Inositol Phospholipid Breakdown Research Articles

Regulation of phospholipid hydrolysis and second messenger formation by protein kinase C

The binding of a variety of agonists to their receptors leads to the breakdown of membrane phospholipids and the formation of intracellular second messengers. Hydrolysis of inositol phospholipids by phospholipase C results in the formation of two second messengers, inositol-1,4,5-trisphosphate which mobilizes intracellular calcium and the neutral lipid diacylglycerol (DAG) which binds to and activates protein kinase C (PKC). PKC is actually a family of homologous serine/threonine protein kinases which play a central role in regulation of growth, differentiation and secretion reactions in a variety of cell types. In addition to these feedforward role of PKC, it is thought to play an important feedback role, regulating early events in signal transduction. To explore these feedback functions we have examined the effect of PKC inhibitors on second messenger formation in thrombin-stimulated human platelets (a rapidly responding system) and the effect of PKC overexpression on second messenger formation and mitogenesis in rat fibroblasts (a system where sustained signaling occurs). Treatment of platelets with inhibitors of PKC potentiates DAG mass formation in response to thrombin while prior activation of PKC with phorbol esters blocks DAG mass formation, consistent with PKC playing a negative feedback role, inhibiting inositol phospholipid breakdown. DAG can also be formed by the sequential hydrolysis of phosphatidylcholine by phospholipase D and phosphatidic acid phosphohydrolase. This is a minor reaction in the rapidly responding platelet system, but may play a role in sustained signaling events. We have found that fibroblasts which overexpress the β1 isozyme of PKC display greatly enhanced DAG formation and phospholipase D activation in response to phorbol ester treatment. Upon stimulation of fibroblasts with thrombin, phospholipase D activation is also enhanced by PKC overexpression while formation of inositol phosphates is suppressed. These data suggest that PKC may act as a switch, terminating inositol phospholipid hydrolysis and activating the hydrolysis of phosphatidylcholine. Furthermore, we have observed a strong correlation between activation of phospholipase D and mitogenesis, suggesting an important role for this enzyme in long-term cellular responses to activation.

The agonist-stimulated accumulation of inositol phosphates is attenuated in neutrophils from male patients under chronic lithium therapy

Neutrophils from 22 patients (11 men, 11 women) under chronic lithium therapy and from 22 age- and sex-matched healthy controls were assessed for the activity of the agonist-stimulated inositol-phospholipid second messenger-producing system. [ 3H]inositol-labeled cells were stimulated with the chemotactic peptide formylmethionylleucylphenylalanin (fMLP). The fMLP-evoked increase in the accumulation of [ 3H]inositol phosphates was significantly attenuated in neutrophils from chronically lithium-treated male but not female patients. Furthermore, the fMLP-stimulated accumulation of inositol phosphates was attenuated in neutrophils from male volunteers, when the labeling of the cells with [ 3H]inositol was performed in the presence of 1mM Li (4 hr, 37°C). However, the presence of lithium ions during the labeling did not further reduce the already diminished response of neutrophils from patients under lithium therapy. These results suggest that lithium treatment induces an inhibition of the agonist-evoked breakdown of inositol phospholipids in human cells, as already shown for rat brain slices.

Contrasting effects of sn-1,2-dioctanoyl glycerol as compared to other protein kinase C activators in adrenal glomerulosa cells

Angiotensin II acts on adrenal glomerulosa cells to induce the phospholipase C-mediated generation of inositol trisphosphate and sn-1,2-diacyglycerol as the major products of inositol phospholipid breakdown. This last product is known to activate protein kinase C, but its role in the action of angiotensin II on steroidogenesis has not been defined. We report herein that, in bovine adrenal glomerulosa cells, protein kinase C activators, such as phorbol 12,13-dibutyrate, 12-O-tetradecanoylphorbol-13-acetate, mezerein and sn 1,2 oleoyl acetoylglycerol, each failed to increase steroidogenesis. These results contrast with our recent report on the enhancement of aldosterone output by sn-1,2-dioctanoylglycerol (DiC8) [ J. Steroid Biochem. 35 (1990) 19–33]. In addition, the difference between DiC8 and the other protein kinase activators was also observed in the pattern of 86Rb efflux from preloaded glomerulosa cells; only DiC8 mimicked the effect of angiotensin II on ion fluxes. Furthermore, staurosporine, a potent inhibitor of protein kinase C, was capable of amplifying the aldosterone output induced by a maximally effective concentration of DiC8 or angiotensin II. These data suggest that the effect of the cell permeant DiC8 on aldosterone biosynthesis either is not mediated by protein kinase C activation, or is mediated by a phorbol ester-insensitive isoenzyme of protein kinase C.

