Basic Amino Acids at the C-Terminus of the Third Intracellular Loop Are Required for the Activation of Phospholipase C by Cholecystokinin-B Receptors

Abstract: In common with other G_q protein-coupled receptors, the third intracellular loop of the cholecystokinin-B (CCK-B) receptor contains three basic amino acids (K333/K334/R335) at the C-terminal segment. To determine the importance of these conserved basic residues in G_q-protein activation and stimulation of phospholipase C, these basic amino acids were mutated. Subsequently, the ability of resulting mutant receptors to activate phospholipase C was investigated by measuring inositol phosphate formation in COS-7 cells and recording Ca²⁺-activated Cl^? currents from Xenopus oocytes. Site-directed mutagenesis was performed to mutate the three basic amino acids, K333/K334/R335, to neutral amino acids, M333/T334/L335. When the resulting mutant CCK-B receptors were expressed in COS-7 cells and Xenopus oocytes, sulfated cholecystokinin octapeptide (CCK-8) failed to induce inositol phosphate formation in COS-7 cells and evoke Ca²⁺-activated Cl^? currents from oocytes. Each basic amino acid was also mutated (K333M, K334T, and R335L). All three single-point mutations resulted in a significant reduction in CCK-8-induced inositol phosphate formation and CCK-8-activated Ca²⁺-dependent Cl^? currents. It is interesting that substituting the basic amino acids, K333/K334/R335, with three other basic residues, R333/R334/K335, did not change the maximal CCK-8-simulated inositol phosphate formation and the amplitude of CCK-8-evoked Ca²⁺-dependent Cl^? currents. Radioligand-binding studies showed that the above-mentioned mutations did not affect the affinity for CCK-8 and receptor expression level in COS-7 cells. These findings suggest that basic amino acids at the C-terminus of the third cytoplasmic loop are required for the signal transduction by CCK-B receptors.     

EP2 receptor mediated cAMP release is augmented by PGF2α activation of the FP receptor via the calcium-calmodulin pathway

Prostaglandins exert their effects on target cells by coupling to specific G protein-coupled receptors (GPCRs) that are often co-expressed in the same cells and use alternate and in some cases opposing intracellular signaling pathways. This study investigated the cross-talk that influences intracellular signaling and gene expression profiling in response to co-activation of the EP2 and FP prostanoid receptors in Ishikawa cells stably expressing both receptors (FPEP2 cells). In this study we show that in FPEP2 cells, PGF alone does not alter adenosine 3′,5′-cyclic monophosphate (cAMP) production, but in combination with Butaprost enhances EP2 receptor mediated cAMP release compared to treatment with Butaprost alone. PGF-mediated potentiation of cAMP release was abolished by antagonism of the FP receptor, inhibition of phospholipase C (PLC) and inositol phosphate receptor (IP3R) whereas inhibition of protein kinase C (PKC) had no effect. Moreover, inhibition of calcium effectors using calmodulin antagonist (W7) or Ca²⁺/calmodulin-dependent kinase II (CaMK-II) inhibitor (KN-93) abolished PGF potentiation of Butaprost-mediated cAMP release. Using siRNA molecules targeted against the adenylyl cyclase 3 (AC3) isoform, we show that AC3 is responsible for the cross-talk between the FP and EP2 receptors. Using gene array studies we have identified a candidate gene, Spermidine/N1-acetyltransferase (SAT1), which is regulated by this cAMP mediated cross-talk. In conclusion, this study demonstrates that co-activation of the FP and EP2 receptors results in enhanced release of cAMP via FP receptor-Gα_q-Ca²⁺-calmodulin pathway by activating calcium sensitive AC3 isoform.     

