The mechanism of calcium control of smooth muscle tonic contraction

It has been shown in the experiments carried out on a fraction of inverted vesicles of myometrium sarcolemma that ATP-dependent Ca2+ transport system prevents dissipation of the calcium gradient directed from the intervesicular space outward with subsequent establishment of the stationary level of cation content inside the membrane vesicles (a blocker of electro-controlled calcium channels diltiasems was present in the incubation medium). Ortovanadatean inhibitor of the sarcolemma calcium pump suppressed Ca2+ stationary exchange in the vesicles fraction. The value of calcium stationary content in the vesicle membrane was regulated both by a change of the calcium pump activity (by varying Mg2+ concentration in the ATP-containing incubation medium), and by modification of calcium permeability of the vesicles (by varying concentration of ionophore A-23187 in this medium). In the presence of diltiasem and ortovanadate the Ca2+ basal current entering the myocytes from hyperpotassium washing solution activated the smooth muscle tonic contraction. In the absence of ortovanadate no contractile response was observed. On the basis of the evidence obtained a mechanism of calcium control of myometrium tonic contraction is proposed. According to this mechanism the Ca2+ current entering the unexcited myocytes under physiological conditions is efficiently compensated by the calcium pump of the sarcolemma. The inhibition of the latter (or an increase of the sarcolemma basal calcium permeability) provides further slow transition of the stationary value of Ca2+ concentration in the myoplasm to a new higher level and activation of the smooth muscle contraction accordingly.     

The two calcium ions initially bound to nonphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase can no longer be kinetically distinguished when they dissociate from phosphorylated ATPase toward the lumen.

Using rapid filtration, we investigated the kinetics of release toward the lumen of sarcoplasmic reticulum vesicles of the two Ca2+ ions transported by the Ca(2+)-dependent ATPase of these vesicles. Release rates at 20 degrees C were measured by three methods, with vesicles previously made leaky with an ionophore. First, we measured the rate at which 45Ca2+ bound to ATPase approached its steady-state level after addition of ATP to the 45Ca(2+)-equilibrated ATPase. At pH 6 in the absence of potassium, the observed kinetics did not reveal any very fast phase of 45Ca2+ dissociation from phosphorylated ATPase. Second, we measured the kinetics of 45Ca2+ dissociation from phosphorylated ATPase in a "chase" experiment, by isotopic dilution of calcium under turnover conditions in the presence of potassium. We found that these kinetics were essentially monophasic. Moreover, when they were measured in the presence of a high concentration of calcium, designed to saturate the low-affinity calcium transport sites on the lumenal side of the ATPase, they only departed slightly from monophasic behavior, irrespective of the experimental pH (pH 6, 7, or 9). This small perturbation by high calcium concentrations of the observed dissociation kinetics was attributed to ADP-facilitated rapid exchange of 40Ca2+ for Mg2+ at the catalytic site of phosphorylated ATPase. The third method was based on the fact that phosphorylation-induced 45Ca2+ occlusion occurred faster than 45Ca2+ dissociation from nonphosphorylated ATPase: here, we measured the rate of 45Ca2+ internalization on addition to 45Ca(2+)-saturated ATPase of an unlabeled ATP-containing medium. This method allowed separate observation of the dissociation kinetics of each of the two 45Ca2+ ions bound to phosphorylated ATPase, after either one or the other had been labeled by a preliminary partial isotopic exchange in the non-phosphorylated state of the ATPase. We found that after ATP-induced phosphorylation, the two 45Ca2+ ions dissociated toward the lumenal medium with virtually identical rate constants; this was observed under different ionic and pH conditions and also in the presence of a high Ca2+ concentration. As a control, the same partial isotopic exchange procedure allowed us to confirm that, in contrast, when ATP was absent from the final dissociation medium, the two 45Ca2+ ions dissociated from nonphosphorylated ATPase toward the cytoplasmic medium at different rates, the one bound more deeply only dissociating after a lag period corresponding to dissociation of the superficial one.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of steady state level of phosphoenzyme and ATP synthesis in sarcoplasmic reticulum vesicles during reversal of the Ca2+ pump.

