Ca2+-ATPase cytochemistry in the spinal cord microvasculature of prenatal rats

Summary Membrane-bound Ca²⁺-ATPase activity was localized cytochemically in the blood vessels of the spinal cord of rat embryos to obtain a better understanding of the membrane activities of vascular cells.The cytochemical method revealed a growth of the parenchymal vasculature. In the parenchyma, reaction product was dense over the entire plasma membrane of voluminous endothelial cells provided with large nuclei and enriched cytoplasmic organelles, suggesting that the endothelial cells may be of a vascular sprout. The parenchymal vessels with a wide lumen were frequently associated with pericytes, and the Ca²⁺-ATPase activity was diminished in intensity on the luminal surface of the flattened endothelial cells. On the other hand, the endothelium of extraparenchymal capillaries exhibited Ca²⁺-ATPase activity primarily on the luminal surface of the plasma membrane. Quercetin, a Ca²⁺-transporting ATPase inhibitor, considerably decreased the abluminal activity in the voluminous endothelial cells with slit-like vascular lumen and the luminal activity of functioning capillary endothelium as well. Thus, a dual activity of Ca²⁺-ATPase, postulating for the activities of Ca²⁺-transporting ATPase and ecto-ATPase, was closely correlated with the maturation processes of the capillary endothelium.     

Luminal localization of blood-brain barrier sodium, potassium adenosine triphosphatase is dependent on fixation.

Cytochemical data in the literature reporting localization of sodium, potassium adenosine triphosphatase (Na(+), K(+)-ATPase) in the blood-brain barrier (BBB) have been contradictory. Whereas some studies showed the enzyme to be located exclusively on the abluminal endothelial plasma membrane, others demonstrated it on both the luminal and abluminal membranes. The influence of fixation on localization of the enzyme was not considered a critical factor, but our preliminary studies showed data to the contrary. We therefore quantitatively investigated the effect of commonly used fixatives on the localization pattern of the enzyme in adult rat cerebral microvessels. Fixation with 1%, 2%, and 4% formaldehyde allowed deposition of reaction product on both the luminal and abluminal plasma membranes. The luminal reaction was reduced with increasing concentration of formaldehyde. Glutaraldehyde at 0.1%, 0.25%, 0.5%, in combination with 2% formaldehyde, drastically inhibited the luminal reaction. The abluminal reaction was not significantly altered in all groups. These results show that luminal localization of BBB Na(+), K(+)-ATPase is strongly dependent on fixation. The lack of luminal localization, as reported in the literature, may have been the result of fixation. The currently accepted abluminal polarity of the enzyme should be viewed with caution.     

Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium

Cardiac plasma membrane Ca²⁺/Mg²⁺ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca²⁺ or Mg²⁺ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca²⁺/Mg²⁺ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca²⁺/Mg²⁺ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or sarcolemmal Ca²⁺-pump ATPase. On the other hand, the cardiac Ca²⁺/Mg²⁺ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or plasma membrane Ca²⁺-pump ATPase. Furthermore, the immune serum inhibited the Ca²⁺/Mg²⁺ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca²⁺/Mg²⁺ ecto-ATPase antibodies indicated the localization of Ca²⁺/Mg²⁺ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca²⁺/Mg²⁺ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca²⁺/Mg²⁺ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.     

Adenosine triphosphate-lead histochemical reactions in ependymal epithelia of murine brains do not represent calcium transport adenosine triphosphatase

Summary The strong enzyme histochemical reactions for adenosine triphosphatase (ATPase) seen in ependymal tanycytes after incubation in calcium-containing media have previously been reported as calcium transport ATPase. Investigation of these reactions showed that: (1) any nucleoside triphosphate can serve as a substrate; (2) diphosphates and monophosphates cannot replace triphosphates; this includes p-nitrophenyl phosphate which is readily hydrolysed by plasma membrane transport ATPases; (3) strong localization occurs in the presence of millimolar concentrations of either calcium or magnesium ions; there is no absolute requirement for calcium ions; (4) they are not inhibited by sulphydryl inhibitors or calmodulin antagonists; (5) lead phosphate precipitates are localized almost entirely on the external face of tanycyte plasma membranes. In addition, the technique gives strong localization to vessels in the choroid plexus but not to the choroidal epithelium. Immunohistochemistry with a primary antibody raised against Ca²⁺,Mg²⁺-ATPase stains the choroidal epithelium but not the vessels or the ependymal tanycytes. These results are inconsistent with identification of the reaction as calcium transport ATPase but support characterization as an ecto-ATPase.     

