Biomedical subjects

B V Potter

Publications and source records attributed to B V Potter.

At least 19 recordsLinked to original sources

PubMed2001

Total synthesis of nucleobase-modified adenophostin A mimics.

The adenophostins exhibit approximately 10-100 times higher receptor binding and Ca2+ mobilising potencies in comparison with the natural second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Despite many synthetic attempts to determine the minimal structural requirement for this unusual behaviour of the adenophostins, few related simplified analogues displaying higher activity than that of Ins(1,4,5)P3 have been reported. However, biological evaluation of such analogues has revealed that one of the key factors for the enhanced biological activity is the adenine moiety. To further understand the effect that the adenine base has upon the activity of the adenophostins, congeners in which this functionality is replaced by uracil, benzimidazole, 2-methoxynaphthalene, 4-methylanisole and 4-methylnaphthalene using the common intermediate 1,2-di-O-acetyl-5-O-benzyl-3-O-(3,4-di-O-acetyl-2,6-di-O-benzyl-alpha-D-glucopyranosyl)-ribofuranose have been synthesised using a base replacement strategy. The synthesis of the uracil and benzimidazole analogues was achieved using the Vorbrüggen condensation procedure. The 1'-C-glycosidic analogues were prepared using Friedel-Crafts type C-aryl glycosidation reactions. Phosphate groups were introduced using the phosphoramidite method with subsequent removal of all-benzyl protecting groups by catalytic hydrogenation or catalytic hydrogen transfer. Apart from one analogue with an alpha-glycosidic linkage all compounds were more potent than Ins(1,4,5)P3 and most tended more towards adenophostin in activity. These analogues will be valuable tools to unravel the role that the adenine moiety plays in the potent activity of the adenophostins and demonstrate that this strategy is effective at producing highly potent ligands.

Adenosine↗

PubMed2001

Synthesis and biological activity of D- and L-chiro-inositol 2,3,4,5-tetrakisphosphate: design of a novel and potent inhibitor of Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase.

The synthesis of a novel and potent Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6 kinase inhibitor and its enantiomer is described. D-chiro-Inositol 2,3,4,5-tetrakisphosphate [D-chiro-Ins(2,3,4,5)P4, 3, Figure 1] and L-chiro-inositol 2,3,4,5-tetrakisphosphate [L-chiro-Ins(2,3,4,5)P4, ent-3] were synthesized from D-1,6-di-O-benzyl-chiro-inositol and L-1,6-di-O-benzyl-chiro-inositol, respectively. We examined inhibition of the multifunctional Ins(3,4,5,6)P4 1-kinase/Ins(1,3,4)P3 5/6-kinase from bovine aorta by 3 and ent-3. Compound 3 was a potent inhibitor with an IC(50) of 1.5 microM, and ent-3 was more than 20-fold less active. The results are compared to those for other inhibitory inositol polyphosphates with structure-activity relationship discussion. Compound 3 is a useful lead for development of further inhibitors of this important enzyme, and ent-3 should find applications in the newly emerging Ins(1,4,5,6)P4 signaling pathway.

Animals↗

PubMed2001

Bicyclic analogues of D-myo-inositol 1,4,5-trisphosphate related to adenophostin A: synthesis and biological activity.

The high affinity of adenophostin A for 1D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors may be related to an alteration in the position of its 2'-phosphate group relative to the corresponding 1-phosphate group in Ins(1,4,5)P(3). To investigate this possibility, two bicyclic trisphosphates 9 and 10, designed to explore the effect of relocating the 1-phosphate group of Ins(1,4,5)P(3) using a novel fused-ring system, were synthesized from myo-inositol. Biological evaluation of 9 and 10 at the Ins(1,4,5)P(3) receptors of hepatocytes showed that both were recognized by hepatic Ins(1,4,5)P(3) receptors and both stimulated release of Ca(2+) from intracellular stores, but they had lower affinity than Ins(1,4,5)P(3). This finding may be explained by considering the three-dimensional structures of 9 and 10 in light of recent studies on the conformation of adenophostin A.

