Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

PTH release stimulated by high extracellular potassium is associated with a decrease in cytosolic calcium in bovine parathyroid cells

We employed the calcium (Ca⁺⁺)-sensitive, intracellular dye QUIN-2 to examine the role of cytosolic Ca⁺⁺ in the stimulation of PTH release by high extracellular potassium (K⁺) concentrations. Addition of 55 mM KCl to cells incubated with 115 mM NaCl and 5 mM KCl lowered cytosolic Ca⁺⁺ at either low (0.5 mM) extracellular Ca⁺⁺ (from 194±14 to 159±9 nM, p<.01, N=6) or high (1.5 mM) extracellular calcium (from 465±38 to 293±20 nM, p<.01, N=10). This reduction in cytosolic Ca⁺⁺ was due to high K⁺$\text{per}\text{se}$ and not to changes in tonicity since addition of 55 mM NaCl was without effect while a similar decrease in cytosolic Ca⁺⁺ occurred when cells were resuspended in 60 mM NaCl and 60 mM KCl. PTH release was significantly (p<.01) greater at 0.5 and 1.5 mM Ca⁺⁺ in QUIN-2-loaded cells incubated with 60 mM NaCl and 60 mM KCl than in those exposed to 115 mM NaCl and 5 mM KCl. In contrast to most secretory cells, therefore, stimulation of PTH release by high K⁺ is associated with a decrease rather than an increase in cytosolic Ca⁺⁺.     

Evidence for a plasma membrane calcium pump in bovine adrenal medulla but not adrenal cortex

Continuous sucrose density gradient subfractions from bovine adrenal medullary microsomes were found to accumulate ⁴⁵Ca²⁺ in the presence of ATP and ammonium oxalate mainly in subfractions of intermediate density. (Na⁺ + K⁺)-ATPase (plasma membrane marker) and Ca²⁺-ATPase activities were also concentrated in these intermediate subfractions but thiamine pyrophosphatase (Golgi apparatus marker) was not. NADH oxidase (endoplasmic reticulum marker) activity was distributed throughout all subfractions.⁴⁵Ca²⁺ accumulation in adrenal cortical microsomes was found to rise and fall in parallel with thiamine pyrophosphatase but not with (Na⁺ + K⁺)-ATPase or NADH oxidase activities.Accumulation of ⁴⁵Ca²⁺ in membrane vesicles in these experiments suggests the existence of a calcium transfer mechanism in plasma membranes of the adrenal medulla but not adrenal cortex.     

Activation of deoxycholate solubilized adenosine triphosphatase by ganglioside and asialoganglioside preparations.

ATPase was prepared from brain microsomes by solubilization with sodium deoxycholate and fractionated at different concentrations of ammonium sulfate. The Mg²⁺-ATPase was activated by total brain gangliosides, disialoganglioside, monosialo — ganglioside, hematoside, total brain gangliosides obtained from a patient with Tay-Sachs disease and asialoganglioside. The effect was smaller on Ca²⁺-ATPase and negligible on (Na⁺ + K⁺)-ATPase. Lactosyl-ceramide, glucosyl-ceramide, galactosyl-ceramide, ceramide and sialyl-lactose failed to produce activation.     

Ca-Mg-ATPase activity in brain coated microvesicles purified on immunosorbents

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca²⁺+Mg²⁺ dependent ATPase activities (about 9 μmol/h per mg protein). The Na⁺+K⁺+Mg²⁺ dependent ATPase activity was 3.2 μmol/h per mg (±1.0, S.D., n=3) when microvesicles were prepared according to (1) and 1.5 μmol/h per mg (±1.0, S.D., n=3) when prepared according to (2).Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca²⁺ transport in mitochondria and erythrocyte membranes had no effect on Ca²⁺+Mg²⁺ dependent ATPase from any of the preparations.As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy.The Ca²⁺+Mg²⁺ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na⁺+K⁺+Mg²⁺ ATPase was decreased.Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na⁺+K⁺+Mg²⁺ dependent ATPase activity in the eluted coated microvescile fraction.It was concluded that Ca²⁺+Mg²⁺ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.     

