Inositol Polyphosphate Production Research Articles

MiRNA-Induced Downregulation of IPMK in Macrophages Mediates Lipopolysaccharide-Triggered TLR4 Signaling.

Inositol polyphosphate multikinase (IPMK) is a pleiotropic enzyme responsible for the production of inositol polyphosphates and phosphoinositide. IPMK in macrophages was identified as a key factor for the full activation of the Toll-like receptor 4 (TLR4) signaling pathway and inflammation by directly interacting with tumor necrosis factor receptor-associated factor 6 (TRAF6). Here, dynamic changes of IPMK levels in lipopolysaccharide (LPS)-stimulated macrophages and their functional significance were investigated. Both the mRNA and protein levels of IPMK were acutely decreased in mouse and human macrophages when cells were stimulated with LPS for between 1 and 6 h. Analysis of the 3' untranslated region (UTR) of mouse IPMK mRNA revealed a highly conserved binding site for miR-181c. Transfection of miR-181c mimics into RAW 264.7 macrophages led to decreased IPMK 3'UTR-luciferase reporter activity and lowered endogenous IPMK levels. When the genomic deletion of a 33-bp fragment containing a putative miR-181c-binding site was introduced within the IPMK 3'UTR of RAW 264.7 macrophages (264.7Δ3'UTR), LPS-triggered downregulation of IPMK levels was prevented. LPS treatment in 264.7Δ3'UTR macrophages decreased TLR4-induced signaling and the expression of proinflammatory cytokines. In response to LPS stimulation, K63-linked ubiquitination of TRAF6 was impaired in 264.7Δ3'UTR macrophages, suggesting an action of IPMK in the suppression of TRAF6 activation. Therefore, our findings reveal that LPS-mediated suppression of IPMK regulates the full activation of TLR4 signaling and inflammation in macrophages.

The inositol polyphosphate kinase Ipk1 transcriptionally regulates mitochondrial functions in Candida albicans.

Inositol polyphosphates (IPs) is an important family of signaling molecules that regulate multiple cellular processes, such as chromatin remodeling, transcription and mRNA export. Inositol polyphosphate kinases, as the critical enzymes for production and transformation of IPs, directly determine the intracellular levels of IPs and therefore are involved in many cellular processes. However, its roles in Candida albicans, the leading fungal pathogen in human beings, remain to be investigated. In this study, we identified the inositol polyphosphate kinase Ipk1 in C. albicans and found that it localizes in the nucleus. Moreover, in the ipk1Δ/Δ mutant, the activity of mitochondrial respiratory chain complexes and the mitochondrial function was severely impaired, which were associated with down-regulation of mitochondrial function-related genes revealed by transcription profiling analysis. The ipk1Δ/Δ mutant also displayed hypersensitivity to a series of environmental stresses, such as antifungal drugs, oxidants, cell wall perturbing agents and macrophage attacks, followed by attenuation of virulence in a mouse systematic infection model. These findings firstly reported the importance of inositol polyphosphate kinase Ipk1 in C. albicans, especially its role in mitochondrial function maintenance and pathogenicity.

Signaling in lymphocyte activation.

The fate of T and B lymphocytes, the key cells that direct the adaptive immune response, is regulated by a diverse network of signal transduction pathways. The T- and B-cell antigen receptors are coupled to intracellular tyrosine kinases and adaptor molecules to control the metabolism of inositol phospholipids and calcium release. The production of inositol polyphosphates and lipid second messengers directs the activity of downstream guanine-nucleotide-binding proteins and protein and lipid kinases/phosphatases that control lymphocyte transcriptional and metabolic programs. Lymphocyte activation is modulated by costimulatory molecules and cytokines that elicit intracellular signaling that is integrated with the antigen-receptor-controlled pathways.

