Formation Of 3H-inositol Phosphates Research Articles

Regulation of transmembrane signaling by ganglioside GM1: interaction of anti-GM1 with Neuro2a cells.

Interaction of antibodies to ganglioside GM1 with Neuro2a cells was studied to investigate the role of GM1 in cell signaling. Binding of anti-GM1 to Neuro2a cells induced the formation of 3H-inositol phosphates (3H-IPs) and elevated the intracellular Ca2+ concentration [Ca2+]i. The rise in [Ca2+]i was due to the influx of Ca2+ from the extracellular medium and release from intracellular Ca2+ pools. The Ca2+ influx pathway did not allow the permeation of Na+ or K+. The influx was inhibited by amiloride, a specific blocker of T-type Ca2+ channels, whereas nifedipine and diltiazem, blockers of L-type Ca2+ channels, did not have any effect. Thus, anti-GM1 appears to activate a T-type Ca2+ channel in Neuro2a cells. The intracellular Ca2+ release was inhibited by pretreatment of cells with neomycin sulfate, phorbol dibutyrate, and pertussis toxin (PTx), which also inhibited the 3H-IP formation in Neuro2a cells. Addition of caffeine neither elevated the [Ca2+]i nor affected the anti-GM1-induced [Ca2+]i rise. The data reveal that the binding of anti-GM1 to Neuro2a cells activates phospholipase C via a PTx-sensitive G protein, which leads to formation of IPs and release of Ca2+ from inositol trisphosphate-sensitive pool of endoplasmic reticulum. Anti-GM1 also arrested the differentiation of Neuro2a cells in culture and significantly stimulated their proliferation. This stimulatory effect of anti-GM1 on cell proliferation was blocked by amiloride but not by PTx, suggesting that the influx of Ca2+ was essentially required for cell proliferation. Our data suggest a role for GM1 in the regulation of transmembrane signaling events and cell growth.

Attenuation of excitatory amino acid toxicity by metabotropic glutamate receptor agonists and aniracetam in primary cultures of cerebellar granule cells.

Activation of glutamate ionotropic receptors represents the primary event in the neurotoxicity process triggered by excitatory amino acids. We demonstrate here that the concentration-dependent stimulation of metabotropic glutamate receptor (mGluR) by the selective agonist trans-1-aminocyclopentane-1,3-dicarboxylate or by quisqualate counteracts both glutamate- and kainate-induced neurotoxicity in primary cultures of rat cerebellar granule cells. The mGluR-evoked responses are potentiated by aniracetam, which per se also elicits neuroprotection. Aniracetam concentration-dependently counteracted glutamate-, kainate-, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death and greatly facilitated neuroprotective response achieved by different concentrations of both quisqualate and trans-1-aminocyclopentane-1,3-dicarboxylate. In addition, aniracetam potentiated the mGluR-coupled stimulation of phospholipase C, as revealed by the measurement of 3H-inositol phosphate formation. Thus, mGluRs could be a suitable target for novel pharmacological strategies pointing to the treatment of neurodegenerative diseases.

Absence of Effects of Class III Antiarrhythmic Agents on Cloned Cardiac K Channels

We have examined the pharmacological subtypes of muscarinic receptors mediating phosphoinositide hydrolysis and contraction in the longitudinal smooth muscle of guinea pig ileum with the use of muscarinic antagonists. Carbachol increased the formation of 3H-inositol phosphates (IPs) in a dose-dependent manner in both ileal smooth muscle and the frontal cortex of rats. The rank order of muscarinic antagonists for inhibition of IP formation induced by carbachol was 4-DAMP = atropine > pirenzepine > AF-DX 116 in guinea pig ileal smooth muscle. In ileal smooth muscle, the inhibition by the M1 antagonist pirenzepine was about 15 times less than that by atropine. However, in the rat frontal cortex, the inhibition by pirenzepine was only about 3 times less than that by atropine. The inhibitory effect of the M2 antagonist AF-DX 116 was weak in both ileal muscle and the frontal cortex. The M3 antagonist 4-DAMP strongly inhibited carbachol-induced IP formation in ileal smooth muscle. The rank order of muscarinic antagonists for inhibition of contraction induced by 10(-7) M carbachol was atropine > or = 4-DAMP > pirenzepine > AF-DX 116. These results suggest that both IP formation and the contractile response induced by muscarinic agonists are mediated through the muscarinic M3 subtype in guinea pig ileum.

