ATP-dependent Calcium Accumulation Research Articles

Comparative calcification of native articular cartilage matrix vesicles and nitroprusside-generated vesicles

Objective Articular cartilage matrix vesicles generate calcium pyrophosphate- and basic calcium phosphate-like mineral in vitro. We sought to determine the morphologic features and calcifying capacity of sodium nitroprusside (SNP)-induced vesicles for comparison to those of controls.Methods Porcine articular cartilage was exposed to 1mM SNP for 24h and vesicles were isolated by enzymatic digestion and serial ultracentrifugation. Control vesicles were derived from an equal weight of untreated articular cartilage. Vesicles-containing fractions pelleted at 2×105g·min (pellet I), 3×106g·min (pellet II, the heavy vesicle fraction) and 8×106g·min (pellet III, the light vesicle fraction) were analysed for Lowry protein content, nucleoside triphosphate pyrophosphohydrolase specific activity (NTPPPH) and ATP-dependent calcifying capacity.Results Electron micrographs (EM) revealed two populations of vesicular structures in both SNP and control pellets. In most experiments, there were no significant differences between the protein contents or ATP-dependent calcium accumulation of SNP vesicles compared to control vesicles. SNP vesicles in pellets I and II had lower NTPPPH activities than their respective controls, P≤ 0.01.Conclusions Our data confirmed that 24-hour treatment with the apoptosis-inducing agent did not increase matrix vesicle protein or alter the calcifying activity of vesicles compared to those from control cartilage. SNP did generate vesicles with lower NTPPPH specific activity in most experiments. SNP vesicles, although morphologically similar to controls, are not biochemically identical to them. Copyright 2002 Published by Elsevier Science Ltd on behalf of OsteoArthritis Research Society International

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.

Canine cardiac sarcoplasmic reticulum is not altered with endurance exercise training

To investigate the effect of exercise training on calcium movements by isolated cardiac sarcoplasmic reticulum (SR), mongrel dogs either remained sedentary (S) or were exercise-trained (E) via running for a period of 8-10 wk. The trained state was confirmed by the increase in skeletal muscle citrate synthase activity and decreases in submaximal exercise heart rates in the E group but not in the S dogs. The properties of isolated cardiac SR were identical between the groups. The variables tested included ATP-dependent calcium transport and calcium-stimulated ATPase activity. Importantly, there was no difference in spontaneous calcium release which occurred after peak ATP-dependent calcium accumulation was reached. Calcium release from passively loaded vesicles induced by calcium and ionophore also did not differ in the SR isolated from the E dogs. The change in the affinity of the SR Ca ATPase for calcium after the addition of the polyanion, heparin, was similar in both groups, indicating that the regulation of calcium-stimulated ATPase activity by the SR protein, phospholamban, is not modified by exercise training. We conclude that exercise training of 8-10 wk duration does not alter the calcium handling properties of cardiac SR isolated from mongrel dogs.

Properties of the residual calcium pools in human red cells exposed to transient calcium loads.

1. Inosine-fed human red cells, pre-loaded with calcium with the use of the ionophore A23187, and reincubated at 37 degrees C after ionophore and external calcium removal, pumped calcium out initially fast and then substantially slower when the residual calcium was still very high (García-Sancho & Lew, 1988 a, b). Particularly surprising was the finding of such residual calcium pools in a subpopulation of cells (L cells, García-Sancho & Lew, 1988b) with normal ATP, high active calcium pumping rates, and K+ channels which remained inactive during the density separation procedure (SCN- treatment; García-Sancho & Lew, 1988a). The purpose of the present experiments was to investigate the properties and possible origins of the residual calcium pools. 2. At each ionophore concentration, the pool size increased with the duration and magnitude of the calcium load. At comparable calcium loads, the pool size was smaller the higher the ionophore concentration used. 3. In cells from the same sample, residual calcium pools were present in cells that became dense after SCN- treatment (H cells) as well as those that remained in the light-cell fraction (L cells). Residual calcium was always higher in H cells than in L cells. 4. Re-exposure of cells to ionophore in Ca2+-free media could rapidly extract over 99% of their residual calcium. Residual calcium is therefore in a rapidly mobilizable form within a membrane-bound compartment. 5. Iodoacetamide-induced ATP depletion of H cells with high residual calcium-stimulated calcium loss. Such stimulation could only occur if ATP depletion inhibited a calcium-retaining process within the cells. 6. L cells with high residual calcium may have failed to dehydrate during SCN- treatment because of irreversible K+ channel inactivation or because K+ permeabilization would no longer generate a dehydrating net cation loss. These possibilities were tested and ruled out since it was found that virtually all cells became dense in low-K+, SCN- media after valinomycin addition or re-exposure to A23187 + calcium. 7. The results suggest that part if not all of the residual calcium is contained within compartments with the properties of endocytic inside-out vesicles capable of ATP-dependent calcium accumulation, such as those found in normal and abnormal human red cells (Lew, Hockaday, Sepúlveda, Somlyo, Somlyo, Ortiz & Bookchin, 1985).

