Metabolism of inositol 1,4,5-trisphosphate in squid photoreceptors

Summary Inositol 1,4,5-trisphosphate (InsP₃) is rapidly formed in squid photoreceptors in response to light, where it is converted sequenctially into inositol bisphosphate (InsP₂) and inositol monophosphate (InsP₁). All of the InsP₃ appears to be degraded to inositol 1,4-bisphosphate via an InsP₃-phosphatase, which is characterized in this study. The enzyme is water-soluble and present in the light-transducing distal segments of squid photoreceptors. It has a K_m of 50 M for InsP₃, requires Mg⁺⁺ for its activity, is maximally active at neutral pH, specifically hydrolyses the 5-phosphate and is inhibited by 2,3-diphosphoglycerate. In these respects, InsP₃-phosphatase of squid is very similar to the enzymes of other cells. Since no InsP₄ or more highly phosphorylated inositols are found in squid photoreceptors, the InsP₃-phosphatase may be important in the regulation of InsP₃ concentration within these cells.Abbreviations InsP ₁ , InsP ₂ , InsP ₃ , InsP ₄ , InsP ₆ inositol monobis-, tris-, tetrakis-, hexakisphosphate, respectively - 2,3-DPG 2,3-diphosphoglycerate - EDTA ethylene diamine tetraacetic acid - DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMSF phenylmethylsulfonyl fluoride     

Radio-label and mass determinations of inositol 1,3,4,5-tetrakisphosphate formation in rat cerebral cortical slices: Differential effects ofmyo-inositol

To investigate the effects of increasing concentrations ofmyo-inositol (inositol) on receptor stimulated [³H]inositol polyphosphate formation in the absence of lithium, slices of rat cerebral cortex were incubated with various concentrations of [³H]inositol (1 to 30 M). Carbachol stimulated formation of [³H]inositol trisphosphate (InsP₃) and [³H]inositol 1,3,4,5-tetrakisphosphate {Ins(1,3,4,5)P₄} increased several fold when the inositol concentration was increased reaching a plateau at approximately 12 M inositol. Time course studies revealed that in the presence of low concentrations of inositol (1 M), [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation in response to carbachol stimulation increased slowly over a 10 to 20 min time period, whereas in the presence of 4 and 12 M inositol, carbachol stimulated [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation was rapid and essentially complete within 3 to 5 min after carbachol addition. Although the carbachol dose response in 12 M inositol had a much greater maximal efficacy, there was no change in potency. Similar to the effects of carbachol on [³H]Ins(1,3,4,5)P₄ formation from prelabeled phosphoinositides, muscarinic receptor stimulation increased Ins(1,3,4,5)P₄ mass formation by seven fold. Furthermore, Li⁺ (8 mM) completely inhibited carbachol stimulated increases in Ins(1,3,4,5)P₄ mass formation. In contrast to the effects of increasing inositol on carbachol stimulated formation of radiolabeled inositol phosphates, increasing inositol had no effect upon mass formation of Ins(1,3,4,5)P₄. These results show that when measuring inositol polyphosphate formation by the radiolabeling technique in the absence of Li⁺, increasing the inositol concentration greatly increases the stimulated component of [³H]InsP₃ and [³H]Ins(1,3,4,5)P₄ formation. However, this inositol induced increase in agonist stimulated Ins(1,3,4,5)P₄ formation is not reflected as an increase in mass formation.     

Effects of ischaemia,reperfusion and α1 receptor stimulation on the inositoltrisphosphate receptor population in rat heart atria and ventricles

Binding sites specific for inositol 1,4,5-trisphosphate (InsP₃) have been demonstrated in sarcoplasmic reticulum vesicles isolated from heart muscle. Scatchard analysis of a binding isotherm indicated a high as well as a low affinity binding site [1]. In this study a comparison was made between InsP₃ binding to crude microsomal membranes prepared from rat heart atria and ventricles respectively. Results obtained showed a four-fold higher incidence of binding to atrial membranes. Furthermore, the receptor populations of the atria and ventricles behaved differently during conditions causing fluctuations in tissue InsP₃ levels, viz. ischaemia, reperfusion and ₁-adrenergic stimulation. Reperfusion, as well as phenylephrine stimulation, caused an increase in InsP₃ levels associated with down-regulation of the ventricular InsP₃ receptor population while binding to atrial binding sites was elevated. In the ventricular population this down-regulation was the result of a reduction in B_max alone with no changes in the Kd values of the high- or the low-affinity binding sites. The reason(s) for the differential response of the atrial and ventricular InsP₃ receptor populations to changes in InsP₃ levels, remains to be established.     

