Aluminium differentially modifies lipid metabolism from the phosphoinositide pathway in Coffea arabica cells

The effect of aluminium (Al) on phosphoinositide-specific phospholipase C (PLC) and lipid kinase activities was examined in a cellular suspension of coffee. Two main effects were seen when cells were treated with AlCl3. In periods as short as 1 minute, Al-exposed cells increased the activity of PLC and IP3 formation up to two fold. Over longer periods PLC activity was inhibited by more than 50%. The activity of phosphatidylinositol 4-kinase (Pl 4-K), phosphatidylinositol phosphate 5-kinase (PIP 5-K) and diacylglycerol kinase increased when cells were incubated in the presence of different concentrations of AlCl3. The present study reports for the first time that Al may have different effects on the Pl-signaling pathway depending on the time of exposure. Our results strongly support the hypothesis that Al disrupts the metabolism of membrane phospholipids regulating not only PLC but also other enzymes that have key roles in signal-transduction pathways.     

A Gq-type G protein couples muscarinic receptors to inositol phosphate and calcium signaling in exocrine cells from the avian salt gland

Summary Muscarinic acetylcholine receptor (mAChR) activation in isolated cells from the nasal salt gland of the domestic duck (Anas platyrhynchos) results in a rapid increase in the rate of phosphatidylinositol hydrolysis and pronounced intracellular calcium signals. Both responses can be elicited by treating these cells with fluoroaluminate (AlF ₄ ^– ) indicating the involvement of a heterotrimeric G protein in the transmembrane signaling process. To characterize this G protein, electrophoretically separated membrane proteins were blotted onto nitrocellulose filters and probed with peptide-antibodies raised against portions of different -subunits of mammalian G proteins. We could demonstrate the presence of at least four different G proteins in salt gland cell membranes. Two of these proteins (40 and 41 kD) were ADP-ribosylated by pertussis toxin and were recognized by an antiserum against a common sequence in all G protein -subunits. One protein (46 kD) was a cholera toxin-substrate and was recognized by a G_s-specific antiserum; the other (42 kD) was recognized by G_q-specific antisera and was resistant to ADP-ribosylation. Since the initial inositol phosphate production upon receptor activation with carbachol and the resulting calcium signals were not affected by pertussis toxin-pretreatment of salt gland cells, we conclude that muscarinic receptors are coupled to phospholipase C by a G_q-type G protein.This study was supported by National Institutes of Health Grant GM-40457 (to T.J.S.) and, in part, by a fellowship (Hi 448/1) from the Deutsche Forschungsgemeinschaft (to J.-P.H.). We are grateful to Drs. D.R. Manning and P.C. Sternweis for their kind gifts of antisera.     

Enhanced phosphoinositide metabolism in colorectal carcinoma cells derived from familial adenomatous polyposis patients

The production of the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate is mediated by activated phosphatidylinositol-specific phospholipase C (PLC) enzymes. We report the enhancement of the phosphoinositide metabolism pathway in KMS-4 and KMS-8 cells, both of which are human colorectal carcinoma cell lines derived from familial adenomatous polyposis patients. In these cells, the cellular contents of diacylglycerol and inositol 1,4,5-trisphosphate were constitutively increased and the PLC activity in vitro was significantly high, as compared with those in normal colon cells or in other sporadic colorectal carcinoma cells. Northern and Western analyses showed the high expression levels of both PLC-γ1 and PLC-δ1 in KMS-4 and KMS-8 cells. Moreover, we detected the enhancement of protein–tyrosine kinase activity and tyrosine phosphorylation of PLC-γ1 in these KMS cells. These results suggest the involvement of activated phosphoinositide signaling pathways in the colorectal tumorigenesis of familial adenomatous polyposis. © 1994 Wiley-Liss, Inc.     

