2 receptor antagonists, or PD98059, an inhibitor of ERK1/2 activation. Cell viability, DNA, protein synthesis and surface area were evaluated by the MTT test, [(3)H]thymydine, [(3)H]leucine and morphometric analysis, respectively. Type 1 receptor levels were measured by western blot analysis. KEY RESULTS: Basal uptakes of glucose and palmitic acid by HL-1 cells (0.37+/-0.07 and 7.31+/-0.22 pmol per 10(4)cells per min, respectively) were both stimulated by 100 nM insulin (+91 and +64%, respectively). Cells exposed to angiotensin II remained viable and did not show signs of hypertrophy. In these conditions, the basal palmitic acid uptake of the cells increased (11.41+/-0.46 pmol per 10(4) cells per min) and insulin failed to stimulate the uptake of glucose and fatty acids. Changes in the rate of uptake of energy substrates were prevented or significantly reduced by irbesartan or PD98059. CONCLUSIONS AND IMPLICATIONS: Angiotensin II is a candidate for increasing insulin resistance in cardiomyocytes. Our results suggest a further mechanism for the cardiovascular protection offered by the angiotensin II type 1 receptor blockers.

Cardiac volume overload rapidly induces oxidative stress-mediated myocyte apoptosis and hypertrophy.

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated the role of oxidative stress in the initial phases of cardiac remodeling induced in an animal model by volume overload. As plausible candidates for a connection between oxidative stress and cardiomyocyte apoptosis or hypertrophy, we explored the behaviour of two MAPKs, specifically JNK and ERK. At 48 h of overload, the greatest increase in oxidative stress coincided with a peak of cardiomyocyte apoptosis. This was possibly induced through the mitochondrial metabolism, as evidenced by the release of cytochrome c and a significant increase in the active forms of caspase-9 and -3, but not caspase-8. Oxidative stress markers significantly decreased at 96 h of overload, combined with a marked attenuation of apoptosis and the appearance of hypertrophy. The highest levels of JNK and the lowest levels of ERK phosphorylation were observed at 48 h of overload. Conversely, a sharp increase in ERK phosphorylation was detected at 96 h of overload coinciding with the hypertrophic response. Together these results show that oxidative stress is an early and transient event in myocardial volume overload. They suggest that oxidative stress mediates amplitude dependent apoptotic and hypertrophic responses in cardiomyocytes through the selective activation of, respectively, JNK and ERK.

Possible role of acylphosphatase, Bcl-2 and Fas/Fas-L system in the early changes of cardiac remodeling induced by volume overload.

To identify early adaptive processes of cardiac remodeling (CR) in response to volume overload, we investigated the molecular events that may link intracellular Ca(2+) homeostasis alterations and cardiomyocyte apoptosis. In swine heart subjected to aorto-cava shunt for 6, 12, 24, 48 and 96 h sarcoplasmic reticulum (SR) Ca(2+) pump activity was reduced until 48 h (-30%), but a recovery of control values was found at 96 h. The decrease in SR Ca(2+)-ATPase (SERCA2a) expression at 48 h, was more marked (-60%) and not relieved by a subsequent recovery, while phospholamban (PLB) concentration and phosphorylation were unchanged at all the considered times. Conversely, acylphosphatase activity and expression significantly increased from 48 to 96 h (+40%). Bcl-2 expression increased significantly from 6 to 24 h, but at 48 h, returned to control values. At 48 h, microscopic observations showed that overloaded myocardium underwent substantial damage and apoptotic cell death in concomitance with an enhanced Fas/Fas-L expression. At 96 h, apoptosis appeared attenuated, while Fas/Fas-L expression was still higher than control values and cardiomyocyte hypertrophy became to develop. These data suggest that in our experimental model, acylphosphatase could be involved in the recovery of SERCA2a activity, while cardiomyocyte apoptosis might be triggered by a decline in Bcl-2 expression and a concomitant activation of Fas.