Parathyroid cell polarity as revealed by cytochemical localization of ATPases,alkaline phosphatase and 5′-nucleotidase

Summary Activities of Ca²⁺-dependent ATPase, Mg²⁺-dependent ATPase, Na⁺-K⁺-dependent ATP-ase, alkaline phosphatase, and 5-nucleotidase were demonstrated after incubation of 40-m vibratome sections of bovine parathyroids and subsequent visualization by electron microscopy. Prior to sectioning, parathyroid tissue was fixed with 1% glutaraldehyde for localization of alkaline phosphatase, and with 2% formaldehyde and 1% glutaraldehyde for demonstration activities of ATPases and 5-nucleotidase. The activities of the five enzymes were found at the apicolateral domain of the plasma membrane in parathyroid cells, i.e. at the site parathyroid cells face neighbouring parenchymal cells. Ca²⁺-ATPase activity was also seen on mitochondria, Golgi complex and RER. The presence of these plasma membrane associated enzymes at the apicolateral domain only indicate polarity in parathyroid cells. It further suggests that many processes including transmembrane transport take place at the apicolateral domain, the site of parathyroid cells opposing blood capillaries.     

Parathyroid cell variants may be provoked during immersion fixation

Summary Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO₄ in the same buffers or, alternatively, in s-collidine.Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5–2% formaldehyde and 2.5–5% acrolein in 0.1 M Na-cacodylate with or without Ca²⁺ and Mg²⁺, Na-phosphate or Na/K-phosphate at 4°C followed by postfixation with 1% OsO₄ in the same buffers or in s-collidine containing sucrose, Ca²⁺ and Mg²⁺. This procedure largely abolished the occurence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

骨骼肌内质网Ca2+泵转运Ca2+的结构基础

Ca²⁺泵(Ca²⁺-ATPase)是调节细胞内Ca²⁺浓度的重要蛋白质之一. Ca²⁺泵在转运Ca²⁺的过程中经历一系列构象变化. 其中,E1状态为外向的Ca²⁺高亲和状态,E2状态则为内向的Ca²⁺低亲和状态. 目前,骨骼肌内质网Ca²⁺泵转运Ca²⁺过程中的几个中间状态,包括E1-2Ca²⁺,E1-ATP,E1-P-ADP,E2-Pi和E2状态的三维晶体结构已经解析. 介绍这几种状态的晶体结构,并分析Ca²⁺泵在执行功能过程中结构与功能的关系.     

Fibre typing using sarcoplasmic reticulum Ca2+-ATPase and myoglobin immunohistochemistry in rat gastrocnemius muscle

Summary Skeletal muscle fibre types were identified by using immunohistochemical detection of sarcoplasmic reticulum Ca²⁺-ATPase and myolobin content in rat gastrocnemius muscle. The strong Ca²⁺-ATPase-reactive fibres were identical with the fast-twitch population, while the fibres with weak reactivity represented the slow-twitch type. Strong myoglobin immunoreactivity reflected the fast oxidative glycolytic (FOG) and slow oxidative (SO) types. Slight to moderate myoglobin immunostaining was found in the fast glycolytie (FG) fibres. The staining intensity of the different fibre types differed as follows: for Ca²⁺-ATPase FG>FOG>SO, and for myoglobin FOG>SO>FG.The immunoreactivity of Ca²⁺-ATPase and myoglobin were well preserved after fixation of the muscles in Bouin's solution, or in formol/acetic acid fixative, and paraffin embedding. Detection of the primary antibodies was carried out by using the avidin-biotin-peroxidase complex, and the immunogold-silver-staining methods. The latter was found to be more sensitive and suitable for postembedding ultrastructural demonstration of the Ca²⁺-pump enzyme on Durcupan-embedded muscles. The method, using 5 nm immunogold conjugate with silver enhancement, offered the advantages of high sensitivity and excellent visualization of the reaction product.The postembedding detection of sarcoplasmic reticulum Ca²⁺-ATPase also proved to be useful in the restrospective identification of the main fibre classes in human muscle biopses.     

