Synthesis and iron binding studies of myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate, and iron binding studies of all myo-inositol tetrakisphosphates

The first syntheses of the natural products myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate are described. The protected key intermediates 4,5,6-tri-O-benzoyl-myo-inositol and (+/-)-3,4,5,6-tetra-O-benzyl-myo-inositol were phosphorylated with dibenzyl N,N-di-isopropylphosphoramidite in the presence of 1H-tetrazole and subsequent oxidation of the phosphite. The crystal structures of the synthetic intermediates (+/-)-1-O-(tert-butyldiphenylsilyl)-2,3,O-cyclohexylidene-myo-inos itol and (+/-)-4,5,6-tri-O-benzoyl-1-O-(tert-butyldiphenylsilyl)-2,3-O-cycl ohexylidene- myo-inositol are reported. myo-Inositol 1,2,3-trisphosphate (+/-)-myo-inositol 1,2-bisphosphate, and all isomeric myo-inositol tetrakisphosphates were evaluated for their ability to alter HO. production in the iron-catalysed Haber-Weiss reaction. The results demonstrated that a 1,2,3-grouping of phosphates in myo-inositol was necessary for inhibition also that (+/-)-myo-inositol 1,2-bisphosphate potentiated HO. production. myo-Inositol 1,2,3-trisphosphate resembled myo-inositol hexakisphosphate (phytic acid) in its ability to act as a siderophore by promoting iron-uptake into Pseudomonas aeruginosa.

Fluorescent probe:complexation of Fe3+ with the myo-inositol 1,2,3-trisphosphate motif

Natural myo-inositol phosphate antioxidants containing the 1,2,3-trisphosphate motif bind Fe3+ in the unstable penta-axial conformation.

Conformational analysis of the natural iron chelator myo-inositol 1,2,3-trisphosphate using a pyrene-based fluorescent mimic

myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P3], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P3 is considered to be important to complex Fe3+ in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P4], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe3+, evident from excimer fluorescence induced by π-π stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca 2+ cations exceeding a 1:1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe3+ have been determined. The complexes formed between Fe 3+ and 4,6-bispyrenoyl Ins(1,2,3,5)P4 display similar stability to those formed with Ins(1,2,3)P3, indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P4, which closely resemble those obtained for Ins(1,2,3)P3. The data presented confirms that Fe3+ binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif. © 2010 The Royal Society of Chemistry.

Inositol polyphosphate-mediated iron transport in Pseudomonas aeruginosa

It has previously been shown that myo-inositol hexakisphosphate (myo- InsP6) mediates iron transport into Pseudomonas aeruginosa and overcomes iron-dependent growth inhibition. In this study, the iron transport properties of myo-inositol trisphosphate and tetrakisphosphate regio-isomers were studied. Pseudomonas aeruginosa accumulated iron (III) at similar rates whether complexed with myo-Ins(1,2,3)P3 or myo-InsP6. Iron accumulation from other compounds, notably D/L myo-Ins(1,2,4,5)P4 and another inositol trisphosphate regio-isomer, D-myo-Ins(1,4,5)P3, was dramatically increased. Iron transport profiles from myo-InsP6 into mutants lacking the outer membrane porins oprF, oprD and oprP were similar to the wild-type, indicating that these porins are not involved in the transport process. The rates of reduction of iron (III) to iron (II) complexed to any of the compounds by a Ps. aeruginosa cell lysate were similar, suggesting that a reductive mechanism is not the rate-determining step.

Activation of calcium-sensitive potassium channels in L6 skeletal myocytes by arginine vasopressin requires extracellular calcium

The effects of extracellular application of arginine vasopressin (AVP) upon membrane currents in L6 skeletal myocytes was investigated using the whole-cell configuration of the patch-clamp technique. At O mV AVP produced large amplitude, transient outward currents that reversed when the clamping potential was changed to -100 mV (negative to EK) The effects of alterations in the extracellular K+ concentration upon the current reversal potential suggested that the current elicited by AVP was carried mainly by K+ ions. Intracellular dialysis with 10 μM inositol 1,4,5-trisphosphate (InsP3) elicited similar currents but only in 6/14 cells. Inclusion of 5 mg ml-1 heparin in the intracellular solutions was ineffective at inhibiting the current responses to AVP. The AVP-induced current was totally abolished when the intracellular EGTA concentration was increased from 0.05 mM to 10 mM or Ca2+ was removed from the extracellular perfusing solution. These results suggest that AVP produces activation of a Ca2+-sensitive K+ conductance in L6 skeletal myocytes by a process dependent upon extracellular Ca2+ and not intracellular Ca2+ release. © 1995 Academic Press. All rights reserved.