Role of protein phosphorylation and inositol phospholipid turnover in rat parotid gland proliferation

The involvement of protein phosphorylation in isoproterenol (ISO)-mediated proliferation in the rat parotid gland was investigated by labeling the cells with [³²P] orthophosphate. An increased (4–6 fold) incorporation of the radiolabel was noted in the total parotid gland homogenates of ISO-treated animals when compared to controls. Plasma membrane, nuclear membrane and cytoplasm were isolated, the proteins separated by SDS/PAGE and the phosphoproteins detected by autoradiography. Two phosphoproteins with apparent M_r of 45 and 170 kDa were identified in the cytoplasm while the 170 kDa phosphoprotein also appeared as part of plasma membrane. Transfer of these proteins to nitrocellulose followed by Western blot detection with an antiphosphotyrosine monoclonal antibody showed reactivity with the 170 kDa region of the plasma membrane and cytoplasm. Separate in vitro studies involving incubations of rat parotid slices with 0.2 mM ISO and [³H] myo-inositol for 1 min induced inositol phosphate hydrolysis resulting in a significant increase in inositol-bis and -tris phosphate production. Inositol phosphate production can be blocked by pre-incubation with a mixed -adrenergic receptor antagonist but not with physiological concentrations of - or ₁-specific adrenergic receptor antagonists, indicating the ISO effects are mediated through the ₂-adrenergic receptors. The inclusion of calmodulin antagonists along with ISO prevented the expression of cell-surface galactosyltransferase and retarded gland hypertrophy and hyperplasia. These results suggest that ISO treatment leads to the phosphorylation of target proteins which may be involved in signal transduction pathways leading to cell proliferation.Abbreviations InsP₁, InsP₂, InSP₃ inositol mono-, bis-, and tris-phosphates - UDP Uridine diphosphate - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate - TFP Trifluoperazine - P-tyr phosphotyrosine - Gal Tase galactosyltransferase     

The latency of the response of Limulus photoreceptors to inositol trisphosphate lacks the calcium-sensitivity of that to light

Summary The latent period before depolarization of Limulus ventral photoreceptors by light flashes was compared with that following brief, intracellular, pressure-injection of d-myo-inositol 1,4,5 trisphosphate. At temperatures between 18 °C and 22 °C and with an extracellular calcium concentration of 10 mM, the responses of 4 cells to light and to injections of 100 M inositol trisphosphate displayed average latencies of 71 and 56 ms, respectively. The latencies of responses to InsP₃ included an estimated 20 ms dead-time inherent in the injection method. Reducing the temperature lengthened the latency of the response to light (Q₁₀ approximately 3.2 between 7 and 22 °C) more than that to inositol trisphosphate (Q₁₀ approximately 2.3). Bathing the photoreceptors in seawater containing no added calcium and 1 mM of the calcium chelator EGTA greatly increased the latency of the light response at all temperatures, but did not increase the latency of the response to inositol trisphosphate. We conclude that the response to inositol trisphosphate lacks the calcium- and temperature-sensitive latent period which characterizes the response to light. If inositol trisphosphate acts, via the release of stored calcium, to stimulate an intermediate in the visual cascade, then that intermediate would appear to be downstream from the latency-generating mechanism.Abbreviations InsP ₃ D-myo-inositol 1,4,5 trisphosphate - ASW Artificial seawater - Ca _i Cytosolic free calcium ion concentration - Ca ₀ Extracellular calcium ion concentration     

Inositol Pyrophosphates Modulate S Phase Progression after Pheromone-induced Arrest in Saccharomyces cerevisiae

Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Plc) in nuclei of mammalian cells during synchronous progression through the cell cycle, but the downstream targets of Plc-generated inositol 1,4,5-trisphosphate are poorly described. Phospholipid signaling in the budding yeast Saccharomyces cerevisiae shares similarities with endonuclear phospholipid signaling in mammals, and many recent studies point to a role for inositol phosphates, including InsP₅, InsP₆, and inositol pyrophosphates, in mediating the action of Plc. In this study, we investigated the changes in inositol phosphate levels in α-factor-treated S. cerevisiae, which allows cells to progress synchronously through the cell cycle after release from a G₁ block. We found an increase in the activity of Plc1 early after release from the block with a concomitant increase in the levels of InsP₇ and InsP₈. Treatment of cells with the Plc inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 prevented increases in inositol phosphate levels and blocked progression of cells through S phase after pheromone arrest. The enzymatic activity of Kcs1 in vitro and HPLC analysis of [³H]inositol-labeled kcs1Δ cells confirmed that Kcs1 is the principal kinase responsible for generation of pyrophosphates in synchronously progressing cells. Analysis of plc1Δ, kcs1Δ, and ddp1Δ yeast mutants further confirmed the role that a Plc1- and Kcs1-mediated increase in pyrophosphates may have in progression through S phase. Our data provide genetic, metabolic, and biochemical evidence that synthesis of inositol pyrophosphates through activation of Plc1 and Kcs1 plays an important role in the signaling response required for cell cycle progression after mating pheromone arrest.     