Adenosine↗

PubMed2001

[3H]-Methyllycaconitine: a high affinity radioligand that labels invertebrate nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChR) of insect and other invertebrates are heterogeneous and new tools are needed to dissect their multiplicity. [(3)H]-Methyllycaconitine ([(3)H]-MLA) is a novel radioligand which is a potent antagonist at vertebrate alpha7-type nAChR. Putative invertebrate nAChR of the aphid Myzus persicae, the moths Heliothis virescens and Manduca sexta, the fly Lucilia sericata, and the squid Loligo vulgaris were investigated in radioligand binding studies with [(3)H]-MLA. Saturable binding was consistent with a single class of high affinity binding sites for each of these invertebrates, characterised by a dissociation constant, K(d), of approximately 1 nM and maximal binding capacities, B(max), between 749 and 1689 fmol/mg protein for the insects and 14,111 fmol/mg protein for squid. [(3)H]-MLA binding to M. persicae membranes was characterised in more detail. Kinetic analysis demonstrated rapid association in a biphasic manner and slow, monophasic dissociation. Displacement studies demonstrate the nicotinic character of [(3)H]-MLA binding sites. Data for all nicotinic ligands, except MLA itself, are consistent with displacement from a high and a low affinity site, indicating that displacement is occurring from two or more classes of nicotinic binding site that are not distinguished by MLA itself. Autoradiographic analysis of the distribution of [(3)H]-MLA binding sites in Manduca sexta shows discrete labelling of neuropil areas of the optic and antennal lobes.

Aconitine↗

PubMed2001

Selective recognition of inositol phosphates by subtypes of the inositol trisphosphate receptor.

Synthetic analogues of inositol trisphosphate (IP(3)), all of which included structures equivalent to the 4,5-bisphosphate of (1,4,5)IP(3), were used to probe the recognition properties of rat full-length type 1, 2 and 3 IP(3) receptors expressed in insect Spodoptera frugiperda 9 cells. Using equilibrium competition binding with [(3)H](1,4,5)IP(3) in Ca(2+)-free cytosol-like medium, the relative affinities of the receptor subtypes for (1,4,5)IP(3) were type 3 (K(d)=11+/-2 nM)>type 2 (K(d)=17+/-2 nM)>type 1 (K(d)=24+/-4 nM). (1,4,5)IP(3) binding was reversibly stimulated by increased pH, but the subtypes differed in their sensitivity to pH (type 1>type 2>type 3). For all three subtypes, the equatorial 6-hydroxy group of (1,4,5)IP(3) was essential for high-affinity binding, the equatorial 3-hydroxy group significantly improved affinity, and the axial 2-hydroxy group was insignificant; a 1-phosphate (or in its absence, a 2-phosphate) improved binding affinity. The subtypes differed in the extents to which they tolerated inversion of the 3-hydroxy group of (1,4,5)IP(3) (type 1>type 2>type 3), and this probably accounts for the selectivity of (1,4,6)IP(3) for type 1 receptors. They also differed in their tolerance of inversion, removal or substitution (by phosphate) of the 2-hydroxy group (types 2 and 3>type 1), hence the selectivity of (1,2,4,5)IP(4) for type 2 and 3 receptors. Removal of the 3-hydroxy group or its replacement by fluorine or CH(2)OH was best tolerated by type 3 receptors, and accounts for the selectivity of 3-deoxy(1,4,5)IP(3) for type 3 receptors. Our results provide the first systematic analysis of the recognition properties of IP(3) receptor subtypes and have identified the 2- and 3-positions of (1,4,5)IP(3) as key determinants of subtype selectivity.

Amino Acid Sequence↗

PubMed2001

Non-steroidal and steroidal sulfamates: new drugs for cancer therapy.

The development of inhibitors to block the formation of estrone and 5-androstenediol from sulfated precursors is an important new strategy for the treatment of breast cancer. In this study a series of tricyclic coumarin sulfamates (665-668 COUMATE) and a tricyclic oxepin sulfamate have been synthesised and tested for their ability to inhibit estrone sulfatase activity (E1-STS). In addition the effect of the steroid-based E1-STS inhibitor, 2-methoxyestrone-3-O-sulfamate (2-MeOEMATE) on the morphology of MDA-MB-231 cells and breast tumour-derived fibroblasts was also examined. The tricyclic coumarin sulfamates and oxepin sulfamate were potent inhibitors of E1-STS activity with IC(50)s ranging from 8 to 250 nM. Of this series 667 COUMATE was the most potent inhibitor (IC(50)=8 nM) and was three-times more potent than estrone-3-O-sulfamate (EMATE, IC(50)=25 nM). 667 COUMATE did not stimulate the growth of MCF-7 breast cancer cells and is therefore devoid of estrogenicity. In vivo, 667 COUMATE inhibited E1-STS activity in rat liver tissue to a similar extent to that of EMATE. 2-MeOEMATE had a marked effect on the morphology of MDA-MB-231 cells and breast tumour-derived fibroblasts causing a significant increase in the number of rounded cells. 667 COUMATE and 2-MeOEMATE therefore offer considerable potential for development for cancer therapy.

