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Adeno-associated viruses (AAVs) are dependoparvoviruses that have proven useful for therapeutic gene transfer; however, our understanding of host factors that influence AAV trafficking and transduction is still evolving. Here, we investigated the role of cellular calcium in the AAV infectious pathway. First, we demonstrated a critical role for the host Golgi compartment-resident ATP-powered calcium pump (secretory pathway calcium ATPase 1 [SPCA1]) encoded by the ATP2C1 gene in AAV infection. CRISPR-based knockout (KO) of ATP2C1 decreases transduction by different AAV serotypes. ATP2C1 KO does not appear to inhibit AAV binding, cellular uptake, or nuclear entry; however, capsids within ATP2C1 KO cells demonstrate dispersed and punctate trafficking distinct from the perinuclear, trans -Golgi pattern observed in normal cells. In addition, we observed a defect in the ability of AAV capsids to undergo conformational changes and support efficient vector genome transcription in ATP2C1 KO cells. The calcium chelator BAPTA-AM, which reduces cytosolic calcium, rescues the defective ATP2C1 KO phenotype and AAV transduction in vitro Conversely, the calcium ionophore ionomycin, which disrupts calcium gradients, blocks AAV transduction. Further, we demonstrated that modulating calcium in the murine brain using BAPTA-AM augments AAV gene expression in vivo Taking these data together, we postulate that the maintenance of an intracellular calcium gradient by the calcium ATPase and processing within the Golgi compartment are essential for priming the capsid to support efficient AAV genome transcription. IMPORTANCE Adeno-associated viruses (AAVs) have proven to be effective gene transfer vectors. However, our understanding of how the host cell environment influences AAV transduction is still evolving. In the present study, we investigated the role of ATP2C1 , which encodes a membrane calcium transport pump, SPCA1, essential for maintaining cellular calcium homeostasis on AAV transduction. Our results indicate that cellular calcium is essential for efficient intracellular trafficking and conformational changes in the AAV capsid that support efficient genome transcription. Further, we show that pharmacological modulation of cellular calcium levels can potentially be applied to improve the AAV gene transfer efficiency., (Copyright © 2020 Madigan et al.)

Recombination is one of the driving forces of viral evolution. RNA recombination events among similar RNA viruses are frequent, although RNA recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, carnation Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral recombination events. Most interviral recombinants are minus-strand recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca 2+ /Mn 2+ pump, regulating intracellular Ca 2+ and Mn 2+ influx into the Golgi apparatus from the cytosol. The interviral recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of recombinants among related and unrelated viruses. This is the first time that a host factor's role in affecting interviral recombination is established. IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca 2+ /Mn 2+ pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca 2+ /Mn 2+ pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses., (Copyright © 2019 American Society for Microbiology.)

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca 2+ -ATPase 4 (Pmca4) gene which encodes the highly conserved Ca 2+ efflux pump, PMCA4. This is the major Ca 2+ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co-immunoprecipitation (Co-IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca 2+ ] in capacitated and Ca 2+ ionophore-treated sperm and with neuronal (nNOS) at basal [Ca 2+ ] (ucapacitated sperm). FRET efficiencies for PMCA4-eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4-nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co-immunoprecipitated with eNOS in a Ca 2+ -dependent manner. In Pmca4 -/- sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild-type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co-ordinates Ca 2+ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4 -/- males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance., (© 2017 Wiley Periodicals, Inc.)

Background: Hailey-Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare, chronic and recurrent blistering disorder, histologically characterized by suprabasal acantholysis. HHD has been linked to mutations in ATP2C1, the gene encoding the human adenosine triphosphate (ATP)-powered calcium channel pump., Methods: In this work, the genetically tractable yeast Kluyveromyces lactis has been used to study the molecular basis of Hailey-Hailey disease. The K. lactis strain depleted of PMR1, the orthologue of the human ATP2C1 gene, was used to screen a Madin-Darby canine kidney (MDCK) cDNA library to identify genetic interactors able to suppress the oxidative stress occurring in those cells., Results: We have identified the Glutathione S-transferase ϴ-subunit (GST), an important detoxifying enzyme, which restores many of the defects associated with the pmr1Δmutant. GST overexpression in those cells suppressed the sensitivity to calcium chelating agents and partially re-established calcium (Ca 2+ ) homeostasis by decreasing the high cytosolic Ca 2+ levels in pmr1Δstrain. Moreover, we found that in the K. lactis mutant the mitochondrial dysfunction was suppressed by GST overexpression independently from calcineurin. In agreement with yeast results, a decreased expression of the human GST counterpart (GSTT1/M1) was observed in lesion-derived keratinocytes from HHD patients., Conclusions: These data highlighted the Glutathione S-transferase as a candidate gene associated with Hailey-Hailey disease., General Significance: Kluyveromyces lactis can be considered a good model to study the molecular basis of this pathology., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition.

Objective: Vascular endothelial growth factor (VEGF) has been identified as a crucial regulator of physiological and pathological angiogenesis. Among the intracellular signaling pathways triggered by VEGF, activation of the calcineurin/nuclear factor of activated T cells (NFAT) signaling axis has emerged as a critical mediator of angiogenic processes. We and others previously reported a novel role for the plasma membrane calcium ATPase (PMCA) as an endogenous inhibitor of the calcineurin/NFAT pathway, via interaction with calcineurin, in cardiomyocytes and breast cancer cells. However, the functional significance of the PMCA/calcineurin interaction in endothelial pathophysiology has not been addressed thus far., Approach and Results: Using in vitro and in vivo assays, we here demonstrate that the interaction between PMCA4 and calcineurin in VEGF-stimulated endothelial cells leads to downregulation of the calcineurin/NFAT pathway and to a significant reduction in the subsequent expression of the NFAT-dependent, VEGF-activated, proangiogenic genes RCAN1.4 and Cox-2. PMCA4-dependent inhibition of calcineurin signaling translates into a reduction in endothelial cell motility and blood vessel formation that ultimately impairs in vivo angiogenesis by VEGF., Conclusions: Given the importance of the calcineurin/NFAT pathway in the regulation of pathological angiogenesis, targeted modulation of PMCA4 functionality might open novel therapeutic avenues to promote or attenuate new vessel formation in diseases that occur with angiogenesis., (© 2014 American Heart Association, Inc.)

