Antagonists show GTP-sensitive high-affinity binding to the sigma-1 receptor.

BACKGROUND AND PURPOSE: Sigma-1 receptors are atypical receptors with potentially two transmembrane domains. Antagonists require doses significantly higher than their published affinities to have biological effects. We have reassessed the binding characteristics of these ligands and found antagonists bind to high- and low-affinity states not distinguished by agonists. EXPERIMENTAL APPROACH: The affinities of sigma-1 receptor ligands was assessed using radioligand saturation and competition binding of [³H]-(+)-pentazocine to permeabilized MDA-MB-468 cells. This was compared with the effect of ligands on metabolic activity using an MTS-based assay and calcium signalling using cells loaded with the calcium dye, Fura-2. KEY RESULTS: Sigma-1 receptor antagonists, but not agonists, show GTP- and suramin-sensitive high-affinity binding. Functional responses (calcium signalling and metabolic activity), while associated with sigma-1 receptor binding, required binding to an unidentified, low-affinity target. CONCLUSIONS AND IMPLICATIONS: Sigma-1 receptors are coupled to G proteins. This interaction is only observed when analysing antagonist binding. The identity of the G protein remains to be resolved. The concept of agonist and antagonist at the sigma-1 receptor needs to be revisited.

The regulation of membrane to cytosol partitioning of signalling proteins by phosphoinositides and their soluble headgroups.

Inositol phospholipids [PIs (phosphoinositides)] represent a group of membrane-tethered signalling molecules which differ with respect to the number and distribution of monoester phosphate groups around the inositol ring. They function by binding to proteins which possess one of several domains that bind a particular PI species, often with high affinity and specificity. PH (pleckstrin homology) domains for example possess ligand-binding pockets that are often lined with positively charged residues and which bind PIs with varying degrees of specificity. Several PH domains bind not only PIs, but also their cognate headgroups, many of which occur naturally in cells as relatively abundant cytosolic inositol phosphates. The subcellular distributions of proteins possessing such PH domains are therefore determined by the relative levels of competing membrane-bound and soluble ligands. A classic example of the latter is the PH domain of phospholipase Cdelta1, which binds both phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate. We have shown that the N-terminal PH domain of the Rho family guanine nucleotide-exchange factor, Tiam 1, binds PI ligands promiscuously allowing multiple modes of regulation. We also recently analysed the ligand-binding specificity of the PH domain of PI-dependent kinase 1 and found that it could bind abundant inositol polyphosphates such as inositol hexakisphosphate. This could explain the dual distribution of this key signalling component, which needs to access substrates at both the plasma membrane and in the cytosol.

Protocols for regulation and study of diphosphoinositol polyphosphates.

The roles of diphosphoinositol polyphosphates (DIPs) in mammalian cell biology have been difficult to determine because of the lack of tools known to regulate their levels. I have determined a series of protocols that regulate these DIPs, and these can be used to further our understanding of these molecules. Sorbitol and sucrose significantly raised levels of bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4) but slightly lowered levels of diphosphoinositol pentakisphosphate (PP-InsP5) in DDT1 MF-2 cells. These effects correlate with the ability of hyperosmotic stress to interfere with protein trafficking described previously and suggest that [PP]2-InsP4 specifically impedes protein trafficking. The effects on [PP]2-InsP4 were not regulated by extracellular signal-regulated kinase or phospholipase D, as exemplified by the lack of effect of U0126 and butan-1-ol. I have also found that genistein potently and rapidly lowers levels of [PP]2-InsP4, whereas a similar inhibitor, herbimycin, was without effect. Thapsigargin, a sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase pump inhibitor previously shown to selectively lower PP-InsP5 after short-term treatment, also selectively raises PP-InsP5 after a longer treatment. The calmodulin inhibitors N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and chlorpromazine significantly lowered all higher inositol phosphates, as well as DIPs, whereas the calmodulin-dependent kinase inhibitors methyl 9-(S)-12-(R)-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-2,3,9,10,11,12-hexahydro-10-(R)hydroxy-9-methyl-1-oxo-10-carboxylate (K-252a) and 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-93) were without effect. W-7 and chlorpromazine also lowered levels of phosphatidylinositol 4,5-bisphosphate and ATP but greatly increased levels of phosphatidylinositol 4-phosphate. Trypan blue exclusion deemed that these doses were not cytotoxic. These results identify an increasing number of reagents that regulate DIP levels. Using these tools, and those described previously, we can further understand the roles of the DIPs in cell biology.

