Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32

Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.     

NCX3 is a major functional isoform of the sodium-calcium exchanger in osteoblasts

The calcium phosphate-based skeleton of vertebrates serves as the major reservoir for metabolically available calcium ions. The skeleton is formed by osteoblasts which first secrete a proteinaceous matrix and then provide Ca++ for the calcification process. The two calcium efflux ports found in most cells are the plasma membrane Ca-ATPase (PMCA) and the sodium-calcium exchanger (NCX). In osteoblasts, PMCA and NCX are located on opposing sides of the cell with NCX facing the mineralizing bone surface. Two isoforms of NCX have been identified in osteoblasts NCX1, and NCX3. The purpose of this study was to determine the extent to which each of the two NCX isoforms support delivery of Ca++ into sites of calcification and to discern if one could compensate for the other. SiRNA technology was used to knockdown each isoform separately in MC3T3-E1 osteoblasts. Osteoblasts in which either NCX1 or NCX3 was impaired were tested for Ca++ efflux using the Ca++ specific fluorophore, fluo-4, in a sodium-dependent calcium uptake assay adapted for image analysis. NCX3 was found to serve as a major contributor of Ca++ translocation out of osteoblasts into calcifying bone matrix. NCX1 had little to no involvement.     

Expression and role of calcium-ATPase pump and sodium-calcium exchanger in differentiated trophoblasts from human term placenta

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are not completely elucidated. Especially, mechanisms of syncytiotrophoblast Ca(2+) extrusion into fetal circulation remain to be established. In the current study we have investigated the characteristics of Ca(2+) efflux in syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblasts isolated from human term placenta. Time-courses of Ca(2+) uptake by differentiated human trophoblasts displayed rapid initial entry (initial velocity (V(i)) of 8.82 +/- 0.86 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid decline of cell-associated (45)Ca(2+) with a V(i) of efflux (V(ie)) of 8.90 +/- 0.96 nmol/mg protein/min. Expression of membrane systems responsible for intracellular Ca(2+) extrusion from differentiated human trophoblast were investigated by RT-PCR. Messenger RNAs of four known isoforms of PMCA (PMCA 1-4) were detected. Messenger RNAs of two cloned human NCX isoforms (NCX1 and NCX3) were also revealed. More specifically, both splice variants NCX1.3 and NCX1.4 were amplified by PCR with total RNA of differentiated human trophoblast cells. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. However, erythrosine B (inhibitor of PMCA) time- and dose-dependently increased cell associated (45)Ca(2+) suggesting a principal role of plasma membrane Ca(2+)-ATPase (PMCA) in the intracellular Ca(2+) extrusion of syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblast cells isolated from human term placenta.     

RNA--induced silencing of the plasma membrane Ca2+-ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

Intraneuronal calcium ([Ca(2+)](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca(2+) transporters. We previously reported that the levels of the plasma membrane Ca(2+)-ATPase (PMCA) and the V(max) for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro--transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca(2+)](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca(2+)](i), slower recovery from KCl-induced Ca(2+) transients, and incomplete return to pre-stimulation Ca(2+) levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca(2+) signaling and enhanced susceptibility to stresses that can alter the regulation of Ca(2+) homeostasis.     

Calcium fluxes in human trophoblast (BeWo) cells: calcium channels,calcium-ATPase,and sodium-calcium exchanger expression

