ATP synthesis by Ca2+ + Mg2+-ATPase in detergent solution at constant Ca2+ levels.

ATP has been synthesized by the purified Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum (SR) solubilized in nonionic detergent dodecyloctaoxyethylenglycol-monoether in a solution containing inorganic phosphate and glycerol by changing pH upon addition of ADP. The Ca2+ concentration is kept constant during the experiment. Optimum synthesis is found at CaCl2 = 0.6 mM and the delta pH = 2.9 +/- 0.2. The enzyme has been digested by trypsin for 1 and 20 min, and it is found that synthesis of ATP is correlated with the Ca2+-uptake into SR. The data indicate that the enzyme alone is responsible for active transport of Ca2+ in SR. The driving force for the ATP synthesis of the process may be due to various ion-protein interactions. H+ cannot substitute for Ca2+ in the synthesis of ATP but acts probably through a modification of the Ca2+ binding sites. The data give support that the integrity of the enzyme molecule between its hydrolytic site and the Ca2+-binding sites is essential for the overall Ca2+ transport.     

Mechanism of action of Mg2+ and Zn2+ on rat placental alkaline phosphatase. I. Studies on the soluble Zn2+ and Mg2+ alkaline phosphatases.

Rat placental alkaline phosphatase (EC 3.1.3.1), a dimer of 135,000 daltons, is strongly activated by Mg2+. However, Zn2+ has to be present on the apoenzyme to obtain this activation. Mg2+ alone is unable to reconstitute functional active sites. Excess Zn2+ which competes for the Mg2+ site leads to a phosphatase with little catalytic activity at alkaline pH but with normal active sites at acidic pH as shown by covalent incorporation of ortho-[32P]phosphate. Two enzyme species with identical functional active sites have been reconstituted that only differ by the presence of Zn2+ or Mg2+ at the effector site. A mechanism is presented by which alkaline phosphatase activity of rat placenta would be controlled by a molecular process involving the interaction of Mg2+ and Zn2+ with the dimeric enzyme molecule.     

Conformational transitions in the Ca2+ + Mg2+-activated ATPase and the binding of Ca2+ ions.

We have studied the fluorescence of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate. The change in intensity of fluorescein fluorescence caused by addition of Ca2+ to the labelled ATPase can be interpreted in terms of a two-conformation model for the ATPase, one conformation (E1) having a high affinity for Ca2+, the other (E2) a low affinity. Effects of Ca2+ as a function of pH allow an estimate of the effect of pH on the E1/E2 ratio, consistent with kinetic studies. A model is presented for binding of Ca2+ to the ATPase as a function of pH that is consistent both with the data on the E1/E2 equilibrium and with literature data on Ca2+ binding.     

Fluxes of Ca2+, Sr2+ and Mg2+ in synaptosomes.

Synaptosomes isolated from sheep brain cortex accumulate Ca²⁺, Sr²⁺ and Mg²⁺ when incubated in isosmotic sucrose media containing 5 mM of either of these cations. The maximal levels of cations retained per mg of protein are 100 nmol of Ca²⁺, 85 nmol of Mg²⁺ and 80 nmol of Sr²⁺. The loss of Ca²⁺ or Sr²⁺ from the preloaded synaptosomes is increased by monovalent cations in the following order: Na⁺> K⁺ > Li⁺> choline, whereas for the loss of Mg²⁺ this order is different: K⁺ > Na⁺ > Li ～ choline. The efflux of Ca²⁺ or Sr²⁺ induced by monovalent cations decreases as the temperature is lowered and it is nearly abolished at 0°C, whereas the efflux of Mg²⁺ is much less influenced by temperature. The results suggest that the mechanism of exchange of Ca²⁺ for Na⁺ in synaptosomes operates similarly for Sr²⁺, but not for Mg²⁺.     

Saltatory propagation of Ca2+ waves by Ca2+ sparks.

