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PubMed1993

Phosphoinositide metabolism in a polyoma-BK-virus-transformed pancreatic islet cell line: evidence for constitutively activated phospholipase C.

We have characterized the phosphoinositide metabolism in a polyoma-BK-virus-transformed rat pancreatic islet cell line which has highly malignant characteristics, expresses viral T-antigen and has lost insulin-secreting capacity. After incorporation with [3H]inositol to isotopic equilibrium, all inositol metabolites were analyzed. When compared with normal pancreatic islets, increased levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), inositol 1,3,4-trisphosphates and inositol tetrakisphosphate (Ins-P4), and decreased levels of phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) were found. The Ins-1,4,5-P3/PIP2 ratio increased, whereas the PIP2/PIP ratio was not altered after the transformation. In the pancreatic islet cell line there was a stable accumulation of inositol phosphates at 3.3 mM glucose. Glucose, KCl, cholecystokinin (CCK) and carbachol with and without LiCl were all without effect on the accumulation of inositol phosphates. Somatostatin inhibited the accumulation of inositol phosphates but a Ca(2+)-free/EDTA solution did not. Preincubation with cholera toxin or pertussis toxin inhibited the accumulation of inositol phosphates at 3.3 mM glucose except for Ins-P4, whereas no effect was observed on the phosphoinositides. NaF stimulated the accumulation of inositol phosphates, with a concomitant decrease in the phosphoinositides, whereas neomycin was without effect on the inositol phosphates. In normal pancreatic islets, pertussis toxin inhibited the CCK-induced increase in Ins-1,4,5-P3, whereas no effect was seen at 3.3 mM glucose. Finally, pertussis toxin inhibited the CCK-induced increase in the Ins-1,4,5-P3/PIP2 ratio in normal pancreatic islets. The same inhibition was also found in the pancreatic islet cell line at 3.3 mM glucose. We conclude that in the transformed pancreatic islet cell line the phosphoinositide hydrolysis is constitutively activated at the level of phospholipase C, with a substantial loss of regulatory control.

3T3 Cells↗

PubMed2005

Sustained diacylglycerol accumulation resulting from prolonged G protein-coupled receptor agonist-induced phosphoinositide breakdown in hepatocytes.

Studies in various cells have led to the idea that agonist-stimulated diacylglycerol (DAG) generation results from an early, transient phospholipase C (PLC)-catalyzed phosphoinositide breakdown, while a more sustained elevation of DAG originates from phosphatidylcholine (PC). We have examined this issue further, using cultured rat hepatocytes, and report here that various G protein-coupled receptor (GPCR) agonists, including vasopressin (VP), angiotensin II (Ang.II), prostaglandin F2alpha, and norepinephrine (NE), may give rise to a prolonged phosphoinositide hydrolysis. Preincubation of hepatocytes with 1-butanol to prevent conversion of phosphatidic acid (PA) did not affect the agonist-induced DAG accumulation, suggesting that phospholipase D-mediated breakdown of PC was not involved. In contrast, the GPCR agonists induced phosphoinositide turnover, assessed by accumulation of inositol phosphates, that was sustained for up to 18 h, even under conditions where PLC was partially desensitized. Pretreatment of hepatocytes with wortmannin, to inhibit synthesis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP2), prevented agonist-induced inositol phosphate and DAG accumulation. Upon VP stimulation the level of PIP) declined, but only transiently, while increases in inositol 1,4,5-trisphosphate (InsP3) and DAG mass were sustained, suggesting that efficient resynthesis of PIP2 allowed sustained PLC activity. This was confirmed when cells were pretreated with wortmannin to prevent resynthesis of PIP2. Furthermore, metabolism of InsP3 was rapid, compared to that of DAG, with a more than 20-fold difference in half-life. Thus, rapid metabolism of InsP3 and efficient resynthesis of PIP2 may account for the larger amount of DAG generated and the more sustained time course, compared to InsP3. The results suggest that DAG accumulation that is sustained for many hours in response to VP, Ang.II, NE, and prostaglandin F2alpha in hepatocytes is mainly due to phosphoinositide breakdown.