Cross-complementation studies offer the possibility to overcome limitations imposed by the inherent complexity of multicellular organisms in the study of human diseases, by taking advantage of simpler model organisms like the budding yeast Saccharomyces cerevisiae. This review deals with, (1) the use of S. cerevisiae as a model organism to study human diseases, (2) yeast-based screening systems for the detection of disease modifiers, (3) Hailey-Hailey as an example of a calcium-related disease, and (4) the presentation of a yeast-based model to search for chemical modifiers of Hailey-Hailey disease. The preliminary experimental data presented and discussed here show that it is possible to use yeast as a model system for Hailey-Hailey disease and suggest that in all likelihood, yeast has the potential to reveal candidate drugs for the treatment of this disorder. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons, which arises from a yet elusive concurrence between genetic and environmental factors. The protein α-synuclein (αSyn), the principle toxic effector in PD, has been shown to interfere with neuronal Ca(2+) fluxes, arguing for an involvement of deregulated Ca(2+) homeostasis in this neuronal demise. Here, we identify the Golgi-resident Ca(2+)/Mn(2+) ATPase PMR1 (plasma membrane-related Ca(2+)-ATPase 1) as a phylogenetically conserved mediator of αSyn-driven changes in Ca(2+) homeostasis and cytotoxicity. Expression of αSyn in yeast resulted in elevated cytosolic Ca(2+) levels and increased cell death, both of which could be inhibited by deletion of PMR1. Accordingly, absence of PMR1 prevented αSyn-induced loss of dopaminergic neurons in nematodes and flies. In addition, αSyn failed to compromise locomotion and survival of flies when PMR1 was absent. In conclusion, the αSyn-driven rise of cytosolic Ca(2+) levels is pivotal for its cytotoxicity and requires PMR1.

Neural cell differentiation involves a complex regulatory signal transduction network in which Ca(2+) ions and the secretory pathway play pivotal roles. The secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) is found in the Golgi apparatus where it is actively involved in the transport of Ca(2+) or Mn(2+) from the cytosol to the Golgi lumen. Its expression during brain development in different types of neurons has been documented recently, which raises the possibility that SPCA1 contributes to neuronal differentiation. In the present study, we investigated the potential impact of SPCA1 on neuronal polarization both in a cell line and in primary neuronal culture. In N2a neuroblastoma cells, SPCA1 was immunocytochemically localized in the juxtanuclear Golgi. Knockdown of SPCA1 by RNA interference markedly delayed the differentiation in these cells. The cells retarded in differentiation showed increased numbers of neurites of reduced length compared with control cells. Ca(2+) imaging assays showed that the lack of SPCA1 impaired Golgi Ca(2+) homeostasis and resulted in disturbed trafficking of different classes of proteins including normally Golgi-localized cameleon GT-YC3.3, bearing a Golgi-specific galactosyltransferase N terminus, and a normally plasma membrane-targeted, glycosyl phosphatidyl inositol-anchored cyan fluorescent protein construct. Also in hippocampal primary neurons, which showed a differential distribution of SPCA1 expression in Golgi stacks depending on differentiation stage, partial silencing of SPCA1 resulted in delayed differentiation, whereas total suppression drastically affected the cell survival. The disturbed overall cellular Ca(2+) homeostasis and/or the altered targeting of organellar proteins under conditions of SPCA1 knockdown highlight the importance of SPCA1 function for normal neural differentiation.

Objective: To assess the effect of RNA interference-mediated gene silencing of plasma membrane-related Ca(2+)-ATPase-1 (PMR1) gene on the insulin secretion in islet beta cells NIT-1 in vitro., Methods: A small interfering RNA duplex (siPMR1) corresponding to the nucleotides 337-357 of mouse PMR1 cDNA was introduced into NIT-1 cells via liposomes. The gene silencing effect was assessed by RT-PCR, and the total insulin level in the transfected cells was measured by radioimmunoassay., Results: Transfection with siPMR1 resulted in obviously reduced PMR1 expression and increased insulin secretion in NIT-1 cells., Conclusion: The synthesized siPMR1 can significantly silence the expression of PMR1 and promote the secretion of insulin in the islet cells in vitro, which shed light on further studies of RNAi-based therapy of diabetes.

Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.

The main role of the plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA) is in the removal of Ca2+ from the cytosol. Recently, we and others have suggested a new function for PMCA as a modulator of signal transduction pathways. This paper shows the physical interaction between PMCA (isoforms 1 and 4) and alpha-1 syntrophin and proposes a ternary complex of interaction between endogenous PMCA, alpha-1 syntrophin, and NOS-1 in cardiac cells. We have identified that the linker region between the pleckstrin homology 2 (PH2) and the syntrophin unique (SU) domains, corresponding to amino acids 399-447 of alpha-1 syntrophin, is crucial for interaction with PMCA1 and -4. The PH2 and the SU domains alone failed to interact with PMCA. The functionality of the interaction was demonstrated by investigating the inhibition of neuronal nitric-oxide synthase-1 (NOS-1); PMCA is a negative regulator of NOS-1-dependent NO production, and overexpression of alpha-1 syntrophin and PMCA4 resulted in strongly increased inhibition of NO production. Analysis of the expression levels of alpha-1 syntrophin protein in the heart, skeletal muscle, brain, uterus, kidney, or liver of PMCA4-/- mice, did not reveal any differences when compared with those found in the same tissues of wild-type mice. These results suggest that PMCA4 is tethered to the syntrophin complex as a regulator of NOS-1, but its absence does not cause collapse of the complex, contrary to what has been reported for other proteins within the complex, such as dystrophin. In conclusion, the present data demonstrate for the first time the localization of PMCA1b and -4b to the syntrophin.dystrophin complex in the heart and provide a specific molecular mechanism of interaction as well as functionality.

