Inositol Trisphosphate Formation Research Articles

Ablation of optineurin impairs metabotropic glutamate receptor 5 signaling in mouse hippocampus

Optineurin (OPTN) is a ubiquitously expressed autophagy adaptor that forms a functional interactome with Group I metabotropic glutamate receptors (mGluR1 and 5), members of the Gαq/11protein receptor family. OPTN attenuates mGluR1 coupling to phospholipase Cβ (PLCβ) and subsequent inositol trisphosphate (IP3) formation in response to agonist stimulation. We have also demonstrated that pathological mGluR5 signaling impairs autophagic machinery via GSK3β/ZBTB16 pathway in both Alzheimer's and Huntington's disease mouse models. In this study, we investigated whether mGluR5 signaling is regulated in an OPTN-dependent manner. We knocked out Optn gene in STHdhQ7/Q7 striatal cells using CRISPR/Cas9 approach and employed OPTN null mice to probe the impact of OPTN loss on mGluR5 canonical and autophagic signaling. We show that OPTN deletion enhances Ca2+ mobilization following mGluR5 activation in STHdhQ7/Q7 cells and impairs mGluR5-mediated ERK1/2 phosphorylation in both STHdhQ7/Q7 cells and acute hippocampal slices. In addition, OPTN loss enhanced basal GSK3β activity and increased the breakdown of ZBTB16 in STHdhQ7/Q7 cells, and abolished mGluR5-mediated inactivation of GSK3β/ZBTB16 pathway in hippocampal slices. OPTN deletion also increased the expression levels of autophagy adaptor P62 in hippocampal slices, an effect that was reversed by mGluR5 antagonism. Overall, the data suggest that mGluR5 and OPTN crosstalk is essential for mGluR5 signaling and therefore can potentially contribute to the pathophysiology of neurodegenerative diseases.

Role of cyclic AMP and cyclic GMP as modulators of platelet cytosolic calcium

Studies from our laboratory at the University of Minnesota as well as that of others have demonstrated that agonist-mediated receptor stimulation leads to the activation of phospholipase C and formation of second messengers, diacyl glycerol and inositol trisphosphate, and mobilization of cytosolic free calcium. Elevation in intracellular calcium activates phospholipase A2 and release of arachidonic acid. This fatty acid is further converted to active metabolites, prostaglandin endoperoxides, and thromboxanes (TXA 2 ). TXA 2 promote fibrinogen binding, irreversible aggregation, and the release of granule contents, including serotonin and ADP. Agents that promote irreversible aggregation, facilitate fibrinogen binding, and those drugs that dissociate this process do so by lowering the cytosolic calcium levels. Endogenous antagonists such as PGE 1 , PGI 2 , and NO exert their inhibitory effect on platelet function by the action of cyclic nucleotides, cyclic AMP and cyclic GMP. Epinephrine-induced modulation of alpha-adrenergic receptor restores the action of agonists in drug-induced refractory platelets. As far as the role of cyclic nucleotides is considered, the observations are consistent with the concept that cyclic nucleotides participation in the biologic regulation may be modulated by cytoplasmic cations, and that a change in intracellular cyclic nucleotides and appropriate cations serves to promote the participation of both regulatory effectors. Anti-platelet drugs such as aspirin, indomethacin, and PGE 1 do not inhibit platelet interactions with the vessel wall. Based on this collective knowledge, novel antiplatelet drugs should be developed for the prevention of acute vascular events.

An intact helical domain is required for Gα14 to stimulate phospholipase Cβ.

BackgroundStimulation of phospholipase Cβ (PLCβ) by the activated α-subunit of Gq (Gαq) constitutes a major signaling pathway for cellular regulation, and structural studies have recently revealed the molecular interactions between PLCβ and Gαq. Yet, most of the PLCβ-interacting residues identified on Gαq are not unique to members of the Gαq family. Molecular modeling predicts that the core PLCβ-interacting residues located on the switch regions of Gαq are similarly positioned in Gαz which does not stimulate PLCβ. Using wild-type and constitutively active chimeras constructed between Gαz and Gα14, a member of the Gαq family, we examined if the PLCβ-interacting residues identified in Gαq are indeed essential.ResultsFour chimeras with the core PLCβ-interacting residues composed of Gαz sequences were capable of binding PLCβ2 and stimulating the formation of inositol trisphosphate. Surprisingly, all chimeras with a Gαz N-terminal half failed to functionally associate with PLCβ2, despite the fact that many of them contained the core PLCβ-interacting residues from Gα14. Further analyses revealed that the non-PLCβ2 interacting chimeras were capable of interacting with other effector molecules such as adenylyl cyclase and tetratricopeptide repeat 1, indicating that they could adopt a GTP-bound active conformation.ConclusionCollectively, our study suggests that the previously identified PLCβ-interacting residues are insufficient to ensure productive interaction of Gα14 with PLCβ, while an intact N-terminal half of Gα14 is apparently required for PLCβ interaction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12900-015-0043-3) contains supplementary material, which is available to authorized users.

