Divalent cation transport by vesicular nucleotide transporter

The vesicular nucleotide transporter (VNUT) is a secretory vesicle protein that is responsible for the vesicular storage and subsequent exocytosis of ATP (Sawada, K., Echigo, N., Juge, N., Miyaji, T., Otsuka, M., Omote, H., and Moriyama, Y. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 5683-5686). Because VNUT actively transports ATP in a membrane potential (Δψ)-dependent manner irrespective of divalent cations such as Mg(2+) and Ca(2+), VNUT recognizes free ATP as a transport substrate. However, whether or not VNUT transports chelating complexes with divalent cations remains unknown. Here, we show that proteoliposomes containing purified VNUT actively took up Mg(2+) when ATP was present, as detected by atomic absorption spectroscopy. The VNUT-containing proteoliposomes also took up radioactive Ca(2+) upon imposing Δψ (positive-inside) but not ΔpH. The Δψ-driven Ca(2+) uptake required ATP and a millimolar concentration of Cl(-), which was inhibited by Evans blue, a specific inhibitor of SLC17-type transporters. VNUT in which Arg-119 was specifically mutated to alanine, the counterpart of the essential amino acid residue of the SLC17 family, lost the ability to take up both ATP and Ca(2+). Ca(2+) uptake was also inhibited in the presence of various divalent cations such as Mg(2+). Kinetic analysis indicated that Ca(2+) or Mg(2+) did not affect the apparent affinity for ATP. RNAi of the VNUT gene in PC12 cells decreased the vesicular Mg(2+) concentration to 67.7%. These results indicate that VNUT transports both nucleotides and divalent cations probably as chelating complexes and suggest that VNUT functions as a divalent cation importer in secretory vesicles under physiological conditions.     

Distribution of endoplasmic reticulum and calciosome markers in membrane fractions isolated from different regions of the canine brain

Four regions of the canine brain (frontal lobe, parieto-occipital lobe, brainstem, and cerebellum) were each fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). Markers of endoplasmic reticulum (glucose-6-phosphate phosphatase and rotenone-insensitive NADPH cytochrome c reductase) and markers of the 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store ([3H]IP3 binding and IP3-induced Ca2+ release) were measured. No correlation was found between the two classes of markers, which suggests that the IP3 receptor does not belong to the endoplasmic reticulum in canine brain. Cerebellum P2 and P3 fractions displayed levels of [3H]IP3 binding 10- to 30-fold higher, and rates of IP3-induced Ca2+ release greater than 15-fold faster than the homologous cerebrum and brainstem fractions. Actively accumulated Ca2+ was only partially released by IP3, both before and after saponin disruption of the plasma membrane compartment. The proportion of the IP3-sensitive Ca2+ store relative to that of the total (IP3-sensitive and IP3-insensitive) Ca2+ store was variable; i.e., it was larger in cerebellum P2 (approximately 90%) than in cerebrum fractions (less than 30%). Cerebellum fractions constitute the best source from which an IP3-sensitive Ca2+ storing organelle can be purified.     

Developmental Changes in the Calcium Dependency of γ-Aminobutyric Acid Release from Isolated Growth Cones: Correlation with Growth Cone Morphology

We have investigated the development of Ca2+-dependent gamma-[3H]aminobutyric acid [( 3H]GABA) release in superfused growth cone fractions isolated from rats between the postnatal ages of 1 and 11 days. We have compared this release with the overall morphology of the subcellular fractions, and identified those structures taking up [3H]GABA by electron microscopical autoradiography. In fractions isolated from rats between 1 and 5 days, K+-evoked [3H]GABA release was completely independent of extracellular Ca2+. After 5 days a Ca2+ dependency appeared, which increased with age, such that by 10 days approximately 50% of the K+-evoked release was Ca2+ dependent. Electron microscopical analysis showed that, at all ages, large numbers of GABAergic growth cones were present in the subcellular fractions. Up to postnatal day 5, the growth cones were synaptic vesicle sparse but, after this age, increasing numbers of synaptic vesicle-containing growth cones were seen. These results suggest that during maturation of GABAergic growth cones into synapses there is, initially, a mechanism for release that is independent of extracellular Ca2+ and that the appearance of a Ca2+-dependent [3H]GABA release from growth cones correlates with the appearance of synaptic vesicles.     