Identification of a novel inositol phosphate recognition site: specific [3H]inositol hexakisphosphate binding to brain regions and cerebellar membranes

[3H]Inositol hexakisphosphate (InsP6) binds with a heterogeneous distribution to frozen sections of unfixed rat brain and is displaced by unlabelled InsP6. The pattern of binding correlates with binding to neuronal cell bodies. [3H]InsP6 binding to cerebellar membranes has been further characterised, is reversible, and saturable, and exhibits high specificity for inositol polyphosphates. The IC50 for competition by unlabelled InsP6 is approximately 100nM, whereas inositol 1,3,4,5,6 pentakisphosphate (Ins(13456)P5), inositol 1,3,4,5 tetrakisphosphate (Ins(1345)P4), and inositol 1,4,5 trisphosphate (Ins(145)P3) bind with an affinity at least one order of magnitude lower. [3H]InsP6 binding is clearly distinct from previously characterised Ins(145)P3 (ref. 1, 2) and Ins(1345)P4 (ref. 3) binding, both in terms of pharmacology and brain distribution.

Interventions against obesity through increased lipolysis in adipose tissue

Obesity is a disease of excess adiposity affecting> 17% of men and >20% of women in Britain. Clinically, it is defined by a Body Mass Index (BMI, kg/m2) of 2:30. Obesity is a confounding factor that promotes insulin resistance, hyperinsulinaemia and type 2 diabetes. Type 2 diabetes accounts for >90% of all cases of diabetes, with a prevalence of 2-6% of adults in most western societies, a majority of which are overweight or obese. Weight loss in obese patients reduces the risk of developing diabetes by >50%. This thesis has investigated the first part of a two-stage therapeutic intervention against obesity in which adipose tissue lipolysis will be combined with increased energy expenditure: the approach is also designed to consider agents that will benefit glycaemic control in coexistent obesity and diabetes by improving insulin sensitivity. Rodent and human in vitro models of adipocyte biology and skeletal muscle have been developed, characterised and evaluated. They include isolated epididymal and parametrial adipocytes of lean and obese diabetic ob/ob mice, cultured 3T3-Ll preadipocytes, isolated human omental and subcutaneous adipocytes and rat L6 cultured muscle cells. Compounds investigated for anti-obesity and anti-diabetic properties include M2 (sibutramine metabolite), 3-guanidinopropionic acid and mazindol. In vivo studies were undertaken to investigate these compounds further in lean and ob/ob mice. In vivo studies indicated that M2 and 3-guanidinopropionic acid reduced body weight gain in ob/ob mice. The three compounds increased lipolysis in adipocytes isolated from lean and ob/ob mice and human adipose depots. The direct action of these compounds was mediated via a pathway involving the f3 adrenoceptors and components of the lipolytic signalling pathway, including protein kinase A and p38 MAP kinase. In addition, M2 and mazindol were capable of increasing glucose uptake into insulin sensitive tissues. M2 and mazindol can act directly on adipose tissue and skeletal muscle to increase glucose uptake via a pathway involving new protein synthesis and activation of the glucose transporters. The M2-stimulated pathway is activated by the conversion of phosphatidylinositol bisphosphate to phosphatidylinositol trisphosphate by phosphatidylinositol 3-kinase. Thus, M2, mazindol and 3-GPA showed pharmacodynamic properties which suggested they might be potential therapeutic treatments for obesity and diabetes.

