Ca2(+)-mobilising properties of synthetic fluoro-analogues of myo-inositol 1,4,5-trisphosphate and their interaction with myo-inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase

The ability of two 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. DL-2-deoxy-2-fluoro-scyllo-Ins(1,4,5)P3 (2F-Ins(1,4,5)P3) and DL-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 (2,2-F2-Ins(1,4,5)P3) were full agonists (EC50s 0.77 and 0.41 microM respectively) and slightly less potent than D-Ins(1,4,5)P3 (EC50 0.13 microM), indicating that the axial 2-hydroxyl group of Ins(1,4,5)P3 is relatively unimportant in receptor binding and stimulation of Ca2+ release. Both analogues mobilized Ca2+ with broadly similar kinetics and were substrates for Ins(1,4,5)P3 3-kinase but, qualitatively, were slightly poorer than Ins(1,4,5)P3. 2F-Ins(1,4,5)P3 was a weak substrate for Ins(1,4,5)P3 5-phosphatase but 2,2-F2-Ins(1,4,5)P3 was apparently not hydrolysed by this enzyme, although it inhibited its activity potently (Ki = 26 microM).     

Interaction of synthetic D-6-deoxy-myo-inositol 1,4,5-trisphosphate with the Ca2(+)-releasing D-myo-inositol 1,4,5-trisphosphate receptor, and the metabolic enzymes 5-phosphatase and 3-kinase.

The ability of D-6-deoxy-myo-inositol 1,4,5-trisphosphate [6-deoxy-Ins(1,4,5)P3], a synthetic analogue of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to mobilise intracellular Ca2+ stores in permeabilised SH-SY5Y neuroblastoma cells was investigated. 6-Deoxy-Ins(1,4,5)P3 was a full agonist (EC50 = 6.4 microM), but was some 70-fold less potent than Ins (1,4,5)P3 (EC50 = 0.09 microM), indicating that the 6-hydroxyl group of Ins(1,4,5)P3 is important for receptor binding and stimulation of Ca2+ release, but is not an essential structural feature. 6-Deoxy-Ins(1,4,5)P3 was not a substrate for Ins (1,4,5)P3 5-phosphatase, but inhibited both the hydrolysis of 5-[32P]+ Ins (1,4,5)P3 (Ki 76 microM) and the phosphorylation of [3H]Ins(1,4,5)P3 (apparent Ki 5.7 microM). 6-Deoxy-Ins (1,4,5)P3 mobilized Ca2+ with different kinetics to Ins(1,4,5)P3, indicating that it is probably a substrate for Ins (1,4,5)P3 3-kinase.     

Synthesis and application of photoaffinity analogues of inositol 1,4,5-trisphosphate selectively substituted at the 1-phosphate group.

We have synthesized two photolabile arylazido-analogues of Ins(1,4,5)P3 selectively substituted at the 1-phosphate group for determination of Ins(1,4,5)P3-binding proteins. These two photoaffinity derivatives, namely N-(4-azidobenzoyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AbaIP3) and N-(4-azidosalicyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AsaIP3), bind to high affinity Ins(1,4,5)P3-specific binding sites at a 9-fold lower affinity (Kd = 66 and 70 nM) than Ins(1,4,5)P3 (Kd = 7.15 nM) in a fraction from rat pancreatic acinar cells enriched in endoplasmic reticulum (ER). Other inositol phosphates tested showed comparable (DL-myo-inositol 1,4,5-trisphosphothioate, Kd = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P4, Kd = 4 microM; Ins(1,4)P2, Kd = 80 microM]. Binding of AbaIP3 was also tested on a microsomal preparation of rat cerebellum [Kd = 300 nM as compared with Ins(1,4,5)P3, Kd = 45 nM]. Ca2+ release activity of the inositol derivatives was tested with AbaIP3. It induced a rapid and concentration-dependent Ca2+ release from the ER fraction [EC50 (dose producing half-maximal effect) = 3.1 microM] being only 10-fold less potent than Ins(1,4,5)P3 (EC50 = 0.3 microM). From the two radioactive labelled analogues ([3H]AbaIP3 and 125I-AsIP3) synthesized, the radioiodinated derivative was used for photoaffinity labelling. It specifically labelled three proteins with apparent molecular masses of 49, 37 and 31 kDa in the ER-enriched fraction. By subfractionation of this ER-enriched fraction on a Percoll gradient the 37 kDa Ins(1,4,5)P3 binding protein was obtained in a membrane fraction which showed the highest effect in Ins(1,4,5)P3-inducible Ca2+ release (fraction P1). The other two Ins(1,4,5)P3-binding proteins, of 49 and 31 kDa, were obtained in fraction P2, in which Ins(1,4,5)P3-induced Ca2+ release was half of that obtained in fraction P1. We conclude from these data that the 37 kDa and/or the 49 and 31 kDa proteins are involved in Ins(1,4,5)P3-induced Ca2+ release from the ER of rat pancreatic acinar cells.     