Inositol trisphosphate mediates the action of hypertrehalosemic hormone on fat body of the American cockroach, Periplaneta americana

The rate of synthesis of inositol trisphosphate (InsP₃) in trophocytes derived from disaggregated cockroach (Periplaneta americana) fat body increases following treatment of the cells with hypertrehalosemic hormone I or II (HTH-I, -II) in vitro. Trophocytes preloaded with [³H]inositol display a significant increase in InsP₃ synthesis as early as 15 s after addition of the hormone. When the trophocytes are pre-incubated with LiCl and subsequently incubated with HTH the [³H] content of the InsP₃ fraction is greater than that found with HTH alone. This is taken as evidence that inositol monophosphate phosphatase is part of the mechanism for clearing InsP₃ from the cytosol. In contrast to HTH, octopamine, which is also capable of exerting a hypertrehalosemic effect in the cockroach, does not increase the synthesis of InsP₃. 1-Octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃), a potent and selective inhibitor of phosphatidylinositol phospholipase C, blocks the activation of phosphorylase by HTH-I as well as the hypertrehalosemic effect induced by the hormone.     

Effects of ginsenosides on carbachol-stimulated formation of inositol phosphates in rat cortical cell cultures

We examined the effect of ginseng total saponins (GTS) on phosphoinositide metabolism stimulated by activation of muscarinic receptor using rat cortical cultures. Carbachol stimulated formation of [³H]inositol phosphates ([³H]InsPs) by 3.3-fold over basal level in [³H]inositol-prelabeled cells. Pretreatment of GTS inhibited formation of [³H]InsPs evoked by carbachol by 70%–90%. Addition of GTS alone had no effect on the basal formation of [³H]InsPs. The inhibitory effect of the GTS on carbachol-stimulated formation of [³H]InsPs was dose- and time-dependent. IC₅₀ was 6.0 ± 2.8 g/ml. We also examined the effect of GTS on [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation evoked by carbachol. Although GTS had no effect on the basal [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation, pretreatment of GTS inhibited [³H]InsP₁, [³H]InsP₂, or [³H]InsP₃ formation evoked by carbachol, respectively. Addition of individual ginsenosides such as ginsenoside Rb₁, Rc, Rd, Re, or Rg₂ had no effect on the basal formation of [³H]InsPs, whereas pretreatment of ginsenoside Rb₂, Rc, Rd, Re, Rf, Rg₁ or Rg₂ inhibited formation of [³H]InsPs evoked by carbachol by 79%–89%. The results suggest that the inhibitory effect of GTS and its individual ginsenosides on carbachol-stimulated formation of [³H]InsPs in cortical neurons could be one pharmacological action of Panax ginseng.     

InsP6-Sensitive Variants of the Gle1 mRNA Export Factor Rescue Growth and Fertility Defects of the ipk1 Low-Phytic-Acid Mutation in Arabidopsis

Myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP₆), also known as phytic acid, accumulates in large quantities in plant seeds, serving as a phosphorus reservoir, but is an animal antinutrient and an important source of water pollution. Here, we report that Gle1 (GLFG lethal 1) in conjunction with InsP₆ functions as an activator of the ATPase/RNA helicase LOS4 (low expression of osmotically responsive genes 4), which is involved in mRNA export in plants, supporting the Gle1-InsP₆-Dbp5 (LOS4 homolog) paradigm proposed in yeast. Interestingly, plant Gle1 proteins have modifications in several key residues of the InsP₆ binding pocket, which reduce the basicity of the surface charge. Arabidopsis thaliana Gle1 variants containing mutations that increase the basic charge of the InsP₆ binding surface show increased sensitivity to InsP₆ concentrations for the stimulation of LOS4 ATPase activity in vitro. Expression of the Gle1 variants with enhanced InsP₆ sensitivity rescues the mRNA export defect of the ipk1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) InsP₆-deficient mutant and, furthermore, significantly improves vegetative growth, seed yield, and seed performance of the mutant. These results suggest that Gle1 is an important factor responsible for mediating InsP₆ functions in plant growth and reproduction and that Gle1 variants with increased InsP₆ sensitivity may be useful for engineering high-yielding low-phytate crops.     

Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants

Inositol pyrophosphates are unique cellular signaling molecules with recently discovered roles in energy sensing and metabolism. Studies in eukaryotes have revealed that these compounds have a rapid turnover, and thus only small amounts accumulate. Inositol pyrophosphates have not been the subject of investigation in plants even though seeds produce large amounts of their precursor, myo‐inositol hexakisphosphate (InsP₆). Here, we report that Arabidopsis and maize InsP₆ transporter mutants have elevated levels of inositol pyrophosphates in their seed, providing unequivocal identification of their presence in plant tissues. We also show that plant seeds store a little over 1% of their inositol phosphate pool as InsP₇ and InsP₈. Many tissues, including, seed, seedlings, roots and leaves accumulate InsP₇ and InsP₈, thus synthesis is not confined to tissues with high InsP₆. We have identified two highly similar Arabidopsis genes, AtVip1 and AtVip2, which are orthologous to the yeast and mammalian VIP kinases. Both AtVip1 and AtVip2 encode proteins capable of restoring InsP₇ synthesis in yeast mutants, thus AtVip1 and AtVip2 can function as bonafide InsP₆ kinases. AtVip1 and AtVip2 are differentially expressed in plant tissues, suggesting non‐redundant or non‐overlapping functions in plants. These results contribute to our knowledge of inositol phosphate metabolism and will lay a foundation for understanding the role of InsP₇ and InsP₈ in plants.     