Altered Cx43 expression during myocardial adaptation to acute and chronic volume overloading.

Gap-junctions are specialized regions of intercellular contacts allowing electrical impulse propagation among adjacent cardiomyocytes. Connexin43 (Cx43) is the predominant gap-junction protein in the working ventricular myocardium and its reduced expression has been extensively implicated in the genesis of conduction abnormalities and re-entry arrhythmia of chronically hypertrophied hearts. In contrast, data on the role played by this protein during cardiac remodeling and early phases of developing hypertrophy are lacking. Therefore, in the present study, we investigated this issue using an experimental model of pig left ventricle (LV) volume overloading consisting in the creation of an aorto-cava fistula. At scheduled times (6, 24, 48, 96, 168 h, and 2, 3 months after surgery) echocardiographic and haemodynamic measurements were performed and myocardial biopsies were taken for the morphological and biochemical analyses. When faced with the increased load, pig myocardium underwent an initial period (from 6 up to 48 h) of remarkable tissue remodeling consisting in the occurrence of cardiomyocyte damage and apoptosis. After that time, the tissue developed a hypertrophic response that was associated with early dynamic changes (up-regulation) in Cx43 protein expression, as demonstrated by Western blot and confocal immunofluorescence analyses. However, an initial transient increase of this protein was also found after 6 h from surgery. With the progression of LV hypertrophy (from 168 hr up to 3 months), a reduction in the myocardial Cx43 expression was, instead, observed. The increased expression of Cx43 protein during acute hypertrophic response was associated with a corresponding increase in the levels of its specific mRNA, as detected by RT-PCR. We concluded that up-regulation of Cx43 gap-junction protein could represent an immediate compensatory response to support the new working conditions in the early stages of ventricular overloading.

Biochemical changes and their relationship with morphological and functional findings in pig heart subjected to lasting volume overload: a possible role of acylphosphatase in the regulation of sarcoplasmic reticulum calcium pump.

We evaluated the changes in sarcoplasmic reticulum (SR) function and the parallel hemodynamic and morphological modifications in a heart subjected to volume overload. We also determined the levels of acylphosphatase, a cytosolic enzyme, that could play a regulatory effect on SR Ca(2+) pump by hydrolyzing the phosphorylated intermediate of this transport system. For this, swine hearts were subjected to volume overload by aorta-cava shunt for 1, 2, or 3 months. Changes in heart contractility reflected modifications of SR function, whose reduction after 1 month of overload was followed by a gradual recovery. A decrease in SERCA2a protein and mRNA content was shown from 1 month and remained for the following 2 months. Phospholamban content and its phosphorylation status were not modified. Acylphosphatase was unchanged at 1 month, but at 2 months this enzyme exhibited an increased activity, protein and mRNA expression. Morphological alterations consisting of the cytoskeletal architectures, intermyofibrillar oedema, swollen mitochondria and abnormality of the membrane system (T-tubule and SR cisternae) were particularly evident after 1 month but almost disappeared after 3 months. These results suggest that our overloaded hearts underwent a substantial recovery of their structural and biochemical properties at 3 months after surgery. A possible involvement of acylphosphatase in the modification of SR function is discussed.

Are macrophages involved in early myocardial reperfusion injury?

