Inositol Phospholipid Turnover Research Articles

Nerve Extracts and Substance P Activate the Phosphatidylinositol Signaling Pathway and Mitogenesis in Newt Forelimb Regenerates

We investigated the inositol phospholipid transmembrane signaling pathway as a possible mediator of neurotrophic (mitogenic) signals in the newt limb regeneration blastema. Blastema mesoderm tissues were prelabeled with myo-[ 3H]inositol, treated with 10 m M LiCl and then exposed to substance P or to extracts of spinal ganglia, brain, or spinal cord. Stimulation with substance P resulted in a rapid dose-dependent reduction of [ 3H]phosphatidylinositol 4,5-bisphosphate and [ 3H]phosphatidylinositol 4-phosphate, correlated with a rapid accumulation of inositol 1,4,5-triphosphate. This effect was inhibited when the blastema tissue was treated with neomycin, a known inhibitor of inositol phospholipid turnover. In addition, substance P stimulated the incorporation of [ 3H]thymidine into DNA of blastema mesoderm cells, and this effect was also suppressed by neomycin, at a dose corresponding to that required to inhibit inositol phosphate accumulation. Extracts of neural tissues, especially spinal ganglia, induced the formation of inositol phosphates and extract activity was attenuated following treatment with heat or trypsin. These findings suggest a role for mitogen-activated inositol phospholipid signaling, initiating events that ultimately lead to cell proliferation.

G-protein involvement in muscarinic receptor-stimulation of inositol phosphates in longitudinal smooth muscle from the small intestine of the guinea-pig.

1. Aluminium fluoride (AlF), pertussis toxin (PTX) and cholera toxin (ChTX) have been used to examine the involvement of G-proteins during muscarinic acetylcholine receptor (AChR) stimulation of inositol phospholipid hydrolysis in fragments of longitudinal smooth muscle from the small intestine of the guinea-pig. 2. Carbachol (CCh) induced time- and concentration-dependent increases in [3H]-inositol monophosphates, [3H]-inositol (1,4) bisphosphate, [3H]-inositol (1,3,4) trisphosphate, [3H]-inositol (1,4,5) trisphosphate ([3H]-Ins (1,4,5)P3) and [3H]-inositol tetrakisphosphates measured by h.p.l.c. These increases were inhibited > 95% in the presence of the muscarinic AChR antagonist atropine (0.5 microM). 3. AlF transiently increased the basal levels of [3H]-Ins (1,4,5)P3 but increases in the levels of the other [3H]-inositol phosphates occurred more slowly. CCh-induced increases in the levels of all the [3H]-inositol phosphates were strongly inhibited in the presence of AlF. 4. PTX had no effect on basal levels of any of the [3H]-inositol phosphates but reduced the effects of CCh on these; ChTX had no effects on either basal or CCh-stimulated levels. 5. It was concluded that muscarinic AChR-stimulated increases in the levels of [3H]-inositol phosphates occur via both a PTX-sensitive G-protein and a PTX-insensitive mechanism. The actions of AlF may suggest the involvement of an inhibitory G-protein in the regulation of muscarinic AChR-stimulated inositol phospholipid turnover.

A study of the synergism between metabotropic glutamate receptor activation and arachidonic acid in the rat hippocampus.

We report that activation of the metabotropic glutamate receptor by the specific agonist, trans-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD), increases release of glutamate only in the presence of a low concentration of arachidonic acid (AA). To identify the molecular mechanism underlying this effect, cAMP accumulation, inositol phospholipid metabolism and protein kinase C (PKC) activation were examined in synaptosomes prepared from hippocampus. ACPD increased cAMP accumulation, but this increase was not further enhanced in the presence of AA. ACPD and AA stimulated both inositol phospholipid turnover and PKC activity; a synergistic action was indicated by the additional stimulation in the presence of both agents. The increase in PKC activity, either directly, or indirectly by increased inositol phospholipid turnover, might therefore underlie the enhanced glutamate release.