Endogenous protease mediated manifestation of a Ca2+-stimulated ATPase in purified dog gastric microsomes

An endogenous soluble protease has been demonstrated to unmask a Ca²⁺-stimulated ATPase activity in purified dog gastric microsomes. The presence of ATP during protease treatment appears essential for the manifestation of the gastric Ca²⁺-stimulated ATPase activity. The endogenous protease appears to have trypsin-like activity, since soybean trypsin inhibitor completely blocks the protease effect. Manifestation of the Ca²⁺-stimulated ATPase occurs without affecting the microsomal (H⁺ +K⁺)-ATPase activity and associated H⁺ uptake ability. The unmasked Ca²⁺-stimulated ATPase appears insensitive to calmodulin. Possible roles of the enzyme in the regulation of gastric H⁺ transport have been discussed.     

Role of MMP-2 in inhibiting Na+ dependent Ca2+ uptake by H2O2 in microsomes isolated from pulmonary smooth muscle

Treatment of microsomes (preferentially enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with H₂O₂ (1 mM) markedly stimulated matrix metalloproteinase activity and also inhibited Na⁺ dependent Ca²⁺ uptake. Electron micrograph revealed that H₂O₂ (1 mM) does not cause any damage to the microsomes. MMP-2 and TIMP-2 were determined to be the ambient protease and corresponding antiprotease of the microsomes. Pretreatment with vitamin E (1 mM) and TIMP-2 (50 g/ml) reversed the effect produced by H₂O₂ (1 mM) on Na⁺ dependent Ca²⁺ uptake in the microsomes. However, H₂O₂ (1 mM) caused changes in MMP-2 activity and Na⁺ dependent Ca²⁺ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 g/ml of TIMP-2 which otherwise reversed MMP-2 (1 g/ml) mediated increase in ¹⁴C-gelatin degradation and inhibition of Na⁺ dependent Ca²⁺ uptake. Combined treatment of the microsomes with a low dose of MMP-2 (0.5 g/ml) and H₂O₂ (0.5 mM) inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes compared to the respective low dose of either of them. Direct treatment of TIMP-2 (5 g/ml) with H₂O₂ (1 mM) abolished the inhibitory effect of the inhibitor on ¹⁴C-gelatinolytic activity elicited by 1 g/ml of MMP-2. Thus, one of the mechanisms by which H₂O₂ activates MMP-2 could be due to inactivation of TIMP-2 by the oxidant. The resulting activation of MMP-2 subsequently inhibits Na⁺ dependent Ca²⁺ uptake in the microsomes. (Mol Cell Biochem 270: 79–87, 2005)     

Trypsinization unmasks a Ca+2- stimulated ATPase activity from purified pig gastric microsomes

Trypsinization of gastric microsomal K⁺- stimulated ATPase in absence of ATP nearly abolished the K⁺- stimulated component of the enzyme activity without any significant effect on the basal (with Mg⁺² alone) activity. The K⁺- stimulated component, however, was completely restored by the ‘activator protein” partially purified form the soluble supernatant fraction of the pig gastric cells. On the other hand, trypsinization of the microsomes in presence of ATP significantly increased (2–3 fold) the basal rate with virtual elimination of the K⁺- stimulated component. Assay of the trypsinized microsomes in presence of the activator protein not only demonstrated complete restoration of the K⁺- stimulated ATPase but also revealed an additional activity which has been characterized as a Ca⁺²- stimulated ATPase.Tryptic digestion has recently been used as a tool to understand the mechanism of action of various transport enzymes such as Na⁺, K⁺- ATPase (1), Ca⁺²- ATPase (2,3) and gastric H⁺, K⁺- ATPase (4). Controlled tryptic digestion of purified enzymes under various conditions of ligand binding may provide us with many valuable informations regarding the molecular architecture of the enzyme protein. However, when dealing with a membrane system containing a host of many different intrinsic and extrinsic proteins one must be cautious about the interpretation of the trypsin effects. In the present paper we report the effects of trypsin digestion of the purified pig gastric microsomes on the microsomal K⁺- stimulated ATPase activity. Our studies demonstrated that digestion of the microsomes with trypsin in absence of ATP inactivated the K⁺- stimulated ATPase but the activity could be fully restored by the addition of partially purified activator protein (5). Microsomes treated with trypsin in presence of ATP responded to the activator protein to the same extent as that without ATP but in addition demonstrated the manifestation of another enzymatic activity which has been characterized as a Ca⁺²- stimulated ATPase. This is a preliminary report dealing primarily with the unmasking of a new ATPase after trypsin treatment. Detailed reports on the characterization and mechanism of action of the gastric Ca⁺²- stimulated ATPase will be published elsewhere.     