Dimers of D-myo-inositol 1,4,5-trisphosphate: design, synthesis, and interaction with Ins(1,4,5)P3 receptors

The design and synthesis of dimeric versions of the intracellular signaling molecule d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] are reported. Ins(1,4,5)P(3) dimers in a range of sizes were constructed by conjugation of a partially protected 2-O-(2-aminoethyl)-Ins(1,4,5)P(3) intermediate with activated oligo- and poly(ethylene glycol) (PEG) tethers, to give benzyl-protected dimers with amide or carbamate linkages. After deprotection, the resulting water-soluble Ins(1,4,5)P(3) dimers were purified by ion-exchange chromatography. The interaction of the Ins(1,4,5)P(3) dimers with tetrameric Ins(1,4,5)P(3) receptors was explored, using equilibrium [(3)H]Ins(1,4,5)P(3)-binding to membranes from cerebellum, and (45)Ca(2+)-release from permeabilized hepatocytes. The results showed that dimers, even when they incorporate large PEG tethers, interact potently with Ins(1,4,5)P(3) receptors, and that the shorter dimers are more potent than Ins(1,4,5)P(3) itself. A very small dimer, consisting of two Ins(1,4,5)P(3) motifs joined by a short N,N'-diethylurea spacer, was synthesized. Preliminary studies of (45)Ca(2+) release from the intracellular stores of permeabilized hepatocytes showed this shortest dimer to be almost as potent as adenophostin A, the most potent Ins(1,4,5)P(3) receptor ligand known. Possible interpretations of this result are considered in relation to the recently disclosed X-ray crystal structure of the type 1 Ins(1,4,5)P(3) receptor core binding domain.     

Differential stereoselectivity of D- and L-myo-inositol 1,2,4, 5-tetrakisphosphate binding to the inositol 1,4,5-trisphosphate receptor and 3-kinase

D- and L-myo-inositol 1,2,4,5-tetrakisphosphate (Ins(1,2,4,5)P(4)) were investigated for their ability to bind to the D-myo-inositol 1, 4,5-trisphosphate (Ins(1,4,5)P(3)) receptor in a bovine adrenal cortical membrane fraction, to mobilize intracellular Ca(2+) stores in Xenopus oocytes, and to bind to the rat brain Ins(1,4,5)P(3) 3-kinase overexpressed and purified in E. coli. In competitive binding experiments with the Ins(1,4,5)P(3) receptor, D-Ins(1,2,4, 5)P(4) effectively displaced [(3)H]Ins(1,4,5)P(3) in a concentration-dependent manner with a potency comparable to that of D-Ins(1,4,5)P(3), while L-Ins(1,2,4,5)P(4) was approximately 50-fold less effective than D-Ins(1,4,5)P(3) and D-Ins(1,2,4,5)P(4). The DL-Ins(1,2,4,5)P(4) racemate bound to the Ins(1,4,5)P(3) receptor with an apparent intermediate efficiency. Injection of D-Ins(1,2,4, 5)P(4) into oocytes evoked a chloride current dependent on intracellular Ca(2+) mobilization in which the agonists ranked in a similar order of potency as in the Ins(1,4,5)P(3) receptor binding. On the other hand, D-Ins(1,2,4,5)P(4) only inhibited the binding of [(3)H]Ins(1,4,5)P(3) to 3-kinase very weakly with a markedly reduced potency compared to D-Ins(1,4,5)P(3), indicating that D-Ins(1,2,4, 5)P(4) is not an effective competitor in the phosphorylation of [(3)H]-Ins(1,4,5)P(3) by 3-kinase. The results, therefore, clearly indicate that D-Ins(1,2,4,5)P(4) is as effective as D-Ins(1,4,5)P(3) in the binding to the receptor but not 3-kinase, and access of Ins(1, 2,4,5)P(4) over the Ins(1,4,5)P(3) receptor calls for stringent stereospecificity with D-Ins(1,2,4,5)P(4) being the active form in DL-Ins(1,2,4,5)P(4)-mediated Ca(2+) mobilization.     