Anaphylatoxin C3a induces rapid protein phosphorylation in guinea pig platelets

We investigated C3a signal transduction using guinea pig platelets. Anaphylatoxin C3a induced cytosolic calcium influx and turnover of inositol phospholipids and caused protein phosporylation via specific C3a receptors on guinea pig platelets. Phosphorylation of 40 kDa and 20 kDa proteins was detected within 30 s after C3a stimulation. From phosphoamino acid analysis of both proteins, it was found that about 80% was serine and 20% was threonine. Phosphorylation was decreased by pretreatment of platelets with calcium blockers, diltiazem and TMB-8. A protein kinase C inhibitor, staurosporine, effectively blocked phosphorylation of both proteins, but H-7 did not. The effects of these inhibitors on platelet aggregation were correlated with their effects on protein phosphorylation. These results suggest that protein-serine/ threonine phosphorylation is important for C3a signal transduction in guinea pig platelets and that the isoenzyme of protein kinase C or another kinase probably participates in such protein phosphorylation. C3a also caused protein-tyrosine phosphorylation of 56 kDa and 33–36 kDa proteins within 30 s. Staurosporine effectively blocked phosphorylation of these bands. It is suggested that tyrosine kinase also may be involved in C3a signalling in guinea pig platelets.

Impaired spatial memory in aged rats is associated with alterations in inositol phospholipid metabolism.

WE have investigated changes in inositol phospholipid turnover and membrane arachidonic acid concentration in the dentate gyrus and frontal cortex of animals aged 3-6 months, 14 months or 22 months which were trained in the Morris water maze. Ageing was associated with poorer performances in the behavioural test, characterized by increased variability in retention and acquisition amongst individuals, a decrease in membrane arachidonic acid concentration and increased unstimulated inositol phospholipid metabolism in synaptosomes prepared from frontal cortex and dentate gyrus. Arachidonic acid stimulated inositol phospholipid metabolism in synaptosomes, but in the older groups, stimulation was associated with good performance in the Morris water maze. In slices prepared from frontal cortex, responsiveness of inositol phospholipid metabolism to glutamate was also age- and performance-dependent. The findings highlight a correlation between age, inositol phospholipid metabolism and performance in the Morris water maze.

Visualization of agonist-stimulated inositol phospholipid turnover in individual neurons of the rat cerebral cortex and hippocampus

A novel autoradiographic method to identify individual neurons responding to neurotransmitter stimulation with increased phosphoinositide turnover is described. When phosphoinositide-coupled receptors are activated, phosphatidylinositol 4, 5-bisphosphate is hydrolysed by phospholipase C generating the two second messengers, inositol 1, 4, 5-trisphosphate and diacylglycerol. During prolonged receptor stimulation, both second messengers are actively recycled to maintain the effective intracellular levels of agonist-sensitive phosphoinositides. Lithium ions inhibit this recycling pathway by blocking the recovery of free inositol from inositol 1, 4, 5-trisphosphate thus leading to the accumulation of phosphatidyl cytidine monophosphate, a membrane bound molecule which is the activated precursor of the synthesis of phosphoinositides. Therefore, addition of excess myo-inositol reverts the effects of lithium inhibition. Thus, taking advantage of this fact and using [ 3H]cytidine as precursor, phosphatidyl [ 3H]cytidine monophosphate accumulation was induced in rat neocortical and hippocampal slices after muscarinic or metabotropic glutamate receptor stimulation. The labelled slices were then fixed, dehydrated and embedded in Durcupan resin. Semithin sections (1 μm thick) were cut and exposed to autoradiographic emulsion for several weeks. Biochemical analysis of the incorporation of [ 3H]cytidine into the chloroform extracted (containing lipids) and the alkali-solubilized (containing nucleic acids and proteins) fractions were carried out in parallel with morphological studies. The stimulation of both receptor types induced labelling of neurons in neocortex and hippocampus. In labelled cells silver grains were characteristically observed over the cytoplasm surrounding the nucleus and main dendritic processes. The anatomical location and distribution of labelled cells as well as the levels of response obtained in both brain regions studied, was found to be receptor specific. Inclusion of 30 mM myo-inositol in the incubation media reversed completely both the accumulation of phosphatidyl [ 3H]cytidine monophosphate and the labelling of cells, thus demonstrating that the label detected autoradiographically corresponds to phosphatidyl [ 3H]cytidine monophosphate. It is concluded that the method is sensitive and specific, allowing identification of individual neurons in both neocortical and hippocampal slices and after stimulation of both muscarinic and metabotropic glutamate receptor subtypes. The method may open a new means to study the phosphoinositide second messenger signalling pathway and the cells in which it takes place.