Melatonin and IP3-induced Ca2+ Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Inositol polyphosphates: a new frontier for regulating gene expression

Highly phosphorylated, soluble inositides are an emerging family of potential eukaryotic second messengers. The mechanisms for generating an outstanding diversity of mono- and pyrophosphorylated inositides have been recently elucidated and require a series of conserved lipases, kinases, and phosphatases. With several of the inositol kinases and the phospholipase C having access to the nucleus, roles for inositides in nuclear functions have been suggested. In support of this hypothesis, multiple studies have revealed the protein machines that are modulated by these inositides and found specific roles in nuclear physiology. In this paper, we review a novel paradigm for regulating gene expression at distinct steps by different inositide isomers. We discuss discoveries showing inositol polyphosphate regulation of gene expression at the level of transcription, chromatin remodeling, messenger ribonucleic acid (mRNA) editing, and mRNA export. Recent structural studies of inositol polyphosphate-binding proteins suggest the inositides modulate protein function as essential structural cofactors, triggers for allosteric or induced fit structural changes, and direct antagonistic competitors for other inositide ligands. We propose that the cell orchestrates the localized production of soluble inositol polyphosphates and inositol pyrophosphates to direct decisive and rapid signaling events. These insights also illustrate how extracellular stimuli might faithfully trigger the correct synchrony between gene expression steps and coordinate nuclear responses to changes in cellular environments.

A Molecular Basis for Inositol Polyphosphate Synthesis in Drosophila melanogaster

Metabolism of inositol 1,4,5-trisphosphate (I(1,4,5)P3) results in the production of diverse arrays of inositol polyphosphates (IPs), such as IP4, IP5, IP6) and PP-IP5. Insights into their synthesis in metazoans are reported here through molecular studies in the fruit fly, Drosophila melanogaster. Two I(1,4,5)P3 kinase gene products are implicated in initiating catabolism of these important IP regulators. We find dmIpk2 is a nucleocytoplasmic 6-/3-kinase that converts I(1,4,5)P3 to I(1,3,4,5,6)P5, and harbors 5-kinase activity toward I(1,3,4,6)P4, and dmIP3K is a 3-kinase that converts I(1,4,5)P3 to I(1,3,4,5)P4. To assess their relative roles in the cellular production of IPs we utilized complementation analysis, RNA interference, and overexpression studies. Heterologous expression of dmIpk2, but not dmIP3K, in ipk2 mutant yeast recapitulates phospholipase C-dependent cellular synthesis of IP6. Knockdown of dmIpk2 in Drosophila S2 cells and transgenic flies results in a significant reduction of IP6 levels; whereas depletion of dmIP3K, either alpha or beta isoforms or both, does not decrease IP6 synthesis but instead increases its production, possibly by expanding I(1,4,5)P3 pools. Similarly, knockdown of an I(1,4,5)P3 5-phosphatase results in significant increase in dmIpk2/dmIpk1-dependent IP6 synthesis. IP6 production depends on the I(1,3,4,5,6)P5 2-kinase activity of dmIpk1 and is increased in transgenic flies overexpressing dmIpk2. Our studies reveal that phosphatase and kinase regulation of I(1,4,5)P3 metabolic pools directly impinge on higher IP synthesis, and that the major route of IP6 synthesis depends on the activities of dmIpk2 and dmIpk1, but not dmIP3K, thereby challenging the role of IP3K in the genesis of higher IP messengers.

Lipids and signal transduction in the nucleus.

During the last few years a growing amount of data has accumulated showing phospholipid participation in nuclear signal transduction. Very recent data strongly support the hypothesis that signal transduction in the nucleus is autonomic. Local production of inositol polyphosphates, beginning with the activation of phospholipase C is required for their specific function in the nucleus. Enzymes which modify polyphosphoinositols may control gene expression. Much less information is available about the role of other lipids in nuclear signal transduction. The aim of this minireview is to stress what is currently known about nuclear lipids with respect to nuclear signal transduction.

Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+.

alpha-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca2+-dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca2+-dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca2+ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca2+ and is blocked by drugs depleting Ca2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca2+-independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin's effects and suggest that LTX-stimulated exocytosis depends upon the co-operative action of external and intracellular Ca2+ involving G proteins and PLC, whereas the Ca2+-independent release is largely non-vesicular.

