Inositol Tris Research Articles

Human NCI-H295 adrenocortical carcinoma cells: a model for angiotensin-II-responsive aldosterone secretion.

Excessive secretion of aldosterone from the adrenal results in the most common form of endocrine hypertension. An understanding of the regulatory processes involved in aldosterone synthesis and release is needed to define the biomolecular mechanisms controlling excessive production of aldosterone. However, in vitro studies regarding the regulatory mechanisms of human aldosterone production have been limited because of difficulties in obtaining tissue and the subsequent isolation of aldosterone-secreting glomerulosa cells. Herein we describe an adrenocortical carcinoma cell line, NCI-H295, which provides a suitable angiotensin-II (AII)-responsive model system to investigate the acute and chronic regulation of aldosterone synthesis. The cells were characterized with regard to the effects of AII on second messenger systems, aldosterone release, and levels of aldosterone synthase (P450c18) mRNA. In the presence of lithium, AII caused a rapid, but transient, increase in the production of inositol tris- and bisphosphates, whereas a prolonged gradual accumulation of inositol monophosphate occurred. Treatment with AII resulted in a 4.5-fold increase in total inositol phosphates in a concentration-dependent manner and an increase in intracellular cytoplasmic free Ca2+. Significant increases in aldosterone (3.5-fold) were detected within 1 h of AII addition. Aldosterone release occurred in a concentration-and time-dependent manner. The type 1 AII (AT1) receptor was shown to be responsible for activation of phosphoinositidase-C, increased intracellular free Ca2+, and aldosterone production, as determined by use of the AT1 receptor antagonist DuP753. In addition, AII treatment resulted in a time-dependent increase in levels of P450c18 mRNA, as detected by RNAse protection assay. In summary, NCI-H295 cells provide a valuable model system to define mechanisms regulating human aldosterone production.

Increase in inositol tris-, pentakis- and hexakisphosphates by high K + stimulation in cultured rat cerebellar granule cells

Effects of high K + stimulation on inositol polyphosphate accumulations and intracellular free calcium concentration ([Ca 2+] i) were investigated in cultured rat cerebellar granule cells. When the [ 3H]inositol-labelled cells were stimulated with KCl, concentration-dependent accumulations of [ 3H]Ins(1,4,5)P 3, [ 3H]InsP 5 and [ 3H]InsP 6 were observed. Nifedipine (3 μM), a calcium channel antagonist, inhibited the high (KCl, 90 mM) K +-induced accumulations of these inositol polyphosphates. In Ca 2+-depleted and EGTA-containing (0.1 mM) medium, the high K +-induced inositol polyphosphate accumulation were completely inhibited. Similar results were also observed in the case of [Ca 2+] i. These results suggest that the rise in [Ca 2+] i caused by activation of voltage-dependent calcium channels plays an important roles in the high K +-induced accumulation of [ 3H]Ins(1,4,5)P 3, [ 3H]InsP 5 and [ 3H]InsP 6 in cultured rat cerebellar granule cells.

Receptor-recognized α2-macroglobulin-methylamine elevates intracellular calcium, inositol phosphates and cyclic AMP in murine peritoneal macrophages

Human plasma alpha 2-macroglobulin (alpha 2M) is a tetrameric proteinase inhibitor, which undergoes a conformational change upon reaction with either a proteinase or methylamine. As a result, a receptor recognition site is exposed on each subunit of the molecule enabling it to bind to its receptors on macrophages. We have used Fura-2-loaded murine peritoneal macrophages and digital video fluorescence microscopy to examine the effects of receptor binding on second messenger levels. alpha 2M-methylamine caused a rapid 2-4-fold increase in intracellular Ca2+ concentration ([Ca2+]i) within 5 s of binding to receptors. The agonists induced a focal increase in [Ca2+]i that spread out to other areas of the cell. The increase in [Ca2+]i was dependent on the alpha 2M-methylamine concentration and on the extracellular [Ca2+]. Both sinusoidal and transitory oscillations were observed, which varied from cell to cell. Neither alpha 2M nor boiled alpha 2M-methylamine, forms that are not recognized by the receptor, affected [Ca2+]i in peritoneal macrophages under identical conditions of incubation. The alpha 2M-methylamine-induced rise in [Ca2+]i was accompanied by a rapid and transient increase in macrophage inositol phosphates, including inositol tris- and tetrakis-phosphates. Native alpha 2M did not stimulate a rise in inositol phosphates. Finally, binding of alpha 2M-methylamine to macrophages increased cyclic AMP transiently. Thus receptor-recognized alpha-macroglobulins behave as agonists whose receptor binding causes stimulation of signal transduction pathways.

