Inositol Lipid Signalling System Research Articles

Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes.

Upon stimulation of renal cortical slices with hepatocyte growth factor (HGF), inositol lipid metabolism was studied in basal-lateral plasma membranes (BLM) and brush-border plasma membranes (BBM). Whereas in BLM rapid increases in 1,2-diacylglycerol, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) were observed, suggesting that in BLM HGF activates both phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), in BBM only HGF-induced transient accumulation of PtdIns3P was seen, which was temporarily delayed from signalling events in BLM and could be blocked by the PtdIns-specific-PLC inhibitor ET-18-OCH(3) and the calpain inhibitor calpeptin, suggesting that 3-kinase activation in BBM lies downstream of PLC activation in BLM and is a calpain-mediated event. Moreover, the increase in immunoprecipitable PI3K-C2 beta activity, which is sensitive to wortmannin (10 nM) and shows strong preference for PtdIns over PtdIns4P as a substrate, was observed only in BBM upon stimulation of renal cortical slices with HGF and could be mimicked by the Ca(2+) ionophore A23187 and blocked by the cell-penetrant Ca(2+) chelator BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)]. On Western blots PI3K-C2 beta revealed a single immunoreactive band of 180 kDa in BLM and BBM, while after stimulation with HGF a gel shift of 18 kDa was noticed only in BBM, suggesting that the observed enzyme activation is achieved by proteolysis. When BBM were subjected to short-term (15 min) exposure to mu-calpain, a similar gel shift together with an increase in PI3K-C2 beta activity was observed, when compared with the BBM harvested after HGF stimulation. The above-mentioned gel shift and increase in PI3K-C2 beta activity could be prevented by the calpain inhibitor calpeptin. The data presented in this report show that in renal cells there is a spatial separation of the inositol lipid signalling system between BLM and BBM, and that HGF causes activation of PLC and PI3K primarily in BLM, which leads to calpain-mediated activation of PI3K-C2 beta in BBM with a concomitant increase in PtdIns3P.

Bioactive lipids and gene expression in neuronal plasticity.

Excitable membranes are a reservoir of bioactive lipids that are contained as part of the structure of phospholipids. Signals such as those elicited by receptor occupancy trigger, via the activation of phospholipases, the release of phospholipid moieties. These enzymes are under control of receptors, calcium ions, protein kinase/phosphatase cascades, and of other signaling mechanisms which still need to be better characterized. The best defined regulatory events of these enzymes are those controlled by the seven transmembrane domain superfamily of receptors that, through G proteins, activate phospholipase C (PLC). This enzyme predominantly hydrolyzes phosphatidylinositol 4′, 5′bisphosphate (PIP2), resulting in the production of the second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). These two messengers of inositol lipids represent a bifurcating, informational system inside the cell. The synaptic inositol lipid signaling system is activated by neurotransmitters, growth factors and drugs. IP3 promotes intracellular Ca2+ mobilization by binding to a specific receptor in the endoplasmic reticulum, which in turn enhances calcium-dependent cellular processes.

Isolation, cloning, and characterisation of a trp homologue from squid (Loligo forbesi) photoreceptor membranes.

The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92-kDa component isolated from a detergent-insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full-length cDNA, encoding the 92-kDa component, using both cDNA library screening and 5'-rapid amplification of cDNA ends (5'-RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of the Drosophila trp and trpl genes. Greatest variation from the Drosophila Trp protein is seen in the carboxyl-terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl-terminal domain has been shown to be in the regulation of several ligand-gated channels. The carboxyl-terminal domain has been expressed and shown to interact with calmodulin in a calcium-dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels involved in calcium signalling.