Discovery of molecular and catalytic diversity among human diphosphoinositol-polyphosphate phosphohydrolases. An expanding Nudt family.

The turnover of the "high energy" diphosphoinositol polyphosphates by Ca(2+)- and cyclic nucleotide-modulated enzymes is considered a regulatory, molecular switching activity. Target processes may include intracellular trafficking. Following our earlier identification of a prototype human diphosphoinositol-polyphosphate phosphohydrolase (hDIPP1), we now describe new 21-kDa human isoforms, hDIPP2alpha and hDIPP2beta, distinguished from each other solely by hDIPP2beta possessing one additional amino acid (Gln(86)). Candidate DIPP2alpha and DIPP2beta homologues in rat and mouse were also identified. The rank order for catalytic activity is hDIPP1 > hDIPP2alpha > hDIPP2beta. Differential expression of hDIPP isoforms may provide flexibility in response times of the molecular switches. The 76% identity between hDIPP1 and the hDIPP2s includes conservation of an emerging signature sequence, namely, a Nudt (MutT) motif with a GX(2)GX(6)G carboxy extension. Northern and Western analyses indicate expression of hDIPP2s is broad but atypically controlled; these proteins are translated from multiple mRNAs that differ in the length of the 3'-untranslated region because of utilization of an array of alternative (canonical and noncanonical) polyadenylation signals. Thus, cells can recruit sophisticated molecular processes to regulate diphosphoinositol polyphosphate turnover.

Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphates and diphosphoinositol polyphosphates.

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)). Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP. Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) >95%; thus, all three substrates are hydrolyzed at the same active site. Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding. GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP. Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP)(2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His(91) and Phe(84). This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.

Diphosphoinositol polyphosphates: the final frontier for inositide research?

The diphosphoinositol polyphosphates comprise a group of highly phosphorylated compounds which have a rapid rate of metabolic turnover through tightly-regulated kinase/phosphohydrolase substrate cycles. The phosphohydrolases occur as multiple isoforms, the expression of which is apparently carefully controlled. Cellular levels of the diphosphoinositol polyphosphates are regulated by cAMP and cGMP in a protein kinase-independent manner. These inositides can also sense a specific mode of intracellular Ca2+ pool depletion. In this review, we will argue that these are characteristics of highly significant cellular molecules.

The diadenosine hexaphosphate hydrolases from Schizosaccharomyces pombe and Saccharomyces cerevisiae are homologues of the human diphosphoinositol polyphosphate phosphohydrolase. Overlapping substrate specificities in a MutT-type protein.

Aps1 from Schizosaccharomyces pombe (Ingram, S. W., Stratemann, S. A. , and Barnes, L. D. (1999) Biochemistry 38, 3649-3655) and YOR163w from Saccharomyces cerevisiae (Cartwright, J. L., and McLennan, A. G. (1999) J. Biol. Chem. 274, 8604-8610) have both previously been characterized as MutT family hydrolases with high specificity for diadenosine hexa- and pentaphosphates (Ap(6)A and Ap(5)A). Using purified recombinant preparations of these enzymes, we have now discovered that they have an important additional function, namely, the efficient hydrolysis of diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition, this finding provides important new information pertinent to the structure, function, and evolution of the MutT motif. Moreover, we report that the human protein previously characterized as a diphosphorylated inositol phosphate phosphohydrolase represents the first example, in any animal, of an enzyme that degrades Ap(6)A and Ap(5)A, in preference to other diadenosine polyphosphates. The emergence of Ap(6)A and Ap(5)A as extracellular effectors and intracellular ion-channel ligands points not only to diphosphorylated inositol phosphate phosphohydrolase as a candidate for regulating signaling by diadenosine polyphosphates, but also suggests that diphosphorylated inositol phosphates may competitively inhibit this process.

A novel context for the 'MutT' module, a guardian of cell integrity, in a diphosphoinositol polyphosphate phosphohydrolase.

