IP3 Accumulation Research Articles

Effects of 2-Azafluorenones on Phosphatidyl-Inositol Specific Phospholipase C Activation in C6 Glioma Cells

The purpose of this study was to determine the effects of an extract from Moringa oleifera (MO) on the development of monocrotaline (MCT)-induced pulmonary hypertension (PH) in Wistar rats. An ethanol extraction was performed on dried MO leaves, and HPLC analysis identified niaziridin and niazirin in the extract. PH was induced with a single subcutaneous injection of MCT (60 mg/kg) which resulted in increases in pulmonary arterial blood pressure (Ppa) and in thickening of the pulmonary arterial medial layer in the rats. Three weeks after induction, acute administration of the MO extract to the rats decreased Ppa in a dose-dependent manner that reached statistical significance at a dose of 4.5 mg of freeze-dried extract per kg body weight. The reduction in Ppa suggested that the extract directly relaxed the pulmonary arteries. To assay the effects of chronic administration of the MO extract on PH, control, MCT and MCT+MO groups were designated. Rats in the control group received a saline injection; the MCT and MCT+MO groups received MCT to induce PH. During the third week after MCT treatment, the MCT+MO group received daily i.p. injections of the MO extract (4.5 mg of freeze-dried extract/kg of body weight). Compared to the control group, the MCT group had higher Ppa and thicker medial layers in the pulmonary arteries. Chronic treatments with the MO extract reversed the MCT-induced changes. Additionally, the MCT group had a significant elevation in superoxide dismutase activity when normalized by the MO extract treatments. In conclusion, the MO extract successfully attenuated the development of PH via direct vasodilatation and a potential increase in antioxidant activity.

Modulating Drug Effects of Metabotropic Glutamate Receptor 1 Alpha (mGluR1α) by Extracellular Ca2+

Metabotropic glutamate receptor 1α (mGluR1α), known as a member of the family C GPCRs, couples to Gq and modulates consequent PLC activity, IP3 accumulation and intracellular Ca2+ extrusion from ER lumen. The mGluR1α is abundantly expressed in central nervous system and has been shown to be responsive to the slow phase of the action potential in post-synapses, and to be involved in chronic neuronal degenerative diseases, like Parkinson's disease, Huntington's disease and Alzheimer's disease. We have predicted a potential Ca2+ binding site adjacent to the binding site to reported endogenous agonist glutamate and antagonists. In this study, we have applied single cell imaging, IP1 binding, and radioactive assay to probe the effect of extracellular calcium in modulating various types of the drugs of modulating mGluR1a such as agonists, antagonists and allosteric modulators. We have shown that extracellular Ca2+ enhances the agonist's activation of intracellular calcium responses of mGluR1α by increasing the drug binding to the receptor. In addition, extracellular Ca2+ also differentially modulates the inhibition of the receptor by antagonists and allosteric modulators. Our studies open a new avenue for modulating drug effects and developing novel drugs against neurodegenerative diseases.

Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling.

Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on β-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.

Defining protein kinase/phosphatase isoenzymic regulation of mGlu5 receptor‐stimulated phospholipase C and Ca2+ responses in astrocytes

Cyclical phosphorylation and dephosphorylation of a key residue within the C-terminal domain of the activated type 5 metabotropic glutamate (mGlu₅) receptor is believed to cause the synchronous, oscillatory changes in inositol 1,4,5-trisphosphate and Ca²⁺ levels observed in a variety of cell types. Here, we have attempted to better define the kinase and phosphatase enzymes involved in this modulation. Ca²⁺ and [³H]inositol phosphate ([³H]IP(x) ) measurements in astrocyte preparations have been used to evaluate the effects of pharmacological inhibition of protein kinase C (PKC) and protein phosphatase activities and small interfering RNA-mediated specific PKC isoenzymic knock-down on mGlu₅ receptor signalling. Ca²⁺ oscillation frequency or [³H]IP(x) accumulation in astrocytes stimulated by mGlu₅ receptors, was concentration-dependently decreased by protein phosphatase-1/2A inhibition or by PKC activation. PKC inhibition also increased [³H]IP(x) accumulation two- to threefold and changed the Ca²⁺ response into a peak-plateau response. However, selective inhibition of conventional PKC isoenzymes or preventing changes in [Ca²⁺](i) concentration by BAPTA-AM loading was without effect on mGlu₅ receptor-stimulated [³H]IP(x) accumulation. Selective knock-down of PKCδ was without effect on glutamate-stimulated Ca²⁺ responses; however, selective PKCε knock-down in astrocytes changed Ca²⁺ responses from oscillatory into peak-plateau type. These data confirm the acute regulation of mGlu₅ receptor signalling by protein kinases and protein phosphatases and provide novel data pinpointing the isoenzymic dependence of this regulation in the native mGlu₅ receptor-expressing rat cortical astrocyte. These data also highlight a potential alternative mechanism by which mGlu₅ receptor signalling might be therapeutically manipulated.