Receptor-induced heterologous desensitization of receptor-regulated phospholipase C

Activation of the P2Y purinoceptor on turkey erythrocytes results in a G11-mediated activation of a phospholipase C-β isoenzyme and hydrolysis of polyphosphoinositides. The role of the protein kinase C and Ca2+-mobilizing arms of the inositol lipid signalling cascade in P2Y purinoceptor-induced desensitization of phospholipase C has been examined using erythrocytes as a model system. Preincubation of intact erythrocytes with either the P2Y purinoceptor agonist, ADPβS, or the protein kinase C-activating phorbol ester, phorbol 12-myristate, 13 acetate (PMA), resulted in a time of preincubation-dependent decrease in guanine nucleotide-, P2Y purinoceptor-, and β-adrenoceptor-stimulated phospholipase C activities in membranes isolated from these cells. The extent of heterologous desensitization induced by ADPβS and PMA were additive suggesting that they did not share a common mechanism. A lack of involvement of activation of protein kinase C in P2Y purinoceptor-induced heterologous desensitization was further supported by the observation that although protein kinase C inhibitors or down-regulation of protein kinase C resulted in a loss of PMA-induced desensitization, neither treatment affected the extent of P2Y purinoceptor-induced desensitization. In addition, elevation of intracellular Ca2+ or prevention of its elevation did not induce heterologous desensitization and had no effect on the desensitization induced by ADPβS. Thus, neither the protein kinase C nor Ca2+ mobilizing arms of the inositol lipid signaling pathway appear to be involved in P2Y purinoceptor promoted heterologous desensitization of phospholipase C. These results are consistent with the existence of a novel feedback pathway for agonist-induced heterologous desensitization of a second messenger generating enzyme. Preincubation of cells with ADPβS or the β-adrenoceptor agonist, isoproterenol, followed by rechallenge with each of the receptor agonist revealed that receptor-specific desentization occurs in addition to heterologous desensitization. Thus, multiple mechanisms account for agonist-induced desentization of the inositol lipid signalling system of turkey erythrocytes.

Receptor-induced heterologous desensitization of receptor-regulated phospholipase C

Activation of the P 2Y purinoceptor on turkey erythrocytes results in a G 11-mediated activation of a phospholipase C-β isoenzyme and hydrolysis of polyphosphoinositides. The role of the protein kinase C and Ca 2+-mobilizing arms of the inositol lipid signalling cascade in P 2Y purinoceptor-induced desensitization of phospholipase C has been examined using erythrocytes as a model system. Preincubation of intact erythrocytes with either the P 2Y purinoceptor agonist, ADPβS, or the protein kinase C-activating phorbol ester, phorbol 12-myristate, 13 acetate (PMA), resulted in a time of preincubation-dependent decrease in guanine nucleotide-, P 2Y purinoceptor-, and β-adrenoceptor-stimulated phospholipase C activities in membranes isolated from these cells. The extent of heterologous desensitization induced by ADPβS and PMA were additive suggesting that they did not share a common mechanism. A lack of involvement of activation of protein kinase C in P 2Y purinoceptor-induced heterologous desensitization was further supported by the observation that although protein kinase C inhibitors or down-regulation of protein kinase C resulted in a loss of PMA-induced desensitization, neither treatment affected the extent of P 2Y purinoceptor-induced desensitization. In addition, elevation of intracellular Ca 2+ or prevention of its elevation did not induce heterologous desensitization and had no effect on the desensitization induced by ADPβS. Thus, neither the protein kinase C nor Ca 2+ mobilizing arms of the inositol lipid signaling pathway appear to be involved in P 2Y purinoceptor promoted heterologous desensitization of phospholipase C. These results are consistent with the existence of a novel feedback pathway for agonist-induced heterologous desensitization of a second messenger generating enzyme. Preincubation of cells with ADPβS or the β-adrenoceptor agonist, isoproterenol, followed by rechallenge with each of the receptor agonist revealed that receptor-specific desentization occurs in addition to heterologous desensitization. Thus, multiple mechanisms account for agonist-induced desentization of the inositol lipid signalling system of turkey erythrocytes.