Role of MMP-2 in PKCδ-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: Involvement of a pertussis toxin sensitive protein

Treatment of bovine pulmonary artery smooth muscle with the O₂^•− generating system hypoxanthine plus xanthine oxidase stimulated MMP-2 activity and PKC activity; and inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment of the smooth muscle with SOD (the O₂^•− scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 activity and PKC activity, and reversed the inhibition of Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment with calphostin C (a general PKC inhibitor) and rottlerin (a PKCδ inhibitor) prevented the increase in PKC activity and reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without causing any change in MMP-2 activity in the microsomes of the smooth muscle. Treatment of the smooth muscle with the O₂^•− generating system revealed, respectively, 36 kDa RACK-1 and 78 kDa PKCδ immunoreactive protein profile along with an additional 38 kDa immunoreactive fragment in the microsomes. The 38 kDa band appeared to be the proteolytic fragment of the 78 kDa PKCδ since pretreatment with TIMP-2 abolished the increase in the 38 kDa immunoreactive fragment. Co-immunoprecipitation of PKCδ and RACK-1 demonstrated O₂^•− dependent increase in PKCδ-RACK-1 interaction in the microsomes. Immunoblot assay elicited an immunoreactive band of 41 kDa G_iα in the microsomes. Treatment of the smooth muscle tissue with the O₂^•− generating system causes phosphorylation of G_iα in the microsomes and pretreatment with TIMP-2 and rottlerin prevented the phosphorylation. Pretreatment of the smooth muscle tissue with pertussis toxin reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without affecting the protease activity and PKC activity in the microsomes. We suggest the existence of a pertussis toxin sensitive G protein mediated mechanism for inhibition of Na⁺ dependent Ca²⁺ uptake in microsomes of bovine pulmonary artery smooth muscle under O₂^•− triggered condition, which is regulated by PKCδ dependent phosphorylation and sensitive to TIMP-2 for its inhibition. (Mol Cell Biochem xxx: 107–117, 2005)     

Biochemical heterogeneity of skeletal-muscle microsomal membranes. Membrane origin, membrane specificity and fibre types

