Guillermo L. Alonso

The interaction of magnesium ions with the calcium pump of sarcoplasmic reticulum

1. In the presence of Ca2+, ATP phosphorylates the Ca2+ pump of sarcoplasmic reticulum at the same site and to the same extent regardless of whether Mg2+ is added or not to the incubation media, the main effect of added Mg2+ being to increase the rate of phosphorylation. 2. When phosphoenzyme is made in Mg2+-containing media it dephosphorylates about 30-times faster than when it is made in the absence of added Mg2+. Addition of Mg2+ after phosphorylation is uneffective in accelerating the hydrolysis of phosphoenzyme even in solubilized enzyme, suggesting that phosphorylation of the Ca2+ pump results in occlusion of the site at which Mg2+ combines to accelerate the release of phosphate. 3. Occlusion of the site for Mg2+ can be partially reversed by trans-1,2-diaminocyclohexonetetraacetic acid (CDTA). Use was made of this property to demonstrate that for the rapid release of phosphate to occur Mg2+ has to be bound to the enzyme. 4. Results seem to indicate that Mg2+ combines with the Ca2+ pump prior to phosphorylation.

Effect of Tris(hydroxymethyl)aminomethane on isolated sarcoplasmic reticulum vesicles

Abstract The preincubation of isolated sarcoplasmic reticulum vesicles in Tris-Cl (pH 7.3) increases their (Ca2+ + Mg2+)-dependent adenosine triphosphatase activity and decreases their ATP-dependent Ca uptake capacity. These effects of Tris are dependent on the preincubation time and the Tris concentration; they are maximal below 10 μ m Ca and decrease upon the increase of Ca concentration in the preincubation media, and they increase upon the increase of the preincubation pH. Differences in ATPase activity between preincubated and control vesicles are abolished by A23187 but not by carbonyl cyanide p-trifluoromethoxy phenyl hydrazone. The results suggest that: (i) Preincubation of the vesicles in Tris causes an increase of their permeability for Ca, or a membrane damage. (ii) Tris must diffuse within the vesicles to promote these effects. (iii) Ca prevents these effects by decreasing the membrane permeability for Tris. The basic findings were reproduced replacing Tris by imidazole.

Effect of haloperidol on the sarcoplasmic reticulum Ca-dependent adenosine triphosphatase

Several effects of the neuroleptic agent haloperidol on the sarcoplasmic reticulum (SR) Ca-dependent adenosine triphosphatase (Ca-ATPase) and Ca transport are described. Haloperidol inhibits the Ca-ATPase activity in the presence of calcimycin. The effect depends on the conditions of preexposure of the membranes to the drug: the inhibition increases with the preincubation time; Ca and Mg protect the enzyme against the effect of the drug. The inhibitory effect of haloperidol decreases upon increasing [Ca2+], at constant [Mg], and disappears at 20 mM [Mg] for any [Ca2+], and at 0.5 mM [Ca2+] for any [Mg2+]. Haloperidol also inhibits phosphorylation of the enzyme by Pi, and ATP-dependent Ca2+ uptake, in both cases with apparent Ki = 0.10-0.15 mM, and increases the rate of Ca efflux from preloaded vesicles in this concentration range. The results suggest that haloperidol interacts with the catalytic site, interfering with the effect of the divalent catalytic cation, but not at other steps of the enzymatic cycle, where Mg2+ and Ca2+ are also activators. They are consistent with a reaction model where haloperidol interacts with the E2 conformers of the enzyme, with lower affinity for the phosphoenzyme than for the dephospho species. The inhibition of Ca uptake by SR vesicles is ascribed to an increased Ca2+ permeability rather than to the inhibition of the Ca-ATPase, which requires higher concentrations of the drug.

