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Ca2+ Release and Heat Production by the Endoplasmic Reticulum Ca2+-ATPase of Blood Platelets

1999; Elsevier BV; Volume: 274; Issue: 40 Linguagem: Inglês

10.1074/jbc.274.40.28344

1083-351X

Fernanda Mitidieri, Leopoldo de Meis,

Cardiac electrophysiology and arrhythmias

Different sarco/endoplasmic reticulum Ca2+-ATPases isoforms are found in blood platelets and in skeletal muscle. The amount of heat produced during ATP hydrolysis by vesicles derived from the endoplasmic reticulum of blood platelets was the same in the absence and presence of a transmembrane Ca2+ gradient. Addition of platelets activating factor (PAF) to the medium promoted both a Ca2+ efflux that was arrested by thapsigargin and an increase of the yield of heat produced during ATP hydrolysis. The calorimetric enthalpy of ATP hydrolysis (ΔH cal) measured during Ca2+transport varied between −10 and −12 kcal/mol without PAF and between −20 and −24 kcal/mol with 4 μm PAF. Different from platelets, in skeletal muscle vesicles a thapsigargin-sensitive Ca2+ efflux and a high heat production during ATP hydrolysis were measured without PAF and the ΔH cal varied between −10 and −12 kcal/mol in the absence of Ca2+ and between −22 up to −32 kcal/mol after formation of a transmembrane Ca2+ gradient. PAF did not enhance the rate of thapsigargin-sensitive Ca2+ efflux nor increase the yield of heat produced during ATP hydrolysis. These findings indicate that the platelets of Ca2+-ATPase isoforms are only able to convert osmotic energy into heat in the presence of PAF. Different sarco/endoplasmic reticulum Ca2+-ATPases isoforms are found in blood platelets and in skeletal muscle. The amount of heat produced during ATP hydrolysis by vesicles derived from the endoplasmic reticulum of blood platelets was the same in the absence and presence of a transmembrane Ca2+ gradient. Addition of platelets activating factor (PAF) to the medium promoted both a Ca2+ efflux that was arrested by thapsigargin and an increase of the yield of heat produced during ATP hydrolysis. The calorimetric enthalpy of ATP hydrolysis (ΔH cal) measured during Ca2+transport varied between −10 and −12 kcal/mol without PAF and between −20 and −24 kcal/mol with 4 μm PAF. Different from platelets, in skeletal muscle vesicles a thapsigargin-sensitive Ca2+ efflux and a high heat production during ATP hydrolysis were measured without PAF and the ΔH cal varied between −10 and −12 kcal/mol in the absence of Ca2+ and between −22 up to −32 kcal/mol after formation of a transmembrane Ca2+ gradient. PAF did not enhance the rate of thapsigargin-sensitive Ca2+ efflux nor increase the yield of heat produced during ATP hydrolysis. These findings indicate that the platelets of Ca2+-ATPase isoforms are only able to convert osmotic energy into heat in the presence of PAF. Heat generation plays a key role in the regulation of the metabolic activity and energy balance of the cell. In animals lacking brown adipose tissue, the principal source of heat during nonshivering thermogenesis is derived from the hydrolysis of ATP by the sarcoplasmic reticulum Ca2+-ATPase of skeletal muscles (1Clausen T. Hardeveld C.V. Everts M.E. Physiol. Rev. 1991; 71: 733-774Crossref PubMed Scopus (210) Google Scholar, 2Chinet A.E. Decrouy A. Even P.C. J. Physiol. ( Lond. ). 1992; 455: 663-678Crossref PubMed Scopus (43) Google Scholar, 3Janský L. Physiol. Rev. 1995; 75: 237-259Crossref PubMed Scopus (123) Google Scholar, 4Dumonteil E. Barré H. Meissner G. Am. J. Physiol. 1993; 265: C507-C513Crossref PubMed Google Scholar, 5Block B.A. Annu. Rev. Physiol. 1994; 56: 535-577Crossref PubMed Scopus (223) Google Scholar, 6Dumonteil E. Barré H. Meissner G. Am. J. Physiol. 1995; 269: C955-C960Crossref PubMed Google Scholar). This enzyme translocates Ca2+ from the cytosol to the lumen of the sarcoplasmic/endoplasmic vesicles by using the chemical energy derived from ATP hydrolysis (7Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 8de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. Vol. 2. John Wiley and Sons, New York1981Google Scholar, 9Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 10de Meis L. Arch. Biochem. Biophys. 1993; 306: 287-296Crossref PubMed Scopus (37) Google Scholar). Calorimetric measurements of rat soleus muscle (2Chinet A.E. Decrouy A. Even P.C. J. Physiol. ( Lond. ). 1992; 455: 663-678Crossref PubMed Scopus (43) Google Scholar) indicate that 25 to 45% of heat produced in resting muscle is related to Ca2+ circulation between sarcoplasm and sarcoplasmic reticulum. Three distinct genes encode the sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) 1The abbreviations used are:SERCAsarco/endoplasmic reticulum Ca2+-ATPasePAFplatelet activating factorMOPS4-morpholinepropanesulfonic acid isoforms, but the physiological meaning of isoforms diversity is not clear. The SERCA 1 gene is expressed exclusively in fast skeletal muscle (11MacLennan D.H. Brandl C.J. Korczak B. Green N.M. Nature. 