There is no ‘Conundrum’ of InsP6

Indirect assays have claimed to quantify phytate (InsP₆) levels in human biofluids, but these have been based on the initial assumption that InsP₆ is there, an assumption that our more direct assays disprove. We have shown that InsP₆ does not and cannot (because of the presence of an active InsP₆ phosphatase in serum) exist in mammalian serum or urine. Therefore, any physiological effects of dietary InsP₆ can only be due either to its actions in the gut as a polyvalent cation chelator, or to inositol generated by its dephosphorylation by gut microflora.We are grateful to Dr Vucenik for bringing up a number of interesting points.It is true that we have not quantified the dietary intakes of our human donors any more (but also hardly any less) than has been done by those groups claiming that InsP₆ is present in bodily fluids. As a qualitative observation we should point out that in fact all our donors for ref. [1] do have a regular intake of dietary cereals and indeed, one is a strict vegetarian on a high cereal diet. But it is quantification that reveals this to be a specious issue. The limits of detection in our two relevant publications [1,2] for InsP₆ in plasma and urine were, respectively, around two and three orders of magnitude lower than the levels claimed to be present by Grases et al. [3] in the fluids of experimentally phytate-deprived human subjects. These numbers make the argument that we could not detect any InsP₆ simply because we chose donors on the ‘wrong’ diet untenable.So how have those many claims that InsP₆ is present in body fluids come about? For most of them, the simple answer appears to be that the assays used are indirect and are based entirely on the assumption that InsP₆ is present in the first place. Thus, for example, Valiente and co-workers [4,5] and Chen and co-workers [6,7] measured organic phosphate remaining after a series of fractionations of urine samples and simply assumed it was due to InsP₆, as did March et al. measuring inorganic phosphate after a similar protocol [8]. Grases co-workers [9] have used extensively a less indirect assay, which, after initial ion chromatography and dephosphorylation by a phytase, measures myo-inositol by mass spectrometry, but nevertheless the assay starts with the assumption that InsP₆ is there and that this is what they are quantifying. More recently, direct quantification of InsP₆ in plasma by mass spectrometry has been claimed [10] on the basis that there are peaks in plasma at m/z 624 running near where InsP₆ standards elute in two different HPLC separations [10,11]. But no evidence is presented to show even that these peaks are the same compound, let alone any data to establish firmly that InsP₆ is present, e.g. a minimal requirement of m/z quantified to two decimal places with allowance for C₁₃ content or a full disintegration fingerprint (see also [12]). Any quantified misidentification is likely to have a stochastic element to it, and it is noteworthy that Perelló & Grases have stated [11, p. 255]: ‘…we have found some humans and rats having undetectable [InsP₆], probably depending on their diet or other unknown factors’. In the light of the preceding discussion, we can offer a simpler explanation: the InsP₆ was never there in the first place.In contrast to these claims we have, using two entirely independent specific and sensitive assays with quantified spiking recovery, unambiguously shown that InsP₆ is not present in plasma or urine. This is crucial and central to the whole debate about the actions of dietary InsP₆, because it means that InsP₆ never enters the blood. It is only absorbed after being dephosphorylated, principally to inositol (see [1,2] for further discussion). Ironically, the most direct evidence for this lies in Dr Vucenik''s own data in experiments examining the fate of radioactive InsP₆ fed to animals, in which only inositol was detected in the blood [13]. This particular study was, as Dr Vucenik points out in her letter, conducted on mice. However, exactly the same conclusion (i.e. InsP₆ does not enter the circulation from the gut) is equally clear in her earlier study [14], which she did not cite and which was indeed on rats; does this omission ‘reflect poorly’ on Dr Vucenik''s own ‘report and the author''s credibility in culling scientific data’?In short, dietary InsP₆ can have only two fates: it can stay in the gut, ultimately to be defecated [15], and while it is there it can chelate metal ions to alter their uptake from the gut into the body. This is no ‘straw-man’ and is certainly the most likely explanation for all of the effects of InsP₆ on cultured cells, which comprise the majority of the reports cited by Dr Vucenik. Alternatively, InsP₆ can be converted to inositol (principally by the gut microflora [15]) and be taken up as such into the circulation; were any InsP₆ to get into the blood it would in any case be rapidly dephosphorylated by the phosphatase activity we have shown to be present in human plasma [1].For animal studies, we have raised the possibility [1,2] that it is the inositol so generated (Vitamin Bh, harmless as far as we know) that is the active mediator of any reported beneficial effects of dietary InsP₆. We note that most of the websites touting InsP₆ as a dietary supplement advocate inositol as an important (essential?) co-supplement; that the only human cancer study highlighted as important by Dr Vucenik that we could examine [16] did not administer InsP₆ alone, but only in conjunction with inositol; and that in the few studies where the separate contributions of inositol and InsP₆ have been considered, there are data suggesting that it may be the inositol that matters (e.g. fig. 1 of [17]). Moreover, we are not the only ones to suggest this idea. In the Discussion of their paper (on mice) in which InsP₆ was shown not to enter the blood from the gut [13], Dr Vucenik and her colleagues state: ‘Inositol may be responsible for the antitumor actions observed in both chemopreventitive and efficacy studies of IP₆ … A question remains as to whether the activity of IP₆ in animal models can be replicated by administration of inositol alone because only inositol was detected in plasma and tumor after oral gavage’. Precisely.Finally, returning to InsP₆ itself, which, incidentally, is officially classified by the FDA as a ‘fake’ cancer cure (http://www.fda.gov/drugs/guidancecomplianceregulatoryinformation/enforcementactivitiesbyfda/ucm171057.htm), our data lead inevitably to the conclusion that while InsP₆ might impact on the gut environment and thus indirectly on its microflora [2,12], its only plausible direct action on the body will be to inhibit cation uptake from the diet. Although InsP₆ binds trivalent cations with a higher affinity than divalents [18], it is nevertheless comparatively non-specific in this action. Administering chemicals to the diet to manipulate ion uptake is not unknown in modern medicine; for treatment of iron disorders such as haemochromatosis, as an alternative to injection of Desferral, oral administration of the closely related chelator Deferasirox is now sometimes recommended [19]. But Deferasirox is a highly iron-specific chelator, administered under close medical supervision for a directly iron-related pathology. Recommending unmonitored, widespread administration of InsP₆ to address a veritable multitude of different pathologies [20] seems to us to be an entirely different matter.In a well-fed human, where the cation to InsP₆ ratio in the diet is high, InsP₆ may very well do no harm (it is, after all, a natural component of our diet) and there is much evidence to support this idea, as argued by Dr Vucenik. But if InsP₆ is not impacting on cation uptake from the diet to do any harm it is difficult to understand how at exactly the same time it can impact on the same uptake to do good. (See reference [21] for the studies Dr Vucenik requested ‘unequivocally demonstrating the toxicity of pure Ca-Mg-InsP₆ as it occurs naturally’ in humans with low dietary cation uptake.) In the light of the above discussion and our rigorous data, we stand unreservedly by our original closing statement [1]: ‘…that chronically altering cation absorption from the gut by artificially loading the diet with a non-specific chelator … in the hope that it might impact indirectly on cancer or other pathologies seems highly inadvisable’.     

