PMCA Protein Research Articles

Estrogen and androgen regulation of plasma membrane calcium pump activity in immortalized distal tubule kidney cells

The ATP dependent plasma membrane calcium pump (PMCA) is a regulator of renal calcium reabsorption. The effect of estrogen and dihydrotestosterone to increase the activity of the PMCA in membrane vesicle preparations from a distal tubule cell line was investigated. 17β Estradiol (10 −10 M) increased PMCA activity (1.5±0.2-fold increase compared to control) with 24 h, but not 1 or 5 h, of exposure, an effect that was blocked by the addition of the estrogen antagonist ICI 164384. α Estradiol did not increase PMCA activity. Dihydrotestosterone (10 −11 M ) resulted in a dose dependent increase in PMCA activity (1.5±0.1-fold increase compared to control) with 24 h, but not 1 or 5 h, of exposure, an effect that was blocked by the androgen receptor agonist flutamide. Testosterone (10 −5 M) also increased PMCA activity (1.9±0.3-fold increase compared to control). Neither estrogen nor dihydrotestosterone increased PMCA protein expression in MDBK cells, indicating that these hormones increase PMCA activity by regulating PMCA activity rather than PMCA expression. These results demonstrate receptor dependent stimulatory effects of both estrogen and dihydrotestosterone to increase PMCA activity. and have significance for our understanding of estrogen and androgen deficient states on calcium transport.

Plasma membrane calcium ATPase overexpression in arterial smooth muscle increases vasomotor responsiveness and blood pressure.

In vascular smooth muscle cells (SMCs), several mechanisms act in concert to regulate the intracellular calcium concentration [Ca2+]i, which may in turn affect vascular tone. One such mechanism is the extrusion of Ca2+ by the plasma membrane calcium ATPase (PMCA). To address, in particular, the role of the neuronal nitric oxide synthase (nNOS)-associating isoform PMCA4b in regulating vascular tone, a doxycycline-responsive transgene for human PMCA4b was overexpressed in arterial SMCs of mice. Overexpression of hPMCA4b resulted in a 2-fold increase in total aortic PMCA4 protein expression and significant real-time RT-PCR-documented differences in the levels of endogenous mouse PMCA1, PMCA4, SERCA2, and IP3R1 gene expression in arterial SMCs. Whereas no significant difference in basal [Ca2+]i or Ca2+ sensitivity was observed in vascular SMCs or mesenteric arteries, respectively, from hPMCA4b-overexpressing versus control mice, hPMCA4b-overexpressing mice revealed a reduced set-point and increased extent of myogenic response and heightened sensitivity to vasoconstrictors. Treatment of arteries with an nNOS inhibitor resulted in a reduced set-point and increased extent of the myogenic response in control but not hPMCA4b-overexpressing mice. Moreover, aortic SMCs from hPMCA4b-overexpressing mice exhibited reduced levels of cGMP under both basal and phenylephrine-stimulated conditions. These changes were associated with significant doxycycline-reversible elevations in blood pressure. Taken together, these data show that overexpression of hPMCA4b in arterial SMCs increases vascular reactivity and blood pressure, an effect that may be mediated in part by negative regulation of nNOS.

Upregulation of cardiac cell plasma membrane calcium pump in a canine model of Chagas disease.

We have previously demonstrated that cardiac myocytes isolated from the hearts of adult dogs develop rapid repetitive cytosolic Ca2+ transients, membrane depolarization, and cell contraction by mobilization of sarcoplasmic reticulum Ca2+ stores when exposed to a soluble factor from the trypomastigotes of Trypanosoma cruzi. These findings led us to investigate the regulatory mechanisms of cytosolic Ca2+ in cardiac tissues from dogs chronically infected with T. cruzi. Expression of the plasma membrane calcium pump (PMCA) RNA and protein was determined by Northern and Western blotting, respectively, followed by densitometric analyses. A 642-bp PMCA 1b complementary DNA probe derived from canine epicardial tissue hybridized to 2 major transcripts (7.3 and 5.3 kb) in canine epicardium. Expression of the dominant transcript (7.3 kb) was 77% greater in cardiac tissues obtained from dogs with chronic T. cruzi infection (140 days after inoculation) in comparison with constitutive expression levels in normal dogs. Monoclonal antibody 5F10, known to recognize all isoforms of the PMCA, was used to detect expression of the PMCA protein in epicardial tissue. Expression of a 142-kDa protein was increased by 58% in the cardiac tissues of infected dogs when compared with those from uninfected dogs. To establish a link between the upregulation of PMCA in dogs chronically infected with Chagas disease and the ventricular-based arrhythmias and myocardial failure that occur during this stage of disease both in dogs and humans, further study will be required.

