Distribution of Secretory Pathway Ca 2+ ATPase (SPCA1) in Neuronal and Glial Cell Cultures

1. Secretory pathway Ca²⁺ ATPase type 1 (SPCA1) is a newly recognized Ca²⁺/Mn²⁺-transporting pump localized in membranes of the Golgi apparatus.2. The expression level of SPCA1 in brain tissue is relatively high in comparison with other tissues.3. With the aim to determine the expression of SPCA1 within the different types of neural cells, we investigated the distribution of SPCA1 in neuronal, astroglial, oligodendroglial, ependymal, and microglial cell cultures derived from rat brains.4. Western Blot analysis with rabbit anti-SPCA1 antibodies revealed the presence of SPCA1 in homogenates derived from neuronal, astroglial, ependymal, and oligodendroglial, but not from microglial cells.5. Cell cultures that gave rise to positive signal in the immunoblot analysis were also examined immunocytochemically.6. Immunocytochemical double-labeling experiments with anti-SPCA1 serum in combination with antibodies against cell-type specific proteins showed a localization of the SPCA1signal within cells stained positively also for GFAP, α-tubulin or MBP.7. These results definitely established the expression of SPCA1 in astroglial, ependymal, and oligodendroglial cells.8. In addition, the evaluation of neuronal cultures for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal in cells identified as neurons.     

Immunocytochemical localization of 3-methylcrotonyl-CoA carboxylase in cultured ependymal, microglial and oligodendroglial cells

To evaluate the ability of ependymal, microglial and oligodendroglial cells to degrade leucine, the presence of 3-methylcrotonyl-CoA carboxylase (MCC) was investigated in cultures of these cells. MCC is a biotin-containing heterodimeric enzyme that is specific for the irreversible part of the leucine catabolic pathway. It has been reported previously that in cell culture MCC is expressed in astrocytes and a subpopulation of neurones. In the present study ependymal, microglial and oligodendroglial cell cultures, derived from the brains of newborn rats, were examined for the expression of MCC by RT-PCR, western blotting and immunocytochemistry. The results of RT-PCR and western blotting showed the presence of mRNA as well as protein of both subunits of MCC in ependymal, microglial and oligodendroglial cell cultures. Immunocytochemical investigation of the cellular and subcellular distribution of MCC demonstrated a mitochondrial location of MCC in all neuroglial cell types investigated. The ubiquitous expression of MCC in glial cells demonstrates the ability of the cells to engage in the catabolism of leucine transported into the brain, mainly for the generation of energy.     

Striatal dopamine-NMDA receptor interactions in the modulation of glutamate release in the substantia nigra pars reticulata in vivo: opposite role for D1 and D2 receptors

To investigate the antioxidative capacities of oligodendrocytes, rat brain cultures enriched for oligodendroglial cells were prepared and characterized. These cultures contained predominantly oligodendroglial cells as determined by immunocytochemical staining for the markers galactocerebroside and myelin basic protein. If oligodendroglial cultures were exposed to exogenous hydrogen peroxide (100 micro m), the peroxide disappeared from the incubation medium following first order kinetics with a half-time of approximately 18 min. Normalization of the disposal rate to the protein content of the cultures by calculation of the specific hydrogen peroxide detoxification rate constant revealed that the cells in oligodendroglial cultures have a 60% to 120% higher specific capacity to dispose of hydrogen peroxide than cultures enriched for astroglial cells, microglial cells or neurones. Oligodendroglial cultures contained specific activities of 133.5 +/- 30.4 nmol x min(-1) x mg protein(-1) and 27.5 +/- 5.4 nmol x min(-1) x mg protein(-1) of glutathione peroxidase and glutathione reductase, respectively. The specific rate constant of catalase in these cultures was 1.61 +/- 0.54 min(-1) x mg protein(-1). Comparison with data obtained by identical methods for cultures of astroglial cells, microglial cells and neurones revealed that all three of the enzymes which are involved in hydrogen peroxide disposal were present in oligodendroglial cultures in the highest specific activities. These results demonstrate that oligodendroglial cells in culture have a prominent machinery for the disposal of hydrogen peroxide, which is likely to support the protection of these cells in brain against peroxides when produced by these or by surrounding brain cells.     