Inositol 1,4,5-trisphosphate as fertilization signal in plants: testcase Chlamydomonas eugametos

Within the first 2 h of sexual reproduction, gametes of the green alga Chlamydomonas eugametos agglutinate, fuse via their mating structures, de-agglutinate and swim off as vis-à-vis pairs. During this period, increases in intracellular inositol 1,4,5-trisphosphate levels and changes in polyphosphoinositide synthesis were associated with cell fusion. The protein-kinase-C inhibitor staurosporine (0.1–0.2 M) inhibited the de-agglutination of pairs and therefore prevented them swimming away, while earlier stages of mating such as agglutination or cell fusion were unaffected. The results suggest that inositol 1,4,5-trisphosphate and diacylglycerol are fertilization signals in C. eugametos. The idea that they could also be fertilization signals in higher plants is discussed in relation to in vitro embryogenesis.Abbreviations DAG diacylglycerol - InsP₃ inositol 1,4,5-trisphosphate - mt⁺/mt^– mating-type plus or minus - PKC protein kinase C - PtdOH phosphatidic acid - PtdInsP phosphatidylinositol - 4-phosphate PtdInsP₂ phosphatidylinositol 4,5-bisphosphate     

ATP Regulation of Type-1 Inositol 1,4,5-Trisphosphate Receptor Activity Does Not Require Walker A-type ATP-binding Motifs

ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP₃R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP₃R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP₃R1, and the ATPB site is conserved among all three InsP₃R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP₃R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP₃R1. ATP augmented InsP₃-induced Ca²⁺ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP₃R1. Similarly, ATP increased the single channel open probability of the mutated InsP₃R1 to the same extent as wild type. ATP likely exerts its effects on InsP₃R1 channel function via a novel and as yet unidentified mechanism.Inositol 1,4,5-trisphosphate receptors (InsP₃R)³ are a family of large, tetrameric, InsP₃-gated cation channels. The three members of this family (InsP₃R1, InsP₃R2, and InsP₃R3) are nearly ubiquitously expressed and are localized primarily to the endoplasmic reticulum (ER) membrane (1–3). Numerous hormones, neurotransmitters, and growth factors bind to receptors that stimulate phospholipase C-induced InsP₃ production (4). InsP₃ subsequently binds to the InsP₃R and induces channel opening. This pathway represents a major mechanism for Ca²⁺ liberation from ER stores (5). All three InsP₃R isoforms are dynamically regulated by cytosolic factors in addition to InsP₃ (1). Ca²⁺ is perhaps the most important determinant of InsP₃R activity besides InsP₃ itself and is known to regulate InsP₃R both positively and negatively (6). ATP, in concert with InsP₃ and Ca²⁺, also regulates InsP₃R as do numerous kinases, phosphatases, and protein-binding partners (7–10). This intricate network of regulation allows InsP₃R activity to be finely tuned by the local cytosolic environment (9). As a result, InsP₃-induced Ca²⁺ signals can exhibit a wide variety of spatial and temporal patterns, which likely allows Ca²⁺ to control many diverse cellular processes.Modulation of InsP₃-induced Ca²⁺ release (IICR) by ATP and other nucleotides provides a direct link between intracellular Ca²⁺ signaling and the metabolic state of the cell. Metabolic fluctuations could, therefore, impact Ca²⁺ signaling in many cell types given that InsP₃R are expressed in all cells (11, 12). Consistent with this, ATP has been shown to augment IICR in many diverse cell types including primary neurons (13), smooth muscle cells (14), and exocrine acinar cells (15) as well as in immortalized cell lines (16–18). The effects of ATP on InsP₃R function do not require hydrolysis because non-hydrolyzable ATP analogues are as effective as ATP (7, 14). ATP is thought to bind to distinct regions in the central, coupling domain of the receptors and to facilitate channel opening (2, 19). ATP is not required for channel gating, but instead, increases InsP₃R activity in an allosteric fashion by increasing the open probability of the channel in the presence of activating concentrations of InsP₃ and Ca²⁺ (7, 8, 20).Despite a wealth of knowledge regarding the functional effects of ATP on InsP₃R function, there is relatively little known about the molecular determinants of these actions. ATP is thought to exert effects on channel function by direct binding to glycine-rich regions containing the consensus sequence GXGXXG that are present in the receptors (2). These sequences were first proposed to be ATP-binding domains due to their similarity with Walker A motifs (21). The neuronal S2⁺ splice variant of InsP₃R1 contains two such domains termed ATPA and ATPB. A third site, ATPC, is formed upon removal of the S2 splice site (2, 22). The ATPB site is conserved in InsP₃R2 and InsP₃R3, while the ATPA and ATPC sites are unique to InsP₃R1. Our prior work examining the functional consequences of mutating these ATP-binding sites has yielded unexpected results. For example, mutating the ATPB site in InsP₃R2 completely eliminated the enhancing effects of ATP on this isoform while mutating the analogous site in InsP₃R3 failed to alter the effects of ATP (23). This indicated the presence of an additional locus for ATP modulation of InsP₃R3. In addition, mutation of the ATPC in the S2⁻ splice variant of InsP₃R1 did not alter the ability of ATP to modulate Ca²⁺ release, but instead impaired the ability of protein kinase A to phosphorylate Ser-1755 of this isoform (22).The ATPA and ATPB sites in InsP₃R1 were first identified as putative nucleotide-binding domains after the cloning of the full-length receptor (24). Early binding experiments with 8-azido-[α-³²P]ATP established that ATP cross-linked with receptor purified from rat cerebellum at one site per receptor monomer (19). Later, more detailed, binding experiments on trypsinized recombinant rat InsP₃R1 showed cross-linking of ATP to two distinct regions of the receptor that corresponded with the ATPA and ATPB sites (17). We and others (16, 22, 23) have also reported the binding of ATP analogues to purified GST fusions of small regions of InsP₃R1 surrounding the ATPA and ATPB sites. It is widely accepted, in the context of the sequence similarity to Walker A motifs and biochemical data, that the ATPA and ATPB sites are the loci where ATP exerts its positive functional effects on InsP₃R1 function (1–3, 16). Furthermore, the higher affinity of the ATPA site to ATP is thought to confer the higher sensitivity of InsP₃R1 to ATP versus InsP₃R3, which contains the ATPB site exclusively (25, 26). The purpose of this study, therefore, was to examine the contributions of the ATPA and ATPB sites to ATP modulation of the S2⁺ splice variant of InsP₃R1. We compared the effects of ATP on InsP₃R1 and on ATP-binding site mutated InsP₃R1 using detailed functional analyses in permeabilized cells and in single channel recordings. Here we report that InsP₃R1 is similar to InsP₃R3 in that ATP modulates IICR even at maximal InsP₃ concentrations and that neither the ATPA nor the ATPB site is required for this effect.     

