Variation of InsP(4),InsP(5) and InsP(6) levels in tissues and biological fluids depending on dietary phytate

Due to the increasing interest of InsP(6) on human health, the aim of this paper is to compare the levels of highly phosphorilated inositols (InsP(4), InsP(5) and InsP(6)) in organs and biological fluids of rats and to study the influence of the presence and absence of InsP(6) in diets. Thus, for this purpose, the variation of InsP(4), InsP(5) and InsP(6) levels in organs and biological fluids of rats submitted to two different diets were studied. In the AIN-76A diet no InsP(6) was present, yet the other was a 1% InsP(6) modified diet (AIN-76A + 1% InsP(6)). The highest InsP(4), InsP(5) and InsP(6) levels were found to be 10-fold superior in the brain than those found in the kidney. When the InsP(6) was eliminated from the diet, the InsP(6) levels decreased dramatically (97.2% in kidney, 89.8% in brain, 100% in bone, 90.5% in plasma and 98.1% in urine), the InsP(5) levels showed an important decrease (61.2% in kidney, 45.5% in brain, 28.1% in bone, 30% in plasma and 88.6% in urine) and the InsP(4) levels in organs only changed slightly. From these results, it can be deduced that the majority of InsP(6) present in the organism is of dietary origin and its endogenous synthesis is not important. According to the results, it can be evidenced that the endogenous synthesis of InsP(5) can occur, besides InsP(6) can be transformed by enzymatic dephosphorilation in InsP(5).     

Dietary myo-inositol hexaphosphate prevents dystrophic calcifications in soft tissues: a pilot study in Wistar rats

Myo-inositol hexaphosphate (InsP6) is an abundant component of plant seeds. It is also found in significant levels in blood and mammalian tissues, but they are totally dependent on their dietary intake. In the present paper, we describe studies on the effect of InsP6 on a model of dystrophic calcification, which was chemically induced by subcutaneous injection of a 0.1% KMnO4 solution. Male Wistar rats were randomly divided into four groups for treatment over 31 days. A: animals consuming a purified diet in which InsP6 was absent but to which 1% of InsP6 (as sodium salt) was added. In this group, the InsP6 plasma levels (0.393 +/- 0.013 microM) were similar to those observed in rats consuming a standard diet. B: animals consuming only the purified diet in which InsP6 was absent. In this case the InsP6 plasma levels decreased (0.026 +/- 0.006 microM); C: animals consuming the same purified diet as group B but received daily subcutaneous injections of 50 microg kg(-1) etidronate during the last 14 days. In this case the InsP6 plasma levels were also very low (0.025 +/- 0.007 microM); D: animals consuming the same diet as group B but a 6% of carob germ (InsP6 rich product) was added. The InsP6 plasma levels (0.363 +/- 0.035 microM) were also similar to those observed in rats consuming a standard diet. After 21 days plaque formation was induced. Calcification plaques were allowed to proceed for 10 days, after which the plaque material present was excised, dried and weighed. It was found that the presence of myo-inositol hexaphosphate (phytate) in plasma at normal concentrations (0.3-0.4 microM) clearly inhibited the development of dystrophic calcifications in soft tissues. These results demonstrates that myo-inositol hexaphosphate acts as an inhibitor of calcium salt crystallization.     

Spontaneous channel activity of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Application of allosteric modeling to calcium and InsP3 regulation of InsP3R single-channel gating

The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po ( approximately 0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2-4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].     