1985; 316: 696-700Crossref PubMed Scopus (847) Google Scholar) whereas blood platelets and lymphoid tissues express SERCA 3 and SERCA 2b genes (12Enouf J. Bredoux R. Papp B. Djaffar I. Lompré A.M. Kieffer N. Gayet O. Clemetson K. Wuytack F. Rosa J.P. Biochem. J. 1992; 286: 135-140Crossref PubMed Scopus (47) Google Scholar, 13Wuytack F. Papp B. Verboomen H. Raeymaekers L. Dode L. Bobe R. Enouf J. Bokkala S. Authi K.S. Casteels R. J. Biol. Chem. 1994; 269: 1410-1416Abstract Full Text PDF PubMed Google Scholar, 14Bobe R. Bredoux R. Wuytack F. Quarck R. Kovàcs T. Papp B. Corvazier E. Magnier C. Enouf J. J. Biol. Chem. 1994; 269: 1417-1424Abstract Full Text PDF PubMed Google Scholar). The catalytic cycle of the different SERCA can be reversed after a Ca2+ gradient has been formed across the vesicles membrane. During this reversal, Ca2+ leaves the vesicles through the ATPase in a process coupled with the synthesis of ATP from ADP and Pi (7Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 8de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. Vol. 2. John Wiley and Sons, New York1981Google Scholar, 9Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 10de Meis L. Arch. Biochem. Biophys. 1993; 306: 287-296Crossref PubMed Scopus (37) Google Scholar, 15Makinose M. Hasselbach W. FEBS Lett. 1971; 12: 267-268Crossref PubMed Scopus (112) Google Scholar). For vesicles derived from skeletal muscle, a part of the Ca2+ retained during transport leaks through the Ca2+-ATPase without promoting synthesis of ATP (16Inesi G. de Meis L. J. Biol. Chem. 1989; 264: 5929-5936Abstract Full Text PDF PubMed Google Scholar, 17de Meis L. J. Biol. Chem. 1991; 266: 5736-5742Abstract Full Text PDF PubMed Google Scholar, 18Galina A. De Meis L. J. Biol. Chem. 1991; 266: 17978-17982Abstract Full Text PDF PubMed Google Scholar, 19de Meis L. Inesi G. FEBS Lett. 1992; 299: 33-35Crossref PubMed Scopus (57) Google Scholar, 20de Meis L. Suzano V.A. J. Biol. Chem. 1994; 269: 14525-14529Abstract Full Text PDF PubMed Google Scholar, 21de Meis L. Wolosker H. Engelender S. Biochim. Biophys. Acta. 1996; 1275: 105-110Crossref Scopus (22) Google Scholar). This efflux is referred to as uncoupled Ca2+efflux. Recently it was shown that the SERCA 1 of skeletal muscle is able to convert osmotic energy into heat. Calorimetric measurements revealed that the amount of heat produced after the hydrolysis of each ATP molecule hydrolyzed increases 2–3-fold when a Ca2+gradient is formed across the vesicles membrane (22de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (55) Google Scholar, 23de Meis L. Biochem. Biophys. Res. Commun. 1998; 243: 598-600Crossref PubMed Scopus (17) Google Scholar, 24de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar). The extra heat produced during ATP hydrolysis seems to be promoted by the uncoupled Ca2+ efflux during which the energy derived from the Ca2+ gradient is converted by the SERCA 1 into heat. sarco/endoplasmic reticulum Ca2+-ATPase platelet activating factor 4-morpholinepropanesulfonic acid In the present study we have measured the heat production during ATP hydrolysis, Ca2+ transport, and Ca2+ efflux in vesicles derived from the sarco/endoplasmic reticulum of skeletal muscle and blood platelets, both in the absence and presence of PAF. Vesicles derived from the dense tubules of human blood platelets and the light fraction of rabbit skeletal muscle sarcoplasmic reticulum were prepared as described previously (25Eletr S. Inesi G. Biochim. Biophys. Acta. 1972; 282: 174-179Crossref PubMed Scopus (324) Google Scholar, 26Le Peuch C. Le Peuch D.A.M. Kats L. Demaille J.G. Hincke M.T. Bredoux R. Enouf J. Levy-Toledano S. Caen J.P. Biochim. Biophys. Acta. 1983; 731: 456-464Crossref PubMed Scopus (53) Google Scholar). The muscle vesicle preparation does not contain significant amounts of ryanodine/caffeine-sensitive Ca2+-channels nor does it exhibit the phenomenon of activation of Ca2+ efflux by external Ca2+,i.e. Ca2+-induced Ca2+ release found in the heavy fraction of the sarcoplasmic reticulum (16Inesi G. de Meis L. J. Biol. Chem. 1989; 264: 5929-5936Abstract Full Text PDF PubMed Google Scholar). Both the muscle and blood platelet vesicles were stored in liquid nitrogen until use. This was measured by the filtration method using 45Ca and Millipore filters (27Chiesi M. Inesi G. J. Biol. Chem. 1979; 254: 10370-10377Abstract Full Text PDF PubMed Google Scholar). After filtration, the filters were washed five times with 5 ml of 3 mm La(NO3)3and the radioactivity remaining on the filters was counted on a liquid scintillation counter. The free Ca2+ concentration was calculated using the association constants of Schwartzenbach et al. (28Schwartzenbach G. Senn H. Anderegg G. Helv. Chim. Acta. 1957; 40: 1886-1900Crossref Scopus (221) Google Scholar) in a computer program described by Fabiato and Fabiato (29Fabiato A. Fabiato F. J. Physiol. ( Paris ). 1979; 75: 463-505PubMed Google Scholar) and modified by Sorenson et al. (30Sorenson M.M. Coelho H.S.L. Reuben J.P. J. Membr. Biol. 1986; 90: 219-230Crossref PubMed Scopus (72) Google Scholar). For Ca2+ efflux experiments, the vesicles were preloaded with45Ca in a medium containing 50 mm MOPS-Tris (pH 7.0), 10 mm MgCl2, 100 mm KCl, 20 mm Pi, 0.3 mm CaCl2, 3 mm ATP, and 60 μg of vesicles protein/ml. After 30 (muscle vesicles) or 60 (platelet vesicles) min incubation at 35 °C, the vesicles were centrifuged at 40,000 × g for 30 min, the supernatant was discarded, and the walls of the tubes were blotted to minimize the volume of residual loading medium. The pellet was kept on ice and resuspended in ice-cold water immediately before use. The efflux was arrested as described above for the Ca2+ uptake. This was assayed measuring the release of32Pi from [γ-32P]ATP. The32Pi produced was extracted from the medium with ammonium molybdate and a mixture of isobutyl alcohol and benzene (31de Meis L. Methods Enzymol. 