Inositol Lipid Pathway Research Articles

The role of inositol 1,4,5-trisphosphate 5-phosphatase in inositol signaling in the CNS of larval Manduca sexta

Production of inositol 1,4,5-trisphosphate (IP 3) in cells results in the mobilization of intracellular calcium. Therefore, the dynamics of IP 3 metabolism is important for calcium dependent processes in cells. This report investigates the coupling of mAChRs to the inositol lipid pathway in the CNS of the larval Manduca sexta. Stimulation of intact abdominal ganglia prelabeled with [ 3H]-inositol using a muscarinic agonist, oxotremorine-M (oxo-M), increased total inositol phosphate levels in a dose dependent manner (EC 50=4.23 μM). These inositol phosphates consisted primarily of inositol 1,4-bisphosphate (IP 2) and inositol monophosphate (IP 1). Similarly, when nerve cord homogenates were provided with [ 3H]-phosphatidylinositol 4,5-bisphosphate ([ 3H]-PIP 2) (10–13 μM) the predominant products were IP 2 and IP 1. In contrast, incubation of purified membranes with 1 mM oxo-M in the presence of 100 μM GTPγS and [ 3H]-PIP 2 increased IP 3 levels, suggesting that the direct activation of phospholipase C (PLC) by mAChRs occurs in a membrane delimited process. Together, these results suggest that in the intact nerve cord and in crude homogenates, a cytosolic 5-phosphatase quickly metabolizes IP 3 to produce to IP 2 and IP 1. This enzyme was kinetically characterized using IP 3 (K m=43.7 μM, V max=864 pmoles/min/mg) and IP 4 (K m=0.93 μM, V max=300pmoles/min/mg) as substrates. The enzyme activity can be potently inhibited by two IP thiol compounds; IP 3S 3 (1,4,6) and IP 3S 3 (2,3,5), that show complex binding kinetics (Hill numbers<1) and can distinguish different forms of the 5-phosphatase in purified membranes. These two inhibitors could be very useful tools to determine the role of the inositol lipid pathway in neuroexcitability.

Nuclear lipid-dependent signal transduction in human osteosarcoma cells

The enzymes and substrates involved in phosphoinositide signal transduction which have been detected in the nucleus of several cell types have been demonstrated to be responsive to agonists. The complexity of this aspect of inositide function has been previously analyzed in some cell models characterized by a mitogenic or differentiating response to specific factors. An interesting experimental model is represented by human derived osteosarcoma Saos-2 cells, characterized by the expression of high affinity receptors for interleukin 1α (IL-1α), which is one of the most potent stimulators of bone resorption. In particular, we investigated the earliest intracellular events following the binding of IL-1α to its receptor, involving the inositide signal transduction pathway. Saos-2 cells present a partitioning of the phosphoi-nositidase (PLC) isoforms; in fact, the nucleus contains both PLC β 1 and γ 1, while the cytoplasm contains almost exclusively the γ 1 isoform. IL-1α evokes a rapid and transient increase of the PLC β 1 activity in the nucleus, which causes the hydrolysis of phosphatidylinositol mono- and bis-phosphate. In response to IL-1α, not only the canonical inositol lipid pathway appears to be involved; also the 3′-phosphorylated lipids generated by phosphatidylinositol 3-kinase (PI 3-K), which may act as second messengers, appear to be affected. In fact, Saos-2 cells present a nuclear PI 3-K activity which can be enhanced by the IL-1α treatment. Among the possible targets of the second messengers released by the nuclear PLC β 1 activation, we found that some protein kinase C isoforms, namely the ϵ and ζ, which are present within the nucleus, are activated after IL-1α exposure. These activated PKC isoforms, in turn, could modulate the activity of the transcription factor NFkB, which, 5 min after IL-lα treatment, has already translocated to the nucleus and bound to DNA to promote gene activation. The actual role of the inositide pathway in the Saos-2 cell function has also been investigated by utilizing cell clones transfected with the mouse sequence of the PLC β 1.