Abnormal Ca(2+) cycling in the failing heart might be corrected by enhancing the activity of the cardiac Ca(2+) pump, the sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) isoform. This can be obtained by increasing the pump's affinity for Ca(2+) by suppressing phospholamban (PLB) activity, the in vivo inhibitor of SERCA2a. In SKO mice, gene-targeted replacement of SERCA2a by SERCA2b, a pump with a higher Ca(2+) affinity, results in cardiac hypertrophy and dysfunction. The stronger PLB inhibition on cardiac morphology and performance observed in SKO was investigated here in DKO mice, which were obtained by crossing SKO with PLB(-/-) mice. The affinity for Ca(2+) of SERCA2 was found to be further increased in these DKO mice. Relative to wild-type and SKO mice, DKO mice were much less spontaneously active and showed a reduced life span. The DKO mice also displayed a severe cardiac phenotype characterized by a more pronounced concentric hypertrophy, diastolic dysfunction and increased ventricular stiffness. Strikingly, beta-adrenergic or forced exercise stress induced acute heart failure and death in DKO mice. Therefore, the increased PLB inhibition represents a compensation for the imposed high Ca(2+)-affinity of SERCA2b in the SKO heart. Limiting SERCA2's affinity for Ca(2+) is physiologically important for normal cardiac function. An improved Ca(2+) transport in the sarcoplasmic reticulum may correct Ca(2+) mishandling in heart failure, but a SERCA pump with a much higher Ca(2+) affinity may be detrimental.

Recent proteomic applications have demonstrated their potential for revealing the molecular mechanisms underlying neurodegeneration. The present study quantifies cerebellar protein changes in mice that are deficient in plasma membrane calcium ATPase 2 (PMCA2), an essential neuronal pump that extrudes calcium from cells and is abundantly expressed in Purkinje neurons. PMCA2-null mice display motor dyscoordination and unsteady gait deficits observed in neurological diseases such as multiple sclerosis and ataxia. We optimized an amine-specific isobaric tags (iTRAQ)-based shotgun proteomics workflow for this study. This workflow took consideration of analytical variance as a function of ion signal intensity and employed biological repeats to aid noise reduction. Even with stringent protein identification criteria, we could reliably quantify nearly 1000 proteins, including many neuronal proteins that are important for synaptic function. We identified 21 proteins that were differentially expressed in PMCA2-null mice. These proteins are involved in calcium homeostasis, cell structure and chromosome organization. Our findings shed light on the molecular changes that underlie the neurological deficits observed in PMCA2-null mice. The optimized workflow presented here will be valuable for others who plan to implement the iTRAQ method.

Darier disease is a genodermatosis with a pattern of autosomal dominant inheritance, which occasionally presents by following a linear or segmental pattern. We present the case of a 22-year-old woman who had presented for three years with small, linearly distributed, yellowish-brown papules on the right lower extremity, with no other skin, mucous membrane or appendage involvement. She had no personal or family history of any interest. Histologically, suprabasal acantholysis and abundant dyskeratotic cells were seen. All of this data led to the diagnosis of linear Darier disease.

Objective: To verify whether a deficiency in the cardiac sarcoplasmic reticulum pump SERCA2a causes cardiac dysfunction in humans., Design: Cardiac performance was measured in a serendipitous human model of primary SERCA2a deficiency, Darier's disease, an autosomal dominant skin disorder caused by mutations inactivating one copy of the ATP2A2 gene, which encodes SERCA2a., Methods: Systolic and diastolic function and contractility were assessed by echocardiography at rest and during exercise in patients with Darier's disease with known mutations. Fourteen patients with Darier's disease were compared with 14 normal controls and six patients with dilated cardiomyopathy with stable heart failure., Results: Resting systolic and diastolic function was normal in patients with Darier's disease and in controls. The increase in systolic function during exercise was not different between patients with Darier's disease and normal controls; neither was there a difference in contractility. As expected, patients with dilated cardiomyopathy had impaired diastolic and systolic function with depressed contractility at rest and during exercise., Conclusion: Contrary to expectations, heterozygous disruption of SERCA2a is not associated with the impairment of cardiac performance in humans. Attempts to increase SERCA2a levels in heart failure, although showing promise in rodent studies, may not be addressing a critical causal pathway in humans.

The neocortex of congenitally deaf mice was examined using electrophysiological recording techniques combined with cortical myeloarchitecture. Our results indicate that relative activity patterns across sensory systems during development contribute to modality assignment of cortical fields as well as the size of cortical fields. In congenitally deaf mice, "auditory cortex" contained neurons that responded to somatosensory, visual, or both somatosensory and visual stimulation; the primary visual area contained a larger proportion of neurons that responded to somatosensory stimulation than in normal animals, and the primary visual area had significantly increased in size. Thus, cortical architecture and functional specification were de-correlated. When results are considered in the light of molecular studies and studies in which peripheral activity is altered in development, it becomes clear that similar types of changes to the neocortex, such as alterations in cortical field size, can be achieved in more than one way in the developing and evolving neocortex.

PMR1, the Ca2+/Mn2+ ATPase of the secretory pathway in Saccharomyces cerevisiae was the first member of the secretory pathway Ca2+ ATPases (SPCA) to be characterized. In the past few years, pmr1Delta yeast have received more attention due to the recognition that the human homologue of this protein, hSPCA1 is defective in chronic benign pemphigus or Hailey-Hailey disease (HHD). Recent publications have described pmr1Delta S. cerevisiae as a useful model organism for studying the molecular pathology of HHD. Some observations indicated that the high Ca2+ sensitive phenotype of PMR1 defective yeast strains may be the most relevant in this respect. Here we show that the total cellular calcium response of a pmr1Delta S. cerevisiae upon extracellular Ca2+ challenge is decreased compared to the wild type strain similarly as observed in keratinocytes. Additionally, the novel magnesium sensitivity of PMR1 defective yeast is revealed, which appears to be a result of competition for uptake between Ca2+ and Mg2+ at the plasma membrane level. Our findings indicate that extracellular Ca2+ and Mg2+ competitively influence the intracellular Ca2+ homeostasis of S. cerevisiae. These observations may further our understanding of HHD.

In elderly patients, an inadequately treated high blood pressure often leads to hypertrophied cardiomyocytes with various defects in gene expression. Due to a decreased expression of the transcription factor PPARalpha, fatty acid oxidation is reduced. If it can be compensated by an increased glucose oxidation, it has been considered as a favorable process. Nonetheless, reduced PPARalpha influences ensue involving e. g. anti-inflammatory mechanisms. The question arises thus whether drugs can normalize reduced PPARalpha effects without increasing fatty acid oxidation. As lead compound of these "fatty acid oxidation inhibitors with PPARalpha activation", the carnitine palmitoyltransferase-1 inhibitor etomoxir was characterized. An increased expression and activity of the Ca (2+) pump of sarcoplasmic reticulum, a faster relaxation and a slowed progression of heart failure was observed in animal experiments. It should, therefore, be examined whether the impaired function of pressure overloaded hypertrophied cardiomyocytes of particularly elderly patients should be a therapeutic target before progression of heart failure, neuroendocrine activation and symptoms such as shortness of breath occur.