Biophysical characterization of rat cardiac Ca2+/Mg2+ ecto-ATPase (Myoglein)

Sarcolemmal Ca²⁺/Mg²⁺ ecto-ATPase (Myoglein; MW 180 kD) is a membrane bound enzyme which requires a millimolar concentration of either Ca²⁺ or Mg²⁺ for maximal hydrolysis of ATP. The isoelectric point (pI) of the cardiac ecto-ATPase was 5.7. The purified Ca²⁺/Mg²⁺ ecto-ATPase from the rat heart sarcolemmal appeared as a single band with MW 90 kD in the SDS-PAGE. In order to understand the nature of this enzyme, the 90 kD band in the SDS-PAGE was electroeluted; the analysis of the eluate showed 2 prominent bands with MW 90 and 85 kD. The presence of 2 bands was further confirmed by gradient gel (10-20%) electrophoresis in 0.375 M Tris-HCl buffer, pH 8.8. Analysis of the purified Ca²⁺/Mg²⁺ ecto-ATPase as well as the electroeluted protein in a non-equilibrium linear two dimensional electrophoresis (Ampholyte pI 3.0-10.0) also showed two distinct bands. Mass spectroscopic analysis of the enzyme using different matrix combinations revealed the presence of multi-components indicating microheterogeneity in the protein structure. Treatment of the ecto-ATPase with DL-dithiothreitol did not alter the pattern of mass spectroscopic analysis and this indicated that the microheterogeneity may be due to some posttranslational modifications. It is concluded that rat cardiac Ca²⁺/Mg²⁺ ecto-ATPase is an acidic protein having two subunits. Furthermore, the enzyme shows microheterogeneity in its molecular structure.     

Renaissance of cytochemical localization of membrane ATPases in the myocardium

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca²⁺-pump ATPase, Ca²⁺/Mg²⁺-ecto ATPase, Na⁺,K⁺-ATPase and Mg²⁺-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO₂)₃, 5 mM MgSO₄, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca²⁺-pump ATPase, Na⁺–K⁺ ATPase and Ca²⁺/Mg²⁺-ecto ATPase in the cardiac membrane.     

Comparison of the effects of the membraneassociated Ca2+/calmodulin-dependent protein kinase on Ca2+-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca²⁺-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca²⁺-ATPase. Studies comparing, the effects of PKA and CaM kinase on cardiac Ca²⁺-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca²⁺-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca²⁺-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 M) directly to the Ca²⁺ transport assay medium results in minimal ( 112–130% of control) stimulation of Ca²⁺ uptake activity when the Ca²⁺ uptake reaction is initiated by the addition of either ATP or Ca²⁺/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca²⁺ transport assay medium results in stimulation of Ca²⁺ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca²⁺ uptake markedly (215% of control) when the Ca²⁺ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca²⁺/EGTA but minimally (130% of control) when the Ca²⁺ uptake reaction is initiated by the addition of ATP. These findings imply that (a) phospholamban is coupled to the Ca²⁺-ATPase in slow-twitch skeletal muscle SR (as in cardiac SR), and (b) the amount of Ca²⁺ uptake stimulation seen upon the addition of calmodulin or PKA depends strongly on the assay conditions employed. Our observations help to explain the wide range of effects of calmodulin or PKA addition reported in previous studies. It should be noted that, since CaM kinase is now known to phosphorylate the Ca²⁺-ATPase in addition to phospholamban, further studies are required to determine the relative contributions of phospholambanversus Ca²⁺-ATPase phosphorylation in the stimulation of Ca²⁺-ATPase function by CaM kinase. Also, earlier studies attributing all of the effects of CaM kinase stimulation of Ca²⁺ uptake and Ca²⁺-ATPase activity to phospholamban phosphorylation need to be re-examined.     

Evidence that both Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase activities in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme molecule

• 1.1. Evidence was obtained that activities of both low-affinity Ca²⁺-ATPase and high-affinity (Ca²⁺ + Mg²⁺)-ATPase in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme.
• 2.2. Two solubilized ATPases were purified by four steps of HPLC; and both activities eluted at the same fractions from each column, and the specific activity ratio of the two enzymes at each step was constant.
• 3.3. By non-denaturing PAGE, the final preparation gave a single band for both protein staining and activity staining for the two ATPases; and the Ca²⁺-ATPase activity comigrated with that of (Ca²⁺ + Mg²⁺)-ATPase.
• 4.4. In SDS-PAGE, each activity staining for the ATPases also gave a single band, and both activities comigrated.
• 5.5. These findings suggest that Ca²⁺-ATPase and (Ca²⁺ + Mg²⁺)-ATPase are a single enzyme.