1988; 157: 190-206Crossref PubMed Scopus (68) Google Scholar). The Mg2+ dependent activity was measured in the presence of 5 mm EGTA. The Ca2+-ATPase activity was determined by subtracting the Mg2+ dependent activity from the activity measured in the presence of both Mg2+ and Ca2+. This was measured as described previously (31de Meis L. Methods Enzymol. 1988; 157: 190-206Crossref PubMed Scopus (68) Google Scholar). [32P]Pi was obtained from the Brazilian Institute of Atomic Energy. This was measured using an OMEGA Isothermal Titration Calorimeter from Microcal Inc. (Northampton, MA) (22de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (55) Google Scholar, 24de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar). The calorimeter cell was filled with a reaction medium, and the reference cell was filled with Milli-Q water. After equilibration at the desired temperature, the reaction was started by injecting sarcoplasmic reticulum vesicles into the reaction cell and the heat change due to ATP hydrolysis was recorded starting from 2 min after the injection up to a maximum of 30 min. The calorimetric enthalpy of hydrolysis (ΔH cal) was calculated by dividing the amount of heat released by the net amount of ATP hydrolyzed. The units used were moles for ATP hydrolyzed and kcal for the heat released. A negative value indicates that the reaction was exothermic and a positive value indicates that it was endothermic. The PAF used wasdl-α-phosphatidylcholine β-acetyl-γ-O-hexadecyl (1-O-hexadecyl-2-acetyl-rac-glycero-3-phosphocholine), obtained from Sigma. PAF was dissolved in ethanol. Thapsigargin (LC Service, Woburn, MA) was dissolved in dimethyl sulfoxide. After dilution, the final concentrations of ethanol and dimethyl sulfoxide in the assay medium were less than 1%. 45Ca was purchased from Dupont (Wilmington, DE). All other reagents were of analytical grade. In agreement with previous reports, we found that the vesicles derived from blood platelets (Fig. 1 A and TableI) were able to accumulate a smaller amount of Ca2+ than the vesicles derived from muscle (Fig. 2 A and TableII). During transport the two vesicle preparations catalyze simultaneously the hydrolysis (Figs.1 B and 2 B) and the synthesis of ATP from ADP and Pi. The rate of synthesis was severalfold slower than the rate of hydrolysis. Using the same experimental conditions as those described in Tables I and II and in the presence of 1 μmfree Ca2+, the rates of ATP synthesis for platelets and muscle vesicles were 0.08 ± 0.01 (6Dumonteil E. Barré H. Meissner G. Am. J. Physiol. 1995; 269: C955-C960Crossref PubMed Google Scholar) and 2.57 ± 0.22 (4Dumonteil E. Barré H. Meissner G. Am. J. Physiol. 1993; 265: C507-C513Crossref PubMed Google Scholar) μmol of ATP/mg protein·30 min−1, respectively. These values are the average ± S.E. of the number of experiments shown in parentheses. The kinetics of Ca2+ transport and ATP synthesis have been analyzed in detail in previous reports (7Hasselbach W. Biochim. Biophys. Acta. 1978; 515: 23-53Crossref PubMed Scopus (153) Google Scholar, 8de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. Vol. 2. John Wiley and Sons, New York1981Google Scholar, 9Inesi G. Annu. Rev. Physiol. 1985; 47: 573-601Crossref PubMed Google Scholar, 10de Meis L. Arch. Biochem. Biophys. 1993; 306: 287-296Crossref PubMed Scopus (37) Google Scholar). In this study we focused on heat produced during transport. The overall reaction of Ca2+ transport was exothermic regardless of whether muscle or platelet vesicles were used (Figs. 1 C and2 C). The amount of heat released during the different incubation intervals was proportional to the amount of ATP cleaved. This could be visualized by either plotting the heat released as a function of the amount of ATP hydrolyzed (Fig.3) or calculating the ΔH cal using the values of heat release and Pi produced at different incubation intervals (Fig.4). Two different ATPase activities can be distinguished in both platelet and muscle vesicles. The Mg2+ dependent activity requires only Mg2+ for its activation and is measured in the presence of EGTA to remove contaminant Ca2+ from the assay medium. The ATPase activity which is correlated with Ca2+ transport requires both Ca2+ and Mg2+ for full activity. In both vesicle preparations, the Mg2+-dependent ATPase activity represents a small fraction of the total ATPase activity measured in presence of Mg2+ and Ca2+ (Figs.1 B and 2 B). The amount of heat produced during the hydrolysis of ATP by the Mg2+-dependent ATPase was the same regardless of whether muscle or platelet vesicles were used (Fig. 3 C) and the ΔH calvalue (Fig. 4 and Tables I and II) calculated in the two conditions was the same as that previously measured with soluble F1 mitochondrial ATPase (23de Meis L. Biochem. Biophys. Res. Commun. 