Determination of the Response of Melanoma Cells to Melanocyte Stimulating Hormone By31p Nuclear Magnetic Resonance Spectroscopy

Using living cells or tissues 31P nuclear magnetic resonance (NMR) spectroscopy can uniquely provide a real-time panoramic view of the major intracellular phosphate metabolites and continuously monitor changes in their concentrations. Hormone regulated cascades in many instances influence intracellular phosphate metabolism at various levels. Regulation of the respective key control enzymes is often mediated by second messengers, themselves phosphate metabolites, such as 3'5' cyclic adenosine monophosphate (cAMP), 3'5' cyclic guanosine monophosphate (cGMP), and inositol Tris phosphate (IP3). Moreover, protein phosphorylation/dephosphorylation reactions are also extensively involved in hormonal regulation. The consequent changes in the rates of the regulated processes, best known in the cases of glycogen and fat metabolism, are reflected in the rates of ATP synthesis and utilization as well as in the levels of phosphate containing intermediary metabolites. In this paper we describe an application of non-invasive 31P NMR spectroscopy for the examination of a signal transducing process and responsive cascades regulated by the melanocyte stimulating hormone (MSH) in live cultured M2R mouse melanoma cells. With proper modifications this technical approach can be adapted to the study of other cell systems.

Aminoglycoside antibiotics inhibit adriamycin uptake in rat peritoneal mast cells

The effects of cholecystokinin octapeptide (CCK8), bombesin and manganese (Mn2+) on phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis were studied in AR42J cells. One-half maximal stimulation of inositol monophosphate (InsP1) accumulation occurred at either 5 nM CCK8 or 5 nM bombesin, and maximal stimulation occurred at 30 nM for each agonist. Mn2+ did not alter basal PIP2 hydrolysis. However, addition of Mn2+ 5 min prior to stimulation with either CCK8 or bombesin for 60 min significantly attenuated [3H]InsP1 accumulation. Following brief periods of incubation with CCK8 (15 sec) Mn2+ significantly reduced inositol tris- and tetrakisphosphate accumulation. These data suggest that Mn2+ may participate in the regulation of CCK8- and bombesin-mediated generation of phosphoinositides.

Determination of inositol polyphosphates from human T-lymphocyte cell lines by anion-exchange high-performance liquid chromatography and post-column derivatization

The intracellular amounts of several inositol tris-, tetrakis- and pentakisphosphates and inositol hexakisphosphate were determined in resting and stimulated cells from human T-lymphocyte lines. The inositol polyphosphates were separated by anion-exchange high-performance liquid chromatography and were detected on-line by a recently developed post-column dye system. In the human T-lymphocyte cell line Jurkat, basal intracellular concentrations ranged between 25±10 pmol per 10 9 cells for inositol 1,4,5-trisphosphate to 6380±355 pmol per 10° cells for inositol hexakisphosphate. Similar basal concentrations were observed in the human T-lymphocyte cell line HPB.ALL, with the exception that inositol hexakisphosphate was approximately 665±10 pmol per 10 9 cells. Stimulation of the human T-lymphocyte cell line Jurkat via the T-cell receptor by a monoclonal antibody directed against the T-cell receptor-CD3 complex induced time-dependent changes in the intracellular concentrations of multiple inositol polyphosphate isomers, including inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4,6-tetrakisphosphate, an as yet unidentified inositol tetrakisphosphate isomer, inositol 1,3,4,5,6-pentakisphosphate, inositol 1,2,3,4,6-pentakisphosphate and dl-inositol 1,2,4,5,6-pentakisphosphate. Inositol 1,4,5-trisphosphate increased only transiently after 5 min, whereas dl-inositol 1,4,5,6-tetrakisphosphate (determined as the enantiomeric mixture) increased after 20 min.