Accumulation of inositol phosphates in low-passage human meningioma cells following treatment with epidermal growth factor

In order to elucidate some of the signal transduction processes in human meningioma cells, the authors studied the effect of epidermal growth factor (EGF) and bromocriptine on inositol phospholipid hydrolysis, using low-passage human meningioma cells in culture. Epidermal growth factor is a well-studied mitogenic factor for meningioma cells, whereas bromocriptine is known to have an inhibitory effect on meningioma cell proliferation. The addition of EGF to meningioma cells caused stimulation of inositol phosphate accumulation in a dose-dependent manner at 60 minutes posttreatment, with the maximum effect (120% to 167% of control) achieved at a concentration of 10 ng/ml. Extraction of separate inositol phosphates accumulation in a dose-dependent manner at 60 minutes posttreatment, with the maximum effect (120% to 167% of control) achieved at a concentration of 10 ng/ml. Extraction of separate inositol phosphates revealed that inositol monophosphate (IP1) and inositol bisphosphate (IP2), but not inositol trisphosphate (IP3), accounted for the increase at 60 minutes. Kinetic analysis of EGF-stimulated inositol phospholipid hydrolysis showed that a sharp and transient increase in IP3 from 5 to 12 minutes post-EGF and a transient but more gradual increase in IP2 from 2 to 12 minutes post-EGF were followed by a gradual and steady increase in IP1, which was significantly greater than control after 5 minutes. On the other hand, long-term studies showed a down-regulation of inositol phosphate accumulation (a 64% decrease vs. control) after 7 days of treatment with EGF (10 ng/ml). Bromocriptine (5 microM) exhibited no significant effect on inositol phosphate accumulation at 60 minutes in four of five meningiomas studied. However, of two meningiomas studied with bromocriptine in combination with EGF, both showed a significant additive increase in inositol phosphate accumulation compared to those treated with EGF alone. The results suggest a close involvement of inositol phospholipid turnover in human meningioma cells in response to mitogenic stimulation by EGF.

Activation of inositol phospholipid turnover and calcium signaling in rat Sertoli cells by P2-purinergic receptors: modulation of follicle- stimulating hormone responses

Activation of inositol phospholipid turnover and calcium signaling in rat Sertoli cells by P2-purinergic receptors: modulation of follicle- stimulating hormone responses

Activation of inositol phospholipid turnover and calcium signaling in rat Sertoli cells by P2-purinergic receptors: modulation of follicle-stimulating hormone responses.