Involvement of non-muscarinic receptors in phosphoinositide signalling during soman-induced seizures

Previous investigations have indicated that soman-induced convulsions involve the inositol lipid signalling system. We previously reported that 10 min after the onset of seizures, inositol, 1,4,5-triphosphate (IP 3) build-up was coupled to activation of non-muscarinic receptor subtypes. In the present study, we demonstrate that (1) in addition to muscarinic receptors, histamine H 1 subtypes and glutamate metabotropic receptors contribute to the first IP 3 increase (first 10 min of seizures) and (2) the histamine H 1 subtype and glutamate metabotropic receptors are also involved in the second step of inositol phosphate response (after 10 min of seizures). α 1-adrenoceptor and 5-HT 2 receptors, known to be coupled to phosphoinositide turnover, did not participate in soman-induced IP 3 response. Neurochemical interactions between cholinergic, histamine H 1 and glutamate metabotropic systems, responsible of the phosphoinositide hydrolysis under soman are envisaged.

Coordination of Ca2+ Signaling by Intercellular Propagation of Ca2+ Waves in the Intact Liver

Activation of the inositol lipid signaling system results in cytosolic Ca2+ oscillations and intra- and intercellular Ca2+ waves in many isolated cell preparations. However, this form of temporal and spatial organization of signaling has not been demonstrated in intact tissues. Digital imaging fluorescence microscopy was used to monitor Ca2+ at the cellular and subcellular level in intact perfused rat liver loaded with fluorescent Ca2+ indicators. Perfusion with low doses of vasopressin induced oscillations of hepatocyte Ca2+ that were coordinated across entire lobules of the liver by propagation of Ca2+ waves along the hepatic plates. At the subcellular level these periodic Ca2+ waves initiated from the sinusoidal domain of cells within the periportal region and propagated radially across cell-cell contacts into the pericentral region, or until terminated by annihilation collision with other Ca2+ wave fronts. With increasing agonist dose, the frequency but not the amplitude of the Ca2+ waves increased. Intracellular Ca2+ wave rates were constant, but transcellular signal propagation was determined by agonist dose, giving rise to a dose-dependent increase in the rate at which Ca2+ waves spread through the liver. At high vasopressin doses, a single Ca2+ wave was observed and the direction of Ca2+ wave propagation was reversed, initiating in the pericentral region and spreading to the periportal region. It is concluded that intercellular Ca2+ waves may provide a mechanism to coordinate responses across the functional units of the liver.

Antioxidants impair the coupling of cell-surface ligand receptors to the inositol lipid signalling pathway in human T lymphocytes but not in jurkat T lymphoblastic leukemia cells. Evidence that leukotrienes are not involved in the coupling mechanism

Ligands including phytohaemagglutinin (PHA) and anti-CD3 monoclonal antibodies trigger the generation of inositol lipid-derived second messengers following their binding to cell-surface structures of human T lymphoid cells. Previous evidence has suggested that the generation of leukotrienes may play an intermediary role in coupling the ligation of T lymphoid cell-surface structures to the inositol lipid signalling system in these cells (A.R. Mire-Sluis et al. (1989) FEBS Lett. 258, 84–88). Here we have studied the actions of two novel selective leukotriene biosynthesis inhibitors, MK 886 and BW A4C and of two general lipid soluble antioxidants, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) on this pathway. Neither MK 886 nor BW A4C abrogated stimulation of inositol lipid breakdown following PHA or anti CD3 treatment of T lymphocytes. By contrast, this pathway was inhibited by BHT and BHA. These observations, together with our failure to demonstrate the generation of lipoxygenase products following PHA stimulation of T lymphocytes, suggests that an antioxidant-sensitive step other than the generation of leukotrienes plays a critical role in coupling cell-surface receptors to the inositol lipid signalling system in these cells. By contrast none of these inhibitors abrogated ligand-stimulated inositol lipid signalling in Jurkat T acute lymphoblastic leukaemia cells. These results suggest a heterogeneity in the organization of the signal transduction machinery in lymphoid cells at different stages of differentiation.