Calcineurin-independent regulation of plasma membrane Ca2+ ATPase-4 in the vascular smooth muscle cell cycle

Calcineurin mediates repression of plasma membrane Ca2+-ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which 45Ca efflux rates decreased 50%, fura 2-AM-based intracellular Ca2+ concentrations ([Ca2+]i) increased twofold, and real-time RT-PCR and Western blot revealed a approximately 40% decrease in PMCA4 expression levels from G0 to G1/S in the cell cycle, where PMCA4 constituted approximately 20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G0 to G1/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G1/S. By contrast, Ca2+-independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G0 to G1/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G1/S-associated repression of PMCA4. These data show that G1/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca2+]i through alternate targets.

Antisense-Inhibition of Plasma Membrane Ca2+ Pump Induces Apoptosis in Vascular Smooth Muscle Cells

The effect of antisense oligodeoxynucleotides (ODNs) of plasma membrane Ca(2+)-pumping ATPase (PMCA) on rat aortic vascular smooth muscle cells (VSMCs) in primary culture was examined. More than 80% of the PMCA expressed in cultured VSMCs was the PMCA-1B subtype. Exposed to antisense ODNs against PMCA-1, not only the expression of the PMCA protein but also mRNA of PMCA-1B was diminished in a concentration-dependent manner. Extracellular Na(+)-independent (45)Ca(2+) efflux catalyzed via PMCA was inhibited with antisense ODNs. Both the resting and ionomycin- or ATP-stimulated levels of intracellular Ca(2+) were increased by antisense ODNs. Furthermore, prolonged treatment with antisense ODNs caused apoptosis in VSMCs. The occurrence of apoptosis was inhibited by FK506, a potent immunosuppressant. These results demonstrate that the PMCA was specifically inhibited by antisense ODNs and suggest that PMCA plays an important role in regulation of intracellular Ca(2+) concentrations, especially at the resting condition to prevent an occurrence of apoptosis that may be induced through the activation of calcineurin.

Calcitriol Upregulates Expression and Activity of the 1b Isoform of the Plasma Membrane Calcium Pump in Immortalized Distal Kidney Tubular Cells

The plasma membrane calcium pump (PMCA) is ubiquitously expressed in calcium transporting epithelia. PMCA is encoded by four distinct genes (PMCA1–4) which can be further posttranscriptionally modified. PMCA1b is the only isoform of PMCA1 expressed in kidney and intestine. Calcitriol upregulates PMCA protein expression and activity and PMCA1 mRNA expression in the intestine. Calcitriol has a similar effect on kidney distal tubule PMCA activity in vivo but the cellular basis for this effect has not been studied. PMCA expression in Madin–Darby bovine kidney (MDBK) cells, a distal kidney tubule cell line, was compared with a proximal tubule (LLC-PK1) and embryonic (HEK 293) kidney cell line. Only MDBK cells express PMCA1b mRNA and PMCA protein. In MDBK cells, calcitriol increased steady state expression of PMCA1b mRNA and protein and upregulated the functional activity of PMCA on calcium transport to a similar degree. Furthermore, calcitriol enhanced PMCA1b mRNA stability. These data are consistent with in vivo localization studies demonstrating the distal kidney tubule localization of PMCA protein. Furthermore, they indicate that calcitriol is an important regulator of PMCA activity in the kidney distal tubule by a pathway that includes translation and posttranscriptional modification of PMCA1b.

The Effect of Ethanol on the Plasma Membrane Calcium Pump Is Isoform-specific

The effect of ethanol has been studied on four different isoforms of the plasma membrane Ca2+-ATPase expressed in Sf9 cells with the help of the baculovirus system. The PMCA2CI protein was maximally activated by 0.5% ethanol, a concentration 8-10 times lower than that needed to obtain the same effect on the PMCA4 protein or on the pump of erythrocyte membranes, which is a mixture of isoforms 1 and 4. Experiments performed with truncated pumps indicated that the stimulation by ethanol was lost if the C-terminal region between Lys1065 and Lys1161, encompassing the calmodulin binding domain, was removed. These observations indicate that the stimulation is the result of a direct interaction of ethanol with the C-terminal regulatory domain of the Ca2+ pump.

Analysis of mRNA expression and cloning of a novel plasma membrane Ca(2+)-ATPase splice variant in human heart.

Four different plasma membrane Ca(2+)-ATPase (PMCA) genes and three sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) genes have been previously cloned and characterized. In this study we have investigated the expression of the mRNA encoding the various PMCA and SERCA proteins in fetal and adult human heart and placenta by the reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cDNA cloning. We have found that PMCA1 and PMCA4 genes were expressed in 8-, 12- and 20-week fetal heart and in adult heart. PMCA2 gene was expressed at low levels in adult heart but was not detected in fetal heart. PMCA3 mRNA was not detected in the heart nor placenta. In contrast, the mRNA encoding SERCA2a, SERCA2b and SERCA3 were expressed in all cardiac developmental stages. Multiple alternatively spliced mRNA transcripts which differ at splice site A and B/C of the PMCA1, PMCA2 and PMCA4 genes were detected in the human heart. Interestingly, a novel tissue specific variant of the PMCA4 gene was detected in both fetal and adult human heart but not in placenta that accounts for about 30% of the total PMCA4 mRNA variant expression. DNA sequence analysis of this novel variant revealed that it corresponds to the equivalent of the PMCA1d variant and accordingly we have named it PMCA4d. We cloned and sequenced eight cDNA inserts encoding for the PMCA1 and PMCA4 variants from a fetal human heart cDNA library confirming that these are the two main PMCA genes expressed in cardiac muscle.