BACKGROUND: Neutrophils are the predominant phagocytes in the early stages of myocardial ischemia-reperfusion response and are also implicated in the development of tissue damage. This study examined the role of recruited macrophages in the evolution of this tissue injury. METHODS: Farm pigs were subjected to 30 minutes of myocardial ischemia followed by 30 minutes of reperfusion. Biopsy samples were taken from the control, ischemic, and ischemic-reperfused left ventricle wall and processed for both morphologic and biochemical analyses. In situ production of tumor necrosis factor-alpha was evaluated by Western blot and immunofluorescence. A full hemodynamic evaluation was also performed. RESULTS: Myocardial ischemia and early reperfusion caused marked neutrophil and macrophage tissue accumulation and tumor necrosis factor-alpha production by the injured tissue. Immunofluorescence studies allowed us to localize tumor necrosis factor-alpha predominantly in tissue-infiltrating macrophages. No depression in the global myocardial contractile function was observed, either during ischemia or after reperfusion. CONCLUSIONS: These data suggest that the newly recruited macrophages within the ischemic and early post-ischemic myocardium may play a role in promoting neutrophil tissue infiltration and subsequent neutrophil-induced tissue dysfunction by producing tumor necrosis factor-alpha.

Interaction between acylphosphatase and SERCA in SH-SY5Y cells.

Ca2+ transport by sarco/endoplasmic reticulum, tightly coupled with the enzymatic activity of Ca2+ -dependent ATPase, controls the cell cycle through the regulation of genes operating in the critical G, to S checkpoint. Experimental studies demonstrated that acylphosphatase actively hydrolyses the phosphorylated intermediate of sarco/endoplasmic reticulum calcium ATPase (SERCA) and therefore enhances the activity of Ca2+ pump. In this study we found that SH-SY5Y neuroblastoma cell division was blocked by entry into a quiescent G0-like state by thapsigargin, a high specific SERCA inhibitor, highlighting the regulatory role of SERCA in cell cycle progression. Addition of physiological amounts of acylphosphatase to SY5Y membranes resulted in a significant increase in the rate of ATP hydrolysis of SERCA. In synchronized cells a concomitant variation of the level of acylphosphatase isoenzymes opposite to that of intracellular free calcium during the G1 and S phases occurs. Particularly, during G1 phase progression the isoenzymes content declined steadily and hit the lowest level after 6 h from G0 to G1 transition with a concomitant significant increase of calcium levels. No changes in free calcium and acylphosphatase levels upon thapsigargin inhibition were observed. Moreover, a specific binding between acylphosphatase and SERCA was demonstrated. No significant change in SERCA-2 expression was found. These findings suggest that the hydrolytic activity of acylphosphatase increase the turnover of the phosphoenzyme intermediate with the consequences of an enhanced efficiency of calcium transport across endoplasmic reticulum and a subsequent decrease in cytoplasmic calcium levels. A hypothesis about the modulation of SERCA activity by acylphosphatase during cell cycle in SY5Y cells in discussed.

Early changes induced in the left ventricle by pressure overload. An experimental study on swine heart.

The purpose of this study was to evaluate the early changes in sarcoplasmic reticulum (SR) function and the parallel morphological and hemodynamic modifications occurring in the heart following pressure overload. As regards SR function, we also explored the levels of acylphosphatase, an enzyme which might have a regulatory effect on the SR Ca(2+) pump by hydrolyzing the phosphorylated intermediate of this transport system. Pigs subjected to pressure overload by aortic stenosis for 6, 12, 24, 48, 72, and 96 h were compared to sham-operated controls. At each of the considered times both SR Ca(2+)-ATPase activity and Ca(2+) uptake, as well as acylphosphatase activity, were significantly enhanced in the pressure overloaded compared to the control hearts, with a maximal increase at 6 h; moreover, a positive and significant correlation was found between these parameters. The modifications in the activities of Ca(2+)-ATPase and acylphosphatase reflected an increased expression of these proteins, while phospholamban did not show significant changes in its concentration nor in its phosphorylation status. As for hemodynamic parameters, rapid changes in the left ventricular function were observed and especially the early hours following the aortic stenosis appeared to be crucial for the adjustment of heart function. The most relevant morphological finding was a focal disarrangement of the myofibrillar pattern which was very evident at 6 h, and progressively attenuated at later times. Taken together our data suggest that an early adaptation to the increased hemodynamic working overload is a consistent activation of the contractile apparatus which reflects, at least in part, an enhanced SR function. Besides the changes in Ca(2+) pump protein expression, increased acylphosphatase levels might also contribute to this effect.