Beyond Vasodilator: Anti-Apoptotic Effect of Endothelin Receptor Antagonist

Refer to the page 97-104 Treatment of pulmonary arterial hypertension (PAH) has changed with development of PAH targeted therapy, which includes use of specific vasodilator in pulmonary arterial system. Bosentan is an endothelin receptor antagonist (ERA) and vasodilator that was the first approved oral drug for PAH treatment. The clinical benefit of bosentan has been established in several multicenter clinical trials1),2) and shown to develop slowly over several months. Therefore, beyond its vasodilator effect, the effect of ERA on PAH progression has been investigated. Pathology of pulmonary artery in PAH patients shows vascular smooth muscle proliferation and obliteration of pulmonary vessels. Endothelial dysfunction is commonly found, and inflammatory response and endothelial cell apoptosis have also been reported. After apoptosis of normal endothelial cell line, surviving cells proliferate. This abnormal proliferation forms unique plexiform lesions that obliterate arterioles and capillaries at the end stage of disease. Vascular smooth muscle cells are prone to hyperplasia and unbalanced apoptosis (decreased apoptosis) leads to hypertrophy of the vessel wall. Endothelin (ET) is produced in endothelial cells of various tissues. Especially, ET-1 is predominantly expressed in pulmonary circulation. ET-2 mainly expressed in the kidney and ET-3 in the brain and intestine.3) ET is secreted from the abluminal side of the cells towards the adjacent vascular smooth muscle cells. There are two types of ET receptor including ETA and ETB. ETA is located in pulmonary vascular smooth muscle cells where it mediates vasoconstriction through inositol trisphosphate (IP3) formation and release of intracellular calcium. The ETA receptor is associated with proliferation of vascular smooth muscle cells in pulmonary arteries. In PAH patients, expression of the ETA receptor is increased and is associated with vasoproliferation. Inflammation also plays an important role in PAH patients and ETA is elevated in this setting. Imbalance between proliferation and apoptosis in vascular smooth muscle cells is associated with vascular hypertrophy in PAH.4) However, before initiation of vascular hypertrophy, inflammation of endothelial cells is observed and is associated with apoptosis of endothelial cells. Therefore, apoptosis of endothelial cells can trigger the initial event for progression of PAH. Vascular endothelial growth factor receptor blockade has been noted as a possible mechanism for endothelial cell apoptosis, followed by the selection of apoptosis-resistant clones, which lead to the formation of plexiform lesions with severe angio-obliterating lesions.5) The article by Hong et al.6) suggests a role for bosentan in attenuating apoptosis and inflammation of endothelial cells. Their results indicate decreases in both caspase-3 and vascular endothelial growth factor in the bosentan-treated group. The proinflammatory cytokine, interleukin-6, was decreased and mRNA expressions of tumor necrosis factor (TNF)-α were decreased. Potential effects of bosentan to reduce apoptosis have been also reported in neuronal cells through the up-regulation of bcl-2 gene expression and decreased expression of the apoptotic protein, caspase.7) These observations suggest that bosentan may prevent disease progression in an early stage by preventing endothelial apoptosis and inflammatory response. Another interesting suggestion is the expanding indication of bosentan to other diseases. Overexpression of endothelin-1 in mice resulted in a cardiomyopathy resembling doxorubicin-induced cardiomyopathy. Bien et al.8) reported that bosentan inhibits doxorubicin-induced cardiomyopathy in a mouse model. Pretreatment with bosentan resulted in reduction of TNF-α and Bax expression. Interestingly, ET antagonists are proapoptotic and antiproliferative in human colon cancer cells.9) Therefore, beyond its role as a vasodilator, bosentan has the potential to attenuate apoptotic cells, and this property can be applied to other disease associated with cell apoptosis.

Reduction of Urinary Bladder Activity in Transgenic Sickle Cell Disease Mice.