A novel isoform of the secretory pathway Ca2+,Mn(2+)-ATPase, hSPCA2, has unusual properties and is expressed in the brain

Unlike lower eukaryotes, mammalian genomes have a second gene, ATP2C2, encoding a putative member of the family of secretory pathway Ca2+,Mn(2+)-ATPases, SPCA2. Human SPCA2 shares 64% amino acid identity with the protein defective in Hailey Hailey disease, hSPCA1. We show that human SPCA2 (hSPCA2) has a more limited tissue distribution than hSPCA1, with prominent protein expression in brain and testis. In primary neuronal cells, endogenous SPCA2 has a highly punctate distribution that overlaps with vesicles derived from the trans-Golgi network and is thus different from the compact perinuclear distribution of hSPCA1 seen in keratinocytes and nonpolarized cells. Heterologous expression in a yeast strain lacking endogenous Ca2+ pumps reveals further functional differences from hSPCA1. Although the Mn(2+)-specific phenotype of hSPCA2 is similar to that of hSPCA1, Ca2+ ions are transported with much poorer affinity, resulting in only weak complementation of Ca(2+)-specific yeast phenotypes. These observations suggest that SPCA2 may have a more specialized role in mammalian cells, possibly in cellular detoxification of Mn2+ ions, similar to that in yeast. We point to the close links between manganese neurotoxicity and Parkinsonism that would predict an important physiological role for SPCA2 in the brain.     

Similar Ca(2+)-signaling properties in keratinocytes and in COS-1 cells overexpressing the secretory-pathway Ca(2+)-ATPase SPCA1

Mutations in the ubiquitously expressed secretory-pathway Ca(2+)-ATPase (SPCA1) Ca(2+) pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca(2+) signaling in human keratinocytes by measuring the free Ca(2+) concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) and SPCA1 can mediate Ca(2+) uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca(2+) pump to load the Golgi complex with Ca(2+) whereas the SERCA Ca(2+) pump was mainly used in control COS-1 cells. Cytosolic Ca(2+) signals in keratinocytes induced by extracellular ATP or capacitative Ca(2+) entry were characterized by an unusually long latency reflecting extra Ca(2+) buffering by an SPCA1-containing Ca(2+) store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca(2+) elicited spontaneous cytosolic Ca(2+) transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca(2+) signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca(2+) in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients.     

Store-independent activation of Orai1 by SPCA2 in mammary tumors

Ca(2+) is an essential and ubiquitous second messenger. Changes in cytosolic Ca(2+) trigger events critical for tumorigenesis, such as cellular motility, proliferation, and apoptosis. We show that an isoform of Secretory Pathway Ca(2+)-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuates basal Ca(2+) levels and tumorigenicity. Contrary to its conventional role in Golgi Ca(2+) sequestration, expression of SPCA2 increased Ca(2+) influx by a mechanism dependent on the store-operated Ca(2+) channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca(2+) stores or STIM1 and STIM2 sensors and uncoupled from Ca(2+)-ATPase activity of SPCA2. Binding of the SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca(2+) influx. Our findings reveal a signaling pathway in which the Orai1-SPCA2 complex elicits constitutive store-independent Ca(2+) signaling that promotes tumorigenesis.     

Functional comparison between secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and sarcoplasmic reticulum Ca2+-ATPase (SERCA) 1 isoforms by steady-state and transient kinetic analyses

Steady-state and transient kinetic studies were performed to functionally analyze the overall and partial reactions of the Ca(2+) transport cycle of the human secretory pathway Ca(2+)/Mn(2+)-ATPase 1 (SPCA1) isoforms: SPCA1a, SPCA1b, SPCA1c, and SPCA1d (encoded by ATP2C1, the gene defective in Hailey-Hailey disease) upon heterologous expression in mammalian cells. The expression levels of SPCA1 isoforms were 200-350-fold higher than in control cells except for SPCA1c, whose low expression level appears to be the effect of rapid degradation because of protein misfolding. Relative to SERCA1a, the active SPCA1a, SPCA1b, and SPCA1d enzymes displayed extremely high apparent affinities for cytosolic Ca(2+) in activation of the overall ATPase and phosphorylation activities. The maximal turnover rates of the ATPase activity for SPCA1 isoforms were 4.7-6.4-fold lower than that of SERCA1a (lowest for the shortest SPCA1a isoform). The kinetic analysis traced these differences to a decreased rate of the E(1) approximately P(Ca) to E(2)-P transition. The apparent affinity for inorganic phosphate was reduced in the SPCA1 enzymes. This could be accounted for by an enhanced rate of the E(2)-P hydrolysis, which showed constitutive activation, lacking the SERCA1a-specific dependence on pH and K(+).     