Decrease by cycloheximide of calcium binding and nonesterified fatty acids in rat-intestinal Golgi-enriched membrane fractions

Rat intestinal Golgi-enriched membrane fractions bind more Ca2+ than do basolateral and microvillus-enriched membrane fractions, and this uptake is reduced by vitamin D-deficiency. The effect of the protein synthesis inhibitor, cycloheximide, on this Ca2+ binding was determined in rat fed a normal, vitamin D-sufficient diet. Cycloheximide, 1.5 mg/kg, rapidly reduced protein synthesis (measured by [3H]leucine incorporation) to 12% of control values within 15 min, but Ca2+ binding diminished gradually to 50% of control values by 60 min. Ca2+ transport across gut sacs was also decreased. The reduction in Ca2+ binding was not due to an alteration in vesicle morphology or to a direct effect of cycloheximide. Nonesterified (free) fatty acids, the probable binding sites for Ca2+ in these membrane fractions, were reduced by cycloheximide to 48% of control values by 60 min. There was no significant change in total lipid phosphate. Cycloheximide may affect the synthesis of proteins necessary for the presence of nonesterified fatty acids in these Golgi membranes.     

Evidence that the erythrocyte calcium pump catalyzes a Ca2+:nH+ exchange

Treatment of whole erythrocytes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) results in inhibition of ATP and phosphate-dependent Ca2+ transport in subsequently prepared inside-out vesicles (IOV). Accumulation of phosphate into IOV in the presence of Ca2+ is virtually abolished by prior DIDS treatment, consistent with the presumed inhibition of the band III anion-exchange protein by this agent. No inhibition of Ca2+-activatable ATP hydrolysis is observed following DIDS treatment when open membranes are used to prevent development of ion gradients. This indicates that DIDS does not affect the inherent ATPase activity of the calcium pump (Waisman, D. M., Smallwood, J., Lafreniere, D., and Rasmussen, H. (1982) FEBS Lett. 145, 337-340). In IOV prepared from untreated cells, ATP-dependent Ca2+ uptake is stimulated by phosphate, sulfate, or chloride. Rates of Ca2+ uptake into DIDS-IOV are not increased by these anions. Lipid-permeable organic acids such as acetate, however, do promote Ca2+ transport in DIDS-IOV. Lipophilic anions incapable of transporting protons into the vesicle interior (nitrate and thiocyanate) support sustained uptake only when the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone is also added. These results support a model of the (Ca2+-Mg2+)-ATPase as a pump exchanging Ca2+ for protons, not transporting Ca2+ alone. Band III protein appears to promote Ca2+ transport in the presence of phosphate, sulfate, or chloride by exchanging external anion for the accumulating OH- (or HCO3-) produced by the calcium pump.     

Guanosine 3',5'-cyclic monophosphate stimulates release of actively accumulated calcium in purified disks from rod outer segments of bovine retina