Abstract 2580Poster Board II-557Genitourinary infection and urinary retention have been related to impaired urinary concentrating ability and altered urinary volume in sickle cell disease (SCD) patients. This condition may be associated with changes in the detrusor smooth muscle (DSM) reactivity. Berkeley transgenic SCD mice display several clinical manifestations similar to those seen in humans, but no studies assessing the alterations of DSM reactivity in SCD mice have been performed. DSM contractions are a consequence of the activation of muscarinic receptors that promote stimulation of phospholipase C, with increased formation of inositol trisphosphate and diacylglycerol to release calcium (Ca2+) from intracellular stores, leading to bladder emptying. The Ca2+-independent RhoA/Rho-kinase pathway is also involved in the regulation of bladder smooth muscle tone by alteration of the sensitivity of contractile proteins for Ca2+. Changes in the contraction mechanisms of DSM may contribute to bladder dysfunction. The aim of this study was to evaluate the contractile mechanisms of isolated DSM of transgenic SCD mouse. SCD (SS) and C57BL/6 (CTL) mice tissues were removed and mounted in 10ml organ baths containing Krebs solution at 37°C and continuously bubbled with a mixture of 95%O2:5%CO2 (pH 7.4). Tissues were stretched to a resting tension of 10 mN and allowed to equilibrate for 60 minutes. Changes in isometric force were recorded using a PowerLab400 Data Acquisition System. Cumulative concentration-response curves to muscarinic agonist carbachol (CCh; 0.01–100 μM) were obtained in the absence and presence of the Rho-kinase inhibitor Y-27632 (1–10 μM). Cumulative concentration-response curves were constructed to hyperpolarizing solution, potassium chloride (KCl; 1–300 mM) and calcium chloride (CaCl2; 0.01–100 mM). Frequency-response curves for electrical field stimulation (EFS; 1–32 Hz) were obtained in strips in the absence and presence of non-selective muscarinic antagonist atropine (1 μM) or purinergic receptor antagonist suramin (100 μM). Cumulative addition of the CCh produced concentration-dependent contractile responses in DSM, although the potency (pEC50) did not change in SS mice (5.80±0.02) compared with CTL mice (5.69±0.05). In contrast, the maximal response (Emax) was significantly reduced in SS mice (4.20±0.42mN) compared with CTL mice (9.96±1.77mN). In addition, the pEC50 and Emax elicited by CCh were reduced in the presence of Y-27632; however, no change was observed among all group studies. Cumulative addition of the KCl produced concentration-dependent contraction in DSM, and both pEC50 (0.98±0.08) and Emax (7.63±1.03mN) were significantly reduced in SS mice, compared with CTL mice (1.24±0.07; 11.24±0.84mN, respectively). CaCl2 produced concentration-dependent contractile responses, the pEC50 did not change in SS mice (2.00±0.06); however, the Emax was significantly reduced (10.1±0.25mN) when compared to CTL mice (1.92±0.05; 17.8±2.3mN). EFS-induced contractions in DSM of SS mice were decreased at significantly higher frequencies than compared to CTL mice. In addition, EFS-induced contractions in DSM were significantly decreased in the presence of atropine and suramin in both SS mice and CTL mice, but no change was observed among all group studies. Our study shows that SS mice exhibit impaired contractile responses to muscarinic agonist, KCl, CaCl2 and EFS, with no involvement of the Rho-kinase signaling pathway. This bladder dysfunction may contribute to the increased risk of urinary tract infections observed in SCD patients. Further studies are necessary to better understand the relationship between DSM reactivity alterations and genitourinary manifestations in SCD. Disclosures:No relevant conflicts of interest to declare.

Prostanoids in health and disease

The prostanoids are a family of lipid mediators generated by the action of cyclooxygenase on a 20-carbon unsaturated fatty acid, arachidonic acid. Prostanoids are generated widely in response to diverse stimuli and, acting in a paracrine or autocrine manner, play important roles in normal physiology and disease. This review summarizes the current knowledge on prostanoid generation and the roles of individual mediators, their biosynthetic pathways, and their receptors in health and disease.