Partial purification and some properties of rat brain inositol 1,4,5-trisphosphate 3-kinase.

An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 x 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5'-[gamma-thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.     

Detection of inositol 1,4,5-trisphosphate kinase in retina. A direct demonstration of phosphorylation of inositol 1,4,5-trisphosphate by ATP.

The metabolism of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] consists of two pathways: dephosphorylation by 5-phosphomonoesterase(s) produces inositol 1,4-bisphosphate, and phosphorylation by Ins(1,4,5)P3 3-kinase yields inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. The requirements for Ins(1,4,5)P3 kinase activity in retina were characterized. Apparent Km values for ATP and Ins(1,4,5)P3 are 1.4 mM and 1.3 microM respectively. A direct demonstration of phosphorylation of Ins(1,4,5)P3 by [gamma-32P]ATP was achieved. Characterization of the 32P-labelled product revealed that it had the expected chromatographic and electrophoretic properties of Ins(1,3,4,5)P4.     

Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase in rat and bovine brain tissues

Inositol 1,4,5-trisphosphate (Ins P3) 3-kinase catalyzes the ATP-dependent phosphorylation of Ins P3 to Inositol 1,3,4,5-tetrakisphosphate (Ins P4). Ca2+/calmodulin (CaM)-sensitivity of Ins P3 3-kinase was measured in the crude soluble fraction from rat brain and different anatomic regions of bovine brain. Kinase activity was inhibited in the presence of EGTA (free Ca2+ below 1 nM) as compared to Ca2+ (10 microM free Ca2+) or Ca2+ (10 microM free Ca2+) and CaM (1 microM). Ca2+-sensitivity was also seen for the cAMP phosphodiesterase measured under the same assay conditions, but was not for the Ins P3 5-phosphatase. DEAE-cellulose chromatography of the soluble fraction of rat brain or bovine cerebellum resolved a Ca2+/CaM-sensitive Ins P3 3-kinase (maximal stimulation at 1 microM Ins P3 substrate level was 2.0-3.0 fold).     

Inositol 1,4,5-trisphosphate and inositol 1,2-cyclic 4,5-trisphosphate are minor components of total mass of inositol trisphosphate in thrombin-stimulated platelets. Rapid formation of inositol 1,3,4-trisphosphate

Stimulation of human platelets by thrombin leads to rises of both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) within 10 s. The mass of Ins(1,4,5)P3 was measured in platelet extracts after conversion to [3-32P]Ins(1,3,4,5)P4 with Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Basal levels were equivalent to 0.2 microM and rose to 1 microM within 10 s of stimulation by thrombin. The mass of Ins(1,3,4)P3 was more than 10-fold greater than that of Ins(1,4,5)P3 between 10 and 60 s of thrombin stimulation. These results indicate that the majority of InsP3 liberated by phospholipase C in stimulated platelets must be the non-cyclic Ins(1,4,5)P3 in order to allow rapid phosphorylation by Ins(1,4,5)P3 3-kinase to Ins(1,3,4,5)P4 and then dephosphorylation to Ins(1,3,4)P3 by 5-phosphomonoesterase. A significant proportion of the InsP3 extracted from thrombin-stimulated platelets under neutral conditions is resistant to Ins(1,4,5)P3 3-kinase but susceptible after acid treatment, implying the presence of inositol 1,2-cyclic 4,5-trisphosphate (Ins(1,2cyc4,5)P3. The relative proportion of Ins(1,2cyc4,5)P3 increases with time. We suggest that such gradual accumulation is attributable to the relative insensitivity of this compound to hydrolytic and phosphorylating enzymes. Therefore, early Ca2+ mobilization in platelets is more likely to be effected by Ins(1,4,5)P3 than by Ins(1,2cyc4,5)P3.     