The role of the Ca2+ concentration gradient in ATP synthesis and membrane phosphorylation by Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Pi concentration required to attain 50% of the maximal membrane phosphorylation varies significantly in the pH range of 5.5 to 4.5, the optimal being at pH 6.0. In the pH range of 6.0 to 7.0, this concentration of Pi was 4- to 10-fold higher in empty vesicles than in vesicles loaded with calcium phosphate, i.e. having transmembrane Ca2+ concentration gradient. ATP, ADP, and Ca2+ inhibit the membrane phosphorylation by Pi, the inhibition being greater at pH 7.0 than at pH 6.0. The pH profile for ATP synthesis shows a higher optimum than for membrane phosphorylation. The optimum pH for synthesis, but not for phosphorylation depends on whether the vesicles were previously loaded with calcium phosphate or with calcium oxalate. Addition of Ca2+ to the assay medium inhibits the extent of membrane phosphorylation and the rate of ATP synthesis to different extents. Evidence is presented that the rate of membrane phosphorylation by Pi is higher than the rate by which the phosphoprotein transfers its pohsphate to ADP for the ATP synthesis.     

Effects of the fraction (1-10 kDa) of the brain of a Yakut horse on kinetic parameters of Ca2+ transport system in sarcolemma vesicles of cardiomyocytes]

The effect of the fraction (1-10 kDa) obtained from the brain of cold-adapted animal (Yakut horse) on Ca2+ transport in sarcolemma vesicles of cardiomyocytes was investigated. It was shown that during insertion of Yakut horse brain fraction into incubation medium at the concentration from 10(-9) M to 3.10(-5) M at Ca2+ transport substrate concentration from 0.1 mM to 1.0 mM, the rate of Ca2+ passive penetration into vesicles slightly increased and at Ca2+ transport substrate concentration 3 mM, which is physiologic, a decrease of rate values was established for all concentrations of the fraction (1-10 kDa) of Yakut horse. While studying the kinetics of an active Ca2+ transport for all investigated concentrations of the fraction (1-10 kDa) of Yakut horse brain from 10(-9) M to 3.10(-5) M at Ca2+ concentration in incubation medium from 10(-7) to 3.10(-6) M, calcium accumulation rates by vesicles exceeded control values. So we can suppose that application of brain fraction (1-10 kDa) of genotypically cold-adapted animal, results in a decrease of intracellular Ca2+ concentration.     

Passive calcium influx by plasma membrane vesicles isolated from rat liver

Passive Ca2+ influx independent of ATP addition to the incubation medium, took place in plasma membrane vesicles isolated from rat liver. The rate of Ca2+ influx was found to depend on the concentration of added Ca2+, and on the incubation temperature, and was inhibited by La3+, Hg2+ and by p-chloromercuribenzoate. Influx was not blocked by calcium channel blockers, or affected by a range of uncouplers. Addition of the Ca2+ ionophore A23187 to vesicles that had taken up the ion induced a rapid efflux of Ca2+ especially when EGTA also was added to the incubation medium. A number of divalent cations inhibited Ca2+ influx. The vesicles could be frozen and stored overnight with little loss in activity. The kinetics of Ca2+ influx could be related to that which occurs in the unstimulated perfused rat liver. The data suggest that the plasma membrane vesicle preparation may be useful for further studies on the basal liver cell Ca2+ influx system in vitro.     

Matrix free Ca2+ in isolated chromaffin vesicles

Isolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187. During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the vesicles. Intravesicular Mg2+ is not released from the vesicles by A23187, as determined by atomic emission spectroscopy. In the presence of NH4Cl, which causes the collapse of the secretory vesicle transmembrane proton gradient (delta pH), Ca2+ uptake decreases. Under these conditions A23187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 microM. Below this concentration Ca2+ is even released from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4Cl we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 microM. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 microM was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4Cl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium control of passive permeability to calcium in sarcoplasmic reticulum vesicles