Diphosphoinositol pentakisphosphate (PP-InsP5 or 'InsP7') and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') are the most highly phosphorylated members of the inositol-based cell signaling family. We have purified a rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP) that cleaves a beta-phosphate from the diphosphate groups in PP-InsP5 (Km = 340 nM) and [PP]2-InsP4 (Km = 34 nM). Inositol hexakisphophate (InsP6) was not a substrate, but it inhibited metabolism of both [PP]2-InsP4 and PP-InsP5 (IC50 = 0.2 and 3 microM, respectively). Microsequencing of DIPP revealed a 'MutT' domain, which in other contexts guards cellular integrity by dephosphorylating 8-oxo-dGTP, which causes AT to CG transversion mutations. The MutT domain also metabolizes some nucleoside phosphates that may play roles in signal transduction. The rat DIPP MutT domain is conserved in a novel recombinant human uterine DIPP. The nucleotide sequence of the human DIPP cDNA was aligned to chromosome 6; the candidate gene contains at least four exons. The dependence of DIPP's catalytic activity upon its MutT domain was confirmed by mutagenesis of a conserved glutamate residue. DIPP's low molecular size, Mg2+ dependency and catalytic preference for phosphoanhydride bonds are also features of other MutT-type proteins. Because overlapping substrate specificity is a feature of this class of proteins, our data provide new directions for future studies of higher inositol phosphates.

Turnover of bis-diphosphoinositol tetrakisphosphate in a smooth muscle cell line is regulated by beta2-adrenergic receptors through a cAMP-mediated, A-kinase-independent mechanism.

Bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4 or 'InsP8') is a 'high-energy' inositol phosphate; we report that its metabolism is receptor-regulated in DDT1 MF-2 smooth muscle cells. This conclusion arose by pursuing the mechanism by which F- decreased cellular levels of [PP]2-InsP4 up to 70%. A similar effect was induced by elevating cyclic nucleotide levels, either with IBMX or by application of either Bt2cAMP (EC50 = 14.7 microM), Bt2cGMP (EC50 = 7.9 microM) or isoproterenol (EC50 = 0.4 nM). Isoproterenol (1 microM) decreased [PP]2-InsP4 levels 25% by 5 min, and 71% by 60 min. This novel, agonist-mediated regulation of [PP]2-InsP4 turnover was very specific; isoproterenol did not decrease the cellular levels of either inositol pentakisphosphate, inositol hexakisphosphate or other diphosphorylated inositol polyphosphates. Bradykinin, which activated phospholipase C, did not affect [PP]2-InsP4 levels. Regulation of [PP]2-InsP4 turnover by both isoproterenol and cell-permeant cyclic nucleotides was unaffected by inhibitors of protein kinases A and G. The effectiveness of the kinase inhibitors was confirmed by their ability to block phosphorylation of the cAMP response element-binding protein. Our results indicate a new signaling action of cAMP, and furnish an important focus for future research into the roles of diphosphorylated inositol phosphates in signal transduction.

Biological variability in the structures of diphosphoinositol polyphosphates in Dictyostelium discoideum and mammalian cells.

Previous structural analyses of diphosphoinositol polyphosphates in biological systems have relied largely on NMR analysis. For example, in Dictyostelium discoideum, diphosphoinositol pentakisphosphate was determined by NMR to be 4- and/or 6-PPInsP5, and the bisdiphosphoinositol tetrakisphosphate was found to be 4, 5-bisPPInsP4 and/or 5,6-bisPPInsP4 [Laussmann, Eujen, Weisshuhn, Thiel and Vogel (1996) Biochem. J. 315, 715-720]. We now describe three recent technical developments to aid the analysis of these compounds, not just in Dictyostelium, but also in a wider range of biological systems: (i) improved resolution and sensitivity of detection of PPInsP5 isomers by microbore metal-dye-detection HPLC; (ii) the use of the enantiomerically specific properties of a rat hepatic diphosphatase; (iii) chemical synthesis of enantiomerically pure reference standards of all six possible PPInsP5 isomers. Thus we now demonstrate that the major PPInsP5 isomer in Dictyostelium is 6-PPInsP5. Similar findings obtained using the same synthetic standards have been published [Laussmann, Reddy, Reddy, Falck and Vogel (1997) Biochem. J. 322, 31-33]. In addition, we show that 10-25% of the Dictyostelium PPInsP5 pool is comprised of 5-PPInsP5. The biological significance of this new observation was reinforced by our demonstration that 5-PPInsP5 is the predominant PPInsP5 isomer in four different mammalian cell lines (FTC human thyroid cancer cells, Swiss 3T3 fibroblasts, Jurkat T-cells and Chinese hamster ovary cells). The fact that the cellular spectrum of diphosphoinositol polyphosphates varies across phylogenetic boundaries underscores the value of our technological developments for future determinations of the structures of this class of compounds in other systems.

