Orai1 and Orai2 mediate store-operated calcium entry that regulates HL60 cell migration and FAK phosphorylation.

Store-operated Ca2+ entry (SOCE) is a major mechanism for the regulation of intracellular Ca2+ homeostasis and cellular function. Emerging evidence has revealed that altered expression and function of the molecular determinants of SOCE play a critical role in the development or maintenance of several cancer hallmarks, including enhanced proliferation and migration. Here we show that, in the acute myeloid leukemia cell line HL60, Orai2 is highly expressed at the transcript level, followed by the expression of Orai1. Using fluorescence Ca2+ imaging we found that Orai2 silencing significantly attenuated thapsigargin-induced SOCE, as well as knockdown of Orai1, while silencing the expression of both channels almost completely reduced SOCE, thus suggesting that SOCE in these cells is strongly dependent on Orai1 and Orai2. On the other hand, the expression of TRPC1, TRPC3 and TRPC6 is almost absent at the transcript and protein level. Bromodeoxyuridine cell proliferation assay revealed that Orai1 and Orai2 expression silencing significantly reduced HL60 cell proliferation. Furthermore, knockdown of Orai1 and Orai2 significantly attenuated the ability of HL60 to migrate in vitro as determined by transwell migration assay, probably due to the impairment of FAK tyrosine phosphorylation. These findings provide evidence for a role for Orai1 and Orai2, in SOCE and migration in the human HL60 promyeloblastic cell line. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Relationship between calcium mobilization and platelet α- and δ-granule secretion. A role for TRPC6 in thrombin-evoked δ-granule exocytosis.

Changes in cytosolic Ca(2+) concentration ([Ca(2+)]c) regulate granule secretion in different cell types. Thrombin activates PAR1 and PAR4 receptors and promotes release of Ca(2+) from distinct intracellular stores, which, in turn, activates store-operated Ca(2+) entry (SOCE). A crucial step during platelet function is the release of physiological agonists stored in secretory granules to the extracellular compartment during activation. We aim to study the role of Ca(2+) mobilization from the extracellular compartment or from different intracellular stores in platelet granule secretion. By using flow cytometry, we have found that α- and δ-granules are secreted in thrombin-stimulated platelets in the absence of extracellular Ca(2+), and in a concentration-dependent manner. Our findings show that thrombin-stimulated granule secretion depends on Ca(2+) mobilization from intracellular stores. Analysis of the kinetics of granule secretion reveals that platelet stimulation with thrombin results in rapid release of α-granules which precedes the secretion of δ-granules. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 573-586 of TRPC6, inhibited thrombin-evoked δ-granule exocytosis. Our results indicate that the mechanisms underlying thrombin-induced α- and δ-granule secretion show differences in dependency on Ca(2+) mobilization.

Orais and STIMs: physiological mechanisms and disease.

The stromal interaction molecules STIM1 and STIM2 are Ca(2+) sensors, mostly located in the endoplasmic reticulum, that detect changes in the intraluminal Ca(2+) concentration and communicate this information to plasma membrane store-operated channels, including members of the Orai family, thus mediating store-operated Ca(2+) entry (SOCE). Orai and STIM proteins are almost ubiquitously expressed in human cells, where SOCE has been reported to play a relevant functional role. The phenotype of patients bearing mutations in STIM and Orai proteins, together with models of STIM or Orai deficiency in mice, as well as other organisms such as Drosophila melanogaster, have provided compelling evidence on the relevant role of these proteins in cellular physiology and pathology. Orai1-deficient patients suffer from severe immunodeficiency, congenital myopathy, chronic pulmonary disease, anhydrotic ectodermal dysplasia and defective dental enamel calcification. STIM1-deficient patients showed similar abnormalities, as well as autoimmune disorders. This review summarizes the current evidence that identifies and explains diseases induced by disturbances in SOCE due to deficiencies or mutations in Orai and STIM proteins.

TMEM97 facilitates the activation of SOCE by downregulating the association of cholesterol to Orai1 in MDA-MB-231 cells.