Expression of 3-hydroxyisobutyrate dehydrogenase in cultured neural cells

The branched-chain amino acids (BCAAs) – isoleucine, leucine, and valine – belong to the limited group of substances transported through the blood–brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched-chain alpha-keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate-limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched-chain amino acids. Unlike the acyl-CoA derivates in the irreversible part of valine catabolism, 3-hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine-derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3-hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity-purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT-PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double-labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy.     

Functional and immunocytochemical evidence for the expression and localization of the secretory pathway Ca2+-ATPase isoform 1 (SPCA1) in cerebellum relative to other Ca2+ pumps

Membrane fractions of pig cerebellum show Ca2+-ATPase activity and Ca2+ transport due to the presence of the secretory pathway Ca2+-ATPase (SPCA). The SPCA1 isoform shows a wide distribution in the neurons of pig cerebellum, where it is found in the Golgi complex of the soma of Purkinje, stellate, basket and granule cells, and also in more distal components of the secretory pathway associated with a synaptic localization such as in cerebellar glomeruli. The SPCA1 may be involved in loading the Golgi complex and the secretory vesicles of these specific neuronal cell types with Ca2+ and also Mn2+. This study of the cellular and subcellular localization of SPCA1 pumps relative to the sarco(endo) plasmic reticulum Ca2+-ATPase and plasma membrane Ca2+-ATPase pumps hints to a possible specific role of SPCA1 in controlling the luminal secretory pathway Ca2+ (or Mn2+) levels as well as the local cytosolic Ca2+ levels. In addition, it helps to specify the zones that are most vulnerable to Ca2+ and/or Mn2+ dyshomeostasis, a condition that is held responsible of an increasing number of neurological disorders.     

Comparison of Primary and Secondary Rat Astrocyte Cultures Regarding Glucose and Glutathione Metabolism and the Accumulation of Iron Oxide Nanoparticles

Astrocyte-rich primary cultures (APCs) are frequently used as a model system for the investigation of properties of brain astrocytes. However, as APCs contain a substantial number of microglial and oligodendroglial cells, biochemical parameters determined for such cultures may at least in part reflect also the presence of the contaminating cell types. To lower the potential contributions of microglial and oligodendroglial cells on properties of the astrocytes in APCs we prepared rat astrocyte-rich secondary cultures (ASCs) by subculturing of APCs and compared these ASCs with APCs regarding basal metabolic parameters, specific enzyme activities and the accumulation of iron oxide nanoparticles. Immunocytochemical characterization revealed that ASCs contained only minute amounts of microglial and oligodendroglial cells. ASCs and APCs did not significantly differ in their specific glucose consumption and lactate production rates, in their specific iron and glutathione contents, in their specific activities of various enzymes involved in glucose and glutathione metabolism nor in their accumulation of iron oxide nanoparticles. Thus, the absence or presence of some contaminating microglial and oligodendroglial cells appears not to substantially modulate the investigated metabolic parameters of astrocyte cultures.     

Proliferation of microglial cells induced by 1-methyl-4-phenylpyridinium in mesencephalic cultures results from an astrocyte-dependent mechanism: role of granulocyte macrophage colony-stimulating factor

There is evidence that an inflammatory microglial reaction participates in the pathophysiology of dopaminergic neuronal death in Parkinson's disease and in animal models of the disease. However, this phenomenon remains incompletely characterized. Using an in vitro model of neuronal/glial mesencephalic cultures, we show that the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) stimulates the proliferation of microglial cells at concentrations that selectively reduce the survival of DA neurones. The mitogenic action of MPP+ was not the mere consequence of neuronal cell demise as the toxin produced the same effect in a model system of neuronal/glial cortical cultures, where target DA neurones are absent. Consistent with this observation, the proliferative effect of MPP+ was also detectable in neurone-free microglial/astroglial cultures. It disappeared, however, when MPP+ was added to pure microglial cell cultures suggesting that astrocytes played a key role in the mitogenic mechanism. Accordingly, the proliferation of microglial cells in response to MPP+ treatment was mimicked by granulocyte macrophage colony-stimulating factor (GM-CSF), a proinflammatory cytokine produced by astrocytes and was blocked by a neutralizing antibody to GM-CSF. Thus, we conclude that the microglial reaction observed following MPP+ exposure depends on astrocytic factors, e.g. GM-CSF, a finding that may have therapeutic implications.     

Bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)-methane (bis-phenol) is a potent and selective inhibitor of the secretory pathway Ca(2+) ATPase (SPCA1)

The secretory pathway Ca(2+) ATPase (SPCA) provides the Golgi apparatus with a Ca(2+) supply essential for Ca(2+)-dependent enzymes involved in the post-translational modification of proteins in transit through the secretory pathway. Ca(2+) in the Golgi apparatus is also agonist-releasable and plays a role in hormone-induced Ca(2+) transients. Although the Ca(2+) ATPase inhibitors thapsigargin is more selective for the sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase (SERCA) than for SPCA, no inhibitor has been characterised that selectively inhibits SPCA. A number of inhibitors were assessed for their selectivity to the human SPCA1d compared to the more ubiquitous human SERCA2b. Each isoform was over-expressed in COS-7 cells and the Ca(2+)-dependent ATPase activity measured in their microsomal membranes. Both bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane(bis-phenol) and 2-aminoethoxydiphenylborate (2-APB) selectively inhibited SPCA1d (with IC(50) values of 0.13μM and 0.18mM, respectively), which were of 62- and 8.3-fold greater potency than the values for hSERCA2b (IC(50) values; 8.1μM and 1.5mM, respectively). Other inhibitors tested such as bis-phenol-A, tetrabromobisphenol-A and trifluoperazine inhibited both Ca(2+) ATPases similarly. Furthermore, bis-phenol was able to mobilize Ca(2+) in cells that had been pre-treated with thapsigargin. Therefore we conclude that given the potency and selectivity of bis-phenol it may prove a valuable tool in further understanding the role of SPCA in cellular processes.     

Functional importance of inositol-1,4,5-triphosphate-induced intracellular Ca2+ mobilization in galanin-induced microglial migration

Galanin (GAL) is a neuropeptide which is up-regulated following neuronal axotomy or inflammation. One subtype of GAL receptor (GalR2) is reported to be expressed in the brain's immune cell population, microglia. In the present study, we investigated the effect of GAL on microglial migration and compared the mechanism with that of bradykinin (BK). GAL significantly increased the migration of rat cultured microglia at 0.1 pM. The GAL-induced signal cascade was partly similar to that induced by BK. It was not dependent on G(i/o) protein but involved activation of protein kinase C, phosphoinositide 3-kinase and Ca(2+)-dependent K(+) channels. However, reverse-mode activation of the Na(+) /Ca(2+) -exchanger 1 was not involved in GAL-induced microglial migration, unlike BK-induced migration. Likewise, nominally-free extracellular Ca(2+) inhibited BK-induced migration but not GAL-induced migration. An inositol-1,4,5-triphosphate receptor antagonist significantly inhibited GAL-induced migration. GAL-induced Ca(2+) signaling did not induce nitric oxide synthase expression, but up-regulated class II major histocompatibility complex expression. These results indicate that activation of inositol-1,4,5-triphosphate receptor and increase in intracellular Ca(2+) are important for GAL-induced migration and immunoreactivity in microglia. The differences in down-stream signal transduction induced by GAL and BK suggest that GAL and BK may control distinct microglial functions under pathological conditions.     

Microglial calcium signal acts as a rapid sensor of single neuron damage in vivo

In the healthy adult brain microglia, the main immune-competent cells of the CNS, have a distinct (so-called resting or surveying) phenotype. Resting microglia can only be studied in vivo since any isolation of brain tissue inevitably triggers microglial activation. Here we used in vivo two-photon imaging to obtain a first insight into Ca(2+) signaling in resting cortical microglia. The majority (80%) of microglial cells showed no spontaneous Ca(2+) transients at rest and in conditions of strong neuronal activity. However, they reliably responded with large, generalized Ca(2+) transients to damage of an individual neuron. These damage-induced responses had a short latency (0.4-4s) and were localized to the immediate vicinity of the damaged neuron (< 50 μm cell body-to-cell body distance). They were occluded by the application of ATPγS as well as UDP and 2-MeSADP, the agonists of metabotropic P2Y receptors, and they required Ca(2+) release from the intracellular Ca(2+) stores. Thus, our in vivo data suggest that microglial Ca(2+) signals occur mostly under pathological conditions and identify a Ca(2+) store-operated signal, which represents a very sensitive, rapid, and highly localized response of microglial cells to brain damage. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Comparison of prostanoid forming capacity of neuronal and astroglial cells in primary cultures

