Agonist-mediated Ca2 Research Articles

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP3Rs) and the mitochondrial Ca2+ uniporter (MCU) are key proteins that regulate intracellular Ca2+ concentration. Mitochondrial Ca2+ accumulation activates Ca2+-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP3R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca2+ signaling. Our results show that TKO cells (exhibiting total loss of Ca2+ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca2+ signaling is dispensable for the bioenergetic needs of both IP3R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.

Human GPR17 missense variants identified in metabolic disease patients have distinct downstream signaling profiles

GPR17 is a G-protein-coupled receptor (GPCR) implicated in the regulation of glucose metabolism and energy homeostasis. Such evidence is primarily drawn from mouse knockout studies and suggests GPR17 as a potential novel therapeutic target for the treatment of metabolic diseases. However, links between human GPR17 genetic variants, downstream cellular signaling, and metabolic diseases have yet to be reported. Here, we analyzed GPR17 coding sequences from control and disease cohorts consisting of individuals with adverse clinical metabolic deficits including severe insulin resistance, hypercholesterolemia, and obesity. We identified 18 nonsynonymous GPR17 variants, including eight variants that were exclusive to the disease cohort. We characterized the protein expression levels, membrane localization, and downstream signaling profiles of nine GPR17 variants (F43L, V96M, V103M, D105N, A131T, G136S, R248Q, R301H, and G354V). These nine GPR17 variants had similar protein expression and subcellular localization as wild-type GPR17; however, they showed diverse downstream signaling profiles. GPR17-G136S lost the capacity for agonist-mediated cAMP, Ca2+, and β-arrestin signaling. GPR17-V96M retained cAMP inhibition similar to GPR17-WT, but showed impaired Ca2+ and β-arrestin signaling. GPR17-D105N displayed impaired cAMP and Ca2+ signaling, but unaffected agonist-stimulated β-arrestin recruitment. The identification and functional profiling of naturally occurring human GPR17 variants from individuals with metabolic diseases revealed receptor variants with diverse signaling profiles, including differential signaling perturbations that resulted in GPCR signaling bias. Our findings provide a framework for structure–function relationship studies of GPR17 signaling and metabolic disease.

Human GPR17 Nonsynonymous Variants Identified in Individuals with Metabolic Diseases Have Distinct Functional Signaling Profiles

GPR17 is a G protein-coupled receptor (GPCR) implicated in the regulation of glucose metabolism and energy homeostasis. Our genetic knockout studies in rodents suggest that GPR17 is a potential therapeutic target for the treatment of metabolic diseases. However, the contributions of GPR17 to human metabolism and metabolic deficits are not well understood. Here, we analyzed the human GPR17 coding sequences of individuals from control and metabolic disease cohorts that were comprised of patients with clinical phenotypes including severe insulin resistance, hypercholesterolemia, and obesity. Across cohorts, 18 nonsynonymous GPR17 variants were identified, including eight variants that were exclusive to the disease cohort. We characterized the protein expression levels, cellular localization, and downstream functional signaling profiles of nine human GPR17 variants (F43L, V96M, V103M, D105N, A131T, G136S, R248Q, R301H, and G354V). We found that the protein expression levels and subcellular localization for each of the nine GPR17 variants were similar to that of the wild type GPR17. As the endogenous GPR17 ligand is still elusive, we used a synthetic GPR17 agonist to quantitatively measure the functional signaling profiles of GPR17 variants. We found some of the variants had distinctly altered signaling profiles. GPR17-G136S lost agonist-mediated cAMP, Ca2+, and beta-arrestin signaling. GPR17-V96M retained cAMP inhibition similar to GPR17-WT but had impaired Ca2+ and beta-arrestin signaling. GPR17-D105N displayed impaired cAMP and Ca2+ signaling but enhanced agonist-stimulated beta-arrestin recruitment. Also, GPR17-G354V retained cAMP and Ca2+ signaling function but had attenuated beta-arrestin recruitment. The identification and functional profiling of naturally occurring human GPR17 variants from individuals with metabolic diseases revealed receptor variants with distinct signaling profiles, including differential signaling perturbations that resulted in receptor signaling bias. These results are expected to contribute to our understanding of the molecular signaling mechanisms underlying GPR17 in metabolic regulation.