1998; 243: 598-600Crossref PubMed Scopus (17) Google Scholar) and soluble myosin at pH 7.2 (32Gajewski E. Steckler D.K. Goldberg R.N. J. Biol. Chem. 1986; 261: 12733-12737Abstract Full Text PDF PubMed Google Scholar). For the vesicles derived from muscle (SERCA 1) the formation of a Ca2+ gradient increased the yield of heat production during ATP hydrolysis (Figs.3 B and 4 B and Table II). This was not observed with the use of platelet vesicles (SERCA 2b and 3) where the yield of heat produced during ATP cleavage was the same in the presence and absence of a transmembrane Ca2+ gradient (Figs.3 A and 4 A and Table I). For the muscle vesicles (Table II), there was no difference in the ΔH cal value of the Mg2+-dependent ATPase and the Ca2+-ATPase when the vesicles were rendered leaky (no gradient). With intact vesicles, the ΔH calvalue was more negative, i.e. more heat was produced during the hydrolysis of each ATP molecule when the free Ca2+concentration in the medium was decreased from 10 to 1 μm(Fig. 4 B and Table II). During transport, the Piavailable in the assay medium diffuses through the membrane to form Ca2+ phosphate crystals inside the vesicles. These crystals operate as a Ca2+ buffer that maintains the free Ca2+ concentration inside the vesicles constant (∼5 mm) at the level of the solubility product of calcium phosphate (8de Meis L. Bittar E. The Sarcoplasmic Reticulum: Transport and Energy Transduction. Vol. 2. John Wiley and Sons, New York1981Google Scholar, 33de Meis L. Hasselbach W. Machado R.D. J. Cell Biol. 1974; 62: 505-509Crossref PubMed Scopus (45) Google Scholar). The energy derived from the gradient depends on the difference between the Ca2+ concentrations inside and outside the vesicles. Thus, the different values of ΔH cal measured with the muscle vesicles with 1 and 10 μm Ca2+ suggest that when the free Ca2+ concentration in the medium is lower, the gradient formed across the vesicles membrane is steeper; thus more heat was produced and a more negative value of the ΔH cal for ATP hydrolysis was observed. With vesicles derived from blood platelets, there was no extra heat production during Ca2+ transport regardless of the free Ca2+ concentration in the medium (Figs. 3 A and4 A and Table I). Similar to muscle, Pi diffuses through the membrane of the platelet vesicles forming calcium phosphate crystal inside the vesicles that ensures the maintenance of the free Ca2+ concentration in the vesicles lumen at the same level as that of the muscle (∼5 mm). Thus during transport, the Ca2+ gradient formed across the membrane in the presence of 1 and 10 μm Ca2+ should be the same in muscle and platelet vesicles. These findings indicate that different from the muscle, the Ca2+-ATPase of platelets is not able to convert the osmotic energy derived from the gradient into heat.Table IHeat production during Ca2+ transport and ATP hydrolysis by the platelets Ca2+-ATPaseCa2+Ca2+ uptakeATPase activityHeat releasedΔH calμmμmol/mg, 30 minμmol/mg, 30 minmcal/mg, 30 minkcal/mol0—0.35 ± 0.08 (7)4.17 ± 0.87 (7)−12.30 ± 0.71 (7)0.10.047 ± 0.007 (3)0.80 ± 0.11 (6)9.80 ± 1.30 (6)−13.43 ± 2.27 (6)0.40.129 ± 0.038 (6)1.45 ± 0.26 (9)14.34 ± 1.67 (9)−10.73 ± 0.86 (9)1.00.144 ± 0.020 (3)1.77 ± 0.29 (4)15.93 ± 0.75 (4)−9.91 ± 1.93 (4)10.00.215 ± 0.023 (9)1.61 ± 0.23 (15)17.11 ± 2.59 (15)−10.99 ± 1.09 (15)The assay medium composition and experimental conditions were as described in Fig. 1 using 5 mm EGTA (zero Ca2+) or 0.1 mm EGTA and 0.029, 0.063, 0.082 or 0.112 mmCaCl2. The free Ca2+ concentrations calculated with the different EGTA and CaCl2 concentrations used are shown. Values are mean ± S.E. of the number of experiments shown in parentheses. Open table in a new tab Figure 2Skeletal muscle vesicles.Ca2+ uptake (A), ATPase activity (B), and heat released (C). The assay medium composition and other experimental conditions were as described in the legend to Fig. 1using 10 μg/ml rabbit intact sarcoplasmic reticulum vesicles in the absence of Ca2+ (Δ) or in the presence of either 10 (○) or 1 (●) μm free Ca2+.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIHeat production during Ca2+ transport and ATP hydrolysis by the skeletal muscle Ca2+-ATPase in the presence and absence of a transmembrane Ca2+ gradientAdditionsCa2+ uptakeATPase activityHeat releasedΔH calμmol/mg, 30 minmcal/mg, 30 minkcal/molIntact vesicles,2.11 ± 0.1220.56 ± 2.3−10.20 ± 1.38 EGTA, 2 mm(11)(11)(11)Leaky vesicles,042.60 ± 2.41518.87 ± 27.8−12.18 ± 1.29 Ca2+, 10 μm(16)(16)(16)Intact vesicles,1.85 ± 0.1640.28 ± 2.511,270.40 ± 62.91−31.88 ± 1.22 Ca2+, 1 μm(5)(5)(5)(5)Intact vesicles,2.65 ± 0.4346.11 ± 2.181,054.06 ± 147.39−22.67 ± 2.14 Ca2+, 10 μm(5)(5)(5)(5)The assay medium composition and experimental conditions were as described in Fig. 2. For the leaky vesicles, 4 μm of the divalent cation ionophore A23187 was included in the assay medium. Values are mean ± S.E. of the number of experiments shown in parentheses. With intact vesicles, the difference of ΔH cal measured with EGTA and 10 μmCa2+ and the difference between the values measured with 1 and 10 μm Ca2+ were significant (t test) with p < 0.001 and p < 0.005, respectively. Open table in a new tab Figure 3Heat released during ATP hydrolysis by muscle and platelet vesicles. The values of heat released measured in Figs. 1 and 2 were plotted as a function of ATP hydrolyzed.A, platelets, or B, muscle vesicles in the absence of Ca2+ and 5 mm EGTA (Δ) and either 10 (○) or 1.0 (●) μm free Ca2+. The data obtained with the use of 5 mm EGTA in A andB were plotted in C, where open symbols represent platelet vesicles and closed symbolsrepresent muscle vesicles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Effect of Ca2+ gradient on the ΔH cal values measured with platelet (A) and skeletal muscle (B) vesicles. The experimental conditions were the same as those of Figs. 1 and 2 with 1 μm free Ca2+ (●), 10 μm free Ca2+(○), or 5 mm EGTA (Δ).