Codeine-induced hepatic injury is via oxido-inflammatory damage and caspase-3-mediated apoptosis.

Codeine (3-methylmorphine) is a known analgesic, antitussive, and antidiarrheal drug that is often abused for recreational purposes. It is metabolized in the liver via the cytochrome P450 system and thus hypothesized to induce hepatic injury especially when misused. Thus, the present study aimed at investigating changes in liver function, hepatic enzyme biomarker, proton pumps, antioxidant status, free radicals and TNF-α levels, as well as caspase 3 activities and hepatic DNA fragmentation after 6 weeks of oral codeine administration. Twenty-one male rabbits were randomized into 3 groups (n = 7). The control group had 1 ml of normal saline, while the low-dose and high-dose codeine groups received 4 and 10 mg/kg b.w of codeine respectively daily. The codeine-treated animals had significantly lower levels of serum proteins, increased activities of hepatic enzyme biomarkers and caspase 3, raised hepatic concentrations of free radicals and TNF-α, as well as increased hepatic DNA fragmentation. Codeine treatment also led to a significant decline in hepatic weight, activities of hepatic enzymatic antioxidant, Na+-K+-ATPase and Ca2+-ATPase. These alterations were more pronounced in high-dose codeine treated animals than in the low-dose group. Histopathological study showed moderate fatty degeneration of hepatic parenchyma, infiltration of the portal tract by inflammatory cells with dense collagen fibre deposition in codeine-treated animals. The present study revealed that codeine induced liver injury and hepatic DNA damage via caspase 3-dependent signaling by suppressing hepatic antioxidant status and enhancing free radical and TNF-α generation.

Investigating the functional link between TMEM165 and SPCA1.

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1.

Yeast-Based Screen to Identify Natural Compounds with a Potential Therapeutic Effect in Hailey-Hailey Disease.

The term orthodisease defines human disorders in which the pathogenic gene has orthologs in model organism genomes. Yeasts have been instrumental for gaining insights into the molecular basis of many human disorders, particularly those resulting from impaired cellular metabolism. We and others have used yeasts as a model system to study the molecular basis of Hailey-Hailey disease (HHD), a human blistering skin disorder caused by haploinsufficiency of the gene ATP2C1 the orthologous of the yeast gene PMR1. We observed that K. lactis cells defective for PMR1 gene share several biological similarities with HHD derived keratinocytes. Based on the conservation of ATP2C1/PMR1 function from yeast to human, here we used a yeast-based assay to screen for molecules able to influence the pleiotropy associated with PMR1 deletion. We identified six compounds, Kaempferol, Indirubin, Lappaconite, Cyclocytidine, Azomycin and Nalidixic Acid that induced different major shape phenotypes in K. lactis. These include mitochondrial and the cell-wall morphology-related phenotypes. Interestingly, a secondary assay in mammalian cells confirmed activity for Kaempferol. Indeed, this compound was also active on human keratinocytes depleted of ATP2C1 function by siRNA-treatment used as an in-vitro model of HHD. We found that Kaempferol was a potent NRF2 regulator, strongly inducing its expression and its downstream target NQO1. In addition, Kaempferol could decrease oxidative stress of ATP2C1 defective keratinocytes, characterized by reduced NRF2-expression. Our results indicated that the activation of these pathways might provide protection to the HHD-skin cells. As oxidative stress plays pivotal roles in promoting the skin lesions of Hailey-Hailey, the NRF2 pathway could be a viable therapeutic target for HHD.

Plasma Membrane-Localized Calcium Pumps and Copines Coordinately Regulate Pollen Germination and Fertility in Arabidopsis.

Calcium plays an important role in plant growth, development, and response to environmental stimuli. Copines are conserved plasma membrane-localized calcium-binding proteins which regulate plant immune responses and development. In this study, we found that copine proteins BON2 and BON3, the paralogs of BON1, physically interact with calcium pumps ACA8 and ACA10 in Arabidopsis. Notably, ACA9, the closest homologue of ACA8 and ACA10 functioning in pollen tube growth, interacts with all three copines. This is consistent with the protein⁻protein interactions between the two protein families, the aca8, aca10, aca8/aca10, bon1/2/3 mutants as well as aca9 mutant exhibited defects on pollen germination and seed production. Taken together, plasma membrane-localized interacting calcium pumps and copines coordinately control pollen tube growth, likely through manipulating calcium efflux.