Parallel lines of evidence have suggested that light initiates changes in both cGMP metabolism and calcium levels in rod outer segments (ROS). We report that cGMP stimulates release of a pool of Ca2+ actively accumulated within purified ROS disks. Disks were purified and actively loaded with 45Ca2+ by an associated ATP-dependent calcium uptake activity as previously described [Puckett, K.L., Aronson, E.T., & Goldin, S.M. (1985) Biochemistry 24, 390-400]. Spikes of 45Ca2+ released from disks were observed in a rapid superfusion system. The Ca2+ release was specifically stimulated by physiological levels of cGMP (Kapp approximately 20 microM; Hill coefficient = 1.7). 8-Bromo-cGMP could also activate the release mechanism, but cAMP was ineffective. At cGMP levels of greater than or equal to 100 microM, approximately 20% of the loaded Ca2+ was released. The Ca2+ release rate at saturating cGMP levels reached a maximum within the 10-s time resolution of the assay system. In contrast to other recent reports of cGMP activation of ROS ion conductances, the majority of the release activity terminated in a spontaneous manner, suggestive of an intrinsic inactivation process. The amount of Ca2+ released and the release kinetics were similar to the presence or absence of an unbleached pool of rhodopsin. Cyclic nucleotides did not stimulate release from disks passively equilibrated with 45Ca2+, i.e., in the absence of ATP but otherwise under identical conditions. Preincubation of the disks with cGMP also reduced the level of ATP-dependent Ca2+ uptake (approximately 30%); this apparent inhibition may be due to activation of the release mechanism, rather than direct modulation of the uptake activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible role of protein phosphorylation in the inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum

The Ca2+-induced Ca2+ release channel in the heavy fraction of the sarcoplasmic reticulum (SR) from rabbit skeletal muscle is inactivated during ATP-dependent Ca2+ uptake (Morii, H., Takisawa, H., & Yamamoto, T. (1985) J. Biol. Chem. 260, 11536-11541). AMP, one of the adenine nucleotides which activate the Ca2+ release, delayed the onset of the channel inactivation when added early during the course of the Ca2+ uptake. However, AMP could no longer activate the channel but accelerated the inactivation when added during the later phase of the Ca2+ uptake. In SR passively loaded with Ca2+, the Ca2+ channel which had been activated by AMP and Ca2+ was not spontaneously inactivated. Similarly, during GTP-dependent Ca2+ uptake, the channel activated by AMP was not inactivated. In addition acid phosphatase markedly delayed the onset of the inactivation during ATP-dependent Ca2+ uptake, without affecting Ca2+ ATPase activity or GTP-dependent Ca2+ uptake by heavy SR. The effect of the phosphatase was completely blocked by ruthenium red, a potent inhibitor of the channel. These results suggest that the channel is inactivated through an ATP-dependent process, presumably phosphorylation of proteins in the SR membrane. This was supported by the findings that the reactivation of the inactivated channel by added Ca2+ was markedly accelerated by the addition of acid phosphatase and that several proteins of heavy SR were phosphorylated during ATP-dependent Ca2+ uptake.     

Studies on canine gastric antrum smooth muscle: preparation and characterization of a plasma membrane enriched fraction

A method is described for preparation of large amounts of a plasma membrane (PM) enriched fraction from the smooth muscle of dog antrum. It consists of preparing microsomes, treating them with ATP + EGTA + Mg, centrifuging in 30% sucrose and then centrifuging the resulting supernatant in 15% sucrose to yield the plasma membrane enriched fraction P6. The subcellular fractions obtained at various steps during purification were characterized by: 5'-nucleotidase and phosphodiesterase I as plasma membrane markers; cytochrome c oxidase as an inner mitochondrial marker; NADPH-cytochrome c reductase as a putative endoplasmic reticulum marker; electron microscopy; polyacrylamide sodium dodecyl sulfate slab gel electrophoresis. The distribution of ATP-dependent and independent Ca uptake in presence and absence of azide and the effect of 5 mM oxalate or 25 mM phosphate on this uptake was also examined. The fraction P6 consists of mostly smooth surface vesicles 164.3 +/- 7.2 nm in diameter, has an exclusion volume of 9.7 microL/mg for [3H]inulin and 11.1 microL/mg for [3H]sucrose. P6 is maximally enriched in the ATP-dependent azide-insensitive Ca-uptake capacity and as compared with the postnuclear supernatant (S1) it shows a very small percent stimulation by oxalate and phosphate. The ATP-dependent Ca uptake by the P6 fraction occurs optimally at pH 7.0-7.4 and is much larger than the ATP-independent Ca uptake. At pH 7.1, the ATP-dependent Ca uptake occurs with a Km of 0.27 microM and a Hill coefficient greater than 2 for Ca2+. Half maximum binding of Ca2+ occurred at 300 microM Ca2+. Ca ionophores A23187 and ionomycin inhibited the ATP-dependent Ca uptake, and if added after the uptake, these caused a release of the accumulated Ca2+. From these and other data it is concluded that this PM preparation contains a Ca transport system which can lead to formation of greater than 1000-fold Ca2+ concentration gradient across the vesicle membrane in 1 min when extravesicular Ca2+ concentration is 0.3 microM. Thus this preparation is an extremely useful material for studying the mechanism of action of the Ca pump in smooth muscle plasma membrane.     

