Plasma membrane Ca(2+)-ATPase associates with the cytoskeleton in activated platelets through a PDZ-binding domain

The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca(2+) in resting platelets. During platelet activation PMCA is phosphorylated transiently on tyrosine residues resulting in inhibition of the pump that enhances elevation of Ca(2+). Tyrosine phosphorylation of many proteins during platelet activation results in their association with the cytoskeleton. Consequently, in the present study we asked if PMCA interacts with the platelet cytoskeleton. We observed that very little PMCA is associated with the cytoskeleton in resting platelets but that approximately 80% of total PMCA (PMCA1b + PMCA4b) is redistributed to the cytoskeleton upon activation with thrombin. Tyrosine phosphorylation of PMCA during activation was not associated with the redistribution because tyrosine-phosphorylated PMCA was not translocated specifically to the cytoskeleton. Because PMCA b-splice isoforms have C-terminal PSD-95/Dlg/ZO-1 homology domain (PDZ)-binding domains, a C-terminal peptide was used to disrupt potential PDZ domain interactions. Activation of saponin-permeabilized platelets in the presence of the peptide led to a significant decrease of PMCA in the cytoskeleton. PMCA associated with the cytoskeleton retained Ca(2+)-ATPase activity. These results suggest that during activation active PMCA is recruited to the cytoskeleton by interaction with PDZ domains and that this association provides a microenvironment with a reduced Ca(2+) concentration.     

Characteristics of a store-operated calcium-permeable channel: sarcoendoplasmic reticulum calcium pump function controls channel gating

We examined the single channel properties and regulation of store-operated calcium channels (SOCC). In human submandibular gland cells, carbachol (CCh) induced flickery channel activity while thapsigargin (Tg) induced burst-like activity, with relatively lower open probability (NP(o)) and longer mean open time. Tg- and CCh-activated channels were permeable to Na(+) and Ba(2+), but not to NMDG, in the absence of Ca(2+). The channels exhibited similar Ca(2+), Na(+), and Ba(2+) conductances and were inhibited by 2-aminoethoxydiphenylborate, xestospongin C, Gd(3+), and La(3+). CCh stimulated flickery activity changed to burst-like activity by (i) addition of Tg, (ii) using Na(+) instead of Ca(2+), (iii) using Ca(2+)-free bath solution, or (iv) buffering [Ca(2+)](i) with BAPTA-AM. Buffering [Ca(2+)](i) induced a 2-fold increase in NP(o) of Tg-stimulated SOCC. Reducing free [Ca(2+)] in the endoplasmic reticulum with the divalent cation chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), induced burst-like channel activity similar to that seen with CCh + Tg. Thus, SOCC is activated by stimulation of muscarinic receptors, inhibition of the sarcoendoplasmic Ca(2+) pump, and lowering [Ca(2+)] in the internal store. Importantly, SOCC activity depends on [Ca(2+)](i) and the free [Ca(2+)] in the internal store. These novel findings reveal that SERCA plays a major role in the gating of SOCC by (i) refilling the internal Ca(2+) store(s) and (ii) decreasing the [Ca(2+)](i)-dependent inhibition.     

Homer proteins accelerate Ca2+ clearance mediated by the plasma membrane Ca2+ pump in hippocampal neurons

The plasma membrane Ca(2+) ATPase (PMCA) is responsible for maintaining basal intracellular Ca(2+) concentration ([Ca(2+)](i)) and returning small increases in [Ca(2+)](i) back to resting levels. The carboxyl terminus of some PMCA splice variants bind Homer proteins; how binding affects PMCA function is unknown. Here, we examined the effects of altered expression of Homer proteins on PMCA-mediated Ca(2+) clearance from rat hippocampal neurons in culture. The kinetics of PMCA-mediated recovery from the [Ca(2+)](i) increase evoked by a brief train of action potentials was determined in the soma of single neurons using indo-1-based photometry. Exogenous expression of Homer 1a, Homer 1c or Homer 2a did not affect PMCA function. However, shRNA mediated knockdown of Homer 1 slowed PMCA mediated Ca(2+) clearance by 28% relative to cells expressing non-silencing shRNA. The slowed recovery rate in cells expressing Homer 1 shRNA was reversed by expression of a short Homer 2 truncation mutant. These results indicate that constitutively expressed Homer proteins tonically stimulate PMCA function in hippocampal neurons. We propose a model in which binding of short or long Homer proteins to the carboxyl terminus of the PMCA stimulates Ca(2+) clearance rate. PMCA-mediated Ca(2+) clearance may be stimulated following incorporation of the pump into Homer organized signaling domains and following induction of the Homer 1a immediate early gene.     