Lipoxygenase-induced lipid peroxidation of isolated cardiac microsomes modulates their calcium-transporting function

We demonstrated previously that products of linoleic and arachidonic acids, arising from enzymatic or non-enzymatic oxidation, inhibit ATP-dependent calcium accumulation into and promote release of calcium from vesicles derived from sarcoplasmic reticulum of guinea-pig heart. In the present study, direct enzymatic peroxidation of cardiac membrane lipids was performed and the effect on calcium transport was examined. Vesicles were preincubated at 37°C with soybean lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12) for up to l h prior to the initiation of calcium accumulation. The extent of membrane peroxidation was assessed by monitoring the production of malondialdehyde. Pretreatment of vesicles with lipoxygenase for 40 and 60 min markedly depressed calcium accumulation. The lipoxygenase-induced suppression of calcium transport was completely antagonized by nordihydroguaiaretic acid (1 μM), not at all by indomethacin (1 μM), and only partially by 5,8,11,14-eicosatetraynoic acid (0.3 μM). Low concentrations of calcium (10 −5-5 · 10 −5 M) enhanced, and a high concentration (10 −3 M) inhibited lipoxygenase-induced peroxidation of membrane lipids. The calcium-accumulating ability of the vesicles was inversely related to the extent of membrane peroxidation. The vesicles which showed the highest degree of peroxidation in the presence of 5 · 10 −5 M calcium, accumulated the lowest amount of calcium. In contrast, calcium at 10 −3 M suppressed lipid peroxidation, resulting in higher calcium uptake than in vesicles peroxidized in the absence of calcium. Thus, calcium transport is depressed in microsomes undergoing lipoxygenase-induced peroxidation, a process which in turn is modulated by calcium.

The effect of linoleic and arachidonic acid derivatives on calcium transport in vesicles from cardiac sarcoplasmic reticulum

The effect of linoleic and arachidonic acid derivatives on ATP-dependent calcium transport was studied in the isolated vesicles from cardiac sarcoplasmic reticulum of guinea-pigs. Oxidation products of linoleic and arachidonic acids, obtained either by autoxidation or incubation with soybean lipoxygenase, effectively blocked in a dose-dependent manner, the net influx of calcium in the absence or presence of 5 mM of oxalate. Unoxidized fatty acids were much weaker at lower concentrations as compared to their oxidized counterparts, except the lipoxygenase-generated product of arachidonic acid which had only a marginal effect even at high concentrations. Autoxidation products of arachidonic acid were the most potent inhibitors of calcium transport. Likewise, autoxidation products of linoleic and arachidonic acids and lipoxygenase-generated products of linoleic acid induced a dose-dependent release of calcium from vesicles previously loaded with 45Ca, and release was further enhanced in the presence of 0.5 mM of EGTA. In contrast, lipoxygenase metabolites of arachidonic acid caused a transient increase in net calcium content. The effect of the fatty acid derivatives on calcium transport did not appear to be due either to the inhibition of Ca2+-ATPase activity or to a non-specific detergent-like action. The effects of oxidized fatty acids, on ATP-dependent calcium accumulation into and release from cardiac microsomal fraction were similar but less potent than those of classical calcium ionophores, X537A or A23187.

Activation and inhibition of the calcium gate of sarcoplasmic reticulum by high-affinity ryanodine binding

The occupancy of high-affinity ryanodine-binding sites of isolated heavy sarcoplasmic reticulum vesicles occurring in concentrated salt solutions affects ATP-dependent calcium accumulation and caffeine-induced calcium release. The initial suppression of calcium uptake is followed by a marked uptake activation resulting in a reduction of the final calcium level in the medium. Simultaneously, caffeine-induced calcium release is blocked. The dependence of inhibition of calcium uptake and caffeine-induced calcium release observed in assay media containing physiological concentrations of magnesium and ATP on the concentration of ryanodine corresponds to the drug's effectiveness in living muscles.

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes.

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.

Reconstitution of ATP-dependent calcium transport from streptococci.