Animals↗

PubMed2001

Synthesis of the enantiomers of 6-deoxy-myo-inositol 1,3,4,5-tetrakisphosphate, structural analogues of myo-inositol 1,3,4,5-tetrakisphosphate.

D-myo-Inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] is produced rapidly from the established second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P4] in stimulated cells. Despite extensive investigations, in particular concerning its potential role in mediating cellular Ca2+ influx, no exact cellular function has been described for this inositol phosphate; however, binding sites have been identified in a number of tissues and it has been shown to act synergistically with Ins(1,4,5)P3. To assist in the elucidation of the mechanism of action and structural requirements within the Ins(1,3,4,5)P4 moiety that are necessary for recognition and activation of the receptor, structural analogues of this tetrakisphosphate are required. Routes for the synthesis of racemic 6-deoxy-myo-inositol 1,3,4,5-tetrakisphosphate [6-deoxy-DL-Ins(1,3,4,5)P4] and the chiral antipodes D- and L-6-deoxy-myo-inositol 1,3,4,5-tetrakisphosphate are described here. The racemic tetrakisphosphate was synthesised from DL-1,2-O-isopropylidene-myo-inositol in eight steps. Deoxygenation at C-6 was achieved following the Barton-McCombie procedure. Both chiral tetrakisphosphates were synthesised through resolution of racemic cis-diol 6-deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-inositol with the chiral auxiliary (S)-(+)-O-acetylmandelic acid. Absolute configuration was confirmed by synthesis of the known D-6-deoxy-myo-inositol. Both D-6-deoxy-Ins(1,3,4,5)P4 and its enantiomer will be useful tools to unravel the enigmatic role of Ins(1,3,4,5)P4 in the polyphosphoinositide pathway of signal transduction.

Animals↗

PubMed2001

Mechanisms involved in alpha6beta1-integrin-mediated Ca(2+) signalling.

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting alpha6beta1-integrin-mediated Ca(2+) signalling. Both Ca(2+) release from thapsigargin-sensitive Ca(2+) stores and capacitative Ca(2+) entry via Ca(2+) channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of alpha6beta1-integrin-mediated Ca(2+) signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca(2+)-releasing second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cADPR.

Adenosine Diphosphate Ribose↗

PubMed2001

InsP(3)-induced Ca(2+) release in permeabilized invertebrate photoreceptors: a link between phototransduction and Ca(2+) stores.

Using the low-affinity fluorescent Ca(2+) indicators, Mag-Fura-2 and Mag-Fura Red, we studied light- and InsP(3)-induced Ca(2+) release in permeabilized microvillar photoreceptors of the medicinal leech, Hirudo medicinalis. Two major components of the phosphoinositide signaling pathway, phospholipase-C and the InsP(3) receptor, were characterized immunologically and appropriately localized in photoreceptors. Whereas phospholipase-C was abudantly expressed in photoreceptive microvilli, InsP(3) receptors were found mostly in submicrovillar endoplasmic reticulum (SER). Permeabilization of the peripheral plasma membrane with saponin allowed direct measurements of luminal free Ca(2+) concentration (Ca(L)) changes. Confocal Ca(2+) imaging using Mag-Fura Red demonstrated that Ins(1,4,5)P(3) mobilizes Ca(2+) from SER. As detected with Mag-Fura-2, a brief 50ms light flash activated rapid Ca(2+) depletion of SER, followed by an effective refilling within 1min of dark adaptation after the light flash. Sensitivity to Ins(1,4,5)P(3) of the Ca(2+) release from SER in leech photoreceptors was accompanied by irreversible uncoupling of phototransduction from Ca(2+) release. Depletion of Ca(2+) stores was induced by Ins(1,4,5)P(3)(EC(50)= 4.75 microM) and the hyper-potent agonist adenophostin A (EC(50)/40nM) while the stereoisomer L-myo Ins(1,4,5)P(3) was totally inactive. Ins(1,4,5)P(3)- or adenophostin A-induced Ca(2+) release was inhibited by 0.1-1mg/ml heparin. The Ca(2+) pump inhibitors, cyclopiazonic acid and thapsigargin, in the presence of Ins(1,4,5)P(3), completely depleted Ca(2+) stores in leech photoreceptors.