Beta gamma-subunit activation of G-protein-regulated phospholipase C.

The availability of purified G alpha 11 and the G-protein-regulated phospholipase C from turkey erythrocytes has allowed an examination of the direct effects of G-protein beta gamma-subunit on the components of the inositol lipid signaling system. Reconstitution of purified turkey erythrocyte or bovine brain beta gamma-subunit into phospholipid vesicles containing G alpha 11 inhibited AlF4- induced activation of phospholipase C. However, beta gamma-subunit at higher concentrations increased phospholipase C activity. This stimulatory effect of beta gamma-subunit on phospholipase C did not require the presence of the alpha-subunit. G alpha o had no effect on the catalytic activity of phospholipase C. However, coreconstitution of G alpha o and beta gamma-subunit shifted to the right the concentration-effect curve for beta gamma-subunit-promoted activation of phospholipase C. As was observed with G alpha 11, the increase in activity observed in the presence of beta gamma-subunit occurred as an increase in the maximal activity and with no change in the apparent affinity for Ca2+ for phospholipase C activation. The concentration dependence of G alpha 11 for activation of turkey erythrocyte phospholipase C and bovine brain phospholipase C-beta, as well as the concentration dependence of the two enzymes for activation by G alpha 11, were very similar. In contrast, beta gamma-subunit was a much less effective activator of bovine brain phospholipase C-beta than the turkey erythrocyte enzyme. The observation of direct effects of free beta gamma-subunit on phospholipase C extend the possibilities for receptor-mediated regulation of this signaling pathway.

The trp gene is essential for a light-activated Ca 2+ channel in Drosophila photoreceptors

Invertebrate phototransduction is an important model system for studying the ubiquitous inositol-lipid signaling system. In the transient receptor potential ( trp) mutant, one of the most intensively studied transduction mutants of Drosophila, the light response quickly declines to baseline during prolonged intense light. Using whole-cell recordings from Drosophila photoreceptors, we show that the wild-type response is mediated by at least two functionally distinct classes of light-sensitive channels and that both the trp mutation and a Ca 2+ channel blocker (La 3+) selectively abolish one class of channel with high Ca 2+ permeability. Evidence is also presented that Ca 2+ is necessary for excitation and that Ca 2+ depletion mimics the trp phenotype. We conclude that the recently sequenced trp protein represents a class of light-sensitive channel required for inositide-mediated Ca 2+ entry and suggest that this process is necessary for maintained excitation during intense illumination in fly photoreceptors.

Evidence for an inositol lipid signal pathway in the yeast-mycelium transition of Ophiostoma ulmi, the Dutch elm disease fungus

Results presented in this paper indicate the involvement of an inositol lipid signalling system, including protein kinase C, in the yeast-mycelium transition of Ophiostoma ulmi . The exogenous supply of inositol 1,4,5-trisphosphate (Inauthor,4,5P 3 ) induced germ tube formation in O. ulmi , maximum germ tube formation resulting at a concentration of 30 μ m . The addition of Inauthor,4,5P 3 10 min after initiation of Ca 2+ uptake by non-growing yeast cells of O. ulmi resulted in a slight increase in Ca 2+ uptake. The protein kinase C-activating phorbol ester, phorbol 12-myristate 13-acetate (PMA), also induced a significant germination response in O. ulmi and approximately 50% of the population exhibited germ tube formation after 12 h incubation in the presence of 16 n m -PMA. This indicated a possible role for protein kinase C, and therefore diacylglycerol, in the germination response. Ca 2+ uptake by yeast extract-(1% w/v) induced germ tubes of O. ulmi was slower than that which occurred in PMA- or Inauthor,4,5P 3 -induced germ tubes under similar conditions, PMA-induced germ tubes showing the highest levels of Ca 2+ uptake. The anti-calmodulin drug R24571, at 3 μ m , caused almost complete suppression of a yeast extract-induced yeast-mycelium (Y-M) transition which confirmed the involvement of calmodulin in germ tube formation. The addition of 16 n m -PMA in conjunction with yeast extract had no effect on germ tube formation yet, when added with R24571, the inhibitory effect of R24571 was reversed and an almost complete Y-M transition resulted after 16 h incubation. This provided further evidence that activation of protein kinase C was required for germ tube formation in O. ulmi . Ca 2+ uptake by yeast cells or during the growth of yeast extract- and PMA-induced germ tubes of O. ulmi increased markedly over the first 12 h of growth with highest Ca 2+ uptake occurring in yeast extract-induced germ tubes. Ca 2+ uptake by 12 h yeast cells of O. ulmi was inhibited after preincubation for 10 min with PMA suggesting either inhibition of Ca 2+ influx or stimulation of Ca 2+ efflux. Use of Ca 2+ -loaded cells showed that efflux of Ca 2+ from Ca 2+ -loaded yeast cells of O. ulmi was similar in the absence or presence of 70 m m glucose but increased rapidly on addition of PMA to the incubation medium.