1. Microsomes were isolated from rabbit fast-twitch and slow-twitch muscle and were separated into heavy and light fractions by centrifugation in a linear (0.3–2m) sucrose density gradient. The membrane origin of microsomal vesicles was investigated by studying biochemical markers of the sarcoplasmic-reticulum membranes and of surface and T-tubular membranes, as well as their freeze-fracture properties. 2. Polyacrylamide-gel electrophoresis showed differences in the Ca²⁺-dependent ATPase/calsequestrin ratio between heavy and light fractions, which were apparently consistent with their respective origin from cisternal and longitudinal sarcoplasmic reticulum, as well as unrelated differences, such as peptides specific to slow-muscle microsomes (mol.wts. 76000, 60000, 56000 and 45000). 3. Freeze-fracture electron microscopy of muscle microsomes demonstrated that vesicles truly derived from the sarcoplasmic reticulum, with an average density of 9nm particles on the concave face of about 3000/μm² for both fast and slow muscle, were admixed with vesicles with particle densities below 1000/μm². 4. As determined in the light fractions, the sarcoplasmic-reticulum vesicles accounted for 84% and 57% of the total number of microsomal vesicles, for fast and slow muscle respectively. These values agreed closely with the percentage values of Ca²⁺-dependent ATPase protein obtained by gel densitometry. 5. The T-tubular origin of vesicles with a smooth concave fracture face in slow-muscle microsomes is supported by their relative high content in total phospholipid and cholesterol, compared with the microsomes of fast muscle, and by other correlative data, such as the presence of (Na⁺+K⁺)-dependent ATPase activity and of low amounts of Na⁺-dependent membrane phosphorylation. 6. Among intrinsic sarcoplasmic-reticulum membrane proteins, a proteolipid of mol.wt. 12000 is shown to be identical in the microsomes of both fast and slow muscle and the Ca²⁺-dependent ATPase to be antigenically and catalytically different, though electrophoretically homogeneous. 7. Basal Mg²⁺-activated ATPase activity was found to be high in light microsomes from slow muscle, but its identification with an enzyme different from the Ca²⁺-dependent ATPase is still not conclusive. 8. Enzyme proteins that are suggested to be specific to slow-muscle longitudinal sarcoplasmic reticulum are the flavoprotėin NADH:cytochrome b₅ reductase (mol.wt. 32000), cytochrome b₅ (mol.wt. 17000) and the stearoyl-CoA desaturase, though essentially by criteria of plausibility.     

Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism

The parathyroid hormone (PTH) release and cytosolic Ca²⁺ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca²⁺ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca²⁺ activity. Both the hormone release and the cytosolic Ca²⁺ activity were lower in cells from human adenomas and hyperplastic glands~ and in comparison with the bovine preparations these ceils had higher set points for the cytosolic Ca²⁺ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca²⁺ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca²⁺ activity.     

Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

Inositol 1,4,5-trisphosphate (IP₃) evokes release of Ca²⁺ from the endoplasmic reticulum (ER), but the resulting Ca²⁺ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A₁, which prevents lysosomal Ca²⁺ uptake by inhibiting H⁺ pumping into lysosomes, increased the amplitude of the initial Ca²⁺ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca²⁺ release from distinct IP₃-sensitive Ca²⁺ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A₁ similarly exaggerated the Ca²⁺ signals evoked by carbachol or carbachol with PTH, indicating that Ca²⁺ released from distinct IP₃-sensitive Ca²⁺ stores is sequestered by lysosomes. The Ca²⁺ signals resulting from store-operated Ca²⁺ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A₁. Using Gd³⁺ (1 mM) to inhibit both Ca²⁺ entry and Ca²⁺ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca²⁺ recycling to the ER after IP₃-evoked Ca²⁺ release. Blocking lysosomal Ca²⁺ uptake with bafilomycin A₁ increased the amplitude of each carbachol-evoked Ca²⁺ signal without affecting the rate of Ca²⁺ recycling to the ER. This suggests that Ca²⁺ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca²⁺ released by IP₃ receptors residing within distinct Ca²⁺ stores, but not the Ca²⁺ entering cells via receptor-regulated, store-operated Ca²⁺ entry pathways.     

Gel electrophoretic and density gradient analysis of the (K + Ca)-ATPase and the (Na + K)-ATPase activities of cardiac membrane vesicles