The expression of TMEM97, a regulator of cholesterol transport, has been reported to be enhanced in some tumour cells. We have recently shown that TMEM97 is involved in the proliferation of the breast cancer cell line MDA-MB-231, probably through changes in store-operated calcium entry (SOCE). By using silencing and overexpression of TMEM97 in MDA-MB-231 cells (two manoeuvres that either reduce or increase the calcium influx, respectively), we show enhanced cholesterol uptake in these cells as compared to the non-tumoral breast cell line, MCF10A. The enhanced cholesterol uptake in MDA-MB-231 cells was inhibited by silencing TMEM97, while overexpression of this protein increased cholesterol uptake in MCF10A cells and, therefore, indicating that this protein plays a role in the enhanced cholesterol uptake in MDA-MB-231 cancer cell line. TMEM97 silencing and overexpression resulted in an increase and decrease in the association of cholesterol to the SOCE calcium channel Orai1, respectively. Interestingly, silencing of TMEM97 in MDA-MB-231 cells significantly reduced the co-localization of Orai1 with the SOCE regulatory protein STIM1. Finally, neither silencing nor overexpression of TMEM97 altered SOCE in MDA-MB-231 cells transfected with the cholesterol insensible mutant of Orai1(Y80E). Our results reveal a novel regulatory mechanism of SOCE that relies on TMEM97 activity that courses through the reduction of the cholesterol content in the plasma membrane, and subsequently, by impairing its interaction with Orai1.

Population genetic screening efficiently identifies carriers of autosomal dominant diseases.

Three inherited autosomal dominant conditions-BRCA-related hereditary breast and ovarian cancer (HBOC), Lynch syndrome (LS) and familial hypercholesterolemia (FH)-have been termed the Centers for Disease Control and Prevention Tier 1 (CDCT1) genetic conditions, for which early identification and intervention have a meaningful potential for clinical actionability and a positive impact on public health1. In typical medical practice, genetic testing for these conditions is based on personal or family history, ethnic background or other demographic characteristics2. In this study of a cohort of 26,906 participants in the Healthy Nevada Project (HNP), we first evaluated whether population screening could efficiently identify carriers of these genetic conditions and, second, we evaluated the impact of genetic risk on health outcomes for these participants. We found a 1.33% combined carrier rate for pathogenic and likely pathogenic (P/LP) genetic variants for HBOC, LS and FH. Of these carriers, 21.9% of participants had clinically relevant disease, among whom 70% had been diagnosed with relevant disease before age 65. Moreover, 90% of the risk carriers had not been previously identified, and less than 19.8% of these had documentation in their medical records of inherited genetic disease risk, including family history. In a direct follow-up survey with all carriers, only 25.2% of individuals reported a family history of relevant disease. Our experience with the HNP suggests that genetic screening in patients could identify at-risk carriers, who would not be otherwise identified in routine care.

STIM1, Orai1 and hTRPC1 are important for thrombin- and ADP-induced aggregation in human platelets.

Ca(2+) entry, particularly store-operated Ca(2+) entry (SOCE), has been reported to be crucial for a variety of cellular functions. SOCE is a mechanism regulated by the Ca(2+) content of the stores, where the intraluminal Ca(2+) sensor STromal Interaction Molecule 1 (STIM1) has been reported to communicate the filling state of the intracellular Ca(2+) stores to the store-operated Ca(2+)-permeable channels in the plasma membrane, likely involving Orai1 and TRPC proteins, such as TRPC1. Here we have investigated the role of Orai1, STIM1 and TRPC1 in platelet aggregation, an event that occurs during the process of thrombosis and hemostasis. Electrotransjection of cells with anti-STIM1 (25-139) antibody, directed towards the Ca(2+)-binding motif, significantly reduced thrombin-induced aggregation and prevented ADP-evoked response. Extracellular application of the anti-STIM1 antibody, in order to block the function of plasma membrane-located STIM1, reduced thrombin- and ADP-stimulated platelet aggregation to a lesser extent. Introduction of an anti-Orai1 (288-301) antibody, which binds the STIM1-binding site located in the Orai1 C-terminus, or extracellular application of anti-hTRPC1 (557-571) antibody to impair hTRPC1 channel function, significantly reduced thrombin- and ADP-induced platelet aggregation. These findings suggest a role of STIM1, Orai1 and hTRPC1 in thrombin- and ADP-induced platelet aggregation probably through the regulation of Ca(2+) entry, which might become targets for the development of therapeutic strategies to treat platelet hyperactivity and thrombosis disorders.