Long-term effects of lithium on receptor-mediated inositol lipid metabolism

Larsson C, Simonsson P, Vecsei L, Ailing C. Long-term effects of lithium on receptor-mediated inositol lipid metabolism.Many neurotransmitter receptors are coupled to a signal transduction system that generates two second messengers, inositol trisphosphate and diacylglycerol. Lithium is known to alter this system by inhibiting the dephosphorylation of inositol monophosphate (IP1). In the brain 50% of maximal inhibition is reached at therapeutic concentrations of lithium. It is therefore tempting to suggest that the inositol lipid signalling system could be a cellular target for lithium treatment. Our aim was to study the long-term effects of lithium on receptor-mediated inositol lipid metabolism by incubating neuroblastoma glioma hybrid cells (NG 108-15) with lithium. The lithium concentration used was the IC50 for the IP1 breakdown in these cells, thereby mimicking the situation in the brain during therapy. Long-term treatment with lithium decreased the basal labelling of [3H]-inositol lipids. When cells...

Molecular characterization of the drosophila trp locus: A putative integral membrane protein required for phototransduction

Recent studies suggest that the fly uses the inositol lipid signaling system for visual excitation and that the Drosophila transient receptor potential (trp) mutation disrupts this process subsequent to the production of IP 3. In this paper, we show that trp encodes a novel 1275 amino acid protein with eight putative transmembrane segments. Immunolocalization indicates that the trp protein is expressed predominantly in the rhabdomeric membranes of the photoreceptor cells.

Soman-induced convulsions affect the inositol lipid signaling system: Potentiation by lithium; Attenuation by atropine and diazepam

Effects of atropine or diazepam pretreatment on soman-induced convulsions and brain phosphoinositide (PI) metabolism, as assessed by brain regional inositol-1-phosphate (IP 1) levels, were studied in saline and LiCl-pretreated rats. IP 1, an intermediate in PI turnover, was measured in cortex, caudate, thalamus, hippocamupus, and cerebellum. Soman (100 μg/kg; sc) produced convulsions in 63% of the saline-pretreated rats, whereas with LiCl pretreatment all rats exposed to 100 μg/kg of soman had tonic-clonic convulsions. Thus, LiCl pretreatment potentiated soman-induced convulsions. Tissue IP 1 increased severalfold in soman-exposed convulsing rats with the highest increases being in frontal cortex and caudate. In contrast, no marked increases of IP 1 occurred in similarly treated nonconvulsing rats. LiCl treatment itself increased IP 1 levels without causing convulsions. In LiCl-pretreated rats, soman again markedly elevated IP 1 levels without causing convulsions. In LiCl-pretreated rats, soman again markedly elevated IP 1 levels above LiCl alone in convulsing rats, whereas no such effect occurred in nonconvulsing rats. In LiCl-pretreated rats, the increased IP 1 levels associated with soman-induced convulsions were greatest in hippocampus and piriform cortex. Thus, LiCl appears to lower the threshold for the spread of seizure activity through limbic structures, thereby potentiating cholinergic-induced convulsions. Diazepam and atropine both blocked soman-induced convulsions, and brain regional IP 1 elevations were concomitantly abolished as well. These results indicate that soman-induced convulsions involve the inositol lipid signaling system. This involvement is potentiated by lithium but attenuated by atropine and diazepam.