Muscarinic Receptor-Mediated Inositol 1,4,5-Trisphosphate Formation in SH-SY5Y Neuroblastoma Cells Is Regulated Acutely by Cytosolic Ca2+ and by Rapid Desensitization

Abstract: Stimulation of muscarinic receptors expressed in SH-SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5-trisphosphate (InsP₃) accumulation, with a dramatic increase (six- to eightfold) over the first 10 s (the “peak” phase) and subsequently, from ～60 s onward, maintained at a lower but sustained level (the “plateau” phase). Chelation of extracellular Ca²⁺ with EGTA or inhibition of Ca²⁺ channels with Ni²⁺ showed that the plateau phase was dependent upon Ca²⁺ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca²⁺ from intracellular stores revealed that Ca²⁺ from this source was capable of supporting the peak phase of the InsP₃ response. Carbachol-stimulated phosphoinositidase C activity in permeabilized SH-SY5Y cells was also shown to be highly dependent on free Ca²⁺ concentration (20–100 nM) and suggests that under normal conditions, InsP₃ formation is enhanced by increases in cytosolic free Ca²⁺ concentration that accompany muscarinic receptor activation. Measurement of carbachol-stimulated total inositol phosphate accumulation in the presence of Li⁺ indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor-mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP₃ accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free Ca²⁺ concentration amplify InsP₃ formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP₃ accumulation is rapid, but partial, desensitization. Such desensitization does not appear to be mediated by changes in cytosolic Ca²⁺ or protein kinase C activity.     

Synergistic effects of adenosine A1 and P2Y receptor stimulation on calcium mobilization and PKC translocation in DDT1 MF-2 cells

1. The effect of adenosine analogues and of nucleotides, alone or in combination, on intracellular calcium, accumulation of inositol (1,4,5) trisphosphate (InsP₃), and on activation of protein kinase C (PKC) was studied in DDT₁ MF2 cells derived from a Syrian hamster myosarcoma. These cells were found to express mRNA for A₁ and some as yet unidentified P2Y receptor(s).2. Activation of either receptor type stimulated the production of InsP₃ and raised intracellular calcium in DDT₁ MF2 cells. Similarly, the A₁ selective agonist N⁶-cyclopentylade- nosine (CPA) increased PKC-dependent phosphorylation of the substrate MBP_4–14 and induced a PKC translocation to the plasma membrane as determined using [³H]-phorbol dibutyrate (PDBu) binding in DDT₁ MF-2 cells. However, neither adenosine nor CPA induced a significant translocation of transiently transfected -PKC-GFP from the cytosol to the cell membrane. In contrast to adenosine analogues, ATP and UTP also caused a rapid but transient translocation of -PKC-GFP and activation of PKC.3. Doses of the A₁ agonist CPA and of ATP or UTP per se caused barely detectable increases in intracellular Ca²⁺ but when combined, they caused an almost maximal stimulation. Similarly, adenosine (0.6 M) and UTP (or ATP, 2.5 M), which per se caused no detectable translocation of either - or -PKC-GFP, caused when combined a very clear-cut translocation of both PKC subforms, albeit with different time courses. These results show that simultaneous activation of P2Y and adenosine A₁ receptors synergistically increases Ca²⁺ transients and translocation of PKC in DDT₁ MF-2 cells. Since adenosine is rapidly formed by breakdown of extracellular ATP, such interactions may be biologically important.     