Punctate releases of Ca2+, called Ca2+ sparks, originate at the regular array of t-tubules in cardiac myocytes and skeletal muscle. During Ca2+ overload sparks serve as sites for the initiation and propagation of Ca2+ waves in myocytes. Computer simulations of spark-mediated waves are performed with model release sites that reproduce the adaptive Ca2+ release observed for the ryanodine receptor. The speed of these waves is proportional to the diffusion constant of Ca2+, D, rather than D, as is true for reaction-diffusion equations in a continuous excitable medium. A simplified "fire-diffuse-fire" model that mimics the properties of Ca2+-induced Ca2+ release (CICR) from isolated sites is used to explain this saltatory mode of wave propagation. Saltatory and continuous wave propagation can be differentiated by the temperature and Ca2+ buffer dependence of wave speed.     

The Ca2+-induced membrane transition in mitochondria. III. Transitional Ca2+ release.

The efflux of Ca²⁺ from mitochondria respiring at steady state, and much of uncoupler-induced Ca²⁺ efflux, is shown to be a consequence of the Ca²⁺-induced membrane transition (the Ca²⁺-induced transition is the Ca²⁺-dependent sudden increase in the nonspecific permeability of the mitochondrial inner membrane which occurs spontaneously when mitochondria are incubated under a variety of conditions (D. R. Hunter, R. A. Haworth, and J. H. Southard, 1976, J. Biol. Chem.251, 5069–5077)). Ca²⁺ release from mitochondria respiring at steady state is shown to be transitional by four criteria: (1) Ca²⁺ release is inhibited by Mg²⁺, ADP, and bovine serum albumin (BSA), all inhibitors of the transition; (2) release is selective for Ca²⁺ over Sr²⁺, a selectivity also found for the transition; (3) the time course of Ca²⁺ release is identical to the time course of the change in the mitochondrial population from the aggregated to the orthodox configuration; and (4) from kinetics, Ca²⁺ release from individual mitochondria is shown to occur suddenly, following a lag period during which no release occurs. Ca²⁺ release induced by uncoupler is shown to be mostly by a transitional mechanism, as judged by four criteria: (1) release of Ca²⁺ is ruthenium red-insensitive and is an order of magnitude faster than Sr²⁺ release which is ruthenium red-sensitive; (2) release of Ca²⁺ is strongly inhibited by keeping the mitochondrial NAD⁺ reduced; (3) the kinetics of Ca²⁺ release indicates a transitional release mechanism; and (4) uncoupler addition triggers the aggregated to orthodox configurational transition which, at higher levels of Ca²⁺ uptake, occurs in the whole mitochondrial population at a rate equal to the rate of Ca²⁺ release. Na²⁺-induced Ca²⁺ release was not accompanied by a configurational change; we therefore conclude that it is not mediated by the Ca²⁺-induced transition.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. II. Releases involving a Ca2+-induced Ca2+ release channel

Calcium ions that have been preloaded into isolated sarcoplasmic reticulum subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances. We report here that not only caffeine, but also Ca2+ ions, thymol, quercetin, menthol, halothane, chloroform, 1-ethyl-2-methylbenzimidazole, ryanodine, tetraphenylboron, ketoconazole, miconazole, clotrimazole, W-7, doxorubicin, 5,5'-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoic acid, and low concentrations of Ag+ induce Ca2+ release from such triadic sarcoplasmic reticulum. All these drugs induce increased undirectional Ca2+ efflux. We believe all these drug-induced Ca2+ releases are mediated by Ca2+ efflux through the same ion channel since these releases are all greatly attenuated when light sarcoplasmic reticulum is substituted for triads and are even more pronounced when transverse tubule-free terminal cisternae are substituted for triads, and all these forms of drug-induced Ca2+ release are inhibited by submicromolar concentrations of ruthenium red, and by submillimolar concentrations of tetracaine, 9-aminoacridine, and Ba2+, yet they are not affected by nifedipine even at a concentration of 50 microM.     