Animals↗

PubMed2001

Structural determinants of adenophostin A activity at inositol trisphosphate receptors.

Adenophostin A is the most potent known agonist of inositol 1,4,5-trisphosphate (InsP(3)) receptors. Ca(2+) release from permeabilized hepatocytes was 9.9 +/- 1.6-fold more sensitive to adenophostin A (EC(50), 14.7 +/- 2.4 nM) than to InsP(3) (145 +/- 10 nM), consistent with the greater affinity of adenophostin A for hepatic InsP(3) receptors (K(d) = 0.48 +/- 0.06 and 3.09 +/- 0.33 nM, respectively). Here, we systematically modify the structures of the glucose, ribose, and adenine moieties of adenophostin A and use Ca(2+) release and binding assays to define their contributions to high-affinity binding. Progressive trimming of the adenine of adenophostin A reduced potency, but it fell below that of InsP(3) only after complete removal of the adenine. Even after substantial modifications of the adenine (to uracil or even unrelated aromatic rings, retaining the beta-orientation), the analogs were more potent than InsP(3). The only analog with an alpha-ribosyl linkage had massively decreased potency. The 2'-phosphate on the ribose ring of adenophostin A was essential and optimally active when present on a five-membered ring in a position stereochemically equivalent to its location in adenophostin A. Xylo-adenophostin, where xylose replaces the glucose ring of adenophostin A, was only slightly less potent than adenophostin A, whereas manno-adenophostin (mannose replacing glucose) had similar potency to InsP(3). These results are consistent with the relatively minor role of the 3-hydroxyl of InsP(3) (the equivalent is absent from xylo-adenophostin) and greater role of the equatorial 6-hydroxyl (the equivalent is axial in manno-adenophostin). This is the first comprehensive analysis of all the key structural elements of adenophostin A, and it provides a working model for the design of related high-affinity ligands of InsP(3) receptors.

Adenosine↗

PubMed2000

Regulation of steroid sulphatase expression and activity in breast cancer.

Steroid sulphatase (STS) catalyzes the conversion of oestrone sulphate (E1S) to oestrone (E1) and its action in breast tumours makes a major contribution to in situ oestrogen production in this tissue. Although expression of STS mRNA and STS activity are increased in malignant breast tissues compared with that in non-malignant tissues, little is known about the regulation of its expression or activity. In the present study we have used a RT-PCR technique to investigate the regulation of STS mRNA expression in cultured breast tissue fibroblasts and MCF-7 cells. STS mRNA expression was readily detectable in fibroblasts derived from breast tissue proximal to tumours, breast tumour tissue and reduction mammoplasty tissue. For two pre-menopausal subjects, STS mRNA expression was similar in proximal and tumour fibroblasts whereas for a third, post-menopausal subject, expression in breast tumour fibroblasts was 2.4-fold that in proximal fibroblasts. The cytokine tumour necrosis factor alpha (TNFalpha) or the STS inhibitor, 2-methoxyoestrone-3-O-sulphamate, had no effect on STS mRNA expression in fibroblasts. STS mRNA was detectable in MCF-7 cells but neither TNFalpha nor interleukin 6 (IL-6) affected its expression. Transient transfection of COS-1 and MCF-7 cells with a STS cDNA lacking STS 5' and 3' sequences increased activity 17-fold and 2-fold, respectively. TNFalpha plus IL-6 increased STS activity in mock transfected MCF-7 cells and further increased STS activity in transfected MCF-7 cells. This indicates that activation can occur independently of STS promoter and enhancer elements. In conjunction with the lack of regulation of STS mRNA it suggest that TNFalpha and IL-6 may increase STS activity via a post-translational modification of the enzyme or by increasing substrate availability.

Animals↗

PubMed2000

Inhibition of steroid sulphatase activity by tricyclic coumarin sulphamates.