Characterization of inositol 1,4,5-trisphosphate-stimulated calcium release from rat cerebellar microsomal fractions. Comparison with [3H]inositol 1,4,5-trisphosphate binding.

The abilities of D-myo-inositol phosphates (InsPs) to promote Ca2+ release and to compete for D-myo-[3H]-inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) binding were examined with microsomal preparations from rat cerebellum. Of the seven InsPs examined, only Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 stimulated the release of Ca2+. Ca2+ release was maximal in 4-6 s and was followed by a rapid re-accumulation of Ca2+ into the Ins(1,4,5)P3-sensitive compartment after Ins(1,4,5)P3, but not after Ins(2,4,5)P3 or Ins(4,5)P2. Ca2+ re-accumulation after Ins(1,4,5)P3 was also faster than after pulse additions of Ca2+, and coincided with the metabolism of [3H]Ins(1,4,5)P3. These data suggest that Ins(1,4,5)P3-induced Ca2+ release and the accompanying decrease in intraluminal Ca2+ stimulate the Ca2+ pump associated with the Ins(1,4,5)P3-sensitive compartment. That this effect was observed only after Ins(1,4,5)P3 may reflect differences in either the metabolic rates of the various InsPs or an effect of the Ins(1,4,5)P3 metabolite Ins(1,3,4,5)P4 to stimulate refilling of the Ins(1,4,5)P3-sensitive store. InsP-induced Ca2+ release was concentration-dependent, with EC50 values (concn. giving half-maximal release) of 60, 800 and 6500 nM for Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 respectively. Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 also competed for [3H]Ins(1,4,5)P3 binding, with respective IC50 values (concn. giving 50% inhibition) of 100, 850 and 13,000 nM. Comparison of the EC50 and IC50 values yielded a significant correlation (r = 0.991). These data provide evidence of an association between the [3H]Ins(1,4,5)P3-binding site and the receptor mediating Ins(1,4,5)P3-induced Ca2+ release.     

Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) regulates intracellular Ca2+ by mobilizing Ca2+ from a non-mitochondrial store. We have investigated the effects of Ca2+ on the binding of [32P]Ins (1,4,5)P3 to permeabilized rat hepatocytes and a liver plasma membrane-enriched fraction. Increasing the free Ca2+ concentration in the medium from 0.1 nM to 0.7 microM increased the capacity of a high affinity binding component (KD = 2-3 nM) in permeabilized cells by a factor of 10. If the membrane fraction was preincubated at 37 degrees C before binding was measured at 4 degrees C, all of the Ins(1,4,5)P3 receptors were transformed to a low affinity state (KD = 65 +/- 12 nM, Bmax = 3.1 +/- 0.1 fmol/mg, n = 4). When 0.7 microM of Ca2+ was added, the receptors were totally transformed to a high affinity state (KD = 2.8 +/- 0.4 nM, Bmax = 2.7 +/- 0.4 fmol/mg, n = 4). The EC50 of the Ca2(+)-induced interconversion of the Ins(1,4,5)P3 receptor was 140 nM. This Ca2(+)-induced transformation of the Ins(1,4,5)P3 receptor from a low affinity to a high affinity state was associated with an inhibition of the Ins(1,4,5)P3-induced Ca2+ release in permeabilized hepatocytes. These data suggest that the Ins(1,4,5)P3-dependent hormones, by increasing the intracellular Ca2+ concentration, induce a reversible transformation of the receptor from its low affinity state, coupled to the Ca2+ release, to a desensitized high affinity state. Transformation of the receptor may play a role in the oscillatory release of Ca2+ observed in single isolated hepatocytes.     