Vanadate amplifies receptor-mediated accumulation of inositol trisphosphates and inhibits inositol tris- and tetrakis-phosphatase activities

Lithium ion, which inhibits hydrolytic degradation of inositol monophosphates, is the most common therapeutic agent used in the control of bipolar disorder. There exists evidence that elevated elemental vanadium levels may play an etiological role in at least some forms of manic-depression. Here we demonstrate that vanadate treatment of intact cells from several different clonal lines synergistically induces substantial augmentation in neurotransmitter receptor-mediated or growth factor receptor-triggered inositol trisphosphate accumulation in situ. Furthermore, studies done using cellular extracts indicate that effects of vanadate treatment in situ may be due to its ability to inhibit hydrolysis of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate in vitro. These results suggest that vanadate treatment may facilitate characterization of inositol phosphate metabolism and intracellular signaling.

Ion Chromatographic Determination of Inositol Tris- and Tetrakisphosphates in Rat Brain

Ion chromatography has been applied to the determination of inositol 2,4,5-trisphosphate (Ins 2,4,5-P3), inositol 1,3,4-trisphosphate (Ins 1,3,4-P3), inositol 1,4,5-trisphosphate (Ins 1,4,5-P3), and inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4). Other common polyanionic metabolites, including ATP and GTP, do not interfere with the determinations even at concentrations exceeding those normally found in tissue extracts. Assay of rat brain for Ins 1,4,5-P3 and Ins 1,3,4,5-P4 by the method of standard addition gives values which are within 10% of the amount calculated by external calibration. When rat brain homogenates are spiked with Ins 1,4,5-P3, the recoveries are greater than 90%. Treatment of animals with lithium chloride and pilocarpine produces increases in total inositol trisphosphates from 14 to 64 nMol/g wet weight, and increases in Ins 1,3,4,5-P4 from 0.9 to 18 nMol/g wet weight in tissue extracts obtained from the cerebral cortex. Using chemically suppressed conductivity, detection limits in brain are estimated to be 50 pMol/g wet weight for inositol trisphosphates and 22 pMol/g wet weight for inositol tetrakisphosphate.

Interactions between inositol tris- and tetrakis-phosphates. Effects on intracellular Ca2+ mobilization in SH-SY5Y cells.

The potential Ca2(+)-releasing activity of the inositol tetrakisphosphates Ins(1,3,4,6)P4 and DL-Ins(1,4,5,6)P4 and the inositol pentakisphosphate Ins(1,3,4,5,6)P5 and their effect on Ins(1,4,5)P3- and DL-Ins (1,3,4,5)P4-mediated Ca2+ release were examined in permeabilized SH-SY5Y human neuroblastoma cells. Neither DL-Ins(1,4,5,6)P4 nor Ins(1,3,4,5,6)P5 exhibit Ca2(+)-releasing activity at concentrations up to 10 microM, but Ins(1,3,4,6)P4 releases Ca2+ dose-dependently, with an EC50 value (conen, giving half-maximal effect) of 5.92 +/- 0.47 microM. Maximal response by this tetrakisphosphate (49 +/- 2.5%) is significantly less than that seen with Ins(1,4,5)P3 (60 +/- 3%) and is achieved at a concentration of 30 microM. In the presence of this concentration of Ins(1,3,4,6)P4 the EC50 value for Ins(1,4,5)P3-mediated Ca2+ release increases from 0.12 +/- 0.02 microM to 2.11 +/- 0.51 microM, providing evidence that this naturally occurring inositol tetrakisphosphate may recognize and exhibit its Ca2(+)-releasing activity via the Ins(1,4,5)P3 receptor. DL-Ins(1,3,4,5)P4, however, at its maximally effective concentration (10 microM) does not significantly affect Ins(1,4,5)P3-mediated Ca2+ release, and therefore appears to mediate its Ca2(+)-mobilizing action through a receptor distinct from that for Ins(1,4,5)P3.