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are poorly understood. In the current study, we have investigated the characteristics of Ca(2+) fluxes in relation with cell Ca(2+) homeostasis in the human placental trophoblast cell line BeWo. Time-courses of Ca(2+) uptake by BeWo cells displayed rapid initial entry (initial velocity (V(i)) of 3.42 +/- 0.35 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid declined of cell-associated (45)Ca(2+) with a V(i) of efflux (Ve(i)) of 3.30 +/- 0.08 nmol/mg protein/min. Further identification of membrane gates for Ca(2+) entry in BeWo cells was carried out. Expression of Ca(2+) transporter/channel CaT1 and L-type alpha(1S) subunit was showed by RT-PCR. However, mRNA for CaT2 channel and L-type alpha(1C) and alpha(1D) subunits were not revealed. Membrane systems responsible for intracellular Ca(2+) extrusion from BeWo cells were also investigated. Plasma membrane Ca(2+)-ATPases (PMCA) and Na/Ca exchangers (NCX) were detected by Western blot in BeWo cells. Expression of specific isoforms of PMCA and NCX was further investigated by RT-PCR. Messenger RNAs of four isoforms of PMCA (PMCA 1-4) were detected. The presence of messenger RNAs of two NCX isoforms (NCX1 and NCX3) was observed. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. Inorganic ions such as cadmium and manganese did not modify the Ca(2+) fluxes, however, barium increased cell-associated (45)Ca(2+) by, in part, by reducing radiolabel exit.     

Expression of the Na(+)/Ca(2+) exchanger in skeletal muscle

The expression of the Na(+)/Ca(2+) exchanger was studied in differentiating muscle fibers in rats. NCX1 and NCX3 isoform (Na(+)/Ca(2+) exchanger isoform) expression was found to be developmentally regulated. NCX1 mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of newborn rats but increased dramatically during the first 2 wk of postnatal life. Immunocytochemical analysis showed that NCX1 was uniformly distributed along the sarcolemmal membrane of undifferentiated rat muscle fibers but formed clusters in T-tubular membranes and sarcolemma of adult muscle. NCX3 appeared to be more uniformly distributed along the sarcolemma and inside myoplasm. In the adult, NCX1 was predominantly expressed in oxidative (type 1 and 2A) fibers of both slow- and fast-twitch muscles, whereas NCX3 was highly expressed in fast glycolytic (2B) fibers. NCX2 was expressed in rat brain but not in skeletal muscle. Developmental changes in NCX1 and NCX3 as well as the distribution of these isoforms at the cellular level and in different fiber types suggest that they may have different physiological roles.     

Dynamic regulation of [Ca2+]i by plasma membrane Ca(2+)-ATPase and Na+/Ca2+ exchange during capacitative Ca2+ entry in bovine vascular endothelial cells.

The dynamic regulation of Ca2+ extrusion by the plasma membrane Ca(2+)-ATPase (PMCA) and Na+/Ca2+ exchange (NCX) was investigated in single cultured calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorimetry to measure cytoplasmic Ca2+ concentration ([Ca2+]i). The quantitative analysis of the recovery from an increase of [Ca2+]i elicited by activation of capacitative Ca2+ entry (CCE) served to characterize kinetic parameters of these Ca2+ extrusion systems in the intact cell. In CPAE cells the PMCA is activated in a Ca(2+)- and time-dependent manner. Full activation of the pump occurs only after [Ca2+]i has been elevated for at least 1 min which results in an increase of the affinity of the pump for Ca2+ and an increase in the apparent maximal extrusion rate (Vmax). Application of calmodulin antagonists W-7 and calmidazolium chloride (compound R 24571) revealed that calmodulin is a major regulator of PMCA activity in vivo. Sequential and simultaneous inhibition of PMCA and NCX suggested that both contribute to Ca2+ extrusion in a non-additive fashion. The activity of one system is dynamically adjusted to compensate for changes in the extrusion rate by the alternative transporter. It was concluded that in vascular endothelial cells, the PMCA functions as a calmodulin-regulated, high-affinity Ca2+ removal system. The contribution by the low-affinity NCX to Ca2+ clearance became apparent at [Ca2+]i > approximately 150 nM under conditions of submaximal activation of the PMCA.     