Attenuation of LPA-Mediated Calcium Signaling and Inositol Polyphosphate Production in RAT-1 Fibroblasts Transformed by the V-SRC Oncogene

Alterations in cellular signaling underlie the transforming actions of many oncogenes. The vsrc oncogene tyrosine kinase, pp60vsrc, is known to alter multiple signal transduction pathways, including those involving phosphatidylinositol (PI) metabolism. In this study, we investigated the effects of vsrc-transformation on lysophosphatidic acid (LPA) receptor coupling to intracellular free calcium [Ca2+]iand PI turnover in rat-1 fibroblasts. In normal rat-1 cells, LPA rapidly elevated [Ca2+]i(EC50= 10nM). In contrast, the ability of LPA to mobilize calcium was markedly attenuated in rat-1-vsrccells. Further study revealed that the LPA-mediated generation of inositol (1,4,5)P3and other inositol polyphosphates was also markedly attenuated in the vsrc-transformed cells. Although LPA caused a transient reduction in the level of PI(4,5)P2in normal rat-1 cells, the agonist elevated the level of PI(4,5)P2in the vsrc-transformed cells. These findings demonstrate that vsrc-transformation alters the coupling of LPA receptors to PI turnover and calcium signaling in rat-1 cells, and point to G protein-coupled receptor systems as targets for modulation by the vsrc kinase.

Association of 1 alpha,25-dihydroxyvitamin D3-occupied vitamin D receptors with cellular membrane acceptance sites.

We previously reported nongenomic activation of ROS 17/2.8 cells by vitamin D metabolites (1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], 25-hydroxyvitamin D3, 22-oxa-calcitriol, etc.). The hormone 1 alpha,25-(OH)2D3, or calcitriol, mediated rapid transient changes in intracellular free calcium levels and concomitant stimulation of inositol polyphosphate and diacylglycerol production. These effects resemble the mechanism of cell activation induced by ligands with plasma membrane (PM) receptors. As preliminary studies indicated that PM isolated from ROS 17/2.8 cells lacked specific binding sites for calcitriol alone, we studied the association between calcitriol-occupied vitamin D receptors (VDR) and ROS 17/2.8 cellular membranes. Saturable binding to the PM and the endoplasmic reticulum (ER) of calcitriol-occupied VDR was demonstrated. Binding of the VDR-[3H]calcitriol complex was displaceable by nonradioactive VDR/calcitriol, but not by the unoccupied VDR or by calcitriol alone. ER binding, but not PM binding, was competitively inhibited by a peptide from the VDR sequence recognized by an ER protein, calreticulin, and by an anticalreticulin antibody. The monoclonal antibody (9A7) against the VDR inhibited PM and ER binding of the hormone-occupied VDR. These results were substantiated by studies using baculovirus-expressed human VDR for binding studies with the PM and ER and for immunoblot analysis. We conclude that specific PM and ER sites of association for calcitriol-occupied VDR exist and suggest that these associations could participate in the nongenomic rapid actions of 1 alpha,25-(OH)2D3.

Involvement of pertussis toxin-sensitive GTP-binding proteins in sphingosine 1-phosphate-induced activation of phospholipase CCa 2+ system in HL60 leukemia cells

Exogenous sphingosine 1-phosphate (S1P) induced Ca 2+ mobilization, in association with an increase in inositol polyphosphate production reflecting activation of phospholipase C in HL60 leukemia cells. The increase in intracellular Ca 2+ concentration ([Ca 2+] i) induced by S1P was inhibited by an appropriate treatment of the cells with pertussis toxin (PTX), U73122 (a phospholipase C inhibitor) or phorbol 12-myristate 13-acetate (PMA). In parallel with the Ca 2+ response, these agents also inhibited inositol polyphosphate production. The S1P-induced Ca 2+ response was also attenuated in the dibutyryl cAMP-induced differentiated cells, where GTP-binding protein-induced Ca 2+ response is suggested to be enhanced. Lysophosphatidic acid (LPA) also increased [Ca 2+] i in the cells, but the maximal response was about half of that of S1P, and furthermore PTX and dibutyryl cAMP treatment hardly affected the LPA-induced Ca 2+ mobilization. We conclude that exogenous S1P mobilizes Ca 2+ through phospholipase C activation. The S1P-induced enzyme activation is at least partly mediated by PTX-sensitive GTP-binding protein-coupled receptors which may be different from LPA receptors.

