Calcium sequestration activity in rat liver microsomes. Evidence for a cooperation of calcium transport with glucose-6-phosphatase

Mechanisms regulating the energy-dependent calcium sequestering activity of liver microsomes were studied. The possibility for a physiologic mechanism capable of entrapping the transported Ca2+ was investigated. It was found that the addition of glucose 6-phosphate to the incubation system for MgATP-dependent microsomal calcium transport results in a marked stimulation of Ca2+ uptake. The uptake at 30 min is about 50% of that obtained with oxalate when the incubation is carried out at pH 6.8, which is the pH optimum for oxalate-stimulated calcium uptake. However, at physiological pH values (7.2-7.4), the glucose 6-phosphate-stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. The Vmax of the glucose 6-phosphate-stimulated transport is 22.3 nmol of calcium/mg protein per min. The apparent Km for calcium calculated from total calcium concentrations is 31.9 microM. After the incubation of the system for MgATP-dependent microsomal calcium transport in the presence of glucose 6-phosphate, inorganic phosphorus and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. In the system for the glucose 6-phosphate-stimulated calcium uptake, glucose 6-phosphate is actively hydrolyzed by the glucose-6-phosphatase activity of liver microsomes. The latter activity is not influenced by concomitant calcium uptake. Calcium uptake is maximal when the concentration of glucose 6-phosphate in the system is 1-3 mM, which is much lower than that necessary to saturate glucose-6-phosphatase. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose-6-phosphatase multicomponent system. The physiological implications of such a cooperation are discussed.     

Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca2+ uptake in permeabilized hepatocytes and Ca2+ release by inositol trisphosphate

The relationships between Ca2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca2+ uptake. A progressive increase in the stimulation of Ca2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20-40 microM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca2+.     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

Ca2+- or Mg2+-Dependent Enzymic ATP Hydrolysis Associated with the Microsomal Fraction of Frog Sciatic Nerves

The microsomal fraction of frog sciatic nerves was found to contain Ca2+- or Mg2+-dependent hydrolytic activity toward different nucleoside di- and triphosphates. In the presence of Ca2+ substrate specificity was in the order CTP > UTP > GTP > ATP. When Mg2+ was used, the triphosphates were approximately equally good substrates. ATP hydrolytic activity was very similar with Ca2+ or Mg2+ as the cofactor, whereas Ca2+ was the more potent activator of hydrolysis of the other triphosphates tested. The preparation showed some activity toward the nucleoside diphosphates but none toward the monophosphates or p-nitrophenylphosphate. The enzymic properties of ATP hydrolysis were more closely studied. The hydrolysis was optimal at 18--24 degrees C in the presence of 1 mM-Ca2+ or 1 mM-Mg2+. Ca2+- and Mg2+-ATP hydrolysis displayed pH maxima around 8.0--8.5 and 7.4--8.0, respectively. Vmax values for Ca2+- and Mg2+-ATP hydrolysis similar: approx. 12 mumol Pi per h per mg protein with a Km value of approx. 0.05 mM. The ATP hydrolysis activity was inhibited by NaF but unaffected by ouabain, vanadate, cytochalasin B, and various drugs known to influence ATPase activity of mitochondria. Zn2+ stimulated the ATP hydrolysis activity at low concentrations (10(-6)-10(-5) M) and inhibited it at higher concentrations. The possibility that these observations account for stimulation and inhibition of axonal transport in frog sciatic nerves exposed to similar concentrations of Zn2+ is discussed.     

Role of a nonmitochondrial Ca2+ pool in the synergistic stimulation by cyclic AMP and vasopressin of Ca2+ uptake in isolated rat hepatocytes.