Silencing or knocking out the Na(+)/Ca(2+) exchanger-3 (NCX3) impairs oligodendrocyte differentiation

Changes in intracellular [Ca(2+)](i) levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation.     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

Relative contribution of the Na(+)/Ca(2+) exchanger, mitochondria and endoplasmic reticulum in the regulation of cytosolic Ca(2+) and catecholamine secretion of bovine adrenal chromaffin cells

The relative importance of mitochondria, the Na(+)/Ca(2+) exchanger (NCX) and the endoplasmic reticulum (ER) in the regulation of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) were examined in bovine chromaffin cells using fura-2 for average [Ca(2+)](i) and amperometry for secretory activity, which reflects the local Ca(2+) concentration near the exocytotic sites. Chromaffin cells were stimulated by a high concentration of K(+) when the three Ca(2+) removal mechanisms were individually or simultaneously inhibited. When the mitochondrial Ca(2+) uptake was inhibited, the [Ca(2+)](i) decayed at a significantly slower rate and the secretory activity was higher than the control cells. The NCX appears to function only in the initial phase of [Ca(2+)](i) decay and when the ER Ca(2+) pump is blocked. Similarly, the ER had a significant effect on the [Ca(2+)](i) decay and on the secretion only when the NCX was blocked. Inhibition of all three mechanisms leads to a substantial delay in [Ca(2+)](i) recovery and an increase in the secretion. The results suggest that the three mechanisms work together in the regulation of the Ca(2+) near the Ca(2+) channels and exocytotic sites and therefore modulate the secretory activity. When Ca(2+) diffuses away from the exocytotic sites, the mitochondrial Ca(2+) uptake becomes the dominant mechanism.     

Allosteric inhibitors of plasma membrane Ca pumps: Invention and applications of caloxins

Plasma membrane Ca2+pumps(PMCA)play a major role in Ca2+homeostasis and signaling by extruding cellular Ca2+with high affinity.PMCA isoforms are encoded by four genes which are expressed differentially in various cell types in normal and disease states.Therefore, PMCA isoform selective inhibitors would aid in delineating their role in physiology and pathophysiology.We are testing the hypothesis that extracellular domains of PMCA can be used as allosteric targets to obtain a novel class of PMCA-specific inhibitors termed caloxins. This review presents the concepts behind the invention of caloxins and our progress in this area.A section is also devoted to the applications of caloxins in literature. We anticipate that isoform-selective caloxins will aid in understanding PMCA physiology in health and disease. With strategies to develop therapeutics from bioactive peptides,caloxins may become clinically useful in car diovascular diseases,neurological disorders,retinopathy,cancer and contraception.     

Glucocorticoids prolong Ca(2+) transients in hippocampal-derived H19-7 neurons by repressing the plasma membrane Ca(2+)-ATPase-1

Calcium ions (Ca(2+)) play an important role in mediating an array of structural and functional responses in cells. In hippocampal neurons, elevated glucocorticoid (GC) levels, as seen during stress, perturb calcium homeostasis and result in altered neuronal excitability and viability. Ligand- and voltage-gated calcium channels have been the presumed targets of hormonal regulation; however, circumstantial evidence has suggested the possibility that calcium extrusion might be an important target of GC regulation. Here we demonstrate that GC-induced repression of the plasma membrane Ca(2+)-ATPase-1 (PMCA1) is an essential determinant of intracellular Ca(2+) levels ([Ca(2+)](i)) in cultured hippocampal H19-7 cells. In particular, GC treatment caused a prolongation of agonist-evoked elevation of [Ca(2+)](i) that was prevented by the expression of exogenous PMCA1. Furthermore, selective inhibition of PMCA1 using the RNA interference technique caused prolongation of Ca(2+) transients in the absence of GC treatment. Taken together, these observations suggest that GC-mediated repression of PMCA1 is both necessary and sufficient to increase agonist-evoked Ca(2+) transients by down-regulating Ca(2+) extrusion mechanisms in the absence of effects on calcium channels. Prolonged exposure to GCs, resulting in concomitant accumulation of [Ca(2+)](i), is likely to compromise neuronal function and viability.     