Store-independent Orai1-mediated Ca2+ entry and cancer.

Ca2+ channels play an important role in the development of different types of cancer, and considerable progress has been made to understand the pathophysiological mechanisms underlying the role of Ca2+ influx in the development of different cancer hallmarks. Orai1 is among the most ubiquitous and multifunctional Ca2+ channels. Orai1 mediates the highly Ca2+-selective Ca2+ release-activated current (ICRAC) and participates in the less Ca2+-selective store-operated current (ISOC), along with STIM1 or STIM1 and TRPC1, respectively. Furthermore, Orai1 contributes to a variety of store-independent Ca2+ influx mechanisms, including the arachidonate-regulated Ca2+ current, together with Orai3 and the plasma membrane resident pool of STIM1, as well as the constitutive Ca2+ influx processes activated by the secretory pathway Ca2+-ATPase-2 (SPCA2) or supported by physical and functional interaction with the small conductance Ca2+-activated K+ channel 3 (SK3) or the voltage-dependent Kv10.1 channel. This review summarizes the current knowledge concerning the store-independent mechanisms of Ca2+ influx activation through Orai1 channels and their role in the development of different cancer features.

(-)­Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells.

Triple negative breast cancer is an aggressive type of cancer that does not respond to hormonal therapy and current therapeutic strategies are accompanied by side effects due to cytotoxic actions on normal tissues. Therefore, there is a need for the identification of anti-cancer compounds with negligible effects on non-tumoral cells. Here we show that (-)­oleocanthal (OLCT), a phenolic compound isolated from olive oil, selectively impairs MDA-MB-231 cell proliferation and viability without affecting the ability of non-tumoral MCF10A cells to proliferate or their viability. Similarly, OLCT selectively impairs the ability of MDA-MB-231 cells to migrate while the ability of MCF10A to migrate was unaffected. The effect of OLCT was not exclusive for triple negative breast cancer cells as we found that OLCT also attenuate cell viability and proliferation of MCF7 cells. Our results indicate that OLCT is unable to induce Ca2+ mobilization in non-tumoral cells. By contrast, OLCT induces Ca2+ entry in MCF7 and MDA-MB-231 cells, which is impaired by TRPC6 expression silencing. We have found that MDA-MB-231 and MCF7 cells overexpress the channel TRPC6 as compared to non-tumoral MCF10A and treatment with OLCT for 24-72 h downregulates TRPC6 expression in MDA-MB-231 cells. These findings indicate that OLCT impairs the ability of breast cancer cells to proliferate and migrate via downregulation of TRPC6 channel expression while having no effect on the biology of non-tumoral breast cells.

Protein complex immunological separation assay (ProCISA): a technique for investigating single protein properties.

Analysis of the posttranslational modification of proteins, such as phosphorylation, might yield misleading results due to the presence of other proteins with similar electrophoretic properties that coimmunoprecipitate with the target protein. The aim of the present work was to develop a reliable, easy and economical technique to completely isolate a protein from its complex. Here we present a new assay developed to fully isolate proteins from macromolecular complexes that consists of an initial SDS/PAGE (under reducing conditions), which isolates the target protein, followed by transfer of the proteins to a buffer, from which the target protein is recaptured by conventional immunoprecipitation. This technique, that we have termed "Protein Complex Immunological Separation Assay" (ProCISA), successfully separated proteins of different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes. We show that ProCISA allows the investigation of the tyrosine phosphorylation state of isolated proteins. This technique could also be used to study other posttranslational modifications without risk of misleading results resulting from contamination with other proteins of similar electrophoretic mobility which complex with the protein of interest.

Thrombin induces apoptotic events through the generation of reactive oxygen species in human platelets.