Relatively few clues have been uncovered to elucidate the cell biological role(s) of mammalian ATP2C1 encoding an inwardly directed secretory pathway Ca2+/Mn2+ pump that is ubiquitously expressed. Deficiency of ATP2C1 results in a human disease (Hailey-Hailey), which primarily affects keratinocytes. ATP2C1-encoded protein is detected in the Golgi complex in a calcium-dependent manner. A small interfering RNA causes knockdown of ATP2C1 expression, resulting in defects in both post-translational processing of wild-type thyroglobulin (a secretory glycoprotein) as well as endoplasmic reticulum-associated protein degradation of mutant thyroglobulin, whereas degradation of a nonglycosylated misfolded secretory protein substrate appears unaffected. Knockdown of ATP2C1 is not associated with elevated steady state levels of ER chaperone proteins, nor does it block cellular activation of either the PERK, ATF6, or Ire1/XBP1 portions of the ER stress response. However, deficiency of ATP2C1 renders cells hypersensitive to ER stress. These data point to the important contributions of the Golgi-localized ATP2C1 protein in homeostatic maintenance throughout the secretory pathway.

Dysfunction and death of spinal cord neurons are critical determinants of neurological deficits in various pathological conditions, including multiple sclerosis (MS) and spinal cord injury. Yet, the molecular mechanisms underlying neuronal/axonal damage remain undefined. Our previous studies raised the possibility that a decrease in the levels of plasma membrane calcium ATPase isoform 2 (PMCA2), a major pump extruding calcium from neurons, promotes neuronal pathology in the spinal cord during experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and after spinal cord trauma. However, the causal relationship between alterations in PMCA2 levels and neuronal injury was not well established. We now report that inhibition of PMCA activity in purified spinal cord neuronal cultures delays calcium clearance, increases the number of nonphosphorylated neurofilament H (SMI-32) immunoreactive cells, and induces swelling and beading of SMI-32-positive neurites. These changes are followed by activation of caspase-3 and neuronal loss. Importantly, the number of spinal cord motor neurons is significantly decreased in PMCA2-deficient mice and the deafwaddler(2J), a mouse with a functionally null mutation in the PMCA2 gene. Our findings suggest that a reduction in PMCA2 level or activity leading to delays in calcium clearance may cause neuronal damage and loss in the spinal cord.

The contribution of Ca(2+) release from intracellular stores to the rise in the free cytosolic Ca(2+) concentration ([Ca(2+)](c)) triggered by Ca(2+) influx was investigated in mouse pancreatic beta-cells. Depolarization of beta-cells by 45 mm K(+) (in the presence of 15 mm glucose and 0.1 mm diazoxide) evoked two types of [Ca(2+)](c) responses: a monotonic and sustained elevation; or a sustained elevation superimposed by a transient [Ca(2+)](c) peak (TCP) (40-120 s after the onset of depolarization). Simultaneous measurements of [Ca(2+)](c) and voltage-dependent Ca(2+) current established that the TCP did not result from a larger Ca(2+) current. Abolition of the TCP by thapsigargin and its absence in sarco-endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3) knockout mice show that it is caused by Ca(2+) mobilization from the endoplasmic reticulum. A TCP could not be evoked by the sole depolarization of beta-cells but required a rise in [Ca(2+)](c) pointing to a Ca(2+)-induced Ca(2+) release (CICR). This CICR did not involve inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) because it was resistant to heparin. Nor did it involve ryanodine receptors (RyRs) because it persisted after blockade of RyRs with ryanodine, and was not mimicked by caffeine, a RyR agonist. Moreover, RyR1 and RyR2 mRNA were not found and RyR3 mRNA was only slightly expressed in purified beta-cells. A CICR could also be detected in a limited number of cells in response to glucose. Our data demonstrate, for the first time in living cells, the existence of an atypical CICR that is independent from the IP(3)R and the RyR. This CICR is prominent in response to a supraphysiological stimulation with high K(+), but plays little role in response to glucose in non-obese mouse pancreatic beta-cells.

In yeast the P-type Ca(2+)-ATPase of the Golgi apparatus, Pmr1p, is the most important player in calcium homeostasis. In Kluyveromyces lactis KlPMR1 inactivation leads to pleiotropic phenotypes, including reduced N-glycosylation and altered cell wall morphogenesis. To study the physiology of K. lactis when KlPMR1 was inactivated microarrays containing all Saccharomyces cerevisiae coding sequences were utilized. Alterations in O-glycosylation, consistent with the repression of KlPMT2, were found and a terminal N-acetylglucosamine in the O-glycans was identified. Klpmr1Delta cells showed increased expression of PIRs, proteins involved in cell wall maintenance, suggesting that responses to cell wall weakening take place in K. lactis. We found over-expression of KlPDA1 and KlACS2 genes involved in the Acetyl-CoA synthesis and down-regulation of KlIDP1, KlACO1, and KlSDH2 genes involved in respiratory metabolism. Increases in oxygen consumption and succinate dehydrogenase activity were also observed in mutant cells. The described approach highlighted the unexpected involvement of KlPMR1 in energy-yielding processes.

Mutations in the ATP2A1 gene, encoding isoform 1 of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1), are one cause of Brody disease, characterized in humans by exercise-induced contraction of fast twitch (type II) skeletal muscle fibers. In an attempt to create a model for Brody disease, the mouse ATP2A1 gene was targeted to generate a SERCA1-null mutant mouse line. In contrast to humans, term SERCA1-null mice had progressive cyanosis and gasping respiration and succumbed from respiratory failure shortly after birth. The percentage of affected homozygote SERCA1(-/-) mice was consistent with predicted Mendelian inheritance. A survey of multiple organs from 10-, 15-, and 18-day embryos revealed no morphological abnormalities, but analysis of the lungs in term mice revealed diffuse congestion and epithelial hypercellularity and studies of the diaphragm muscle revealed prominent hypercontracted regions in scattered fibers and increased fiber size variability. The V(max) of Ca(2+) transport activity in mutant diaphragm and skeletal muscle was reduced by 80% compared with wild-type muscle, and the contractile response to electrical stimulation under physiological conditions was reduced dramatically in mutant diaphragm muscle. No compensatory responses were detected in analysis of mRNAs encoding other Ca(2+) handling proteins or of protein levels. Expression of ATP2A1 is largely restricted to type II fibers, which predominate in normal mouse diaphragm. The absence of SERCA1 in type II fibers, and the absence of compensatory increases in other Ca(2+) handling proteins, coupled with the marked increase in contractile function required of the diaphragm muscle to support postnatal respiration, can account for respiratory failure in term SERCA1-null mice.