Synthesis, crystallography and biological activity of myo-inositol phosphates

The antioxidant property of myo-inositol hexakisphosphate is important in the prevention of hydroxyl radical formation which may allow it to act as a 'safe' carrier of iron within the cell. Here, the hypothesis that the recently discovered natural product, myo-inositol 1,2,3-trisphosphate represents the simplest structure to mimic phytate's antioxidant activity has been tested. The first synthesis of myo-inositol 1,2,3-trisphosphate has been completed, along with its X-ray structure determination and that of key synthetic intermediates. Iron binding studies of myo-inositol 1,2,3-trisphosphate demonstrated that phosphate groups with the equatorial-axial-equatorial conformation are required for complete inhibition of hydroxyl radical formation. myo-Inositol monophosphatase is a key enzyme in recycling myo-inositol from its monophosphates in the brain and its inhibition is implicated in lithium's antimanic properties. Current synthetic strategies require inositol compounds to be protected (often with more than one group), resolved, phosphorylated and deprotected to produce the desired optically active myo-inositol phosphates. Here, the synthesis of myo-inositol 3-phosphate has been achieved in only 4 steps from myo-inositol. The stereoselective addition of the chiral phosphorylating agent (2R,4S,5R)-2-chloro-3,4-dimethyl-5-phenyl-1,3,2-oxazaphospholidin-2-one to a protected inositol intermediate allowed separation of diastereoisomers and easy deprotection to myo-inositol 3-phosphate. This strategy also allows the possible introduction of labels of oxygen and sulphur to give a thiophosphate of known stereochemistry at phosphorus which would be useful for the analysis of the stereochemical course of phosphate hydrolysis catalysed by inositol monophosphatase.

Mouse splenocyte proliferation and its modulation by sex steroids

Concanavalin A, a T cell mitogen enhanced DNA synthesis in murine splenocytes. Amongst the early signals prior to this event was an increase in cytosolic calcium derived from both intra- and extracellular sources. The requirements for extracellular calcium persisted for four hours after the lectin administration which itself was needed for six hours. Putative calcium channel antagonists and calmodulin inhibitors blocked ihe increase in DNA synthesis. The calcium signal was mimicked by application of the ionophore, A23187, although no increase in DNA synthesis occurred. An activator of protein kinase C, 12-0- tetradecanoylphorbol 13-acetate, had little effect in isolation but the combined application of these two agents greatly enhanced DNA synthesis. The natural mediators of these events are presumed to be inositol trisphosphate and diacylglycerol derived from phosphatidylinositol bisphosphate hydrolysis. Lectin application and protein kinase C activation both increased intracellular pH possibly as a result of Na'l'/H"'' exchange since amiloride an inhibitor of this antiporter inhibited lectin induced DNA synthesis. The calcium and hydrogen ionic changes occur within minutes of lectin application; the protracted requirement for this mitogen suggests further signalling mechanisms occur to elicit maximum DNA synthesis in these cells. Gonadectomy caused an increase in thymic and splenic weight. Spleno-cytes derived from castrated mice showed no change in mitogen response whereas those from ovariectomised mice demonstrated a reduced lectin sensitivity. Testosterone, 5 a dihydrotestosterone, a and 0 oestradiol all inhibited lectin induced DNA synthesis but only at pharmacological concentrations. Testosterone glucuronide and cholesterol were without effect Studies with mouse serum fractions of differing steroidal status were unable to confirm the presence or absence of serum factors which might mediate the effects of steroid on lymphoid cells, all fractions tested inhibited lymphocyte transformation. Both interleukin-2 and lipopolysaccharide induced splenocyte mitogene-sis was also impaired by high steroid concentrations in vitro, suggesting that steroids mediate their effect by a non-specific, non-receptor-mediated event.

An Ins(1,4,5)P3 receptor in Paramecium is associated with the osmoregulatory system

In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca2+ signaling, although the molecular identity of the channels responsible for the Ca2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5)P3-binding domain binds [3H]Ins(1,4,5)P3, whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5)P3-receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca2+ (Paramecium cells rapidly adjust their Ca2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5)P3. Considering the ongoing expulsion of substantial amounts of Ca2+ by the osmoregulatory system, we propose here that Ins(1,4,5)P3 receptors serve a new function, i.e. a latent, graded reflux of Ca2+ to fine-tune [Ca2+] homeostasis.