Evidence that cyclic AMP may regulate Ca2+-mobilization and phospholipases in thrombin-stimulated human platelets

The regulation of human platelet responses by cyclic AMP (cAMP) has been investigated by measuring thrombin-stimulated serotonin release, Ca²⁺ uptake and phospholipase activity. Thrombin-induced 1,2-diacylglycerol (DG) formation as a result of phospholipase C activation was inhibited by pretreatment with dibutyryl cAMP (dbcAMP) in a dose-dependent manner. Subsequent failure to produce phosphatidic acid (PA), which is converted from 1,2-DG by phosphorylation and would serve as intracellular Ca²⁺ ionophore, appeared to parallel the decrease in Ca²⁺ uptake activity. Phospholipase A₂ activity, monitored by the production of [³H]lysophosphatidylcholine and [³H]lysophosphatidylethanolamine, was also suppressed by dbcAMP. These data indicate that the intracellular cAMP level may be closely associated with Ca²⁺ uptake and phospholipases activation. In addition, it is suggested that alteration of intracellular cAMP regulates phospholipase activation and consequently platelet responses, perhaps by controlling available Ca²⁺ content.     

Activation of the Human FPRL-1 Receptor Promotes Ca<Superscript>2+</Superscript> Mobilization in U87 Astrocytoma Cells

The human formyl peptide receptor like 1 (FPRL-1) is a variant of the G_i-coupled formyl-peptide receptor. Functional FPRL-1 is endogenously expressed in the U87 astrocytoma cell line and there is accumulating evidence to suggest that FPRL-1 may be involved in neuroinflammation associated with the pathogenesis of Alzheimer’s disease. In this study, we examined the ability of FPRL-1 to mobilize intracellular Ca²⁺ in U87 astrocytoma cells, as well as in Chinese hamster ovary (CHO) cells stably expressing FPRL-1. We showed that Trp–Lys–Tyr–Met–Val–Met–NH₂ (WKYMVM), a specific agonist for FPRL-1, stimulated Ca²⁺ influx in both U87 and FPRL-1/CHO cells. These effects can be inhibited by the FPRL-1 selective antagonist, WRW⁴. Involvement of G_i proteins was demonstrated with the use of pertussis toxin, while inhibitors of store-operated channels (SOC) including 1-[2-(4-methoxyphenyl)]-2-[3-(4-methpxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride (SKF96365) and 2-aminoethoxydiphenyl borate (2-APB) were found to abolish the WKYMVM-induced Ca²⁺ increase. However, intracellular Ca²⁺ mobilization in both cell lines were unaffected by the phospholipase Cβ inhibitor U73122 or selective ryanodine receptor inhibitors. Our data demonstrated that activation of G_i-coupled FPRL-1 can lead to Ca²⁺ influx possibly via SOCs in U87 and FPRL-1/CHO cells.     

Isolation of Zn2+ as an Endogenous Agonist of GPR39 from Fetal Bovine Serum

We attempted to determine natural agonists of GPR39 in fetal bovine serum (FBS). FBS was conditioned to extract peptides and fractionated by two types of HPLC. The activity of each fraction was monitored by intracellular calcium mobilization. Then the purified active ingredient was analyzed by inductively coupled plasma mass spectrometry. In this fashion, Zn^{2 +} ion was identified as an agonist of GPR39, though no peptidergic molecules were found. The calcium-mobilizing activity of Zn^{2 +} was not abolished by pertussis toxin but was by a phospholipase C (PLC) inhibitor, U73122, indicating that the activity of GPR39 is mediated through the G_qα -PLC pathway. In addition, Zn^{2 +} also activated mouse and rat GPR39, showing that the function of GPR39 as a Zn^{2 +} receptor is conserved across species. This study is the first exploration of GPR39 agonists in FBS and indicates that GPR39 functions as a Gq-coupled Zn^{2 +}-sensing receptor.     