It is known that plasma membrane Ca(2+)-transporting ATPases (PMCAs) extrude Ca(2+) from the cell and that sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) and secretory pathway Ca(2+)-ATPases (SPCAs) sequester Ca(2+) in intracellular organelles; however, the specific physiological functions of individual isoforms are less well understood. This information is beginning to emerge from studies of mice and humans carrying null mutations in the corresponding genes. Mice with targeted or spontaneous mutations in plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) are profoundly deaf and have a balance defect due to the loss of PMCA2 in sensory hair cells of the inner ear. In humans, mutations in SERCA1 (ATP2A1) cause Brody disease, an impairment of skeletal muscle relaxation; loss of one copy of the SERCA2 (ATP2A2) gene causes Darier disease, a skin disorder; and loss of one copy of the SPCA1 (ATP2C1) gene causes Hailey-Hailey disease, another skin disorder. In the mouse, SERCA2 null mutants do not survive to birth, and heterozygous SERCA2 mutants have impaired cardiac performance and a high incidence of squamous cell cancers. SERCA3 null mutants survive and appear healthy, but endothelium-dependent relaxation of vascular smooth muscle is impaired and Ca(2+) signaling is altered in pancreatic beta cells. The diversity of phenotypes indicates that the various Ca(2+)-transporting ATPase isoforms serve very different physiological functions.

The LDB1 gene of Saccharomyces cerevisiae was identified by complementation of the ldb1 mutant phenotype with a genomic library. We found that the ldb1 defect is complemented by PMR1 which codes for the yeast secretory pathway/Golgi Ca(2+)/Mn(2+)-ATPase. Besides that, the analysis of a null mutation of the PMR1 gene revealed a phenotype identical to that of ldb1 mutant. Thus, LDB1 must be considered a synonym of PMR1.

Two sarcoendoplasmic reticulum Ca(2+)-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to beta-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in beta-cell cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) regulation, insulin secretion, and glucose homeostasis. Basal [Ca(2+)](c) was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [Ca(2+)](c) that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. Ca(2+) mobilization by acetylcholine was normal in SERCA3-deficient beta-cells. In contrast, [Ca(2+)](c) oscillations resulting from intermittent glucose-stimulated Ca(2+) influx and [Ca(2+)](c) transients induced by pulses of high K(+) were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases Ca(2+) that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [Ca(2+)](c) regulation in beta-cells. SERCA3 becomes operative when [Ca(2+)](c) rises and is required for normal [Ca(2+)](c) oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice.

We analyzed mutant mice showing behavioral defects such as severe tremor, up-and-down and side-to-side wriggling of neck without coordination, and found that the gene causing the defects was located between 46 and 60.55 centimorgans (cM) on the mouse chromosome 6. In this region, nucleotide transition of the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) gene was found, which caused a glutamic acid to change into lysine. Since PMCA2 is expressed in the cerebellum and plays an important role to maintain the homeostasis of the intracellular Ca2+ as a Ca2+ pump, the behavioral defect can be ascribed to the impairment of Ca2+ regulation in neurons of the cerebellum. To confirm the defect of Ca2+ homeostasis in the mutant mice, we measured high K+-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in the cerebellar neurons. Contrary to our expectation, the extent of the [Ca2+]i increase in all the regions tested in the cerebellar slice was far smaller than that of the wild type mice, while the resting [Ca2+]i remained almost unaltered. The rate of rise in [Ca2+]i during high K+-induced depolarization was significantly reduced, and the extrusion rate of increased [Ca2+]i was also reduced. These results suggested that voltage-gated Ca2+ channels were down-regulated in the mutant mice in order to regulate [Ca2+]i toward the normal homeostasis. The behavioral defects may be ascribed to the down-regulated Ca2+ homeostasis since dynamic changes in [Ca2+]i are important for various neuronal functions.

The discovery and biochemical characterization of the secretory pathway Ca(2+)-ATPase, PMR1, in Saccharomyces cerevisiae, has paved the way for identification of PMR1 homologues in many species including rat, Caenorhabditis elegans, and Homo sapiens. In yeast, PMR1 has been shown to function as a high affinity Ca(2+)/Mn(2+) pump and has been localized to the Golgi compartment where it is important for protein sorting, processing, and glycosylation. However, little is known about PMR1 homologues in higher organisms. Loss of one functional allele of the human gene, hSPCA1, has been linked to Hailey-Hailey disease, characterized by skin ulceration and improper keratinocyte adhesion. We demonstrate that expression of hSPCA1 in yeast fully complements pmr1 phenotypes of hypersensitivity to Ca(2+) chelators and Mn(2+) toxicity. Similar to PMR1, epitope-tagged hSPCA1 also resides in the Golgi when expressed in yeast or in chinese hamster ovary cells. (45)Ca(2+) transport by hSPCA1 into isolated yeast Golgi vesicles shows an apparent Ca(2+) affinity of 0.26 microm, is inhibitable by Mn(2+), but is thapsigargin-insensitive. In contrast, heterologous expression of vertebrate sarcoplasmic reticulum and plasma membrane Ca(2+)-ATPases in yeast complement the Ca(2+)- but not Mn(2+)-related phenotypes of the pmr1-null strain, suggesting that high affinity Mn(2+) transport is a unique feature of the secretory pathway Ca(2+)-ATPases.

Susceptibility to noise-induced hearing loss (NIHL) is poorly understood at the genetic level. Mice homozygous for a null mutation in the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) gene are deaf (Kozel et al., 1998). PMCA2 is expressed on outer hair cell stereocilia (Furuta et al., 1998). Fridberger et al. (1998) observed that the outer hair cell cytoplasmic Ca2+ concentration rises following acoustic overstimulation. We hypothesized that Pmca2+/- mice may be more susceptible to NIHL. Since the auditory brainstem response (ABR) thresholds of Pmca2+/- mice vary with the presence of a modifier locus (Noben-Trauth et al., 1997), Pmca2+/- mice were outcrossed to normal hearing CAST/Ei mice. The pre-exposure ABR thresholds of the resulting Pmca2+/+ and Pmca2+/- siblings were indistinguishable. Groups of these mice were exposed to varying intensities of broadband noise, and ABR threshold shifts were calculated. Fifteen days following an 8 h, 113 dB noise exposure, the Pmca2+/- mice displayed significant (P < or = 0.0007) permanent threshold shifts at 16 and 32 kHz that were 15 or 25 dB greater than those observed in Pmca2+/+ littermates. Pmca2 may be the first gene with a known mutated protein product that confers increased susceptibility to NIHL.

The cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase gene (ATP2A2) encodes the following two different protein isoforms: SERCA2a (muscle-specific) and SERCA2b (ubiquitous). We have investigated whether this isoform specificity is required for normal cardiac function. Gene targeting in mice successfully disrupted the splicing mechanism responsible for generating the SERCA2a isoform. Homozygous SERCA2a(-/-) mice displayed a complete loss of SERCA2a mRNA and protein resulting in a switch to the SERCA2b isoform. The expression of SERCA2b mRNA and protein in hearts of SERCA2a(-/-) mice corresponded to only 50% of wild-type SERCA2 levels. Cardiac phospholamban mRNA levels were unaltered in SERCA2a(-/-) mice, but total phospholamban protein levels increased 2-fold. The transgenic phenotype was characterized by a approximately 20% increase in embryonic and neonatal mortality (early phenotype), with histopathologic evidence of major cardiac malformations. Adult SERCA2a(-/-) animals (adult phenotype) showed a reduced spontaneous nocturnal activity and developed a mild compensatory concentric cardiac hypertrophy with impaired cardiac contractility and relaxation, but preserved beta-adrenergic response. Ca(2+) uptake levels in SERCA2a(-/-) cardiac homogenates were reduced by approximately 50%. In isolated cells, relaxation and Ca(2+) removal by the SR were significantly reduced. Comparison of our data with those obtained in mice expressing similar cardiac levels of SERCA2a instead of SERCA2b indicate the importance of the muscle-specific SERCA2a isoform for normal cardiac development and for the cardiac contraction-relaxation cycle.

Darier's disease (DD) is a high penetrance, autosomal dominant mutation in the ATP2A2 gene, which encodes the SERCA2 Ca2+ pump. Here we have used a mouse model of DD, a SERCA2(+/-) mouse, to define the adaptation of Ca2+ signaling and Ca2+-dependent exocytosis to a deletion of one copy of the SERCA2 gene. The [Ca2+]i transient evoked by maximal agonist stimulation was shorter in cells from SERCA2(+/-) mice, due to an up-regulation of specific plasma membrane Ca2+ pump isoforms. The change in cellular Ca2+ handling caused approximately 50% reduction in [Ca2+]i oscillation frequency. Nonetheless, agonist-stimulated exocytosis was identical in cells from wild-type and SERCA2(+/-) mice. This was due to adaptation in the levels of the Ca2+ sensors for exocytosis synaptotagmins I and III in cells from SERCA2(+/-) mice. Accordingly, exocytosis was approximately 10-fold more sensitive to Ca2+ in cells from SERCA2(+/-) mice. These findings reveal a remarkable plasticity and adaptability of Ca2+ signaling and Ca2+-dependent cellular functions in vivo, and can explain the normal function of most physiological systems in DD patients.

Work performing heart preparations from hypercontractile, phospholamban deficient mouse hearts showed no change in parameters of contraction or relaxation in response to isoproterenol stimulation. Thus, the aim of the present study was to investigate whether or not changes at the level of the contractile apparatus occur in addition to the altered expression of Ca2+-regulating proteins observed in these mouse models, e.g., phospholamban, ryanodine receptors. Triton-X skinned fiber preparations from phospholamban deficient (n = 9) and wild-type (n = 10) mice were used and the Ca2+-activated force as well as the myosin ATPase-activity were simultaneously measured. The tension dependent ATPase-activity was unchanged in phospholamban deficient animals when compared to controls. The SERCA 2a-inhibitor cyclopiazonic acid did not affect myosin ATPase-activity in this system. The Ca2+-sensitivity of Ca2+-activated force and myosin ATPase were unchanged as well. Comparison of the concentrations needed to achieve half maximal activation of the myosin ATPase-activity and force demonstrated that the Ca2+-sensitivity of the myosin ATPase was higher compared to the Ca2+-sensitivity of tension development. This holds true for phospholamban deficient mice (EC50 ATPase: 0.9 +/- 0.2 micromol/l; tension: 1.7 +/- 0.3 micromol/l; p < 0.001) and wild-type controls (1.1 +/- 0.01 micromol/l; 2.2 +/- 0.4 micromol/l; p < 0.01). The myosin ATPase-activity and force were correlated to each other in both, phospholamban deficient mice and controls and did not change at submaximal Ca2+ concentrations. The ATPase/ force-ratio, as a parameter of tension cost, was similar in either phospholamban deficient mice or controls. Thus, the present study provides evidence that at the level of the contractile proteins regulation of Ca2+-activated force and energy demand of force development are not altered in phospholamban deficient mice with enhanced myocardial performance. At the level of the regulation of crossbridge interaction, no adaptive or compensatory mechanisms have been initiated by ablation of phospholamban.

Adductor pollicis muscle function of a 21-year-old man with genetically confirmed Brody's disease (sarcoplasmic reticulum [SR] -Ca2+ATPase deficiency) was investigated to study the possible effects of reduced SR-Ca2+ATPase activity on muscle relaxation and force production. Following maximal electrical activation of the ulnar nerve, tetanic muscle half-relaxation time was greater in the patient (246 +/- 10 ms) than control subjects (97 +/- 4 ms, n = 8). During repetitive activation, there was a similar decline in maximal shortening velocity in the patient and controls, indicating a comparable reduction in cross-bridge cycling rate. The finding that the slowing of relaxation was greater in the patient (329 ms versus 138 +/- 20 ms) suggests that there was a further reduction of SR-Ca2+ATPase activity in the patient's muscle during fatigue. Following a voluntary contraction, involuntary activity of the antagonist muscles facilitated force decline and masked the impaired relaxation in the patient. This antagonist-induced relaxation indicates that it might be difficult to establish impaired muscle relaxation with voluntary contractions.