Phosphoinositide breakdown is associated with Fc-gamma RII-mediated activation of 5'-lipoxygenase in murine eosinophils.

In this report we present data on the ability of murine eosinophils to generate inositol phosphate derivatives, and their relationship with the activation of 5'-lipoxygenase by a Fc-gamma R-dependent mechanism. The addition of anti-IgG F(ab')2 to mouse eosinophils, previously sensitized with IgG, induces inositol phosphate generation after 2 min and after 10 min of stimulation. Maximal generation of inositol tris and inositol tetrakis phosphate has been detected after 15 min of stimulation, and the optimal concentration of anti-IgG F(ab')2 was found to be 25 micrograms. Inositol tris phosphate formation is also observed at 5 min after the addition of the calcium ionophore A23187 (5 microM). We also report that neomycin, an inhibitor of phosphoinositide-phospholipase C, inhibits Fc-gamma R-mediated phosphoinositide breakdown in a dose-dependent manner (88% inhibition at 150 microM of neomycin). The possible involvement of phosphoinositide breakdown in the activation of 5'-lipoxygenase has been investigated. Using streptolysin-O permeabilized cells and different doses of neomycin that inhibit phosphoinositide breakdown, we have demonstrated a parallel decrease in LTC4 released by these cells, using either A23187 (86% inhibition at 200 microM of neomycin) or anti-IgG F(ab')2 (82.4% inhibition at 100 microM of neomycin). [Ca2+]i elevation has been observed by loading the cells with the fluorescent calcium indicator Fura-2 penta-acetoxy methyl ester and after stimulating with the anti-Fc-gamma RII mAb (2.4G2). It is likely that the activation of murine eosinophils by a Fc-gamma R mechanism stimulates phosphoinositide breakdown as a primary step that leads to the activation of murine 5'-lipoxygenase, producing the formation of leukotriene C4.

Glycosyl phosphatidylinositol in thyroid: cell signalling or protein anchor?

During the last 10 years, attention has been focused on the stimulation by various agonists of the hydrolysis of phosphatidylinositol bis-phosphate into the second messengers inositol tris phosphate and diacylglycerol. Two other aspects of the metabolism of phosphoinositides were therefore not paid sufficient attention. The first one was the release by insulin of a glycosyl inositolphosphate from a glycosyl phosphatidylinositol, the hydrosoluble product being able to reproduce some of the hormone effects; the second was the discovery that several membrane proteins were anchored via a glycosyl phosphatidylinositol. For over 20 years, we have been interested in the effect of thyreostimulin (TSH) on the turnover of phosphatidylinositol in pig thyrocyte. As this effect did not seem to result from the hydrolysis of phosphatidylinositol bis-phosphate we explored another possibility, the synthesis of glycosyl inositol-phosphate. We have shown that, in cultured pig thyrocytes, TSH stimulates the release of the polar head of a glycosyl phosphatidylinositol. This soluble glycosyl inositol-phosphate which acts as insulin on adipocyte, modulates the cAMP accumulation and iodine metabolism in thyrocytes and could be held responsible for the cAMP independent effects of TSH. However, we do not yet know if there is a link between the glycosyl phosphatidylinositol sensitive to TSH and the anchor membrane protein. To date, the amount of 2 of these proteins: NAD glyco-hydrolase in thyroid cell membranes and heparan sulfate proteoglycan have been shown to be increased by TSH treatment of thyroid cells.

Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain

We have previously shown that Ins(1,3,4,5)P4 is degraded to Ins(1,4,5)P3 by a soluble Ins(1,3,4,5)P4 3-phosphatase from pig brain [Höer, Kwiatkowski, Seib, Rosenthal, Schultz & Oberdisse (1988) Biochem. Biophys. Res. Commun. 154, 668-675]. Here we present some properties of this enzyme using [5-32P]Ins(1,3,4,5)P4 as substrate. The molecular mass, estimated by gel filtration chromatography on a Superose 6 column, was determined to be 36 kDa. The 3-phosphatase showed a high affinity towards the substrate Ins(1,3,4,5)P4 (Km approximately 400 nM); the Vmax. of the freshly prepared enzyme was 2 nmol/min per mg of protein. The influence of Ins(1,4,5)P3 and Ins(1,3,4)P3, the reaction products of Ins(1,3,4,5)P4 hydrolysis by either 3- or 5-phosphatase respectively, on the 3-phosphatase was tested. Both isomers inhibited the enzyme, with Ki values of about 2 microM and 1.75 microM for Ins(1,3,4)P3 and Ins(1,4,5)P3 respectively. Enzyme activity was not influenced by Mg2+ up to 30 mM or Ca2+ up to 1 mM. Commercially available Ins(3,4,5,6)P4 from turkey erythrocytes produced a marked inhibition of the 3-phosphatase (Ki approximately 500 nM). Significant inhibitory effects on enzyme activity were also found with GTP and the pyrimidine nucleotides UTP and CTP. The kinetic data presented here suggest that the Ins(1,3,4,5)P4 3-phosphatase may be regulated by the intracellular concentrations of inositol tris- and tetrakis-phosphates.

Ca2+]i rises via G protein during regulatory volume decrease in rabbit proximal tubule cells

Although animal cells swell in hypotonic medium, their volume is subsequently regulated by a net loss of KCl via Ca2(+)-dependent channels. A rise in intracellular free calcium ([Ca2+]i) thus appears to be an initial event in the adaptation of external tonicity, although details of this mechanism are not known. To investigate cell volume regulation, we measured [Ca2+]i (by use of fura-2) and cell diameters in single cells of cultured renal proximal convoluted tubule. We found that a rapid rise in [Ca2+]i occurred after cells were exposed to hypotonic solution (250 mosM) from 95.8 +/- 3.8 to 468.2 +/- 24 nM (n = 16). The rise in [Ca2+]i was not observed in cells exposed to Ca2(+)-free medium, and exposure to isotonic high-K or low-Na medium did not elicit a rise in [Ca2+]i, suggesting that this rise was a result of Ca2+ influx and not via voltage-dependent Ca2+ influx or decrease of Ca2+ efflux via Na(+)-Ca2+ pump. Pretreatment of cells with pertussis toxin dose dependently blocked the rise in [Ca2+]i. The hypotonic solution enhanced accumulations of inositol tris- and tetra-phosphate after a 1-min exposure. Studies that measured cell diameters suggest that recovery of cell volume may include the rise in [Ca2+]i. These data suggest that the regulatory volume decrease of proximal tubule cells involves a pertussis toxin-sensitive guanine nucleotide binding protein-operated Ca2+ influx.

Analysis of free inositol trisphosphate in heart tissue by FPLC and isotachophoresis

Recent, increasing interest in inositol tris(1,4,5)phosphate (IP 3) turnover and metabolism has led to a need for a fast and quantitative determination of this compound in various tissues. These requirements are fullfilled by separation in different steps (FPLC and Sephadex G-10) culminating in further separation and quantification by isotachophoresis. Isotachophoresis means a migration of ion species of the same sign in an electrical field with all ions moving with the same velocity. The migration takes place when an electrical field is applied to a system of electrolytes of specific design. Detection was carried out by monitoring conductivity changes. The method is highly sensitive and allows measurements of IP 3 in the pmolar range. Linearity was demonstrated over a wide range, 50–4500 pmol. The total imprecision was low with a coefficient of variation of 3%. When determining in biological tissue the recovery was estimated to be close to 100%. The mean content of IP 3 in 15 rat heart specimens was 44.3 pmol/mg dry weight corresponding to tissue samples in the order of 4 mg wet weight. By noradrenaline stimulation myocardial content of IP 3 increased by 50%.