2-O-(2-Aminoethyl)-myo-inositol 1,4,5-trisphosphate as a novel ligand for conjugation: physicochemical properties and synthesis of a new Ins(1,4,5)P(3) affinity matrix

2-O-(2-Aminoethyl)-Ins(1,4,5)P(3), (5), a novel derivative of the Ca(2+)-mobilising second messenger d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], was synthesised from myo-inositol. 5 was found to be a potent mobiliser of intracellular Ca(2+), and an Ins(1,4,5)P(3) affinity matrix synthesised from 5 was effective at selectively binding N-terminal fragments of the Ins(1,4,5)P(3) receptor containing the intact Ins(1,4,5)P(3) binding site. The microprotonation scheme for 5 was resolved and the related constants were determined in comparison with Ins(1,4,5)P(3) and another reactive Ins(1,4,5)P(3) analogue 1-O-(2-aminoethyl-1-phospho)-Ins(4,5)P(2), (2a), by potentiometric and NMR titration methods. The (31)P and (1)H NMR titration curves for compound 5 and Ins(1,4,5)P(3) are remarkably close, indicating analogous acid-base properties and intramolecular interactions for the two compounds. The 1-phosphate-modified Ins(1,4,5)P(3) derivative 2a, on the contrary, behaves as a bisphosphorylated rather than a trisphosphorylated inositol. Thus, 5 is a new reactive Ins(1,4,5)P(3) analogue of considerable potential for investigation of the chemical biology of Ins(1,4,5)P(3)-mediated cellular signalling.     

The size of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores depends on inositol 1,4,5-trisphosphate concentration.

An explanation of the complex effects of hormones on intracellular Ca2+ requires that the intracellular actions of Ins(1,4,5)P3 and the relationships between intracellular Ca2+ stores are fully understood. We have examined the kinetics of 45Ca2+ efflux from pre-loaded intracellular stores after stimulation with Ins(1,4,5)P3 or the stable phosphorothioate analogue, Ins(1,4,5)P3[S]3, by simultaneous addition of one of them with glucose/hexokinase to rapidly deplete the medium of ATP. Under these conditions, a maximal concentration of either Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 evoked rapid efflux of about half of the accumulated 45Ca2+, and thereafter the efflux was the same as occurred under control conditions. Submaximal concentrations of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 caused a smaller rapid initial efflux of 45Ca2+, after which the efflux was similar whatever the concentration of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 present. The failure of submaximal concentrations of Ins(1,4,5)P3 and Ins(1,4,5)P3[S]3 to mobilize fully the Ins(1,4,5)P3-sensitive Ca2+ stores despite prolonged incubation was not due either to inactivation of Ins(1,4,5)P3 or to desensitization of the Ins(1,4,5)P3 receptor. The results suggest that the size of the Ins(1,4,5)P3 sensitive Ca2+ stores depends upon the concentration of Ins(1,4,5)P3.     

Turkey erythrocytes possess a membrane-associated inositol 1,4,5-trisphosphate 3-kinase that is activated by Ca2+ in the presence of calmodulin.

Turkey erythrocytes contain soluble and particulate kinase activities which catalyse the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The particle-bound activity accounts for approximately one-quarter of the total cellular Ins(1,4,5)P3 kinase, when assayed at a [Ca2+] of 10 nM. The particle-bound Ins(1,4,5)P3 kinase is not washed from the membrane by 0.6 M-KCl, yet may be solubilized by a variety of detergents. This suggests that it is an intrinsic membrane protein. The product of the membrane-bound Ins(1,4,5)P3 kinase is inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], identifying the enzyme as an Ins(1,4,5)P3 3-kinase. In the presence of calmodulin, the membrane-associated Ins(1,4,5)P3 3-kinase is activated as [Ca2+] is increased over the range 0.2-1.0 microM. Under these conditions, the rates of dephosphorylation of Ins(1,3,4,5)P4 and Ins(1,4,5)P3 by phosphatases in the membrane fraction are unchanged.     

Comparative studies of D-myoinositol 1,4,5-trisphosphate and synthetic 6-deoxy D-myoinositol 1,4,5-trisphosphate: binding and calcium release activity in rat parotid microsomes.