Molecular cloning and expression of a rat hepatic multiple inositol polyphosphate phosphatase.

The characterization of the multiple inositol polyphosphate phosphatase (MIPP) is fundamental to our understanding of how cells control the signalling activities of 'higher' inositol polyphosphates. We now describe our isolation of a 2.3 kb cDNA clone of a rat hepatic form of MIPP. The predicted amino acid sequence of MIPP includes an 18 amino acid region that aligned with approximately 60% identity with the catalytic domain of a fungal inositol hexakisphosphate phosphatase (phytase A); the similarity encompassed conservation of the RHGXRXP signature of the histidine acid phosphatase family. A histidine-tagged, truncated form of MIPP was expressed in Escherichia coli and the enzymic specificity of the recombinant protein was characterized: Ins(1,3,4,5,6)P5 was hydrolysed, first to Ins(1,4,5,6)P4 and then to Ins(1,4,5)P3, by consecutive 3- and 6-phosphatase activities. Inositol hexakisphosphate was catabolized without specificity towards a particular phosphate group, but in contrast, MIPP only removed the beta-phosphate from the 5-diphosphate group of diphosphoinositol pentakisphosphate. These data, which are consistent with the substrate specificities of native (but not homogeneous) MIPP isolated from rat liver, provide the first demonstration that a single enzyme is responsible for this diverse range of specific catalytic activities. A 2.5 kb transcript of MIPP mRNA was present in all rat tissues that were examined, but was most highly expressed in kidney and liver. The predicted C-terminus of MIPP is comprised of the tetrapeptide SDEL, which is considered a signal for retaining soluble proteins in the lumen of the endoplasmic reticulum; the presence of this sequence provides a molecular explanation for our earlier biochemical demonstration that the endoplasmic reticulum contains substantial MIPP activity [Ali, Craxton and Shears (1993) J. Biol. Chem. 268, 6161-6167].

Design of potent and selective inhibitors of myo-inositol 1,4,5-trisphosphate 5-phosphatase.

The interactions of synthetic analogues of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with the Ins(1,4,5)P3 receptor in permeabilized SH-SY5Y cells and with two key metabolic enzymes, Ins(1,4,5)P(3)3-kinase from a supernatant preparation of rat brain homogenates and Ins(1,4,5)P(3)5-phosphatase from human erythrocyte ghosts, have been examined. L-chiro-Inositol 2,3,5-trisphosphorothioate [L-chiro-Ins(2,3,5)PS3], which we have previously identified as a partial agonist at the Ins(1,4,5)P3 receptor [Safrany, S. T., Wilcox, R. A., Liu, C., Dubreuil, D., Potter, B. V. L., & Nahorski S. R. (1993) Mol. Pharmacol. 43, 499-503], is identified as the most potent 5-phosphatase inhibitor yet described (inhibiting dephosphorylation of [3H]Ins(1,4,5)P3 with Ki = 230nM). L-chiro-Ins(2,3,5)PS3 was also found to be the most potent small-molecule inhibitor of 3-kinase (Ki = 820 nM). The properties of three novel, potent, and selective inhibitors of 5-phosphatase are described. L-myo-Inositol 1,4,5-trisphosphorothioate inhibited 5-phosphatase with Ki = 430 nM, showing 250-fold selectivity over 3-kinase (Ki = 108 microM); myo-inositol 1,3,5-trisphosphorothioate inhibited 5-phosphatase with 475-fold selectivity over 3-kinase (Ki = 520 nM and 247 microM, respectively). The most potent, selective inhibitor of 5-phosphatase was L-chiro-inositol 1,4,6-trisphosphorothioate [L-chiro-Ins(1,4,6)PS3]. L-chiro-Ins(1,4,6)PS3 inhibited 5-phosphatase with Ki = 300 nM and did not interact with the Ins(1,4,5)P3 receptor or 3-kinase at doses tested. These studies, therefore, identify a highly potent and selective inhibitor of 5-phosphatase, which should be considered the tool of choice when inhibiting this enzyme in a broken cell or cell-free system.