A role for myosin Va in cerebellar plasticity and motor learning: a possible mechanism underlying neurological disorder in myosin Va disease.

Mutations of the myosin Va gene cause the neurological diseases Griscelli syndrome type 1 and Elejalde syndrome in humans and dilute phenotypes in rodents. To understand the pathophysiological mechanisms underlying the neurological disorders in myosin Va diseases, we conducted an integrated analysis at the molecular, cellular, electrophysiological, and behavioral levels using the dilute-neurological (d-n) mouse mutant. These mice manifest an ataxic gait and clonic seizures during postnatal development, but the neurological disorders are ameliorated in adulthood. We found that smooth endoplasmic reticulum (SER) rarely extended into the dendritic spines of Purkinje cells (PCs) of young d-n mice, and there were few, if any, IP(3) receptors. Moreover, long-term depression (LTD) at parallel fiber-PC synapses was abolished, consistent with our previous observations in juvenile lethal dilute mutants. Young d-n mice exhibited severe impairment of cerebellum-dependent motor learning. In contrast, adult d-n mice showed restoration of motor learning and LTD, and these neurological changes were associated with accumulation of SER and IP(3) receptors in some PC spines and the expression of myosin Va proteins in the PCs. RNA interference-mediated repression of myosin Va caused a reduction in the number of IP(3) receptor-positive spines in cultured PCs. These findings indicate that myosin Va function is critical for subsequent processes in localization of SER and IP(3) receptors in PC spines, LTD, and motor learning. Interestingly, d-n mice had defects of motor coordination from young to adult ages, suggesting that the role of myosin Va in PC spines is not sufficient for motor coordination.

Intracellular Signaling in Human Iridial Fibroblasts and Iridial Melanocytes in Response to Prostaglandins, Endothelin, Isoproterenol, and Other Pharmacological Agents

The receptor-coupled signal transduction systems present in isolated human iridial fibroblasts (HIF) and in human iridial melanocytes (HIM) were investigated. Cell responsiveness to numerous prostaglandins (PGs), and other compounds of interest, was profiled in order to better understand their involvement in the iridial hyper-pigmentation process observed during treatment of elevated intraocular pressure with FP-receptor against PG analogs. [(3)H]-inositol phosphates ([(3)H]-IPs) generated in the cells were measured by ion-exchange chromatography followed by liquid scintillation spectroscopy. cAMP generated in the cells was quantified using an enzyme immunoassay. HIF cells exhibited a robust phosphoinositide (PI) hydrolysis response to FP-class PG analogs, such as cloprostenol (potency, EC(50) = 2.4 ± 0.5 nM, n = 5), fluprostenol (EC(50) = 5.3 ± 0.6 nM, n = 3), PGF(2α) (EC(50) = 54 ± 18 nM, n = 5), and latanoprost acid (EC(50) = 121 ± 17 nM, n = 4). Other PGs exhibited the following potencies (EC(50)) for stimulating [(3)H]-IPs accumulation in HIF cells: PGD(2) EC(50) = 327 ± 195 nM, n =3; PGE(2) EC(50) = 550 ± 50 nM, n = 3; and two TP-receptor agonists (I-BOP, EC(50) = 23 ± 8 nM, n = 3; U-46619 EC(50) = 1.1 ± 0.4 µM, n = 3). Endothelin-1 (ET-1) and histamine increased [(3)H]-IPs production in HIF and HIM cells. HIM cells exhibited minimal PI turnover response to cloprostenol, latanoprost acid, latanoprost, PGF(2α), PGE(2), and histamine, but there were robust responses to ET-1 (EC(50) = 4.6 nM, n = 2) and an ET(B)-receptor agonist (BQ-3020, EC(50) = 5 nM, n = 2) that were blocked by an ET(B)-antagonist (BQ-788, IC(50) = 21 ± 6 nM, n = 3). In the adenylyl cyclase activation assay, numerous PGs (1 and 10 µM) stimulated cAMP production in HIF cells yielding the following rank order of efficacy: PGI(2) > PGE(2) > misoprostil > isoproterenol = BW245C > PGD(2) = PGF(2α) = fluprostenol. In HIM cells, PGE(2) (EC(50) = 1.3 ± 0.3 nM) and isoproterenol (β-agonist; EC(50) = 89 ± 13 nM) potently and efficaciously stimulated cAMP production and ICI-118851 (β(2)-antagonist) attenuated the effects of isoproterenol. However, latanoprost acid, latanoprost, ET-1, and BW245C (DP-receptor agonist) were relatively less efficacious than isoproterenol and PGE(2) in HIM cells at stimulating cAMP production. These studies have shown that while HIF cells express FP prostaglandin and histamine receptors coupled to phospholipase C to produce [(3)H]-IPs, the HIM cells lack such functionally active FP-receptors. In contrast, HIF and HIM cells express functional ET-1 receptors coupled to [(3)H]-IPs production and both cell-types respond to PGE(2), BW245C, and isoproterenol by generating cAMP. It is concluded that human iridial fibroblasts and melanocytes respond differently to PGs and histamine, but in the same manner to ET-1, isoproterenol and BW245C. This may have relevance to the intercellular communication within the iris relative to the melanogenic processes.