Transcriptional down-regulation of poly(ADP-ribose) polymerase gene expression by E1A binding to pRb proteins protects murine keratinocytes from radiation-induced apoptosis.

Adenovirus E1A confers enhanced cell sensitivity to radiation and drug-induced DNA damage by a mechanism involving the binding to cellular proteins. Mutant analysis in E1A-transfected murine keratinocytes demonstrates that increased sensitivity to DNA damage requires at least E1A binding to the p300/CREB-binding protein (CBP) transcriptional coactivators and to pRb family members, indicating that this biological activity of E1A is the result of the concomitant perturbation of different cell pathways. Here we show that in the same cells E1A binding to members of the retinoblastoma protein family induces transcriptional down-regulation of the poly(ADP-ribose) polymerase (PARP) gene, coding for a NAD-dependent enzyme stimulated by DNA breaks. Inhibition of PARP expression is accompanied by a decrement of gamma-irradiation-induced apoptosis, which is overridden by reconstitution of wild type levels of PARP. Hence, E1A effects on PARP transcription are central determinant of the apoptotic sensitivity of E1A-expressing keratinocytes. Conversely, E1A binding to only p300/CBP results in an increase in PARP enzyme activity and consequently in cell death susceptibility to irradiation, which is effectively counteracted by the PARP chemical inhibitor 3-aminobenzamide. Therefore, our results identify in the E1A-mediated effects on PARP expression and activity a key molecular event involved in E1A-induced cell sensitization to genotoxic stress.

A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps.

In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect.

Oxidative stress and antioxidant defenses in renal patients receiving regular haemodialysis.

Patients with chronic renal failure, and particularly those receiving regular haemodialysis, have a high incidence of premature cardiovascular disease. Oxidative stress, which causes lipid peroxidation, may contribute to increase the risk of atherosclerosis. The results of the present study indicate that lipid peroxidation products (malonaldehyde and 4-hydroxyalkenals) are significantly increased in plasma of renal patients before dialysis and, although reduced, remained above the normal range after this treatment. Moreover, production of free radicals and reactive oxygen metabolites was increased in chronic renal failure patients, especially after dialysis. On the other hand, the antioxidant defenses of those patients were higher than those of normal subjects, as judged from the plasma levels of specific antioxidant molecules and from the plasma antioxidant capacity. We also found that triglycerides were significantly higher in renal patients, both before and after dialysis, than in the control group. These results suggest that patients on chronic haemodialysis are particularly prone to oxidative stress and that dialysis itself may worsen this condition. Rather than to a weakening of antioxidant defenses, the susceptibility of chronic renal failure patients to oxidative stress might be ascribed to an increased free radical and reactive oxygen metabolite production and to increased levels of oxidizable substrates, notably triglycerides with their unsaturated fatty acids.

Beneficial effects of the 21-aminosteroid U 74389G on the ischemia-reperfusion damage in pig hearts.