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.     

Carbachol-stimulated transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T(84) cells is mediated by intracellular ca(2+), PYK-2, and p60(src)

Ca(2+)-dependent agonists, such as carbachol (CCh), stimulate epidermal growth factor receptor (EGFR) transactivation and mitogen-activated protein kinase activation in T(84) intestinal epithelial cells. This pathway constitutes an antisecretory mechanism by which CCh-stimulated chloride secretion is limited. Here, we investigated mechanisms underlying CCh-stimulated epidermal growth factor receptor (EGFR) transactivation. Thapsigargin (TG, 2 microM) stimulated EGFR and extracellular signal-regulated kinase (ERK) phosphorylation in T(84) cells. Inhibition of either EGFR or ERK activation, with tyrphostin AG1478 (1 microM) and PD 98059 (20 microM), respectively, potentiated chloride secretory responses to TG, as measured by changes in short-circuit current (I(sc)) across T(84) cells. CCh (100 microM) stimulated tyrosine phosphorylation and association of the Ca(2+)-dependent tyrosine kinase, PYK-2, with the EGFR, which was inhibited by the Ca(2+) chelator, BAPTA (20 microM). The calmodulin inhibitor, fluphenazine (50 microM) inhibited CCh-stimulated PYK-2 association with the EGFR and phosphorylation of EGFR and ERK. CCh also induced tyrosine phosphorylation of p60(src) and association of p60(src) with both PYK-2 and the EGFR. The Src family kinase inhibitor, PP2 (20 nM-20 microM) attenuated CCh-stimulated EGFR and ERK phosphorylation and potentiated chloride secretory responses to CCh. We conclude that CCh-stimulated transactivation of the EGFR is mediated by a pathway involving elevations in intracellular Ca(2+), calmodulin, PYK-2, and p60(src). This pathway represents a mechanism that limits CCh-stimulated chloride secretion across intestinal epithelia.     

The Ca(2+): H(+) coupling ratio of the plasma membrane calcium ATPase in neurones is little sensitive to changes in external or internal pH

To explore the effects of both external and internal pH (pH(o) and pH(i)) on the coupling between Ca(2+) extrusion and H(+) uptake by the PMCA activity in snail neurones H(+) uptake was assessed by measuring surface pH changes (ΔpH(s)) with pH-sensitive microelectrodes while Ba(2+) or Ca(2+) loads were extruded. Ru360 or ruthenium red injection showed that injected Ca(2+) was partly taken up by mitochondria, but Ca(2+) entering through channels was not. External pH was changed using a mixture of three buffers to minimise changes in buffering power. With depolarisation-induced Ca(2+) or Ba(2+) loads the ΔpH(s) were not changed significantly over the pH range 6.5-8.5. With Ca(2+) injections into cells with mitochondrial uptake blocked the ΔpH(s) were significantly smaller at pH 8.5 than at 7.5, but this could be explained in part by the slower rate of activity of the PMCA. Low intracellular pH also changed the ΔpH(s) responses to Ca(2+) injection, but not significantly. Again this may have been due to reduced pump activity at low pH(i). I conclude that in snail neurones the PMCA coupling ratio is either insensitive or much less sensitive to pH than in red blood cells or barnacle muscle.     

Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons.