Lysophosphatidic acid‐induced Ca2+ mobilization in the neural retina of chick embryo

Lysophosphatidic acid (LPA) plays various roles in the regulation of cell growth as a lipid mediator. We studied the effect of LPA on intracellular Ca²⁺ concentration ([Ca²⁺]_i) with Fura‐2 in the neural retina of chick embryo during neurogenesis. Bath application of LPA (1–100 μM) to the embryonic day 3 (E3) chick retina caused an increase in [Ca²⁺]_i in a dose‐dependent manner, with an EC₅₀ value of 9.2 μM. The Ca²⁺ rise was also evoked in a Ca²⁺‐free medium, suggesting that release of Ca²⁺ from intracellular Ca²⁺ stores (Ca²⁺ mobilization) was induced by LPA. U‐73122, a blocker of phospholipase C (PLC), inhibited the Ca²⁺ rise to LPA. Pertussis toxin partially inhibited the Ca²⁺ rise to LPA, indicating that G_i/G_o protein was at least partially involved in the LPA response. The developmental profile of the LPA response was studied from E3 to E13. The Ca²⁺ rise to LPA declined drastically from E3 to E7, in parallel with decrease in mitotic activity of retinal progenitor cells. The signal transduction pathway and developmental profile of the Ca²⁺ response to LPA were the same as those of the Ca²⁺ response to adenosine triphosphate (ATP), which enhances the proliferation of retinal progenitor cells. The coapplication of LPA with ATP resulted in enhancement of Ca²⁺ rise in the E3 chick retina. Our results show that LPA induces Ca²⁺ mobilization in the embryonic chick retina during neurogenesis. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 495–504, 1999     

Different mechanisms are involved in intracellular calcium increase by insulin-like growth factors 1 and 2 in articular chondrocytes: Voltage-gated calcium channels,and/or phospholipase C coupled to a pertussis-sensitive G-protein

This study describes the mechanisms involved in the IGF-1 and IGF-2-induced increases in intracellular calcium concentration [Ca²⁺]i in cultured chondrocytes and the involvement of type 1 IGF receptors. It shows that IGF-1, IGF-2, and insulin increased the cytosolic free calcium concentration [Ca²⁺]i in a dose-dependent manner, with a plateau from 25 to 100 ng/ml for both IGF-1 and IGF-2 and from 1 to 2 μg/ml for insulin. The effect of IGF-1 was twice as great as the one of IGF-2, and the effect of insulin was 40% lower than IGF-1 effect. Two different mechanisms are involved in the intracellular [Ca²⁺]i increase. 1) IGF-1 and insulin but not IGF-2 involved a Ca²⁺ influx through voltage-gated calcium channels: pretreatment of the cells by EGTA and verapamil diminished the IGF-1 or insulin-induced[Ca²⁺]i but did not block the effect of IGF-2.2)IGF-1, IGF-2, and insulin also induced a Ca²⁺ mobilization from the endoplasmic reticulum: phospholipase C (PLC) inhihitors, neomycin, or U-73122 partially blocked the intracellular [Ca²⁺]i increase induced by IGF-1 and insulin and totally inhibited the effect of IGF-2. This Ca²⁺ mobilization was pertussis toxin (PTX) dependent, suggesting an activation of a PLC coupled to a PTX-sensitive G-protein. Lastly, preincubation of the cells with IGF₁ receptor antibodies diminished the IGF-1-induced Ca²⁺ spike and totally abolished the Ca²⁺ influx, but did not modify the effect of IGF-2. These results suggest that IGF-1 action on Ca²⁺ influx involves the IGF₁ receptor, while part of IGF-1 and all of IGF-2 Ca²⁺ mobilization do not implicate this receptor. J. Cell. Biochem. 64:414–422. © 1997 Wiley-Liss, Inc.     

The WD40 repeat protein,WDR36, orchestrates sphingosine kinase-1 recruitment and phospholipase C-β activation by Gq-coupled receptors

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that G_q-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed G_q regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gα_q to phospholipase C (PLC)-β and the thromboxane A₂ receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M₃ and bradykinin B₂ receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca²⁺]_i increases. Surprisingly, WDR36 also augmented M₃ receptor-induced [Ca²⁺]_i increases, which was due to increased Ca²⁺ mobilization while the Ca²⁺ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by G_q-coupled M₃, B₂ and H₁ receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B₂ receptors. It is concluded that WDR36 reduces signalling of G_q-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gα_q and PLCβ. Our results support a role of WDR36 in orchestration of G_q signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.     