Stimulation of Phospholipase D Activity in Human Neuroblastoma (LA‐N‐2) Cells by Activation of Muscarinic Acetylcholine Receptors or by Phorbol Esters: Relationship to Phosphoinositide Turnover

We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.

A Method to Measure Simultaneously Cyclic AMP and Inositol Phosphate Accumulation in Rat Brain Slices

The simultaneous measurement of the accumulation of cyclic AMP and inositol phosphates in rat cerebral cortical slices is described. After stimulation, the separation of cyclic AMP and inositol phosphates was achieved using ion-exchange chromatography and their concentrations were determined by means of a double-labeling technique, the substrates adenine and inositol being labeled with 14C and 3H, respectively. The recoveries were 70-80% for inositol phosphates and 40-50% for cyclic AMP. To test the applicability of the method, norepinephrine was chosen as an agonist, because it is known to stimulate the production of these two second messengers by interacting with alpha- and beta-adrenergic receptors. This procedure is an improvement over existing methods, because we obtained the simultaneous formation of 3H-inositol phosphates and [14C]cyclic AMP in a concentration-dependent process. EC50 values were similar for the two, 8.5 +/- 3.9 microM for 3H-inositol phosphates and 20.2 +/- 6.3 microM for [14C]cyclic AMP, and close to the values obtained when each process was studied alone. The action of adrenergic antagonists was also tested. Propranolol blocked the norepinephrine stimulation of [14C]cyclic AMP, but did not inhibit the norepinephrine stimulation of 3H-inositol phosphates. The opposite results were observed with prazosin. Our results suggest that this method could be a useful tool to examine the interaction between these two receptor-coupled effectors.

Calcium‐ Versus G Protein‐Mediated Phosphoinositide Hydrolysis in Rat Cerebral Cortical Synaptoneurosomes

The role of calcium and sodium in stimulating phosphoinositide hydrolysis in brain was investigated in rat cerebral cortical synaptoneurosomes. In buffer containing 136 mM sodium and various concentrations of added calcium (0-1.0 mM), basal, potassium-stimulated, and norepinephrine-stimulated formation of 3H-inositol phosphates decreased with decreasing extracellular calcium. Potassium- and norepinephrine-stimulated formation of 3H-inositol phosphates was reduced to basal levels by addition of EGTA. Isosmotically replacing sodium with choline chloride or N-methyl-D-glucamine to disrupt Na+/Ca2+ exchange resulted in a large increase in the formation of 3H-inositol phosphates. Measurement of cytosolic calcium with fura-2 revealed that the cytosolic calcium concentration was sensitive to changes in the extracellular calcium concentration and increased on resuspension of synaptoneurosomes in sodium-free rather than sodium-containing medium. In the absence of sodium, potassium-stimulated formation of 3H-inositol phosphates was reduced or eliminated, depending on the extracellular calcium concentration. Subtraction of basal formation of 3H-inositol phosphates from that in the presence of 1 mM carbachol or 100 microM norepinephrine revealed that the carbachol-stimulated component was the same in the presence and absence of sodium, whereas the norepinephrine-stimulated component was reduced in the absence of sodium. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate inhibited norepinephrine- and, to a lesser extent, carbachol but not basal or aluminum fluoride-stimulated formation of 3H-inositol phosphates in sodium-free medium. These results suggest that an increase in intracellular calcium, via disruption of Na+/Ca2+ exchange or depolarization-induced calcium influx, may explain previous demonstrations that agents that stimulate Na+ influx can also stimulate phosphoinositide hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of Muscarinic Receptor Density and of Guanine Nucleotide‐Stimulated Phosphoinositide Hydrolysis in Human SH‐SY5Y Neuroblastoma Cells Following Long‐Term Treatment with 12‐O‐Tetradecanoylphorbol 13‐Acetate or Mezerein