Clotting of bovine fibrinogen. Calcium binding to fibrin during clotting and its dependence on release of fibrinopeptide B

Polymerization of bovine fibrinogen acted upon by thrombin is accompanied by binding of Ca2+ and a concomitant decrease of the free Ca2+ concentration. The latter can be recorded by a Ca2+-selective electrode as a shift in the electrode potential. The shift shows marked dependence on the initial free Ca2+ concentration, being maximal at about 10(-4.1) M and decreasing sharply on either side of this. Thus, the effect is limited to the 10(-3)-10(-5) M free Ca2+ concentration range. From the initial and the final value of the electrode potential during a clotting experiment, the amount of Ca2+ bound to fibrinogen and fibrin, respectively, can be calculated. The difference between the two, plotted against free Ca2+ concentration, gives a bell-shaped curve. This indicates that the reason for the Ca2+ binding is a shift of the pK of some groups from a lower to higher value. The recordings can be used for evaluation of the kinetics of the Ca2+ uptake. However, they have to be corrected for the effect of the continuous shift in the free Ca2+ concentration during the experiment. The reaction does not follow simple kinetics, showing a lag period. Therefore, rates were estimated from inverse half-reaction times. Half-times of the corrected curves show that the reaction is first order with respect to thrombin. Moreover, the rate of Ca2+ uptake is identical with that of the conformational change seen in differential scanning calorimetry [Donovan, J.W., & Mihalyi, E. '1985) Biochemistry 24, 3434]. The inverse rate and the final corrected Ca2+ uptake increase linearly with the initial fibrinogen concentration. Concomitant estimates of fibrinopeptide A and B release showed that the Ca2+ uptake runs parallel to the release of fibrinopeptide B. Fibrinopeptide A was released largely during the lag period of the Ca2+ uptake. In agreement with this, clotting with Ancrod, an enzyme that liberates only fibrinopeptide A, was not accompanied by binding of Ca2+. Thus, polymerization is not sufficient for the Ca2+ uptake to occur; liberation of fibrinopeptide B seems to be obligatory. Further support for this was obtained with experiments with the polymerization inhibitor Gly-Pro-Arg-Pro. The tetrapeptide inhibits polymerization and also, proportional to this, release of fibrinopeptide B [Hurlet-Jensen, A., Cummins, H.Z., Nossel, H.L., & Liu, C.Y. (1982) Thromb. Res. 27, 419; Lewis, S.D., Shields, P.P., & Shafer, J.A. (1985) J. Biol. Chem. 260, 10192]. Calcium uptake was also depressed by the tetrapeptide in a way similar to its effect upon fibrinopeptide B release.     