Ontogeny and Effects of Hypothalamic Pituitary Disconnection on Formation of Inositol Trisphosphate in Fetal Sheep Pituitary Cells

In late gestation fetal sheep, the pituitary becomes increasingly responsive to stimulation by arginine vasopressin (AVP). This change appears to be one important factor mediating the plasma cortisol surge, a critical developmental event. It is not known precisely why pituitary corticotropes become more responsive at this time. In this study we examined the possibility that changes in second messenger generation [inositol trisphosphate (IP(3))] are responsible. Two studies were undertaken. The first was an ontogeny study, where pituitaries were isolated from 100-, 120-, and 140-d gestational age (dGA) fetal sheep. Cells were cultured, stimulated with AVP, and the formation of IP(3) assessed. The amount of IP(3) generated increased with gestational age (percent increases from unstimulated controls were 4.6, 11.5, and 21.5 for 100, 120, and 140 dGA, respectively), with significant differences between the 140-dGA group and both earlier groups apparent. The second study examined the impact of 120-dGA hypothalamo-pituitary disconnection (HPD), which prevents corticotrope maturation, on responsiveness of pituitary cells isolated from 140-dGA fetuses. Cells were stimulated with AVP, and the formation of IP(3) and secretion of ACTH were assessed. Significantly less IP(3) was formed, and ACTH secreted in cells from HPD compared with control fetuses (IP(3) and ACTH levels were 50% and 35% lower, respectively). Results from the HPD study demonstrate that the ontogenic changes in IP(3) after AVP require an intact hypothalamic-pituitary-adrenal axis. These findings suggest that heightened second messenger generation may be a key reason for increased ACTH secretory responsiveness to AVP in the late gestation sheep fetus.

RANTES stimulates Ca2+ mobilization and inositol trisphosphate (IP3) formation in cells transfected with G protein‐coupled receptor 75

RANTES is an inflammatory chemokine with a critical role in T-lymphocyte activation and proliferation. Its effects are mediated through G protein-coupled heptahelical receptors (GPCRs). We show for the first time that RANTES activates the orphan G protein-coupled receptor 75 (GPR75). To identify a ligand for GPR75 we have used three different and independent methods, namely luciferase assay, bioluminescence assay and IP3 accumulation assay. Treatment of cells expressing GPR75 with subnanomolar concentrations of RANTES led to stimulation of the luciferase activity in a reporter-gene assay, an increase in inositol trisphosphate, and intracellular Ca2+. The latter effect was blocked by the phospholipase-C inhibitor (PLC) U73122 indicating that Gq proteins mediate GPR75 signaling. RANTES enhanced the phosphorylation of AKT and mitogen-activated protein kinase (MAPK) in GPR75-transfected cells and this effect was blocked by the PLC inhibitor U73122 and the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. The hippocampal cell line HT22, which expresses GPR75 endogenously, but not the other known RANTES receptors, was used to study the effects of RANTES and GPR75 on neuronal survival. Treatment of HT22 cells with RANTES significantly reduced the neurotoxicity of amyloid-beta peptides, by activating PLC and PI3K. This demonstrate clearly and undoubtedly the ability of RANTES to act on GPR75. Defects in the RANTES/GPR75-signaling pathway may contribute to neuroinflammatory and neurodegenerative processes as observed in Alzheimer's disease.

Plasma membrane requirements for 1α,25(OH) 2D 3 dependent PKC signaling in chondrocytes and osteoblasts

1,25-Dihydroxyvitamin D 3 [1α,25(OH) 2D 3] acts on chondrocytes and osteoblasts through traditional nuclear Vitamin D receptor (VDR) mechanisms as well as through rapid actions on plasma membranes that initiate intracellular signaling pathways. We have investigated the mechanisms involved in activation of protein kinase C (PKC) and downstream biological responses that depend on the latter pathway. These studies show that PKC activation depends on presence of a membrane receptor ERp60 and rapid increases in phospholipase A 2 (PLA 2) activity. Cells that are responsive to 1α,25(OH) 2D 3 express PLA 2 activating protein (PLAA), suggesting a link between ERp60 and PLA 2. Increased PLA 2 results in increased arachidonic acid release and formation of lysophospholipid, which then activates phospholipase C beta (PLCβ), leading to rapid formation of inositol-trisphosphate (IP3) and diacylglycerol (DAG). PLA 2, PLC, and DAG are all associated with lipid rafts including caveolae in many cells, suggesting that the caveolar environment may be an important mediator of PKC activation by 1α,25(OH) 2D 3. Here, we use the VDR −/− mouse costochondral cartilage growth plate to examine the expression of ERp60 and PLAA in vivo in 1α,25(OH) 2D 3-responsive hypertrophic chondrocytes (growth zone cells) and in resting zone cells that do not respond to this Vitamin D metabolite in vitro. In addition, we determined if intact lipid rafts are required for the response of rat costochondral cartilage growth zone cells to 1α,25(OH) 2D 3. The results show that ERp60 and PLAA are localized to 1α,25(OH) 2D 3-responsive growth zone cells and metaphyseal osteoblasts, even in VDR −/− mice. Disruption of lipid rafts using beta-cyclodextrin blocks the activation of PKC by 1α,25(OH) 2D 3 and reduces the ability of 1α,25(OH) 2D 3 to regulate [ 35S]-sulfate incorporation.