Effect of the inositol polyphosphate InsP(6) on DNA-PK-dependent phosphorylation

Inositol hexakisphosphate (InsP(6)) is a member of the inositol polyphosphate group that participates in numerous intracellular signaling pathways. Cheung and colleagues previously reported that InsP(6) stimulated double-strand break repair by nonhomologous end joining (NHEJ) in cell-free extracts and that InsP(6) binding by the Ku70/80 subunit of the DNA-dependent protein kinase (DNA-PK) was required for stimulation of NHEJ in vitro. This report describes InsP(6)-dependent phosphorylation of two NHEJ factors, XRCC4 and XLF, in partially purified human cell extracts. XRCC4 and XLF are known substrates for DNA-PK, which does not require InsP(6) for protein kinase activity. Consistent with a role for DNA-PK in these reactions, InsP(6)-dependent phosphorylation of XRCC4 and XLF was DNA dependent and not observed in the presence of DNA-PK inhibitors. Depletion of the Ku70/80 DNA-, InsP(6)-binding subunit of DNA-PK resulted in loss of InsP(6)-dependent phosphorylation and showed a requirement for Ku70/80 in these reactions. Complementation of Ku70/80-depleted reactions with recombinant wild-type Ku70/80 restored InsP(6)-dependent phosphorylation of XRCC4 and XLF. In contrast, addition of a Ku70/80 mutant with reduced InsP(6) binding failed to restore InsP(6)-dependent phosphorylation. While additional protein kinases may participate in InsP(6)-dependent phosphorylation of XRCC4 and XLF, data presented here describe a clear requirement for DNA-PK in these phosphorylation events. Furthermore, these data suggest that binding of the inositol polyphosphate InsP(6) by Ku70/80 may modulate the substrate specificity of the phosphoinositide-3-kinase-related protein kinase DNA-PK.     

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) has InsP(3)-independent gating and disrupts intracellular Ca(2+) homeostasis

The type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) is a ubiquitous intracellular Ca(2+) release channel that is vital to intracellular Ca(2+) signaling. InsP(3)R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca(2+) homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP(3)R1 and utilized a recombinant truncated form of the channel (capn-InsP(3)R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP(3)R1 revealed InsP(3)-independent gating and high open probability (P(o)) under optimal cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) conditions. However, some [Ca(2+)](i) regulation of the cleaved channel remained, with a lower P(o) in suboptimal and inhibitory [Ca(2+)](i). Expression of capn-InsP(3)R1 in N2a cells reduced the Ca(2+) content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca(2+) loading compared with control cells expressing full-length InsP(3)R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP(3)R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP(3)R1 generates a dysregulated channel that disrupts cellular Ca(2+) homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP(3)R1 in a clinically relevant injury model, suggesting that Ca(2+) leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.     

Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.     

Priming action of inositol hexakisphosphate (InsP6) on the stimulated respiratory burst in human neutrophils.

After priming by a number of different host, bacterial and chemical agents, human neutrophils may be stimulated to produce a greater respiratory burst than would be elicited by the stimulus alone. Other neutrophil functions may be similarly enhanced by pre-exposure to a priming agent. We describe here a new extracellular role for inositol hexakisphosphate (InsP6) as a priming agent for a variety of human neutrophil functional responses. Preincubation of the cells with InsP6 alone (up to 250 microM) has no stimulatory effect upon the basal production of reactive oxygen intermediates but the response to a subsequent stimulus (FMLP, PMA or phagocytic particles) is substantially enhanced. Levels 100-200% higher than 'stimulus only' controls have been recorded. Peak enhancement of the FMLP-induced oxidative response occurs after 1-2 min preincubation with InsP6 and the effect is dose-dependent (maximum at approx. 100 microM InsP6). As others have shown FMLP stimulation of superoxide anion production has no external Ca2+ dependence but the presence of low levels of Ca2+ and Mg2+ (0.1 mM) during priming appears to be an essential requirement for full expression. Reports of intracellular concentrations of InsP6 in mammalian cells in the 30-100 microM range suggest that the local release of this inositol polyphosphate from damaged or effect cells could have a physiologically important modulatory role on neutrophil functions.     

Increased levels of inositol hexakisphosphate (InsP6) protect HEK293 cells from tumor necrosis factor (alpha)- and Fas-induced apoptosis

The overexpression of inositol 1,3,4-trisphosphate 5/6-kinase has recently been shown to protect HEK293 cells from tumor necrosis factor alpha (TNF(alpha))-induced apoptosis. This overexpression leads to an increase in the levels of both inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol 1,2,3,4,5,6-hexakisphosphate (InsP6). Cells that overexpress InsP5 2-kinase have increased levels of InsP6 and are also protected from TNFalpha-induced apoptosis; furthermore, cells that express an RNA interference construct to the 2-kinase are deficient in InsP6 and are sensitized to TNFalpha-induced apoptosis. Therefore the protective effect of 5/6-kinase on TNFalpha-mediated apoptosis is due to an increase of InsP6 or to a metabolite derived from InsP6. Furthermore, we find that the InsP6 also protects from Fas-mediated apoptosis. No effect was seen in the endocytic rate of transferrin receptor, caspase 8 activity, or TNF receptor number at the cell surface. Cells that overexpress 2-kinase do show an increase in the amount of receptor-interacting protein (RIP), while cells with reduced InsP6 levels show relatively less RIP, providing a possible mechanism for the effect on apoptosis.     