In this study, we report our data on the binding of D-myoinositol(1,4,5)P3 and of 6-deoxy D-myoinositol(1,4,5)P3 to a rat parotid microsomal fraction and their effect on Ca2+ release. The binding affinity and the potency of 6-deoxy Ins(1,4,5)P3 to induce Ca2+ release are about 100 times lower than those of Ins(1,4,5)P3. However, maximal concentrations of both inositol trisphosphates induce similar calcium efflux and present comparable displacement of radioligand binding. Experiments were performed to exclude that the microsomal preparations used display rapid metabolism of Ins(1,4,5)P3 or 6-deoxy Ins(1,4,5)P3 during binding and Ca2+ release. We also report that, in permeabilized rat parotid acini preparations, 6-deoxy Ins(1,4,5)P3 is about 100 times less potent than Ins(1,4,5)P3 in inducing Ca2+ release. These data indicate that removal of the hydroxyl group in position 6 of the Ins(1,4,5)P3 molecule severely reduces its binding affinity which seems, in a large part at least, responsible for the reported loss of potency in mobilizing Ca2+. Nevertheless, 6-deoxy Ins(1,4,5)P3 seems to be a full agonist for the release of Ca2+.     

Metabolic and functional consequences of introducing inositol 1,4,5-trisphosphate into saponin-permeabilized human platelets.

In an earlier study we reported the effect of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in releasing Ca2+ from highly purified human platelet intracellular membrane vesicles. [Authi & Crawford (1985) Biochem. J. 230, 247-253]. We have now investigated the metabolic and functional consequences of introducing Ins(1,4,5)P3 into saponin-permeabilized platelets. Washed human platelets when resuspended in a suitable medium were permeabilized with saponin (10-14 micrograms/ml) to allow entry of low-Mr water-soluble molecules without significant release of the cytoplasmic marker enzyme protein lactate dehydrogenase. Saponin-permeabilized platelets show identical platelet responses (shape change, aggregation and release of 5-hydroxy[14C]tryptamine) to both collagen (5 micrograms/ml) and thrombin (0.1 unit/ml) as obtained with intact cells, indicating that there is minimal disturbance to the surface membrane receptor topography for these two agonists. Ins(1,4,5)P3 (1-10 microM) added to saponin-treated platelets (but not to intact platelets) induced dose-related shape change, aggregation and release of 5-hydroxy[14C]tryptamine which at maximal doses was comparable with responses obtained with thrombin or collagen. The cyclo-oxygenase inhibitors indomethacin and aspirin, if added prior to saponization and Ins(1,4,5)P3 addition, completely inhibited both aggregation and release of 5-hydroxy[14C]tryptamine (EC50 for indomethacin, 50 nM; for aspirin, 30 microM). We believe that Ins(1,4,5)P3 induces the release of Ca2+ from intracellular storages sites which stimulates the Ca2+-dependent phospholipase A2 releasing arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to the aggregatory prostanoids (prostaglandin H2 and thromboxane A2) resulting in the observed responses. This concept is supported by the use of the thromboxane receptor antagonists EPO 45 and EPO 92, both of which also completely inhibit Ins(1,4,5)P3-induced responses in saponin-permeabilized platelets. Electron microscopy of the platelet preparations revealed that thrombin- and collagen-induced platelet aggregates of intact and saponized cells were identical, showing extensive pseudopod formation and dense granule release. The Ins(1,4,5)P3-induced aggregates also showed similar dense granule release but an almost total absence of pseudopod formation. These results are discussed in the light of the second messenger role of Ins(1,4,5)P3 in stimulus-response coupling in platelets.     

Stimulation of hepatic inositol 1,4,5-trisphosphate kinase activity by Ca2+-dependent and -independent mechanisms.

A cytosolic fraction derived from rat hepatocytes was used to investigate the regulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] kinase, the enzyme which converts Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. The activity was doubled by raising the free Ca2+ concentration of the assay medium from 0.1 microM to 1.0 microM. A 5 min preincubation of the hepatocytes with 100 microM-dibutyryl cyclic AMP (db.cAMP) plus 100 nM-tetradecanoylphorbol acetate (TPA) resulted in a 40% increase in Ins(1,4,5)P3 kinase activity when subsequently assayed at 0.1 microM-Ca2+. This effect was smaller at [Ca2+] greater than 0.5 microM, and absent at 1.0 microM-Ca2+. Similar results were obtained after preincubation with 100 microM-db.cAMP plus 300 nM-vasopressin (20% increase at 0.1 microM-Ca2+; no effect at 1.0 microM-Ca2+). Preincubation with vasopressin, db.cAMP or TPA alone did not alter Ins(1,4,5)P3 kinase activity. It is proposed that these results, together with recent evidence implicating Ins(1,3,4,5)P4 in the control of Ca2+ influx, could be relevant to earlier findings that hepatic Ca2+ uptake is synergistically stimulated by cyclic AMP analogues and vasopressin.     