Retinoic Acid-Induced Granulocytic Differentiation of HL60 Human Promyelocytic Leukemia Cells Is Preceded by Downregulation of Autonomous Generation of Inositol Lipid-Derived Second Messengers

Inositol phosphates (InsPs) and diacyglycerol (DAG) are second messengers derived via the breakdown of inositol phospholipids, and which play important signalling roles in the regulation of proliferation of some cell types. We have studied the operation of this pathway during the early stages of retionic acid (RA)-induced granulocytic differentiation of HL60 myeloid leukemia cells. The autonomous breakdown of inositol lipids that occurred in HL60 cells labeled with [3H] inositol was completely abolished following 48 hours of RA treatment. The rate of influx of 45Ca2+ was also significantly decreased at 48 hours, consistent with the role of inositol lipid-derived second messengers in regulating Ca2+ entry into cells. The downregulation of inositol lipid metabolism clearly preceded the onset of reduced proliferation induced by RA treatment, and was therefore not a consequence of decreased cell growth. The generation of InsPs in RA-treated cells was reactivated by the fluoroaluminate ion, a direct activator of guanine nucleotide-binding protein(s) (G proteins) that regulate the inositol lipid signalling pathway. Subtle alterations to a regulatory mechanism may therefore mediate the RA-induced downregulation of this pathway. The data are consistent with the hypothesis that the autonomous generation of inositol lipid-derived second messengers may contribute to the continuous proliferation of HL60 cells, and that the RA-induced downregulation of this pathway may, in turn, play a role in signalling the cessation of proliferation that preceedes granulocytic differentiation.

Retinoic acid-induced granulocytic differentiation of HL60 human promyelocytic leukemia cells is preceded by downregulation of autonomous generation of inositol lipid-derived second messengers

Inositol phosphates (InsPs) and diacyglycerol (DAG) are second messengers derived via the breakdown of inositol phospholipids, and which play important signalling roles in the regulation of proliferation of some cell types. We have studied the operation of this pathway during the early stages of retionic acid (RA)-induced granulocytic differentiation of HL60 myeloid leukemia cells. The autonomous breakdown of inositol lipids that occurred in HL60 cells labeled with [3H] inositol was completely abolished following 48 hours of RA treatment. The rate of influx of 45Ca2+ was also significantly decreased at 48 hours, consistent with the role of inositol lipid- derived second messengers in regulating Ca2+ entry into cells. The downregulation of inositol lipid metabolism clearly preceded the onset of reduced proliferation induced by RA treatment, and was therefore not a consequence of decreased cell growth. The generation of InsPs in RA- treated cells was reactivated by the fluoroaluminate ion, a direct activator of guanine nucleotide-binding protein(s) (G proteins) that regulate the inositol lipid signalling pathway. Subtle alterations to a regulatory mechanism may therefore mediate the RA-induced downregulation of this pathway. The data are consistent with the hypothesis that the autonomous generation of inositol lipid-derived second messengers may contribute to the continuous proliferation of HL60 cells, and that the RA-induced downregulation of this pathway may, in turn, play a role in signalling the cessation of proliferation that preceedes granulocytic differentiation.

Temporal studies of the inhibition of voluntary ethanol intake in the rat induced by intermittent ethanol treatment and some long term neurochemical consequences

Male rats were injected with ethanol (groups 3 and 5; 2.0 g/kg i.p.) or saline (groups 2 and 4) once a week for 52 weeks. The rats had access to ethanol as a voluntary choice for 24 h either once 6 days after the injection (groups 2 and 3) or twice 3 and 6 days after the injection (groups 4 and 5). At the beginning of the treatment ethanol injections inhibited voluntary ethanol intake if tested 6 days later (groups 3 and 5), but a tolerance developed to this inhibition. During tolerance development the rats in group 5 also drank less ethanol on day 3 than on day 6. No corresponding behaviour was seen in group 4. Thus part of the tolerance was a gradual reduction of the duration of inhibition. During the evaluation period (25 weeks) after the treatment, ethanol exposure (20 weeks) consisted of a continuous choice between ethanol and water. Of different ethanol concentrations both ethanol-injected groups (3 and 5) took the same voluntary dose of ethanol independent of the offered concentration. After 5 weeks without ethanol all rats were killed and a number of neurochemical variables were determined. Compared with almost unexposed rats (group 1) changes were seen in inositol phospholipid breakdown, muscarinic binding sites in hippocampus, noradrenaline concentrations in frontal cortex, hippocampus and hypothalamus, dopamine concentration in frontal cortex and 5-hydroxytryptamine concentration in hypothalamus. In most cases the largest changes were seen in group 5. None of the variables had a constant relation to ethanol intake in the total population. However, significant correlations were found in some of the groups.