Incorporation of ortho-Carbaboranyl-Nε-Modified l-Lysine into Neuropeptide Y Receptor Y1- and Y2-Selective Analogues

Nontoxic ortho-carbaborane is one of the most promising structure for boron neutron capture therapy (BNCT). For directed uptake of ortho-carbaborane by tumor cells, receptor-subtype selective neuropeptide Y (NPY) and its derivatives were modified with ortho-carbaborane. The derivative [F(7), P(34)]-NPY has been shown to be a breast cancer selective ligand that binds to the Y(1)-receptor subtype, whereas [Ahx(5-24)]-NPY selectively addresses Y(2)-receptor subtypes that are found in neuroblastoma cells. ortho-Carbaboranyl propionic acid was synthesized and linked to the ε-amino group of N(α)-Fmoc protected L-lysine. The characterization of the compounds was performed by NMR, IR, and MS studies. The carbaborane-modified amino acid was incorporated into NPY, [F(7), P(34)]-NPY, and [Ahx(5-24)]-NPY by an optimized solid phase peptide synthesis using Fmoc protection. Binding studies and IP accumulation assays confirmed nanomolar affinity and activity of the modified analogues despite of the large carbaborane cluster. Internalization studies revealed excellent and receptor subtype specific uptake of the conjugates into respective cells.

In Vitro Antagonistic Properties of a New Angiotensin Type 1 Receptor Blocker, Azilsartan, in Receptor Binding and Function Studies

The angiotensin II (AII) antagonistic action of azilsartan (AZL) [2-ethoxy-1-{[2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylic acid] was investigated in radioligand binding and function studies. AZL inhibited the specific binding of ¹²⁵I-Sar¹-Ile⁸-AII to human angiotensin type 1 receptors with an IC₅₀ of 2.6 nM. The inhibitory effect of AZL persisted after washout of the free compound (IC(50) value of 7.4 nM). Olmesartan, telmisartan, valsartan, and irbesartan also inhibited the specific binding with IC₅₀ values of 6.7, 5.1, 44.9, and 15.8 nM, respectively. However, their inhibitory effects were markedly attenuated with washout (IC₅₀ values of 242.5, 191.6, >10,000, and >10,000 nM). AZL also inhibited the accumulation of AII-induced inositol 1-phosphate (IP1) in the cell-based assay with an IC₅₀ value of 9.2 nmol; this effect was resistant to washout (IC₅₀ value of 81.3 nM). Olmesartan and valsartan inhibited IP1 accumulation with IC₅₀ values of 12.2 and 59.8 nM, respectively. The activities of these compounds were markedly reduced after washout (IC₅₀ value of 908.5 and 22,664.4 nM). AZL was defined as an inverse agonist in an experiment by using a constitutively active mutant of human angiotensin type 1 receptors. In isolated rabbit aortic strips, AZL reduced the maximal contractile response to AII with a pD'₂ value of 9.9. The inhibitory effects of AZL on contractile responses induced by AII persisted after the strips were washed; these inhibitory effects were more potent than those of olmesartan. These results suggest that AZL is a highly potent and slowly dissociating AII receptor blocker. Its tight receptor binding might be expected to produce potent and long-lasting antihypertensive effects in preclinical and clinical settings.