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The assay medium composition and experimental conditions were as described in Fig. 1 using 5 mm EGTA (zero Ca2+) or 0.1 mm EGTA and 0.029, 0.063, 0.082 or 0.112 mmCaCl2. The free Ca2+ concentrations calculated with the different EGTA and CaCl2 concentrations used are shown. Values are mean ± S.E. of the number of experiments shown in parentheses. The assay medium composition and experimental conditions were as described in Fig. 2. For the leaky vesicles, 4 μm of the divalent cation ionophore A23187 was included in the assay medium. Values are mean ± S.E. of the number of experiments shown in parentheses. With intact vesicles, the difference of ΔH cal measured with EGTA and 10 μmCa2+ and the difference between the values measured with 1 and 10 μm Ca2+ were significant (t test) with p < 0.001 and p < 0.005, respectively. Kinetics evidence described in previous reports (22de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (55) Google Scholar, 24de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar) indicate that the extra heat measured in muscle vesicles was related to the uncoupled Ca2+ efflux mediated by the Ca2+-ATPase. This can be measured arresting the pump by the addition of an excess EGTA to the medium (Fig.5). In this condition, the free calcium available in the medium is chelated but Mg·ATP and other reagents remain at the same concentration as those used in the experiments of Figs. 1 and 2. For the muscle vesicles, the efflux promoted by EGTA decreased when thapsigargin, a specific inhibitor of the Ca2+-ATPase (34Thastrup O. Foder B. Scharff O. Biochem. Biophys. Res. Commun. 1987; 142: 654-660Crossref PubMed Scopus (117) Google Scholar, 35Sagara Y. Fernandez-Belda F. de Meis L. Inesi G. J. Biol. Chem. 1992; 267: 12606-12613Abstract Full Text PDF PubMed Google Scholar), was added to the medium simultaneously with EGTA. The difference between the total efflux and the efflux measured in the presence of thapsigargin represents the uncoupled efflux mediated by the Ca2+-ATPase (19de Meis L. Inesi G. FEBS Lett. 1992; 299: 33-35Crossref PubMed Scopus (57) Google Scholar, 36Wolosker H. de Meis L. Am. J. Physiol. 1994; 266: C1376-C1381Crossref PubMed Google Scholar) and in muscle vesicles it represents about 70% of the total Ca2+ efflux (Fig. 5 and TableIII). The uncoupled efflux can also be measured diluting vesicles previously loaded with Ca2+ in a medium containing only buffer and EGTA (Fig.6 B and Tables III andIV). For the muscle vesicles, this leakage was also decreased by thapsigargin to the same extent as that measured in the conditions of Fig. 5. The Ca2+ efflux of platelet vesicles was slower than that of muscle and was not impaired by thapsigargin, regardless of the method used to measure the efflux (Fig. 6 A and Table IV). These data suggest that Ca2+ leaks through the SERCA 1 of skeletal muscle but not through the SERCA 2B and 3 found in blood platelets. Therefore, the difference of heat production measured in muscle and platelet vesicles after formation of a transmembrane gradient (Fig. 3 and Tables I andII) could be due to the absence of uncoupled Ca2+ leakage through the Ca2+-ATPase in platelet vesicles (thapsigargin-sensitive efflux in Table III).Table IIICa2+ efflux from skeletal muscle vesiclesMethod usednCa2+ effluxTotal (A)5 μm TG (B)TG-sensitive (A-B)nmol/mg min−1Without PAF 1) Preloaded and diluted8206 ± 3366 ± 14139 ± 24 2) EGTA added during uptake7203 ± 2663 ± 19140 ± 22With 4 μm PAF 3) Preloaded and diluted8196 ± 34109 ± 1994 ± 23 4) PAF added during uptake4228 ± 3897 ± 20130 ± 38In 1 and 3 the vesicles were previously loaded with 45Ca as described under “Experimental Procedures” and then diluted in a medium containing 50 mm MOPS/Tris (pH 7.0) and 0.1 mm EGTA as described in Fig. 6 B. The assay medium composition and experimental conditions used to measure Ca2+ release in 2 and 4 were as described in Figs. 5 and8 B, respectively. As shown, TG refers to thapsigargin. Values are mean ± S.E. of the number of experiments shown undern. Open table in a new tab Figure 6Ca2+ efflux from platelet (A) and skeletal muscle (B) vesicles. The vesicles were preloaded with 45Ca as described under “Experimental Procedures” and diluted to a final concentration of 30 μg of protein/ml into a medium containing 50 mm MOPS/Tris (pH 7.0) and 0.1 mm EGTA either in the absence (●) or presence (○) of 1 μmthapsigargin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IVCa2+ efflux from blood platelets vesiclesVesiclesnCa2+ effluxTotal (A)5 μm TG (B)TG-sensitive (A-B)nmol/mg min−11) Preloaded and diluted640 ± 341 ± 602) EGTA added during uptake444 ± 1279 ± 2503) PAF added during uptake4>273 ± 961 ± 3>212 ± 10In A the vesicles were previously loaded with 45Ca as described under “Experimental Procedures” and then diluted in a medium containing 50 mm MOPS/Tris (pH 7.0) and 0.5 mmEGTA. The assay medium composition and experimental conditions used to measure Ca2+ release after the addition of either 5 mm EGTA (2Chinet A.E. Decrouy A. Even P.C. J. Physiol. ( Lond. ). 