     

Phosphorylation and dephosphorylation of Mg2+-independent Ca2+-ATPase from goat spermatozoa

We reported previously that a Ca²⁺-ATPase in rat testes and goat spermatozoa could be activated by Ca²⁺ alone without Mg²⁺, though it has a lot of similarities with the well known Ca²⁺, Mg²⁺-ATPase. Recently, we were successful in isolating the phosphorylated intermediate of the former enzyme under control conditions i.e., in the presence of low concentration of Ca²⁺ and at low temperature. Increase of the concentration of Ca²⁺ and/or temperature lead to dephosphorylation. Based on our observations, we proposed a reaction scheme comparable to that of Ca²⁺, Mg²⁺-ATPase. The findings strengthened our previous report that Mg²⁺-independent Ca²⁺-ATPase is involved in Ca²⁺ transport and Ca²⁺ uptake like Ca²⁺, Mg²⁺-ATPase.     

Activating effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group

The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase in the plasma membranes of rat liver was investigated. (Ca²⁺–Mg²⁺)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5–5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25–1.0 M) significantly increased (Ca²⁺-Mg²⁺)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca²⁺–Mg²⁺)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 M) increasing (Ca²⁺–Mg²⁺)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca²⁺–Mg²⁺)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme.     

Relationship between plasma membrane Ca2+-ATPase activity and acrosome reaction in guinea pig sperm

The results obtained by biochemical measurement demonstrated for the first time that significant decrease of the plasma membrane Ca²⁺-ATPase activity occurred during capacitation and acrosome reaction of guinea pig sperm. Ethaorynic acid, one kind of Ca²⁺-ATPase antagonists, inhibited the plasma membrane Ca²⁺-ATPase activity, but calmodulin (50μg/mL) and trifluoperazine (200- 500μmol/L) did not, suggesting that calmodulin is not involved in ATP-driven Ca²⁺ efflux from sperm. However, calmodulin is involved in the control of Ca²⁺ influx. TFP, one kind of calmodulin antagonists, accelerated the acrosome reaction and Ca²⁺ uptake into sperm cells significantly. Ca²⁺-ATPase antagonists, quercetin, sodium orthovandate, furosemide and ethacrynic acid promoted the acrosome reaction, but inhibited Ca2+ uptake, which cannot be explained by their inhibitory effects on the plasma membrane Ca²⁺-ATPase activity. It is speculated that this phenomenon might be caused by simultaneous inhibitions of the activities of Ca²⁺-ATPase present in the plasma membrane, the outer acrosome membrane and the outer mitochondrion membrane resulting in Ca²⁺ accumulation in the cytoplasm, which in turn blocks further Ca2+ entry through some negative feedback mechanism(s). The inhibitory effect of Ca²⁺-ATPase antagonist on glycolytic activity may also be the reason for Ca²⁺ accumulation in cytoplasm and inhibition of Ca²⁺ uptake.     

Distinct Roles of the C-terminal 11th Transmembrane Helix and Luminal Extension in the Partial Reactions Determining the High Ca2+ Affinity of Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2b (SERCA2b)

The molecular mechanism underlying the characteristic high apparent Ca²⁺ affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11-amino acid luminal extension (LE). The effects of each of these parts and their interactions with the SERCA environment were examined by transient kinetic analysis of the partial reaction steps in the Ca²⁺ transport cycle in mutant and chimeric Ca²⁺-ATPase constructs. Manipulations to the LE of SERCA2b markedly increased the rate of Ca²⁺ dissociation from Ca₂E1. Addition of the SERCA2b tail to SERCA1a slowed Ca²⁺ dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca₂E1. The interaction of LE with L7/8 is also important for the low rate of the Ca₂E1P → E2P conformational transition. These findings can be rationalized in terms of stabilization of the Ca₂E1 and Ca₂E1P forms by docking of the LE near L7/8. By contrast, low rates of E2P dephosphorylation and E2 → E1 transition in SERCA2b depend critically on TM11, particularly in a SERCA2 environment, but do not at all depend on the LE or L7/8. This indicates that interaction of TM11 with SERCA2-specific sequence element(s) elsewhere in the structure is critical in the Ca²⁺-free E2/E2P states. Collectively these properties ensure a higher Ca²⁺ affinity of SERCA2b relative to other SERCA isoforms, not only on the cytosolic side, but also on the luminal side.     