Calcium Release Mediated by Redox-Sensitive RyR2 Channels Has a Central Role in Hippocampal Structural Plasticity and Spatial Memory.

Previous studies indicate that hippocampal synaptic plasticity and spatial memory processes entail calcium release from intracellular stores mediated by ryanodine receptor (RyR) channels. In particular, RyR-mediated Ca2+ release is central for the dendritic spine remodeling induced by brain-derived neurotrophic factor (BDNF), a neurotrophin that stimulates complex signaling pathways leading to memory-associated protein synthesis and structural plasticity. To examine if upregulation of ryanodine receptor type-2 (RyR2) channels and the spine remodeling induced by BDNF entail reactive oxygen species (ROS) generation, and to test if RyR2 downregulation affects BDNF-induced spine remodeling and spatial memory. Downregulation of RyR2 expression (short hairpin RNA [shRNA]) in primary hippocampal neurons, or inhibition of nitric oxide synthase (NOS) or NADPH oxidase, prevented agonist-mediated RyR-mediated Ca2+ release, whereas BDNF promoted cytoplasmic ROS generation. RyR2 downregulation or inhibitors of N-methyl-d-aspartate (NMDA) receptors, or NOS or of NADPH oxidase type-2 (NOX2) prevented RyR2 upregulation and the spine remodeling induced by BDNF, as did incubation with the antioxidant agent N-acetyl l-cysteine. In addition, intrahippocampal injection of RyR2-directed antisense oligodeoxynucleotides, which caused significant RyR2 downregulation, caused conspicuous defects in a memorized spatial memory task. The present novel results emphasize the key role of redox-sensitive Ca2+ release mediated by RyR2 channels for hippocampal structural plasticity and spatial memory. Based on these combined results, we propose (i) that BDNF-induced RyR2-mediated Ca2+ release and ROS generation via NOS/NOX2 are strictly required for the dendritic spine remodeling and the RyR2 upregulation induced by BDNF, and (ii) that RyR2 channel expression is crucial for spatial memory processes. Antioxid. Redox Signal. 29, 1125-1146.

CALCIUM/CALMODULIN DEPENDENT PROTEIN KINASE II δ MODULATES INTRACELLULAR Ca2+ RELEASE AND CELL PROLIFERATION IN ADULT CARDIAC FIBROBLASTS

Calcium/calmodulin dependent protein kinase II δ (CaMKIIδ) is a well-recognised mediator of cardiomyocyte function and dysfunction, however little is known of its role in the cardiac fibroblast (CF). [Ca2+]i plays a crucial role in cell proliferation, yet the mechanism by which this is regulated in adult CFs remains elusive. We hypothesised that CaMKIIδ can regulate intracellular Ca2+ release channel activity and proliferation in adult CFs. Using Fura-2 loaded cells, we initially confirmed agonist-mediated intracellular Ca2+ release using Angiotensin II (1 µM). We demonstrated that CaMKIIδ and Inositol 1,4,5-triphosphate receptor type 2 (IP3R2) are both highly expressed in adult rat CFs but the ryanodine receptor (RyR2) is not. This identifies IP3R2 as the main intracellular Ca2+ release channel in these cells. To investigate whether CaMKIIδ modulates IP3R activity in CFs, cells were pre-treated with inhibitors autocamtide inhibitory peptide (AIP) (1 µM) and KN-93 (5 µM) prior to Angiotensin II application. Ca2+ release was significantly inhibited in the presence of both inhibitors (81±6.5% (AIP) vs 123±6% (control), p

Characterization of Store-Operated Calcium Channels in Pancreatic Duct Epithelia

Store-operated Ca2+ channels (SOCs) are activated by depletion of intracellular Ca2+ stores following agonist-mediated Ca2+ release. In pancreatic duct epithelial cells (PDEC), the activation of GPCR coupled to phospholipase C stimulated SOC-mediated Ca2+ influx. Direct measurement of [Ca2+] in the ER showed that SOCs slowed ER depletion. SOC-mediated currents were inwardly rectifying and greatly increased in the absence of divalent cations, as typical for SOCs in other cell types. Pharmacology of epithelial SOCs was consistent with that of some types of SOCs. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. Both STIM and Orai proteins were expressed in PDEC and were colocalized after store depletion. Furthermore, knockdown of Orai3 expression, the most abundant Orai subtype, reduced SOC-mediated Ca2+ influx significantly. In conclusion, basolateral Ca2+ entry through SOCs fills internal Ca2+ stores depleted by external stimuli and facilitates Ca2+-dependent cellular processes such as salt and mucin secretion from the exocrine pancreatic ducts.