Inhibition of Protein Prenylation Down-Regulates Signalling by Inflammatory Mediators in Human Keratinocytes

Several inflammatory mediators have been shown to activate phospholipase C in human keratinocytes via GTP-binding protein-coupled receptors. Since GTP-binding proteins are prenylated proteins, we have examined the role of prenylation in signal transduction in HaCaT keratinocytes. Indirect inhibition of prenylation with the HMG CoA reductase inhibitors fluvastatin or compactin decreased bradykinin-stimulated inositol 1,4,5-triphosphate generation. This effect was abolished by mevalonic acid but not by serum, indicating a requirement for a non-sterol metabolite for signal generation. The BK response was also inhibited by zaragozic acids B and C, known inhibitors of prenyl protein transferases. These results suggest that protein prenylation may be a novel therapeutic target in dermatological conditions where an up-regulation of the inositol lipid pathway has been demonstrated.

The cloning and sequence of the C isoform of PtdIns4P 5-kinase.

In this study we describe the purification and sequencing of the C isoform of platelet PtdIns4P 5-kinase. Subsequently a cDNA was isolated from a human circulating-leucocyte library, which when sequenced was shown to contain all of the peptides identified in the purified protein. In addition, expression of this cDNA in bacteria led to the production of a protein which was recognized by specific monoclonal antibodies raised to the bovine brain enzyme [Brooksbank, Hutchings, Butcher, Irvine and Divecha (1993) Biochem. J. 291, 77-82] and also led to the appearance of PtdIns4P 5-kinase activity in the bacterial lysates. Interestingly, the cDNA showed no similarity to any of the previously cloned inositide kinases. A search of the DNA databases showed that two proteins from Saccharomyces cerevisiae shared close similarity to this enzyme, one of which, the mss4 gene product, has been implicated in the yeast inositol lipid pathway. These data suggest that the PtdIns4P 5-kinases are a new family of inositide kinases unrelated to the previously cloned phosphoinositide 3/4-kinases.

Organic Solvents Activate Human Platelets Through the Inositol Lipid-linked Signal Transduction System

It has recently been proposed that occupational exposure to organic solvents in vivo may lead to platelet activation and this has been substantiated by exposure of platelets to solvents in vitro. The present work was undertaken to study the effects of organic solvents on the platelet inositol lipid signal transduction system. Human platelets that had been prelabelled with [(32)P] P, were exposed to a saturated atmosphere of the organic solvents toluene, xylene or hexane. Extracts were analyzed for metabolites of the polyphosphoinositide cycle and ATP. All solvents studied induced a decrease in radioactivity in phosphatidylinositol 4,5-bisphosphate together with an increase in radioactivities in phosphatidylinositol 4-phosphate and phosphatidic acid. This is compatible with solvent-induced activation of the cells through the inositol lipid pathway. In cells exposed to toluene or xylene we could detect an increased level in inositol trisphosphates at 3 min of exposure. The solvent-induced changes in metabolic ATP could not explain the solvent-induced effects on the inositol lipid metabolism. It is concluded that the organic solvents toluene, xylene and hexane can activate human platelets through the inositol lipid-linked transmembrane signal system.

Age-Related Events in Human Active T Lymphocytes: Changes in the Phosphoinositidase C Activity

Since PHA-stimulated active T lymphocytes from aged humans showed changes in the metabolic pattern of inositol lipids in comparison with young subjects, we studied the possible role of phosphoinositidase C (PIC) in the generation of this phenomenon. The breakdown of exogenous [ 3H]phosphatidylinositol 4, 5-bisphosphate was found to be optimal at neutral pH and Ca ++ concentrations close to millimolar levels. Under these conditions PIC activity of resting lymphocytes did not differ in aged and young subjects, while, after short periods of PHA stimulation (up to 4 hr) the substrate hydrolysis was lower and delayed in the elderly group in comparison with that of controls. Our findings support the hypothesis that the age-related default of this enzyme, responsible for the age-related changes in the inositol lipid pathway of this peculiar subpopulation, could be involved, as a primary event, in the mechanisms leading to the reduced proliferative response of aged active T lymphocytes.