Effect of phorbol 12-myristate 13-acetate on Ca2+-ATPase activity in rat liver nuclei

The effect of phorbol 12-myristate 13-acetate (PMA) on Ca²⁺-ATPase activity in rat liver nuclei was investigated. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺-Mg²⁺)-ATPase activity. The nuclear Ca²⁺-ATPase activity was significantly increased by the presence of PMA (2–20 μM) in the enzyme reaction mixture; the maximum effect was seen at 10 μM. The PMA (10 μM)-increased Ca²⁺-ATPase activity was not blocked by the presence of staurosporine (2 μM) or dibucaine (2 and 10 μM), an inhibitor of protein kinase. Meanwhile, vanadate (20 and 100 μM) caused a significant reduction in the nuclear Ca²⁺-ATPase activity increased by PMA (10 μM). The present finding suggests that PMA has an activating effect on liver nuclear Ca²⁺-ATPase independent of protein kinase. © 1994 Wiley-Liss, Inc.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

The alteration of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl₄) solution was investigated. Rats received a single oral administration of CCl₄ (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl₄ administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity, indicating that the increased Ca²⁺ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity increased by CCl₄ administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25–1.0 uM) to activate (Ca²⁺-Mg²⁺)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl₄-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver obtained from CCl₄-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca²⁺ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl₄ administration.     

Ca2+-ATPase in mucous and oxyntico-peptic cells of the fowl proventriculus

Summary Calcium adenosine triphosphatase (Ca²⁺-ATPase) was localized by means of histo- and ultracytochemistry in the secretory cells of the proventriculus of the domestic fowl. The mucous cells exhibited plasmalemmal-associated enzyme activity on the external aspect of the basolateral cell membrane. Intracellularly, the luminal aspect of Golgi-membranes and of secretory vesicle membranes reacted positively for Ca²⁺-ATPase activity, as did the apical cytosol and the matrix of lysosomes. Oxyntico-peptic cells were characterized by apical and apico-lateral plasmalemmal activity and by an organelle-associated distributional pattern similar to that in the mucous cells. In addition, Ca²⁺-ATPase was associated either with the matrix of mitochondria or with tubuli of the rough-surfaced endoplasmic reticulum. The results are discussed with respect to messenger and effector functions of calcium in the process of proventricular mucus secretion. In addition, Ca²⁺-ATPase distributional patterns in the oxyntico-peptic cell are related to the unique structure and function of these cells.     

SPCA2 Regulates Orai1 Trafficking and Store Independent Ca2+ Entry in a Model of Lactation

An unconventional interaction between SPCA2, an isoform of the Golgi secretory pathway Ca²⁺-ATPase, and the Ca²⁺ influx channel Orai1, has previously been shown to contribute to elevated Ca²⁺ influx in breast cancer derived cells. In order to investigate the physiological role of this interaction, we examined expression and localization of SPCA2 and Orai1 in mouse lactating mammary glands. We observed co-induction and co-immunoprecipitation of both proteins, and isoform-specific differences in the localization of SPCA1 and SPCA2. Three-dimensional cultures of normal mouse mammary epithelial cells were established using lactogenic hormones and basement membrane. The mammospheres displayed elevated Ca²⁺ influx by store independent mechanisms, consistent with upregulation of both SPCA2 and Orai1. Knockdown of either SPCA2 or Orai1 severely depleted Ca²⁺ influx and interfered with mammosphere differentiation. We show that SPCA2 is required for plasma membrane trafficking of Orai1 in mouse mammary epithelial cells and that this function can be replaced, at least in part, by a membrane-anchored C-terminal domain of SPCA2. These findings clearly show that SPCA2 and Orai1 function together to regulate Store-independent Ca²⁺ entry (SICE), which mediates the massive basolateral Ca²⁺ influx into mammary epithelia to support the large calcium transport requirements for milk secretion.