The subcellular distribution of 45Ca2+ accumulated by isolated rat hepatocytes exposed to dibutyryl cyclic AMP (dbcAMP) followed by vasopressin (Vp) was studied by means of a nondisruptive technique. When treated with dbcAMP followed by vasopressin, hepatocytes obtained from fed rats accumulated an amount of Ca2+ approximately fivefold higher than that attained under control conditions. Ca2+ released from the mitochondrial compartment by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) accounted for only a minor portion of the accumulated Ca2+. The largest portion was released by the Ca2+ ionophore A23187 and was attributable to a nonmitochondrial compartment. DbcAMP + Vp-treatment also caused a maximal stimulation of glucose production and a twofold increase in cellular glucose 6-phosphate levels. In hepatocytes obtained from fasted rats, dbcAMP + Vp-stimulated Ca2+ accumulation was lower, although with the same subcellular distribution, and was associated with a minimal glucose production. In the presence of gluconeogenetic substrates (lactate plus pyruvate) hepatocytes from fasted rats were comparable to cells isolated from fed animals. However, Ca2+ accumulation and glucose 6-phosphate production could be dissociated in the absence of dbcAMP, in the presence of lactate/pyruvate alone. Under this condition in fact Vp induced only a minimal accumulation of Ca2+ in hepatocytes isolated from fasted rats, although glucose production was markedly increased. Moreover, treatment of fed rat hepatocytes with 1 mM ATP caused a maximal activation of glycogenolysis, but only a moderate stimulation of cellular Ca2+ accumulation. In this case, sequestration of Ca2+ occurred mainly in the mitochondrial compartment. By contrast, the addition of ATP to dbcAMP-pretreated hepatocytes induced a large accumulation of Ca2+ in a nonmitochondrial pool. Additional experiments using the fluorescent Ca2+ indicator Fura-2 showed that dbcAMP pretreatment can enlarge and prolong the elevation of cytosolic free Ca2+ caused by Vp. A nonmitochondrial Ca2+ pool thus appears mainly responsible for the Ca2+ accumulation stimulated by dbcAMP and Vp in isolated hepatocytes, and cyclic AMP seems able to activate Ca2+ uptake in such a nonmitochondrial pool.     

Evidence for an alteration in the microsomal Ca2+ release mechanism in senescent rat parotid acinar cells

Previously, we have shown that Ca2+ mobilization following an alpha 1-adrenergic receptor stimulus is reduced in parotid acinar cells from senescent rats as a result of an altered ability of inositol 1,4,5-trisphosphate (IP3) to induce Ca2+ release from a non-mitochondrial, intracellular Ca2+ store (Ishikawa, Y., et al. Biochim. Biophys. Acta 968, 203-210). We have used this model to examine the IP3-induced Ca2+ release mechanism in these cells. 45Ca2+ efflux, after exposure to (-) epinephrine, from cells of young adult (3-6 months) rats was approx. 2-fold that observed from cells from older animals (approx. 24 months) either in the presence or absence of extracellular Ca2+. Similarly, cytosolic Ca2+ levels were greater in cells of young adult rats under these same incubation conditions. However, microsomal membrane preparations, from both age groups displayed similar IP3 binding sites (Kd approximately 90 nM, Bmax approximately 850 fmol/mg protein) and ATP-dependent Ca2+ transport ability (approx. 8 nmol/mg protein.min -1). These data suggest that there is an alteration in the IP3-induced Ca2+ release mechanism in microsomal membranes of parotid glands from senescent rats which may account for the decreased Ca2+ release seen after agonist stimulation of this tissue.     

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.     

Characterization of the hormone-sensitive Ca2+ uptake activity of the hepatic endoplasmic reticulum