1-Butanol↗

PubMed2006

Nuclear phosphoinositide kinases and inositol phospholipids.

The presence of inositol phospholipids in the nuclei of mammalian cells has by now been well established, as has the presence of the enzymes responsible for their metabolism. However, our understanding of the role of these nuclear phosphoinositides in regulating cellular events has lagged far behind that for its cytosolic counterpart. It is clear, though, that the nuclear phosphoinositide pool is independent of the cytosolic pool and is, therefore, likely to be regulating a unique set of cellular events. As with its cytosolic phosphoinositides, many nuclear phosphoinositides and their metabolic enzymes are located at distinct sub-cellular structures. This arrangement spatially limits the production and activity of inositol phospholipids and is believed to be a major mechanism for regulating their function. Here, we will introduce the components of nuclear inositol phospholipid signal transduction and discuss how their spatial arrangement may dictate which nuclear functions they are modulating.

1-Phosphatidylinositol 4-Kinase↗

PubMed1999

Enhanced phosphoinositide hydrolysis via overexpression of phospholipase C beta1 or delta1 inhibits stimulus-induced insulin release in insulinoma MIN6 cells.

To study the effects of enhanced phosphoinositide hydrolysis on insulin secretion, phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1) or PLCdelta1 was overexpressed in insulinoma MIN6 cells via adenoviral vectors. Inositol phosphate production stimulated by NaF (with AlCl3) in PLCbeta1-overexpressing cells and that stimulated by KCl or glucose in both PLCbeta1- and PLCdelta1-overexpressing cells were greater than that in control cells. In addition, reduced phosphatidylinositol-4,5-bisphosphate levels were observed in these cells stimulated by NaF or KCl. The greater phosphoinositide hydrolysis was accompanied by 25-45% inhibition of insulin secretion. These data suggest that excessive phosphoinositide hydrolysis inhibits secretagogue-induced insulin release in MIN6 cells.

Adenoviridae↗

PubMed1998

Inhibition of phosphoinositide metabolism or chelation of intracellular calcium blocks FSH-induced but not spontaneous meiotic resumption in mouse oocytes.

Mammalian oocytes are arrested at the diplotene phase of the first meiotic division until ovulation. In the mouse, germinal vesicle breakdown (GVBD) and progression to metaphase II is thought to be triggered by a positive signal originating in the follicular cells following stimulation by the luteinizing hormone (LH) surge. Isolated, fully grown oocytes can also undergo spontaneous reinitiation of meiosis in vitro in the absence of gonadotrophin stimulation. To investigate the mechanism of meiotic resumption, inhibitors of phosphoinositide metabolism and an intracellular calcium chelator were used during maturation in vitro under different conditions. In a series of experiments, isolated cumulus cell-oocyte complexes (COCs) maintained in meiotic arrest by hypoxanthine were induced to resume meiosis by treatment with follicle-stimulating hormone (FSH). Under these conditions, both LiCl and neomycin, which inhibit phosphoinositide hydrolysis, produced a dose-dependent inhibitory effect on meiotic resumption. Similar results were obtained when FSH-induced meiotic resumption was observed in the presence of the acetoxymethyl ester form of 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), an intracellular calcium chelator. In hypoxanthine-arrested oocytes, GVBD induced by epidermal growth factor (EGF), which mimics FSH action in in vitro maturation, was also repressed by LiCl and neomycin. Conversely, meiotic resumption triggered by a pulse of 8-bromo-cyclic adenosine monophosphate (8-Br cAMP) was not affected by these two inhibitors. In experiments in which oocytes were cultured under conditions which permit spontaneous meiotic maturation, resumption of meiosis was not affected by either inhibition of phosphoinositide hydrolysis or chelation of intracellular calcium. Therefore, it appears that meiotic resumption induced by hormone stimulation requires activation of the phosphoinositide pathway and mobilization of intracellular calcium. In contrast, spontaneous maturation probably occurs through a different mechanism because it is not affected by inhibition of this signaling pathway.

Animals↗

PubMed1996

SK & F 96365 inhibits carbachol-induced phosphoinositide turnover in human neuroblastoma SH-SY5Y and rat cerebellar granule cells.