Zebrafish slc30a10 deficiency revealed a novel compensatory mechanism of Atp2c1 in maintaining manganese homeostasis.

Recent studies found that mutations in the human SLC30A10 gene, which encodes a manganese (Mn) efflux transporter, are associated with hypermanganesemia with dystonia, polycythemia, and cirrhosis (HMDPC). However, the relationship between Mn metabolism and HMDPC is poorly understood, and no specific treatments are available for this disorder. Here, we generated two zebrafish slc30a10 mutant lines using the CRISPR/Cas9 system. Compared to wild-type animals, mutant adult animals developed significantly higher systemic Mn levels, and Mn accumulated in the brain and liver of mutant embryos in response to exogenous Mn. Interestingly, slc30a10 mutants developed neurological deficits in adulthood, as well as environmental Mn-induced manganism in the embryonic stage; moreover, mutant animals had impaired dopaminergic and GABAergic signaling. Finally, mutant animals developed steatosis, liver fibrosis, and polycythemia accompanied by increased epo expression. This phenotype was rescued partially by EDTA- CaNa2 chelation therapy and iron supplementation. Interestingly, prior to the onset of slc30a10 expression, expressing ATP2C1 (ATPase secretory pathway Ca2+ transporting 1) protected mutant embryos from Mn exposure, suggesting a compensatory role for Atp2c1 in the absence of Slc30a10. Notably, expressing either wild-type or mutant forms of SLC30A10 was sufficient to inhibit the effect of ATP2C1 in response to Mn challenge in both zebrafish embryos and HeLa cells. These findings suggest that either activating ATP2C1 or restoring the Mn-induced trafficking of ATP2C1 can reduce Mn accumulation, providing a possible target for treating HMDPC.

Manganese-induced turnover of TMEM165.

TMEM165 deficiencies lead to one of the congenital disorders of glycosylation (CDG), a group of inherited diseases where the glycosylation process is altered. We recently demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi manganese homeostasis defect and that Mn2+ supplementation was sufficient to rescue normal glycosylation. In the present paper, we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn2+ concentrations, TMEM165 was degraded in lysosomes. Remarkably, while the variant R126H was sensitive upon manganese exposure, the variant E108G, recently identified in a novel TMEM165-CDG patient, was found to be insensitive. We also showed that the E108G mutation did not abolish the function of TMEM165 in Golgi glycosylation. Altogether, the present study identified the Golgi protein TMEM165 as a novel Mn2+-sensitive protein in mammalian cells and pointed to the crucial importance of the glutamic acid (E108) in the cytosolic ELGDK motif in Mn2+-induced degradation of TMEM165.

Dicranostiga leptopodu (Maxim.) Fedde extracts attenuated CCl4-induced acute liver damage in mice through increasing anti-oxidative enzyme activity to improve mitochondrial function.

Dicranostiga Leptodu (Maxim.) fedde (DLF), a poppy plant, has been reported have many benefits and medicinal properties, including free radicals scavenging and detoxifying. However, the protective effect of DLF extracts against carbon tetrachloride (CCl4)-induced damage in mice liver has not been elucidated. Here, we demonstrated that DLF extracts attenuated CCl4-induced liver damage in mice through increasing anti-oxidative enzyme activity to improve mitochondrial function. In this study, the mice liver damage evoked by CCl4 was marked by morphology changes, significant rise in lipid peroxidation, as well as alterations of mitochondrial respiratory function. Interestingly, pretreatment with DLF extracts attenuated CCl4-induced morphological damage and increasing of lipid peroxidation in mice liver. Additionally, DLF extracts improved mitochondrial function by preventing the disruption of respiratory chain and suppression of mitochondrial Na+K+-ATPase and Ca2+-ATPase activity. Furthermore, administration with DLF extracts elevated superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) levels and maintained the balance of redox status. This results showed that toxic protection effect of DLF extracts on mice liver is mediated by improving mitochondrial respiratory function and keeping the balance of redox status, which suggesting that DLF extracts could be used as potential toxic protection agent for the liver against hepatotoxic agent.