The actions of tumor promoters on the coupling of muscarinic receptors to the hydrolysis of inositol lipids and the generation of Ca2+ signals were examined in the human neuroblastoma SH-SY5Y cell line. Pretreatment of SH-SY5Y cells with 50 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 5 days resulted in neuronal differentiation, a 28% decrease in both N-[3H]methylscopolamine and [3H]-scopolamine binding, and a significantly larger reduction (48%) in agonist-stimulated 3H-inositol phosphate generation. Whereas mezerein could mimic the effects produced by TPA, the biologically inactive 4 alpha-phorbol 12,13-didecanoate was without effect on both antagonist binding and agonist-stimulated phosphoinositide (PPI) turnover. A decline (approximately 50%) in the agonist-mediated rise in cytoplasmic Ca2+ and a substantial loss of protein kinase C activity also were observed following pretreatment with TPA or mezerein. The ability of fluoride, an agent capable of direct activation of guanine nucleotide binding proteins, to stimulate 3H-inositol phosphate release was significantly reduced in SH-SY5Y cells treated with these agents. Furthermore, pretreatment of SH-SY5Y neuroblastoma cells with TPA or mezerein impaired 3H-inositol phosphate formation induced by the addition of either guanosine 5'-O-(3-thiotriphosphate) or carbamylcholine to digitonin-permeabilized cells, but not that elicited by the addition of 2 mM CaCl2. Although cells cultured in the presence of serum-free media also exhibited neuronal differentiation, no significant alteration in either muscarinic receptor number or agonist-stimulated PPI hydrolysis was observed. The results suggest that TPA and mezerein decrease agonist-stimulated PPI hydrolysis and Ca2+ signaling in SH-SY5Y cells not only by a reduction in muscarinic receptor number but also through an inhibition of guanine nucleotide-stimulated PPI turnover.

Regulation of Muscarinic Agonist‐Induced Activation of Phosphoinositidase C in Electrically Permeabilized SH‐SY5Y Human Neuroblastoma Cells by Guanine Nucleotides

myo-[3H]Inositol-labelled SH-SY5Y cells were permeabilized with electrical discharges. 3H-Inositol phosphate formation in cells shown to be fully permeable was stimulated by the muscarinic agonist carbachol, by guanosine 5'-(gamma-thio)triphosphate [GTP(S)], and by guanosine 5'-(beta gamma-imido)diphosphate (GppNHp). Synergism was observed on coincubation of these GTP analogues with carbachol. GTP was also stimulatory and guanosine 5'-(beta-thio)diphosphate was inhibitory in the presence of agonist. Atropine blocked the effects of carbachol. Stimulation by GTP(S) (0.1 mM) occurred after a 1-2-min lag, whereas Ca2+ (0.5 mM), carbachol (1 mM), and carbachol plus GTP(S) stimulated without delay. The effects of carbachol plus GTP(S) but not those of Ca2+ were inhibited by spermine (4 mM). Accumulation of 3H-inositol phosphates was enhanced by Li+ (4 mM) only in intact cells. In intact or permeabilized cells, the "partial" agonist arecoline was maximally 40-50% as efficacious as carbachol. In permeabilized cells, the maximal effects of carbachol and arecoline were enhanced 2.8- and 5.3-fold, respectively, by 0.1 mM GTP(S), but only the EC50 for carbachol was substantially reduced. The binding affinity of carbachol but not that of arecoline in permeabilized cells was significantly reduced by 0.1 mM GppNHp. These data indicate that a guanine nucleotide-binding regulatory protein is involved in coupling muscarinic receptors to phosphoinositidase C in SH-SY5Y cells and that the activity of this protein influences the relationship between receptor occupation and phosphoinositide response.

Tumor necrosis factor-α/cachectin activates the [formula omitted] system of human neutrophils independently of the hydrolysis of phosphoinositides and the release of arachidonic acid

We have investigated the mechanisms of transmembrane signalling implicated in the activation of the respiratory burst of adherent neutrophils by tumor necrosis factor-α/cachectin (TNF). The activation of the respiratory burst by TNF is insensitive to pertussis toxin and weakly sensitive to protein kinase C inhibitors. Cytochalasin B and dibutyryl cyclic AMP have an inhibitory effect. The activation of the respiratory burst by TNF takes place in the absence of formation of 3H-inositol phosphates, 32P-phosphatidic acid, and 3H-arachidonic acid. These results demonstrate that the activation of the respiratory burst by an endogenous, physiologic stimulus can be independent of the formation of messengers derived from hydrolysis of phosphoinositides.