The role of plasma membrane STIM1 and Ca2 +entry in platelet aggregation. STIM1 binds to novel proteins in human platelets

Ca2+ elevation is essential to platelet activation. STIM1 senses Ca2+ in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca2+ entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca2+ entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca2+ entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca2+-sensing role of STIM1 is served by the protein in the ER.

Characterization of store-operated Ca2+ channels in pancreatic duct epithelia

Store-operated Ca2+ channels (SOCs) are activated by depletion of intracellular Ca2+ stores following agonist-mediated Ca2+ release. Previously we demonstrated that Ca2+ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca2+ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca2+ influx as Ca2+ was released from intracellular stores. Direct measurement of [Ca2+] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP3 or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La3+ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca2+ entry through SOCs fills internal Ca2+ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca2+ such as salt and mucin secretion from the exocrine pancreatic ducts.

Staurosporine maintains the activation of store-operated Ca2+ entry even after the refilling of Ca2+ stores

Store-operated Ca2+ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca2+ signaling in non-excitable cells. Here we show that SOCE is enhanced in COS-7 cells treated with staurosporine (ST), a protein kinase inhibitor. In COS-7 cells, stimulation with ATP induced Ca2+ release from intracellular Ca2+ stores and Ca2+ entry from the extracellular space. Ca2+ release was not affected by treatment with ST, but Ca2+ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca2+ sequestration into Ca2+ stores. The Ca2+ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca2+ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca2+ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La3+ and 2-APB. The large increase in Ca2+ entry produced in the cells co-expressing Venus-Orai1 and STIM1-mKO1 was stabilized with ST treatment, and confocal imaging of these cells suggested that the complex between Orai1 and STIM1 did not completely dissociate following the refilling of Ca2+ stores. These results show that SOCE remains activated even after the refilling of Ca2+ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1–STIM1 interaction.

PLCδ: Johnny‐come‐lately to ORAI and the ups and downs of calcium signalling

PLCδ: Johnny‐come‐lately to ORAI and the ups and downs of calcium signalling

Effect of cGMP on Pharmacomechanical Coupling in the Uterine Artery of Near-Term Pregnant Sheep

The present study examined the role of cGMP in the regulation of alpha(1)-adrenoceptor-mediated pharmacomechanical coupling in the uterine artery of near-term pregnant sheep. The cell-permeable cGMP analog 8-bromo-cGMP produced a dose-dependent relaxation of the uterine artery and shifted norepinephrine (NE) dose-response curve to the right with a decreased maximal contraction. Accordingly, 8-bromo-cGMP significantly decreased the potency and the maximal response of NE-induced inositol 1,4,5-trisphosphate (IP(3)) synthesis in the uterine artery. In addition, 8-bromo-cGMP significantly reduced the binding affinity of IP(3) to the IP(3) receptor. The density of IP(3) receptors was not affected. Simultaneous measurement of intracellular Ca2+ concentrations ([Ca2+](i)) and tensions in the same tissue indicated that 8-bromo-cGMP decreased NE-induced contractions by 92% but only blocked 44% [Ca2+](i). In accordance, 8-bromo-cGMP significantly decreased tension generation for a given [Ca2+](i) (g/R(f340/380), 24.87 +/- 3.43 versus 3.10 +/- 0.35). In the absence of extracellular Ca2+, NE produced a transient increase in [Ca2+](i) and contraction, which were inhibited by 8-bromo-cGMP by 47 and 76%, respectively. In contrast to NE-induced responses, 8-bromo-cGMP had no significant effects on KCl-induced [Ca2+](i) and contractions. The results indicate that cGMP suppresses alpha(1)-adrenoceptor-mediated pharmacomechanical coupling in the uterine artery by inhibiting IP(3) synthesis and Ca2+ release from intracellular stores, as well as inhibiting the agonist-mediated Ca2+ sensitization of myofilaments, which is likely to play an important role in the adaptation of uterine artery contractility during pregnancy.