Refilling state of internal Ca 2+ stores is not the only intracellular signal stimulating Ca 2+ influx in human endothelial cells

To further analyse the role of the refilling state of internal Ca 2+ pools in the stimulation of Ca 2+ influx in human endothelial cells, we investigated the combined effect of thapsigargin (TG) and histamine on cytosolic Ca 2+ concentration ([Ca 2+] i) and inositol polyphosphate production. At normal extracellular Ca 2+ levels, TG induced a progressive and sustained elevation in [Ca 2+] i which was dose-dependently prevented by pretreatment with 1–10 μM histamine. Similarly, pretreatment with 0.1 and 1 μM TG suppressed histamine-induced Ca 2+ transients partially and totally, respectively. TG pretreatment did not alter the inositol triphosphate (IP 3) level liberated by histamine, but modified IP 3 metabolism by decreasing inositol biphosphate (IP 2) and increasing inositol monophosphate (IP i) contents. In the absence of Ca 2+ influx, 1 μM TG only induced a small transient increase in [Ca 2+] i whereas the Ca 2+ mobilization evoked by 10 μM histamine was unchanged. In both cases, the absence of any additional effect of either TG, histamine or 2 μM ionomycin indicated the complete depletion of Ca 2+ stores. The re-establishment of the transmembrane Ca 2+ gradient induced a transient rise in [Ca 2+] i. Its amplitude differed between histamine- and TG-treated cells. It was imposed by cell pretreatment and was selectively affected by changes in the membrane potential. At 5 mM external K +, the transient rise in [Ca 2+] i was more marked in histamine- than in TG-stimulated cells; this difference was suppressed by TG pretreatment. The presence of 130 mM external K + increased Ca 2+ entry in TG-treated cells but reduced it in histamine-stimulated cells. These results indicate that the refilling state of internal Ca 2+ stores does not constitute the single regulator of Ca 2+ influx. TG and histamine seem to activate Ca 2+ influx through distinct but interdependent pathways regulated by membrane potential.

Agonist-induced Ca2+ influx in human neutrophils is not mediated by production of inositol polyphosphates but by emptying of the intracellular Ca2+ stores.

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Myometrial characteristics of the syrian hamster uterine smooth muscle cell line, SHM.

We describe several characteristics of a novel smooth muscle cell line, SHM (Syrian hamster myometrium) derived from a primary uterine leiomyosarcoma which was induced by chronic estrogen plus androgen treatment of a female Syrian (golden) hamster. To determine the usefulness of the SHM cell line as a model for understanding myometrial function and its regulation, we have examined the morphologic and immunocytochemical properties of these cells, and the ability of uterotonic agonists to activate transmembrane signaling via phosphoinositide hydrolysis. The SHM cells exhibited a spindle-shape, smooth musclelike morphology when subconfluent, and a more compact, stellate shape at confluence. Like primary myocytes, SHM cells expressed the intermediate filament desmin and the contractile protein alpha smooth muscle actin, but not the epithelial antigen cytokeratin. Norepinephrine and bradykinin, which stimulate contraction and inositol polyphosphate production in the uterus, also stimulated inositol polyphosphate production in SHM cells. The maximal phosphoinositide signaling responses were lower in SHM cells compared with primary hamster uterine myocytes. We conclude that the SHM cell line exhibits primary uterine myocyte characteristics, and may therefore be a useful system for examining the mechanisms through which myometrial functions are regulated.

Enhancement of noradrenaline-induced inositol polyphosphate formation by glucocorticoids in rat vascular smooth muscle cells.

Glucocorticoids are known to regulate the contractility of vascular smooth muscle by increasing its response to noradrenaline. The molecular mechanisms for achieving this remain unclear. Recent results in our laboratory have demonstrated that glucocorticoids affect both alpha 1-adrenoceptor number and coupling to G proteins. Whether this leads to an increase in second-messenger production has to be established. The present experiments, therefore, report the effects of dexamethasone on inositol polyphosphate production in vascular smooth muscle cells in culture. Noradrenaline induced the release of inositol polyphosphates from prelabelled [3H]inositol phosphoinositides in the membrane in a dose-dependent manner. The concentration of noradrenaline which caused half-maximal response was 1.26 mumol/l. Prazosin inhibited noradrenaline-induced inositol monophosphate formation to 10.26 +/- 3.67% (mean +/- S.E.M.; P less than 0.01, n = 5) of control value whereas yohimbine reduced it to only 61.74 +/- 11.82% (P less than 0.05, n = 5), suggesting an action primarily through alpha 1-adrenergic receptors. Dexamethasone (100 nmol/l, 48 h) enhanced noradrenaline-induced inositol monophosphate, bisphosphate and trisphosphate formation up to twofold (P less than 0.001, n = 5). The enhancement of the response occurred despite the fact that dexamethasone reduced [3H]inositol prelabelling of membrane phosphoinositides by 49.5 +/- 9.9% (P less than 0.05, n = 3). The present results suggest that the potential action of glucocorticoids on vascular smooth muscle contractility is, at least in part, through controlling alpha 1-adrenoceptor-mediated second-messenger production.