SK & F 96365, a receptor-mediated Ca2+ entry inhibitor, has been reported to inhibit Ca2+ responses to various agonists without affecting internal Ca2+ release and phosphoinositide turnover. Since muscarinic acetylcholine receptor-mediated phosphoinositide turnover shows a marked dependence on factors affecting cytosolic Ca2+ concentration, the effects of SK & F 96365 on the coupling of muscarinic receptors to the phosphoinositide hydrolysis were examined in human neuroblastoma SH-SY5Y and rat cerebellar granule cells. SK & F 96365 concentration-dependently (3-30 microM) inhibited the inositol phosphate formation elicited by carbachol in both cellular systems. Moreover, SK & F 96365 inhibited the carbachol-induced inositol phosphate formation in the absence of extracellular Ca2+, and similar extent of inhibition was achieved in the presence or absence of extracellular Ca2+. In ligand binding studies, we found that the binding affinities for [3H] N-methyl-scopolamine in both cells were attenuated by SK & F 96365 (3-30 microM), while Bmax values for the ligand were not changed. The competition curves of SK & F 96365 showed a Ki value of 28.4 uM in SH-SY5Y cells. The results indicated that the decrease of carbachol-stimulated phosphoinositide hydrolysis by SK & F 96365 is due to the competitive inhibition of agonist binding to the M3 muscarinic receptors.

Animals↗

PubMed1989

Dissociation of phosphoinositide hydrolysis and positive inotropic effect of histamine mediated by H1-receptors in guinea-pig left atria.

The present study was undertaken to determine whether the phosphoinositide hydrolysis is responsible for the positive inotropic effect of histamine in guinea-pig left atria. Histamine induced hydrolysis of phosphoinositides and a positive inotropic effect in a concentration-dependent manner. These effects were antagonized by chlorpheniramine (0.1 mumol/l) but not by cimetidine (10 mumol/l). At a concentration of 1 mumol/l histamine produced a dual-component positive inotropic response composed of an initial increasing phase and a second and late developing, greater positive inotropic phase. Histamine (10 mumol/l) caused a gradual increase in the formation of [3H]inositol trisphosphate (IP3) and a significant increase in the [3H]IP3 level was detected 10 min after the stimulation. Thus, the increase in IP3 did not precede the increase in force of contraction. The phospholipase C inhibitors 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (100 mumol/l) and neomycin (100 mumol/l) significantly reduced the histamine-induced [3H]inositol monophosphate accumulation. However, pretreatment with the phospholipase C inhibitors did not affect the positive inotropic effect of histamine, either in its extent or in its pattern. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nmol/l) and phorbol-12,13-dibutyrate (PDBu) (100 nmol/l) also significantly inhibited the phosphoinositide hydrolysis induced by histamine. The inhibitory effect of the phorbol esters on the phosphoinositide response was completely abolished in the presence of 10 mumol/l 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1989

Effect of calcium overload on the phosphoinositide breakdown in the rat left ventricular papillary muscle.

We investigated the effect of Ca2+ overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca2+ overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca2+-containing medium following either Ca2+-containing or Ca2+-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [3H]inositol phosphates ([3H]IPs) in the tissues prelabeled with [3H]inositol. Ca2+ repletion following Ca2+-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [3H]IPs accumulations. Treatment with ouabain following Ca2+-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [3H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [3H]IPs were significantly potentiated by prior Ca2+-free superfusion instead of Ca2+-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [3H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca2+-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca2+, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol.

Animals↗

PubMedDate not supplied

Phosphoinositide-generated messengers in cardiac signal transduction.

A multitude of agonists like e.g. endothelin-1, angiotensin-II, serotonin, thrombin, histamine and vasopressin as well as alpha 1-adrenergic and muscarinic stimulation lead to stimulation of the phosphoinositide cycle in the heart. Besides the seven membrane spanning-domain receptor-coupled stimulation of the key enzyme of the phosphoinositide cycle, phospholipase C-beta, another class of hormones, growth factors, also couple to the phosphoinositide cycle, now through receptors with intrinsic tyrosine kinase activity that can phosphorylate and stimulate the phospholipase C-gamma isozyme. In this review we summarize the multitude of receptor (sub)types, G-protein-subunit- and phospholipase C-isozymes that are present in the heart. Furthermore, generation of second messengers and cellular responses are described together with the (patho)physiological implications for the heart of phosphoinositide cycle activation and second messenger accumulation.