Mutational analysis of antigen receptor regulation of B lymphocyte growth. Evidence for involvement of the phosphoinositide signaling pathway.

Stimulation of the antigen receptor of WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-IgM) induces irreversible growth arrest. Anti-IgM stimulates two kinds of transmembrane signaling events, phosphorylation of proteins on tyrosyl residues and breakdown of inositol phospholipids, which results in increases of inositol phosphates, diacylglycerol, and calcium. The roles of these reactions in mediating the growth arrest of the B lymphoma cells have not been established. To examine this issue, we took a genetic approach. Mutants of WEHI-231 cells were isolated that were resistant to anti-IgM-induced growth arrest. Five out of seven independent mutants analyzed had normal cell-surface expression of antigen receptors. Although each of these five mutants had tyrosine protein phosphorylation patterns comparable to wild-type cells, they exhibited alterations in the phosphoinositide signaling pathway. Four of the mutants had decreased phosphoinositide breakdown, probably due to an alteration in phospholipase C. Decreased second messenger production may be responsible for the growth-resistant phenotype. Full growth arrest was restored upon addition of the calcium ionophore ionomycin, suggesting that the limiting second messenger was intracellular free calcium. The final mutant appeared to be altered in a component(s) that responds to diacylglycerol and calcium. Taken together, these results provide further evidence that the phosphoinositide pathway is at least partly responsible for mediating antigen receptor regulation of B lymphoma cell growth.

Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts.

We are using a Rat-6 fibroblast cell line that stably overexpresses the beta 1 isozyme of protein kinase C (PKC) to study regulation of phospholipid hydrolysis by PKC. Stimulation of control (R6-C1) or overexpressing (R6-PKC3) cells with phorbol ester results in an increase in diacylglycerol (DAG) mass with no increase in inositol phosphates, indicating that DAG is not formed by inositol phospholipid breakdown. A more dramatic DAG increase occurs in R6-PKC3 cells (4.0-fold over basal) compared to R6-C1 cells (1.5-fold over basal). To further define the source of DAG, phosphatidylcholine (PC) pools were labeled with [3H]myristic acid or with [3H]- or [32P]alkyllyso-PC and formation of labeled phosphatidylethanol, an unambiguous marker of phospholipase D activation, was monitored. Phorbol ester-stimulated phosphatidylethanol formation is 5-fold greater in the R6-PKC3 cell line. Formation of radiolabeled phosphatidic acid (PA) is also enhanced by PKC overexpression. In cells double-labeled with [3H]- and [32P]-alkyl-lysoPC, the 3H/32P ratio of PA and PC are identical 15 min after stimulation, suggesting that a phospholipase D mechanism predominates. In support of this, the PA phosphohydrolase inhibitor propranolol decreased phorbol 12-myristate 13-acetate-stimulated DAG formation by 72%. Increases in DAG and phosphatidylethanol were inhibited by the PKC inhibitors K252a and staurosporine. These results indicate that phospholipase D is regulated by the action of PKC. Enhanced phospholipase D activity may contribute to the growth abnormalities seen in PKC-overexpressing cells.

Antagonism by 8-hydroxy-2 (DI-N-propylamino) tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells

IMR-32 and SK-N-MC cells were found to contain [ 3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [ 3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC 50 value of about 50 μM in both cases. Pirenzepine inhibited the carbachol (100 μM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC 50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT 1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA 2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [ 3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (−)alprenolol. 5-HT (10–100 μM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.

The Na +, K +-ATPase inhibitor ouabain and the Na + ionophore monensin have opposite effects upon carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms

The effects of ouabain and monensin upon basal and carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms have been investigated. Basal inositol phospholipid breakdown was increased by both compounds at both 6 and 18 mM K +. Enhancement of the carbachol reponse at 6 mM, but not at 18 mM K +, was found with high concentrations of ouabain. On the other hand, monensin blocked the response to carbachol. Monensin also inhibited the specific binding of [ 3H]pirenzepine to cerebral cortical membranes, but this was found only at concentrations higher than required to affect the basal and carbachol-stimulated inositol phospholipid breakdown responses. Quabain did not affect [ 3H]pirenzepine binding at any of the concentrations tested (6–600 μM). It is concluded that agents that increase the intracellular sodium ion concentration affect the inositol phospholipid breakdown response to carbachol, but that the modulation can be both to potentiate and to inhibit the response.