The identification of the active pharmacophore required for potent inhibition of steroid sulphatase activity, i.e. an aryl-O-sulphamate structure, has led to the synthesis and testing of a large number of 1-4 ring-based inhibitors. 4-Methylcoumarin-7-O-sulphamate (COUMATE) was one of the first non-steroid based inhibitors identified. In an attempt to increase the potency of this class of inhibitor a series of tricyclic COUMATEs (665-6615 COUMATEs) have been synthesised and evaluated. Using placental microsomes as a source of oestrone sulphatase (E1-STS) the size of the third ring of the tricyclic COUMATEs was found to have a marked effect on inhibitor potency. Whereas 665- and 6615-COUMATEs had IC(50)s of 200 and 370 nM, respectively, the most potent inhibitor in vitro in this series was 6610 COUMATE with an IC(50) of 1 nM. Selected inhibitors were tested for their in vivo potency by administration of a single dose (0.1 or 1 mg/kg, p.o.) to female rats. Surprisingly, in vivo 6615 COUMATE proved to be the most active drug, inhibiting rat liver E1-STS activity by 23 and 94% when assayed 24 h after administration of the 0.1 and 1 mg/kg doses. E1-STS activity in brain tissue and white blood cells was also found to be inhibited when selected drugs were tested. These studies have identified a number of tricyclic COUMATEs with therapeutic potential.

Animals↗

PubMed2000

Rapid activation and partial inactivation of inositol trisphosphate receptors by adenophostin A.

Adenophostin A, the most potent known agonist of inositol 1,4, 5-trisphosphate (InsP(3)) receptors, stimulated (45)Ca(2+) release from the intracellular stores of permeabilized hepatocytes. The concentration of adenophostin A causing the half-maximal effect (EC(50)) was 7.1+/-0.5 nM, whereas the EC(50) for InsP(3) was 177+/-26 nM; both responses were positively co-operative. In rapid superfusion analyses of (45)Ca(2+) release from the intracellular stores of immobilized hepatocytes, maximal concentrations of adenophostin A or InsP(3) evoked indistinguishable patterns of Ca(2+) release. The Ca(2+) release evoked by both agonists peaked at the same maximal rate after about 375 ms and the activity of the receptors then decayed to a stable, partially (60%) inactivated state with a half-time (t(1/2)) of 318+/-29 ms for adenophostin A and 321+/-22 ms for InsP(3). Dissociation rates were measured by recording rates of InsP(3)-receptor channel closure after rapid removal of agonist. The rate of adenophostin A dissociation (t(1/2), 840+/-195 ms) was only 2-fold slower than that of InsP(3) (t(1/2), 436+/-48 ms). We conclude that slow dissociation of adenophostin A from InsP(3) receptors does not underlie either its high-affinity binding or the reported differences in the Ca(2+) signals evoked by InsP(3) and adenophostin A in intact cells.

Adenosine↗

PubMed2000

InsP4 facilitates store-operated calcium influx by inhibition of InsP3 5-phosphatase.

Receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) initiates Ca2+ release from intracellular stores and the subsequent activation of store-operated calcium influx. InsP3 is metabolized within seconds by 5-phosphatase and 3-kinase, yielding Ins(1,4)P2 and inositol 1,3,4,5-tetrakisphosphate (InsP4), respectively. Some studies have suggested that InsP4 controls Ca2+ influx in combination with InsP3 (refs 3 and 4), but another study did not find the same result. Some of the apparent conflicts between these previous studies have been resolved; however, the physiological function of InsP4 remains elusive. Here we have investigated the function of InsP4 in Ca2+ influx in the mast cell line RBL-2H3, and we show that InsP4 inhibits InsP3 metabolism through InsP3 5-phosphatase, thereby facilitating the activation of the store-operated Ca2+ current I(CRAC) (ref. 9). Physiologically, this mechanism opens a discriminatory time window for coincidence detection that enables selective facilitation of Ca2+ influx by appropriately timed low-level receptor stimulation. At higher concentrations, InsP4 acts as an inhibitor of InsP3 receptors, enabling InsP4 to act as a potent bi-modal regulator of cellular sensitivity to InsP3, which provides both facilitatory and inhibitory feedback on Ca2+ signalling.

Animals↗

PubMed2000

Contribution of the adenine base to the activity of adenophostin A investigated using a base replacement strategy.