Adrenergic alpha-Agonists↗

PubMed1987

Maitotoxin stimulates phosphoinositide breakdown in neuroblastoma hybrid NCB-20 cells.

1. Maitotoxin (MTX) was an extraordinarily potent stimulant of phosphoinositide breakdown in the neuroblastoma hybrid NCB-20 cells. 2. Maximal responses were obtained at 0.25-0.5 ng MTX/ml, and resulted in increased formation of [3H]inositol mono-, bis-, and trisphosphates. Increased formation of [3H]inositol bis- and trisphosphate was observed as early as 15 sec after the addition of MTX. 3. MTX-induced phosphoinositide breakdown in NCB-20 cells was not antagonized by organic (nifedipine, methoxyverapamil) or inorganic (Mn2+, Co2+, Cd2+) calcium channel blockers. However, the response on phosphoinositide breakdown was completely eliminated in the absence of extracellular calcium. 4. The results suggest that MTX either directly stimulates phosphoinositide breakdown in a calcium-dependent manner or acts indirectly through calcium channels insensitive to organic/inorganic calcium channel blockers.

Animals↗

PubMed1994

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 (t1/2 = 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.

Chemical Fractionation↗

PubMed1995

Effect of prenatal treatment with methylazoxymethanol on carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism in the neonatal, young, and adult offspring.

Carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism was investigated in the neonatal, young and adult cerebral cortex slices of rats prenatally treated with methylazoxymethanol (MAM) on gestational day 15 (GD15) or GD19. In rat offspring treated on GD15 there was a significant reduction in the accumulation of [3H]inositol phosphates induced by carbachol and a significant increase in the accumulation of [3H]inositol phosphates induced by norepinephrine on day 7, whereas no changes were observed at the other ages. No significant changes, on the other hand, were observed for glutamate-stimulated phosphoinositide metabolism in GD15 treated rats and for carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism in animals treated on GD19 at any of the different ages evaluated. These results indicate that treatment with MAM on GD15, which results in a marked microencephaly, causes a marked alteration of muscarinic and alpha 1-adrenergic receptor-stimulated phosphoinositide metabolism during brain development and that these alterations undergo adaptive changes in the adult brain.

Animals↗

PubMed1993

Studies on the metabolism of metallothionein and alkaline phosphatase of adult rat primary hepatocyte cultures: role of fetal calf serum and agonists of the phosphoinositide cascade.

Adult rat primary hepatocytes maintained in DMEM/F12 (Ham) media were used as a model system for studying the role of fetal calf serum (FCS) and agonists of the phosphoinositide cascade in the metabolism of metallothionein (MT) and alkaline phosphatase (ALP). Experiments were performed both after a 24 h preincubation with FCS and with bovine serum albumin (BSA). Hepatocytes were treated with dexamethasone (DEX), zinc (Zn) and with the agonists of the phosphoinositide cascade A23187, 1,2-dioctanoyl-sn-glycerol (DiC8), 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II (AT), platelet activating factor (PAF), Arg8-vasopressin (VP) and were analyzed for MT and ALP activity in cell homogenates. Cell viability was evaluated by lactate dehydrogenase (LDH) liberation into culture medium, induction of tyrosine aminotransferase (TAT) through DEX and by trypan blue exclusion. Overall, cell viability was improved by the FCS pretreatment and by DEX. Exposure of hepatocytes to the established direct inducers Zn and DEX of MT resulted in a manifold increase in MT, independent of whether the cultures were FCS pretreated or not. The FCS preincubation produced a moderate elevation of ALP activity by stimulating cell viability. However, ALP was unaltered in response to Zn and DEX. None of the experiments conducted with agonists of the phosphoinositide cascade led to an elevation of MT and ALP. Only the incubation of hepatocytes with A23187 resulted in a concentration dependent significant decrease of MT and ALP. This observation was due to a cytotoxic effect of A 23187, displayed by LDH leakage and an increase in the number of cells stained with trypan blue. In conclusion, in primary hepatocyte cultures agonists of the phosphoinositide did not have an effect on the metabolism of MT and ALP. Previous in vivo results indicating alterations of Zn metabolism in liver, therefore seem to be caused by indirect systemic responses.