The two major ATPase activities of intact and leaky cardiac membrane vesicles (microsomes) were characterized with respect to ionic activation requirements. The predominant ATPase activity of intact vesicles was (K⁺ + Ca²⁺)-ATPase, an enzymic activity localized to sarcoplasmic reticulum, whereas the predominant ATPase activity of leaky, sodium dodecyl sulfate-pretreated vesicles was (Na⁺ + K⁺)-ATPase, an enzymic activity localized to sarcolemma. The (K⁺ + Ca²⁺)-ATPase activity was stimulated 4- to 5-fold by 100 mM K⁺ in the presence of 50 μM Ca²⁺. Phosphorylation of the (K⁺ + Ca²⁺)-ATPase of intact vesicles with [γ-³²P]ATP was Ca²⁺ dependent, and monovalent cations including K⁺ increased the level of [³²P]phosphoprotein by up to 50% when phosphorylation was measured at 5°C. After the intact vesicles were treated with SDS (0.30 mg/ml), (K⁺ + Ca²⁺)-ATPase was inactivated, as was Ca²⁺-dependent ³²P incorporation. The monovalent cation-stimulated ATPase activity of the particulate residue (SDS-extracted membrane vesicles) displayed the usual characteristics of ouabain-sensitive (Na⁺ + K⁺)-ATPase and the activity was increased 9- to 14-fold over the small amount of patent (Na⁺ + K⁺)-ATPase activity of intact membrane vesicles. ³²P incorporation by the (Na⁺ + K⁺)-ATPase of SDS-extracted vesicles was Na⁺ dependent, and Na⁺-stimulated incorporation was increased 7- to 9-fold over that of intact vesicles.Slab gel polyacrylamide electrophoresis of both intact and SDS-extracted crude vesicle preparations revealed at least 40 distinct Coomassie Blue-positive protein bands and provided evidence for a possible heterogeneous membrane origin of the vesicles. Periodic acid-Schiff staining of the gels revealed at least two major glycoproteins. Simultaneous electrophoresis of the ³²P-intermediates of the (K⁺ + Ca²⁺)-ATPase and the (Na⁺ + K⁺)-ATPase in the same gels did not resolve the two enzymes clearly. With sucrose gradient centrifugation of intact membrane vesicles, it was possible to physically resolve the two ATPase activities. Latent (Na⁺ + K⁺)-ATPase activity (unmasked by exposing the various fractions to SDS) was found in the higher regions of the gradient, whereas (K⁺ + Ca²⁺)-ATPase activity was primarily in the denser regions. A reasonable interpretation of the data is that cardiac microsomes consist of membrane vesicles derived both from sarcolemma and sarcoplasmic reticulum. (Na⁺ + K⁺)-ATPase is localized to intact vesicles of sarcolemma but is mainly latent, whereas (K⁺ + Ca²⁺)-ATPase is mostly patent and is localized to vesicles of sarcoplasmic reticulum.     

Mapping of a carboxyl-terminal active site of parathyroid hormone by calciummimaging

We recently showed that the C-terminal fragment PTH (52–84) effectively increases intracellular free calcium ([Ca²⁺]_i in a subset of growth plate chondrocytes not activated by the N-terminal PTH fragment (1–34). Here we characterize the active site on C-terminal PTH (52–84) with respect to calcium (Ca²⁺)-signaling and the mechanism involved by using synthetic PTH-subfragments in digital CCD ratio-imaging experiments. Our results show amino acids 73–76 to be the core region for increasing [Ca²⁺]_i. Ryanodine (1 μM), caffeine (10 mM), lithium (2 mM), or cyclopiazonic acid (2–5 μMI), agents that interfere with intracellular Ca²⁺ release, all failed to block PTH (52–84) induced [Ca²⁺]_i increases. Depletion of extracellular calcium ([Ca²⁺]_o) blocked PTH (52–84) induced [Ca²⁺]; increases, indicating a transmembrane Ca²⁺ influx. In contrast to voltage-gated and Ca²⁺ release activated Ca²⁺ influx, PTH (52–84) evoked Ca²⁺ influx was not blocked by nickel (1 mM). We conclude that PTH amino acids 73–76 are essential for activation of a nickel-insensitive Ca²⁺ influx pathway in growth plate chondrocytes that is likely to be of relevance for matrix calcification, a key step in endochondral bone formation.     

Interaction of Ca 2+ with endoplasmic reticulum of rat liver: a standardized procedure for the isolation of rat liver microsomes

A standardized method has been developed for the rapid isolation of rat liver microsomes using Ca²⁺ and its advantages over other available methods have been outlined. In addition to hydrolytic enzymes and chemical composition, the important enzymes in the electron transport system were determined in Ca²⁺ microsomes and normal 105,000g microsomes and indicate only minor differences between the two preparations. Two classes of microsomes—smooth and rough particles prepared with or without the addition of Ca²⁺—were compared for their chemical and biochemical properties and indicated little differences within each microsomal fraction. The ability of other divalent cations like Mg²⁺, Fe²⁺, Ba²⁺, Zn²⁺, and Hg²⁺ to aggregate the microsomes was observed while the monovalent and trivalent cations tested did not appreciably sediment the microsomes under the present experimental conditions.     