A BASIC program for the numerical solution of the transient kinetics of complex biochemical models

A highly optimized software for the kinetic analysis of complex chemical models is presented. The program is applied to the analysis of a vectorial biochemical reaction, where many species are linked by multiple equilibria of any order. The reaction stimulates the Ca2(+)-transport-linked ATPase reaction taking place in a suspension of vesicular fragments of isolated sarcoplasmic reticulum membranes, as described in many experimental reports. The model includes 12 reactants and intermediate chemical species, 14 kinetic constants, compartmentalization, and thermodynamic adjustment. The concentrations of all the model components, at any time, starting from a known initial condition, are calculated. The transient concentrations of the species are obtained by numerical integration of the appropriate differential equations, using an optimized version of the Runge-Kutta-Gill algorithm, with the aid of a Digital PDP11/23 computer and a standard BASIC-11 software, which could be fast and easily fitted to work with any microcomputer and/or alternative language or faster working compiled BASIC version. The errors of the calculations are evaluated.

Local Anesthetics Inhibit Ca-ATPase in Masticatory Muscles

Local anesthetics have myotoxic effects and inhibit Ca-ATPase activity and Ca transport in skeletal muscles. Such effects have not been fully elucidated in masticatory muscles. We tested the hypothesis that local anesthetics increase myoplasmic calcium in masticatory muscles by inhibiting Ca-ATPase at a concentration similar to that of dental cartridges. The effects of lidocaine and bupivacaine on Ca-ATPase from rabbit masseter and medial pterygoid muscles were tested with radioisotopic and colorimetric methods. Bupivacaine had an action similar to that of lidocaine on Ca-ATPase activity, but less effect on calcium transport. The pre-exposure of the membranes to the anesthetics enhanced the Ca-ATPase activity in the absence of calcium ionophore, supporting their permeabilizing effect. The results demonstrate that amide-type anesthetics do not inhibit calcium binding, but do reduce calcium transport and enzyme phosphorylation by ATP, and suggest that the myoplasmic calcium increase induced by lidocaine and bupivacaine might promote masticatory muscle contraction and eventual rigidity.

Calcium uptake, calcium release and adenosinetriphosphatase activity in sarcoplasmic reticulum fragments deposited on millipore filters.

Abstract 1. 1. A semi-rapid perfusion technique is used for the study of ATP-dependent Ca uptake, Ca release and ATPase activity in sarcoplasmic reticulum fragments. It involves the deposition of the biological material on millipore filters through which solutions containing the substrate, cofactors and/or inhibitors are perfused. The results show well-known properties of isolated sarcoplasmic reticulum vesicles, Ca uptake calls for ATP and Mg, and is greatly enhanced by oxalate and Pi. 2. 2. Ca uptake and ATPase activity are independent of the perfusion flow rate. 3. 3. The method allows the detection of Ca uptake in less than a second, using Ca concentrations within the sarcoplasmic range. 4. 4. While no differences are detected between ATPase activity measuredby our or other common techniques, ATP-dependent Ca uptake is systematically lower with the perfusion method. 5. 5. The addition of EGTA causes a decrease of ATPase activity and a sudden release of Ca. Very fast Ca release is also observed upon Ca depletion from the perfusing solutions. 6. 6. Very fast Ca release occurs upon stopping the Ca pumping mechanism, either by subtraction of ATP or Mg from the perfusing solution. The rate ofCa release is no so fast when Ca uptake proceeds in the presence of oxalate or inorganic phosphate. After Ca release, ATP elicits Ca uptake again. 7. 7. 45Ca taken up in the presence of ATP is rapidly exchanged with 40Ca, at a constant Ca concentration. Very fast 45Ca release is also observed when Ca concentration is raised from 5 μM to 5 mM. It comprises all 45Ca taken up in the presence of ATP. Lower amounts of 45Ca are released when the perfusing Ca concentration is raised for 5 to 8 μM. Under these conditions, 45Ca is probably exchanged with 40Ca. 8. 8. Microsomes loaded with Ca in their suspension medium, release Ca at a very low rate when deposited on millipore filters and perfused with an ATP-containing solution. 9. 9. The rapid rate of Ca release reported in this article may serve as a model of the rapid Ca relase from sarcoplasmic reticulum, in vivo, which triggers muscle contraction, since it occurs by effect of physiological agents.