The characteristics and kinetics of calcium uptake activity were studied in isolated hepatic microsomes. The sustained accumulation of calcium was ATP- and oxalate-dependent. Glucagon increased microsomal Ca2+ uptake upon either in vivo injection, or in vitro perfusion of the hormone in the liver. In contrast, the effect of insulin depended on the route of administration. Calcium accumulation by subsequently isolated hepatic microsomes increased when insulin was injected intraperitoneally whereas it decreased when the hormone was perfused directly into the liver. These effects of glucagon and insulin were dose dependent. When insulin was added to the perfusate prior to the addition of glucagon, insulin blocked the glucagon-stimulated increase in microsomal Ca2+ uptake. Cyclic AMP mimicked the effect of glucagon on microsomal Ca2+ accumulation when the cyclic nucleotide was perfused into the liver. The effects of glucagon and insulin on the kinetics of hepatic microsomal Ca2+ uptake were investigated. In microsomes isolated from perfused rat livers treated with glucagon the V of the uptake was significantly increased over the control values (12.2 vs. 8.6 nmol Ca2+ per min per mg protein, P less than 0.02). In contrast, the addition of insulin to the perfusate significantly decreased the V of Ca2+ uptake by subsequently isolated microsomes (6.8 vs. 8.3 nmol Ca2+ per min per mg protein, P less than 0.05). However, neither hormone had an effect on the apparent Km for Ca2+ (4.1 +/- 0.5 microM) of the reaction. The effect of these hormones on the activity of Ca2+-stimulated ATPase was also studied. No significant changes in either V or Km for Ca2+ of the enzymatic reaction were detected.     

Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands.

Ca2+ transport across mammary-gland Golgi membranes was measured after centrifugation of the membrane vesicles through silicone oil. In the presence of 2.3 microM free Ca2+ the vesicles accumulated 5.8 nmol of Ca2+/mg of protein without added ATP, and this uptake was complete within 0.5 min. In the presence of 1 mM-ATP, Ca2+ was accumulated at a linear rate for 10 min after the precipitation of intravesicular Ca2+ with 10 mM-potassium oxalate. ATP-dependent Ca2+ uptake exhibited a Km of 0.14 microM for Ca2+ and a Vmax. of 3.1 nmol of Ca2+/min per mg of protein. Ca2+-dependent ATP hydrolysis exhibited a Km of 0.16 microM for Ca2+ and a Vmax. of 10.1 nmol of Pi/min per mg of protein. The stoichiometry between ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase varied between 0.3 and 0.7 over the range 0.03-8.6 microM-Ca2+. Both Ca2+ uptake and Ca2+-stimulated ATPase were strongly inhibited by orthovanadate, which suggests that the major mechanism by which Golgi vesicles accumulate Ca2+ is through the action of the Ca2+-stimulated ATPase. However, Ca2+ uptake was also decreased by the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone), indicating that it may occur by other mechanisms too. The effect of CCCP may be related to the existence of transmembrane pH gradients (delta pH) in these vesicles: the addition of 30 microM-CCCP reduced delta pH from a control value of 1.06 to 0.73 pH unit. Golgi vesicles also possess a Ca2+-efflux pathway which operated at an initial rate of 0.5-0.57 nmol/min per mg of protein.     

Evidence that ATP-dependent Ca2+ transport in rat parotid microsomal membranes requires charge compensation.

ATP-dependent Ca2+ transport was investigated in a rat parotid microsomal-membrane preparation enriched in endoplasmic reticulum. Ca2+ uptake, in KCl medium, was rapid, linear with time up to 20 s, and unaffected by the mitochondrial inhibitors NaN3 and oligomycin. This Ca2+ uptake followed Michaelis-Menten kinetics, and was of high affinity (Km approximately 38 nM) and high capacity (approximately 30 nmol/min per mg of protein). In the presence of oxalate, Ca2+ uptake continued to increase for at least 5 min, reaching an intravesicular accumulation approx. 10 times higher than without oxalate. Ca2+ uptake was dependent on univalent cations in the order K+ = Na+ greater than trimethylammonium+ greater than mannitol and univalent anions in the order Cl- greater than acetate- greater than Br- = gluconate- = NO3- greater than SCN-. Ca2+ uptake was not elevated if membranes were incubated in the presence of a lipophilic anion (NO3-) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Ca2+ transport was altered by changes in the K+-diffusion potential of the membranes. A relatively negative K+-diffusion potential increased the initial rate of Ca2+ accumulation, whereas a relatively positive potential decreased Ca2+ accumulation. In the presence of an outwardly directed K+ gradient, nigericin had no effect on Ca2+ uptake. In aggregate, these studies suggest that the ATP-dependent Ca2+-transport mechanism present in rat parotid microsomal membranes exhibits an electrogenic Ca2+ flux which requires the movement of other ions for charge compensation.     