BACKGROUND: Thrombin is a major physiological platelet agonist that activates a number of cell functions including aggregation. Platelet stimulation with thrombin has been shown to result in the development of apoptotic events, including activation of caspases-3 and -9, cytochrome c release and phosphatidylserine (PS) exposure; however, the mechanism underlying the activation of apoptosis remains unclear. OBJECTIVES: In the present study, we aim to investigate whether endogenously generated reactive oxygen species upon thrombin stimulation is required for the activation of apoptosis in human platelets. METHODS: Changes in the mitochondrial membrane potential were registered using the dye JC-1; caspase-3 and -9 activity was determined from the cleavage of their respective specific fluorogenic substrates; PS externalization was estimated using annexin V-fluorescein isothicyanate and cytochrome c release was detected by Western blotting in samples from the mitochondrial and cytosolic fractions. RESULTS: Treatment of platelets with thrombin stimulates mitochondrial membrane potential depolarization and endogenous generation of H(2)O(2) . Platelet exposure to exogenous H(2)O(2) results in cytochrome c release and activation of caspases-9. In addition, H(2)O(2) induces the activation of caspase-3 and PS exposure by a mechanism dependent on cytochrome c release and caspase-9 activation. Finally, thrombin-evoked development of apoptotic events was impaired by treatment with catalase. CONCLUSION: Our results indicate that thrombin-induced apoptosis is likely mediated by endogenous generation of H(2)O(2) in human platelets.

hTRPC1-associated alpha-actinin, and not hTRPC1 itself, is tyrosine phosphorylated during human platelet activation.

BACKGROUND: Canonical transient receptor potential channels (TRPCs), which are regulated by several processes, including tyrosine phosphorylation, are candidates for the conduction of store-operated Ca(2+) entry (SOCE). OBJECTIVES: To assess hTRPC phosphotyrosine content upon platelet stimulation. METHODS: A new protein complex immunological separation assay (ProCISA) was developed to allow assessment of isolated hTRPC tyrosine phosphorylation by Western blotting. RESULTS: Classical immunoprecipitation suggested that thrombin (Thr) evoked an initial decrease in hTRPC1 phosphotyrosine content, which reached a minimum at 1 s, and then increased again, exceeding basal levels after 3 min. However, TRPC isolation from protein complexes using ProCISA revealed that hTRPC1, 4 and 5 were not tyrosine phosphorylated at rest or after Thr stimulation. Stimulation with Thr for 3 min increased the phosphotyrosine content of alpha-actinin, which shows similar electrophoretic properties to hTRPCs and coimmunoprecipitates with hTRPC1. Thr-evoked alpha-actinin tyrosine phosphorylation was increased by inhibiting the alpha-actinin phosphatase, SHP-1, which enhanced phosphorylation of the TRPC complex and SOCE. Inhibition of tyrosine phosphorylation impaired the interaction between hTRPC1 and the intracellular Ca(2+) sensor STIM1. CONCLUSIONS: hTRPC1, 4 and 5 are not tyrosine phosphorylated during SOCE in human platelets although tyrosine phosphorylation is important for SOCE. The results obtained using ProCISA caution the use of classical immunoprecipitation for the determination of the tyrosine phosphorylation state of a given protein, where the presence of other proteins with similar electrophoretic mobilities may give misleading results.

[Synaptic tagging and memory trace]. / La 'marca sinaptica' y la huella de la memoria.

AIM: To present a panorama of the main features and possible identity of the synaptic tag, such as to discuss some of its functional implications. DEVELOPMENT: Long-term potentiation (LTP) constitutes a very attractive synaptic/cellular memory model. LTP, like memory, can manifest itself early (essentially depending on the modification of pre-existing proteins at synapse) and late (depending on new protein synthesis). As LTP is a highly specific phenomenon, a dilemma arises: how can the proteins, required to plastic change stabilization, that are synthesized at the soma of a neuron containing thousands of synaptic contacts--all depending of the same nucleus--go to the appropriate synapses? In this review, we present some of the models that intend to explain this question, making emphasis on synaptic tagging hypothesis. Some of the main findings that have contributed to tagging hypothesis are exposed. The local protein synthesis and the activation of protein kinases are analyzed as candidates to be the synaptic tag. Additionally, some of the functional implications of synaptic tagging are discussed. CONCLUSIONS: The synaptic tagging hypothesis offers a very flexible and reasonable solution to the specificity of long-lasting synaptic changes. Although some of the tagging features are known, the synaptic tag identity has not yet been elucidated. It seems that there is not a unique synaptic tag, but there are rather multiple molecular synaptic tags involved. Each of them might function as a synaptic tag under particular circumstances. Each might be differentially recruited by specific stimuli and mediate plasticity over different time domains.