Alkaline Phosphatase↗

PubMed1988

Histamine H1-receptors mediate phosphoinositide and calcium response in cultured smooth muscle cells--interaction with cicletanine (CIC).

Smooth muscle cells were cultured from guinea-pig aorta and labelled with 45Ca++ and 32Pi to investigate the possible effect of cicletanine, a new antihypertensive drug, on the release of intracellular Ca++ and the metabolism of phosphoinositide induced by histamine. In 45Ca++ labelled cells, histamine increased in a dose-dependent manner the 45Ca++ efflux in the first two minutes. Stimulation of 45Ca++ release was observed with H1-agonist [2-pyridylethylamine dihydrochloride (2-PEA)] but not with H2-agonist (dimaprit). In addition, histamine- or 2-PEA- induced 45Ca++ efflux was inhibited by the H1-antagonists (mepyramine and terfenadine) whereas the H2-antagonist (cimetidine) was without effect. Similar results were obtained in 32Pi labelled cells; both H1-agonists (histamine and 2-PEA) increased the labelling of phosphoinositides. This effect was completely blocked by mepyramine. These results demonstrate that the histamine-induced stimulation of 45Ca++ efflux and phosphoinositide metabolism are mediated through H1-receptors. In the above systems, cicletanine was as effective as the H1-antagonist (mepyramine) with an IC50 of 10(-6) M for both 45Ca++ efflux and phosphoinositide metabolism. Blockade of these systems by cicletanine may be part of the mechanism by which this drug produces relaxation of blood vessels and may account for its in vivo antihypertensive action.

Animals↗

PubMed1994

Effects of the amphiphilic peptides mastoparan and adenoregulin on receptor binding, G proteins, phosphoinositide breakdown, cyclic AMP generation, and calcium influx.

1. The amphiphilic peptide mastoparan is known to affect phosphoinositide breakdown, calcium influx, and exocytosis of hormones and neurotransmitters and to stimulate the GTPase activity of guanine nucleotide-binding regulatory proteins. Another amphiphilic peptide, adenoregulin was recently identified based on stimulation of agonist binding to A1-adenosine receptors. 2. A comparison of the effects of mastoparan and adenoregulin reveals that these peptides share many properties. Both stimulate binding of agonists to receptors and binding of GTP gamma S to G proteins in brain membranes. The enhanced guanyl nucleotide exchange may be responsible for the complete conversion of receptors to a high-affinity state, complexed with guanyl nucleotide-free G proteins. 3. Both peptides increase phosphoinositide breakdown in NIH 3T3 fibroblasts. Pertussis toxin partially inhibits the phosphoinositide breakdown elicited by mastoparan but has no effect on the response to adenoregulin. N-Ethylmaleimide inhibits the response to both peptides. 4. In permeabilized 3T3 cells, both adenoregulin and mastoparan inhibit GTP gamma S-stimulated phosphoinositide breakdown. Mastoparan slightly increases basal cyclic AMP levels in cultured cells, followed at higher concentrations by an inhibition, while adenoregulin has minimal effects. 5. Both peptides increase calcium influx in cultured cells and release of norepinephrine in pheochromocytoma PC12 cells. The calcium influx elicited by the peptides in 3T3 cells is not markedly altered by N-ethylmaleimide. 6. Multiple sites of action appear likely to underlie the effects of mastoparan/adenoregulin on receptors, G proteins, phospholipase C, and calcium.

3T3 Cells↗

PubMed2004

Phosphoinositide lipid second messengers: new paradigms for transepithelial signal transduction.