Modulation of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms by excitatory amino acids and by bay K-8644 is dependent upon the assay calcium and potassium concentrations used

The calcium and potassium ion dependencyof the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 (10 μM) potentiated the response to carbachol at 6 mM K + when Ca 2+-free, but not when 2.52 mM Ca 2+ assay buffer was used. In Ca 2+-free buffer, verapamil (10 μM) inhibited the response to carbachol at both 6 and 18 mM K +, but higher concentrations (30–300 μM) were needed when 2.52 mM Ca 2+ was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM [ 3H]pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA, 100 μM) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K + at 1.3 mM Ca 2+, but was without effect on the basal rate at other K + and Ca 2+ concentrations. In the presence of NMDA (100 μM) or quisqualate (100 μM), the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K + and Ca 2+ concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used.

Muscarinic receptors in isolated canine gastric parietal cells involved in cyclic AMP formation and inositol phospholipid breakdown

Muscarinic receptors in isolated canine gastric parietal cells involved in cyclic AMP formation and inositol phospholipid breakdown

Modulation of receptor-mediated inositol phospholipid breakdown in the brain

Modulation of receptor-mediated inositol phospholipid breakdown in the brain

Multiple actions of fluoride ions upon the phosphoinositide cycle in the rat brain

The effects of sodium fluoride upon basal and agonist-stimulated inositol phospholipid breakdown have been investigated in rat brain miniprisms. NaF concentration dependently increased basal inositol phospholipid breakdown, with a maximum effect being seen at 20 mM. NaF reduced the inositol phospholipid breakdown responses to stimulation by carbachol, noradrenaline, serotonin and quisqualate, but not to the stimulation produced by raising the assay [K +] from 6 to 18 mM. More detailed study demonstrated NaF to have a ‘levelling’ effect, reducing all InsP/(Lipid + InsP) valuesn 0.15 (i.e. produce by carbachol at raised [K +], noradrenline and by 50 mM K +) to about this Time-course experiments indicated that NaF treatment reduced the rate of carbachol-stimulated inositol phospholipid breakdown up to this InsP/(Lipid + InsP) level and thereafter blocked further breakdown. Inhibitory effects upon carbachol-stimulated inositol phospholipid phosphoinositides from [ 3H]myo-inositol, NaF reduced the total Lipid + InsP labelling by about 20%. NaF in addition inhibits the activity of Ins(1,4)P 2-phosphatase in cerebral cortical homogenates. It is concluded that fluoride ions inhibit agonist-stimulated inositol phospholipid breakdown via actions not only on G-proteins but also on phosphoinositide-specific phospholipase C substrate availability.

Pharmacological characterization of tachykinin‐stimulated inositol phospholipid hydrolysis in peripheral tissues

1. Tachykinin-stimulated inositol phospholipid hydrolysis was examined in slices of rat parotid gland, hamster urinary bladder and guinea-pig ileum longitudinal muscle. 2. In the presence of lithium, substance P and other naturally-occurring and synthetic tachykinins induced large, dose-dependent increases in [3H]-inositol monophosphate accumulation. 3. In slices of rat parotid gland, [pGlu6,L-Pro9]SP(6-11) was considerably more potent in stimulating inositol phospholipid hydrolysis than [pGlu6,D-Pro9]SP(6-11). 4. In contrast, in slices of hamster urinary bladder, [pGlu6,D-Pro9]SP(6-11) exhibited greater potency in evoking inositol phospholipid breakdown than [pGlu6,L-Pro9]SP(6-11). 5. The differential selectivity of these C-terminal fragments of substance P suggests that they may be useful tools for distinguishing between NK1 and NK2 receptors. 6. L-659,837 and L-659,874 antagonized eledoisin-stimulated inositol phospholipid hydrolysis in slices of hamster urinary bladder. Neither compound significantly reduced substance-P evoked inositol phospholipid breakdown in slices of rat parotid gland, or senktide-induced inositol phospholipid hydrolysis in slices of guinea-pig ileum. 7. L-659,837 and L-659,874 had no effect on the atropine-sensitive, carbachol-stimulated inositol phospholipid hydrolysis in slices of rat parotid gland. 8. These data further support the notion that L-659,837 and L-659,874 are potent and selective NK2 receptor antagonists.

Pertussis toxin produces differential inhibitory effects on basal, P2-purinergic, and chemotactic peptide-stimulated inositol phospholipid breakdown in HL-60 cells and HL-60 cell membranes.