Divergent Behavioral Roles of Angiotensin Receptor Intracellular Signaling Cascades

Central injections of angiotensin II (AngII) increase both water and NaCl intake. These effects of AngII occur largely through stimulation of the AngII type 1 (AT(1)) receptor. Stimulation of the AT(1) receptor leads to a number of intracellular events, including phospholipase C (PLC) activation and the subsequent formation of diacylglycerol and inositol trisphosphate (IP(3)), which then activate protein kinase C (PKC) and increase intracellular calcium, respectively. In addition, AT(1) receptor stimulation leads to the activation of MAPK family members. Recent experiments using mutated AT(1) receptor constructs or the AngII analog Sar(1),Ile(4),Ile(8)-AngII (SII) revealed that MAPK activation can occur independent of PLC/PKC/IP(3) activation. The present experiments used in vitro and in vivo approaches to clarify the cellular and behavioral responses to SII. Specifically, SII mimicked AngII stimulation of MAPK in AT(1) receptor-transfected COS-1 cells and rat brain but blocked the effects of AngII in two distinct settings: in vitro stimulation of IP(3) and in vivo increases in water intake. Moreover, SII increased intake of 1.5% NaCl, despite the SII blockade of IP(3) formation and water intake. Examination of brain tissue showed increases in Fos expression in several AngII-sensitive brain areas after injection of AngII, but not SII. The lack of SII-induced IP(3) production, water intake, and Fos expression strongly suggest that the PLC/PKC/IP(3) pathway is required for water intake, but not NaCl consumption stimulated by AngII. Collectively, these results support the hypothesis that divergent intracellular signals from a single receptor type can give rise to separable behavioral phenomena.

Synthesis of pyridoxal phosphate derivatives with antagonist activity at the P2Y 13 receptor

We have synthesized a series of derivatives of the known P2 receptor antagonist PPADS (pyridoxal-5′-phosphate-6-azo-phenyl-2,4-disulfonate) and examined their ability to inhibit functional activity of the recombinant human P2Y 13 nucleotide receptor expressed in 1321N1 human astrocytoma cells co-expressing Gα 16 protein (AG32). Analogues of PPADS modified through substitution of the phenylazo ring, including halo and nitro substitution, and 5′-alkyl phosphonate analogues were synthesized and tested. A 6-benzyl-5′-methyl phosphonate analogue was prepared to examine the effect of stable replacement of the azo linkage. The highest antagonistic potency was observed for 6-(3-nitrophenylazo) derivatives of pyridoxal-5′-phosphate. The 2-chloro-5-nitro analogue (MRS 2211) and 4-chloro-3-nitro analogue (MRS 2603) inhibited ADP (100 nM)-induced inositol trisphosphate (IP 3) formation with pIC 50 values of 5.97 and 6.18, respectively, being 45- and 74-fold more potent than PPADS. The antagonism of MRS 2211 was competitive with a pA 2 value of 6.3. MRS2211 and MRS2603 inhibited phospholipase C (PLC) responses to 30 nM 2-methylthio-ADP in human P2Y 1 receptor-mediated 1321N1 astrocytoma cells with IC 50 values of >10 and 0.245 μM, respectively. Both analogues were inactive (IC 50 > 10 μM) as antagonists of human P2Y 12 receptor-mediated PLC responses in 1321N1 astrocytoma cells. Thus, MRS2211 displayed >20-fold selectivity as antagonist of the P2Y 13 receptor in comparison to P2Y 1 and P2Y 12 receptors, while MRS2603 antagonized both P2Y 1 and P2Y 13 receptors.