To study the role of extracellular nucleotides in the regulation of Sertoli cells, the effects of ATP and its analogs on the Ca(2+)-phospholipid- and cAMP-dependent pathways were tested. Cultured Sertoli cells from immature animals were incubated with ATP or structurally related compounds, and phosphoinositide (PI) turnover or cAMP accumulation was measured. Among the several nucleotide phosphate analogs tested, adenosine 5'-O-(3-thiotriphosphate) was the agonist most potent in stimulating inositol phosphate accumulation. The effects of purine nucleotides on PI turnover were time and concentration dependent. Because nonhydrolizable ATP analogs also stimulated PI turnover, ATP metabolites or metabolic products are not responsible for the observed stimulation. The order of potency of the different ATP analogs [adenosine 5'-O-(3-thiotriphosphate) > ATP approximately equal to UTP > beta, gamma-methyleneadenosine 5'-triphosphate, 2-methylthio-ATP > adenosine] was consistent with the presence of P2U receptors (nucleotide receptors) on the surface of the Sertoli cell. Augmented PI turnover was accompanied by a transient increase in Ca2+ concentration, measured in single Sertoli cells loaded with the intracellular Ca2+ indicator fura-2. When used alone, ATP and its analogs did not have a direct effect on cAMP levels in the Sertoli cell. However, ATP or its analogs inhibited FSH-dependent cAMP accumulation by more than 70%. Purine nucleotides also efficiently blocked the effects of FSH distal to cAMP accumulation, because extracellular ATP completely reversed the changes in Sertoli cell shape induced by FSH. The nucleotide-dependent inhibition of cAMP accumulation was blocked by pertussis toxin to a different degree depending on the purine or pirimidine nucleotide used. This indicated that more than one mechanism contributes to the purine nucleotide-dependent inhibition of cAMP accumulation. These data provide evidence that purine nucleotide receptors coupled to multiple pathways are present on the Sertoli cell in culture, and that extracellular ATP has profound biological effects on the FSH responsiveness of the Sertoli cell.

The Nonpeptide Tachykinin NK 2 Receptor Antagonist SR 48968 Interacts with Human, but Not Rat, Cloned Tachykinin NK 3 Receptors

SR 48968, a non-peptide tachykinin NK 2 receptor antagonist, has been shown to possess sub-micromolar affinity for NK 3 receptors present in the guinea pig. In the present study, we have compared the binding affinities of SR 48968 to the cloned human and rat NK 3 receptors expressed in CHO cells. Using [ 125I]-[MePhe 7]-neurokinin B as the radioligand, SR48968 displayed an IC 50 value of 350 nM for the human NK 3 receptor as compared with a value of greater than 10 μM for the rat NK 3 receptor. Exposure of cells transfected with human NK 3 receptor cDNA to [Pro 7]-neurokinin B increased inositol phospholipid turnover in a concentration-dependent manner and this response was blocked competitively by SR 48968. Our results demonstrate that SR 48968 is an antagonist at the human NK 3 receptor and may be a useful tool for elucidating the species-dependent variations in the non-peptide antagonist binding site(s) on the NK 3 receptor.

Abnormal antigen receptor-initiated signal transduction in lpr T lymphocytes

The CD4 -, CD8 - double-negative (DN) T cells that accumulate in an age-dependent manner in peripheral lymphoid organs of lpr-homozygous mice display deficient activation in response to signals initiated by the antigen-specific T cell receptor (TCR)/CD3 complex. Abnormalities which could contribute to this defect include low expression and rapid TCR/CD3 modulation, loss of CD4, CD8, CD2 and Ly-6.2, aberrant expression of CD45, and constitutive elevations of inositol phospholipid turnover, tyrosine phosphorylation, and expression of the p59 fyn protein tyrosine kinase (PTK) and a few other protooncogenes. These properties strongly suggest that DN lpr T cells are chronically activated in vivo, perhaps by some self antigen(s), rendering them refractory to additional in vitro stimulation. Deficient activation of these cells is, therefore, most likely a secondary consequence of the Fas defect whose primary effect is allowing these aberrant cells to escape from the thymus and expand in the periphery.

Protein Kinase Inhibitors Inhibit Stimulation of Inositol Phospholipid Turnover and Induction of Phenylalanine Ammonia-Lyase in Fungal Elicitor-Treated Tobacco Suspension Culture Cells

Protein Kinase Inhibitors Inhibit Stimulation of Inositol Phospholipid Turnover and Induction of Phenylalanine Ammonia-Lyase in Fungal Elicitor-Treated Tobacco Suspension Culture Cells

Stimulation by Fungal Elicitor of Inositol Phospholipid Turnover in Tobacco Suspension Culture Cells

Stimulation by Fungal Elicitor of Inositol Phospholipid Turnover in Tobacco Suspension Culture Cells

Inositol phospholipid turnover in digitonin-permeabilised dispersed mouse pancreatic islet cells.