Inositol 1,4,5-trisphosphate releases Ca2+ from a Ca2+-transporting membrane vesicle fraction derived from human platelets

Human platelet membrane vesicles that accumulated Ca2+ in the presence of ATP were isolated on an isoosmotic KCl-Percoll gradient. ATP-dependent Ca2+ uptake was stimulated by oxalate and phosphate to steady-state levels of greater than 100 nmol/mg protein, and the accumulated Ca2+ could be largely released by ionophore A23187. Inositol 1,4,5-trisphosphate, in a dose-dependent manner (0.5-5.0 microM), caused the rapid release (less than 5 s) of 40-70% of the total A23187-releasable store of accumulated Ca2+. The membrane vesicles that release accumulated Ca2+ in response to inositol 1,4,5-trisphosphate were enriched in enzymes characteristically found in smooth endoplasmic reticulum. These results support the hypothesis that inositol 1,4,5-trisphosphate, produced by the hydrolysis of phosphatidylinositol 1,4-bisphosphate in response to stimulation of cell surface receptors, is a second messenger mediating the release of Ca2+ from intracellular storage sites.     

Calcium-ion-transporting activity in two microsomal subfractions from rat pancreatic acini. Modulation by carbamylcholine

Two microsomal subfractions from isolated rat pancreatic acini were produced by centrifugation through a discontinuous sucrose density gradient and characterized by biochemical markers. The denser fraction ( SF2 ) was a highly purified preparation of rough endoplasmic reticulum; the less-dense fraction ( SF1 ) was heterogeneous and contained Golgi, endoplasmic reticulum and plasma membranes. 45Ca2+ accumulation in the presence of ATP and its rapid release after treatment with the bivalent-cation ionophore A23187 were demonstrated in both fractions. The pH optimum for active 45Ca2+ uptake was approx. 6.8 for the rough endoplasmic reticulum ( SF2 ) and approx. 7.5 for SF1 . Initial rate measurements were used to determine the affinity of the rough-endoplasmic-reticulum uptake system for free Ca2+. An apparent Km of 0.16 +/- 0.06 microM and Vmax. of 21.5 +/- 5.6 nmol of Ca2+/min per mg of protein were obtained. 45Ca2+ uptake by SF1 was less sensitive to Ca2+, half-maximal uptake occurring at 1-2 microM-free Ca2+. When fractions were prepared from isolated acini stimulated with 3 microM-carbamylcholine, 45Ca2+ uptake was increased in the rough endoplasmic reticulum. The increased uptake was due to a higher Vmax. with no significant change in Km. No effect was observed on 45Ca2+ uptake by SF1 . In conclusion, two distinct non-mitochondrial, ATP-dependent calcium-uptake systems have been demonstrated in rat pancreatic acini. One of these is located in the rough endoplasmic reticulum, but the precise location of the other has not been determined. We have shown that the Ca2+-transporting activity in the rough endoplasmic reticulum may have an important role in maintaining the cytosolic free Ca2+ concentration in resting acinar cells and is involved in Ca2+ movements which occur during stimulation of enzyme secretion.     