The type 2 inositol (1,4,5)-trisphosphate (InsP3) receptor determines the sensitivity of InsP3-induced Ca2+ release to ATP in pancreatic acinar cells

Calcium release through inositol (1,4,5)-trisphosphate receptors (InsP(3)R) is the primary signal driving digestive enzyme and fluid secretion from pancreatic acinar cells. The type 2 (InsP(3)R2) and type 3 (InsP(3)R3) InsP(3)R are the predominant isoforms expressed in acinar cells and are required for proper exocrine gland function. Both InsP(3)R2 and InsP(3)R3 are positively regulated by cytosolic ATP, but InsP(3)R2 is 10-fold more sensitive than InsP(3)R3 to this form of modulation. In this study, we examined the role of InsP(3)R2 in setting the sensitivity of InsP(3)-induced Ca(2+) release (IICR) to ATP in pancreatic acinar cells. IICR was measured in permeabilized acinar cells from wild-type (WT) and InsP(3)R2 knock-out (KO) mice. ATP augmented IICR from WT pancreatic cells with an EC(50) of 38 mum. However, the EC(50) was 10-fold higher in acinar cells isolated from InsP(3)R2-KO mice, indicating a role for InsP(3)R2 in setting the sensitivity of IICR to ATP. Consistent with this idea, heterologous expression of InsP(3)R2 in RinM5F cells, which natively express predominately InsP(3)R3, increased the sensitivity of IICR to ATP. Depletion of ATP attenuated agonist-induced Ca(2+) signaling in WT pancreatic acinar cells. This effect was more profound in acinar cells prepared from InsP(3)R2-KO mice. These data suggest that the sensitivity of IICR to ATP depletion is regulated by the particular complement of InsP(3)R expressed in an individual cell. The effects of metabolic stress on intracellular Ca(2+) signals can therefore be determined by the relative amount of InsP(3)R2 expressed in cells.     

Mass measurements of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in a neuronal cell line stimulated with bradykinin: inositolphosphate response shows desensitization.

In a neuronal cell line (108CC15, NG108-15) the levels of inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4), as measured by receptor binding assays, rise transiently after stimulation with bradykinin (EC50 approx. 150 nM). Maximal InsP3 level of 354 pmol/mg protein (15-fold basal level) is obtained at 10-15 s after addition of bradykinin, the InsP4 level rises maximally to 78 pmol/mg protein (14-fold basal level) at 20-30 s. In a rat glioma cell line, bradykinin (2 microM) causes a fast 6-fold increase in InsP3 and InsP4 levels. In the neuronal cells the bradykinin-dependent rise of the inositolphosphate levels is diminished with reduced extracellular Ca2+ concentration. However, depletion of internal Ca2+ stores does not affect the bradykinin-induced rise in InsP3 and InsP4 levels. Homologous desensitization to bradykinin occurs in the signal transduction pathway already at the production of inositolphosphates, since after a 2 min stimulation with bradykinin the rise in cellular masses of InsP3 and InsP4, inducible by a following second bradykinin stimulus, is substantially reduced.     

Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins

Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, including transient expression in COS cells, acute injection of recombinant protein into Xenopus oocytes and (45)Ca(2+) flux from permeabilised COS cells, we demonstrated that CaBPs decrease the sensitivity of InsP(3)-induced Ca(2+) release (IICR). In addition, we found a Ca(2+)-independent interaction between CaBP1 and the NH(2)-terminal 159 amino acids of the type 1 InsP(3)R. This interaction resulted in decreased InsP(3) binding to the receptor reminiscent of that observed for CaM. Unlike CaM, however, CaBPs do not inhibit ryanodine receptors, have a higher affinity for InsP(3)Rs and more potently inhibited IICR. We also show that phosphorylation of CaBP1 at a casein kinase 2 consensus site regulates its inhibition of IICR. Our data suggest that CaBPs are endogenous regulators of InsP(3)Rs tuning the sensitivity of cells to InsP(3).     