GPCR Activation is Measured with Cisbio’s cAMP and IP1 HTRF HTplex Cell-Based Assay

BioTechniquesVol. 49, No. 4 Sponsored Paper - Application ForumOpen AccessGPCR activation is measured with Cisbio's cAMP and IP1 HTRF HTplex cell-based assayE.J. Dell, Franka Ganske & Laurence JacquemartE.J. DellBMG LABTECH GmbH, Offenburg, GermanySearch for more papers by this author, Franka GanskeBMG LABTECH GmbH, Offenburg, GermanySearch for more papers by this author & Laurence JacquemartCisbio Bioassays, Bagnols-sur-Cèze, FranceSearch for more papers by this authorPublished Online:28 Jun 2018https://doi.org/10.2144/000113517AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit IntroductionThe G protein–coupled receptor (GPCR) superfamily has almost 1000 members and thus represents the largest protein family in the human genome. GPCRs carry information within cells via two major signaling pathways: regulation of cAMP levels and increases in intracellular Ca2+ triggered by inositol 1,4,5-triphosphate (IP3). Activation of these pathways via Gs-or Gi-coupled receptors results in the increase or decrease of cAMP levels, respectively, while activation of a Gq-coupled receptor activates phospholipase C (PLC) and triggers the inositol phosphate (IP) cascade.Cisbio Bioassays' new generation Lumi4-Tb TR-FRET cryptate chemistry allows for the detection of cAMP and IP1 levels in one well by using two different acceptors, a green dye (λ = 520 nm) and a red dye (λ = 665 nm). The experiments described here where performed on the PHERAstar Plus HTS microplate reader using Simultaneous Dual Emission detection. This unique feature allows the plate to be read once for dual emission assays, thereby decreasing time and variability.The HTplex assay from CisbioThe HTplex assay uses two antibodies labeled with Lumi4-Tb (anti-cAMP Cryptate and anti-IP1 Cryptate as donors) and two acceptors (cAMP-green dye and IP1-red dye). In the inactivate state, a high TR-FRET signal is seen for both the red and green emissions (Figure 1). CHO cells were stimulated with 10 µM vasopression. Upon GPCR activation, cAMP and IP1 were produced, thereby decoupling the tracer green-cAMP and the tracer red-IP1 from the Tb-cryptate antibody. This leads to a decrease in the TR-FRET signal. Hence specific emission signals are inversely proportional to the concentration of cAMP and IP.Figure 1. Principle of the HTplex assay.Results & DiscussionFigure 2 shows an initial cAMP (Gs) response at lower concentrations of vasopressin presumably through activation of endogenous V1 receptors, and a delayed IP3 (Gq) response at higher concentrations through activation of the transfected V2 receptor (as measured by IP1 accumulation). EC50 for cAMP and IP1 were 15 × 10-6 M and 23 × 10-6 M, respectively, and both signals were well above the reliability range for an HTS assay, giving Z' values > 0.75. The HTPlex assay from Cisbio was successfully employed with BMG LABTECH's PHERAstar Plus HTS microplate reader.Figure 2. IP1 and cAMP Signaling in CHO cells upon stimulation with vasopressin.

GalR2-positive allosteric modulator exhibits anticonvulsant effects in animal models

Galanin receptors type 1 (GalR1) and/or type 2 (GalR2) represent unique pharmacological targets for treatment of seizures and epilepsy. Previous studies have shown that the endogenous peptide ligand galanin exerts powerful anticonvulsant effect through activation of these two G protein-coupled receptors, which are highly expressed in the temporal lobe of rodent brain. Here we report the characterization of a putative GalR2-positive allosteric modulator CYM2503. CYM2503 potentiated the galanin-stimulated IP1 accumulation in HEK293 cells stably expressing GalR2 receptor, whereas it exhibited no detectable affinity for the (125)I galanin-binding site of GalR2 receptor, an effect consistent with that of a positive allosteric modulator. In the rat Li-pilocarpine status epilepticus model, CYM2503, injected intraperitoneally, increased the latency to first electrographic seizure and the latency to first stage 3 behavioral seizure, decreased the latency to the establishment of status epilepticus, and dramatically decreased the mortality. In a Li-pilocarpine seizure model in mice, CYM2503 increased the latency to first electrographic seizure and decreased the total time in seizure. CYM2503 also attenuated electroshock-induced seizures in mice. Thus, CYM2503 provides a starting point for the development of anticonvulsant therapy using the galanin R2 receptor as target.