Ram spermatozoa produce inositol 1,4,5-trisphosphate but not inositol 1,3,4,5-tetrakisphosphate during the Ca 2+/ionophore-induced acrosome reaction

An investigation was made of the production of inositol tris- and tetrakisphosphates concomitant with ionosphere-stimulated breakdown of PtdIns(4,5)P 2 in ram spermatozoa. As spermatozoa displayed very low rates of incorporation of [ 3H]inositol into their phosphoinsitides, the studies were carried out using 32P-labelled cells. Using a specially developed procedure, inositol tris- and tetrakisphosphates were isolated, free of labelled ATP and P i,; they were then separated from each other (and from other minor labelled compounds) and analysed, using ionophoresis and HPLC. Levels of 32P-labelled material with the chromatographic characterizatics of Ins(1,4,5)P 3 were very low in untreated cells, but rose sharply with ionophore treatment, in parallel with rapid PtdInsP 2 breakdown. No 32P-labelled material with the characteristics of Ins(1,3,4,5)P 4 or Ins(1,3,4,)P 3 was found, and there was no evidence for phosphorylation of Ins(1,4,5)P 3 in sperm homogenates. The implications of our findings are discussed with respect to the physiological modulation of ca 2+ influx that is required to initiate the acrosome reaction at fertilization.

Total synthesis of [formula omitted]-inositol 2,3,5-trisphosphate: A [formula omitted]-inositol 1,4,5-trisphosphate analogue from benzene [formula omitted] photo-oxidation

The inositol tris- and tetrakis-phosphate analogues ( 4) and ( 14) have been synthesised from benzene by a sequence including reaction of singlet oxygen with a trans -cyclohexa-3,5-diene-1,2-diol ( 3) derivative.

A Novel Extracellular Nucleotide Receptor Coupled to Phosphoinositidase-C in Pituitary Cells*

In primary cultures of sheep pituitary cells extracellular nucleotides stimulated rapid increases in inositol tris- and bisphosphate, accompanied by intracellular Ca2+ mobilization. A similar stimulation of inositol phosphate production by extracellular nucleotides was observed in rat and baboon pituitary cells. The inositol phosphate response to nucleotides was greater than that elicited by any of the known hypothalamic releasing peptides. UTP, ATP, and ATP gamma S were the most potent agonists, with EC50 values for inositol phosphate production of 1.2, 2.6, and 2.7 microM. The relative potencies of a range of nucleotides indicates that the pharmacological specificity of the pituitary nucleotide receptor is different from that of the previously characterized P2X and P2Y purinoceptors present in other tissues. Increasing extracellular Mg2+ concentrations caused a shift to the right of the ATP dose-response curves, indicating that the predominantly active agonist species is not MgATP and may be ATP4-. In the absence of both Ca2+ and Mg2+ (1 mM EDTA) ATP stimulated inositol phosphate production with high potency (EC50 = 200 nM), indicating that an ectokinase or ecto-ATPase reaction is not involved in its mode of action. Phosphoinositidase-C activation by ATP was insensitive to pertussis toxin. The magnitude of the inositol phosphate and 45Ca2+ responses to extracellular nucleotides indicates that a substantial fraction of the cells in primary pituitary cultures bears nucleotide receptors. None of the major pituitary hormones appear to be released by extracellular nucleotides. The cell types in the pituitary that bear these nucleotide receptors are at present unidentified.