Selective amplification of endothelin-stimulated inositol 1,4,5-trisphosphate and calcium signaling by v-src transformation of rat-1 fibroblasts.

The effects of the expression of the protein tyrosine kinase pp60v-src on endothelin- and thrombin-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and calcium responses were investigated in Rat-1 fibroblasts. The ability of endothelin-1 to induce the accumulation of these second messengers was dramatically amplified by v-src transformation, with 6- and 3-fold enhancements of the peak Ins(1,4,5)P3 and peak calcium responses, respectively. In contrast, thrombin-dependent responses were slightly reduced following v-src transformation, demonstrating that the augmentation of endothelin-stimulated signal transduction is a selective effect. The magnitude of the stimulated accumulation of Ins(1,4,5)P3 presumably depends upon both the functional activation of phospholipase C to produce Ins(1,4,5)P3, and the activity of the enzymes that metabolize Ins(1,4,5)P3. Although the metabolism of Ins(1,4,5)P3 was strikingly altered by expression of pp60v-src, with a bias towards the production of higher inositol polyphosphates that is consistent with an activated Ins(1,4,5)P3 3-kinase, this change could not account for the marked increase in endothelin-stimulated signaling induced by v-src transformation. This suggests that an effect of pp60v-src is expressed at the level of the plasma membrane, through an interaction with one or more components in the receptor/guanine nucleotide binding protein (G protein)/phospholipase C system that transduces the endothelin signal into Ins(1,4,5)P3 production. Preparation of membranes from normal and v-src-transformed cells showed that, while there was no change in the number of high-affinity endothelin binding sites, the release of Ins(1,4,5)P3 in response to guanine nucleotides and endothelin-1 was significantly increased following v-src transformation. In contrast, the Ins(1,4,5)P3 responses to thrombin and high Ca2+ concentrations were unaffected by transformation. Thus the selective interactions within the G protein system that couples the endothelin receptor to phospholipase C are potential sites at which the v-src transformation process may act to amplify endothelin-dependent Ins(1,4,5)P3 production.

P1-Purinergic Receptor-Mediated Modulation of TSH Actions on FRTL-5 Thyroid Cells: Possible Switching From cAMP Pathway to Inositol Phosphate-Ca System

Abstract In thyroid cells, a PI-agonist, via G1 like protein, enhanced a TSH-induced I−-efflux by intensifying a TSH-dependent inositol polyphosphate production followed by a Ca2+ mobilization, but diminished a TSH-dependent DNA synthesis by attenuating a TSH-dependent cAMP accumulation.

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes.

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45-60 s. The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.

Effect of angiotensin II and III on inositol polyphosphate production in differentiated NG108-15 hybrid cells

Neuroblastoma × glioma hybrid cells (NG108-15), differentiated by treatment with 1.5% dimethyl sulfoxide (DMSO) and 0.5% fetal bovine serum, were used to measure the effect of angiotensin II and III (ANG II and ANG III) on the generation of inositol polyphosphates. ANG II increased the synthesis of inositol monophosphates (IP1), inositol diphosphates (IP2), and inositol trisphosphates (IP3) with maximal responses observed at 300, 120, and 30 sec, respectively. The percent increases above basal values at the maximal responses were 140% ± 9% (IP1), 142% ± 4% (IP2), and 132% ± 4% (IP3). This effect was not attenuated by pretreatment of the cells with pertussis toxin. Furthermore, both ANG II and ANG III increased the production of inositol polyphosphates in a dose-dependent manner with ED50 values of 145 nM and 11 nM, respectively. We conclude that differentiated NG108-15 cells express an ANG III selective receptor that mediates phosphatidylinositol breakdown through a pertussis toxin insensitive G-protein.