Syntheses of 3'-O-alpha-D-glucopyranosyl-1-beta-D-ribofuranosidoimidazole 2',3'', 4''-trisphosphate (7) and 3'-O-alpha-D-glucopyranosyl-9-beta-D-ribofuranosidopurine 2',3'',4''- trisphosphate (8), two analogues of the superpotent 1D-myo-inositol 1,4,5-trisphosphate receptor agonist adenophostin A (2), are described. 5-O-Benzyl-1, 2-O-isopropylidene-alpha-D-ribofuranose was prepared by an improved route from 1,2-O-isopropylidene-alpha-D-xylofuranose and was coupled with 3,4-di-O-acetyl-2,6-di-O-benzyl-D-glucopyranosyl dimethyl phosphite to give 3',4'-di-O-acetyl-2',5, 6'-tri-O-benzyl-3-O-alpha-D-glucopyranosyl-1, 2-O-isopropylidene-alpha-D-ribofuranose. Removal of the isopropylidene acetal and subsequent acetylation gave the central disaccharide 1,2,3',4'-tetra-O-acetyl-2',5, 6'-tri-O-benzyl-3-O-alpha-D-glucopyranosyl-D-ribofuranose. Vorbrüggen condensation with activated imidazole or purine gave the required beta-substituted derivatives which were further elaborated to 7 and 8, respectively. Radioligand binding assays to hepatic InsP(3) receptors and functional assays of Ca(2+) release from permeabilized hepatocytes gave a rank order of potency of the ligands 2 approximately 8 > 7 approximately Ins(1,4,5)P(3) indicating that the N(6)-amino group of 2 is of little importance for activity and that a minimum of a two-fused-ring nucleobase is required for activity to exceed that of Ins(1,4,5)P(3). The role of the adenine base in the activity of the adenophostins is discussed. This general method should facilitate ready access to nucleobase-modified adenophostin analogues for SAR studies.

Adenine↗

PubMed2000

C-glycoside based mimics of D-myo-inositol 1,4,5-trisphosphate.

Epimeric C-glycoside based polyphosphates, alpha- and beta-D-glucopyranosylmethanol 3,4,1'-trisphosphates (8 and 9) were prepared from D-glucose. The key intermediate, allyl 2,6-di-O-benzyl-alpha-D-glucopyranoside, was prepared in five steps (67% yield) from allyl alpha-D-glucopyranoside without the need for chromatography. Compounds 8 and 9 were shown to be full agonists at the Ins(1,4,5)P3 receptors of permeabilised hepatocytes, but with markedly different potencies. Such C-glycoside analogues are worthy of further development as Ins(1,4,5)P, receptor ligands.

Adenosine↗

PubMed2000

Synthesis of potent agonists of the D-myo-inositol 1,4, 5-trisphosphate receptor based on clustered disaccharide polyphosphate analogues of adenophostin A.

Clustered disaccharide analogues of adenophostin A (2), i.e. mono-, di-, and tetravalent derivatives 6-8, respectively, were synthesized and evaluated as novel ligands for the tetrameric D-myo-inositol 1,4, 5-trisphosphate receptor (IP(3)R). The synthesis was accomplished via Sonogashira coupling of propargyl 2-O-acetyl-5-O-benzyl-3-O-(3, 4-di-O-acetyl-2, 6-di-O-benzyl-alpha-D-glucopyranosyl)-beta-D-ribofuranoside (16) with iodobenzene 18, 22, or 25, followed by deacetylation, phosphorylation, and deprotection. The abilities of the target compounds 6-8, as well as ribophostin 4, propylphostin 5, and IP(3) (1), to evoke Ca(2+) release from permeabilized hepatocytes or displacement of [(3)H]IP(3) from its receptor in hepatic membranes were compared. Although the binding affinities of 4-8 were similar, there were modest though significant differences in their potencies in Ca(2+) release assays: tetraphostin 8 > IP(3) approximately diphostin 7 > phenylphostin 6 > ribophostin 4 approximately propylphostin 5.

Adenosine↗

PubMed2000

Nicotinic acid adenine dinucleotide phosphate (NAADP(+)) is an essential regulator of T-lymphocyte Ca(2+)-signaling.

Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP(+)) dose-dependently stimulated intracellular Ca(2+)-signaling. At a concentration of 10 nM NAADP(+) evoked repetitive and long-lasting Ca(2+)-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca(2+)-peak followed by trains of smaller Ca(2+)-oscillations was observed. Higher concentrations of NAADP(+) (1 and 10 microM) gradually reduced the initial Ca(2+)-peak, and a complete self-inactivation of Ca(2+)-signals was seen at 100 microM. The effect of NAADP(+) was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4, 5-trisphosphate- and cyclic adenosine diphosphoribose-mediated Ca(2+)-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP(+). Most importantly, microinjection of a self-inactivating concentration of NAADP(+) completely abolished subsequent stimulation of Ca(2+)-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP(+) Ca(2+)-release system is essential for T-lymphocyte Ca(2+)-signaling.

Adenosine Diphosphate Ribose↗