Transport of Ca and Mn by mitochondria from rat liver, heart and brain

The energy-dependent, respiration-supported uptake and the uncoupler- or Na+-induced release of Ca2+ and Mn2+ by mitochondria from rat liver, heart and brain were investigated, using as indicators radioisotopes (45Ca and 54Mn), proton ejection, oxygen consumption, nicotinamide nucleotide oxidation-reduction and, in the case of Ca2+, the metallochromic dye Arsenazo III. Ca2+ uptake in the presence of Pi was rapid in mitochondria from liver and brain, and less rapid in those from heart. Mn2+ uptake was much slower than that of Ca2+ in liver and heart, but only slightly slower in brain. When added together, Ca2+ accelerated the uptake of Mn2+, and Mn2+ retarded the uptake of Ca2+, by mitochondria from all three tissues. When Mn2+ was present during Ca2+ uptake, its own uptake remained accelerated even after Ca2+ uptake was terminated. Mg2+, which was not taken up, inhibited Ca2+ uptake by mitochondria from all three tissues, and, when present during Ca2+ uptake, accelerated the subsequent uptake of Mn2+. The uncoupler CCCP induced a release of both Ca2+ and Mn2+ from all three sources of mitochondria; yet, release of Mn2+ took place only in the absence of Pi. The release followed the same pattern as the uptake, i.e., Ca2+ accelerated the release of Mn2+ and Mn2+ retarded the release of Ca2+. Na+ induced a release of both Ca2+ and Mn2+ from heart and brain but not from liver mitochondria; again, Mn2+ release occurred only in the absence of Pi. The Na+-induced release of Ca2+ was inhibited by Mn2+, but the Na+-induced release of Mn2+ was not accelerated by Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Silver ions trigger Ca2+ release by acting at the apparent physiological release site in sarcoplasmic reticulum

Rapid Ca2+ release from Ca2+ -loaded sarcoplasmic reticulum vesicles (SR) was previously shown to occur upon the addition of micromolar concentrations of heavy metals, and the extent of Ca2+ release was dependent on the binding affinity of the metal to sulfhydryl group(s) on an SR protein (Abramson, J.J., Weden, L., Trimm, J.L., and Salama, G. (1982) Biophys. J. 37, 134a; Abramson, J.J., Trimm, J.L., Weden, L., and Salama, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1526). The nature of this Ca2+ release site was examined further and found to be predominantly distributed in heavy SR (HSR) rather than light SR fractions. Ag+ -induced Ca2+ release from heavy SR was blocked by local anesthetics and ruthenium red which are known to inhibit Ca2+ release in skeletal fibers and in heavy SR, respectively. The rate of Ca2+ efflux from SR triggered by Ag+ was dependent on pH, Mg2+, and ionic strength of the medium. Efflux rates increased by a factor of 4 from pH 6.0 to 7.0 and then decreased in more alkaline reaction mixtures. Efflux rates from actively or passively loaded SR increased by a factor of 2.5 with increasing Mg2+ from 0 to 1 mM and then decreased in the range of 1 to 10 mM Mg2+. ATP-dependent Ca2+ uptake by SR was similar in 100 mM KCl and in 200 mM sucrose solutions, but the extent and rate of Ca2+ efflux induced by Ag+ were dramatically reduced with decreasing ionic strength of the medium. In solutions containing 5 mM Mg2+, the rate of Ca2+ efflux from heavy SR averaged over the first 1.5 s after the addition of Ag+ was 58 nmol of Ca2+/mg of SR/s, a value comparable to the fast initial rate of ATP-dependent Ca2+ uptake. The maximum initial rate of Ag+ -induced Ca2+ efflux from heavy SR in 1 mM Mg2+ may be comparable to the rate of Ca2+ release and tension development in muscle fibers. Our data indicate that Ag+ reacts with a protein or proteins in the SR, probably not the (Ca2+, Mg2+)-ATPase, to induce a rapid release of Ca2+, possibly from the physiological Ca2+ release site.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: a comparison with skinned muscle fiber studies