Bcl-2 suppresses Ca 2+ release through inositol 1,4,5-trisphosphate receptors and inhibits Ca 2+ uptake by mitochondria without affecting ER calcium store content

Cell survival is promoted by the oncoprotein Bcl-2. Previous studies have established that one of the pro-survival actions of Bcl-2 is to reduce cellular fluxes of Ca2+ within cells. In particular, Bcl-2 has been demonstrated to inhibit the release of Ca2+ from the endoplasmic reticulum. However, the mechanism by which Bcl-2 causes reduced Ca2+ release is unclear. In the accompanying paper [C.J. Hanson, M.D. Bootman, C.W. Distelhorst, T. Maraldi, H.L. Roderick, The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect, Cell Calcium (2008)], we described that only stable expression of Bcl-2 allowed it to work in a pro-survival manner whereas transient expression did not. In this study, we have employed HEK-293 cells that stably express Bcl-2, and which are, therefore, protected from pro-apoptotic stimuli, to examine the effect of Bcl-2 on Ca2+ homeostasis and signalling. We observed that Bcl-2 expression decreased the Ca2+ responses of cells induced by application of submaximal agonist concentrations. Whereas, decreasing endogenous Bcl-2 concentration using siRNA potentiated Ca2+ responses. Furthermore, we found that Bcl-2 expression reduced mitochondrial Ca2+ uptake by raising the threshold cytosolic Ca2+ concentration required to activate sequestration. Using a number of different assays, we did not find any evidence for reduction of endoplasmic reticulum luminal Ca2+ in our Bcl-2-expressing cells. Indeed, we observed that Bcl-2 served to preserve the content of the agonist-sensitive Ca2+ pool. Endogenous Bcl-2 was found to interact with inositol 1,4,5-trisphosphate receptors (InsP3Rs) in our cells, and to modify the profile of InsP3R expression. Our data suggest that the presence of Bcl-2 in the proteome of cells has multiple effects on agonist-mediated Ca2+ signals, and can abrogate responses to submaximal levels of stimulation through direct control of InsP3Rs.

Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery

AimsWe investigated the role of src family kinases (srcFK) in agonist-mediated Ca2+-sensitization in pulmonary artery and whether this involves interaction with the rho/rho-kinase pathway.Methods and resultsIntra-pulmonary arteries (IPAs) and cultured pulmonary artery smooth muscle cells (PASMC) were obtained from rat. Expression of srcFK was determined at the mRNA and protein levels. Ca2+-sensitization was induced by prostaglandin F2α (PGF2α) in α-toxin-permeabilized IPAs. Phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light-chain-20 (MLC20) and translocation of rho-kinase in response to PGF2α were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF2α enhanced phosphorylation of three srcFK proteins at tyr-416. In α-toxin-permeabilized IPAs, PGF2α enhanced the Ca2+-induced contraction (pCa 6.9) approximately three-fold. This enhancement was inhibited by the srcFK blockers SU6656 and PP2 and by the rho-kinase inhibitor Y27632. Y27632, but not SU6656 or PP2, also inhibited the underlying pCa 6.9 contraction. PGF2α enhanced phosphorylation of MYPT-1 at thr-697 and thr-855 and of MLC20 at ser-19. This enhancement, but not the underlying basal phosphorylation, was inhibited by SU6656. Y27632 suppressed both basal and PGF2α-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF2α triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656.Conclusions srcFK are activated by PGF2α in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1.