The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered.     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

Deletions in the acidic lipid-binding region of the plasma membrane Ca2+ pump. A mutant with high affinity for Ca2+ resembling the acidic lipid-activated enzyme

The C-terminal segment of the loop between transmembrane helices 2 and 3 (A(L) region) of the plasma membrane Ca(2+) pump (PMCA) is not conserved in other P-ATPases. Part of this region, just upstream from the third transmembrane domain, has been associated with activation of the PMCA by acidic lipids. cDNAs coding for mutants of the Ca(2+) pump isoform h4xb with deletions in the A(L) region were constructed, and the proteins were successfully expressed in either COS or Chinese hamster ovary cells. Mutants with deletions in the segment 296-349 had full Ca(2+) transport activity, but deletions involving the segment of amino acids 350-356 were inactive suggesting that these residues are required for a functional PMCA. In the absence of calmodulin the V(max) of mutant d296-349 was similar to that of the recombinant wild type pump, but its K(0.5) for Ca(2+) was about 5-fold lower. The addition of calmodulin increased the V(max) and the apparent Ca(2+) affinity of both the wild type and d296-349 enzymes indicating that the activating effects of calmodulin were not affected by the deletion. At low concentrations of Ca(2+) and in the presence of saturating amounts of calmodulin, the addition of phosphatidic acid increased about 2-fold the activity of the recombinant wild type pump. In contrast, under these conditions phosphatidic acid did not significantly change the activity of mutant d296-349. Taken together these results suggest that (a) deletion of residues 296-349 recreates a form of PMCA similar to that resulting from the binding of acidic lipids at the A(L) region; (b) the A(L) region acts as an acidic lipid-binding inhibitory domain capable of adjusting the Ca(2+) affinity of the PMCA to the lipid composition of the membrane; and (c) the function of the A(L) region is independent of the autoinhibition by the C-terminal calmodulin-binding region.     

Phospholipase C, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and tyrosine phosphorylation are involved in carbachol-induced phospholipase D activation in Chinese hamster ovary cells expressing muscarinic acetylcholine receptor of Caenorhabditis elegans

Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO-GAR-3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca(2+) elevation and stimulated PLD activity through the mAChR; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after CCh treatment. The CCh-induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down-regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca(2+)-calmodulin-dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca(2+) by EGTA and BAPTA/AM, abolished CCh-induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC-PLD pathway and the CaM kinase II/tyrosine kinase-PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.     

Enhanced activity of a large conductance, calcium-sensitive K+ channel in the presence of Src tyrosine kinase

Large conductance, calcium-sensitive K(+) channels (BK(Ca) channels) contribute to the control of membrane potential in a variety of tissues, including smooth muscle, where they act as the target effector for intracellular "calcium sparks" and the endothelium-derived vasodilator nitric oxide. Various signal transduction pathways, including protein phosphorylation can regulate the activity of BK(Ca) channels, along with many other membrane ion channels. In our study, we have examined the regulation of BK(Ca) channels by the cellular Src gene product (cSrc), a soluble tyrosine kinase that has been implicated in the regulation of both voltage- and ligand-gated ion channels. Using a heterologous expression system, we observed that co-expression of murine BK(Ca) channel and the human cSrc tyrosine kinase in HEK 293 cells led to a calcium-sensitive enhancement of BK(Ca) channel activity in excised membrane patches. In contrast, co-expression with a catalytically inactive cSrc mutant produced no change in BK(Ca) channel activity, demonstrating the requirement for a functional cSrc molecule. Furthermore, we observed that BK(Ca) channels underwent direct tyrosine phosphorylation in cells co-transfected with BK(Ca) channels and active cSrc but not in cells co-transfected with the kinase inactive form of the enzyme. A single Tyr to Phe substitution in the C-terminal half of the channel largely prevented this observed phosphorylation. Given that cSrc may become activated by receptor tyrosine kinases or G-protein-coupled receptors, these findings suggest that cSrc-dependent tyrosine phosphorylation of BK(Ca) channels in situ may represent a novel regulatory mechanism for altering membrane potential and calcium entry.     