A kinetic model for Ca2+ efflux mediated by the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

We present a model for Ca2+ efflux from vesicles of sarcoplasmic reticulum (SR). It is proposed that efflux is mediated by the Ca2+ + Mg2+-activated ATPase that is responsible for Ca2+ uptake in this system. In the normal ATPase cycle of the ATPase, phosphorylation of the ATPase is followed by a conformational change in which the Ca2+-binding sites change from being outward-facing and of high affinity to being inward-facing and of low affinity. To mediate Ca2+ efflux, it is proposed that the ATPase can adopt a conformation in which the Ca2+-binding sites are of low affinity but still outward-facing. It is shown that experimental data on the rates of Ca2+ efflux can be simulated in terms of this model, with Ca2+-binding-site affinities previously proposed to explain ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227]. Effects of Mg2+ and adenine nucleotides on efflux rates are explained. It is suggested that Ca2+ efflux from SR mediated by the ATPase could be important in excitation-contraction coupling in skeletal muscle.     

Two roles of Ca2+ in agonist stimulated Ca2+ oscillations.

We propose a mechanism for agonist-stimulated Ca2+ oscillations that involves two roles for cytosolic Ca2+: (a) inhibition of inositol-1,4,5-trisphosphate (IP3) stimulated Ca2+ release from the endoplasmic reticulum (ER) and (b) stimulation of the production of IP3 through its action on phospholipase C (PLC), via a Gq protein related mechanism. Relying on quantitative experiments by Parker, I., and I. Ivorra (1990. Proc. Natl. Acad. Sci. USA. 87:260-264) on the inhibition of Ca2+ release from the ER using caged-IP3, we develop a kinetic model of inhibition that allows us to simulate closely their experiments. The model assumes that the ER IP3 receptor is a tetramer of independent subunits that can bind both Ca2+ and IP3. Upon incorporation of the action of Ca2+ on PLC that leads to production of IP3, we observe in-phase-oscillations of Ca2+ and IP3 at intermediate values of agonist stimulation. The oscillations occur on a time scale of 10-20 s, which is comparable to the time scale for inhibition in Xenopus oocytes. Analysis of the mechanism shows that Ca(2+)-inhibition of IP3-stimulated Ca2+ release from the ER is an essential step in the mechanism. We also find that the effect of Ca2+ on PLC can lead to an indirect increase of cytosolic Ca2+, superficially resembling "Ca(2+)-induced Ca(2+)-release." The mechanism that we propose appears to be consistent with recent experiments on REF52 cells by Harootunian, A. T., J. P. Y. Kao, S. Paranjape, and R. Y. Tsien. (1991. Science [Wash. DC]. 251:75-78.) and we propose additional experiments to help test its underlying assumptions.     

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.     

Cross-linking of the (Ca2+ + Mg2+)-ATPase protein.

The addition of cupric-1,10,-phenanthroline, a cross-linking catalyst, to sarcoplasmic reticulum membranes caused protein sulfhydryl groups to form disulfide bridges. Following a short exposure to the catalyst (15 s, 22 degrees C) most of the protein was in a dimeric form (Mr = 248 000). Longer exposure times resulted in the formation of trimers, tetramers and other oligomers too large to enter the gel. At low temperatures (4 degrees C) dimer formation predominates even for exposure times as long as 5 min. Cross-linking in the presence of 7.5 mM Triton X-100 (a concentration that resulted in clearing of the membrane suspension and thus solubilization of the membrane components) showed the appearance of a considerable dimer fraction, however, most of the (Ca2+ + Mg2+)-ATPase protein appeared as a monomer. Following 1 min of cross-linking at 22 degrees C, freeze-etched membranes showed no alteration in the number or appearance of 80 A intramembranous particles. Thus extensive cross-linking of the (Ca2+ + Mg2+)-ATPase protein can occur without disruption of the normal position of the intramembrane portion of the molecule.     

A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1'PCa2 and to E2 modulate the rates of the transport step E1'PCa-E2'PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.     

Mg2+--Ca2+--Na+ interactions in uterine smooth muscle: studies with cAMP.