1992; 455: 663-678Crossref PubMed Scopus (43) Google Scholar) or 6 μm PAF (3Janský L. Physiol. Rev. 1995; 75: 237-259Crossref PubMed Scopus (123) Google Scholar) were as described in Fig. 8 A. Values are mean ± S.E. of the number of experiments shown under n. Open table in a new tab In 1 and 3 the vesicles were previously loaded with 45Ca as described under “Experimental Procedures” and then diluted in a medium containing 50 mm MOPS/Tris (pH 7.0) and 0.1 mm EGTA as described in Fig. 6 B. The assay medium composition and experimental conditions used to measure Ca2+ release in 2 and 4 were as described in Figs. 5 and8 B, respectively. As shown, TG refers to thapsigargin. Values are mean ± S.E. of the number of experiments shown undern. In A the vesicles were previously loaded with 45Ca as described under “Experimental Procedures” and then diluted in a medium containing 50 mm MOPS/Tris (pH 7.0) and 0.5 mmEGTA. The assay medium composition and experimental conditions used to measure Ca2+ release after the addition of either 5 mm EGTA (2Chinet A.E. Decrouy A. Even P.C. J. Physiol. ( Lond. ). 1992; 455: 663-678Crossref PubMed Scopus (43) Google Scholar) or 6 μm PAF (3Janský L. Physiol. Rev. 1995; 75: 237-259Crossref PubMed Scopus (123) Google Scholar) were as described in Fig. 8 A. Values are mean ± S.E. of the number of experiments shown under n. In previous reports it was shown that some of the lipids derived from the breakdown of membrane phospholipids were able to increase the uncoupled efflux mediated by the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (37Cardoso C.M. de Meis L. Biochem. J. 1993; 296: 49-52Crossref PubMed Scopus (38) Google Scholar). We therefore tested different lipids in platelet vesicles in search of a compound that could promote a thapsigargin-sensitive Ca2+ efflux. The reasoning was that if we could promote the leakage of Ca2+ through the platelet Ca2+-ATPase then, similar to the muscle vesicles, the platelet vesicles should become able to convert osmotic energy into heat. In the course of these experiments we found thatdl-α-phosphatidylcholine β-acetyl-γ-O-hexadecyl could promote such an efflux in platelets but not in muscle vesicles. This phospholipid belongs to a family of acetylated phospholipids known as PAF which are produced when cells involved in inflammatory process are activated. PAF was found to inhibit the Ca2+ uptake of both platelets and muscle vesicles (Figs. 7 and 8 and TableV). With the two vesicles, half-maximal inhibition was obtained with 4 to 6 μm PAF. In contrast with the Ca2+ uptake, the ATPase activity of the two vesicle preparations was not inhibited by PAF (Fig. 7). The discrepancy between Ca2+ uptake and ATPase activity suggests that the decrease of Ca2+ accumulation was promoted by an increase of Ca2+ efflux and not by an inhibition of the ATPase. The amount of Ca2+ retained by the vesicles is determined by the differences between the rates of Ca2+ uptake and Ca2+ efflux. The higher the efflux, the smaller the amount of Ca2+ retained by the vesicles. The addition of PAF during the course of Ca2+ uptake promoted the release of Ca2+ until a new steady state level of Ca2+retention was achieved (Fig. 8 and Table V). With both preparations, when the higher concentration of PAF was added, the lower the new steady state level of Ca2+ filling (Figs. 7 and 8). The release of Ca2+ promoted by PAF was not accompanied by a burst of ATP synthesis. On the contrary, PAF inhibited the synthesis of ATP driven by the coupled Ca2+ efflux (Fig.9). This indicates that Ca2+release promoted by PAF was not promoted by an increase of the reversal of the pump. A major difference between the muscle and platelet vesicles was found when thapsigargin was added to the medium together with PAF. For platelet vesicles, the rate of Ca2+ release measured after the addition of PAF was greatly decreased in the presence of thapsigargin (Fig. 8 A and Table IV) indicating that most of the Ca2+ left the vesicles through the ATPase as an uncoupled Ca2+ efflux. This could be better seen after the initial minute of incubation. In fact, the rate of release in platelet vesicles was so fast that we could not measure the initial velocity of release with the method available in our laboratory. Thus, the values with PAF in Table III differ from the other values in that it does not reflect a true rate, but only the parcel of Ca2+ released during the first incubation minute. In muscle, the rate of Ca2+ efflux measured after the addition of PAF was slower than that measured with platelet vesicles (compare Fig. 8, A and B) and the proportion between the Ca2+ effluxes sensitive and insensitive to thapsigargin measured with PAF was practically the same as that measured when the pump was arrested with EGTA (Table IV).Figure 8Ca2+ release after the addition of PAF . The assay medium composition was 50 mmMOPS/Tris buffer (pH 7.0), 2 mm MgCl2, 10 mm Pi, 40 μm CaCl2, 100 mm KCl, and 3 mm ATP. The reaction was started by the addition of either platelet (A) or muscle (B) vesicles to a final concentration of 30 μg/ml protein. ○, control without additions. The arrows indicate the addition of either 6 μm PAF (Δ) or 6 μmPAF plus 4 μm thapsigargin (▴).