Uptake and release of Ca2+ in heavy and light fractions of fragmented sarcoplasmic reticulum (FSR) isolated from frog and rabbit skeletal muscle was studied under conditions similar to those employed in skinned muscle fiber experiments, where ATP and Mg2+ concentrations were considered to be physiological and free Ca2+ concentration was kept constant during the Ca2+ uptake and release. Ca2+ level in FSR monotonously approached a steady state level which depended only on the final experimental conditions. Heavy fractions, but not light fractions, exhibited characteristics similar to those of Ca2+-induced Ca2+ release reported in skinned fiber studies: i) the rate and steady state level of Ca2+ uptake increased with increase in free Ca2+ concentration in the reaction medium up to 10(-6) M. With further increase in free Ca2+ concentration, the steady state level of Ca2+ taken up decreased while the Ca2+ uptake rate increased. ii) The steady state Ca2+ level was decreased by caffeine but increased by procaine or ruthenium red. Parallel measurement of Ca2+-ATPase activity clearly showed that these drugs modify the Ca2+ efflux but hardly affect the Ca2+-pump activity. It was concluded that the Ca2+-induced Ca2+ release mechanism was in operation at as low as 10(-6) M free Ca2+ concentration. Treatment of FSR with 0.6 M KCl did not have any significant effect.     

Changes in mitochondrial calcium metabolism after treating mastocytoma cells with N6,O2′-dibutyryladenosine 3′,5′ cyclic monophosphate

Comparison of Ca2+ uptake by isolated mouse liver mitochondria, and mitochondria prepared from mastocytoma cells grown with and without N6,O2'-dibutyryladenosine 3',5' cyclic monophosphate (DB cyclic AMP) and theophylline showed several differences in their capacity to take up and retain calcium. In particular mitochondria from DB cyclic AMP-treated mastocytoma cells took up more Ca2+ than mitochondria from untreated mastocytoma cells. Ca2+ uptake by mitochondria from DB cyclic AMP-treated cells was also increased in the presence of oxalate whereas oxalate did not affect Ca2+ uptake by mitochondria from untreated mastocytoma cells and it reduced Ca2+ uptake by mouse liver mitochrondria. The results suggest that inhibiting the growth of mastocytoma cells with DB cyclic AMP alters their mitochondrial Ca2+ metabolism.     

A novel mechanism of cation/substrate cotransport: Na+/H+/adenosine cotransport in Vibrio parahaemolyticus

Adenosine is actively transported with Na+ in Vibrio parahaemolyticus (Sakai, Y., Tsuda, M., Tsuchiya, T. (1987) Biochim, Biophys. Acta 893, 43-48). The proton conductor carbonylcyanide m-chlorophenylhydrazone, CCCP, strongly inhibited active transport of adenosine at pH 8.5 as well as at pH 7.0. This seemed peculiar because the driving force, an electrochemical potential of Na+, is established by the Na(+)-extruding respiratory chain at pH 8.5 in this organism, although it is established by the function of the Na+/H+ antiporter at pH 7.0. This suggested that H+ might be involved in the adenosine transport. We detected H+ uptake induced by adenosine influx in V. parahaemolyticus cells in the presence of Na+, but not in its absence, suggesting the occurrence of Na+/H+/adenosine cotransport. We isolated formycin A-resistant mutants which showed defective adenosine transport. The mutation resulted in simultaneous losses of Na+ uptake and H+ uptake induced by adenosine. In revertants from these mutants the Na+ uptake and H+ uptake were restored simultaneously. The frequencies of reversion were in the order of 10(-7), indicating that the mutations were single mutations; namely that Na+/adenosine cotransport and H+/adenosine cotransport took place via the same carrier. Thus, we conclude that adenosine is transported by the novel mechanism of Na+/H+/adenosine cotransport in V. parahaemolyticus.     

Effect of vanadate, a potent alkaline phosphatase inhibitor, on 45Ca and 32Pi uptake by matrix vesicle-enriched fractions from chicken epiphyseal cartilage