Membrane vesicles of three streptococcal strains (Streptococcus faecalis, Streptococcus lactis, and Streptococcus sanguis) were extracted with octyl-beta-D-glucoside in the presence of Escherichia coli lipid and glycerol. For reconstitution, the detergent extract was mixed with bath-sonicated E. coli lipid, in the presence of octyl-beta-D-glucoside, and proteoliposomes were formed by a 25-fold dilution. ATP-dependent calcium accumulation by proteoliposomes was comparable to that found in parent vesicles. Recovery of this calcium transport activity was dependent on the inclusion of an osmolyte protein stabilant (glycerol, etc.) during solubilization. The properties of ATP-driven calcium transport were studied in the reconstituted system. In proteoliposomes, ATP-linked calcium accumulation was not affected by the protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or by the ionophores, valinomycin and nigericin, in the presence of potassium, or by N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. On the other hand, calcium transport was completely blocked by micromolar levels of orthovanadate; half-maximal inhibitions were observed at 0.4, 4, and 4 microM vanadate, for S. faecalis, S. lactis, and S. sanguis, respectively. This marked sensitivity to orthovanadate suggests operation of an E1E2-type ion-motive pump. These data demonstrate that, in a reconstituted system, calcium transport is not linked to an ATP-dependent proton circulation via the F0F1-ATPase, but rather is driven by a calcium-translocating ATPase. Thus, calcium extrusion from the cytosol of enteric, lactic acid, or oral streptococci is mediated by an ATP-linked process analogous to the ion-motive ATPases of eukaryotic membranes.

ATP-dependent calcium accumulation by non-mitochondrial organelles of axoplasm isolated from Myxicola giant axons

Axoplasm from freshly isolated Myxicola giant axons was mixed with small volumes of ‘artificial axoplasm’ containing 45Ca and either CaEGTA/EGTA or CaDTPA/DTPA buffers giving various nominal values of [Ca 2+]. The axoplasm samples were centrifuged at 100000 x g for 30 min to form a pellet and the percentage of 45Ca bound to the pellet was determined. The fraction of bound calcium rose with increasing values of [Ca 2+] along an S-shaped curve. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used to reveal the presence of mitochondrial Ca uptake. At physiological values of [Ca 2+], around 100 nM, Ca uptake was insensitive to FCCP. As [Ca 2+] was elevated, increasing sensitivity to FCCP was noted above [ Ca 2+] = 0.5 μM. At low values of [Ca 2+], including the physiological range, Ca binding was significantly reduced by vanadate and quercetin, agents known to inhibit Ca uptake mediated by Ca 2+-activated ATPase reactions. Inhibition of Ca binding by these agents was approximately 50% at physiological values of [Ca 2+]. ATP depletion decreased the percentage of Ca binding by the pellet at physiological [Ca 2+]. The results suggest that about 50% of the Ca buffering by particulate matter in axoplasm is via organelles requiring intact Ca 2+-ATPase reaction at physiological values of [Ca 2+].

Calcium transport and protein content in cell plasma membranes of spontaneously hypertensive rats.

In the absence of depolarizing agents, 45Ca uptake by synaptosomes of the brain tissue of spontaneously hypertensive rats (SHR) was 40% greater than in normotensive controls. This difference disappeared after membrane treatment with depolarizing agents or with the addition of the Ca channel blocker verapamil. Increased 45Ca uptake was also observed in isolated platelets of SHR. It can be assumed that in both cases these differences were caused by partial depolarization of the plasma membrane as a result of its increased permeability to Na+. The addition of calmodulin to microsomal fractions of SHR brain tissue resulted in a considerably diminished increase in ATP-dependent calcium accumulation. These data are in accord with previous findings obtained in a study on the erythrocyte membrane Ca pump. Data obtained by means of differential scanning microcalorimetry of erythrocyte membranes and gel electrophoresis of membrane proteins indicated a higher band-3 protein content in the erythrocyte membranes of SHR. These data support the concept of primary hypertension as a type of generalized membrane pathology.

Comparative studies on Ca 2+- and Mg 2+-binding of sarcoplasmic reticulum and chromaffin granule membranes

The binding of calcium and magnesium ions to sarcoplasmic reticulum (SR) and chromaffin granule membranes was comparatively studied. The SR membranes are equipped with equal quantities of binding sites for both calcium and magnesium ions. The binding sites in presence of ATP combine specifically with calcium ions, while in the absence of ATP the binding sites react unspecifically with both ions. The trace amount of magnesium present in the SR membranes preparations is sufficient to drive ATP dependent calcium accumulation. Magnesium binding, however, is not affected by ATP. The chromaffin granule membranes bind calcium and magnesium in the same concentration range as observed for the SR membranes. Magnesium binding, however, is two times higher than that of calcium binding. In the absence of ATP, calcium and magnesium ions mutually compete. In the presence of ATP, magnesium binding values increase 3–5-fold, while the calcium binding isotherm remains unchanged. The appreciable contribution of the lipid phase to ions binding has been investigated, but was found to be of minor importance in this study.