P2-purinergic receptor agonists (UTP) and formylated peptide receptor agonists (FMLP) were found to be equally efficacious in eliciting rapid 6-7-fold increases in inositol polyphosphate accumulation in differentiated HL-60 granulocytes. The activation of this response by either agonist was substantially but incompletely inhibited in cells treated with pertussis toxin. Thus, in cells containing only 1-10% of the control level of non-ADP-ribosylated Gi-2/3, UTP induced rapid 2-fold increases in inositol polyphosphate accumulation whereas smaller 50% increases were observed in FMLP-stimulated cells. Washed membranes prepared from control and toxin-treated HL-60 cells were used to characterize this toxin-insensitive activation of phospholipase C further. The agonist-independent stimulation of phospholipase C by either millimolar Ca2+ or the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was only modestly attenuated by toxin treatment. There was a 70-80% decrease in the rate and extent of phospholipase C activity stimulated by GTP per se in the absence of receptor agonists. The rate and extent of FMLP-induced potentiation of GTP-dependent phospholipase C activity were also inhibited by greater than 80% in toxin-treated membranes. Conversely, the potency and efficacy characterizing UTP-induced potentiation of GTP-dependent phospholipase C activity were only modestly attenuated (less than 20% inhibition). The results indicate that P2-purinergic receptors (and perhaps other Ca2(+)-mobilizing receptors) activate both pertussis toxin-sensitive and toxin-insensitive pathways for phospholipase C regulation in phagocytic leukocytes.

Differential enhancement by potassium ions of M1-type and M2-type muscarinic receptor-mediated phosphoinositide breakdown in the rat brain

Raising the assay [K+] from 6 to 18 mM enhances the inositol phospholipid breakdown response to carbachol in rat brain miniprisms. In the frontal cortex, the degree of enhancement by K+ was independent of the carbachol concentration used, whereas in the striatum, a significantly higher degree of enhancement was seen at 1000 than at 50 microM carbachol. The carbachol-stimulated inositol phospholipid breakdown was antagonized by pirenzepine at both [K+] with potencies suggesting involvement of M1-type muscarinic receptors in the frontal cortex and both M1- and M2-type muscarinic receptors in the striatum. It is suggested that the response mediated by the M1-type receptors is enhanced to a greater degree by raised [K+] than that mediated by the M2-type receptors.

Protein tyrosine phosphorylation is induced in murine B lymphocytes in response to stimulation with anti-immunoglobulin.

Activation of both T and B lymphocytes through their membrane receptors for antigen is known to induce breakdown of inositol phospholipids. In addition, T cell activation by antigen is accompanied by increased protein tyrosine phosphorylation of components of the T cell antigen receptor. We now provide evidence that B cell activation through membrane immunoglobulin is also coupled to stimulation of protein tyrosine kinase activity. One potential candidate for a B lymphocyte protein tyrosine kinase is an 80 kd molecule that is itself phosphorylated at tyrosine residues in response to stimulation with anti-immunoglobulin antibodies.

Enhancement of carbachol-stimulated inositol phospholipid breakdown in rat brain miniprisms by potassium ions: mechanistic studies

Enhancement of carbachol-stimulated inositol phospholipid breakdown in rat brain miniprisms by potassium ions: mechanistic studies

Inhibition of Polyphosphoinositide Breakdown and c‐myc Induction Accompanying Inhibition of Human B‐Cell Activation by Two Monoclonal Antibodies Against the Leucocyte Common Antigen (CD45)

In this report we show that the two monoclonal anti-CD45 antibodies, EO-1 and FN-126, potently inhibit G0 to G1 transition and S phase entry in human B cells stimulated with anti-mu and low molecular weight B-cell growth factor. Both antibodies were found to inhibit anti-mu-induced inositol phospholipid breakdown and c-myc mRNA induction. In contrast, EO-1 and FN-126 only partially inhibited the early anti-mu-induced increase in cytoplasmic Ca2+ levels, both in normal and in Ca2(+)-depleted medium. B-cell activation provoked by 12-O-tetradecanoylphorbol 13-acetate (TPA) was not inhibited by these antibodies, except when using high concentrations of EO-1. In addition, both antibodies were found to inhibit G1 entry induced by the anti-CD20 antibody 1F5, which confers an activation of B cells without any detectable increase in [Ca2+]i or in phospholipid metabolism. This indicates that alternative mechanisms in addition to the inhibition of polyphosphoinositide (PI) breakdown are involved in the inhibitory action of these antibodies.