21-Aminosteroids (Lazaroids), acting as free radical scavengers and as membrane stabilizers, proved to have beneficial effects in various pathological conditions. In the present study we explored the effectiveness of one of these compounds, U 74389 G, in protecting pigs myocardium against the ischemia-reperfusion damage induced by transient coronary occlusion achieved by clampling the left anterior descending coronary artery. Animals were divided into three groups: control, untreated and treated. Control animals were operated but not subjected to ischemia-reperfusion; the untreated group underwent to ischemia-reperfusion without pharmacological treatment; while the treated group received the aminosteroid (4 mg/kg) before coronary occlusion and at the time of reperfusion. Specimens of myocardial tissue and blood samples were taken for morphological and biochemical studies. In the ischemic-reperfused myocardium of the untreated animals, the dominant morphological features were neutrophil infiltration, intercellular edema and severe swelling of mitochondria. All these alterations, notably neutrophil infiltration, were attenuated by aminosteroid treatment. As for the biochemical findings, the changes in adenine nucleotides and nucleosides levels, thus the reduction of energy charge, were reversed in the treated, but not in the untreated group. Myocardial concentration of malondialdehyde, which was undetectable in the control group, was raised in all the animals after reperfusion, but this effect was significantly less marked with aminosteroid treatment. In addition, the higher myocardial content of ascorbic acid and the reduced serum potential peroxidation exhibited by the treated animals compared with untreated group indicate an enhanced antioxidant protection induced by aminosteroid administration. On the other hand, the serum levels of myoglobin, cardiac troponin I and creatine kinase MB isoenzyme suggest the ability of the aminosteroid to attenuate the modifications of membrane permeability induced by ischemia-reperfusion injury. All these results lead to the conclusion that aminosteroid treatment, at least in the conditions of the present study, is effective in reducing the morphological and biochemical alterations occurring in ischemic-reperfused myocardium.

Stimulation of cardiac sarcoplasmic reticulum calcium pump by acylphosphatase. Relationship to phospholamban phosphorylation.

Ca2+ transport by cardiac sarcoplasmic reticulum is tightly coupled with the enzymatic activity of Ca2+-dependent ATPase, which forms and decomposes an intermediate phosphoenzyme. Heart sarcoplasmic reticulum Ca2+ pump is regulated by cAMP-dependent protein kinase (PKA) phospholamban phosphorylation, which results in a stimulation of the initial rates of Ca2+ transport and Ca2+ ATPase activity. In the present studies we found that acylphosphatase from heart muscle, used at concentrations within the physiological range, actively hydrolyzes the phosphoenzyme of cardiac sarcoplasmic reticulum Ca2+ pump, with an apparent Km on the order of 10(-7) M, suggesting an high affinity of the enzyme for this special substrate. In unphosphorylated vesicles acylphosphatase enhanced the rate of ATP hydrolysis and Ca2+ uptake with a concomitant significant decrease in apparent Km for Ca2+ and ATP. In vesicles whose phospholamban was PKA-phosphorylated, acylphosphatase also stimulated the rate of Ca2+ uptake and ATP hydrolysis but to a lesser extent, and the Km values for Ca2+ and ATP were not significantly different with respect to those found in the absence of acylphosphatase. These findings suggest that acylphosphatase, owing to its hydrolytic effect, accelerates the turnover of the phosphoenzyme intermediate with the consequence of an enhanced activity of Ca2+ pump. It is known that phosphorylation of phospholamban results in an increase of the rate at which the phosphoenzyme is decomposed. Thus, as discussed, a competition between phospholamban and acylphosphatase effect on the phosphoenzyme might be proposed to explain why the stimulation induced by this enzyme is less marked in PKA-phosphorylated than in unphosphorylated heart vesicles.

Protection of rat heart from ischaemia-reperfusion injury by the 21-aminosteroid U-74389G.