Depolarization and Agonist-Stimulated Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex

Muscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values for carbachol. Omission of buffer Ca2+ ([Ca2+]e 10-20 microM) reduced basal Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K(+)-evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L-type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3-kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.     

Kinetics of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate generation in dog-thyroid primary cultured cells stimulated by carbachol

The action of carbachol on the generation of inositol trisphosphate and tetrakisphosphate isomers was investigated in dog-thyroid primary cultured cells radiolabelled with [3H]inositol. The separation of the inositol phosphate isomers was performed by reverse-phase high pressure liquid chromatography. The structure of inositol phosphates co-eluting with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] standards was determined by enzymatic degradation using a purified Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. The data indicate that Ins(1,3,4,5)P4 was the only [3H]inositol phosphate which co-eluted with a [32P]Ins(1,3,4,5)P4 standard, whereas 80% of the [3H]InsP3 co-eluting with an Ins(1,4,5)P3 standard was actually this isomer. In the presence of Li+, carbachol led to rapid increases in [3H]Ins(1,4,5)P4. The level of Ins(1,4,5)P3 reached a peak at 200% of the control after 5-10 s of stimulation and fell to a plateau that remained slightly elevated for 2 min. The level of Ins(1,3,4,5)P4 reached its maximum at 20s. The level of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] increased continuously for 2 min after the addition of carbachol. Inositol-phosphate generation was also investigated under different pharmacological conditions. Li+ largely increased the level of Ins(1,3,4)P3 but had no effect on Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin, which stimulates dog-thyroid adenylate cyclase and cyclic-AMP accumulation, had no effect on the generation of inositol phosphates. The absence of extracellular Ca2+ largely decreased the level of Ins(1,3,4,5)P4 as expected considering the Ca2(+)-calmodulin sensitivity of the Ins(1,4,5)P3 3-kinase. Staurosporine, an inhibitor of protein kinase C, increased the levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ins(1,3,4)P3. This supports a negative feedback control of diacyglycerol on Ins(1,4,5)P3 generation.     

Partial purification and reconstitution of inositol 1,4,5-trisphosphate receptor/Ca2+ channel of bovine liver microsomes.

The binding of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] to bovine liver microsomes was characterized. The Ins(1,4,5)P3 receptor of the microsomes was solubilized by 1% Triton X-100 and purified by sucrose density gradient, Heparin-Sepharose, DEAE-Toyopearl, ATP-Agarose, and Ins(1,4,5)P3-Sepharose column chromatographies. More than 1,000-fold enrichment of the Ins(1,4,5)P3-binding activity was achieved. Kd values of the binding activity were 2.8 nM in microsomes and 3.0 nM in the partially purified receptor, respectively, and the binding activity was optimal in the medium containing 100 mM KCl and at pH between 7.5 and 8.5. The presence of Ca2+ failed to inhibit the binding. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PtdIns), and phosphatidylinositol-4-monophosphate [PtdIns(4)P] showed no effect on the Ins(1,4,5)P3 binding. However, soybean phospholipids asolectin and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] strongly inhibited the binding activity. PtdIns(4,5)P2 inhibited the activity competitively with a half-maximal inhibitory concentration of 30 micrograms/ml. The partially purified Ins(1,4,5)P3 receptor was reconstituted into proteoliposomes. Fluorescence measurements using Quin 2 indicated that Ins(1,4,5)P3 stimulated Ca2+ influx into the proteoliposomes. The EC50 of Ins(1,4,5)P3 on Ca2+ influx was 50 nM. This result strongly suggest that Ins(1,4,5)P3 binding protein of liver microsomes acts as a physiological Ins(1,4,5)P3 receptor/Ca2+ channel.