Multiple forms of phosphatidylinositol are generated by differential phosphorylation of the inositol headgroup. These phosphoinositides, specifically PI(4,5)P2, have been implicated as modulators in a variety of transport processes. The data indicate that phosphoinositides can modulate transporters directly or via the activation of down-stream signaling components. The phosphoinositide pathway has been linked to changes in transporter kinetics, intracellular signaling, membrane targeting and membrane stability. Recent results obtained for several of the well-characterized transport systems suggest the need to reassess the role of PI(4,5)P2 and question whether lower abundance forms of the phosphoinositides, notably PI(3,4,5)P3 (PIP3) and PI(3,4)P2, are the pertinent transport regulators. In contrast to PI(4,5)P2, these latter forms represent a dynamic, regulated pool, the characteristics of which are more compatible with the nature of signaling intermediates. A recently described, novel transepithelial signaling pathway has been demonstrated for PIP3 in which a signal initiated on the basolateral membrane is transduced to the apical membrane entirely within the planar face of the inner leaflet of the plasma membrane. The new paradigms emerging from recent studies may be widely applicable to transporter regulation in other cell types and are particularly relevant for signaling in polarized cells.

Animals↗

PubMed1991

Enhancement of the protective qualities of gastric mucus by sucralfate: role of phosphoinositides.

The effects of intragastric administration of sucralfate on the physicochemical properties of gastric mucus, and the mechanism of its protective action against alcohol-induced mucosal injury were investigated using in vivo and in vitro models. The experiments in vivo were conducted with groups of rats receiving a dose of 100 mg sucralfate twice daily for 5 consecutive days. The animals were sacrificed 16 hours after the last dose, their stomachs dissected, and the mucosa subjected to physicochemical measurements. In the in vitro studies, gastric mucosa was cultured in the presence of sucralfate, ethanol, or both. The in vivo results revealed that sucralfate elicited an 8% increase in mucus gel dimension, while its sulfo- and sialomucin content increased by 63% and 81%, respectively. The changes in mucus gel mucin content with sucralfate were accompanied by a 9.5% increase in mucus hydrogen ion (H+) retardation capacity, 1.9-fold increase in viscosity, and a 60% increase in the gel hydrophobicity. The mucus elaborated in the presence of sucralfate exhibited 14% lower protein content and 62% higher content of carbohydrate than that of control, and contained more neutral lipids. Furthermore, the gastric mucus of the sucralfate group showed a marked increase in mucus glycoprotein polymeric form. The data obtained with gastric mucosal culture demonstrated that sucralfate elicited a significant increase in mucin synthesis, which was reflected in the enhanced metabolism of mucosal phosphoinositides. In contrast, ethanol, which exhibited detrimental effects on mucin synthesis, also caused alterations in the phosphoinositide signal pathway. The changes in mucin and phosphoinositide distribution patterns evoked by ethanol were prevented by sucralfate. Our results suggest that the mucosal strengthening action of sucralfate occurs through the stimulation of the metabolism of phosphoinositide-derived messenger molecules.

Animals↗

PubMed1991

Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane.

The distribution of phosphoinositides and phosphatidic acid (PA) between the outer and inner layers of the human erythrocyte membrane was investigated by using two complementary methodologies: hydrolysis by phospholipase A2 (PLA2) and immunofluorescence detection with monoclonal antibodies against polyphosphoinositides. The contents of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and PA were decreased by 15-20% after 60 min incubation with PLA2, while that of phosphatidylinositol (PI) was increased. Studies with 32P-labelled cells revealed that PLA2 treatment led to indirect effects on the metabolism of these phospholipids. Therefore, the asymmetric distribution of phosphoinositides and PA was inferred from the data obtained in ATP-depleted erythrocytes. In these cells with arrested phosphoinositide metabolism, the asymmetric distribution of the major phospholipids was maintained: PLA2 hydrolyzed approx. 20% of PI, PIP2 and PA (but no PIP) indicating their localization in the outer layer of the membrane. This finding was confirmed by immunofluorescence studies with antibodies specific to each phosphoinositide. External addition of anti-PIP2 but not anti-PIP gave a positive reaction both in control and in ATP-depleted erythrocytes. A pretreatment of cells with PLA2 led to a decrease in the intensity of anti-PIP2 staining. These results demonstrate that significant fractions of PIP2, PI and PA are localized on the outer surface of the erythrocyte membrane.

Adenosine Triphosphate↗