Metabolism of inositol pentakisphosphate to inositol hexakisphosphate in Xenopus laevis oocytes

The formation and metabolism of inositol pentakis-and hexakisphosphates (InsP5 and InsP6) were investigated in Xenopus laevis oocytes. After [3H]inositol injection, [3H]InsP5 and subsequently [3H]Insp6 increased progressively over 72 h. In intact oocytes, [3H]InsP5 was progressively converted to [3H]InsP6 from 6 to 72 h of incubation and was not metabolized to lower inositol phosphates. In contrast, [3H]InsP6 remained unmetabolized for up to 72 h. These data are consistent with the kinetics of the increases in [3H]InsP5 and [3H]InsP6 in [3H]inositol-labeled oocytes. The highly phosphorylated inositols showed significant changes during oogenesis and maturation. In oocytes incubated for 48 h after [3H]inositol injection, the radioactive incorporation into polyphosphoinositols increased progressively from stage 3 to stage 6, with 5- and 6-fold rises (cpm/mg protein) for [3H]InsP5 and [3H]InsP6, respectively. These developmental changes were associated with 5-fold increases in [3H]inositol tetrakisphosphate between stages 3 and 6 of oogenesis. Induction of oocyte maturation by progesterone (1 microM) during the last 12 of a 36-h incubation with [3H]inositol doubled the levels of [3H]InsP6 relative to [3H]InsP5, suggesting that the activity of inositol pentakisphosphate kinase increases during maturation. These results provide direct evidence for metabolic conversion of InsP5 to InsP6 in animal cells and show that the higher inositol polyphosphates, unlike the lower phosphoinositols, are extraordinarily stable. These species increase markedly during ovum development and may play a regulatory role in oogenesis and maturation.     

Expression and distribution of InsP(3) receptor subtypes in proliferating vascular smooth muscle cells

The expression and distribution of types 1, 2, and 3 inositol 1,4, 5-trisphosphate receptor (InsP(3)R) in proliferating, primary cultures of rat aortic smooth muscle were compared to fully developed and differentiated rat aortic smooth muscle. Subtype-specific InsP(3)R antibodies revealed that the expression of type 1 InsP(3)R was similar in cultured aortic cells and aorta homogenate but expression of type 2 and 3 InsP(3)R subtypes was increased 3-fold in cultured aortic cells. The distribution of the type 1 InsP(3)R was located throughout the cytoplasm; type 2 InsP(3)R was found closely associated with the nucleus and at the plasma membrane; type 3 InsP(3)R was distributed predominantly around the nucleus. Alterations in InsP(3)R subtype expression and localization may have important functions in regulating intracellular calcium release around the nucleus when vascular smooth muscle cells switch to a more proliferating phenotype.     

Regulation of the type III InsP(3) receptor by InsP(3) and ATP

Many hormones and neurotransmitters raise intracellular calcium (Ca(2+)) by generating InsP(3) and activating the inositol 1,4, 5-trisphosphate receptor (InsP(3)R). Multiple isoforms with distinct InsP(3) binding properties () have been identified (). The type III InsP(3)R lacks Ca(2+)-dependent inhibition, a property that makes it ideal for signal initiation (). Regulation of the type III InsP(3)R by InsP(3) and ATP was explored in detail using planar lipid bilayers. In comparison to the type I InsP(3)R, the type III InsP(3)R required a higher concentration of InsP(3) to reach maximal channel activity (EC(50) of 3.2 microM versus 0.5 microM for the types III and I InsP(3)R, respectively). However, the type III InsP(3)R did reach a 2.5-fold higher level of activity. Although activation by InsP(3) was isoform-specific, regulation by ATP was similar for both isoforms. In the presence of 2 microM InsP(3), low ATP concentrations (<6 mM) increased the open probability and mean open time. High ATP concentrations (>6 mM) decreased channel activity. These results illustrate the complex nature of type III InsP(3)R regulation. Enhanced channel activity in the presence of high InsP(3) may be important during periods of prolonged stimulation, whereas allosteric modulation by ATP may help to modulate intracellular Ca(2+) signaling.     

Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion

In many cell types agonist-receptor activation leads to a rapid and transient release of Ca(2+) from intracellular stores via activation of inositol 1,4,5 trisphosphate (InsP(3)) receptors (InsP(3)Rs). Stimulated cells activate store- or receptor-operated calcium channels localized in the plasma membrane, allowing entry of extracellular calcium into the cytoplasm, and thus replenishment of intracellular calcium stores. Calcium entry must be finely regulated in order to prevent an excessive intracellular calcium increase. Junctate, an integral calcium binding protein of endo(sarco)plasmic reticulum membrane, (a) induces and/or stabilizes peripheral couplings between the ER and the plasma membrane, and (b) forms a supramolecular complex with the InsP(3)R and the canonical transient receptor potential protein (TRPC) 3 calcium entry channel. The full-length protein modulates both agonist-induced and store depletion-induced calcium entry, whereas its NH(2) terminus affects receptor-activated calcium entry. RNA interference to deplete cells of endogenous junctate, knocked down both agonist-activated calcium release from intracellular stores and calcium entry via TRPC3. These results demonstrate that junctate is a new protein involved in calcium homeostasis in eukaryotic cells.     

Chronic muscarinic stimulation of SH-SY5Y neuroblastoma cells suppresses inositol 1,4,5-trisphosphate action. Parallel inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ mobilization and inositol 1,4,5-trisphosphate binding.

The possibility that chronic activation of the phosphoinositide-mediated signaling pathway modifies the Ca(2+)-mobilizing action of inositol 1,4,5-trisphosphate (InsP3) was examined. SH-SY5Y human neuroblastoma cells were exposed to carbachol, permeabilized electrically, loaded with 45Ca2+, and 45Ca2+ mobilization in response to exogenous InsP3 was assessed. In control permeabilized cells, InsP3 released 65 +/- 2% of sequestered 45Ca2+ (EC50 = 0.32 +/- 0.05 microM). Pre-treatment with carbachol reduced both maximal InsP3-induced 45Ca2+ release (to 34 +/- 3%, with half-maximal and maximal inhibition at approximately 3 and 6 h, respectively) and the potency of InsP3 (EC50 = 0.92 +/- 0.13 microM). This inhibitory effect of carbachol was half-maximal at approximately 5 microM, was mediated by muscarinic receptors, and was reversible following withdrawal of agonist. Pretreatment with phorbol 12,13-dibutyrate did not alter the maximal effect of InsP3 but doubled its EC50. Evidence suggesting that the inhibitory effects of carbachol pretreatment resulted from altered Ca2+ homeostasis was not forthcoming; both 45Ca2+ uptake and release induced by ionomycin and thapsigargin were identical in control and pretreated permeabilized cells, as were the characteristics of reuptake of released Ca2+. In contrast, carbachol pretreatment, without altering the affinity of InsP3 (Kd = 64 +/- 7 nM), reduced the density of [32P]InsP3-binding sites from 2.0 +/- 0.1 to 1.0 +/- 0.1 pmol/mg protein with a time course essentially identical to that for the reduction in responsiveness to InsP3. This effect was not mimicked by pretreatment of cells with phorbol 12,13-dibutyrate. These data indicate that chronic activation of phosphoinositide hydrolysis can reduce the abundance of InsP3 receptors and that this causes a reduction in size of the InsP3-sensitive Ca2+ store. This modification, possibly in conjunction with a protein kinase C-mediated event, appears to account for the carbachol-induced suppression of InsP3 action. As intracellular InsP3 mass remained elevated above basal for at least 24 h after addition of carbachol, suppression of the Ca(2+)-mobilizing activity of InsP3 represents an important adaptive response to cell stimulation that can limit the extent to which intracellular Ca2+ is mobilized.     

Single-channel properties in endoplasmic reticulum membrane of recombinant type 3 inositol trisphosphate receptor

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP(3)R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP(3)R-3 are strikingly similar to those of Xenopus type 1 InsP(3)R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP(3)R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP(3)R isoforms is a mechanism to modify the temporal and spatial features of [Ca(2+)](i) signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP(3)Rs that have relatively similar Ca(2+) permeation properties.     