Principles and Determinants of G-Protein Coupling by the Rhodopsin-Like Thyrotropin Receptor

In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR) and different subtypes of G-proteins. The thyrotropin receptor (TSHR) binds G-proteins promiscuously and activates both Gs (cAMP) and Gq (IP). Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts.We systematically investigated the amino acids of intracellular loop (ICL) 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template.We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits Gα as well as Gβγ. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443) and helix 8 (R687) that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only.Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein subtypes.

The G-protein Coupled Receptor Associated Sorting Protein GASP-1 Regulates the Signalling and Trafficking of the Viral Chemokine Receptor US28

Human cytomegalovirus (HCMV) encodes the seven transmembrane (7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Gαq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor–κB (NF-κB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.

Selegiline, an MAO-B inhibitor, attenuates airway smooth muscle contraction in the rat trachea.

Selegiline is widely used for Parkinson's disease and sometimes for Alzheimer's disease. It is reported to affect intracellular Ca(2+) concentration. Since intracellular Ca(2+) is partly regulated by phosphatidylinositol (PI) response and is important for smooth muscle contraction, selegiline may affect airway smooth muscle tension. We examined the effects of selegiline on acetylcholine (ACh)- and KCl-induced contractile and PI responses in rat trachea. The trachea was cut into 3-mm-wide ring segments or 1-mm-wide slices. ACh (3 microM, 50% effective dose) or KCl (40 mM) was added, and ring relaxation was induced by the addition of selegiline. Tracheal slices were incubated with [(3)H]myo-inositol and 3 microM ACh in the presence of selegiline, and [(3)H]inositol monophosphate (IP(1)) was measured. Selegiline dose-dependently attenuated ACh- and KCl-induced tracheal ring contractions. Fifty-percent inhibitory doses (ID50) of selegiline against ACh- and KCl-induced contraction were 120 +/- 30 microM and 80 +/- 20 microM, respectively. Basal and ACh-induced IP(1) accumulation were 2.20 +/- 0.20 Bq and 7.88 +/- 0.23 Bq, respectively, and selegiline at a dose of 1000 microM attenuated ACh-induced IP(1) accumulation (5.44 +/- 0.30 Bq). These results suggest that selegiline inhibits contractile responses through the inhibition of voltage-operated Ca(2+) channels and the PI response.

Clonidine attenuates the carbachol-induced contractile and phosphatidylinositol responses of rat trachea.

Although clonidine is known to affect vascular smooth muscle, its effects on airway smooth muscle are not fully understood. This study was designed to examine the effects of clonidine on carbachol-induced contractile and phosphatidylinositol responses of rat trachea. Clonidine, at a dose of 100 microM or greater, attenuated carbachol-induced contraction and the accumulation of carbachol-induced inositol monophosphate (IP1). Clonidine also attenuated the accumulation of aluminium fluoride-induced IP1. The concentration-effect relationship of IP1 accumulation was similar to that of carbachol-induced contraction; r = 0.797, P < 0.001. These results suggest that clonidine attenuates contractile responses, at least in part, through the inhibition of phospholipase C (coupled with G-proteins) in phosphatidylinositol responses.

The G protein-coupled receptor associated sorting protein GASP-1 regulates the signaling and trafficking of the viral chemokine receptor US28

Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.

Extracellular Ca2+ and Glutamate Modulating the Function of Metabotropic Glutamate Receptor 1 Alpha (mGluR1α)

Metabotropic Glutamate Receptor subtype 1 alpha (mGluR1α) mediates the accumulation of IP3 and DAG, release of intracellular Ca2+ from ER, and activation of PKC and PKA, by interacting with extracellular glutamate and Ca2+. While more than 12 X-ray Structures of different forms of the extracellular domain (ECD) has been determined, the detailed binding locations of Ca2+ still remain unidentified. In this study, we first report our prediction of several Ca2+ binding sites in the ECD of mGluR1a using our newly developed computational algorithms. Putative residues involved in the calcium binding were verified using a grafting approach. Their capability to bind Ca2+ and its trivalent analog, Tb3+ were determined using fluorescence energy transfer and NMR. The substitution of charged or polar ligand binding residues with Ile at Site 1 resulted in up to 99-fold decrease in the Tb3+ binding affinity. In transiently-transfected HEK cells, mutations on these proposed ligand binding residues in the predicted calcium binding site either resulted in increase or decrease of intracellular Ca2+ response toward changes in [Ca2+]o. When treated mGluR1α with glutamate and Ca2+ together, the intracellular Ca2+ response is significantly greater than individual effects by glutamate or Ca2+, suggesting a cooperative effects by calcium binding and glutamate.