MgATP-dependent glucose 6-phosphate-stimulated Ca2+ accumulation in liver microsomal fractions. Effects of inositol 1,4,5-trisphosphate and GTP

Ca2+ release triggered by inositol 1,4,5-trisphosphate (IP3) and/or GTP has been studied with rough and smooth microsomes isolated from rat liver. Microsomes were loaded with Ca2+ in the presence of MgATP and in the presence or in the absence of glucose 6-phosphate (glucose-6-P) which markedly stimulated the MgATP-dependent Ca2+ accumulation in rough and smooth microsomes (5- and 10-fold, respectively). Upon addition of IP3 (5 microM), rough and smooth microsomes rapidly release a part (not exceeding 20%) of the Ca2+ previously accumulated both in the absence and in the presence of glucose-6-P. Under the same experimental conditions, inositol 1,3,4,5-tetrakisphosphate was ineffective in triggering any Ca2+ release. Upon addition of GTP (10 microM) both the microsomal fractions progressively release the Ca2+ previously accumulated in the presence of glucose-6-P, when 3% polyethylene glycol was also present. In the absence of polyethylene glycol, GTP released Ca2+ from rough microsomes only, and GTP plus IP3 caused a Ca2+ release which was the sum of the Ca2+ releases caused by GTP and IP3 independently. Both IP3 and GTP, added to microsomes at the beginning of the glucose-6-P-stimulated Ca2+ uptake, reduced the Ca2+ accumulation into rough and smooth microsomes without modifying the initial rate (3 min) of Ca2+ uptake. Also in these conditions, the effects of GTP and IP3 were merely additive. These results indicate that both rough and smooth liver microsomes are responsive to IP3 and GTP with respect to Ca2+ release and that IP3 and GTP likely act independently.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37 degrees C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 microM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 micrograms/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange.     

Glucose 6-phosphate and hexokinase can be used as an ATP-regenerating system by the Ca(2+)-ATPase of sarcoplasmic reticulum.

In the presence of hexokinase, vesicles derived from the sarcoplasmic reticulum of skeletal muscle are able to accumulate Ca2+ in a medium containing ADP and glucose 6-phosphate. No significant Ca2+ uptake is observed if one of these components is omitted from the assay medium. Due to its high affinity for ATP, the Ca(2+)-ATPase can use the very low concentrations of ATP formed from glucose 6-phosphate and ADP to form a Ca2+ gradient. This finding indicates that glucose 6-phosphate and hexokinase can be used as an ATP-regenerating system. The Ca2+ uptake supported by glucose 6-phosphate and ADP is inhibited by glucose and D-xylose. Half-maximal inhibition is observed in the presence of 0.4 mM glucose and 100 mM D-xylose. The transport ratio (Ca2+ transported:substrate utilized) is the same for glucose 6-phosphate and ATP. The Ca2+ gradient formed when glucose 6-phosphate and ADP are the substrates can be used to synthesize ATP from ADP and Pi. The concentration of ATP formed after reversal of the Ca2+ pump is much higher than that expected from direct equilibration of the reaction between glucose 6-phosphate and ADP.     

Na+/Ca2+ exchange in coated microvesicles.

Coated microvesicles isolated from bovine neurohypophyses could be loaded with Ca2+ in two different ways, either by incubation in the presence of ATP or by imposition of an outwardly directed Na+ gradient. Na+, but not K+, was able to release Ca2+ accumulated by the coated microvesicles. These results suggest the existence of an ATP-dependent Ca2+-transport system as well as of a Na+/Ca2+ carrier in the membrane of coated microvesicles similar to that present in the membranes of secretory vesicles from the neurohypophysis. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ of the ATP-dependent uptake was 0.8 microM. The average Vmax. was 2 nmol of Ca2+/5 min per mg of protein. The total capacity of microvesicles for Ca2+ uptake was 3.7 nmol/mg of protein. Both nifedipine (10 microM) and NH4Cl (50 mM) inhibited Ca2+ uptake. The ATPase activity in purified coated-microvesicles fractions from brain and neurohypophysis was characterized. Micromolar concentrations of Ca2+ in the presence of millimolar concentrations of Mg2+ did not change enzyme activity. Ionophores increasing the proton permeability across membranes activated the ATPase activity in preparations of coated microvesicles from brain as well as from the neurohypophysis. Thus the enzyme exhibits properties of a proton-transporting ATPase. This enzyme seems to be linked to the ion accumulation by coated microvesicles, although the precise coupling of the proton transport to Ca2+ and Na+ fluxes remains to be determined.     