Do mammals make all their own inositol hexakisphosphate?

A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6 with [32P]ATP using a recombinant InsP6 kinase from Giardia lambia, followed by HPLC of the 32P-labelled products with an internal [3H]InsP7 standard. This assay was used to quantify InsP6 levels in a variety of biological samples.Concentrations of InsP6 in rat tissues varied from 10-20 microM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352 +/- 11 microM InsP6. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP6 levels per mg of protein similar to rat tissues. This amounted to 1.952 +/- 0.117 nmol InsP6 per culture dish, despite the cells being grown in serum shown to contain no detectable(less than 20 pmol per dish) InsP6. These results demonstrate that mammalian cells synthesize all their own InsP6. Human blood was analysed, and although the white cell fraction contained InsP6 at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP6 was detected (<1 nM InsP6). Human urine was also examined, and also contained no detectable (<5 nM) InsP6. These results suggest that dietary studies purporting to measure InsP6 at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP6 in the diet or applying it topically can produce health benefits by increasing extracellular InsP6 levels may require reassessment.     

The importance to chondrocyte differentiation of changes in expression of the multiple inositol polyphosphate phosphatase

It is important to both physiological and pathological osteogenesis to understand the significance of changes in gene expression in growth-plate chondrocytes that transit between the proliferative and hypertrophic states. MINPP is one such gene of interest. The Minpp protein dephosphorylates highly phosphorylated inositol signaling molecules InsP(5) and InsP(6). We show here that the ATDC5 chondrocyte progenitor cell line can recapitulate developmentally specific changes in MINPP expression previously only seen in longitudinal bone growth plates-both an initial 2-3-fold increase and a subsequent decrease back to initial levels during transition to hypertrophy. The increase in MINPP expression was accompanied by a 40% decrease in InsP(6) levels in ATDC5 cells. However, InsP(5) levels were not modified. Furthermore, throughout the hypertrophic phase, during which MINPP expression decreased, there were no alterations in InsP(5) and InsP(6) levels. We also created an ATDC5 line that stably overexpressed Minpp at 2-fold higher levels than in wild-type cells. This had no significant effect upon cellular levels of InsP(5) and InsP(6). Thus, substantial changes in MINPP expression can occur without a net effect upon InsP(5) and InsP(6) turnover in vivo. On the other hand, Minpp-overexpressing cells showed impaired chondrogenesis. We noted that the expression of alkaline phosphatase activity was inversely correlated with the expression of MINPP. The ATDC5 cells that overexpress Minpp failed to show an insulin-dependent increase in alkaline phosphatase levels, which presumably affects phosphate balance [J. Biol. Chem. 276 (2001) 33995], and may be the reason cellular differentiation was impaired. In any case, we conclude that Minpp is important to chondrocyte differentiation, but in a manner that is, surprisingly, independent of inositol polyphosphate turnover.     

A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex.

P400 protein is a 250 kd glycoprotein, characteristic of the cerebellum, which is accumulated at the endoplasmic reticulum, at the plasma membrane and at the post-synaptic density of Purkinje cells. In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that P400 protein is identical with cerebellar InsP3 receptor protein. InsP3 receptor was solubilized with Triton X-100 from a post-nuclear fraction of ddY mouse cerebellum and was purified with high yield by sequential column chromatography on DE52, heparin-agarose, lentil lectin-Sepharose and hydroxylapatite. In these chromatographies, P400 protein co-migrated completely with the InsP3 binding activity. The purified receptor is a 250 kd protein with a Bmax of 2.1 pmol/microgram and a KD of 83 nM. It reacted with three different monoclonal antibodies against P400 protein, indicating that P400 protein is the same substance as the InsP3 receptor (P400/InsP3 receptor protein). Electron microscopy of the purified receptor showed a square shape with sides approximately 25 nm long. Binding assays of the cerebella of Purkinje cell-degeneration (pcd) mice with [3H]InsP3 demonstrated that the InsP3 binding sites in the cerebellum are distributed exclusively on the Purkinje cells. Immunohistochemical analysis indicated that P400/InsP3 receptor is present at the dendrites, cell bodies, axons and synaptic boutons of the Purkinje cells.