Partitioning of the plasma membrane Ca2+-ATPase into lipid rafts in primary neurons: effects of cholesterol depletion

Spatial and temporal alterations in intracellular calcium [Ca(2+)](i) play a pivotal role in a wide array of neuronal functions. Disruption in Ca(2+) homeostasis has been implicated in the decline in neuronal function in brain aging and in neurodegenerative disorders. The plasma membrane Ca(2+)-ATPase (PMCA) is a high affinity Ca(2+) transporter that plays a crucial role in the termination of [Ca(2+)](i) signals and in the maintenance of low [Ca(2+)](i) essential for signaling. Recent evidence indicates that PMCA is uniquely sensitive to its lipid environment and is stimulated by lipids with ordered acyl chains. Here we show that both PMCA and its activator calmodulin (CaM) are partitioned into liquid-ordered, cholesterol-rich plasma membrane microdomains or 'lipid rafts' in primary cultured neurons. Association of PMCA with rafts was demonstrated in preparations isolated by sucrose density gradient centrifugation and in intact neurons by confocal microscopy. Total raft-associated PMCA activity was much higher than the PMCA activity excluded from these microdomains. Depletion of cellular cholesterol dramatically inhibited the activity of the raft-associated PMCA with no effect on the activity of the non-raft pool. We propose that association of PMCA with rafts represents a novel mechanism for its regulation and, consequently, of Ca(2+) signaling in the central nervous system.     

Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4.

Modulation of Ca(2+) channels by neurotransmitters provides critical control of neuronal excitability and synaptic strength. Little is known about regulation of the Ca(2+) efflux pathways that counterbalance Ca(2+) influx in neurons. We demonstrate that bradykinin and ATP significantly facilitate removal of action potential-induced Ca(2+) loads by stimulating plasma membrane Ca(2+)-ATPases (PMCAs) in rat sensory neurons. This effect was mimicked in the soma and axonal varicosities by phorbol esters and was blocked by antagonists of protein kinase C (PKC). Reduced expression of PMCA isoform 4 abolished, and overexpression of isoform 4b enhanced, PKC-dependent facilitation of Ca(2+) efflux. This acceleration of PMCA4 underlies the shortening of the action potential afterhyperpolarization produced by activation of bradykinin and purinergic receptors. Thus, isoform-specific modulation of PMCA-mediated Ca(2+) efflux represents a novel mechanism to control excitability in sensory neurons.     

Ca(2+) removal mechanisms in freshly isolated rabbit aortic endothelial cells

Calcium removal from the cytoplasm was investigated in freshly isolated aortic endothelial cells by monitoring changes in intracellular calcium ([Ca(2+)](i)) using ratiometric fura-2 fluorimetry. Blockade of the Na(+)/Ca(2+) exchanger (NCX) by replacement of external sodium with equi-molar N-methyl-D-glutamine (0Na PSS) decreased the removal rate by 52%. Blockade of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) by cyclopiazonic acid (CPA) decreased the removal rate by 50%. Simultaneous application of CPA and 0Na PSS did not reduce the removal rate any further (53%). The lack of additivity of these two procedures, suggests that SERCA and the NCX function in series to lower [Ca(2+)](i). In addition, in the absence of extracellular Ca(2+), removal of external Na(+) markedly reduced the rate of loss of Ca(2+) from the ER further supporting the hypothesis that NCX is functionally linked to ER calcium release channels, and thus, plays an important role in ER calcium unloading. To investigate the mechanism for the coupling of NCX and SERCA, the same protocols as described above were repeated after treating the cells with cytochalasin D, which disrupts the cytoskeleton. This treatment uncoupled the NCX from SERCA, as evidenced by the resulting additive inhibitory effects of application of CPA and removal of extracellular Na(+) on the rate of Ca(2+) removal from the cytoplasm. These data suggest that in endothelial cells NCX and SERCA function in series to remove about half of the free Ca(2+) from the cytosol, while PMCA contributes to the other half of the Ca(2+) removal process.     