Evidence for nuclear phosphoinositidase C activity in the antiproliferative signals produced by interferon in Burkitt lymphoma cells

The influence of interferon alpha on nuclear phosphoinositidase C (PIC) in Daudi cells has been analysed. Results showed an early increase of PIC activity detectable within 90 min of interferon treatment concomitant with an increase of diacylglycerol (DAG) levels. Since the interferon-induced DAG production is not modified by the addition of propranolol, a compound known to inhibit production of DAG from phosphatidylcholine hydrolysis, it is suggested that the interferon antiproliferative signal is transduced into the nucleus via the inositol lipid pathway. A parallel analysis performed on intact cells showed a rapid inhibition of PIC activity accompanied by an increase of DAG level thus suggesting that interferon-generated signals at plasma-membrane level use pathways different from that of inositol lipids. A selected clone of Daudi cells resistant to interferon action provided a control for specificity of results.

A Role for G-proteins and Inositol Phosphate Signalling in Dictyostelium discoideum Slug Behaviour

Phototaxis by Dictyostelium discoideum slugs was disoriented in the presence of pertussis toxin, suggesting a role for pertussis-toxin-sensitive G-proteins in slug behaviour. The decrease in orientation accuracy was dependent on the concentration of this bacterial toxin. Pertussis toxin did not significantly affect slug thermotaxis, while cholera toxin had no significant effects on either slug phototaxis or thermotaxis. The addition of micromolar concentrations of lithium salts (LiCl or LiBr) resulted in disorientation of both phototaxis and positive thermotaxis by slugs. The corresponding sodium salts had no effect on slug behaviour, confirming that the effects were specific to lithium ions. The effects of lithium ions suggest a role for inositol phosphates as possible second messengers in Dictyostelium slug behaviour. Except for slight impairment of thermotaxis by d-sphingosine, we found no effects on slug behaviour of either phorbol 12-myristate 13-acetateorsphingolipids. We thus found little pharmacological evidence for a role in slug signal transduction of the diacylglycerol branch of the inositol lipid pathway.

A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts

Endothelin, a novel vasoactive peptide derived from endothelial cells (Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) Nature 332, 411-415), acts as a potent mitogen in Swiss 3T3 fibroblasts. The effect is dose-dependent with a half-maximal effect obtained at approximately 3 x 10(-11) M and is synergistically enhanced by a low concentration of insulin-like growth factor-I. Endothelin specifically binds to a single class of high affinity receptors in intact Swiss 3T3 cells and stimulates phospholipase C with the production of second messengers inositol trisphosphate and 1,2-diacylglycerol, leading to biphasic increases in the intracellular free Ca2+ concentration, as measured with a fluorescent indicator fura-2, phosphorylation of a putative cellular substrate of 80 kDa for protein kinase C, and transient expression of cellular protoonocogenes, c-fos and c-myc. Mitogenic effect of endothelin is markedly attenuated in phorbol ester-pretreated, protein kinase C-depleted cells. Endothelin-induced inositol phosphates production is not affected by removal of extracellular Ca2+, suggesting that endothelin-induced phospholipase C activation is not the result of stimulation of Ca2+ influx across the plasma membrane. These composite results indicate that the inositol lipid signaling pathway plays an important role in endothelin-induced mitogenesis in Swiss 3T3 fibroblasts. The mitogenic effect of endothelin is considerably smaller than that of bombesin, another well characterized mitogen acting through the inositol lipid pathway, despite comparable potencies in eliciting initial second messenger signals. In endothelin-treated cells, an increase in cellular 1,2-diacylglycerol content is transient, and cellular cyclic AMP content is reduced. By contrast, bombesin induces a more prolonged increase in cellular 1,2-diacylglycerol content and a slight increase in cellular cyclic AMP content. Because both 1,2-diacylglycerol and cyclic AMP are thought to serve as signals for promoting DNA synthesis in Swiss 3T3 fibroblasts, these differences in the signal generation may contribute to the differences in potencies between the two mitogens.

