Invited Symposium: SERCA-Type of Calcium Pumps and Phospholamban
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 (1-3). Calorimetric measurements of rat soleus muscle (4) indicate that 25-45% of heat produced in resting muscle is related to Ca2+ circulation between sarcoplasm and sarcoplasmic reticulum. In cold-acclimated ducklings, 70% of the total heat production is derived from muscle (5,6). The Ca2+-ATPase of the sarcoplasmic reticulum of skeletal muscle is able to interconvert different forms of energy (7). During Ca2+ transport the chemical energy derived from ATP hydrolysis is converted into osmotic energy. After Ca2+ has accumulated inside the reticulum a Ca2+ gradient is formed across the membrane and this promotes the reversal of the catalytic cycle of the enzyme, during which osmotic energy is converted back into chemical energy. During cycle reversal, Ca2+ leaves the reticulum through the Ca2+ -ATPase and this is coupled with the synthesis of ATP from ADP and Pi. The Ca2+ release is referred to as active Ca2+ efflux. Not all the Ca2+ that leaves the vesicles is active; a part of it leaks through the Ca2+-ATPase without promoting synthesis of ATP and this is referred to as uncoupled Ca2+ efflux (8, 9). In our laboratory we observed that heat is produced when Ca2+ leaks from the vesicles through the uncoupled efflux. In this process the Ca2+-ATPase converts osmotic energy into heat (10-12). By varying the fraction of enzyme units that are uncoupled and those which ae coupled during the reversal of the Ca2+ pump it is possible to regulate the amount of heat that is generated during the uncoupled efflux.
Materials and Methods
1-Preparation of vesiclesfrom the sarcoplasmic reticulum of either rabbit (endotherm)or rainbow trout Salmo gairdnerii (poikilotherm) skeletal muscle were prepared as previously described (12). The preparation had practically no junctional protein and the efflux of Ca2+ measured in these vesicles is not altered by ryanodine. Permeated vesicles were prepared by incubating the vesicles at pH 9.0 in the presence of 2 mM EGTA at room temperature for 20 min (13). 2-ATPase activity, Ca2+ uptake, ATP synthesis and heats of reaction. The methods for measuring the ATPase activity using (g-32P) ATP, calcium uptake using 45Ca and ATP synthesis from ADP and 32Pi are described elsewhere(14). Heats of reaction were measured using an OMEGA Isothermal Titration Calorimeter from Microcal Inc. (Northampton, MA) (11-13). 3- The calorimetric enthalpy of ATP hydrolysis (dHcal) was calculated by dividing the amount of heat released by the 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 indicate that it was endothermic.
A transmembrane Ca2+ gradient is formed when intact the vesicles are incubated in a medium containing ATP. This is not observed with permeated vesicles because the membrane was disrupted. Both in the presence and absence of a Ca2+ gradient, the amount of heat produced during the hydrolysis of ATP was found to be proportional to the amount of ATP hydrolyzed. However, in the presence of the gradient, the amount of heat produced by the hydrolysis of each ATP molecule was larger than that measured with permeated vesicles (Table 1).This was observed with both rabbit and trout vesicles but depending on the origin of the vesicles there was a difference on the tempeerature dependence. For the rabbit, the difference of dHcal between intact and leaky vesicles could be detected between 30 and 35 degree C. At 25 degree C it disappeared as if the mechanism that converts osmotic energy into heat were locked. Table 1 - Gradient and heat production in rabbit and trout vesicles (dHcal, kcal/mol).
Conditions Rabbit Trout 35oC 25oC 25oC 15oC Intact vesicles -20.8+ 1.3 -11.5+ 0.5 -21.7+ 1.2 -11.1+ 0.7 (Ca2+ gradient) (41) (17) (18) (9) Leaky vesicles -12.2+ 1.3 -11.9+ 0.4 -10.1 + 1.1 10.4 +1.1 (no gradient) (16) (6) (9) (3)For the trout on the other hand, the difference could be detected between 20 and 25 degree and disappeared difference indicates that the vesicles were able to convert a part of the osmotic energy derived from the gradient into heat and raises the possibility that this conversion could reflect the uncoupled leakage of Ca2+ through the ATPase (Table 1). The coupled and the uncoupled Ca2+ effluxes represent two distinct routes of energy conversion, both mediated by the Ca2+ -ATPase: one route in which the osmotic energy derived from the Ca2+ gradient is used to synthesize ATP (coupled Ca2+ efflux), and one route in which the osmotic energy is converted into heat (uncoupled Ca2+ efflux). According to this reasoning it would be expected that drugs that change the rates of the coupled and uncoupled Ca2+ effluxes should also change the amount of heat produced and the amount of ATP synthesized during the transport of Ca2+. In previous reports it was shown that the Ca2+-ATPase is coupled more tightly by dimethyl sulfoxide and uncoupled by heparin ( ). Table 2 -Rabbit vesicles - Effect of drugs
ATP dHcal Additions n hidrol./ synth* kcal/mol None 27 31.5 + 5.3 - 22.3 + 1.4 DMSO 20% (v/v) 9 5.4 + 1.9 -13.2 + 0.7 Hep 3 mg /ml 4 57.7 + 8.1 -30.2 + 2.1
The addition of dimethyl sulfoxide to the assay medium promoted both a decrease in the amount of heat produced during the hydrolysis of ATP and an increase in the amount of ATP sinthesized. Note that dimethyl sulfoxide did not change the dHcal measured with permeated vesicles (Table 2). The opposite result was obtained with heparin. In the presence of 3ug/ml heparin the vesicles were still able to accumulate Ca2+ but now the heat produced during ATP hydrolysis was higher than that measured in the control without drug, and this was accompanied by a decrease in the amount of ATP resynthesized during transport.
Discussion and Conclusion
During the transport of Ca2+, heat is produced in at least two different steps of the cycle of energy interconversion. The firs step corresponds to the hydrolysis of ATP where chemical energy is directly converted into heat. The second step occur during the reversal of the cycle when 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. Thereforer, during steady state, the Ca2+ concentrations inside the vesicles and in the assay medium remain constant but the ATPase operates simultaneously forward (ATP hydrolysis and Ca2+ uptake) and backwards (Ca2+ efflux, ATP synthesis and heat production). Drugs can modulate the interconversion of osmotic energy either favorin the sinthesys of ATP and decreasing the heat production or, alternatively, decreasing the synthesis of ATP and increasing the heat production.
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|de Meis, L.; (1998). Conversion of Osmotic Energy into Heat by SERCA. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/wuytack/de_meis0488/index.html|
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