Inositol phospholipid turnover in digitonin-permeabilised dispersed mouse pancreatic islet cells.

UTP activates multiple second messenger systems in cultured rat astrocytes

In astrocytes, a number of second messenger systems are activated upon stimulation with ATP. Recently, UTP has been demonstrated to have effects similar to ATP in some cell types. To determine if this was also true in astrocytes, cultured cells were stimulated with UTP which was found to evoke thromboxane release, stimulate inositol phospholipid turnover and increase intracellular free calcium concentration. A 53 kDa protein was identified on astrocyte membranes by immunoblotting with an antibody raised against a putative rat fibroblast P 21 receptor. These data indicate that astrocytes possess a UTP-sensitive receptor which may be distinct from the P 2Y-purinergic receptor.

Pituitary adenomas with high and low basal inositol phospholipid turnover; the stimulatory effect of kinins and an association with interleukin‐6 secretion

We examined basal inositol phospholipid turnover and the response to the kinin, kallidin, in human pituitary adenomas and determined whether or not there was an association between these parameters and interleukin (IL-6) secretion status by the tumours. Pituitary adenoma tissue was dispersed and cells were cultured in monolayer for 96 hours. The medium was then removed and assayed for IL-6 and anterior pituitary hormones. The cells were labelled with 3H-myoinositol for 24 hours and then incubated under basal conditions with kallidin and, in some cases, with TRH and GnRH for 60 minutes. Total inositol phosphate accumulation and pituitary hormone secretion were assessed. Tissue was collected from 29 consecutive patients being treated surgically for pituitary adenomas. Total 3H-inositol phosphates, growth hormone, prolactin, LH, FSH, TSH and immunoreactive IL-6. Two groups of pituitary adenomas were identified, one with high and one with low basal inositol phospholipid turnover. Kallidin stimulated inositol phosphate accumulation in seven of the 29 adenomas studied. The kallidin-responsive adenomas were associated with high basal phosphoinositide turnover. All seven kallidin-responsive adenomas secreted IL-6. The adenomas studied with high basal inositol phosphate production were also responsive to TRH and in two tumours to GnRH. Kallidin stimulated GH release in one GH-secreting adenoma but had no effect on hormone secretion from any other tumour. Two groups of pituitary adenomas have been identified with high and low basal inositol phosphoinositide turnover. Phosphoinositide metabolism is readily stimulated by kallidin and TRH in adenomas with high but not low turnover. Kinin-responsive adenomas secreted IL-6 but IL-6 secretor status does not preclude that they will respond to kallidin.

Functional nonequivalence of structurally homologous domains of neurokinin-1 and neurokinin-2 type tachykinin receptors

The neurokinin-1 (NK-1) and neurokinin-2 (NK-2) receptors are both members of the tachykinin receptor family. Although both receptors bind peptide ligands synthesized from common precursors and activate inositol 1,4,5-triphosphate and cAMP responses, differences between these receptors have been observed in the extent and kinetics of agonist-induced responses. Here, to test if structurally homologous domains of the NK-1 and NK-2 receptors are functionally distinct, stably transfected Chinese hamster ovary (CHO) cell lines expressing receptors that had either their putative third cytoplasmic loop (C3) or carboxyl tail (CT) domains replaced with the equivalent domain of the other receptor were compared with stably transfected CHO cell lines expressing wild-type receptors. Radioligand binding demonstrated that each of these chimeric receptors had agonist binding affinities indistinguishable from wild-type receptors. However, not all chimeric receptors were equivalent in their ability to stimulate inositol phospholipid turnover and cAMP production. The data suggest that the NK-1 C3 and the NK-2 CT domains play important roles in G-protein activation that cannot be replaced by the analogous domain of the other receptor. The characterization of CHO cell lines expressing truncated forms of both receptors supported the hypothesis that the CT domain of the NK-2, but not the NK-1, receptor plays a critical role in G-protein activation. The data suggest a potential mechanism for the differences observed in response characteristics in tissues expressing NK-1 and NK-2 receptors and demonstrate that the mechanisms whereby highly homologous receptors activate G-proteins can be different.