ATP-dependent calcium accumulation in brain microsomes. Enhancement by phosphate and oxalate.

1. ATP-dependent calcium uptake by a rabbit brain vesicular fraction (microsomes) was studied in the presence of phosphate or oxalate. These anions, which are known to form insoluble calcium salts, increased the rate of calcium uptake and the capacity of the vesicles for calcium accumulation. 2. The degree of activation depended on the concentration of phosphate or oxalate. Under optimal conditions, phosphate promoted a 5-fold increase in the amount of calcium stored at steady state. This level was 200-250 nmol Ca-2+/mg protein. 3. Initial rate of calcium uptake followed Michaelis-Menten kinetics with an apparent Km for calcium of 6.7-10-minus 5 M and a V of 44 nmol/min per mg protein. Optimal pH was 7.0. With 2 mM ATP, optimal Mg-2+ concentration was 2 mM. 4. Dintrophenol and NaN3 inhibited calcium uptake in a mitochondria-enriched fraction but not in the microsomal fraction. 5. Calcium uptake activity was compared in the six subfractions prepared from the whole microsomal fraction by means of a sucrose density gradient fractionation. 6. The Mg-2+-dependent ATPase activity of brain microsomes was activated by calcium. Maximal activation was attained with 100 muM CaCl2. Greater calcium concentrations caused a progressive inhibition. 7. The data suggest that the ATP-dependent calcium uptake in brain microsomes, as in muscle microsomes, is brought about by an active transport process, calcium being accumulated as a free ion inside the vesicles.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

The metabolism of lyso-platelet-activating factor (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) by a calcium-dependent lysophospholipase D in rabbit kidney medulla

A Ca2+-dependent lysophospholipase D activity in microsomal preparations from the rabbit kidney medulla hydrolyzes the choline moiety from 1-O-[9,10-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) to form 1-O-[9,10-3H]hexadecyl-2-lyso-sn-glycero-3-P; the latter is subsequently dephosphorylated by a phosphohydrolase to 1-O-[9,10-3H]hexadecyl-sn-glycerol. Sodium vanadate, which is known to inhibit phosphohydrolases, reduces the proportion of hexadecylglycerol and increases the formation of hexadecyl-lysoglycerophosphate. Essentially no hydrolysis occurs when the sn-2 position of the hexadecyllysoGPC substrate contains an acyl moiety. The lysophospholipase D in rabbit kidney is of microsomal origin and has a broad pH optimum between 8.0 and 8.8, with the activity decreasing sharply from pH 7.6 to 7.2. Wykle et al. (Biochim. Biophys. Acta 619 (1980) 58-67) have previously demonstrated the existence of a microsomal lysophospholipase D (specific for ether lipid substrates) in rat tissues that requires Mg2+ and exhibits a pH optimum of 7.2; high activities of the Mg2+-dependent lysophospholipase D were found in liver and brain, but not in kidney. In contrast to the Mg2+-dependent lysophospholipase D in rat tissues, the renal enzyme from rabbits requires Ca2+ (5 mM), whereas Mg2+ (5 mM) exhibits little stimulatory action. Under optimal assay conditions (0.1 M Tris-HCl (pH 8.4)/5 mM CaCl2), lysophospholipase D in the rabbit kidney medulla has an activity of 2.7 nmol/min per mg protein compared to 0.9 nmol/min per mg protein for the lysophospholipase D in the rat kidney medulla (0.1 M Tris-HCl (pH 7.2)/5 mM MgCl2). The Ca2+-dependent lysophospholipase D is highest in the liver and kidney medulla from rabbits, but is very low in rat tissues; similar activities were found in male and female rabbits. Our data indicate that the divalent metal ion requirements for expression of maximum lysophospholipase D activities can differ markedly among animal species and also suggest the microsomal Ca2+-dependent lysophospholipase D is an important catabolic route for lyso-PAF metabolism in rabbit renomedullary tissue.