Role of the ERC motif in the proximal part of the second intracellular loop and the C-terminal domain of the human prostaglandin F2α receptor (hFP-R) in G-protein coupling control

The human FP-R (F2alpha prostaglandin receptor) is a Gq-coupled heptahelical ectoreceptor, which is of significant medical interest, since it is a potential target for the treatment of glaucoma and preterm labour. On agonist exposure, it mediates an increase in intracellular inositol phosphate formation. Little is known about the structures that govern the agonist-dependent receptor activation. In other prostanoid receptors, the C-terminal domain has been inferred in the control of agonist-dependent receptor activation. A DRY motif at the beginning of the second intracellular loop is highly conserved throughout the G-protein-coupled receptor family and appears to be crucial for controlling agonist-dependent receptor activation. It is replaced by an ERC motif in the FP-R and no evidence for the relevance of this motif in ligand-dependent activation of prostanoid receptors has been provided so far. The aim of the present study was to elucidate the potential role of the C-terminal domain and the ERC motif in agonist-controlled intracellular signalling in FP-R mutants generated by site-directed mutagenesis. It was found that substitution of the acidic Glu(132) in the ERC motif by a threonine residue led to full constitutive activation, whereas truncation of the receptor's C-terminal domain led to partial constitutive activation of all three intracellular signal pathways that had previously been shown to be activated by the FP-R, i.e. inositol trisphosphate formation, focal adhesion kinase activation and T-cell factor signalling. Inositol trisphosphate formation and focal adhesion kinase phosphorylation were further enhanced by ligand binding in cells expressing the truncation mutant but not the E132T (Glu132-->Thr) mutant. Thus C-terminal truncation appeared to result in a receptor with partial constitutive activation, whereas substitution of Glu132 by threonine apparently resulted in a receptor with full constitutive activity.

βγ subunits of heterotrimeric G-proteins contribute to Ca2+ release at fertilization in the sea urchin

A cytoplasmic Ca2+ transient is required for egg activation at fertilization in all animals. The pathway leading to release of Ca2+ from the endoplasmic reticulum in echinoderms includes activation of a SRC homolog, followed by phospholipase Cgamma activation, and formation of inositol trisphosphate. However, the upstream activators or modulators of this signaling pathway are not known. We recently identified four Galpha subunits of heterotrimeric G-proteins present in the sea urchin egg, and here we find that activation of G-proteins of the Galphas and Galphaq type, but not Galphai or Galpha12 type, is required for normal Ca2+ dynamics at fertilization. The effects of these G-proteins are mediated by the Gbetagamma subunits, occur upstream of the cytoplasmic Ca2+ release, and influence both the amplitude of Ca2+ release and the duration of the lag phase. We propose integration of the G-protein input into the framework of signaling at sea urchin fertilization.

Heterologous desensitization of the sphingosine-1-phosphate receptors by purinoceptor activation in renal mesangial cells.

1 Sphingosine-1-phosphate (S1P) is considered a potent mitogen for mesangial cells and activates the classical mitogen-activated protein kinase (MAPK) cascade via S1P receptors. In this study, we show that S1P signalling is rapidly desensitized upon S1P receptor activation. A complete loss of S1P sensitivity occurs after 10 min of S1P pretreatment and remains for at least 8 h. A similar desensitization is also seen with the S1P mimetic FTY720-phosphate, but not with the nonphosphorylated FTY720, nor with sphingosine or ceramide. 2 Prestimulating the cells with extracellular ATP or UTP, which bind to and activate P2Y receptors on mesangial cells, a similar rapid desensitization of the S1P receptor occurs, suggesting a heterologous desensitization of S1P receptors by P2Y receptor activation. Furthermore, adenosine binding to P1 receptors triggers a similar desensitization. In contrast, two other growth factors, PDGF-BB and TGFbeta2, have no significant effect on S1P-induced MAPK activation. 3 S1P also triggers increased inositol trisphosphate (IP3) formation, which is completely abolished by S1P pretreatment but only partially by ATP pretreatment, suggesting that IP3 formation and MAPK activation stimulated by S1P involve different receptor subtypes. 4 Increasing intracellular cAMP levels by forskolin pretreatment has a similar effect on desensitization as adenosine. Moreover, a selective A3 adenosine receptor agonist, which couples to phospholipase C and increases IP3 formation, exerted a similar effect. 5 Pretreatment of cells with various protein kinase C (PKC) inhibitors prior to ATP prestimulation and subsequent S1P stimulation leads to a differential reversal of the ATP effect. Whereas the broad-spectrum protein kinase inhibitor staurosporine potently reverses the effect, the PKC-alpha inhibitor CGP41251, the PKC-delta inhibitor rottlerin and calphostin C show only a partial reversal at maximal concentrations. 6 Suramin, which is reported as a selective S1P3 receptor antagonist compared to the other S1P receptor subtypes, has no effect on the S1P-induced MAPK activation, thus excluding the involvement of S1P3 in this response. 7 In summary, these data document a rapid homologous and also heterologous desensitization of S1P signalling in mesangial cells, which is mechanistically triggered by PKC activation and eventually another staurosporine-sensitive protein kinase, as well as by increased cAMP formation.