Phosphatidic acid and phospholipase D both stimulate phosphoinositide turnover in cultured human keratinocytes

Phosphatidic acid (PA) induced a rapid dose-dependent increase in production of inositol phosphates in cultured adult human keratinocytes, peaking at 30 s. Natural and dioleoyl PA were equally effective, while other phospholipid classes had no effect. Lipid A was also active. Lyso-PA also induced inositol phosphate production, but contamination of the PA preparation by lyso-PA could not account for the effect of PA. The effect of PA could not be reproduced by treatment of cells with calcium ionophore. PA-induced inositol phosphate production could be inhibited (> 50%) by pre-treatment of cells with either pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate, suggesting the involvement of a GTP-binding protein and a protein kinase C-mediated negative feedback mechanism. PA also stimulated release of arachidonic acid from keratinocytes. Treatment of cells with exogenous phospholipase D similarly induced inositol phosphate production in the keratinocytes. Since PA may be formed by receptor-mediated activation of phospholipase D, or by phosphorylation of diacylglycerol, the results suggest that PA may play a significant role in signalling mechanisms of human keratinocytes.     

Ethanol-induced pseudohyphal transition in the cells of Candida tropicalis: participation of phosphoinositide signal transduction

Ethanol-induced pseudohyphal development in the cells of Candida tropicalis Pk233 was accompanied by the transient accumulation of inositol 1,4,5-trisphosphate (IP3) that occurred at an early growth stage. The concomitant addition of myo-inositol prevented the activation of IP3 accumulation and cancelled pseudohyphal development in the presence of ethanol. The addition of a specific phospholipase C inhibitor, U73 122, inhibited ethanol-induced pseudohyphal transition at the concentrations of subinhibitory levels of cell growth. Pseudohyphal development was also induced by the Ca2+ ionophore, A23 187 in the absence of ethanol. The effect of A23 187 on the development of pseudohyphae was little influenced by myo-inositol, but stimulated by concomitant addition of 12-O-tetradecanoylphorbol 13-acetate. These results suggest that ethanol activated phospholipase C in competition with myo-inositol, and the resulting IP3-Ca2+ and protein kinase C pathways of PI signal transduction may work in pseudohyphal transition.     

Phospholipid signalling and lipid-derived second messengers in plants

Phospholipid signalling is mediated by phospholipid breakdown products generated by phospholipases. The enzymes from animals and plants generating known or potential lipid-derived second messengers are compared. Plants possess a phospholipase C and a phospholipase A₂ both of which are agonist-activated. These agonists (auxin, elicitors, perhaps others) bind to the external surface of the plasma membrane. The target enzyme for potential plant lipid-derived second messengers is lipid-activated protein kinase but the possibility that other enzymes may be also lipid-modulated should not be precluded.Abbreviations DAG diacylglycerol - CDPK calmodulin-like domain protein kinase - PLA2 phospholipase A₂ - PLC phospholipase C - PLD phospholipase D - PKC protein kinase C - PS phosphatidylserine     

Expression pattern and sub‐cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U‐73122 in rat astrocytoma cells

Phosphoinositide specific phospholipase C (PI‐PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI‐PLC family display different expression and/or sub cellular distribution under non‐physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI‐PLCs compared to untreated cells. Special attention require PI‐PLC beta3 and PI‐PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U‐73122 treatment. The meaning of these modifications is unclear, also because the use of this N‐aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells. J. Cell. Biochem. 110: 1005–1012, 2010. © 2010 Wiley‐Liss, Inc.     

A rapid attenuation of muscarinic agonist stimulated phosphoinositide hydrolysis precedes receptor sequestration in human SH-SY-5Y neuroblastoma cells

Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t_1/2=14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5–10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.Abbreviations I(1,4,5)P₃ inositol 1,4,5-trisphosphate - IP total inositol phosphate fraction - IPL total inositol lipid fraction - mAChR muscarinic acetylcholine receptor - NMS N-methylscopolamine - Oxo-M oxotremorine-M - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PPI phosphoinositide - QNB quinuclidinyl benzilate Special issue dedicated to Dr. Bernard W. Agranoff     

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: Further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDa protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells. we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 μM), on pepsinogen secretion and phosphorylation of the 72-kDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with ³²P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 μM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS “phosphorylation/calmodulin binding domain peptide” indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion. J. Cell. Biochem. 64:514–523. © 1997 Wiley-Liss, Inc.     