The influence of variation in the extracellular concentrations of Na+, Mg2+, and Ca2+ in the depolarizing medium on isoproterenol-induced increases in cAMP levels and relaxation was studied in rat uterus. Isoproterenol (10(-8) M) failed to increase cAMP levels in the high-K+ medium containing no Na+. When 80 mM Na+ was present in the medium, isoproterenol caused increases in cAMP levels similar to those observed in nondepolarized uterus. A similar effect of 2.5 mM Mg2+ was observed on the cAMP response. These effects of Na+ and Mg2+ were antagonized by increasing the extracellular concentration of Ca2+. The simultaneous presence of 80 mM Na+ and 2.5 mM Mg2+ did not produce an additive effect on the cAMP responses.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. III. Block of Ca2+-induced Ca2+ release by organic polyamines

Calcium ions that have been preloaded into isolated SR subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances in a manner that is effectively blocked by ruthenium red and other organic polyamines. Effective blocking substances include certain antibiotics (neomycin, gentamicin, streptomycin, clindamycin, kanamycin, and tobramycin), naturally occurring polyamines (spermine and spermidine), and a number of basic polypeptides and proteins (polylysine, polyarginine, certain histones, and protamine). These agents have only one feature in common: the presence of several amino groups. Ruthenium red, neomycin, spermine, and protamine all appear to act by blocking SR Ca2+ channels since unidirectional 45Ca2+ efflux from the vesicles is strongly inhibited by these agents. Functions ascribable to the SR Ca2+ pump are largely unaffected by these agents. Since inositol 1,4,5-trisphosphate is ineffective at inducing Ca2+ release under these conditions, we conclude that these polyamines may directly block SR Ca2+ channels at very low concentrations by a mechanism unrelated to effects on inositol 1,4,5-trisphosphate production.     

Ca2+ waves in astrocytes.

The glial cell is the most numerous cell type in the central nervous system and is believed to play an important role in guiding brain development and in supporting adult brain function. One type of glial cell, the astrocyte also may be an integral computational element in the brain since it undergoes neurotransmitter-triggered signalling. Here we review the role of the astrocyte in the central nervous system, emphasizing receptor-mediated Ca2+ physiology. One focus is the recent discovery that the neurotransmitter glutamate induces a variety of intracellular Ca2+ changes in astrocytes. Simple Ca2+ spikes or intracellular Ca2+ oscillations often appear spatially uniform. However, in many instances, the Ca2+ rise has a significant spatial dimension, beginning in one part of the cell it spreads through the rest of the cell in the form of a wave. With high enough agonist concentration an astrocyte syncitium supports intercellular waves which propagate from cell to cell over relatively long distances. We present results of experiments using more specific pharmacological glutamate receptor agonists. In addition to describing the intercellular Ca2+ wave we present evidence for another form of intercellular signalling. Some possible functions of a long-range glial signalling system are also discussed.     

Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes.

One current hypothesis for the initiation of Ca2+ entry into nonelectrically excitable cells proposes that Ca2+ entry is linked to the state of filling of intracellular Ca2+ stores. In the human T lymphocyte cell line Jurkat, stimulation of the antigen receptor leads to release of Ca2+ from internal stores and influx of extracellular Ca2+. Similarly, treatment of Jurkat cells with the tumor promoter thapsigargin induced release of Ca2+ from internal stores and also resulted in influx of extracellular Ca2+. Initiation of Ca2+ entry by thapsigargin was blocked by chelation of Ca2+ released from the internal storage pool. The Ca2+ entry pathway also could be initiated by an increase in the intracellular concentration of Ca2+ after photolysis of the Ca(2+)-cage, nitr-5. Thus, three separate treatments that caused an increase in the intracellular concentration of Ca2+ initiated Ca2+ influx in Jurkat cells. In all cases, Ca(2+)-initiated Ca2+ influx was blocked by treatment with any of three phenothiazines or W-7, suggesting that it is mediated by calmodulin. These data suggest that release of Ca2+ from internal stores is not linked capacitatively to Ca2+ entry but that initiation is linked instead by Ca2+ itself, perhaps via calmodulin.