Although alkaline phosphatase has been long associated with the mineralization process, its exact function remains to be elucidated. To clarify its possible role in matrix vesicle-mediated mineralization, we tested the effect of vanadate, a phosphate analogue and powerful competitive inhibitor of alkaline phosphatase activity, on calcium and phosphate uptakes by a matrix vesicle-enriched microsomal fraction. Vanadate was also tested in a hydroxyapatite-seeded ion uptake system to determine possible direct effects on mineral formation. The effect of vanadate on vesicle mineral ion uptake was complex; low dosages of vanadate (2-20 microM) were stimulatory to Ca2+ uptake, but were inhibitory to Pi. Higher dosages (greater than 67 microM) were inhibitory to both ions. The effect of vanadate on ion uptake was strongly influenced by the stage of vesicle loading; major effects were seen during the lag and early uptake phases, and minimal effects were seen in the terminal stages. Concentrations of vanadate highly inhibitory to vesicle ion uptake had minimal effects on ion accretion by a hydroxyapatite-seeded system. Inhibition of alkaline phosphatase activity by vanadate broadly paralleled inhibition of Pi and Ca2+ uptake; however, at low vanadate concentrations, inhibition of Pi uptake closely paralleled that of alkaline phosphatase. The data indicate that vanadate binds with high affinity to Pi-loading sites, blocking initial Pi uptake. Complexation between vanadate and Ca2+ may be responsible for the stimulation of Ca2+ uptake at early stages of vesicle ion loading with low levels of vanadate by enhancing binding of Ca2+ to the vesicles. It may also account for the selective inhibition of Ca2+ uptake during the rapid stage of vesicle ion loading with high levels of vanadate by reducing Ca2+ ion activity. The close parallelism between inhibition of early Pi uptake and of alkaline phosphatase activity supports the concept that alkaline phosphatase is involved in Pi transport during the early stages of matrix vesicle ion loading. However, the fact that only about half of the Pi uptake was affected by vanadate, despite the progressive inhibition of alkaline phosphatase activity, indicates that alkaline phosphatase is not solely responsible for Pi uptake by the matrix vesicle-enriched fraction.     

Effects of exercise of varying duration on sarcoplasmic reticulum function

Sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+-Mg2+-ATPase activity were examined in muscle homogenates and the purified SR fraction of the superficial and deep fibers of the gastrocnemius and vastus muscles of the rat after treadmill runs of 20 or 45 min or to exhaustion (avg time to exhaustion 140 min). Vesicle intactness and cross-contamination of isolated SR were estimated using a calcium ionophore and mitochondrial and sarcolemmal marker enzymes, respectively. Present findings confirm previously reported fiber-type specific depression in the initial rate and maximum capacity of Ca2+ uptake and altered ATPase activity after exercise. Depression of the Ca2+-stimulated ATPase activity of the enzyme was evident after greater than or equal to 20 min of exercise in SR isolated from the deep fibers of these muscles. The lowered ATPase activity was followed by a depression in the initial rate of Ca2+ uptake in both muscle homogenates and isolated SR fractions after greater than or equal to 45 min of exercise. Maximum Ca2+ uptake capacity was lower in isolated SR only after exhaustive exercise. Ca2+ uptake and Ca2+-sensitive ATPase activity were not affected at any duration of exercise in SR isolated from superficial fibers of these muscles; however, the Mg2+-dependent ATPase activity was increased after 45 min and exhaustive exercise bouts. The alterations in SR function could not be attributed to disrupted vesicles or differential contamination in the SR from exercise groups and were reinforced by similar changes in Ca2+ uptake in crude muscle homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of Ca uptake characteristics of microsomal fractions isolated from unfertilized and fertilized sea urchin eggs

The microsomal fraction isolated from sea urchin H. pulcherrimus eggs has the ability to actively accumulate Ca²⁺ in the presence of ATP. The Ca²⁺ uptake was sustained by addition of oxalate and was apparently insensitive to sodium azide. The sequestered microsomal Ca was readily released by the divalent cation ionophore A23187. The microsomal fraction obtained from fertilized eggs accumulated Ca²⁺ about five times more quickly than did that from unfertilized eggs. The increased Ca²⁺ uptake by microsomal fraction obtained from fertilized eggs was due to an increase in the maximum velocity of Ca²⁺ uptake and there was no difference in K_m for calcium between the two fractions.     