Abscisic acid-induced changes in inositol metabolism in Spirodela polyrrhiza

 In response to abscisic acid (ABA), the duckweed Spirodela polyrrhiza (L.) activates a developmental pathway that culminates in the formation of dormant structures known as turions. Levels of the mRNA encoding d-myo-inositol-3-phosphate synthase (EC.5.5.1.4) which converts glucose-6-phosphate to inositol-3-phosphate, increase early in response to ABA. In order to understand the role of this enzyme in turion formation, we have investigated changes in inositol metabolism in ABA-treated plants. Here, we show that ABA-treatment leads to a 3-fold increase in free inositol, which peaks 2 d after treatment. This increase is followed by sequential increases in inositol phosphates and in accumulation of inositol hexakisphosphate (InsP₆), in particular. In addition, we observed an early increase in a novel inositol bisphosphate which is not directly on the pathway to InsP₆. In control plants, we observed synthesis and turnover of both inositol pentakisphosphate and InsP₆. Two compounds more polar than InsP₆ (diphosphoinositol polyphosphates) were present in both ABA-treated and control plants. Together, this suggests that the role of InsP₆ in plants may be more complex than simply that of a storage compound during dormancy. Received: 10 January 2000 / Accepted: 25 February 2000     

Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIβ in the Golgi apparatus in cerebellar granule cells

Background information. Interconnections between the Ca²⁺ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase‐anchoring proteins). In many cell types, the activation of InsP₃R (inositol 1,4,5‐trisphosphate receptor), an endoplasmic reticulum Ca²⁺ channel, is a key event of Ca²⁺ signalling. The phosphorylation of InsP₃R1 by PKA stimulates Ca²⁺ mobilization. This control is thought to be tight, involving the association of PKA with InsP₃R1. The InsP₃R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP₃R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Results. Immunoprecipitation experiments showed that InsP₃R1 associated with PKA type IIβ and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co‐purification of AKAP450 with InsP₃R1 on heparin‐agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP₃R1 and RIIβ (regulatory subunit of PKA IIβ) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP₃R1 was detected in this organelle only in granule cells. Conclusions. Taken together these results suggest that InsP₃R1 forms a complex with AKAP450 and PKAIIβ, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP₃R1 with PKA in Purkinje cells would require a different macromolecular complex.     

Evidence that the TRP-1 protein is unlikely to account for store-operated Ca2+ inflow in Xenopus laevis oocytes

The role of the TRP-1 protein, an animal cell homologue of the Drosophila transient receptor potential Ca²⁺ channel, in store-operated Ca²⁺ inflow in Xenopus laevis oocytes was investigated. A strategy involving RT-PCR and 3 and 5 rapid amplification of cDNA ends (RACE) was used to confirm and extend previous knowledge of the nucleotide and predicted amino acid sequences of Xenopus TRP-1 (xTRP-1). The predicted amino acid sequence was used to prepare an anti-TRP-1 polyclonal antibody which detected the endogenous oocyte xTRP-1 protein and the human TRPC-1 protein expressed in Xenopus oocytes. Ca²⁺ inflow (measured using fura-2) initiated by 3-deoxy-3-fluoroinositol 1,4,5-trisphosphate (InsP₃F) or lysophosphatidic acid (LPA) was completely inhibited by low concentrations of lanthanides (IC₅₀ = 0.5 M), indicating that InsP₃F and LPA principally activate store-operated Ca²⁺ channels (SOCs). Antisense cRNA or antisense oligodeoxynucleotides, based on different regions of the xTRP-1 cDNA sequence, when injected into Xenopus oocytes, did not inhibit InsP₃F-, LPA- or thapsigargin-stimulated Ca²⁺ inflow. Oocytes expressing the hTRPC-1 protein, which is 96% similar to xTRP-1, exhibited no detectable enhancement of either basal or InsP₃F-stimulated Ca²⁺ inflow and only a very small enhancement of LPA-stimulated Ca²⁺ inflow compared with control oocytes. It is concluded that the endogenous xTRP-1 protein is unlikely to be responsible for Ca²⁺ inflow through the previously-characterised Ca²⁺-specific SOCs which are found in Xenopus oocytes. It is considered that xTRP-1 is likely to be a receptor-activated non-selective cation channel such as the channel activated by maitotoxin.