Transient permeabilization of endocrine cells: Inositol lipid metabolism

Publisher Summary Permeabilization by hypoosmotic shock treatment is a gentle method for the rapid labeling of large quantities of clonal endocrine GH 3 cells with [ 3 H] inositol, a substrate for the synthesis of Phosphatidylinositol (Ptdlns). Further work will be required to establish the mechanism for the basal and thyrotropin-releasing hormone (TRH)-stimulated increases in the labeling of Ptdlns, which could occur through enzyme-mediated base exchange as well as through Ptdlns-synthase activity. Both activities have been found in GH 3 cell membranes. The hypo-osmotic shock treatment (HOST) method may be used for the studies of other metabolites in the inositol lipid pathway. For example, preliminary studies in the laboratory have shown increased incorporation of label into CDP-diglyceride after HOST permeabilization in the presence of [ 3 H] CTP. The true strength of this method is that the treated cells grow normally in culture, making it possible to study the metabolism of a wide range of introduced substrates under a variety of growth conditions.

Tyrosine kinase-activating growth factors potentiate thrombin- and AIF4- -induced phosphoinositide breakdown in hamster fibroblasts. Evidence for positive cross-talk between the two mitogenic signaling pathways.

Basic fibroblast growth factor (FGF) and alpha-thrombin can stimulate DNA synthesis in Chinese hamster fibroblasts (CCL39) by two separate signaling pathways (Chambard, J.C., Paris, S., L'Allemain, G., and Pouysségur, J. (1987) Nature 326, 800-803) but can also act synergistically. We have examined whether this synergism might depend upon changes in inositol lipid metabolism. Indeed, FGF, which has no effect on its own on phosphoinositide hydrolysis, potentiates (by up to 2-fold) thrombin-induced formation of inositol phosphates. This enhancing effect is also observed upon direct activation by AIF4- of the GTP-binding protein coupled to phospholipase C, and is best revealed when phospholipase C is weakly stimulated. With low thrombin concentrations or with AIF4-, the formation of inositol phosphates is immediately increased with a marked reduction of the initial lag, whereas at high thrombin concentrations, the stimulation by FGF becomes pronounced only after desensitization of phospholipase C to thrombin. FGF-induced potentiation is not mimicked by calcium ionophores, but is likewise elicited by epidermal growth factor, platelet-derived growth factor, and to a lesser extent by insulin, other growth factors known to activate receptor tyrosine kinases. We therefore propose that the tyrosine kinase-activating growth factors enhance the coupling between GTP-binding protein and phospholipase C, presumably through the phosphorylation of one of these two proteins. Treatment of cells with pertussis toxin attenuates thrombin-induced phospholipase C activity but does not impede the potentiation by FGF. Comparison of the potentiating effects of FGF on inositol phosphate formation and on DNA synthesis suggests than an increased production of second messengers by the inositol lipid pathway in the first hours of stimulation might be, at least in part, responsible for the synergistic actions of FGF and thrombin on DNA synthesis.

Transmembrane signalling pathways initiating cell growth in fibroblasts.

The mechanisms of growth factor action were studied in a fibroblastic cell line capable of reversible growth arrest in G0-G1. This cell line, derived from Chinese hamster lung, can be stimulated to divide by a limited set of purified growth factors, including EGF, FGF, PDGF, alpha-thrombin (THR), serotonin (5-HT) and insulin. THR and 5-HT stimulate, via a G-protein (Gp), a polyphosphoinositide-specific phospholipase C (PtdIns(4,5)P2-PLC). In contrast, the mitogens EGF, FGF, PDGF, and insulin do not stimulate PtdIns(4,5)P2-PLC unless this pathway has been preactivated by THR or AlF-4. Finally, from the specific inhibitory action of pertussis toxin on THR- and 5-HT-induced DNA synthesis, and from the exploitation of the 5-HT pharmacological tools, we conclude that: (i) there are at least two distinct G-proteins involved in signalling growth: Gp, coupling receptors to PtdIns(4,5)P2-PLC, and Gi, coupling receptors negatively to adenylyl cyclase and probably to other unknown effector(s); (ii) activation of receptor-tyrosine kinases provides an alternate growth factor signalling pathway, independent of Gp- and Gi-mediated actions; and (iii) tyrosine kinases positively 'cross-communicate' with the inositol-lipid pathway (phosphorylation of Gp, PLC, PtdIns kinases...?).