Molecular and pharmacological characterization of zebrafish ‘contractile’ and ‘inhibitory’ prostanoid receptors

Prostanoids comprising prostaglandins (PGs) and thromboxanes (TXs) have been shown to play physiological and pathological roles in zebrafish. However, the molecular basis of zebrafish prostanoid receptors has not been established. Here, we demonstrate that there exist at least five ‘contractile’ (Ca²⁺-mobilizing) and one ‘inhibitory’ (G_i-coupled) prostanoid receptors in zebrafish; five ‘contractile’ receptors consisting of two PGE₂ receptors (EP1a and EP1b), two PGF_2α receptors (FP1 and FP2), and one TXA₂ receptor TP, and one ‘inhibitory’ receptor, the PGE₂ receptor EP3. [³H]PGE₂ specifically bound to the membranes of cells expressing zebrafish EP1a, EP1b and EP3 with a Kd of 4.8, 1.8 and 13.6 nM, respectively, and [³H]PGF_2α specifically bound to the membranes of cells expressing zebrafish FP1 and FP2, with a Kd of 6.5 and 1.6 nM, respectively. U-46619, a stable agonist for human and mouse TP receptors, significantly increased the specific binding of [³⁵S]GTPγS to membranes expressing the zebrafish TP receptor. Upon agonist stimulation, all six receptors showed an increase in intracellular Ca²⁺ levels, although the increase was very weak in EP1b, and pertussis toxin abolished only the EP3-mediated response. Zebrafish EP3 receptor also suppressed forskolin-induced cAMP formation in a pertussis toxin-sensitive manner. In association with the low structural conservation with mammalian receptors, most agonists and antagonists specific for mammalian EP1, EP3 and TP failed to work on each corresponding zebrafish receptor. This work provides further insights into the diverse prostanoid actions mediated by their receptors in zebrafish.     

Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y₂ receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y₂ receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca²⁺ mobilization indicating that nucleotides cause Ca²⁺ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca²⁺ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca²⁺ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca²⁺ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca²⁺ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca²⁺ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y₂ receptor can cause Ca²⁺ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins.     

Leukotriene D4-induced Ca2+ Mobilization in Ehrlich Ascites Tumor Cells

Stimulation of Ehrlich ascites tumor cells with leukotriene D₄ (LTD₄) within the concentration range 1–100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca²⁺ concentration, [Ca²⁺] _i . The Ca²⁺ peak time, i.e., the time between addition of LTD₄ and the highest measured [Ca²⁺] _i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD₄ (100 nm), the highest measured increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium is 260 ± 14 nm and the EC₅₀ value for LTD₄-induced Ca²⁺ mobilization is estimated at 10 nm. Neither the peptido-leukotrienes LTC₄ and LTE₄ nor LTB₄ are able to mimic or block the LTD₄-induced Ca²⁺ mobilization, hence the receptor is specific for LTD₄. Removal of Ca²⁺ from the experimental buffer significantly reduces the size of the LTD₄-induced increase in [Ca²⁺] _i . Furthermore, depletion of the intracellular Ins(1,4,5)P₃-sensitive Ca²⁺ stores by addition of the ER-Ca²⁺-ATPase inhibitor thapsigargin also reduces the size of the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium, and completely abolishes the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-free medium containing EGTA. Thus, the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells involves an influx of Ca²⁺ from the extracellular compartment as well as a release of Ca²⁺ from intracellular Ins(1,4,5)P₃-sensitive stores. The Ca²⁺ peak times for the LTD₄-induced Ca²⁺ influx and for the LTD₄-induced Ca²⁺ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD₄ also induces a transient increase in Ins(1,4,5)P₃ generation in the Ehrlich cells, and the Ins(1,4,5)P₃ peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4,5)P₃ content seems to increase before the LTD₄-induced Ca²⁺ release from the intracellular stores but after the LTD₄-induced Ca²⁺ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD₄-induced increase in Ins(1,4,5)P₃ as well as the LTD₄-induced increase in [Ca²⁺] _i , indicating that a U73122-sensitive phospholipase C is involved in the LTD₄-induced Ca²⁺ mobilization in Ehrlich cells. The LTD₄-induced Ca²⁺ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD₄-activated Ca²⁺ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD₄ acts in the Ehrlich cells via a specific receptor for LTD₄, which upon stimulation initiates an influx of Ca²⁺, through yet unidentified Ca²⁺ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P₃ formation and finally release of Ca²⁺ from the intracellular Ins(1,4,5)P₃-sensitive stores. Received: 9 February 1996/Revised: 15 August 1996     