Stimulation by nifedipine of calcium transport by cardiac sarcolemnal vesicles from spontaneously hypertensive rats

The effects of the calcium antagonist nifedipine on the binding and ATP-dependent accumulation of calcium by cardiac plasma membranes from spontaneously hypertensive rats (SHR) and from their normotensive controls (WKY) were studied at free calcium concentrations of 2 × 10 −8 M and 4 × 10 −7 M corresponding to very high affinity and high affinity binding sites respectively. Nifedipine did not significantly modify calcium binding to either class of sites in SHR or WKY membranes. In contrast, in a free calcium concentration of 2 × 10 −8 M, nifedipine enhanced ATP-dependent Ca 2+ transport. The concentration of nifedipine required for significant stimulation was smaller in SHR than in WKY membranes (10 −7 M and 10 −5 M respectively). Another calcium antagonist, D 600, did not modify ATP-dependent calcium accumulation by SHR or WKY vesicles. These results raise the question of the mechanism of action of nifedipine on the calcium pump and confirm the presence of abnormalities in cardiac plasma membranes from young SHR, rendering them more sensitive than WKY membranes to the calcium antagonist nifedipine.

Effects of adrenalectomy and in vivo administration of dexamethasone on ATP-dependent calcium accumulation by sarcoplasmic reticulum from rat heart

ATP-dependent Ca 2+ accumulation and (Mg 2+ + Ca 2+)ATPase activities were determined in cardiac sarcoplasmic reticulum (SR) from control, adrenalectomized and adrenalectomized-dexamethasone-treated rats. SR from adrenalectomized rats displayed significantly diminished rates (∼40 to 50%, P < 0.001) of Ca 2+ accumulation compared to SR from control rats. The decreased Ca 2+ accumulating activity of the membranes from adrenalectomized compared to control rats could be observed at varying Ca 2+ concentrations (0.5 to 11.9 μ m); kinetic analysis showed that the velocity of Ca 2+ transport but not the apparent affinity of the transport system for Ca 2+ was altered by adrenalectomy. ATPase activities (with Mg 2+ or with Mg 2+ + Ca 2+) of SR were not altered significantly following adrenalectomy. In vivo administration of dexamethasone to adrenalectomized rats led to a partial (75 to 80%) yet significant ( P < 0.01 to 0.05) restoration of Ca 2+ accumulating activity of SR towards normal; dexamethasone-treatment caused decrease in basal (Mg 2+)ATPase activity and enhancement in (Mg 2+ + Ca 2+)ATPase activity. When added in vitro, dexamethasone had no effect on Ca 2+ accumulating and ATPase activities of SR from control or adrenalectomized rats. These findings imply an important role for glucocorticoids in the maintenance of membrane Ca 2+ transport function, and therefore, normal myocardial contractility.

ATP-dependent calcium transport and its correlation with Ca 2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca 2+-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH 2)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K m of 0.2 μM Ca 2+ and a V max of 5.3 nmol Ca 2+/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca 2+-ATPase are: K m = 0.25 μM Ca 2+ and V max = 19.2 nmol P i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V max of ATP-dependent calcium uptake amounts to 25 nmol Ca 2+/min per mg protein and of the Ca 2+-ATPase to 23 nmol P i/min per mg protein. The close correlation between kinetic parameters of Ca 2+-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca 2+-pump located in duodenal basolateral plasma membranes.

Abnormal Calcium Handling by Isolated Cardiac Plasma Membrane from Spontaneously Hypertensive Rats

1. Calcium handling by heart sarcolemmal vesicles from young spontaneously hypertensive rats (SHR) and normotensive Wistar--Kyoto (WKY) control rats were compared. 2. Calcium binding was significantly altered in SHR membranes at the physiological cytosolic Ca2+ concentrations which occur in resting and excited cells. 3. ATP-dependent calcium accumulation occurred at a higher rate in SHR than in WKY rat membrane vesicles. 4. Na+-dependent calcium extrusion of loaded vesicles was higher in SHR than in WKY rat membrane vesicles. 5. These alterations may play a significant role in the pathogenesis of hypertension.