2-aminoethoxydiphenyl borate inhibits agonist-induced Ca2+ signals by blocking inositol trisphosphate formation in acutely dissociated mouse pancreatic acinar cells.

Evidence suggests that 2-aminoethoxydiphenyl borate (2-APB) modulates intracellular Ca(2+) signals in a complex manner. 2-APB inhibits or potentiates intracellular Ca(2+) signals in different cell types, perhaps through different mechanisms. Here, we report a novel mechanism underlying 2-APB-induced inhibition of agonist-activated Ca(2+) oscillations in mouse pancreatic acinar cells, using patch-clamp and biochemical techniques. Pre-treatment of the cells with 100 microM 2-APB completely abolished ACh- but not inositol trisphosphate (InsP(3))-induced Ca(2+) oscillations, suggesting that the mechanism of inhibition occurs between cytoplasmic receptors and InsP(3) receptor activation. In addition, 100 microM 2-APB significantly inhibited ACh-induced phospholipase C (PLC) activation. These findings indicate that, in mouse pancreatic acinar cells, in addition to modulating InsP(3) receptors and blocking the store-operated Ca(2+) pathway, high concentrations of 2-APB also inhibit agonist-induced Ca(2+) signals by reducing InsP(3) formation.

Multiple signal transduction pathways mediated by 5-HT receptors.

Among human serotonin (5-HT) receptor subtypes, each G protein-coupled receptor subtype is reported to have one G protein-signaling cascade. However, the signaling may not be as simple as previously thought to be. 5-HT5A receptors are probably the least well understood among the 5-HT receptors, but the authors found that 5-HT5A receptors couple to multiple signaling cascades. When the 5-HT5A receptors were expressed in undifferentiated C6 glioma cells, they modulated the level of second messengers. For example, activation of 5-HT5A receptors inhibited the adenylyl cyclase activity and subsequently reduced the cAMP level, as previously reported. In addition to this known signaling via Gi/Go, 5-HT5A receptors are coupled to the inhibition of ADP-ribosyl cyclase and cyclic ADP ribose formation. On the other hand, activation of 5-HT5A receptors transiently opened the K+ channels, presumably due to the increase in intracellular Ca2+ after formation of inositol (1,4,5) trisphosphate. The K+ currents were inhibited by both heparin and pretreatment with pertussis toxin, suggesting the cross-talk between Gi/Go protein and phopholipase C cascade. Thus, the authors results indicate that 5-HT5A receptors couple to multiple second messenger systems and may contribute to the complicated physiological and pathophysiological states. Although this multiple signaling has been reported only for 5-HT5A/5-HT1 receptors so far, it is possible that other 5-HT receptor subtypes bear similar complexity. As a result, in addition to the wide variety of expression patterns of each 5-HT receptor subtype, it is possible that multiple signal transduction systems may add complexity to the serotonergic system in brain function. The investigation of these serotonergic signaling and its impairment at cellular level may help to understand the symptoms of brain diseases.

Neurotensin stimulates both calcium mobilization from inositol trisphosphate-sensitive intracellular stores and calcium influx through membrane channels in frog pituitary melanotrophs.

Neurotensin (NT) is a potent stimulator of electrical and secretory activities in frog pituitary melanotrophs. The aim of the present study was to characterize the transduction pathways associated with activation of NT receptors in frog melanotrophs. Application of synthetic frog NT (fNT) increased the cytosolic calcium concentration ([Ca2+]c) and stimulated the formation of inositol trisphosphate (IP3). The phospholipase C inhibitor U-73122 blocked the electrophysiological and secretory effects of fNT. Intracellular application of the IP3 receptor antagonist heparin abolished fNT-induced electrical activity. Suppression of Ca2+ in the incubation medium markedly reduced the effect of NT on [Ca2+]c, firing rate, and alpha-melanocyte-stimulating hormone (alphaMSH) secretion. Similarly, the inhibitor of IP3-induced Ca2+ release and store-operated Ca2+ channels, 2-Aminoethoxydiphenylborane, and the nonselective Ca2+ channel blockers GdCl3 and NiCl2, attenuated the [Ca2+]c increase and the electrical and secretory responses evoked by fNT. Coapplication of the L- and N-type Ca2+ channel blockers nifedipine and omega-CgTx GVIA reduced the effects of fNT on action potential discharge, [Ca2+]c increase, and alphaMSH release. The protein kinase C (PKC) inhibitors, PKC-(19-31) and chelerythrine, reduced the electrophysiological and secretory responses induced by iterative applications of fNT. Collectively, these results demonstrate that, in frog melanotrophs, NT stimulates the phospholipase C/PKC pathway and increases [Ca2+]c. Both Ca2+ release from intracellular stores and Ca2+ influx through L- and N-type Ca2+ channels are involved in fNT-induced alphaMSH secretion. In addition, the present data indicate that PKC plays a crucial role in maintenance of the responsiveness of melanotrophs to NT.