Inhibitory effect of Ca(2+) on ATP-mediated stimulation of NPR-A-coupled guanylyl cyclase in renal glomeruli from spontaneously hypertensive and normotensive rats.

Atrial natriuretic peptide (ANP) regulates blood pressure mainly through the occupation of the guanylyl cyclase-coupled receptor NPR-A, which requires ATP interaction for maximal activation. This study investigates the effect of extracellular Ca(2+) on ATP-mediated regulation of NPR-A-coupled guanylyl cyclase activity in glomerular membranes from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). ATP induced a significant increase in basal and ANP(1-28)-stimulated guanylyl cyclase activity that was greater in SHR than in WKY. Extracellular Ca(2+) inhibited ATP-stimulated guanylyl cyclase activity in a concentration-dependent manner, but did not modify basal and ANP(1-28)-stimulated guanylyl cyclase activity. In the presence of ATP, NPR-A showed higher affinity for ANP(1-28) and lower Bmax. Ca(2+) did not modify NPR-A-ANP(1-28) binding properties. The different effects of extracellular Ca(2+) on ANP(1-28)- or ATP-mediated guanylyl cyclase activation suggest that these events are differentially regulated. Addition of extracellular Ca(2+) induced similar effects in hypertensive and normotensive rats, suggesting that it is not responsible for the elevated cGMP production observed in SHR.

CCK causes rapid tyrosine phosphorylation of p125FAK focal adhesion kinase and paxillin in rat pancreatic acini.

Recent studies show CCK stimulates tyrosine phosphorylation (TYR PHOSP) of a number of proteins and evidence from the pancreas and other cellular systems suggest this could be important in mediating some of CCK's growth and secretory effects. In other tissues various neuropeptides such as bombesin can cause tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) and paxillin which are important in mediating their growth effects. The purpose of the present study was to determine the effects of CCK in rat pancreatic acini on the TYR PHOSP of these latter proteins. In dispersed rat pancreatic acini, cell lysates were incubated with an anti-phosphotyrosine mAb (PY20) which was immunoprecipitated and then analyzed by Western blotting with anti-phosphotyrosine mAb (4G10), anti-p125FAK mAb or anti-paxillin mAb. CCK-8 at 5 min increased TYR PHOSP of five proteins of molecular weight > 60,000 including a broad M(r) 110-130,000 and M(r) 70-80,000. An increase in TYR PHOSP of both p125FAK and paxillin was detected within 1 min of adding CCK and reached a maximum at 2.5 min with a 9.1 +/- 1.9-fold increase for p125FAK and 3.6 +/- 0.6-fold for paxillin. CCK-8 caused a half-maximal increase in TYR PHOSP of p125FAK at 0.1 nM and paxillin at 0.03 nM. CCK-JMV also stimulated an increase in TYR PHOSP of both proteins, but was only 50% as efficacious as CCK-8. CCK-JMV caused a half-maximal increase at 10 nM and maximal at 1 microM for both proteins. To investigate whether the low affinity CCK receptor state also caused TYR PHOSP of both proteins, increasing concentrations of CCK-JMV were added to a maximally effective CCK-8 concentration (1 nM). Detectable inhibition of CCK-8-stimulated TYR PHOSP occurred with 1 microM CCK-JMV and with 3 microM CCK-JMV the CCK-8-stimulated response was inhibited 50% and was the same as that seen with CCK-JMV alone. These studies demonstrate that in rat pancreatic acini, CCK causes rapid TYR PHOSP of both p125FAK and paxillin. This stimulation is mediated by both the high affinity and low affinity CCK receptor states. This phosphorylation of these proteins could be important in mediating CCK's effect on the cytoskeleton or growth effects as shown for a number of other agents (oncogenes, neuropeptides, integrins).

Are tyrosine phosphorylation of p125(FAK) and paxillin or the small GTP binding protein, rho, needed for CCK-stimulated pancreatic amylase secretion?