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: Functional coupling of the sphingosine-1-phosphate receptor to PLC-xβ in Xenopus oocytes

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca²⁺-dependent oscillatory Cl⁻ currents by acting through membrane-bound receptors. External application of 50 μM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-μM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca²⁺-dependent Cl⁻ currents were observed in the absence of extracellular Ca²⁺, were blocked by intracellular injection of the Ca²⁺ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca²⁺ ATPase. Intracellular Ca²⁺ release appeared to be from inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store, because Cl⁻ currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xβ), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xβ, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein α subunits that were identified in Xenopus laevis; G_qα, G₁₁α, G₀α, and G_i1α. Among AS-ODNs against the Gαs tested, AS-G_qα and AS-G_i1α to S1P and AS-G_qα and AS-G₁₁α to LPA specifically reduced current responses, respectively, to about 20–30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca²⁺ from the IP₃-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes. J. Cell. Physiol. 176:412–423, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of Ca Activity in Cardiomyocytes through Caveolae-Gαq Interactions

Cardiomyocytes have a complex Ca²⁺ behavior and changes in this behavior may underlie certain disease states. Intracellular Ca²⁺ activity can be regulated by the phospholipase Cβ–Gα_q pathway localized on the plasma membrane. The plasma membranes of cardiomycoytes are rich in caveolae domains organized by caveolin proteins. Caveolae may indirectly affect cell signals by entrapping and localizing specific proteins. Recently, we found that caveolin may specifically interact with activated Gα_q, which could affect Ca²⁺ signals. Here, using fluorescence imaging and correlation techniques we show that Gα_q-Gβγ subunits localize to caveolae in adult ventricular canine cardiomyoctyes. Carbachol stimulation releases Gβγ subunits from caveolae with a concurrent stabilization of activated Gα_q by caveolin-3 (Cav3). These cells show oscillating Ca²⁺ waves that are not seen in neonatal cells that do not contain Cav3. Microinjection of a peptide that disrupts Cav3-Gα_q association, but not a control peptide, extinguishes the waves. Furthermore, these waves are unchanged with rynaodine treatment, but not seen with treatment of a phospholipase C inhibitor, implying that Cav3-Gα_q is responsible for this Ca²⁺ activity. Taken together, these studies show that caveolae play a direct and active role in regulating basal Ca²⁺ activity in cardiomyocytes.     

Pituitary Adenylate Cyclase-Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca²⁺ release and Ca²⁺ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca²⁺ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP₃), cyclic AMP, and the intracellular Ca²⁺ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP₃ and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP₃, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP₃ production, the Ca²⁺ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP₃ channels. The potencies of the peptides to cause Ca²⁺ release in the presence of cinnarizine were similar. The Ca²⁺ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca²⁺ release was unaffected by Rp-adenosine 3′,5′-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca²⁺ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP₃ production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca²⁺ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca²⁺ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP₃ and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca²⁺ in adrenal chromaffin cells.     