Calcium efflux from sarcoplasmic reticulum vesicles that have been equilibrated with 1-100 mM CaCl2 in the absence of ATP has two apparently first order components. The initial calcium content of each component increases with the total Ca content of the sarcoplasmic reticulum, which reaches 5, 24, and 80 nmol/mg of protein after equilibration with 1, 10, and 100 mM CaCl2, respectively. Initial rates of Ca efflux into a medium containing 10 mM EGTA increase in proportion to Ca in the loading medium up to 20 mM. Above 20 mM, efflux from the slow component clearly saturates, whereas efflux from the fast component continues to increase. The rate constant for the smaller, faster component to efflux (k congruent to 0.5 min-1) is not affected by changing the concentration of Ca either inside or outside the vesicles. The rate constant of the larger, slower component (k congruent to 0.05 min-1) is also unaffected by changes in internal Ca concentration. However, external [Ca2+] diminishes the rate constant of the slow component 6-10-fold. Inhibition by external [Ca2+] is characterized by cooperative interaction between two sites with an apparent Kd of 5.3 X 10(-6) M. The two components may represent two populations of sarcoplasmic reticulum vesicles that differ 10-fold in passive permeability to Ca when external [Ca2+] is less than 10(-6) M, and 60-100-fold when external [Ca2+] is greater than 10(-5) M. The passive permeability in one of these populations seems to be regulated by external, high affinity Ca binding sites.     

Ca2+ transport through the brush border membrane of human placenta syncytiotrophoblasts.

The calcium (Ca2+) uptake by brush border membrane vesicles isolated from fresh human placentas has been characterized. This process was saturable and time- and concentration-dependent. It exhibited a double Michaelis-Menten kinetics, with apparent Km values of 0.17 +/- 0.03 and 2.98 +/- 0.17 mM Ca2+, and Vmax values of 0.9 +/- 0.13 and 2.51 +/- 0.45 pmol.micrograms-1.5 s-1. It was not influenced by the presence of Na+ or Mg2+ in the incubation medium. It was not increased by K+ or anion diffusion potentials, inside negative. At a steady state of 1 mM Ca2+ uptake, a large proportion (approximately 94%) of the Ca2+ was bound to the internal surface of the membranes. Preincubation of these membrane vesicles with voltage-dependent Ca2+ channel blockers (nifedipine and verapamil) had no influence on Ca2+ uptake. However, this uptake was very sensitive to pH. In the absence of a pH gradient, the Ca2+ uptake increased with alkalinity. When the intravesicular pH was kept constant while the pH of the incubation medium was increased, Ca2+ uptake was also stimulated by alkaline pH. In contrast, when the pH of the incubation medium was kept constant and the intravesicular pH was progressively increased, Ca2+ uptake was diminished with alkaline pH. Therefore, H+ gradient (H+ in trans-position greater than H+ in cis-position) favored Ca2+ transport, suggesting a H+/Ca2+ exchange mechanism. Finally, in contrast to the basal plasma membrane, the brush border membrane did not show any ATP-dependent Ca2+ transport activity.     

Sequential mechanism of calcium binding and translocation in sarcoplasmic reticulum adenosine triphosphatase

Cooperative calcium binding (apparent Kd = 1.04 X 10(-6) M) to the ATPase of sarcoplasmic reticulum vesicles occurs with a maximal stoichiometry of 2 mols of divalent cation/mol of enzyme in the absence of ATP. The bound calcium is distributed into two pools which undergo fast or slow isotopic exchange, respectively. The two pools retain a 1:1 molar ratio under various conditions and are both located within a protein crevice, as suggested by their cooperative interaction and exchange kinetics. Following enzyme phosphorylation by ATP, both pools of bound calcium are "internalized" (cannot be displaced by quench reagents). If following 45Ca2+ binding, isotopic dilution is obtained in the medium by adding 40Ca2+ with ATP, internalization of both pools of bound 45Ca2+ (2 mol/mol of phosphoenzyme) is still observed within the first enzyme cycle. When the cycle is reversed by addition of excess ADP soon after ATP, only half of the internalized 45Ca2+ is released from the enzyme into the medium outside the vesicles, while the other half remains with the vesicles. If half of the bound 45Ca2+ is exchanged (fast exchange) with 40Ca2+ previous to the addition of ATP, none of the remaining 45Ca2+ is released outside the vesicles upon reversal of the enzyme cycle. Therefore, the pool of bound calcium which undergoes slower exchange with the outside medium, is the first to be released inside the vesicles upon enzyme phosphorylation. A sequential mechanism of calcium binding and translocation is proposed, that accounts for binding cooperativity and exchange kinetics, presteady state transients following addition of ATP, and the Ca2+ concentration dependence of ATPase activity in steady state.     