Studies of a possible role of tyrosine phosphorylation in the secretory process in rat pancreatic acinar cells provide conflicting conclusions. Recent studies show that tyrosine phosphorylation of the focal adhesion kinase, p125FAK and the cytoskeletal protein, paxillin, may mediate a number of cellular changes and this phosphorylation is dependent on the activation of the small GTP binding protein, p21Rho (Rho). In this work we have investigated the role of tyrosine phosphorylation of each of these proteins and of the activation of Rho in pancreatic enzyme secretion. Pretreatment with genistein, a tyrosine kinase inhibitor, decreased CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin and CCK-8-stimulated amylase secretion by more than 60%, raising the possibility that tyrosine phosphorylation of these two proteins could be important in the ability of CCK-8 to stimulate amylase release. However, genistein did not alter the amylase release stimulated by TPA but inhibited TPA-stimulated p125FAK and paxillin tyrosine phosphorylation by 70%. Pretreatment with C3 transferase, which specifically inactivates Rho, causes a decrease in CCK-8-induced maximal amylase release by 33%. Moreover, C3 transferase pretreatment causes a 48% and a 38% decrease in the tyrosine phosphorylation of p125FAK and paxillin by CCK-8, respectively. Pretreatment with different concentrations of cytochalasin D, an actin cytoskeleton assembly inhibitor, completely inhibited CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin without having any effect on either the potency or efficacy of CCK-8 at stimulating amylase release. Furthermore, cytochalasin D completely inhibited TPA-stimulated tyrosine phosphorylation of both proteins without affecting TPA-stimulated amylase release. These results show that tyrosine phosphorylation of p125FAK and paxillin is not required for CCK-8 stimulation of enzyme secretion. However, our results suggest Rho is involved in the CCK-8 stimulation of amylase release by a parallel pathway to its involvement in the CCK-8-stimulated tyrosine phosphorylation of p125FAK and paxillin.

Characteristics of the renal C-type natriuretic peptide receptor in hypertrophied and developing rat kidney.

This study investigates the effect of hypertrophy, using one kidney and one kidney/one clip rats, and development, comparing 3- and 12-week-old rats, on the expression of the 28-amino acid atrial natriuretic peptide (ANP(1-28)) binding sites in rat kidney. Here we report an increased B(max) value of glomerular binding sites for ANP(1-28) and C-type natriuretic peptide 1-22 (CNP(1-22)) in hypertrophied and developing kidney, without modifying their affinity, an effect that was prevented in the presence of the synthetic des[Gln(18), Ser(19), Gly(20), Leu(21), Gly(22)]ANP(4-23)-amide (C-ANF), suggesting that natriuretic peptide receptor (NPR)-C binding sites might be enhanced. The enhanced B(max) was only detected in the high affinity binding site for CNP(1-22), which has been identified as the 67 kDa NPR-C-like protein. A similar effect was observed in renal glomeruli from 3-week-old rats compared with 12-week-old rats. Our results indicate that ANP(1-28), CNP(1-22) and C-ANF inhibited cAMP synthesis stimulated by the physiological agonists histamine and 5-hydroxytryptamine or directly by forskolin. The inhibitory effect was found to be significantly greater in 1-kidney and 1-kidney/1-clip rats than in controls, and in 3-week-old rats compared with 12-week-old rats. Our observations suggest that this effect must be attributed to the 67 kDa NPR-C-like protein due to the enhanced B(max) values and the reported inhibitory role for this receptor on adenylyl cyclase activity. The enhanced inhibitory role of natriuretic peptides on cAMP synthesis in hypertrophied and developing kidney may influence glomerular function in the rat kidney and suggests a role for the 67 kDa NPR-C-like protein in growth.

A role for phosphoinositides in tyrosine phosphorylation of p125 focal adhesion kinase in rat pancreatic acini.