Abstract 379: Enhancement of TRPC6-mediated Ca 2+ Entry in Pulmonary Artery Smooth Muscle Cells of Idiopathic Pulmonary Arterial Hypertension Patients

Background & Hypothesis: Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and sustained pulmonary vasoconstriction are thought to play critical roles in the development of idiopathic pulmonary arterial hypertension (IPAH). Recently, we demonstrated that upregulation of the canonical transient receptor potential 6 (TRPC6) channel contributes to excessive proliferation of PASMCs isolated from IPAH patients. This study aimed at characterizing whether up-regulated TRPC6 expression affects resting cytosolic [Ca 2+ ] ([Ca 2+ ] cyt ) level and Ca 2+ entry in PASMCs of IPAH patients. Methods & Results: [Ca 2+ ] cyt was measured by fluorescence ratio video imaging with the Ca 2+ indicator fura-2. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable diacylglycerol analog that activates TRPC6 channels, was used to stimulate channel activities in the presence of extracellular Ca 2+ . The resting [Ca 2+ ] cyt andOAG-mediated increase in [Ca 2+ ]cyt were significantly higher in PASMCs from IPAH patients compared to PASMCs isolated from secondary pulmonary artery hypertension and normotensive patients. In PASMCs from IPAH patients, inhibition of TRPC6 expression by adenovirus-mediated siRNA specifically targeted the human TRPC6 gene led to an approximately 90% reduction of TRPC6 mRNA and protein levels and significantly attenuated OAG-mediated increase in [Ca 2+ ] cyt . Treatment of the IPAH-PASMCs with siRNA also decreased the resting [Ca 2+ ] cyt and significantly inhibited cell proliferation in comparison to cells treated with scrambled control siRNA. Furthermore, exogenous overexpression of human TRPC6 increased the resting [Ca 2+ ] cyt and enhanced OAG-mediated Ca 2+ entry in normal PASMCs. C onclusions: These results suggest that upregulation of TRPC6 channels in PASMC from IPAH patients serves as an important pathway for agonist-mediated Ca 2+ entry, mitogen-mediated PASMC proliferation and, ultimately, pulmonary vascular remodeling. Targeting TRPC6 expression and function in PASMCs would help develop novel therapeutic approaches for IPAH patients.

ER stress disrupts Ca2+-signaling complexes and Ca2+ regulation in secretory and muscle cells from PERK-knockout mice

Disruption of protein synthesis and folding results in ER stress, which is associated with the pathophysiology of diverse diseases affecting secretory and muscle cells. Cells are protected against ER stress by activation of the unfolded protein response (UPR) that is regulated by the protein kinase PERK, which phosphorylates the translation initiation factor 2 eIF2alpha to attenuate protein synthesis. PERK-/- cells are unable to modulate ER protein load and experience high levels of ER stress. In addition to its role in protein synthesis, the ER also orchestrates many signaling events essential for cell survival, prominent among which is Ca2+ signaling. It is not known, however, whether there is a relationship between ER stress and the function of the Ca2+-signaling pathway in muscle and non-muscle cells. To directly address this question we characterized Ca2+ signaling in the secretory pancreatic and parotid acinar cells and in urinary bladder smooth muscle (UBSM) cells obtained from PERK-/- and wild-type mice. Deletion of PERK that results in high levels of ER stress, and distention and fragmentation of the ER slowed the rate of agonist-mediated Ca2+ release from the ER and reduced Ca2+-induced Ca2+ release, although IP3 production, localization of the IP3 receptors, IP3-mediated Ca2+ release, Ca(v)1.2 current and RyRs activity remained unaltered. On the other hand, ER stress disrupted the integrity of the Ca2+-signaling complexes in both secretory and UBSM cells, as revealed by markedly reduced co-immunoprecipitation of plasma membrane- and ER-resident Ca2+-signaling proteins. These findings establish a relationship between the unfolding protein response, ER stress and Ca2+ signaling and highlight the importance of communication within the terminal ER-plasma membrane microdomain for propagation of the Ca2+ signal from the plasma membrane into the cell.