Modification at C2 of myo-inositol 1,4,5-trisphosphate produces inositol trisphosphates and tetrakisphosphates with potent biological activities.

Novel 2-position-modified D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogues, DL-2-deoxy-2-fluoro-myo-inositol 1,4,5-trisphosphate [DL-2F-Ins(1,4,5)P3], DL-myo-inositol 1,2,4,5-tetrakisphosphate [DL-Ins(1,2,4,5)P4], DL-scyllo-inositol 1,2,4-trisphosphate [DL-sc-Ins(1,2,4)P3], scyllo-inositol 1,2,4,5-tetrakisphosphate [sc-Ins(1,2,4,5)P4] and scyllo-inositol 1,2,4,5-tetrakisphosphorothioate [sc-Ins(1,2,4,5)PS4] were investigated for their ability to bind to the Ins(1,4,5)P3 receptor, mobilise intracellular Ca2+ stores and interact with metabolic enzymes. With the exception of sc-Ins(1,2,4,5)PS4, all the Ins(1,4,5)P3 analogues potently displaced [3H]Ins(1,4,5)P3 from its receptor in bovine adrenal cortex and were apparently potent full agonists at the Ca2+ mobilising Ins(1,4,5)P3 receptor of SH-SY5Y cells, giving respective IC50 and EC50 values of: sc-Ins(1,2,4,5)P4 (IC50 14 nM, EC50 77 nM), DL-2F-Ins(1,4,5)P3 (IC50 25 nM, EC50 105 nM), DL-Ins(1,2,4,5)P4 (IC50 26 nM, EC50 163 nM), DL-sc-Ins(1,2,4)P3 (IC50 52 nM, EC50 171 nM), compared to Ins(1,4,5)P3 (IC50 4 nM, EC50 52 nM). sc-Ins(1,2,4,5)P4 was equipotent to Ins(1,4,5)P3 for Ca2+ release making it the most potent inositol tetrakisphosphate and indeed Ins(1,4,5)P3 analogue yet characterised. In contrast, although sc-Ins(1,2,4,5)P4 (IC50 425 nM, EC50 1603 nM) was a significantly weaker ligand and agonist than Ins(1,4,5)P3, it was a partial agonist of high intrinsic activity with maximally effective concentrations releasing only about 80% of Ins(1,4,5)P3-sensitive Ca2+ stores of SH-SY5Y cells. Ins(1,4,5)P3 and sc-Ins(1,2,4,5)P4 were readily metabolised by Ins(1,4,5)P3 3-kinase and 5-phosphatase activities, DL-2F-Ins(1,4,5)P3 and DL-sc-Ins(1,2,4)P3 were resistant to 5-phosphatase, while sc-Ins(1,2,4,5)PS4 and DL-Ins(1,2,4,5)P4 were resistant to both 3-kinase and 5-phosphatase activity and were potent inhibitors of the 5-phosphatase enzyme (Ki = 300 nM and 2.9 microM, respectively). These results demonstrate that modification of the 2-position of Ins(1,4,5)P3, even with an anionic group, does not critically affect Ins(1,4,5)P3 binding interaction or Ca2+ release, suggesting that the 2-OH of Ins(1,4,5)P3 fails to interact significantly with the binding site of its receptor. However, modification remote from the crucial vicinal 4,5-bisphosphate can affect analogue efficacy in Ca2+ release.

A comparison between muscarinic receptor occupancy, inositol 1,4,5-trisphosphate accumulation and Ca2+ mobilization in permeabilized SH-SY5Y neuroblastoma cells.