Prostaglandin (PG) and thromboxane (TX) biosynthesis in primary neuronal and astroglial cell cultures was studied. Cultures obtained from fetal (15–16 days old) and neonatal rat brain hemispheres were characterized by chemical and immunocytochemical staining techniques as predominantly neurons or mature and immature astrocytes, respectively. Six-day old neuronal cell cultures grown in the presence of cytosine arabinoside (2 μM) from the day 3 onwards were contaminated up to 10% with glioblasts. In astroglial cultures up to 3% of the cells were postively stained with a marker for oligodendroglial cells. Fibroblast contamination was below 1% in both cultures. Prostanoid formation (measured by specific radioimmunoassays) in 6-day old neuronal cell cultures was low (sum of the amount of PGs and TX formed: 1.16 ± 0.17 (ng/mg protein/15 min) as compared to 14-day old cultured astroglial cells: 21.27 ± 2.53 (ng/mg protein/15 min). Also the pattern of prostanoids formed was different in neuronal (PGD₂ ? PGF_2α > TXB₂ ? PGE₂) and astroglial cells (PGD₂ > TXB₂ ? PGF_2α ? PGE₂ ? 6-ketoPGF_1α). Preincubation with arachidonic acid (1 μg/ml) did not affect prostanoid formation in both cultures, whereas it was stimulated 4–6-fold by addition of the calcium ionophore A23187 (1 μM). These results, although found on cultured neuronal and glial cells of different stages of development, support the view that astroglial cells might play a crucial role in brain prostanoid synthesis.     

Transient receptor potential vanilloid subtype 1 mediates microglial cell death in vivo and in vitro via Ca2+-mediated mitochondrial damage and cytochrome c release

The present study examined the expression of transient receptor potential vanilloid subtype 1 (TRPV1) in microglia, and its association with microglial cell death. In vitro cell cultures, RT-PCR, Western blot analysis, and immunocytochemical staining experiments revealed that rat microglia and a human microglia cell line (HMO6) showed TRPV1 expression. Furthermore, exposure of these cells to TRPV1 agonists, capsaicin (CAP) and resiniferatoxin (RTX), triggered cell death. This effect was ameliorated by the TRPV1 antagonists, capsazepine and iodo-resiniferatoxin (I-RTX), suggesting that TRPV1 is directly involved. Further examinations revealed that TRPV1-induced toxicity was accompanied by increases in intracellular Ca(2+), and mitochondrial damage; these effects were inhibited by capsazepine, I-RTX, and the intracellular Ca(2+) chelator BAPTA-AM. Treatment of cells with CAP or RTX led to increased mitochondrial cytochrome c release and enhanced immunoreactivity to cleaved caspase-3. In contrast, the caspase-3 inhibitor z-DEVD-fmk protected microglia from CAP- or RTX-induced toxicity. In vivo, we also found that intranigral injection of CAP or 12-hydroperoxyeicosatetraenoic acid, an endogenous agonist of TRPV1, into the rat brain produced microglial damage via TRPV1 in the substantia nigra, as visualized by immunocytochemistry. To our knowledge, this study is the first to demonstrate that microglia express TRPV1, and that activation of this receptor may contribute to microglial damage via Ca(2+) signaling and mitochondrial disruption.     

The secretory pathway Ca2+/Mn2+-ATPase 2 is a Golgi-localized pump with high affinity for Ca2+ ions

Accumulation of Ca(2+) into the Golgi apparatus is mediated by sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) and by secretory pathway Ca(2+)-ATPases (SPCAs). Mammals and birds express in addition to the housekeeping SPCA1 (human gene name ATP2C1, cytogenetic position 3q22.1) a homologous SPCA2 isoform (human gene name ATP2C2, cytogenetic position 16q24.1). We show here that both genes present an identical exon/intron layout. We confirmed that hSPCA2 has the ability to transport Ca(2+), demonstrated its Mn(2+)-transporting activity, showed its Ca(2+)- and Mn(2+)-dependent phosphoprotein intermediate formation, and documented the insensitivity of these functional activities to thapsigargin inhibition. The mRNA encoding hSPCA2 showed a limited tissue expression pattern mainly confined to the gastrointestinal and respiratory tract, prostate, thyroid, salivary, and mammary glands. Immunocytochemical localization in human colon sections presented a typical apical juxtanuclear Golgi-like staining. The expression in COS-1 cells allowed the direct demonstration of (45)Ca(2+) (K(0.5) = 0.27 microm) or (54)Mn(2+) transport into an A23187-releasable compartment.     

The Ca2+/Mn2+ pumps in the Golgi apparatus

Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca(2+) store. Besides Ca(2+)-release channels and Ca(2+)-binding proteins, the Golgi complex contains Ca(2+)-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca(2+)-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca(2+) and Mn(2+) and, therefore, plays an important role in the cytosolic and intra-Golgi Ca(2+) and Mn(2+) homeostasis. Mutations in the human gene encoding the SPCA1 pump (ATP2C1) resulting in Hailey-Hailey disease, an autosomal dominant skin disorder, are discussed.