Characterization of cell membrane and sarcoplasmic reticulum from bovine uterine smooth muscle

Subcellular fractions, enriched in sarcoplasmic reticulum or in cell membrane, were separated from one another. Starting material was a microsomal pellet (15–40 × 1000 g) obtained by differential centrifugation from the uteri of close-to-term pregnant cows. A microsomal fraction enriched in ATP-dependent calcium accumulation was shown to contain sarcoplasmic reticulum and cell membrane. Only 8% or less of the protein in this fraction could be recovered, using affinity chromatography on Sepharose 6MB wheat germ agglutinin. The small yields did not allow extensive characterization. A method was developed to separate sarcoplasmic reticulum from cell membrane using discontinuous sucrose density gradient centrifugation. Protein was collected at the 24–28, the 28–33, and the 33–45% sucrose interfaces. Characterization was by enzyme assays and by specific receptor assay. ATP-dependent calcium accumulation was fourfold greater in the 24–28% sucrose layer than in the 33–45% layer. In contrast, 5′-nucleotidase was more than threefold as high in the 33–45% sucrose layer as in the 24–28% layer. Ouabain-inhibited p-nitrophenylphosphatase doubled and ouabain-inhibited Na,K-ATPase tripled in the 28–33% layer, compared with the 24–28% layer, specific ouabain binding was also doubled in the 28–33% sucrose layer. 125I-Labeled wheat germ agglutinin binding was greatest in the 33–45% sucrose layer. It is concluded that the 24–28% layer consists primarily of sarcoplasmic reticulum, whereas the 28–33 and the 33–45% layers are concentrated in the cell membrane. Specific prostaglandin (PGE 2) binding was found to be a property of the cell membrane.

Calcium localization in the rat neurohypophysis.

Neurosecretory nerve endings in the rat neurohypophysis release their hormones by exocytosis subsequent to an influx of calcium from the external medium. The nerve endings are characterized by the presence of neurosecretory granules, mitochondria, occasional vacuoles, and a population of microvesicles similar in appearance to spherical synaptic vesicles. The function of the microvesicles has, for a long time, been uncertain. In view of evidence that coated microvesicles isolated from cerebral cortex are capable of ATP-dependent calcium accumulation, a method has now been developed for the visualization of calcium in the neurohypophysis at the ultrastructural level. With this technique, calcium precipitates are consistently seen in the microvesicles, mitochondria and glial cell (pituicyte) nuclei. In addition, the pituicyte cytoplasm and perivascular space show a diffuse precipitate which can be removed by washing the tissue prior to fixation. The function of the microvesicles might therefore be to sequester calcium within the nerve endings.

An improved procedure for the isolation of plasma membranes from rat mesenteric arteries

A plasma membrane enriched fraction has been prepared from rat mesenteric arteries by an improved procedure which eliminated the contamination by cells other than smooth muscle; i.e. those derived from mesenteries and fat tissues. Morphological and enzymatic characterization of the plasma fraction revealed only minimal contamination by other intracellular membrane fragments in the smooth muscle cell. The plasma membrane was concentrated approximately 8-fold from the post nuclear supernatant fraction as suggested by the enrichment of 5′-nucleotidase, alkaline phosphatase and ATP-dependent calcium accumulation activities. Calcium accumulation by various membrane fractions in the presence of ATP was closely associated with plasma membrane marker enzyme activities, and was not affected by azide and oxalate. The ATP-dependent calcium accumulation by plasma membrane enriched fraction was not affected by the sodium ionophore, monensin, but was substantially inhibited by calcium ionophores X537A and A23187. This suggests that the functional properties of the plasma membrane of vascular smooth muscle include calcium extrusion across the cellular membrane via an energy-dependent transport process and that the plasma membrane plays the major role in regulating the intracellular calcium in vascular smooth muscle.

Factors affecting calcium accumulation and release in canine submandibular salivary microsomes

Microsomes isolated from canine submandibular glands were examined for ATP-dependent calcium accumulation and calcium release. Calcium uptake (10 to 20 nmoles per min per mg protein at 37 °C) was linear with time up to 20 min and with the amount of microsomal protein in the range of 15 to 200 μg/ml of incubation medium. Of the nucleotide triphosphates tested, only adenosine triphosphate (ATP) supported calcium accumulation. Ruthenium red and ethacrynic acid, at concentrations of 90 μg/ml and 10 −4 M respectively, almost completely inhibited calcium uptake. Metabolic inhibitors such as 2,4 dinitrophenol, azide and oligomycin only partially affected microsomal calcium uptake. Incubation of prelabelled microsomes in a calcium-free solution resulted in the release of calcium at a rate of 0.025/min. The rate was decreased at 0 °C and by inclusion of 4.5 mM ATP in the medium. Addition of 2 mM calcium or removal of magnesium also reduced release. Ruthenium red at a concentration that decreased calcium uptake by greater than 90 per cent did not affect calcium outflow; whereas ethacrynic acid (5 × 10 −4M) induced a significant increase in calcium loss.