Chapter 17 Analysis of receptor-coupled events in neuropeptide action using clonal cell lines

This chapter discusses the analysis of receptor-coupled events in neuropeptide action using clonal cell lines. Compared to other classes of chemical messengers, the peptides can be differentiated by their special relationship to the brain. The chapter focuses on the biochemical events coupled to peptide receptors to provide an overview of the pathways of receptor occupancy and biological responses. The cellular mechanisms underlying the diverse peripheral and central actions of peptides have unique characteristics that suggest new ways in which peptides can act as neural signals. A number of peptides are identified stimulants of the inositol lipid pathway. Thus, the application of molecular genetics to the expression of neuropeptide receptors in somatic cell lines may be expected to be one of the most promising routes to understanding peptide-mediated communication among cells in the brain.

Transduction in invertebrate photoreceptors: role of pigment bistability.

Transduction in invertebrate photoreceptors: role of pigment bistability.P Hillman, S Hochstein, and B MinkeP Hillman, S Hochstein, and B MinkePublished Online:01 Apr 1983https://doi.org/10.1152/physrev.1983.63.2.668MoreSectionsPDF (14 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Download PDF FiguresReferencesRelatedInformationCited ByThe Drosophila light-activated TRP and TRPL channels - Targets of the phosphoinositide signaling cascadeProgress in Retinal and Eye Research, Vol. 66Molecular and functional identification of a novel photopigment in Pecten ciliary photoreceptors26 January 2018 | The Journal of General Physiology, Vol. 150, No. 3The absence of attenuating effect of red light exposure on pre-existing melanopsin-driven post-illumination pupil responseVision Research, Vol. 124Intrinsically Photosensitive Retinal Ganglion CellsMichael Tri Hoang Do, and King-Wai Yau1 October 2010 | Physiological Reviews, Vol. 90, No. 4Does Melanopsin Bistability Have Physiological Consequences?1 October 2008 | Journal of Biological Rhythms, Vol. 23, No. 5Melanopsin-Dependent Nonvisual Responses: Evidence for Photopigment Bistability In Vivo29 June 2016 | Journal of Biological Rhythms, Vol. 22, No. 5Targeted Mutagenesis of the Farnesylation Site of Drosophila Gγe Disrupts Membrane Association of the G Protein βγ Complex and Affects the Light Sensitivity of the Visual System17 June 2004 | Journal of Biological Chemistry, Vol. 279, No. 35Melanopsin—Shedding Light on the Elusive Circadian Photopigment7 July 2009 | Chronobiology International, Vol. 21, No. 2Heterologous Expression of Limulus Rhodopsin23 June 2003 | Journal of Biological Chemistry, Vol. 278, No. 42Selective Photostimulation of Genetically ChARGed NeuronsNeuron, Vol. 33, No. 1Different ionic conductances are modulated during the late receptor potential and the prolonged depolarizing afterpotential in Hermissenda type A photoreceptorsJournal of Comparative Physiology A, Vol. 172, No. 1The cyclophilin homolog ninaA is a tissue-specific integral membrane protein required for the proper synthesis of a subset of Drosophila rhodopsinsCell, Vol. 65, No. 2Chapter 5 Inositol lipid pathway in fly photoreceptors: Excitation, calcium mobilization and retinal degenerationProgress in Retinal Research, Vol. 11Light and dark adaptation in fly photoreceptors: Duration and time integral of the impulse responseVision Research, Vol. 29, No. 10Ectopic expression of a minor Drosophila opsin in the major photoreceptor cell class: Distinguishing the role of primary receptor and cellular contextCell, Vol. 53, No. 3Lipid fluidity of the outer segment membranes from cephalopod retinaExperimental Eye Research, Vol. 40, No. 4Early processing of colour and motion in a mosaic visual systemNeuroscience Research Supplements, Vol. 2Early events in visual transduction in photoreceptorsNeuroscience Research Supplements, Vol. 2 More from this issue > Volume 63Issue 2April 1983Pages 668-772 Copyright & PermissionsCopyright © 1983 by American Physiological Societyhttps://doi.org/10.1152/physrev.1983.63.2.668PubMed6340134History Published online 1 April 1983 Published in print 1 April 1983 Metrics