Key role of a PtdIns-4,5P2 micro domain in ionic regulation of the mammalian heart Na/Ca exchanger

Phosphatidylinositol biphosphate (PtdIns-4,5P₂) plays a key role in the regulation of the mammalian heart Na⁺/Ca²⁺ exchanger (NCX1) by protecting the intracellular Ca²⁺ regulatory site against H⁺_i and (H⁺_i + Na⁺_i) synergic inhibition. MgATP and MgATP-γ-S up-regulation of NCX1 takes place via the production of this phosphoinositide. In microsomes containing PtdIns-4,5P₂ incubated in the absence of MgATP and at normal [Na⁺]_i, alkalinization increases the affinity for Ca²⁺_i to the values seen in the presence of the nucleotide at normal pH; under this condition, addition of MgATP does not increase the affinity for Ca²⁺_i any further. On the other hand, prevention of Na⁺_i inhibition by alkalinization in the absence of MgATP does not take place when the microsomes are depleted of PtdIns-4,5P₂. Experiments on NCX1–PtdIns-4,5P₂ cross-coimmunoprecipitation show that the relevant PtdIns-4,5P₂ is not the overall membrane component but specifically that tightly attached to NCX1. Consequently, the highest affinity of the Ca²⁺_i regulatory site is seen in the deprotonated and PtdIns-4,5P₂-bound NCX1. Confirming these results, a PtdIns-5-kinase also cross-coimmunoprecipitates with NCX1 without losing its functional competence. These observations indicate, for the first time, the existence of a PtdIns-5-kinase in the NCX1 microdomain.     

Demarcation of Ca2+ transport processes in guinea pig stomach smooth muscle

Summary Microsomal fractions were isolated from gastric antrum and fundus smooth muscle of guinea pigs. Ca²⁺ uptake into and Ca²⁺ release from the membrane vesicles were studied by a rapid filtration method, and Ca²⁺ transport properties of the different regions of the stomach were compared. ATP-dependent Ca²⁺ uptake was similar in microsomes isolated from both regions. This uptake was increased by oxalate and was not affected by NaN₃. Oxalate affected Ca²⁺ permeability of both antrum and fundus microsome vesicles similarly. Fundus microsome vesicles preincubated in 100mm NaCl and then diluted to 1/20 concentration with Na⁺-free medium had significantly higher ATP-independent Ca²⁺ uptake than vesicles preincubated in 100mm KCl and treated the same way. This was not true for antrum vesicles. Monensin abolished Na⁺-dependent Ca²⁺ uptake, and NaCl enhanced Ca²⁺ efflux from fundus microsome vesicles. The halflife values of Ca²⁺ loss from fundus vesicles in the presence of NaCl were significantly smaller than those in the presence of KCl. The release of Ca²⁺ from the vesicles within the first 3 min was accelerated by NaCl to three times that by KCl. However, NaCl had ro effect on Ca²⁺ release from antrum microsome vesicles.Results suggest two distinct mechanisms of stomach membrane Ca²⁺ transport: (1) ATP-dependent Ca²⁺ uptake and (2) Na⁺–Ca²⁺ exchange; the latter in the fundus only.     

Defective calcium pump in the sarcoplasmic reticulum of the hypertrophied rabbit heart.