Effect of carticaine on the sarcoplasmic reticulum Ca2+-adenosine triphosphatase. II. Cations dependence

Ca2+-ATPase is a major intrinsic protein in the sarcoplasmic reticulum (SR) from skeletal muscles. It actively transports Ca2+ from the cytoplasm to the SR lumen, reducing cytoplasmic [Ca2+] to promote muscle relaxation. Carticaine is a local anesthetic widely used in operative dentistry. We previously showed that carticaine inhibits SR Ca2+-ATPase activity and the coupled Ca2+ uptake by isolated SR vesicles, and increases the rate of Ca2+ efflux from preloaded vesicles. We also found that these effects were antagonized by divalent cations, and concluded that they were mainly due to the direct interaction of carticaine with the Ca2+-ATPase protein. Here we present additional results on the modulation of the above effects of carticaine by Ca2+ and Mg2+. The activating effect of Ca2+ on the ATPase activity is competitively inhibited by carticaine, indicating a decreased Ca2+ binding to the high affinity Ca2+ transport sites. The activating effect of Mg2+ on the phosphorylation of Ca2+-ATPase by orthophosphate is also inhibited by carticaine. The anesthetic does not affect the reaction mechanism of the cations acting as cofactors of ATP in the catalytic site. On the basis of the present and our previous results, we propose a model that describes the effect of carticaine on the Ca2+-ATPase cycle.

Thermodynamics of Ca2+ transport through sarcoplasmic reticulum membranes during the transient-state of simulated reactions

The kinetics of a chemical model of Ca2+ transport and coupled ATPase activity in sarcoplasmic reticulum membranes were solved for the transient-state of simulated reactions, using a numerical integration procedure. The simulation conditions reproduced in vitro experiments using either fragmented membranes or vesicles with Ca2+ accumulating ability. The results yielded the concentrations of all the ligands and intermediates of the enzymatic cycle as a function of the reaction time. These results were applied to calculations of several thermodynamic variables: (1) the step by step profile of the standard free energy change of the cycle. (2) The step by profile of the actual free energy change of the cycle, and its evolution with the reaction time. (3) The separate contributions of ATP hydrolysis and Ca2+ transport to the overall free energy change with the reaction. (4) The dependence of the velocity of the free energy change with the reaction time. (5) The efficiency of the transport system, and its change with the reaction time. (6) The separate contributions of the Ca2+ gradient and some enzymatic intermediates as free energy stores. The main findings are: (1) the step by step diagrams of the free energy change calculated from the results of the kinetic analysis better describe the thermodynamic profile of the cycle than previously reported diagrams of the standard free energy and basic free energy changes. The relative contribution of each partial step to the driving force of the whole reactions, as well as their changes upon the advancement of the reactions, are derived from the diagrams. (2) Free energy yielded by ATP hydrolysis is stored by the system, not only as a Ca2+ gradient, but also as enzymatic intermediates of the reaction. The progressive increase of both free energy pools upon the advancement of the reaction is quantitated.

Characteristics of the Sarcoplasmic Reticulum Ca2+-dependent ATPase from Masticatory Muscles

We compared the sarcoplasmic reticulum (SR) Ca-ATPase from masseter (M) and medial pterygoid (MP) muscles with that from fast muscles (FM) to examine whether its calcium transport capability and enzymatic activity are different. SR vesicles from FM, M, and MP muscles were obtained according to Champeil et al.(1985). Assays for characterization of the enzyme properties were performed. The results showed similar optimal conditions for the Ca-ATPase activity and calcium transport in M, MP, and FM. However, the maximal values of calcium transport, Ca-ATPase activity, and Ki for thapsigargin were significantly lower in the masticatory muscles. These findings are likely related to different Ca-ATPase isoforms. Since the local anesthetics used in dentistry inhibit Ca-ATPase and calcium transport in FM, it will be important for the effects of these drugs on the Ca-ATPase of masticatory muscles to be assessed.

Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle.

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.