Isoform PMCA3 of the plasma membrane calcium pump (ATPase) is localized in the inner plexiform layer of rat retina

Regulation of cytosolic Ca2+ is of critical importance for maintaining normal cellular function in the nervous system. Control of calcium homeostasis involves the Ca2+-ATPase of plasma membrane (PMCA) and other transporters. The presence of these regulatory molecules may not be a fixed characteristic of cells in a general sense. Rather, each class of cells may utilize a unique combination of transporters to maintain homeostatic control. In addition, unique distributions of transport molecules and their isoforms may exist in individual parts of cells imparting unique transport signatures to specific cell types. PMCA is a major regulator of calcium transport under normal physiological conditions. In order to understand localization of PMCA isoforms in specific cells we have localized specific PMCA mRNAs by in situ hybridization and translated PMCA protein by immunocytochemistry. The present work is focused on localization of PMCA3, an isoform predominantly known to exist in brain and skeletal muscle, but not previously investigated in retina. We previously showed that mRNA encoding rat plasma membrane Ca2+-ATPase isoform PMCA3 was localized in the granule cell layer of the cerebellum.

Plasma membrane Ca(2+)-pump functional specialization in the brain. Complex of isoform expression and regulation by effectors.

The plasma membrane Ca(2+)-pump (PMCA) is a key element in the removal of intracellular Ca2+. A number of PMCA pumps, encoded by a multigenic family and differing in their regulatory domains, also exist in the neuronal cells. We discuss here an idea regarding a new, higher level of specialization of PMCA protein isoforms with different sensitivities toward phospholipids and calmodulin. The idea is based on the kinetic data from PMCA stimulation by acidic phospholipids, with a combination of results describing an alternative RNA splicing at site A and C coding of regulatory domains of protein. The resulting complex modulation of the Ca(2+)-pump underlies the specific cellular requirements for Ca2+ homeostasis in a tissue-selective manner and is regulated by the level and spatial distribution of enzyme isoforms as well as by the level of their regulatory factors. The possible role of PMCA protein in the neuronal injury is also discussed.

A Signal for Endoplasmic Reticulum Retention Located at the Carboxyl Terminus of the Plasma Membrane Ca2+-ATPase Isoform 4CI

The plasma membrane Ca(2+)-ATPase isoform 4b (PMCA4CI) with truncations in the cytoplasmically exposed COOH-terminal tail was expressed in COS and HeLa cells and in Sf9 cells using the baculovirus system. The truncated protein terminating with the acidic sequence Glu1067-Arg1087 was retained within the endoplasmic reticulum (ER), whereas mutants lacking this sequence or having it at a distance from the COOH terminus were delivered to the plasma membrane. Although the truncated protein retained in the endoplasmic reticulum was still able to form a Ca(2+)-dependent phosphoenzyme, it underwent partial degradation. Substitution of glutamic and aspartic residue(s) in the acidic region promoted rescue of the protein to the plasma membrane. The results suggest that the sequence Glu1067-Arg1087 encodes a masked signal for ER retention and for the degradation of the protein. However, its presence at the COOH terminus was not sufficient to induce ER-retention and degradation; when the sequence was attached to the full-length PMCA protein, normal plasma delivery was observed. Evidently, ER retention and degradation required the presence of the sequence in its specific location within the PMCA structure. The degradation of the protein retained in the endoplasmic reticulum occurred through the proteolytic attack at cytoplasmically exposed residues (amino acid sequence 720-750) by a cytoplasmic PEST sequence-related protease different from calpain.

Localization of plasma membrane Ca2+ pump mRNA and protein in human ameloblasts by in situ hybridization and immunohistochemistry.

The distribution of the plasma membrane Ca(2+)-pump (PMCA) proteins in human ameloblasts was examined immunohistochemically using monoclonal antibodies JA8 and 5F10. Further, the distribution of mRNA transcripts derived from two PMCA genes, PMCA-1 and PMCA-4 was examined using in situ hybridization. In rats, the PMCA-1 gene is purported to code for PMCA proteins with a role in maintaining the intracellular Ca2+ levels in nonepithelial cells. Other genes including the PMCA-4 gene may code for PMCA proteins characteristic of Ca2+ transporting epithelia. The present results show immunohistochemical staining in the Tomes processes and plasma membranes of human ameloblasts. Our studies also demonstrate a gradation of expression of the PMCA-1 and PMCA-4 mRNA transcripts which parallels the onset and progression of enamel mineralization. These studies suggest that PMCA proteins in human ameloblasts may function both in intracellular Ca2+ homeostasis and in regulating the vectorial Ca2+ influx into mineralizing enamel.