Abnormalities in intracellular Ca2+ handling play a crucial role in the pathogenesis of heart failure. The reduced capacity of failing human myocardium to restore low resting Ca2+ levels during diastole has been explained by the impairment of Ca2+ uptake into the sarcoplasmic reticulum (SR) via the SR Ca2+ATPase. It is unclear whether Ca2+ATPase function, protein levels, and mRNA steady-state levels correspond to one other, and whether the cause of heart failure, namely idiopathic dilated or ischemic cardiomyopathy, produces different changes. The present study examined SR Ca2+ATPase activity and both mRNA and protein levels of SR Ca2+ATPase, phospholamban, and Gi alpha 2 in left ventricular myocardium from eight nonfailing hearts, from eight hearts of patients with idiopathic dilated cardiomyopathy (DCM), and from six hearts from patients with ischemic cardiomyopathy (ICM). Compared to nonfailing myocardium, the activity of the SR Ca2+ATPase was significantly reduced in failing myocardium from patients with DCM (36%, P < 0.01) and from patients with ICM (37%, P < 0.001). Significantly lower levels of SR Ca2+ATPase mRNA levels (55% and -56%, P < 0.001 for DCM and ICM, respectively) and phospholamban mRNA (45%, P < 0.001 for DCM; 31%, P < 0.05 for ICM) were observed in failing than in nonfailing myocardium. In contrast, no significant changes were observed at the level of proteins, Gi alpha 2 mRNA and protein levels were both significantly increased in failing myocardium. There were no differences between idiopathic dilated and ischemic cardiomyopathy concerning the examined parameter. It is concluded that reduced SR Ca2+ATPase activity contributes to an altered intracellular Ca2+ handling by the SR in both dilated and ischemic cardiomyopathic hearts. However, changes in SR Ca2+ATPase and phospholamban steady-state protein levels do not contribute to these alterations. The dissociation between protein and mRNA levels provides evidence for a posttranscriptional or post-translational regulation of these proteins. The observed alterations are not dependent on the underlying cause of end-stage heart failure.

Brody's disease, i.e., sarcoplasmic reticulum (SR) Ca(2+)-dependent Mg(2+)-ATPase (Ca(2+)-ATPase) deficiency, is a rare inherited disorder of skeletal muscle function. Pseudo-myotonia is the most important clinical feature. SR Ca(2+)-ATPase and Ca2+ homeostasis are examined in m. quadriceps and/or cultured muscle cells of controls and 10 patients suffering from Brody's disease. In both m. quadriceps and cultured muscle cells of patients, the SR Ca(2+)-ATPase activity is decreased by approximately 50%. However, the concentration of SR Ca(2+)-ATPase and SERCA1 are normal. SERCA1 accounts for 83 and 100% of total SR Ca(2+)-ATPase in m. quadriceps and cultured muscle cells, respectively. This implies a reduction of the molecular activity of SERCA1 in Brody's disease. The cytosolic Ca2+ concentration ([Ca2+]i) at rest and the increase of [Ca2+]i after addition of acetylcholine are the same in cultured muscle cells of controls and patients. The half-life of the maximal response, however, is raised three times in the pathological muscle cells. Addition of dantrolene or verapamil after the maximal response accelerates the restoration of the [Ca2+]i in these muscle cells. The differences in Ca2+ handling disappear by administration of dantrolene or verapamil concomitantly with acetylcholine. The reduced Ca2+ re-uptake from the cytosol presumably due to structural modification(s) of SERCA1 may explain the pseudo-myotonia in Brody's disease. Single cell measurements suggest a beneficial effect of dantrolene or verapamil in treating patients suffering from Brody's disease.

Since defective regulation of ion transport could initiate or contribute to the abnormal cellular function in myotonic dystrophy (MyD), Na+/K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase were examined in skeletal muscle and cultured skeletal muscle cells of controls and MyD patients. Na+/K(+)-ATPase was investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphate (3-O-MFPase). SR Ca(2+)-ATPase was analysed by e.l.i.s.a., Ca(2+)-dependent phosphorylation and its activities with ATP and 3-O-methylfluorescein phosphatase (3-O-MFP). In MyD muscle the K(+)-dependent 3-O-MFPase activity and the activity and concentration of SR Ca(2+)-ATPase were decreased by 40%. In cultured muscle cells from MyD patients the activities as well as the concentration of both Na+/K(+)-ATPase and SR Ca(2+)-ATPase were reduced by about 30-40%. The ouabain-binding constant and the molecular activities, i.e. catalytic-centre activities with ATP or 3-O-MFP, of Na+/K(+)-ATPase and SR Ca(2+)-ATPase were similar in muscle as well as in cultured cells from both controls and MyD patients. Thus the decreased activity of both ATPases in MyD muscle is caused by a reduction in the number of their molecules. To check whether the deficiency of ATP-dependent ion pumps is a general feature of the pathology of MyD, we examined erythrocytes from the same patients. In these cells the Ca2+ uptake rate and the Ca(2+)-ATPase activity were lower than in controls, but the Ca(2+)-ATPase concentration was normal. Thus the reduced Ca(2+)-ATPase activity is caused by a decrease in the molecular activity of the ion pump. The Na+/K(+)-ATPase activity is also lower in erythrocytes of MyD patients. It is concluded that the observed alterations in ion pumps may contribute to the pathological phenomena in the muscle and other tissues in patients with MyD.

A patient with exertional rhabdomyolysis and continuously elevated serum creatine kinase (CK) was investigated. The known causes of recurrent attacks of rhabdomyolysis were ruled out by appropriate histochemical and biochemical investigations. During ischaemic exercise tests an abnormal K(+)-efflux from exercising muscles was observed. The patient was found to have a deficiency of muscular Ca(2+)-ATPase. Dantrolene sodium therapy gave relief of muscle symptoms and improved the exercise tolerance. Both the CK level and the K(+)-efflux in ischaemic forearm testing became normal on this therapy.

Double negative (DN) T cells expanding in peripheral lymphoid tissues in mice bearing lymphoproliferation (lpr) gene are generally unresponsive to mitogens, antigens, and anti-T cell receptor (TCR) or anti-CD3 monoclonal antibodies (mAb). In response to the stimulation with 0.125-5.0 microM ionomycin, control T cells sustained an increase in intracellular free calcium ([Ca2+]i), while DN lpr T cells showed a gradual fall following initial rapid increase in [Ca2+]i. Such gradual fall in [Ca2+]i was overcome by the addition of endoplasmic and sarcoplasmic reticulum Ca(2+)-ATPase inhibitor or high dose (10 microM) of ionomycin. The requirement of high concentration of calcium ionophore for the sustained increase of [Ca2+]i in lpr DN T cells is due to dysfunction of Ca(2+)-ATPase pump.