Two novel ATP2C1 mutations in patients with Hailey-Hailey disease and a literature review of sequence variants reported in the Chinese population.

Hailey-Hailey disease (HHD) is an autosomal dominant disorder in which the ATP2C1 gene has been implicated. Many mutations of this gene have been detected in HHD patients. To analyze such mutations in HHD and summarize all those identified in Chinese patients with this disease, we examined four familial and two sporadic cases and searched for case reports and papers by using the Chinese Biological Medicine Database and PubMed. HHD diagnoses were made based on clinical features and histopathological findings. Polymerase chain reaction and direct sequencing of the ATP2C1 gene were performed using blood samples from HHD patients, unaffected family members, and 120 healthy individuals. Three mutations were identified, including the recurrent mutation c.2126C>T (p.Thr709Met), and two novel missense mutations, c.2235_2236insC (p.Pro745fs*756) and c.689G>A (p.Gly230Asp). Considering our data, 81 different mutations have now been reported in Chinese patients with HHD. In cases of misannotation or duplication, previously published mutations were renamed according to a complementary DNA reference sequence. These mutations are scattered throughout the ATP2C1 gene, with no evident hotspots or clustering. It is of note that some reported "novel" mutations were in fact found to be recurrent. Our findings expand the range of known ATP2C1 sequence variants in this disease.

Aerobic metabolism and cardiac activity in the descendants of zebrafish exposed to pyrolytic polycyclic aromatic hydrocarbons.

The increase of anthropogenic activities on coastal areas induces discharges of polycyclic aromatic hydrocarbons (PAHs) in aquatic ecosystem. PAH effects depend not only on their concentration and the way of contamination but also on the different developmental stages of the organism. Zebrafish were exposed to relevant concentration of pyrolytic PAHs from the first meal (i.e., 5-day post fertilization, dpf) to mature adults. Parental effect of this type of exposure was evaluated through the assessment of aerobic metabolic scope, cardiac frequency, and cardiac mRNA expression on larval and/or embryo progeny of contaminated fish. Our results suggest that cardiac frequency increased in larval descendants of fish exposed to the environmental concentration of pyrolytic PAHs (i.e., 5 ng.g(-1) of food), while a lack of effect on aerobic metabolism in 5 dpf larvae was highlighted. A surexpression of mRNA related to the cardiac calcium transporting ATPase atp2a2a, a protein essential for contraction, is in accordance with this increasing cardiac frequency. Even if cardiac development genes cmlc1 and tnnt2a were not affected at early life stages tested, complementary work on cardiac structure could be interesting to better understand PAHs action.

Successful treatment with narrow-band UVB therapy for a case of generalized Hailey-Hailey disease with a novel splice-site mutation in ATP2C1 gene.

Hailey-Hailey disease (HHD) is a rare autosomal dominant disorder characterized by development of recurrent blisters, erosions, and crustations in the intertriginous areas. The treatment of HHD is often challenging, and various methods have been tried. We report here a case of a 45-year-old woman with a generalized form of HHD that was dramatically improved and well controlled by narrow-band ultraviolet B phototherapy.

Arabidopsis ACA7, encoding a putative auto-regulated Ca(2+)-ATPase, is required for normal pollen development.