In ischaemia-reperfusion syndromes lipid peroxidation appears an important factor contributing to tissue damage. The 21-aminosteroids (lazaroids) exhibit beneficial effects in various pathological conditions, especially in post-traumatic lesions of the central nervous system, where a peroxidative injury seems to be involved. The aim of our study was to ascertain if one of these compounds, U-74389G, plays a significant role in protecting heart muscle from ischaemia-reperfusion damage. Rat hearts used for heterotopic transplantation represented the experimental model in this investigation. Animals (Wistar rats weighing 200-250 g) were divided into five groups: controls, untreated and treated donors, untreated and treated recipients. Donors were anaesthetized and heparinized, and the heart was excised through a bilateral thoracotomy, arrested with St Thomas solution and stored in cold saline for 2 hours. For the recipient preparation, a modified Ono's technique was used, and heart reimplantation was performed with a termino-lateral aorto-aortic anasthomosis and a termino-lateral pulmonary-cava anasthomosis. After the anasthomoses were completed hearts were reperfused for 30 min; then hearts were excised and specimens were taken for biochemical and morphological studies. These were conducted on three groups of hearts: (A) hearts reimplanted and reperfused without treatment of the donor or of the recipient animal; (B) hearts subjected to the same procedure but in the presence of U-74389G treatment of donors and recipient rats; (C) control hearts rapidly excised from normal, non-operated animals. Electron microscopy studies showed, in hearts transplanted without treatment, the typical morphological aspects of lipoperoxidative injury: swollen mitochondria with disrupted cristae, damaged endothelial cells with the nucleous bulging into the lumen and a discontinued endothelial lining with diffuse oedema among the fibers. Lazaroid treatment attenuated most of these damages in hearts of group B. As for the biochemical findings, the hearts transplanted in the presence of U-74389G treatment had significantly higher ATP and creatine phosphate levels (P < 0.01) and lower malondialdehyde concentrations (P < 0.05) with respect to the hearts transplanted without treatment. Furthermore, serum creatine kinase activity was lower in treated than in untreated recipient animals (P < 0.05). Taken together, all these results indicate that U-74389G treatment is effective in protecting cardiac muscle from structural and functional ischaemia-reperfusion injuries, at least from those arising during a heart transplantation procedure.

Acylphosphatase stimulates Ca2+ transport and Ca(2+)-dependent ATPase activity in cardiac sarcoplasmic reticulum.

Acylphosphatase purified from heart muscle actively hydrolyzes the phosphoenzyme intermediate of cardiac sarcoplasmic reticulum Ca(2+)-ATPase. This effect was evident with acylphosphatase concentrations (up to 100 units/mg sarcoplasmic reticulum protein) that fall within the physiological range, and the low value of the apparent Km, on the order of 10(-7)M, suggests a high affinity towards this special substrate. Moreover, acylphosphatase addition to sarcoplasmic reticulum vesicles significantly enhanced the rate of Ca(2+)-dependent ATP hydrolysis. Maximal stimulation, observed with 100 units/mg vesicular protein, resulted in an ATPase activity which was about two folds over basal value. The same acylphosphatase concentration increased at a similar extent the rate of ATP driven Ca2+ influx into sarcoplasmic reticulum vesicles. Taken together these findings lead to suppose that acylphosphatase, owing to its hydrolytic activity, induces an accelerated turnover of the phosphoenzyme intermediate, whence an overall stimulation of heart sarcoplasmic reticulum Ca2+ pump, affecting both ATP hydrolysis and Ca2+ influx.

Acylphosphatase: a potential modulator of heart sarcolemma Na+,K+ pump.

Acylphosphatase, purified from cardiac muscle, catalyzes the hydrolysis of the phosphorylated intermediate of heart sarcolemmal Na+,K(+)-ATPase. This effect was remarkable even using acylphosphatase amounts (100-300 units/mg of membrane protein) near the lower limit of the physiological range; besides the low value of the apparent Km, on the order of 10(-7) M, indicates that the enzyme has a high affinity for this special substrate. The results of a dot-immunobinding assay suggest the possibility of an interaction between acylphosphatase and native, undenaturated Na+,K(+)-ATPase. Moreover, when added to sarcolemmal vesicles, acylphosphatase was found to affect the functional properties of the Na+,K+ pump with regard to the rate of both ATP hydrolysis and cation transport. However, while ATPase activity and Na+ uptake were stimulated, the last at a greater extent, the active K+ transport was inhibited, so that the Na+/K+ ratio, which was calculated as 1.50 without acylphosphatase, rose to 6.68 in the presence of 300 units/mg of vesicle protein of this enzyme. Taken together, the reported results indicate that acylphosphatase, because of its hydrolytic activity on the phosphoenzyme intermediate, induces a sort of "uncoupling" effect on the heart sarcolemmal membrane Na+,K+ pump. Possible mechanisms for such an effect, which suggests a potential role of acylphosphatase in the control of this active transport system, are discussed.