Pasteurella multocida Toxin Facilitates Inositol Phosphate Formation by Bombesin through Tyrosine Phosphorylation of Gαq

The intracellularly acting Pasteurella multocida toxin (PMT) is a potent mitogen that stimulates Gq-dependent formation of inositol trisphosphate. We show that PMT, a nontoxic mutant of PMT (PMTC1165S), and bombesin each stimulate time-dependent phosphorylation of G alpha q at tyrosine 349. Although PMT and PMTC1165S each cause phosphorylation of G alpha q, only the wild-type toxin activates Gq. Pretreatment of cells with wild-type or mutant PMT potentiated the formation of inositol phosphates stimulated by bombesin equally. These data show that PMT potentiates bombesin receptor signaling through tyrosine phosphorylation of Gq and distinguishes between the two proposed models of Gq activation, showing that tyrosine phosphorylation is not linked to receptor uncoupling.

The protein-tyrosine phosphatase DEP-1 modulates growth factor-stimulated cell migration and cell-matrix adhesion.

Density-enhanced protein-tyrosine phosphatase-1 (DEP-1 also CD148) is a transmembrane molecule with a single intracellular PTP domain. It has recently been proposed to function as a tumor suppressor. We have previously shown that DEP-1 dephosphorylates the activated platelet-derived growth factor (PDGF) beta-receptor in a site-selective manner (Kovalenko et al. (2000). J. Biol. Chem. 275, 16219-16226). We analysed cell lines with inducible DEP-1 expression for cellular functions of DEP-1. Several aspects of PDGFbeta-receptor signaling were negatively affected by DEP-1 expression. These include PDGF-stimulated activation of inositol trisphosphate formation, Erk1/2, p21Ras, and Src. Activation of receptor-associated phosphoinositide-3 kinase activity and of Akt/PKB were weakly attenuated at early time points of stimulation. Inhibition of PDGF-stimulated signaling depended on DEP-1 catalytic activity. Importantly, DEP-1 inhibited PDGF-stimulated cell migration. The catalytically inactive DEP-1 C1239S variant enhanced cell migration and PDGF-stimulated Erk1/2 activation, suggesting a dominant negative interference with endogenous DEP-1. In contrast to cell migration, cell-substrate adhesion was promoted by active DEP-1 and delayed or suppressed by DEP-1 C1239S, correlating with positive effects of DEP-1 on adhesion-stimulated Src kinase. We propose that negative regulation of growth-factor stimulated cell migration and promotion of cell-matrix adhesion may be related to the function of DEP-1 as tumor suppressor.

Permissive Effect of Voltage on mGlu 7 Receptor Subtype Signaling in Neurons

G protein-coupled receptors mobilize neuronal signaling cascades which until now have not been shown to depend on the state of membrane depolarization. Thus we have previously shown that the metabotropic glutamate receptor type 7 (mGlu7 receptor) blocks P/Q-type Ca(2+) channels via activation of a G(o) protein and PKC, in cerebellar granule cells. We show here that the transient depolarizations used to evoke the studied Ca(2+) current were indeed permissive to activate this pathway by a mGlu7 receptor agonist. Indeed, sustained depolarization to 0 mV was sufficient to inhibit P/Q-type Ca(2+) channels. This effect involved a conformational change in voltage-gated sodium channel independently of Na(+) flux, activation of a pertussis toxin-sensitive G-protein, inositol trisphosphate formation, intracellular Ca(2+) release, and PKC activity. Subliminal sustained membrane depolarization became efficient in inducing inositol trisphosphate formation, release of intracellular Ca(2+) and in blocking Ca(2+) channels, when applied concomitantly with the mGlu7a receptor agonist, d,l-aminophosphonobutyrate. This synergistic effect of membrane depolarization and mGlu7 receptor activation provides a mechanism by which neuronal excitation could control action of the mGlu7 receptor in neurons.