Characteristics of Ca2+ ion effect on the activity of Mg2+-dependent ATPase system in ghosts and reconstituted erythrocytes]

A good conformity if demonstrated of the kinetics of calcium ions effect on ATPase activity of human and rat erythrocyte ghosts. The increase of calcium concentration in the rat errythrocytes hemolysis medium (above 50-100 micrometer) results in a considerable aggregation of reconstructed vesicles. An activation of ouabaine-sensitive component of Mg2+-dependent ATPase under the increase of intracellular Ca2+ in reconstructed human erythrocytes is observed.     

Lack of effects of calcium X calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium X calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca2+ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium X calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca2+ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium X calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca2+ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent 45Ca2+-40Ca2+ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium X calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca2+ release and 45Ca2+-40Ca2+ exchange.     

Ca 2+ outflow from sarcoplasmic reticulum vesicles during changes in membrane potential, Ca2+ concentration and pH

The concentration gradient Ca2+ outflux from the vesicles of the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied under conditions of the induced membrane potential, the concentrations of Ca2+ and H+ in the medium washing over the vesicles being different. The Ca2+ outflux from vesicles is shown to be the same with a decrease of the membrane potential from--80 down to -10 mV and gets higher with the zero and subsequent positive values of the latter. A significant intensification of the Ca2+ outflux from vesicles under the effect of external-vesicular Ca2+ has been observed at its concentration of 10(-5) M. Against this background of external-vesicular Ca2+ and zero value of the membrane potential either exogenous AMP or the pH increase from 6.5 up to 7.8 favour a release of more than 70% of passively accumulated Ca2+. The pH effect grows with a decrease in the external-vesicular concentration of Ca2+. A conclusion is drawn on the significance of protons in the regulation of the Ca2+ release from the sarcoplasmic reticulum.     

Kinetic effects of calcium and ADP on the phosphorylated intermediate of sarcoplasmic reticulum ATPase

The decomposition of 32P phosphorylated enzyme intermediate formed by incubation of sarcoplasmic reticulum ATPase with [gamma-32P]ATP was studied following dilution of the reaction medium with a large excess of nonradioactive ATP. The phosphoenzyme decomposition includes two kinetic components. The fraction of intermediate undergoing slower decomposition is minimal in the presence of low (microM) Ca2+ and maximal in the presence of high (mM) Ca2+. A large fraction of phosphoenzyme undergoes slow decomposition when the Ca2+ concentration is high inside the vesicles, even if the Ca2+ concentration in the medium outside the vesicles is low. Parallel measurements of ATPase steady state velocity in the same experimental conditions indicate that the apparent rate constant for the slow component of phosphoenzyme decomposition is inadequate to account for the steady state ATPase velocity observed under the same conditions and cannot be the rate-limiting step in a single, obligatory pathway of the catalytic cycle. On the contrary, the steady state enzyme velocity at various Ca2+ concentrations is accounted for by the simultaneous contribution of both phosphoenzyme fractions undergoing fast and slow decomposition. Contrary to its slow rate of decomposition in the forward direction of the cycle, the phosphoenzyme pool formed in the presence of high Ca2+ reacts rapidly with ADP to form ATP in the reverse direction of the cycle. Detailed analysis of these experimental observations is consistent with a branched pathway following phosphoryl transfer from ATP to the enzyme, whereby the phosphoenzyme undergoes an isomeric transition followed by ADP dissociation, or ADP dissociation followed by the isomeric transition. The former path is much faster and is prevalent when the intravesicular Ca2+ concentration is low. When the intravesicular Ca2+ concentration rises, a pool of phosphoenzyme is formed by reverse equilibration through the alternate path. In the absence of ADP this intermediate decays slowly in the forward direction, and in the presence of ADP it decays rapidly in the reverse direction of the cycle.     