α1-Adrenoceptor-mediated phosphorylation of MYPT-1 and CPI-17 in the uterine artery: role of ERK/PKC

We previously demonstrated that ERK/PKC signaling pathways play a key role in regulation of Ca(2+) sensitivity and contractility of the uterine artery. The present study tested the hypothesis that ERK and PKC differentially regulated myosin light chain phosphatase activity by phosphorylation of myosin phosphatase target protein-1 (MYPT-1) and CPI-17. Agonist-induced contractions and phosphorylation of MYPT-1/Thr(696), MYPT-1/Thr(850), and CPI-17/Thr(38) were measured simultaneously in the same tissues of isolated near-term pregnant ovine uterine arteries. Phenylephrine produced time-dependent concurrent increases in the phosphorylation of ERK(44/42) and MYPT-1/Thr(850) that preceded contractions. In addition, phenylephrine induced phosphorylation of CPI-17/Thr(38) that was concurrent with the contractions. In contrast, phenylephrine did not induce phosphorylation of MYPT-1/Thr(696) in the uterine artery. PD-098059 inhibited phosphorylation of ERK(44/42) and the initial peak phosphorylation of MYPT-1/Thr(850) but did not affect CPI-17/Thr(38) phosphorylation. Activation of PKC by phorbol 12,13-dibutyrate induced a time-dependent phosphorylation of CPI-17/Thr(38) that preceded contractions of the uterine artery. In addition, phorbol 12,13-dibutyrate activated PKC-alpha and induced a coimmunoprecipitation of PKC-alpha with caldesmon. The results suggest that phosphorylation of MYPT-1/Thr(850) and CPI-17/Thr(38) play important roles in regulation of agonist-mediated Ca(2+) sensitivity in the uterine artery, in part by ERK and PKC, respectively. In addition, phosphorylated CPI-17 may regulate Ca(2+) sensitivity by interacting with caldesmon and reversing its inhibitory effect on myosin ATPase.

Cystic fibrosis airway epithelial Ca2+i signaling: The mechanism for the laeger agonist-mediated Ca2+i signals in human cystic fibrosis airway epithelia

Cystic fibrosis airway epithelial Ca2+i signaling: The mechanism for the laeger agonist-mediated Ca2+i signals in human cystic fibrosis airway epithelia

Cross talk between P2Y2 nucleotide receptors and CXC chemokine receptor 2 resulting in enhanced Ca2+ signaling involves enhancement of phospholipase C activity and is enabled by incremental Ca2+ release in human embryonic kidney cells.

We have shown previously that activation of endogenously expressed, Galphaq/11-coupled P2Y2 nucleotide receptors with UTP reveals an intracellular Ca2+ response to activation of recombinant, Galphai-coupled CXC chemokine receptor 2 (CXCR2) in human embryonic kidney cells. Here, we characterize further this cross talk and demonstrate that phospholipase C (PLC) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-dependent Ca2+ release underlies this potentiation. The putative Ins(1,4,5)P3 receptor antagonist 2-aminoethoxydiphenyl borane reduced the response to CXCR2 activation by interleukin-8, as did sustained inhibition of phosphatidylinositol 4-kinase with wortmannin, suggesting the involvement of phosphoinositides in the potentiation. Against a Li+ block of inositol monophosphatase activity, costimulation of P2Y2 nucleotide receptors and CXCR2 caused phosphoinositide accumulation that was significantly greater than that after activation of P2Y2 nucleotide receptors or CXCR2 alone, and was more than additive. Thus, PLC activity, as well as Ca2+ release, was enhanced. In these cells, agonist-mediated Ca2+ release was incremental in nature, suggesting that a potentiation of Ins(1,4,5)P3 generation in the presence of coactivation of P2Y2 nucleotide receptors and CXCR2 would be sufficient for additional Ca2+ release. Potentiated Ca2+ signaling by CXCR2 was markedly attenuated by expression of either regulator of G protein signaling 2 or the Gbetagamma-scavenger Galphat1 (transducin alpha subunit), indicating the involvement of Galphaq and Gbetagamma subunits, respectively.

Sphingosine 1-phosphate induces Ca2+ transients and cytoskeletal rearrangement in C2C12 myoblastic cells.

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+ concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349-357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+ channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+ channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.

Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors

Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C2C12 myoblasts SPP elicited significant Ca2+ mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca2+ response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca2+ transients performed with a spectrophotofluorimeter showed that the rise in Ca2+ was strictly dependent on availability of extracellular Ca2+. Cell treatment with pertussis toxin partially prevented the Ca2+ response induced by SPP, indicating that Gi-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca2+ mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca2+, although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca2+ increase, supporting the notion that Ca2+ mobilization in C2C12 cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.