Microgametogenesis is a complex process that involves numerous well-coordinated cell activities, ending with the production of pollen grains. Pollen development has been studied at the cytological level in Arabidopsis and other plant species, where its temporal time course has been defined. However, the molecular mechanism underlying this process is still unclear, since a relative small number of genes and/or processes have been identified as essential for pollen development. We have designed a methodology to select candidate genes for functional analysis, based on transcriptomic data obtained from different stages of pollen development. From our analyses, we selected At2g22950 as a candidate gene; this gene encodes a protein belonging to the auto-regulated Ca(2+)-ATPase family, ACA7. Microarray data indicate that ACA7 is expressed exclusively in developing pollen grains, with the highest level of mRNA at the time of the second pollen mitosis. Our RT-PCR experiments showed that ACA7 mRNA is detected exclusively in developing flowers. Confocal microscopy experiments showed a plasma membrane localization for the recombinant GFP:ACA7 protein. We identified two different insertional mutant lines, aca7-1 and aca7-2; plants from both mutant lines displayed a normal vegetative development but showed large amounts of dead pollen grains in mature flowers assayed by Alexander's staining. Histological analysis indicated that abnormalities are detected after the first pollen mitosis and we found a strong correlation between ACA7 mRNA accumulation and the severity of the phenotype. Our results indicate that ACA7 is a plasma membrane protein that has an important role during pollen development, possibly through regulation of Ca(2+) homeostasis.

[Hepatic energy metabolism on rats by six traditional Chinese medicine with hot property].

OBJECTIVE: To investigate the common rule of hepatic energy metabolism on rats by six traditional Chinese medicine (TCM) with hot property. METHOD: The activity of Na(+)-K(+)-ATPase, Ca2(+)-ATPase and succinate hydrogenase (SDH), the content of hepatic glycogen and the mRNA expression of hepatic uncoupling protein2 (UCP2) were measured after the rats were administrated with water extracts of Radix Aconiti Lateralis Preparata, Rhizoma Zingiberis, Rhizoma Alpiniae Officinarum, Pericarpium Zanthoxyli and Cortex Cinnamomi, Fructus Evodiae at the dose of 10.5, 8.4, 6.0, 4.0, 3.5, 4.2 g x kg(-1). respectively in 30 days, twice a day. RESULT: The activity of Na+(-)K(+)-ATPase has been increased by the six TCM and the statistical significance has been observed in Rhizoma Alpiniae Officinarum, Pericarpium Zanthoxyli, Fructus Evodiae groups. The raising tendency of Ca2(+)-ATPase activity has been observed by the six TCM and the statistical significance has been obtained in Rhizoma Alpiniae Officinarum group. The activity of SDH has been increaseded by six TCM while statistical significance has been observed except in five groups of the six groups except in Radix Aconiti Lateralis Preparata group. The content of hepatic glycogen has been decreased significantly by six TCM. No signiticant change of the mRNA expression of UCP2 has been found. CONCLUSION: TCM has good effects on hepatic energy metabolism by raising the activity of mitochondria SDH to increase the production of ATP and by increasing the activity of Na(+)-K+)-ATPase, Ca2(+)-ATPase to increase the consumption of ATP.

High sensibility to reactivation by acidic lipids of the recombinant human plasma membrane Ca2+-ATPase isoform 4xb purified from Saccharomyces cerevisiae.

The human plasma membrane Ca2+ pump (isoform 4xb) was expressed in Saccharomyces cerevisiae and purified by calmodulin-affinity chromatography. Under optimal conditions the recombinant enzyme (yPMCA) hydrolyzed ATP in a Ca2+ dependent manner at a rate of 15 micromol/mg/min. The properties of yPMCA were compared to those of the PMCA purified from human red cells (ePMCA). The mobility of yPMCA in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of ePMCA. Both enzymes achieved maximal activity when supplemented with acidic phospholipids. However, while ePMCA in mixed micelles of phosphatidylcholine-detergent had 30% of its maximal activity, the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA but the activity in the presence of phosphatidylcholine improved by partially removing the detergent. The reactivation of the detergent solubilized yPMCA required specifically acidic lipids and, as judged by the increase in the level of phosphoenzyme, it involved the increase in the amount of active enzyme. These results indicate that the function of yPMCA is highly sensitive to delipidation and the restitution of acidic lipids is needed for a functional enzyme.

Hepatic cirrhosis, dystonia, polycythaemia and hypermanganesaemia--a new metabolic disorder.