Previous studies have shown that different agonists increase tyrosine phosphorylation of the focal adhesion related proteins p125(FAK), p130(Cas), and paxillin in different cell types and that tyrosine phosphorylation depends on the integrity of the actin cytoskeleton. Because phosphoinositides are important for the maintenance of the cytoskeleton, the role of phosphoinositides in the tyrosine phosphorylation of these proteins in response to occupancy of m3 muscarinic and CCK(A) receptors has been investigated in pancreatic acini. Addition of carbachol or CCK-8 to pancreatic acini resulted in rapid increases in the tyrosine phosphorylation of p125(FAK), p130(Cas), and paxillin. Pretreatment of pancreatic acini with LY294002 or wortmannin resulted in a concentration-dependent inhibition of tyrosine phosphorylation of p125(FAK), p130(Cas), and paxillin stimulated by carbachol or CCK-8. Carbachol- or CCK-8-stimulated tyrosine phosphorylation of these proteins was not inhibited by rapamycin, PD 98059 or SB 203580, and thus it was dissociated from the activation of p70 S6 or MAP kinases. These results indicate that m3 muscarinic and CCK(A) receptor-mediated increase in p125(FAK), p130(Cas), and paxillin tyrosine phosphorylation in pancreatic acini depends on the ability of these cells to synthesise phosphoinositides.

Collaborative effect of SERCA and PMCA in cytosolic calcium homeostasis in human platelets.

Intracellular free Ca2+ concentration ([Ca2+]c) is finely regulated by several mechanisms that either increase or reduce [Ca2+]c. Two different Ca2+ pumps have been described so far as the main mechanisms for Ca2+ removal from the cytosol, either by its sequestration into the stores, mediated by the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) or by Ca2+ extrusion to the extracellular medium, by the plasma membrane Ca2+-ATPase (PMCA). We have used inhibitors of these pumps to analyze their Ca2+ clearance efficacy in human platelets stimulated by the physiological agonist thrombin. Results demonstrate that, after platelet stimulation with thrombin, activation of SERCA precedes that of PMCA, although the ability of PMCA to remove Ca2+ from the cytosol last longer than that of SERCA. The efficacy of SERCA and PMCA removing Ca2+ from the cytosol is reduced when the concentration of thrombin increases. This phenomenon correlates with the greater increase in [Ca2+]c induced by higher concentrations of thrombin, which further confirms that SERCA and PMCA activities are regulated by [Ca2+]c.

A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets.

Store-mediated Ca(2+) entry (SMCE) is a major pathway for Ca(2+) influx in many cells, yet how depletion of the intracellular Ca(2+) stores leads to the activation of Ca(2+) entry across the plasma membrane is not well understood. Recent work in platelets favors a secretion-like conformational coupling mechanism involving proteins in the plasma membrane (PM) and in the membrane of the Ca(2+) store, located in the endoplasmic reticulum (ER). The activation and maintenance of SMCE in platelets has been shown to depend on remodeling of the actin cytoskeleton, which may be required to allow trafficking of the ER toward the PM to permit coupling to occur and to stabilize this coupling once achieved. The coupling itself has been shown to involve one isoform of the inositol 1,4,5-trisphosphate receptor (IP(3)RII) and the Ca(2+)-permeable channel protein, human Trp1 (hTrp1).

The inositol trisphosphate receptor antagonist 2-aminoethoxydiphenylborate (2-APB) blocks Ca2+ entry channels in human platelets: cautions for its use in studying Ca2+ influx.

It has been reported that store-mediated Ca2+ entry (SMCE) in human platelets is likely to be mediated by a secretion-like coupling mechanism. Recently, 2-aminoethoxydiphenylborate (2-APB) has been used in the investigation of SMCE. Here, the mechanism of action of 2-APB is investigated in human platelets. In a Ca2+-free medium (EGTA added), addition of 0.1 U/ml thrombin caused an elevation in [Ca2+]i. Preincubation with 100 microM 2-APB for 170s abolished the release of internal Ca2+. In platelets whose internal Ca2+ stores had been depleted by treatment with 200 nM thapsigargin, addition of extracellular Ca2+ caused an elevation in [Ca2+]i indicative of SMCE. Preincubation with 2-APB decreased SMCE by 95.5+/-1.1%. After activation of SMCE, addition of 2-APB rapidly decreased [Ca2+]i to basal levels; in contrast, the coupling between Trp1 and IP3RII, which has been shown to play an important role in SMCE in platelets, remained intact at the same time points. The rate of decrease of [Ca2+]i and the absence of measurable latency in the effect of 2-APB were comparable to the effects of La3+ (a cation channel blocker). These data suggest that 2-APB may act as a blocker of Ca2+ permeable plasma membrane channels. These data provide further information regarding the mechanism and site of action of 2-APB and highlight the necessity of careful interpretation of work performed using this molecule.