Electrically permeabilized SH-SY5Y neuroblastoma cells have been used to examine the relationship between receptor occupation by muscarinic agonists, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) accumulation and Ca2+ mobilization from intracellular stores. The kinetics, concentration-dependence and guanine nucleotide-sensitivity of these responses have been characterized for the agonists, carbachol, arecoline and oxotremorine. Carbachol stimulated Ins(1,4,5)P3 accumulation and Ca2+ mobilization with an EC50 value approximately 50 microM, only slightly lower than the apparent affinity of this agonist for the "free" receptor (100 microM). Arecoline and oxotremorine were partial agonists, mobilizing 45 and 21% of the Ca2+ mobilized by carbachol, and yielded EC50 values for both Ins(1,4,5)P3 and Ca2+ responses, similar to their binding affinity. Guanosine 5'-O-3 thio-triphosphate (GTP gamma S) markedly enhanced the responses elicited by all three agonists. Carbachol became significantly more potent for both Ins(1,4,5)P3 accumulation (EC50 = 4.1 microM) and Ca2+ mobilization (EC50 = 0.25 microM), revealing a separation of the dose-response relationships. GTP gamma S caused a smaller separation of the responses elicited by arecoline (Ca2+ mobilization EC50 = 0.9 microM; Ins(1,4,5)P3 accumulation EC50 = 3.6 microM), and only enhanced maximal responses for oxotremorine. These data reveal that the functional coupling of muscarinic receptors to activation of phosphoinositidase C and subsequent Ca2+ mobilization from intracellular stores is maintained after electrical permeabilization. Furthermore, this model has been used to reveal differences in the relative activities of muscarinic agonists and how they are influenced by a hydrolysis-resistant guanine nucleotide.

Identification of partial agonists with low intrinsic activity at the inositol-1,4,5-trisphosphate receptor.

The interactions of synthetic L-chiro-inositol-2,3,5-trisphosphorothioate [L-ch-Ins(2,3,5)PS3] and D-6-deoxy-myo-inositol-1,4,5-trisphosphorothioate [D-6-deoxy-Ins(1,4,5)PS3] with D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptors have been examined using radioligand binding assays and Ca2+ mobilization from permeabilized SH-SY5Y cells. The ability of these analogues to compete with [3H]Ins(1,4,5)P3 for specific sites on adrenal cortical membranes indicated that, although weaker than Ins(1,4,5)P3, both ligands competed fully for these sites [L-ch-Ins(2,3,5)PS3,Ki = 0.5 microM; D-6-deoxy-Ins(1,4,5)PS3,Ki = 5.3 microM]. However, in assays examining the amount of Ca2+ mobilized from the stores of permeabilized SH-SY5Y cells, both of these synthetic analogues displayed low intrinsic activity [L-ch-Ins(2,3,5)PS3, 34%; D-6-deoxy-Ins(1,4,5)PS3, 42% of that of Ins(1,4,5)P3]. Moreover, L-ch-Ins(2,3,5)PS3 and D-6-deoxy-Ins(1,4,5)PS3 were able to inhibit the response to Ins(1,4,5)P3 with Ki values (6 microM and 33 microM, respectively) virtually identical to their EC50 values for Ca2+ release. This is consistent with partial agonist behavior, because these compounds exhibit low maximal responses when the extent of Ca2+ release is examined. These compounds represent the first examples of inositol-based analogues with low intrinsic activity and may point the way towards the design of selective antagonists at Ins(1,4,5)P3 receptors. It also seems probable that these may represent the first true affinity values of inositol phosphates at the active receptor.

3-position modification of myo-inositol 1,4,5-trisphosphate: consequences for intracellular Ca2+ mobilisation and enzyme recognition.

The ability of the novel D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogues, L-chiro-inositol 2,3,5-trisphosphate (L-ch-Ins(2,3,5)P3) and D-3-deoxy-3-fluoro-myo-inositol 1,4,5-trisphosphate (3F-Ins(1,4,5)P3), to bind to the Ins(1,4,5)P3 receptor, mobilise intracellular Ca2+ stores and interact with metabolic enzymes has been investigated. L-ch-Ins(2,3,5)P3 and 3F-Ins(1,4,5)P3 were bound by the Ins(1,4,5)P3 receptor from bovine adrenal cortex with relatively high affinity (Ki values 60.4 and 8.0 nM respectively) but with lower affinity than Ins(1,4,5)P3 (KD = 5.9 nM). Both analogues were apparent full agonists at the Ca2+ mobilising receptor in SH-SY5Y cells, but were less potent than Ins(1,4,5)P3 (EC50 L-ch-Ins(2,3,5)P3 = 1.4 microM, 3F-Ins(1,4,5)P3 = 0.37 microM and Ins(1,4,5)P3 = 0.12 microM). L-ch-Ins(2,3,5)P3 and 3F-Ins(1,4,5)P3 were resistant to Ins(1,4,5)P3 3-kinase, and were potent inhibitors of the enzyme (Ki values 7.1 and 8.6 microM respectively). 3F-Ins(1,4,5)P3 was hydrolysed by Ins(1,4,5)P3 5-phosphatase at a similar rate to Ins(1,4,5)P3, but inhibited dephosphorylation of [3H]Ins(1,4,5)P3 with high potency (apparent Ki = 3.9 microM) L-ch-Ins(2,3,5)P3 was also recognised by the enzyme with high affinity (Ki = 7.7 microM) but was resistant to hydrolysis. These results suggest that the environment around C-3 is of major importance for recognition not only by Ins(1,4,5)P3 3-kinase but also by Ins(1,4,5)P3 5-phosphatase.