The effect of non-NMDA antagonists and phorbol esters on excitatory amino acid stimulated inositol phosphate formation in rat cerebral cortex

The effects of various modulators of excitatory amino acid stimulated inositol phosphate (IP) formation were investigated in slices of rat cerebral cortex. Both quisqualic acid (Quis) and ibotenic acid (Ibo) stimulated IP formation in a dose-dependent manner. Quis (0.3mM) stimulated the rapid formation of inositol bis, tris and tetrakis-phosphates, with a slower linear rise in the monophosphate which plateaued after 20 min. The responses to both Ibo (0.3 mM) and Quis (0.3 mM) were dose-dependently inhibited by phorbol dibutyrate (PDBu); the Ibo response was particularly sensitive to PDBu, with a 90% inhibition of the response at 1 ?M and an IC(50) of 200 nM; Quis stimulated IP formation was less sensitive with a 50% inhibition observed at 10 ?M PDBu. PMA (1 ?M) and dioctanoylglycerol (30 ?M) also inhibited Ibo-stimulated IP formation though 4-?-phorbol (10 ?M) was ineffective. The inhibition by PDBu was reversed with staurosporine (10 ?M) and polymyxin (10 ?M), and both protein kinase C inhibitors potentiated the Ibo response in the absence of PDBu. The putative Quis-kainate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and 6-cyano-7-nitroquinoxaline-2,3-dione did not inhibit the responses to either Quis or Ibo at a concentration of 100 ?M. Both responses were, however, inhibited by dl-2-amino-4-phosphonobutyric acid and O- phospho- l -serine (IC(50)s were 1-2 mM), and also by the putative Quis receptor agonist dl-?-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA; IC(50) 10?M). Our data suggest that excitatory amino acid stimulated IP formation in rat cerebral cortex is via a site distinct from previously defined amino acid receptors.

Arachidonic acid evokes inositol phospholipid hydrolysis in astrocytes

Astrocyte cultures prelabelled with [ 3pH]inositol were exposed to arachidonic acid (AA) in the presence and absence of various agonists. AA alone evoked a dose-dependent increase in the accumulation of inositol phosphates (IP), an effect not secondary to eicosanoid synthesis and release but which was abolished by EGTA. Separation of the IP revealed that AA stimulated increases in inositol tris-, bis- and monophosphates. IP formation evoked by carbachol or norepinephrine was additive with AA, whereas IP formation by platelet activating factor (PAF) or ATP was non-additive with AA. These results suggest that AA released upon stimulation of astrocytes or other cells in the CNS could initiate and/or amplify intercellular signalling. Arachidonate; Inositol phosphate; Astrocyte; Phospholipase C; Receptor; Cell signaling

Inositol tris- and polyphosphate formation during chemotaxis of Dictyostelium

Using the technique of HPLC with Partisil SAX columns, we have found that stimulation of amoebae of Dictyostelium discoideum with the chemoattractant cyclic AMP induces the rapid accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 5 s. A smaller HPLC peak (designated P3) that elutes just after the Ins(1,4,5)P3 peak accumulates more slowly to a maximum at 20 s. In control studies, the changes in Ins(1,4,5)P3 were shown not to be due to varying recovery from the cell extracts and a comparison of reverse-phase and Partisil SAX HPLC columns showed similar values for determinations by either method. The involvement of a G-protein in this chemotactic system was confirmed by the finding that accumulation of Ins(1,4,5)P3 was elicited by the addition of GTP gamma S (5'-[gamma-thio]triphosphate) to saponin-permeabilized amoebae. A study of the changes in the lipid-soluble phosphatidyl inositol phosphates demonstrated that cyclic AMP also stimulated a rapid loss of radioactivity from 32P-labelled phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which corresponded in its timing to the rise in Ins(1,4,5)P3, indicating that a phosphoinositidase C (phospholipase C) is present that can be stimulated by occupation of the cell surface cyclic AMP receptors.