Modifications induced by acylphosphatase in the functional properties of heart sarcolemma Na+,K+ pump.

Acylphosphatase purified from cardiac muscle actively hydrolyzes the phosphoenzyme intermediate of heart sarcolemma Na+,K(+)-ATPase. This effect occurred with acylphosphatase amounts (up to 800 units/mg membrane protein) that fall within the physiological range and the low value of the apparent Km (0.69 x 10(-7) M) indicates a considerable affinity of the enzyme towards this specific substrate. Acylphosphatase addition to purified sarcolemmal vesicles significantly increased the rate of Na+,K(+)-dependent ATP hydrolysis. Maximal stimulation, observed with 800 units/mg protein, resulted in an ATPase activity which was about 2-fold over basal value. The same acylphosphatase amounts significantly stimulated, in a similar and to an even greater extent, the rate of ATP driven Na+ transport into sarcolemmal vesicles. These findings lead to suppose that an accelerated hydrolysis of the phosphoenzyme may result in an enhanced activity of heart sarcolemmal Na+,K+ pump, therefore suggesting a potential role of acylphosphatase in the control of this active transport system.

Acylphosphatase induced modifications in the functional properties of erythrocyte membrane sodium pump.

Human red cell acylphosphatase actively hydrolyzes the Na+/K(+)-ATPase phosphoenzyme from erythrocyte membrane. This effect occurred with amounts of acylphosphatase (up to 10 units/mg membrane protein) within the physiological range, and the low value of the apparent Km (0.147 +/- 0.050 microM) indicates that the enzyme has a high affinity for this substrate. When added at the above concentration to inside out vesicles from human erythrocytes, acylphosphatase significantly enhanced the rate of strophantidine-sensitive ATP hydrolysis. The same amounts of acylphosphatase stimulated, although to a lower extent, the rate of ATP-dependent 22Na+ influx (normal efflux). Thus, the calculated stoichiometry for Na+/ATP was 2.68 in the absence of acylphosphatase and 1.06 in the presence of 10 units/mg vesicle protein of the enzyme. Conversely, acylphosphatase addition strongly decreased the rate of ATP-dependent 86Rb+(K+) efflux (normal influx) which, with 10 units/mg vesicle protein, was almost suppressed. As a consequence, the Na+/Rb+ ratio, calculated as 1.52 in the absence of acylphosphatase rose to 72.5 in the presence of 10 units/mg vesicle protein of this enzyme. These results suggest that, because of its hydrolytic activity on the phosphoenzyme intermediate, acylphosphatase 'uncouples' erythrocyte membrane Na+,K+ pump. Possible mechanisms for this effect are discussed.

Alterations induced by acylphosphatase in the activity of heart sarcolemma calcium pump.

We studied the effect of muscle acylphosphatase on the Ca2+ pumping ATPase of heart sarcolemma. Acylphosphatase addition to calmodulin-depleted sarcolemmal vesicles produced a significant increase in the rate of Ca(2+)-dependent ATP hydrolysis, even higher than obtained with exogenously added calmodulin. Maximal stimulation (about four fold over basal value) was obtained with 550 units/mg vesicle protein, a concentration that fall within the physiological range. Conversely, similar amounts of acylphosphatase decreased the rate of ATP-dependent Ca2+ transport into the sarcolemmal vesicles. The maximal statistically significant inhibition of Ca2+ uptake was observed with the same acylphosphatase concentration that gave the maximal stimulation of Ca(2+)-ATPase activity. From these findings acylphosphatase appears to reduce the efficiency of heart sarcolemmal Ca2+ pump with an impairment of the coupling between ATP hydrolysis and Ca2+ transport. A possible mechanism of this effect is discussed.