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table VEffect of PAF on the Ca2+ uptake and the ΔHcal of ATP hydrolysisCa2+PAFSkeletal muscle vesiclesBlood platelets vesiclesCa2+ uptakeΔH calCa2+uptakeΔH calμmμmol/mg, 30 minKcal/molμmol/mgKcal/mol101.83 ± 0.21−32.99 ± 2.900.11 ± 0.03−12.58 ± 1.29(4)(4)(3)(5)40.45 ± 0.19−25.69 ± 1.710.03 ± 0.01−20.04 ± 0.37(4)(4)(3)(3)1002.66 ± 0.44−22.92 ± 2.240.20 ± 0.04−10.70 ± 1.01(3)(3)(3)(3)60.68 ± 0.35−16.91 ± 1.500.06 ± 0.01−23.90 ± 1.06(5)(5)(3)(3)The assay medium and experimental conditions were as in Figs. 1 and 2. The values in the table are the average ± S.E. of the number of experiments shown in parentheses. The differences between the ΔH cal values measured in the absence and in the presence of PAF with skeletal muscle were significant (ttest) with p < 0.05 both with 1 and 10 μm Ca2+ and, with blood platelets were significant with p < 0.005 (1 μmCa2+) and p < 0.001 (10 μmCa2+). Open table in a new tab Figure 9Effect of PAF on the rate of ATP synthesis by platelet (●) and muscle (○) vesicles. The assay medium composition was the same as that described in the legends to Figs. 1and 2 except that trace amounts of [32P]Piwere added to the medium in order to measure the amount of [γ-32P]ATP formed from ADP and Pi after 30 min incubation at 35 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The assay medium and experimental conditions were as in Figs. 1 and 2. The values in the table are the average ± S.E. of the number of experiments shown in parentheses. The differences between the ΔH cal values measured in the absence and in the presence of PAF with skeletal muscle were significant (ttest) with p < 0.05 both with 1 and 10 μm Ca2+ and, with blood platelets were significant with p < 0.005 (1 μmCa2+) and p < 0.001 (10 μmCa2+). Having found a compound that induces the release of Ca2+through the pump, we then measured the heat produced during ATP hydrolysis in the presence and absence of PAF. The PAF concentrations selected were sufficient to enhance the rate of efflux without completely abolishing the retention of Ca2+ by the vesicles, i.e. without abolishing the formation of a Ca2+ gradient through the vesicles membrane (Fig. 8). In such conditions PAF was found to enhance the amount of heat produced during the hydrolysis of ATP by blood platelets (Fig.10 and Table V). In muscle vesicles, however, PAF was found to decrease the amount of heat produced during ATP hydrolysis. The ΔH cal values measured with PAF and muscle vesicles were less negative than those measured in the absence of PAF, but still more negative than the values measured in the absence of Ca2+ gradient. It is generally assumed that the energy released during the hydrolysis of ATP by Ca2+-ATPase can be divided in two non-interchangeable parts, one is converted into heat and the other is used to pump Ca2+ across the membrane (1Clausen T. Hardeveld C.V. Everts M.E. Physiol. Rev. 1991; 71: 733-774Crossref PubMed Scopus (210) Google Scholar, 2Chinet A.E. Decrouy A. Even P.C. J. Physiol. ( Lond. ). 1992; 455: 663-678Crossref PubMed Scopus (43) Google Scholar, 3Janský L. Physiol. Rev. 1995; 75: 237-259Crossref PubMed Scopus (123) Google Scholar, 4Dumonteil E. Barré H. Meissner G. Am. J. Physiol. 1993; 265: C507-C513Crossref PubMed Google Scholar, 5Block B.A. Annu. Rev. Physiol. 1994; 56: 535-577Crossref PubMed Scopus (223) Google Scholar, 6Dumonteil E. Barré H. Meissner G. Am. J. Physiol. 1995; 269: C955-C960Crossref PubMed Google Scholar). Here, this was observed with the platelet vesicles before the addition of PAF (Table I). The recent finding (22de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (55) Google Scholar, 23de Meis L. Biochem. Biophys. Res. Commun. 1998; 243: 598-600Crossref PubMed Scopus (17) Google Scholar, 24de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar) that the SERCA 1 can convert osmotic energy into heat revealed an alternative route that increases 2–3-fold the amount of heat produced during ATP hydrolysis, therefore permitting the maintenance of the cell temperature with a smaller consumption of ATP (Table II). By this route, a part of the energy released during ATP hydrolysis is dissipated into the surrounding medium as heat. The other part is used to pump Ca2+ across the membrane. During uptake, a fraction of the Ca2+ accumulated flows back through the Ca2+-ATPase from the vesicles lumen to the medium driven by the Ca2+ gradient. This efflux is coupled with the production of heat (thapsigargin-sensitive efflux). Thus, for the muscle vesicles, heat would be produced at least in two different steps of the energy interconversion cycle: (i) during the hydrolysis of ATP, where a part of the chemical energy released was directly converted into heat and (ii) during the leakage of Ca2+ through the