Effects of endogenous calcium transport inhibitor from heart muscle on the active calcium uptake and passive calcium release properties of sarcoplasmic reticulum

In the present study, the effects of the cytosolic Ca2+ transport inhibitor on ATP-dependent Ca2+ uptake by, and unidirectional passive Ca2+ release from, sarcoplasmic reticulum enriched membrane vesicles were examined in parallel experiments to determine whether inhibitor-mediated enhancement in Ca2+ efflux contributes to inhibition of net Ca2+ uptake. When assays were performed at pH 6.8 in the presence of oxalate, low concentrations (less than 100 micrograms/mL) of the inhibitor caused substantial inhibition of Ca2+ uptake by SR (28-50%). At this pH, low concentrations of the inhibitor did not cause enhancement of passive Ca2+ release from actively Ca2+-loaded sarcoplasmic reticulum. Under these conditions, high concentrations (greater than 100 micrograms/mL) of the inhibitor caused stimulation of passive Ca2+ release but to a much lesser extent when compared with the extent of inhibition of active Ca2+ uptake (i.e., twofold greater inhibition of Ca2+ uptake than stimulation of Ca2+ release). When Ca2+ uptake and release assays were carried out at pH 7.4, the Ca2+ release promoting action of the inhibitor became more pronounced, such that the magnitude of enhancement in Ca2+ release at varying concentrations of the inhibitor (20-200 micrograms/mL) was not markedly different from the magnitude of inhibition of Ca2+ uptake. In the absence of oxalate in the assay medium, inhibition of Ca2+ uptake was observed at alkaline but not acidic pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kainate-induced uptake of calcium by synaptosomes from rat brain

Kainic acid induces a rapid increase in 45Ca2+ uptake by crude synaptosomal fractions isolated from rat brain. This enhanced Ca2+ permeability occurs with a half-time of approx. 1 s, similar to the fast phase of depolarization-induced calcium uptake. The depolarization-induced uptake of calcium is inhibited 85% by 3 mM CoCl2, 80% by 100 microM quinacrine and 50% by 15 microM trifluoperazine while these agents had little effect on the kainate-induced uptake. It is proposed that kainate induces receptor-mediated opening of a class of calcium channels with properties different from those of the voltage-dependent channels.     

Characterization of Cellular Transport, Subcellular Distribution, and Secretion of the Neurotoxicant 1-Methyl-4-Phenylpyridinium in Bovine Adrenomedullary Cell Cultures

Cultures of bovine adrenomedullary chromaffin cells accumulated 1-[methyl-3H]methyl-4-phenylpyridinium ([3H]MPP+) in a time- and concentration-dependent manner with an apparent Km of 0.7 microM and a Vmax of 3 pmol/min/10(6) cells. The uptake was sodium dependent and sensitive to inhibitors of the cell-surface catecholamine transporter. At low concentrations of MPP+, the subcellular distribution was identical to that of endogenous catecholamines in the catecholamine-containing chromaffin vesicles. However, at a higher concentration of MPP+, a larger proportion of the toxicant was recovered in the cytosolic fraction, with less in the chromaffin vesicle fractions. When cells were prelabeled with [3H]MPP+, at 1 and 300 microM, and then permeabilized with digitonin in the absence of Ca2+, there was a proportionally greater release of MPP+ from the cells labeled at the higher concentration of the toxicant. In the presence of Ca2+, cell permeabilization induced a time-dependent secretion of catecholamines and a parallel secretion of MPP+. Under these conditions, the secretion of endogenous catecholamines was unaffected by the presence of MPP+. When the permeabilization studies were carried out in the presence of tetrabenazine, a massive release of MPP+ was observed in the absence of Ca2+ and was not further increased by Ca2+. In intact cells prelabeled with 300 microM [3H]MPP+, the secretagogues nicotine and veratridine elicited a Ca2+ -dependent secretion of catecholamines and MPP+ from the cells in similar proportions to their cellular contents. Barium-induced release of both species was independent of external Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)