Role of calcium ions in rapid effects of L-thyroxine on phosphoinositide metabolism in rat liver cells

The role of calcium ions in the L-thyroxine-induced initiation of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) and also the course of releasing individual fractions of inositol phosphates and diacylglycerides (DAG) were studied in liver cells during early stages of the hormone effect. L-Thyroxine stimulated a rapid hydrolysis in hepatocytes of PtdInsP₂ labeled with [¹⁴C]linoleic acid and [³H]inositol mediated by phosphoinositide-specific phospholipase C. This was associated with accumulation of [¹⁴C]DAG, total inositol phosphates, [³H]inositol 1,4,5-trisphosphate (Ins1,4,5P₃) and [³H]inositol 1,4-bisphosphate (Ins1,4P₂). Elimination of calcium ions from the incubation medium of hepatocytes did not abolish the effect of thyroxine on the accumulation of [¹⁴C]DAG and total [³H]inositol phosphates. Preincubation of liver cells with TMB-8 increased the stimulatory effect of L-thyroxine on the accumulation of [¹⁴C]DAG. During the incubation of hepatocytes in the presence of the hormone the content of ¹⁴C-labeled fatty acids did not change. The L-thyroxineinduced accumulation of [³H]Ins1,4,5P₃ and [³H]Ins1,4P₂ did not depend on the presence of calcium ions in the incubation medium of the cells.     

Reduction in Gh protein expression is associated with cytodifferentiation of vascular smooth muscle cells

G_h, a high molecular weight GTP-binding protein that couples ₁-adrenoceptors in heart and liver to phosphatidylinositol (Ptdlns)-specific phospholipase C (PLC), has recently been shown to be a tissue transglutaminase type 11. Transglutaminases have been suggested to play a role in the maintenance of blood vessel structure, and therefore it is possible that changes in their expression may accompany pathological states which involve phenotypic modulation of smooth muscle. Hence, we investigated the expression of G_h during differentiation of rat aortic smooth muscle cells in culture. G_h content was reduced markedly in cultured smooth muscle cells compared to freshly isolated cells as determined by Western blotting using a G_h-specific monoclonal antibody. In contrast, the level of G_q, a heterotrimeric G-protein that couples ₁-adrenoceptors to PLC, was maintained throughout the culture period. These findings indicate that changes in G, expression accompany phenotypic modulation of vascular smooth muscle cells. These changes in G_h protein expression may be important in the altered responsiveness of vessels in pathological disease states.     

Effects of insulin,pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.     

Coordinated Regulation of Radioadaptive Response by Protein Kinase C and p38 Mitogen-Activated Protein Kinase

Eukaryotic cells are known to have an inducible or adaptive response that enhances radioresistance after a low priming dose of radiation. This radioadaptive response seems to present a novel cellular defense mechanism. However, its molecular processing and signaling mechanisms are largely unknown. Here, we studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the expression of radioadaptive response in cultured mouse cells. Protein immunoblot analysis using isoform-specific antibodies showed an immediate activation of PKC-alpha upon X-irradiation as indicated by a translocation from cytosol to membrane. A low priming dose caused a prolonged translocation, while a nonadaptive high dose dramatically downregulated the total PKC level. Low-dose X-rays also activated the p38 MAPK. The activation of p38 MAPK and resistance to chromosome aberration formation were blocked by SB203580, an inhibitor of p38 MAPK, and Calphostin C, an inhibitor of PKC. Furthermore, it was demonstrated that p38 MAPK was physically associated with delta1 isoform of phospholipase C (PLC-delta1), which hydrolyzed phosphatidylinositol bisphosphate into diacylglycerol, an activator of PKC, and that SB203580 also blocked the activation of PKC-alpha. These results indicate the presence of a novel mechanism for coordinated regulation of adaptive response to low-dose X-rays by a nexus of PKC-alpha/p38 MAPK/PLC-delta1 circuitry feedback signaling pathway with its breakage operated by downregulation of labile PKC-alpha at high doses or excess stimuli.     