ATPase where part of the energy derived from ATP hydrolysis used to pump Ca2+ is first converted into osmotic energy, and then converted by the enzyme into heat (22de Meis L. Bianconi M.L. Suzano V.A. FEBS Lett. 1997; 406: 201-204Crossref PubMed Scopus (55) Google Scholar, 24de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar). The data reported show that not all SERCA isoforms are able to readily convert osmotic energy into heat. The vesicles of blood platelets, as obtained after cell fractionation, are not able to promote this conversion. These vesicles, however, can be converted by PAF into a system capable of increasing the heat production during ATP hydrolysis, suggesting that the mechanism capable of providing additional heat production can be turned on and off and this could represent a mechanism of thermoregulation specific of the cells expressing SERCA 2b and 3. Both in muscle and platelet vesicles there is a Ca2+ efflux which is not inhibited by thapsigargin. We do not know through which membrane structure this Ca2+ flows, but the data obtained with platelets before the addition of PAF indicate that during this efflux, osmotic energy is not converted into heat. In platelets, PAF promoted simultaneously the appearance of thapsigargin-sensitive efflux and extra heat production during ATP hydrolysis (Tables IV and V). These observations corroborate with the notion that the conversion of osmotic energy into heat cannot be promoted by any kind of Ca2+leakage and that a device is needed for this conversion (24de Meis L. Am. J. Physiol. 1998; 274: C1738-C1744Crossref PubMed Google Scholar). For the endoplasmic/sarcoplasmic reticulum, this device seems to be the Ca2+-ATPase itself, which in addition to interconvert chemical into osmotic energy, could also convert osmotic energy into heat. Regardless of its possible physiological implication, in this study the use of PAF as an experimental tool permitted us to show that the platelet vesicles can be converted from an inactive into an active system capable of converting osmotic energy into heat. Heat generation is implicated in the regulation of several physiological processes including metabolism and energy balance of the cell. The Vmax of most enzymes varies significantly after a discrete temperature change leading to a substantial change of the metabolic activity of the cell (1Clausen T. Hardeveld C.V. Everts M.E. Physiol. Rev. 1991; 71: 733-774Crossref PubMed Scopus (210) Google Scholar, 2Chinet A.E. Decrouy A. Even P.C. J. Physiol. ( Lond. ). 1992; 455: 663-678Crossref PubMed Scopus (43) Google Scholar, 3Janský L. Physiol. Rev. 1995; 75: 237-259Crossref PubMed Scopus (123) Google Scholar). PAF was originally described as a soluble factor in blood, so it is apparent that some cells secrete it following synthesis. Subsequent experimentation revealed that the secretion of PAF varies greatly depending on the cell type. In some cells, for instance, endothelial cells, the PAF synthesized is not secreted and is used by the cell itself (for reviews, see Refs. 38Prescott S.M. Zimmerman A. McIntyre T.M. J. Biol. Chem. 1990; 265: 17381-17384Abstract Full Text PDF PubMed Google Scholar and 39Chao W. Oslo M.S. Biochem. J. 1993; 292: 617-629Crossref PubMed Scopus (425) Google Scholar). The synthesis of PAF is initiated by phospholipase A2. The subsequent steps of synthesis are catalyzed by enzymes located in the endoplasmic reticulum. In metabolic labeling experiments, PAF appears first in the endoplasmic reticulum and then in the plasma membrane. PAF causes an elevation of the cytosolic-free Ca2+ promoted by the release of Ca2+ from both the plasma membrane and from intracellular stores. This was shown in various cells that express SERCA 2B and 3 including platelets, neutrophils, macrophage, endothelial cells, and neuronal cells (38Prescott S.M. Zimmerman A. McIntyre T.M. J. Biol. Chem. 1990; 265: 17381-17384Abstract Full Text PDF PubMed Google Scholar, 39Chao W. Oslo M.S. Biochem. J. 1993; 292: 617-629Crossref PubMed Scopus (425) Google Scholar). PAF can therefore act in two different manners, through a receptor in the plasma membrane or as an intracellular messenger. The affinity for PAF of the cell membrane receptor is very high, and the PAF concentration needed for cell activation varies between 10−12 and 10−9m, a concentration much smaller than that needed in this report to activate the thapsigargin-sensitive Ca2+ efflux in platelet vesicles (38Prescott S.M. Zimmerman A. McIntyre T.M. J. Biol. Chem. 1990; 265: 17381-17384Abstract Full Text PDF PubMed Google Scholar, 39Chao W. Oslo M.S. Biochem. J. 1993; 292: 617-629Crossref PubMed Scopus (425) Google Scholar). The only possibility that the effect of PAF observed in this report could have some physiological implication is if the concentration of PAF reaches the micromolar range in the microenvironment surrounding the endoplasmic reticulum membrane, where the Ca2+-ATPase is located and PAF is synthesized. In this case, the local effect of PAF not only would promote the release of Ca2+ from the endoplasmic reticulum (Fig. 8 A) but also enhance the amount of heat produced during hydrolysis of ATP (Tables IV and V). We are grateful to Valdecir A. Suzano for the technical assistance.