We report a new constellation of clinical features consisting of hypermanganesaemia, liver cirrhosis, an extrapyramidal motor disorder and polycythaemia in a 12 year-old girl born to consanguineous parents. Blood manganese levels were >3000 nmol/L (normal range <320 nmol/L) and MRI revealed signal abnormalities of the basal ganglia consistent with manganese deposition. An older brother with the same phenotype died at 18 years, suggesting a potentially lethal, autosomal recessive disease. This disorder is probably caused by a defect of manganese metabolism with the accumulation of manganese in the liver and the basal ganglia similar to the copper accumulation in Wilson disease. In order to assess the genetic basis of this syndrome we investigated two candidate genes: ATP2C2 and ATP2A3 encoding the manganese-transporting calcium-ATPases, SPCA2 and SERCA3, respectively. Genotyping of the patient and the family for microsatellite markers surrounding ATP2C2 and ATP2A3 excluded these genes. The patient was found to be heterozygous for both gene loci. Despite the unknown pathophysiology, we were able to develop a successful treatment regime. Chelation therapy with disodium calcium edetate combined with iron supplementation is the treatment of choice, lowering blood manganese levels significantly and improving clinical symptoms.

Sequence, annotation and developmental expression of the sea urchin Ca(2+) -ATPase family.

Whole genome sequence data permit the study of protein families regulating cellular homeostasis during development. Here we present a study of the sea urchin Ca(2+)-ATPases made possible by the Sea Urchin Genome Sequencing Project. This is of potential interest because adult sea urchins, their gametes and embryos live in the relatively high Ca(2+) concentration of 10 mM. Three Ca(2+)-ATPases regulate Ca(2+) levels in animal cells: plasma membrane Ca(2+)-ATPase (PMCA), sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and secretory pathway Ca(2+)-ATPase (SPCA). The primary structures of Sp-PMCA and Sp-SERCA in the sea urchin, Strongylocentrotus purpuratus (Sp), have been published. Here, we present the primary structure of Sp-SPCA, which is 912 amino acids and has 66% identity and 80% similarity to human SPCA1. Southern blots and genome analysis show that Sp-SPCA is a single copy gene. Each Sp Ca(2+)-ATPase is highly conserved when compared to its human ortholog, indicating that human and sea urchin share structurally similar energy driven Ca(2+) homeostasis mechanisms that have been maintained throughout the course of deuterostome evolution. Annotation using the assembled sea urchin genome reveals that Sp-SPCA, Sp-PMCA and Sp-SERCA have 23, 17 and 24 exons. RT-Q-PCR shows that transcripts of Sp-SPCA are at low levels compared to Sp-PMCA and Sp-SERCA. Gradual increases in Sp-PMCA and Sp-SERCA mRNA begin at the 18 hour hatched blastula stage and peak 4-5-fold higher by 25 h at the mid to late blastulae stage.

[Effects of acupuncture at the points of the Pericardium Meridian on the activity of myocardial Ca2+-ATPase and gene expression in the rat of myocardial ischemia and reperfusion injury].

OBJECTIVE: To investigate the mechanism of acupuncture at "Neiguan" (PC 6) and "Ximen" (PC 4) of the Pericardium Meridian in treatment of myocardial ischemia. METHODS: Rats were randomly divided into 5 groups, sham-operation group, ischemia-reperfusion model group, Neiguan acupuncture group, Ximen acupuncture group and Zhigou acupuncture group. Electroacupuncture (EA) was given at corresponding acupoints for 20 min in the later 3 groups, followed by ligation of the left anterior descending branch of the coronary artery and 40 min later, acupuncture was given at the points for another 20 min, reperfusion for 60 min, with ECG monitoring. Then the myocardial tissue sample (below the ligation site) of the left cardiac ventricle was taken for preparation of sarcoplasmic reticulum, and determination of Ca2+-ATPase activity according to quantitative analysis of phosphorum. The relative mRNA levels were determined by Northerm Bolt analysis. RESULTS: In the acupuncture groups, both the activities of Ca2+-ATPase and the gene expressions increased significantly as compared with the model group (all P < 0.01). CONCLUSION: The points of the Pericardium Meridian can obviously improve the Ca2+-ATPase activity and the gene expressions, reduce the degree of myocardial ischemia and reperfusion injury, and strength myocardial functions.

Identification and characterization of calcium and manganese transporting ATPase (PMR1) gene of Pichia pastoris.