Zinc homeostasis in C6 glioma cells

Zinc homeostasis in mammalian cells is precisely regulated by cellular signal transduction mechanisms. The main result of this study is the finding that modulators of phospholipase C (PLC) activity affect cellular zinc export. Two different PLC inhibitors caused an increase of the total cellular zinc level whereas two different PLC activators caused a decrease. Furthermore, both the inhibition of cyclic nucleotide phosphodiesterases as well as the administration of 8-bromo-cAMP evoked a drop in the intracellular zinc level, indicating the involvement of cAMP in the control of cellular zinc export. It is concluded that the activity of PLC controls cellular zinc transport and that the effect of elevated zinc concentrations on PLC activity might be mediated by cAMP. However, modulation of other major signaling enzymes did not affect the cellular zinc homeostasis. These include activation and inhibition of guanylate cyclase, activation of protein kinase G, activation of protein kinase A, and activation or inhibition of protein kinase C. Furthermore there was no evidence for the existence of a zinc-sensing receptor in C6 glioma cells, which would stimulate PLC activity and evoke a mobilization of intracellular free-calcium levels.     

alpha Latrotoxin of black widow spider venom binds to a specific receptor coupled to phosphoinositide breakdown in PC12 cells

alpha Latrotoxin of black widow spider is known to bind with high affinity to surface sites of rat pheochromocytoma (PC12) cells, thereby causing depolarization, calcium influx and massive neurotransmitter release. We show here that the toxin causes the accumulation of inositol phosphates, the products of phosphoinositide breakdown. Inositol 1,4,5, trisphosphate was predominantly accumulated shortly after toxin application. Phosphoinositide breakdown appears to be a direct consequence of toxin binding because high K+ and ionophores (which induce depolarization, calcium influx and transmitter release by different mechanisms) were without such effect. Phosphoinositide breakdown is known as an event coupled to the activation of receptors of various hormones and transmitters. We suggest therefore that the alpha latrotoxin binding site is a receptor coupled across the membrane to the phosphoinositide hydrolysing system.     

Nutrients Induce an Increase in Inositol 1,4,5-Trisphosphate in Soybean Cells: Implication for the Involvement of Phosphoinositide-Specific Phospholipase C in DNA Synthesis

Abstract: Phosphoinositide-specific phospholipase C (PI-PLC) hydrolyzes the membrane lipid phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) to generate 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP₃). Both molecules serve as second messengers to carry out various cellular functions in mammals. In the present study, we demonstrate that many organic and inorganic nutrients cause the elevation of InsP₃ concentrations in cultured soybean cells. This elevation of InsP₃ content is sustained for several hours following treatment with Murashige-Skoog (MS) inorganic nutrients. Phosphate and calcium are the major components in MS salts responsible for the increase in InsP₃ levels. DNA synthesis, a measure of cell growth, was significantly suppressed by the PI-PLC-specific inhibitor 1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122), whereas its near-identical analogue 1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-2,5-pyrrolidinedione did not cause any suppression. Activation of PI-PLC by MS salts increased DNA synthesis and abolished the suppression of DNA synthesis caused by U-73122. Thus, we conclude that the higher cellular concentration of InsP₃ induced by MS treatment is involved in DNA synthesis.     

Sodium channel activation does not alter lipid metabolism in cultured neuroblastoma cells

The interaction of voltage-sensitive Na⁺-channels and membrane lipid metabolism was examined by incubating cultured neuroblastoma cells with neurotoxins which alter the voltage-dependent relationship between the closed and open conformation of the channel protein. Guanidinium flux rate, a measure of Na⁺-channel activation, was increased 10-fold by the combined action of veratridine (100 M) and scorpion venom (28 g/ml). This response was completely blocked by tetrodotoxin (1 M). Under the same experimental conditions, the toxins did not increase the efflux of [³H]arachidonic acid from prelabeled cell membrane lipids or stimulate uptake of exogenous [³H]arachidonic acid. In addition, altering membrane fatty acid composition by incubating cells for 24 hr in a medium containing 50 M arachidonic or oleic acid did not alter guanidinium flux rates relative to that of control cultures. When cells were pulsed with³²Pi for 60 min and stimulated by veratridine plus scorpion venom for an additional 30 min, uptake of³²Pi into phosphatidylinositol as reduced; stimulating cells with bradykinin, a receptor agonist which activates the inositol cycle, promoted a 3.8 fold increase. Polyphosphoinositide turnover was not affected by Na⁺-channel activation, but was stimulated by bradykinin. These results suggest that voltage-sensitive Na⁺-channel activation in cultured neuroblastoma cells can function independent of membrane phospholipid and fatty acid metabolism.