PH-induced changes in the reactions controlled by the low- and high-affinity Ca2+-binding sites in sarcoplasmic reticulum.

The effect of pH on the Ca2+-binding sites of high and low affinity, located respectively on the outer and inner surfaces of the sarcoplasmic reticulum membrane, was investigated using intact and leaky sarcoplasmic reticulum vesicles. With the use of intact vesicles, different pH profiles of membrane phosphorylation and rates of nucleoside triphosphate hydrolysis were obtained depending on the assay temperature, on the Ca2+ concentration, and on whether ATP or ITP was used as substrate. The different pH profiles were related to the amount of Ca2+ accumualted by the vesicles, i.e., to different degrees of saturation of the inner, low-affinity Ca2+-binding site. With the use of leaky vesicles, the saturation of the two Ca2+-binding sites could be controlled more precisely since the Ca2+ concentration on both sides of the membrane was equal to the Ca2+ concentration of the assay medium. Using leaky vesicles and measuring the rates of nucleotide hydrolysis, nucleotide-phosphate exchange and membrane phosphorylation by nucleotide as an indication of the degree of saturation of the Ca2+-binding sites, we observed that the affinity of both the high- and low-affinity sites increased three to four orders of magnitude when the pH of the assay medium was increased from 6.1 to 8.65.     

Evidence for a delta pH-driven Ca2+ uptake in EGTA-treated bovine spermatozoa

Calcium efflux from ejaculated bovine spermatozoa occurred upon incubation in Ca2+/EGTA buffers with Ca2+ ion concentrations ranging from 0.1 microM to 1 nM. Both total cellular calcium and cytosol free Ca2+ concentrations, the latter measured with Quin 2, were inversely correlated with the Ca2+ activity of the medium. An influx of radioactive 45Ca2+ parallel to a net efflux of calcium took place in spermatozoa incubated in 45Ca2+/EGTA buffers with 45Ca2+ activity of 0.01 microM or 0.1 microM. The uptake of the radioactive isotope was higher in spermatozoa incubated at pH 7.8 than that found at pH 6.8, increased in the presence of acetate or amiloride but decreased when ammonium chloride or monensin was added to the incubation mixture. Addition of acetate produced a decrease of the cytoplasmic pH, determined with the indicator carboxyfluorescein, whereas addition of NH4Cl or monensin caused a pH increase. Addition of either nigericin or monensin to spermatozoa suspended in a choline medium containing low concentrations of Na+, K+ and Ca2+ produced a cytosolic acidification, the subsequent addition of Ca2+ caused a cytosolic alkalinization parallel to an increase of the cytosolic free Ca2+. Addition of CaCl2 to EGTA-pretreated spermatozoa resuspended in a poorly buffered medium induced an evident decrease of extracellular pH suggesting a cellular proton extrusion. Both monensin and nigericin caused an increase of the calcium transport in spermatozoa suspended in a choline medium containing a physiological concentration of 1.5 mM CaCl2. Taken together the present results indicate that, under the experimental conditions used, a delta pH-driven Ca2+ uptake occurs in ejaculated bovine spermatozoa and suggest that Ca2+ is taken up in exchange with H+.     

The effect of oxytocin and sigetin on Ca2+ transport in the fraction of plasma membranes of myometrial cells

Oxytocin and sigetin were studied for their effect on the active and passive transport of Ca2+ in the fraction of myometrium sarcolemma in women. Oxytocin (5.10(-7) M) introduced into the sarcolemma vesicles and sigetin (5.10(-3) M) added into the incubation medium inhibit Mg2+, ATP-dependent accumulation of Ca2+ in these structures. The both agents in the mentioned concentration do not affect the passive release of cation from vesicles. A conclusion is drawn that inhibition of the calcium pump of myometrium cell plasma membranes underlies the physiological action of oxytocin and sigetin as stimulators of the contractile activity of the myometrium.     