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Pichia pastoris. The entire P. pastoris PMR1 gene (PpPMR1) codes a protein of 924 amino acids. Sequence analysis of the PpPMR1 cDNA and the genomic DNA revealed that there is no intron in the coding region. The putative gene product contains all of the conserved regions observed in P-type ATPases and exhibits 66.2%, 60.3% and 50.6% identity to Pichia angusta (Hansenula polymorpha), Saccharomyces cerevisiae PMR1 and human ATP2C1 gene products, respectively. A pmr1 null mutant strain of P. pastoris exhibited growth defects in media with the addition of EGTA, but with supplementation of Ca2+ to a calcium-deficient media reversed the growth defects of the mutant strain. Manganese reversed the growth defects of the mutant strain; however, the cell growth was not as profound as the Ca2+ -supplemented media. The results demonstrated that the P. pastoris gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+/Mn2+ -ATPase. The DNA sequence of the P. pastoris PMR1 gene has been submitted to GenBank under Accession No. DQ239958.

Conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase mutants: effect of mutations either at Ca(2+)-binding site II or at tryptophan 552 in the cytosolic domain.

By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.

Regulatory role of ovarian sex hormones in calcium uptake activity of cardiac sarcoplasmic reticulum.

Alterations in the intracellular Ca2+ handling in cardiomyocytes may underlie the cardiac dysfunction observed in the ovarian sex hormone-deprived condition. To test the hypothesis that ovarian sex hormones had a significant role in the cardiac intracellular Ca2+ mobilization, the sarcoplasmic reticulum (SR) Ca2+ uptake and SR Ca2+-ATPase (SERCA) activity were determined in 10-wk ovariectomized rat hearts. With the use of left ventricular homogenate preparations, a significant suppression of maximum SR Ca2+ uptake activity, but with an increase in SR Ca2+ responsiveness, was demonstrated in ovariectomized hearts. In parallel measurements of SERCA activity in SR-enriched membrane preparations from ovariectomized hearts, a suppressed maximum SERCA activity with a leftward shift in the relationship between pCa (-log molar free Ca2+ concentration) and SERCA activity was also detected. A significant downregulation of SERCA proteins and reduction in the SERCA mRNA level were observed in association with suppressed maximum SERCA activity. While there were no changes in total phospholamban and phosphorylated Ser16 phospholamban levels, a decrease in phosphorylated Thr17 phospholamban as well as an increase in the suprainhibitory, monomeric form of phospholamban stoichiometry was found. Estrogen and progesterone supplementations were equally effective in preventing changes in ovariectomized hearts. Our data showed for the first time that female sex hormones played an important role in the regulation of the cardiac SR Ca2+ uptake. Under hormone-deficient conditions, there was an adaptive response of SERCA that escaped the regulatory effect of phospholamban.

The expression of genes for transepithelial calcium-transporting proteins in the bovine duodenum.

Intestinal epithelial cells contain calcium-binding proteins and Ca2+-transporting adenosine triphosphatase (Ca2+-ATPase), which play important roles in intestinal Ca transport. However, the factors that affect the expression of these transepithelial Ca-transporting proteins in dairy cattle are unknown. In this study, a semi-quantitative reverse transcription polymerase chain reaction was used to determine the expression of the mRNAs for intestinal Ca-binding protein calbindin-D9k (CaBP9k), two isoforms of plasma membrane Ca2+-ATPase (PMCA1 and PMCA4), and vitamin D receptor (VDR) in duodenal tissue samples from 20 non-lactating, non-pregnant Holstein dairy cattle (0.4-135.9 months old). The correlations between the expressions of transepithelial Ca-transporting proteins, the ages of the cattle, and the presence of several plasma components were evaluated. The duodenal CaBP9k mRNA content had a significant negative correlation with age and positive correlations with plasma inorganic phosphorus (iP) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) concentrations. The PMCA1 mRNA content was negatively correlated with the plasma Ca concentration. The duodenal PMCA4 mRNA content was correlated negatively with the plasma iP. The VDR mRNA content had a positive correlation with the plasma magnesium concentration.