The plasma membrane calcium pump displays memory of past calcium spikes. Differences between isoforms 2b and 4b

To understand how the plasma membrane Ca(2+) pump (PMCA) behaves under changing Ca(2+) concentrations, it is necessary to obtain information about the Ca(2+) dependence of the rate constants for calmodulin activation (k(act)) and for inactivation by calmodulin removal (k(inact)). Here we studied these constants for isoforms 2b and 4b. We measured the ATPase activity of these isoforms expressed in Sf9 cells. For both PMCA4b and 2b, k(act) increased with Ca(2+) along a sigmoidal curve. At all Ca(2+) concentrations, 2b showed a faster reaction with calmodulin than 4b but a slower off rate. On the basis of the measured rate constants, we simulated mathematically the behavior of these pumps upon repetitive changes in Ca(2+) concentration and also tested these simulations experimentally; PMCA was activated by 500 nm Ca(2+) and then exposed to 50 nm Ca(2+) for 10 to 150 s, and then Ca(2+) was increased again to 500 nm. During the second exposure to 500 nm Ca(2+), the activity reached steady state faster than during the first exposure at 500 nm Ca(2+). This memory effect is longer for PMCA2b than for 4b. In a separate experiment, a calmodulin-binding peptide from myosin light chain kinase, which has no direct interaction with the pump, was added during the second exposure to 500 nm Ca(2+). The peptide inhibited the activity of PMCA2b when the exposure to 50 nm Ca(2+) was 150 s but had little or no effect when this exposure was only 15 s. This suggests that the memory effect is due to calmodulin remaining bound to the enzyme during the period at low Ca(2+). The memory effect observed in PMCA2b and 4b will allow cells expressing either of them to remove Ca(2+) more quickly in subsequent spikes after an initial activating spike.     

Calmodulin antagonizes amyloid-β peptides-mediated inhibition of brain plasma membrane Ca(2+)-ATPase

The synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in regulating intracellular Ca(2+) concentration in brain. We have recently found that PMCA is the only Ca(2+) pump in brain which is inhibited by amyloid-β peptide (Aβ), a neurotoxic peptide implicated in the pathology of Alzheimer's disease (AD) [1], but the mechanism of inhibition is lacking. In the present study we have characterized the inhibition of PMCA by Aβ. Results from kinetic assays indicate that Aβ aggregates are more potent inhibitors of PMCA activity than monomers. The inhibitory effect of Aβ could be blocked by pretreating the purified protein with Ca(2+)-calmodulin, the main endogenous activator of PMCA, and the activity of truncated PMCA lacking the calmodulin binding domain was not affected by Aβ. Dot-overlay experiments indicated a physical association of Aβ with PMCA and also with calmodulin. Thus, calmodulin could protect PMCA from inhibition by Aβ by burying exposed sites on PMCA, making them inaccessible to Aβ, and also by direct binding to the peptide. These results suggest a protective role of calmodulin against neuronal Ca(2+) dysregulation by PMCA inhibition induced by Aβ.     

Ceramide is a potent activator of plasma membrane Ca2+-ATPase from kidney-promixal tubule cells with protein kinase A as an intermediate

The kidney-proximal tubules are involved in reabsorbing two-thirds of the glomerular ultrafiltrate, a key Ca(2+)-modulated process that is essential for maintaining homeostasis in body fluid compartments. The basolateral membranes of these cells have a Ca(2+)-ATPase, which is thought to be responsible for the fine regulation of intracellular Ca(2+) levels. In this paper we show that nanomolar concentrations of ceramide (Cer(50) = 3.5 nm), a natural product derived from sphingomyelinase activity in biological membranes, promotes a 50% increase of Ca(2+)-ATPase activity in purified basolateral membranes. The stimulatory effect of ceramide occurs through specific and direct (cAMP-independent) activation of a protein kinase A (blocked by 10 nm of the specific inhibitor of protein kinase A (PKA), the 5-22 peptide). The activation of PKA by ceramide results in phosphorylation of the Ca(2+)-ATPase, as detected by an anti-Ser/Thr specific PKA substrate antibody. It is observed a straight correlation between increase of Ca(2+)-ATPase activity and PKA-mediated phosphorylation of the Ca(2+) pump molecule. Ceramide also stimulates phosphorylation of renal Ca(2+)-ATPase via protein kinase C, but stimulation of this pathway, which inhibits the Ca(2+) pump in kidney cells, is counteracted by the ceramide-triggered PKA-mediated phosphorylation. The potent effect of ceramide reveals a new physiological activator of the plasma membrane Ca(2+)-ATPase, which integrates the regulatory network of glycerolipids and sphingolipids present in the basolateral membranes of kidney cells.     

Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells

This study investigates the role of tyrosine phosphorylation and dephosphorylation in the regulation of the Ca(2+) permeant TRPV6 channel. HEK293 cells co-transfected with TRPV6 and the tyrosine phosphatase PTP1B show a constitutive Ca(2+) entry which was independent of tyrosine phosphorylation under resting conditions. Following depletion of intracellular Ca(2+) stores, TRPV6-mediated Ca(2+) entry could be increased in the presence of a tyrosine phosphatase inhibitor (bis-(N,N-dimethyl-hydroxamido) hydroxo-vanadate; DMHV). Inhibition of Src-kinases completely abolished DMHV-induced increase in TRPV6-mediated Ca(2+) influx. Co-transfection with Src led to tyrosine phosphorylation of TRPV6 which could be dephosphorylated by PTP1B. In vivo interaction of TRPV6 with PTP1B was visualized using the bimolecular fluorescence complementation (BiFC) method and proved by co-immunoprecipitation of both proteins. These data indicate that tyrosine phosphorylation is involved in the regulation of the TRPV6 channel protein.     

Protein kinase C-dependent phosphorylation of transient receptor potential canonical 6 (TRPC6) on serine 448 causes channel inhibition

TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry following the stimulation of a G(q)-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca(2+) entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6(S768A) (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser(448), in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba(2+) and Ca(2+) entry experiments revealed that GF1 did not potentiate TRPC6(S448A) activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6(S448A). Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca(2+) entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca(2+) entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser(448).     

Ca2+-dependent protein kinase--a modulation of the plasma membrane Ca2+-ATPase in parotid acinar cells

Cross-talk between cAMP and [Ca(2+)](i) signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca(2+) release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type II inositol 1,4,5-trisphosphate receptors (Bruce, J. I. E., Shuttleworth, T. J. S., Giovannucci, D. R., and Yule, D. I. (2002) J. Biol. Chem. 277, 1340-1348). The aim of the present study was to evaluate the functional and molecular mechanism whereby cAMP regulates Ca(2+) clearance pathways in parotid acinar cells. Following an agonist-induced increase in [Ca(2+)](i) the rate of Ca(2+) clearance, after the removal of the stimulus, was potentiated substantially ( approximately 2-fold) by treatment with forskolin. This effect was prevented completely by inhibition of the plasma membrane Ca(2+)-ATPase (PMCA) with La(3+). PMCA activity, when isolated pharmacologically, was also potentiated ( approximately 2-fold) by forskolin. Ca(2+) uptake into the endoplasmic reticulum of streptolysin-O-permeabilized cells by sarco/endoplasmic reticulum Ca(2+)-ATPase was largely unaffected by treatment with dibutyryl cAMP. Finally, in situ phosphorylation assays demonstrated that PMCA was phosphorylated by treatment with forskolin but only in the presence of carbamylcholine (carbachol). This effect of forskolin was Ca(2+)-dependent, and protein kinase C-independent, as potentiation of PMCA activity and phosphorylation of PMCA by forskolin also occurred when [Ca(2+)](i) was elevated by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid and was attenuated by pre-incubation with the Ca(2+) chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The present study demonstrates that elevated cAMP enhances the rate of Ca(2+) clearance because of a complex modulation of PMCA activity that involves a Ca(2+)-dependent step. Tight regulation of both Ca(2+) release and Ca(2+) efflux may represent a general feature of the mechanism whereby cAMP improves the fidelity and specificity of Ca(2+) signaling.