Hypophosphatemia revealed in patients with acute disorders of cerebral circulation was one of the causes of a decrease of the content of 2,3-DPG, ATP in red blood cells and of a reduction of Ca2(+)-ATPase activity in red blood cell membranes. In the given group of patients, hypophosphatemia was provoked by complete parenteral feeding, hypocapnia and by loss of phosphorus with urine and congestive gastrointestinal contents.

A pronounced deficiency of Mg2+ and Ca2+-dependent ATPase and of secretory granules and vesicles was demonstrated in the seminal plasma of a patient with infertility problems (person A) and with a lowered serum testosterone level. Both total ejaculates and different fractions of split-ejaculates were examined on repeated occasions. Divalent cations, fructose and protein were also determined in most of the samples. The low activity of the Mg2+ and Ca2+-dependent ATPase and the ultrastructure of the split-ejaculates of the seminal plasma of person A contrasted sharply against the high activity of the Mg2+ and Ca2+-dependent ATPase and the ultrastructure of the split-ejaculates of the seminal plasma of another individual (person B). The latter displayed a normal Mg2+ and Ca2+-dependent ATPase activity as well as an ordinary representation of the secretory granules and vesicles. The findings on the ATPase system in the seminal plasma of person A together with the lowered levels of divalent cations in the order Ca2+ less than Mg2+ less than Zn2+ are proposed to reflect a defective functioning of the prostate gland in person A.

Four male patients from two families were first seen with impaired skeletal muscle relaxation that rapidly worsened during exercise. Muscle biopsies from 2 patients were examined by appropriate biochemical and microscopic immunocytochemical techniques. The adenosine triphosphate (ATP)-dependent Ca2+ transport rate was extremely low in a particulate membrane fraction of skeletal muscle, and there was also a marked reduction of the concentration of 100-kD phosphoprotein, corresponding to Ca2+-ATPase of sarcoplasmic reticulum, in muscle microsomes. The concentration of immunoreactive Ca2+-ATPase of sarcoplasmic reticulum was markedly reduced on immunoblots. Evaluation by microscopic immunocytochemical techniques, using one polyclonal and two monoclonal antibodies against sarcoplasmic reticulum Ca2+ transport protein, revealed that the severe reduction of immunoreactive Ca2+-ATPase was limited to the histochemical type 2 fibers. The deficiency of the Ca2+ transport protein in the sarcoplasmic reticulum of type 2 fibers, which may be the primary expression of a presumed gene defect, can explain the impaired muscle relaxation of the patients. This disease appears to be a clinically, electromyographically, and biochemically distinct metabolic myopathy.

The role of endomyocardial biopsy may be restricted to the diagnosis of myocarditis. On the other hand, a broad utilization of the biopsy technique may be championed by those who believe that the light and electron microscopic characteristics of the tissue are important and that the biochemical features of the tissue may hold important clues to the diagnosis and therapy of dilated cardiomyopathy. The popularity of endomyocardial biopsy arises from the ease and safety of the procedure. Light microscopy of the tissue is useful not only to diagnose myocarditis but also to discover infiltrative cardiomyopathies and to characterize the dilated cardiomyopathies by the amount of fibrosis and cellular hypertrophy. Electron microscopy supplies more detailed information on the myofibrils, nuclei, tubular structures, mitochondria, glycogen, and lipofuscin. At present, these data are clinically applicable only to the anthracycline cardiomyopathies. On an investigational basis, subcellular characteristics may help us better understand the etiology and pathogenesis of dilated cardiomyopathy. The greatest promise lies in the biochemical assessment of tissue, which may uncover single or multiple biochemical abnormalities in heart failure. Although the clinical usefulness of endomyocardial biopsy is quite restricted at present, its future looks quite promising.

31P Magnetic resonance spectroscopy studies were carried out in vivo on skeletal muscle of a patient with verapamil-responsive, chronic, progressive post-exertional muscle pain. A sister suffered from a similar complaint. The results showed that the muscle: (1) decreased its high energy phosphate content more rapidly than normal during exercise, indicating either increased utilisation or decreased production of ATP; (2) acidified more rapidly than normal during exercise suggesting an increased glycolytic rate; (3) continued in some studies to acidify markedly during the first minute after exercise, indicating that glycolysis remained active into the recovery period; (4) had phosphocreatine and ADP recovery rates consistent with normal rates of oxidative phosphorylation. On the basis of these results, it was proposed that the patient suffers from a defect in Ca2+ handling in the muscle. Subsequently, direct measurement of Ca2+-ATPase activity in the sarcoplasmic reticulum fraction from a muscle biopsy sample showed that the activity of this enzyme was reduced by about 90%.

The activities of Na+,K+-, and Ca2+-ATPases were determined in plasma membranes obtained from livers of rats treated acutely and chronically with CCl4. Twenty-four hours after a single oral dose of CCl4 the ATPases decreased below 50% of control values. The activity of Ca2+-ATPase returned to normal after 4 days, and Na+,K+-ATPase activity returned to normal values after 12 days. One week after initiation of the chronic intraperitoneal treatment with CCl4, the Na,K+-ATPase decreased to 40% of control values and continued to decrease further until reaching values below 1%. Ca2+-ATPase followed a pattern similar to that obtained with Na+,K+-ATPase, except that the decrease was not as severe. Colchicine treatment prevented the modifications in ATPases when given simultaneously with CCl4 and reverted the alterations in ATPase activities of the CCl4-cirrhotic animals. Because ATPases are known to be modulated by the lipid composition of the membrane, we also determined the cholesterol to phospholipid ratio in all the isolated membranes. The ratios were increased in membranes with low ATPase activity due to an increase in the total concentration of cholesterol. Plasma membranes of cirrhotic rats treated with colchicine showed a low concentration of cholesterol, a decreased cholesterol to phospholipid ratio, and Na+,K+-ATPase activity was almost normal. When plasma membranes of cirrhotic rats were fused with phosphatidyl serine-containing liposomes, the cholesterol to phospholipid ratio decreased and the ATPase activity increased. The ATPase activity of normal plasma membranes decreased below 20% of control values when enriched with cholesterol. Our results suggest that the decrease in the plasma membrane Na+,K+-ATPase activity of the cirrhotic rat is due in part to an increase in its cholesterol concentration and in the cholesterol to phospholipid ratio.