Prostaglandin Production by Mast Cells and the Regulation of Airway Smooth Muscle Responses

The role of mast cell activation and the generation of mediator release are important factors in determining the reactivity of lung tissue during allergic reactions and asthma. In this article, the advantages of our procedure for isolating mast cells from lung tissue over other methods are discussed. Our studies have demonstrated the importance of cross talk between mast cells and other cell types that also release mediators of bronchoconstriction during activation, thus amplifying the response. In addition, it has been found that inositol phospholipid turnover in response to various mast cell mediators is enhanced in hyperresponsive lung tissue. The consequences to the contractility of the tissue of such alterations in the cellular signaling system in lung tissue are discussed.

Anti-CD9 monoclonal antibody elicits staurosporine inhibitable phosphatidylinositol 4,5-bisphosphate hydrolysis, phosphatidylinositol 3,4-bisphosphate synthesis, and protein-tyrosine phosphorylation in human platelets

Phosphoinositide metabolism elicited by anti-CD9 monoclonal antibody, a well-characterized platelet activator, was studied using acetylsalicylic acid-treated human platelets. TP82, which is an anti-CD9 monoclonal antibody, induced classical phosphatidylinositol 4,5-bisphosphate hydrolysis, as monitored by intracellular Ca 2+ mobilization and phosphatidic acid production, and synthesis of phosphatidylinositol 3,4-bisphosphate, which is a major component of newly-described 3-phosphorylated inositol phospholipids produced during platelet activation. These changes were severely inhibited by 1 μM staurosporine, a potent, though non-selective, protein kinase inhibitor, which also abolished TP82 induction of tyrosine phosphorylation of multiple platelet proteins. Protein-tyrosine phosphorylation appears necessary to initiate both the classical phosphoinositide turnover and synthesis of the newly-described 3-phosphorylated inositol phospholipids in anti-CD9 monoclonal antibody-induced platelet activation.

Differential responses of rat cerebral somatostatinergic and cholinergic cells to glutamate agonists.

Reductions in cortical somatostatin (SRIH) and choline acetyl-transferase (ChAT) are major biochemical deficits in Alzheimer disease (AD). SRIH and ChAT were measured in fetal rat cerebral neurons after exposure to the glutamate agonists N-methyl-D-aspartate (NMDA), kainate (KA), and quisqualate (Q). NMDA (96 h incubation) stimulated SRIH release and content in a dose-dependent manner with a Bmax of 10(-5)M and EC50 of 2-3 x 10(-6)M. KA showed a small stimulation in SRIH levels at 10(-5)M, but produced marked inhibition at 10(-4)M. Q decreased both intracellular and secreted SRIH. KA (51-76% of basal) and Q (27-56% of basal) but not NMDA (91-114% of basal) also inhibited the incorporation of [35S]methionine into proteins. In similar experiments 10(-4)M Q (23 +/- 9% of basal) and KA (20 +/- 3% of basal) but not NMDA (80 +/- 16% of basal) reduced ChAT levels in hypothalamic/septal cultures. These inhibitory actions on ChAT activity by KA and Q were reversed by gamma-glutamyltaurine (GT) but not by 2-amino-5-phosphonopentanoic acid (AP5). Chronic NMDA exposure partially inhibited muscarinic acetylcholine receptor (mAChR) mediated inositol phospholipid (PI) turnover, whereas it was abolished after KA and Q pretreatment. These findings suggest that in cerebral cell cultures, NMDA has a stimulatory action on somatostatinergic neurons and non-NMDA receptor agonism could play an important role in EAA-mediated neural damage.