To evaluate Ca²⁺-uptake in sarcoplasmic reticulum in the hypertrophied rabbit heart, microsomes were prepared from myocardium of rabbits with experimentally induced aortic stenosis. A significant reduction of microsomal Ca²⁺-uptake was observed in hypertrophied left ventricle, 195±10 compared to 280±18 nmol/mg found in control animals. A similar pattern was observed for the Ca²⁺-stimulated ATPase (30±9 and 59±10 nmol/min/mg resp.). A minimal activity difference of the microsomal marker enzyme rotenone-insensitive NADPH cyt. $\text{c}$ reductase was found (7.77±0.05 and 8.17±0.11 nmol/min/mg resp.). The specific activity of the latter enzyme was 5–6 fold increased in microsomes compared to homogenates in both animal groups, which excludes the possibility of increased amounts of contaminant or nonfunctional protein in sarcoplasmic reticulum prepared from hypertrophied myocardium. In addition the yield of microsomal protein did not differ significantly. Maximal phosphorylation by exogenous cyclic AMP and protein kinase increased Ca²⁺-uptake in both microsomal preparations (to 287±27 and 375±26 nmol/mg resp. for hypertrophied and control hearts), but Ca²⁺-transport rate found in pathological hearts remained lower than in controls. These findings indicate that impairment of Ca²⁺-metabolism in the hypertrophied heart is based on a defective Ca²⁺-pump.     

Effects of calcium on potassium and water transport in human erythrocyte ghosts

Bulk water transport in reconstituted ghosts is statistically comparable to that in the parent red cells, and is unaffected by incorporation of Ca²⁺ over the range of 0.01 to 1 mM. Brief exposure of ghosts to p-chloromercuribenzene sulfonate results in a supression of osmotic water flow but leaves K⁺ permeability unchanged. Incorporation of p-chloromercuribenzene sulfonate provokes extremely rapid K⁺ loss which can be counteracted by simultaneous inclusion of Ca²⁺.Erythrocyte ghosts, when prepared with a small amount of Ca²⁺, demonstrate recovery of normal impermeability to choline, sucrose, Na⁺ and inulin and have an improved K⁺ retention over Ca²⁺-free preparations.The rate of passive transport of K⁺ from unwashed erythrocyte ghosts was measured during the initial few minutes of efflux. The initial rates vary in a bimodal fashion with the concentration of Ca²⁺ incorporated at the time of hemolysis. In low concentrations (0.01–0.1 mM), Ca²⁺ protects the K⁺ barrier while at higher concentrations (0.1–1.0 mM) it provokes a K⁺ leakage ranging from 7 to 50 times the normal rate of passive K⁺ loss. The Ca²⁺-induced K⁺ leak is thus a graded response rather than a discrete membrane transport state. The transition from a Ca²⁺-protected to a Ca²⁺-damaged membrane occurs upon an increase in Ca²⁺ concentration of less than 50 μmoles/l.     

Membranes of mammary gland : X. Adenosine triphosphate dependent calcium accumulation by golgi apparatus rich fractions from bovine mammary gland

Golgi apparatus rich fractions from lactating bovine mammary gland had an Mg²⁺-dependent, Ca²⁺-stimulated adenosine triphosphatase. These Golgi apparatus fractions also accumulated Ca²⁺ in vitro. Accumulation of Ca²⁺ required ATP and could be abolished by treatment either with low concentrations of deoxycholate followed by ultrasound, or by heating at 100 °C for 10 min. The adenosine triphosphatase activity of Golgi apparatus was strongly stimulated by low concentrations of Ca²⁺ and moderately stimulated by high concentrations of K⁺. This activity was unaffected by Na⁺ and was not inhibited by ouabain. The pH optimum for the Mg²⁺-dependent hydrolysis of ATP was 7.5, the K_m was 5 × 10⁻⁵ M and the activation energy was 6 000 calories/mole. This Mg²⁺-dependent adenosine triphosphatase activity was also found in rough endoplasmic reticulum, smooth microsomes and milk fat globule membrane, the latter membrane being derived directly from the apical plasma membrane. All of these membrane fractions had the ability to specifically accumulate Ca²⁺. Specific accumulation was highest with smooth microsomes and lowest with milk fat globule membrane with Golgi apparatus and rough endoplasmic reticulum being intermediate. These observations provide one plausible explanation for intracellular Ca²⁺ accumulation and secretion into milk. Further, these results help explain the ultrastructural observations of casein micelle formation in secretory vesicles elaborated by Golgi apparatus.