Muscarinic receptors, phosphoinositide metabolism and intracellular calcium in neuronal cells.

1. We have utilised SH-SY5Y human neuroblastoma cells and primary cultures of rat neonatal cerebellar granule cells, both expressing M3 muscarinic receptors, to examine agonist driven polyphosphoinositide hydrolysis and alterations in intracellular calcium. 2. Stimulation of SH-SY5Y cells leads to a biphasic increase in intracellular calcium, the initial peak being due to the release of calcium from an intracellular store and the second maintained phase being due to calcium entry across the plasma membrane. The channel involved does not appear to be voltage sensitive, to involve a pertussis toxin sensitive G protein, or be opened by inositol polyphosphates. 3. Muscarinic receptor stimulation also leads to increased inositol polyphosphate formation in SH-SY5Y cells. Ins(1,4,5)P3 mass formation was biphasic in profile whereas Ins(1,3,4,5)P4 mass formation was slower and monophasic in profile. These data are consistent with substantial activity of 5-phosphatase (dephosphorylating Ins(1,4,5)P3 to Ins(1,4)P2) and 3-kinase (phosphorylating Ins(1,4,5)P3 to Ins(1,3,4,5)P4) in SH-SY5Y cells. 4. In order to better understand the role of Ins(1,4,5)P3 and its metabolites in calcium homeostasis we have examined the ability of a variety of natural and synthetic analogues to release intracellular sequestered calcium. The Ins(1,4,5)P3 calcium mobilizing receptor displays a remarkable degree of stereo- and positional selectivity with the most potent agonist to date being Ins(1,4,5)P3 (EC50 = 0.09 microM). 5. As an alternative to the continuous SH-SY5Y neuroblastoma (tumour derived) cell line we have used the primary cultured cerebellar granule cell. These cells also display a biphasic increase in Ins(1,4,5)P3 mass and a subsequent release of intracellular stored calcium. In our hands carbachol appears to increase calcium influx, a response which is only visible in the absence of magnesium.

Synthetic D- and L-enantiomers of 2,2-difluoro-2-deoxy-myo-inositol 1,4,5-trisphosphate interact differently with myo-inositol 1,4,5-trisphosphate binding proteins: identification of a potent small molecule 3-kinase inhibitor.

The ability of two enantiomeric fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. (-)-D-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 [D-2,2-F2-Ins(1,4,5)P3] was a full agonist [EC50 0.21 microM] and slightly less potent than D-Ins(1,4,5)P3 [EC50 0.13 microM]. (+)-L-2,2-F2Ins(1,4,5)P3 was a very poor agonist, confirming the stereospecificity of the Ins(1,4,5)P3 receptor. D-2,2-F2-Ins(1,4,5)P3 mobilized Ca2+ with broadly similar kinetics to Ins(1,4,5)P3 and was a substrate for Ins(1,4,5)P3 3-kinase inhibiting Ins(1,4,5)P3 phosphorylation (apparent Ki = 10.2 microM) but was recognised less well than Ins(1,4,5)P3. L-2,2-F2-Ins(1,4,5)P3 was a potent competitive inhibitor of 3-kinase (Ki = 11.9 microM). Whereas D-2,2-F2-Ins(1,4,5)P3 was a good substrate for Ins(1,4,5)P3 5-phosphatase, L-2,2-F2Ins(1,4,5)P3 was a relatively potent inhibitor (Ki = 19.0 microM).