Gαq-mediated plasma membrane translocation of sphingosine kinase-1 and cross-activation of S1P receptors

Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of G_q-coupled receptors induced a profound, rapid (half-life 3–5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein α-subunits specifically of the G_q family. Classical G_q signalling pathways, or phosphorylation at Ser²²⁵, phospholipase D and Ca²⁺/calmodulin were not involved in M₃ receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of G_q-coupled receptors, linking them via cross-activation of S1P receptors to G_i and G_12/13 signalling pathways.     

Cyanide-stimulated inositol 1,4,5-trisphosphate formation: An intracellular neurotoxic signaling cascade

Cyanide-induced neurotoxicity is associated with altered cellular Ca²⁺ homeostasis resulting in sustained elevation of cytosolic Ca²⁺. In order to characterize the effect of cyanide on intracellular signaling mechanisms, the interaction of KCN with the inositol 1,4,5-triphosphate Ca²⁺ signaling system was determined in the PC12 cell line. KCN in the concentration range of 1.0–100 μM produced a rapid rise in intracellular IP₃ levels (peak level occurred within 60 sec); 10 μM KCN elevated intracellular levels of IP₃ to 148% of control levels. This response was mediated by phospholipase C (PLC) since U73122, a specific PLC inhibitor, blocked the response. Removal of Ca²⁺ from the incubation medium and chelation of intracellular Ca²⁺ with BAPTA partially attenuate the cyanide-stimulated IP₃ generation, showing that the response is partially Ca²⁺ dependent. Also, treatment of cells with nifedipine or LaCl₃, Ca²⁺ channel blockers, partially blocked the generation of IP₃. This study shows that cyanide in concentrations as low as 1 μM stimulates IP₃ generation that may be mediated by receptor and nonreceptor IP₃ production since they have differential dependence on Ca²⁺. It is proposed that this response is an early intracellular signaling action that can contribute to altered Ca²⁺ homeostasis characteristic of cyanide neurotoxicity. © 1997 John Wiley & Sons, Inc.     

Adenosine A2A receptors inhibit the conductance of NMDA receptor channels in rat neostriatal neurons

Summary Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70–80%), NMDA-induced inward currents were inhibited by the adenosine AZA receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the AZA receptor selective antagonist 8-(3chlorostyryl) caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP--S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14–24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca²⁺ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, AZA receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca²⁺ pathway rather than the adenylate cyclase/PKA pathway.     

Lysophosphatidic acid induces Ca2+ mobilization and c-Myc expression in mouse embryonic stem cells via the phospholipase C pathway

Embryonic stem cells (ESC) are pluripotent and could be maintained in vitro in a self-renewing state indefinitely, at the same time preserving their potential to differentiate towards more specific lineages. Despite the progress in the field, the complex network of signalling cascades involved in the maintenance of the self-renewing and pluripotent state remains not fully understood. In the present study, we have investigated the role of lysophosphatidic acid (LPA), a potent mitogen present in serum, in Ca²⁺ signalling and early gene activation in mouse ESC (mESC). In these cells, we detected the expression of the G-protein coupled LPA receptor subtypes LPA₁, LPA₂ and LPA₃. Using fluorescence Ca²⁺ imaging techniques, we showed that LPA induced an increase in intracellular Ca²⁺ concentration. This increase was also observed in the absence of extracellular Ca²⁺, suggesting the involvement of internal stores. Pre-treatment with BAPTA-AM, thapsigargin or U-73122 efficiently blocked this Ca²⁺ release, indicating that LPA was evoking Ca²⁺ mobilization from the endoplasmic reticulum via the phospholipase C (PLC) pathway. Interestingly, this signalling cascade initiated by LPA was involved in inducing the expression of the Ca²⁺-dependent early response gene c-myc, a key gene implicated in ESC self-renewal and pluripotency. Additionally, LPA increased the proliferation rate of mESC. Our findings therefore outline the physiological role of LPA in mESC.