Inhibition of Heterotrimeric G Protein Signaling by a Small Molecule Acting on Gα Subunit

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Galpha subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Galpha subunit is feasible and should constitute a new strategy for therapeutic intervention.

PI(3,4,5)P3 potentiates phospholipase C-β activity

Phospholipase C-β (PLC-β) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-β1 and PLC-β3 bound immobilized PIP3. In this study, PIP3 was found to potentiate Ca2+-stimulated PLC-β activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP3 accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP3 accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP3 in directly potentiating PLC-β activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-β3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-β is not important for p110CAAX-induced membrane association.

Localization of prolyl oligopeptidase in the thalamic and cortical projection neurons: A retrograde neurotracing study in the rat brain

Prolyl oligopeptidase (POP) is a serine endopeptidase which hydrolyses proline-containing peptides shorter than 30-mer. POP is believed to be associated with cognitive functions via neuropeptide cleavage. POP has been also connected to the inositol 1,4,5-triphosphate (IP3) signalling but the effects of POP-inhibition to the IP3 accumulation in vivo are still unclear. However, little is known about the physiological role of POP in the brain. We have previously found that in the rat brain POP was specifically expressed in the pyramidal neurons of the cerebral cortex, particularly in the primary motor and somatosensory cortices, and corresponding projection areas in thalamus. Using a retrograde neurotracer we have now visualized the localization of POP in thalamocortical and corticothalamic projection neurons in ventrobasal complex and medial geniculate nucleus of thalamus and somatosensory/motor and auditory cortices. We observed that both in thalamus and cortex over 50% of projection neurons contained POP. These results support the hypothesis that POP is involved in thalamocortical and corticothalamic signal processing. We also propose, based on our neuroanatomical findings and literature, that POP may take part in the thalamocortical oscillations by interacting with IP3 signalling in cells.

Oxidative stress-induced renal angiotensin AT1 receptor upregulation causes increased stimulation of sodium transporters and hypertension

Reactive oxygen species have emerged as important molecules in cardiovascular dysfunction such as diabetes and hypertension. Recent work has shown that oxidative stress and angiotensin II signaling mutually regulate each other by multiple mechanisms and contribute to the development of hypertension. Most of the known biological actions of angiotensin II can be attributed to AT1 receptors. The present study was carried out to investigate the role of renal AT1 receptor signaling in oxidative stress-mediated hypertension. Male Sprague-Dawley rats received tap water (control) or 30 mM L-buthionine sulfoximine (BSO), an oxidant, with and without 1 mM tempol (an antioxidant) for 2 wk. Compared with control rats, BSO-treated rats exhibited increased oxidative stress and reduced antioxidant levels and developed hypertension. BSO treatment also caused increased renal proximal tubular AT1 receptor protein abundance, message levels, and ligand binding. In these rats, angiotensin II caused significantly higher accumulation of inositol trisphosphate (IP3) and phospholipase C (PLC) activation which was sensitive to blockade by AT1 but not to AT2 antagonist. Also, angiotensin II-mediated, AT1-dependent MAP kinase, Na-K-ATPase, and Na/H exchanger 3 activation was higher in BSO-treated rats than in control rats. Tempol supplementation of BSO-treated rats restored redox status, normalized AT1 receptor expression, and decreased blood pressure. Tempol also normalized the angiotensin II-mediated, AT1-dependent IP3 accumulation and PLC, MAP kinase, Na-K-ATPase, and Na/H exchanger 3 stimulation. These data suggest that oxidative stress leads to AT1 receptor upregulation, which in turn causes overstimulation of sodium transporters and subsequently contributes to sodium retention and hypertension. Tempol, while reducing oxidative stress, normalizes AT1 receptor signaling and decreases blood pressure.