Ca(2+) oscillations regulated by Na(+)-Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous [Ca(2+)](i) oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca(2+) from intracellular stores via InsP(3) receptors. In this study, we further investigated functions and contributions of Ca(2+) transporters on plasma membrane to generate [Ca(2+)](i) oscillations. In confocal Ca(2+) imaging experiments, spontaneous [Ca(2+)](i) oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca(2+) free solution and were completely blocked by the application of 0.1mM La(3+). When plasma membrane Ca(2+) pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, [Ca(2+)](i) oscillations were inhibited. Application of Ni(2+) or KBR7943 to block Na(+)-Ca(2+) exchanger (NCX) also inhibited [Ca(2+)](i) oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca(2+) activated outward K(+) currents (I(KCa)) with a conductance of 170+/-21.6pS could be recorded. The amplitudes of I(KCa) and membrane potential (V(m)) periodically fluctuated liked to [Ca(2+)](i) oscillations. These results suggest that in undifferentiated hMSCs both Ca(2+) entry through plasma membrane and Ca(2+) extrusion via PMCAs and NCXs play important roles for [Ca(2+)](i) oscillations, which modulate the activities of I(KCa) to produce the fluctuation of V(m).     

AMPA-mediated excitotoxicity in oligodendrocytes: role for Na(+)-K(+)-Cl(-) co-transport and reversal of Na(+)/Ca(2+) exchanger

We investigated the role of Na(+)-K(+)-Cl(-) co-transporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchanger (NCX(rev)) in glutamate-mediated excitotoxicity in oligodendrocytes obtained from rat spinal cords (postnatal day 6-8). An immunocytochemical characterization showed that these cultures express NKCC1 and Na(+)/Ca(2+) exchanger isoforms 1, 2, and 3 (NCX1, NCX2, NCX3). Exposing the cultures to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) plus cyclothiazide (CTZ) led to a transient rise in intracellular (), which was followed by a sustained overload, NKCC1 phosphorylation, and a NKCC1-mediated Na(+) influx. In the presence of a specific AMPA receptor inhibitor 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), the AMPA/CTZ failed to elicit any changes in . The AMPA/CTZ-induced sustained rise led to mitochondrial Ca(2+) accumulation, release of cytochrome c from mitochondria, and cell death. The AMPA/CTZ-elicited increase, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCX(rev). These data suggest that in cultured oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1-mediated Na(+) influx. The latter triggers NCX(rev) and NCX(rev)-mediated overload and compromises mitochondrial function and cellular viability.     

Differential regulation of the apical plasma membrane Ca(2+) -ATPase by protein kinase A in parotid acinar cells

Cross-talk between intracellular calcium ([Ca(2+)](i)) signaling and cAMP defines the specificity of stimulus-response coupling in a variety of cells. Previous studies showed that protein kinase A (PKA) potentiates and phosphorylates the plasma membrane Ca(2+)-ATPase (PMCA) in a Ca(2+)-dependent manner in parotid acinar cells (Bruce, J. I. E., Yule, D. I., and Shuttleworth, T. J. (2002) J. Biol. Chem. 277, 48172-48181). The aim of this study was to further investigate the spatial regulation of [Ca(2+)](i) clearance in parotid acinar cells. Par-C10 cells were used to functionally isolate the apical and basolateral PMCA activity by applying La(3+) to the opposite side to inhibit the PMCA. Activation of PKA (using forskolin) differentially potentiated apical [Ca(2+)](i) clearance in mouse parotid acinar cells and apical PMCA activity in Par-C10 cells. Immunofluorescence of parotid tissue slices revealed that PMCA1 was distributed throughout the plasma membrane, PMCA2 was localized to the basolateral membrane, and PMCA4 was localized to the apical membrane of parotid acinar cells. However, in situ phosphorylation assays demonstrated that PMCA1 was the only isoform phosphorylated by PKA following stimulation. Similarly, immunofluorescence of acutely isolated parotid acinar cells showed that the regulatory subunit of PKA (RIIbeta) translocated to the apical region following stimulation. These data suggest that PKA-mediated phosphorylation of PMCA1 differentially regulates [Ca(2+)](i) clearance in the apical region of parotid acinar cells because of a dynamic translocation of PKA. Such tight spatial regulation of Ca(2+) efflux is likely important for the fine-tuning of Ca(2+)-dependent effectors close to the apical membrane important for the regulation of fluid secretion and exocytosis.