OBJECTIVETo study the expression of plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) in spiral ganglion cell (SGC) from inner ear of newborn rats and further check PMCA2 splice variants at site A and C.

METHODSSpiral ganglion tissues isolated from cochlea of newborn rats (P3-P4) were cultured and identified in vitro. The cochlea of newborn rats (P3-P4) were isolated and cut into frozen sections. The expression of PMCA2 was detected by immunofluorescence analyses. The SGC cultured in 4 wells of the 6-well culture plate were collected and the total RNA was extracted by Trizol and reverse transcribed to cDNA. The site A and C splice variants of PMCA2 were respectively checked by nested PCR and common PCR.

RESULTSThe SGC grew well with good refraction and showed positive immunoreactivity for neuronal marker NF-200. Strong green fluorescence could be seen in cytomembrane, cytoplasm and neuritis, as well showing SGC immunoreactivity for PMCA2 antibody. In the cochlear sections, the spiral ganglion tissues were strongly stained by PMCA2. PMCA2z was present at splice site A, but PMCA2b and PMCA2c were present at splice site C.

CONCLUSIONSGC from newborn rats strongly expresses PMCA2 and different splice variants are present at PMCA2 splice site A and C.

Animals ; Female ; Male ; Plasma Membrane Calcium-Transporting ATPases ; metabolism ; Protein Isoforms ; metabolism ; RNA Splice Sites ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; cytology ; metabolism

OBJECTIVETo investigate the association between polymorphism of ATP2B1 gene, its interaction with smoking and susceptibility of essential hypertension.

METHODSA case-control study was conducted to elucidate the role of ATP2B1 gene variants related to the risk of essential hypertension. Genomic DNA was extracted from peripheral blood leukocytes, using the QIAamp DNA Mini Kit (QIAGEN,Germany). Two SNPs, - rs17249754 and rs6253, were examined on 1 280 patients and 1 010 healthy controls, using a Snapshot method. Statistical analyses were performed with SPSS Windows software (version 19.0;SPSS, Chicago, IL).

RESULTSA significant difference was found in rs17249754 allele frequency between cases and controls (OR = 1.223, 95%CI: 1.083-1.381, P = 0.001). After adjustment for age, sex, BMI, smoking and drinking, the difference was still statistically significant (OR = 1.212, 95%CI:1.070-1.373, P = 0.003). In addition, data from genotype distribution analysis under different models showed that appeared significant associations between ATP2B1 gene polymorphism and essential hypertension (additive model OR = 1.469, 95%CI: 1.121-1.925, P = 0.005; dominant model OR = 1.324, 95%CI:1.029-1.704, P = 0.029;recessive model OR = 1.123, 95%CI:1.031-1.223, P = 0.008). In this study, the proportion of smokers in cases was significantly higher than that in controls (P = 0.005), but no associations between rs17249754-smoking interaction and essential hypertension were found after the adjustment for gender, age, BMI and alcohol consumption (OR = 1.024, 95% CI:0.614-1.707).

CONCLUSIONOur research findings showed that the polymorphism of ATP2B1 gene rs17249754 was significantly associated with the incidence of essential hypertension in Han population of northeastern China. However, the interaction between rs17249754 and smoking did not seem to have contributed to the occurrence of the essential hypertension.

Aged ; Case-Control Studies ; Essential Hypertension ; Female ; Humans ; Hypertension ; etiology ; Male ; Middle Aged ; Plasma Membrane Calcium-Transporting ATPases ; genetics ; Polymorphism, Single Nucleotide ; Smoking

Intracellular Ca²⁺ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (H₂O₂) on cytosolic Ca²⁺ accumulation in mouse parotid acinar cells. Intracellular Ca²⁺ levels were slowly elevated when 1 mM H₂O₂ was perfused in the presence of normal extracellular Ca²⁺. In a Ca²⁺-free medium, 1 mM H₂O₂ still enhanced the intracellular Ca²⁺ level. Ca²⁺ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H₂O₂. On the other hand, 10 mM H₂O₂ induced more rapid Ca²⁺ accumulation and facilitated Ca²⁺ entry from extracellular fluid. Ca²⁺ refill into intracellular Ca²⁺ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca²⁺ release from Ca²⁺ store was not affected by 1 mM H₂O₂ in permeabilized cells. Ca²⁺ efflux through plasma membrane Ca²⁺-ATPase (PMCA) was markedly blocked by 1 mM H₂O₂ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H₂O₂-induced Ca²⁺ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H₂O₂ under pathological conditions may lead to cytosolic Ca²⁺ accumulation and that the primary mechanism of H₂O₂-induced Ca²⁺ accumulation is likely to inhibit Ca²⁺ efflux through PMCA rather than mobilize Ca²⁺ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.
Acinar Cells* ; Animals ; Antioxidants ; Calcium ; Catalase ; Cell Membrane* ; Cytosol ; Dithiothreitol ; Extracellular Fluid ; Hand ; Hydrogen Peroxide* ; Hydrogen* ; Inositol 1,4,5-Trisphosphate ; Ions ; Manganese ; Mice* ; Perfusion ; Plasma Membrane Calcium-Transporting ATPases ; Plasma* ; Reactive Oxygen Species

OBJECTIVETo investigate the location and distribution of plasma membrane Ca²⁺ -ATPase isoform 2(PMCA2) in the cochleas of C57BL/6J mice at various ages (4w, 14w, 22w, 45w), and to reveal the relationship of PMCA2 and age-related hearing loss (AHL).

METHODSThe distribution of PMCA2 in the cochleas of C57BL/6J mice was detected by immunohistochemistry at various ages (4w, 14w, 22w, 45w). Real-time polymerase chain reaction (Rt-PCR) was used to detect the level of PMCA2 mRNA in the cochleas of C57BL/6J mice at the ages of 4, 14, 22 and 45 weeks old respectively. Using SPSS17.0 software for statistical analysis.

RESULTSPMCA2 was mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 was also observed in spiral ligament. Hair cells missed and the number of spiral ganglion cells reduced with age. Expression of PMCA2 in the cochleas of C57BL/6J mice also showed age-related decreasing. The results of Rt-PCR demonstrated the expression of mRNA of gene (Atp2b2) at 14 weeks age was significantly less than 4 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 22 weeks age was significantly less than 14 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 45 weeks age was significantly less than 14 week-old mice cochlears (P<0.01).

CONCLUSIONSPMCA2 is mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 is also observed in spiral ligament. The expression of PMCA2 demonstrates an age-related decrease with age. The mRNA expression level of PMCA2 gene(Atp2b2) in the cochleas of C57BL/6J mice displayed an age-related decrease. PMCA2 transporters may play a critical role in maintaining the normal morphology of the inner ear and it may be related to AHL.

Aging ; Animals ; Cochlea ; enzymology ; Hair Cells, Auditory ; metabolism ; Isoenzymes ; metabolism ; Mice ; Mice, Inbred C57BL ; Plasma Membrane Calcium-Transporting ATPases ; metabolism ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Spiral Ganglion ; cytology ; metabolism ; Stria Vascularis ; cytology ; metabolism

The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.
Animals ; Brain ; metabolism ; Calcium ; metabolism ; Cells, Cultured ; Cerebellum ; cytology ; Fluorescence Resonance Energy Transfer ; HEK293 Cells ; Humans ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type I ; metabolism ; PDZ Domains ; Plasma Membrane Calcium-Transporting ATPases ; metabolism ; Protein Interaction Maps ; Protein Isoforms ; metabolism ; Rats ; Rats, Sprague-Dawley