The interaction of lithium ions with inositol lipid signalling systems.

There is now a growing consensus that hormones and neurotransmitters, in stimulating or inhibiting cellular function, produce their characteristic changes by utilizing a relatively small number of general receptor transduction systems. One such mechanism, which was initially conceived to have as its end-point, the elevation in cytosolic free Ca*+, is the inositol lipid signalling system. This activation process is now known, in addition, to lead to the activation of protein kinase C, and there is a large body of evidence indicating that the synergistic action of Ca’+ and protein kinase C plays a central role in controlling cell function (for a recent review see Downes & Michell, 1985). Despite the enormous advances that this decade has brought in our understanding of the mechanisms by which receptor agonists increase the cytosolic free Ca2+ concentration and activate cellular protein kinases, our ability to perturb these processes pharmacologically, other than at the level of the receptor itself, has remained somewhat primitive. The ready availability of specific pharmacological research tools would do much to speed our understanding both of the extent to which the signalling system pervades general cell function, and of the detailed control processes which govern the performance of the inositol lipid cycle under conditions of both shortand long-term receptor stimulation. Also, at a time when the long-held notion that second messenger systems are too ubiquitous to be plausible drug targets is receiving at least a partial come-uppance with the advent of tissue-selective cyclic nucleotide phosphodiesterase inhibitors, i t does not seem too fanciful to advance the hope that the inositol lipid cycle and its sequelae might similarily yield potentially valuable therapeutic agents. I t is from this perspective that the present short review is written. One of the few agents that interferes with the inositol lipid cycle in any selective sense is lithium. This agent, in the form of lithium carbonate, has been used for over a quarter of a century in the treatment of manic-depressive illness. Its utility is proven (Schou, 1976), but its mode of action remains a matter of conjecture. Throughout the 1970s, Allison and his colleagues conducted an investigation into the interaction of lithium with inositol metabolism, primarily in the central nervous system (CNS). The brain of animals receiving lithium (and with plasma lithium levels in the

Bidirectional control of cytosolic free calcium by thyrotropin-releasing hormone in pituitary cells.

It is now established that a key step in the action of calcium-mobilizing agonists is stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). The latter substance acts as a second messenger, controlling the release of calcium from intracellular stores (see ref. 3 for review). The bifurcating nature of the signalling system is exemplified by the fact that the other product of PtdIns(4,5)P2 hydrolysis, 1,2-diacylglycerol, can alter cellular function by activating protein kinase C, the cellular target for several tumour-promoting agents such as the phorbol esters. In various tissues, including GH3 pituitary tumour cells, a synergistic interaction between calcium ions and protein kinase C underlies agonist-induced changes in cell activity. The data presented here suggest that when GH3 cells are stimulated by thyrotropin-releasing hormone (TRH), an agonist inducing PtdIns(4,5)P2 hydrolysis, the two limbs of the inositol lipid signalling system interact to control free cytosolic calcium levels [( Ca2+]i). At low levels of TRH receptor occupancy, [Ca2+]i increases rapidly, then declines relatively slowly. As receptor occupancy increases, the calcium signal becomes more short-lived due to the appearance of a second, inhibitory, component. This latter component, which is enhanced when [Ca2+]i is elevated by high potassium depolarization, is mimicked by active phorbol esters and by bacterial phospholipase C. It seems likely that protein kinase C subserves a negative feedback role in agonist-induced calcium mobilization.