Ca2+ transport studied with arsenazo III in Tetrahymena microsomes. Effects of calcium ionophore A23187 and trifluoperazine

Transport of Ca2+ in microsomal membrane vesicles of the Tetrahymena has been investigated using arsenazo III as a Ca2+ indicator. The microsomes previously shown to carry a Mg2+-dependent, Ca2+-stimulated ATPase (Muto, Y. and Nozawa, Y. (1984) Biochim. Biophys. Acta 777, 67-74) accumulated calcium upon addition of ATP and Ca2+ sequestered into microsomal vesicles was rapidly discharged by the Ca2+ ionophore A23187. Kinetic studies indicated that the apparent Km for free Ca2+ and ATP are 0.4 and 59 microM, respectively. The Vmax was about 40 nmol/mg protein per min at 37 degrees C. The calcium accumulated during ATP-dependent uptake was released after depletion of ATP in the incubation medium. Furthermore, addition of trifluoperazine which inhibited both (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ uptake rapidly released the calcium accumulated in the microsomal vesicles. These observations suggest that Tetrahymena microsome contains both abilities to take up and to release calcium and may act as a Ca2+-regulating site in this organism.     

Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells

Upon stimulation with 10(-6) -10(-3) M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx.     

The role of free calmodulin in the regulation of calcium-pump activity in erythrocytes

The activity of Ca-pump in inside-out oriented vesicles obtained from erythrocyte membranes after their 30 min treatment with EGTA at 20 degrees C (membranes A) and 37 degrees C (membranes B) was investigated. It was shown that in membranes A placed into an incubation medium containing 0.1 mM EGTA (pH 7.4) the overall effect of exogenous calmodulin is due to the increase in the maximal activity of the enzyme, its affinity for Ca2+ being unaffected thereby. In membranes B placed into the same medium (pH 6.75) the activation of the Ca-pump by calmodulin is due to the increased affinity for Ca2+ at a constant maximal activity of the enzyme. The dependencies of the value of the calmodulin-stimulated component of membranes A and the Ca2+-binding capacity of calmodulin measured by the intensity of N-phenyl-1-naphthylamine fluorescence on the concentration of free Ca2+ are coincident. In the case of membranes B, the stimulation of Ca-pump by calmodulin occurs at much lower Ca2+ concentrations than the Ca2+ binding-induced conformational shifts in calmodulin. The experimental results suggest that the affinity of the Ca-pump for Ca2+ may affect calmodulin existing in a Ca2+-independent state. The hydrophobic interactions between the Ca-calmodulin complex and the Ca-ATPase molecule are apparently essential for the regulation of the maximal enzyme activity.     

Two components of hormone-evoked calcium release from intracellular stores of pancreatic acinar cells.

Dispersed pancreatic acini loaded with Fura 2 were used to study the effect of hormonal stimulation on [Ca2+]i (free cytosolic Ca2+ concentration). Stimulation of acini with cholecystokinin octapeptide or carbachol resulted in two components of increase in [Ca2+]i. The maximal increase in [Ca2+]i and the time to maximum for both components was dependent on hormone concentration. The first component reached a maximum after 2-10 s of stimulation, whereas the second component required 30-60 s of stimulation for maximal effect. Both components of the [Ca2+]i increase can be observed in the presence or absence of Ca2+ in the incubation medium. The two components of Ca2+ release from intracellular stores showed similar dependency on agonist concentration. Termination of cell stimulation with specific antagonist revealed two, kinetically separated, rates of decrease in [Ca2+]i. The initial decrease in [Ca2+]i, was completed within 2.5-7 s, whereas the secondary decrease in [Ca2+]i, back to resting values, required approx. 40 s. The magnitude of the antagonist-induced initial (rapid) and secondary (slow) decrease in [Ca2+]i was dependent on the duration of cell stimulation. Hence it appears that stimulation of pancreatic acinar cells with Ca2+-mobilizing hormones results in two, kinetically separated, components of Ca2+ release from intracellular stores.