Inhibition of polyphosphoinositide turnover in rat cerebral cortex by clonidine

In the rat brain, a number of receptors are linked to phospholipase C which catalyzes the hydrolysis of membrane inositol phospholipids; stimulation of α 1-adrenergic receptors, for example, increases polyphos-phoinositide turnover, but stimulation of α 2-receptors does not. The hydrolysis of inositol phospholipids in rat cortical slices was investigated using a direct assay involving prelabeling these lipids with 3H-inositol and then measuring the formation of 3H-inositol phosphates in the presence of lithium ions. As expected, clonidine, an α 2-agonist, did not stimulate the formation of 3H-inositol phosphates; however, clonidine antagonized the ability of noradrenaline to stimulate 3H-inositol phosphate formation. This effect was not blocked by antagonists of α 2, 5HT 2, H 2, or muscarinic receptors. Clonidine did not affect carbachol-stimulated 3H-inositol phosphate formation.

Prostaglandin E 2 and F 2α inhibit growth of human gastric carcinoma cell line KATO III with simultaneous stimulation of cyclic AMP production

The effects of prostaglandins(PGs) on the growth of human gastric carcinoma cell line KATO III were investigated. PGE 2 as well as PGF 2α significantly and dosedependently inhibited the growth of this gastric carcinoma cell line (PGE 2 > PGF 2 α ). This inhibition of cell growth by the PGs was associated with the increase in cyclic AMP production (PGE 2 > PGF 2 α ), whereas inositolphospholipid turnover was not affected by either PGE 2 or PGF 2α as assessed by the formation of 3H-inositol phosphates. Furthermore, the proliferation of these gastric carcinoma cells was also suppressed by the administration of forskolin as well as of dibutyryl cyclic AMP. These results suggest that PGE 2 and PGF 2α inhibit the growth of cultured human gastric carcinoma cells KATO III via stimulation of cyclic AMP production.

Muscarinic receptor coupling to inositol phospholipid metabolism in guinea-pig cerebral cortex, parotid gland and ileal smooth muscle

Inositol phospholipid hydrolysis induced by agonist-stimulation of muscarinic receptors has been examined in slices of guinea-pig cerebral cortex, parotid gland and ileal smooth muscle. An assay measuring 3H-inositol phosphate formation from prelabelled lipids in the presence of LiCl, allowed marked stimulation by agonists to be followed. The pD 2-value of carbachol differed markedly, between tissues being more than 10-fold lower in cerebral cortex than in parotid gland. The partial agonist oxotremorine showed the largest relative maximal responsiveness in parotid gland, followed by ileum and cortex. Atropine suppressed the phosphoinositide response to carbachol with an almost similar affinity in each tissue, but pirenzepine was found to have a 20-fold higher affinity in cerebral cortex, p K i = 7.7 than in parotid gland, p K i = 6.3. Carbachol, even in the presence of guanosine triphosphate (GTP), displayed complex binding against 3H-AT-methylscopolamine ( 3H-NMS) in cortical and ileal membranes, though in membranes from the parotid gland a single homogeneous population was found. Atropine inhibition of 3H-NMS parallelled its suppression of the phosphoinositide response, the affinities in each tissue studied being similar. Pirenzepine inhibited binding from two components in cerebral cortex, the high affinity value being similar to that obtained in the phosphoinositide assay. In parotid gland, however, only low affinity pirenzepine binding sites were observed, closely resembling the affinity found for this antagonist in the functional assay. These experiments suggest (a) that there are differences between agonist occupation of muscarinic receptors and phosphoinositide hydrolysis within the different tissues, (b) that both high and low affinity pirenzepine binding sites appear to be linked to phosphoinositide metabolism, and (c) that low affinity pirenzepine sites may be more efficiently coupled to the hydrolysis of phosphoinositides.

Stimulation of phosphoinositide breakdown in rat pancreatic islets by glucose and carbamylcholine

In the presence of Li +, glucose, 2-ketoisocaproate and carbamylcholine induced the rapid formation of 3H-inositol phosphates in rat pancreatic islets prelabelled with 3H-inositol. The production of labelled inositol phosphates continued up to 20 min of incubation. Glibenclamide and ionophore A23187 had no significant effect on labelled inositol phosphate production. The effects of carbamylcholine and to a lesser extent, glucose were found to persist in the absence of added Ca 2+, but both were strongly inhibited by excess EGTA. In general, the rise in 3H-inositol phosphate production was associated with a fall in lipid bound radioactivity, although the latter was found to occur more slowly, and was of a smaller magnitude than labelled inositol phosphate formation. The results suggest that nutrient secretagogues and cholinergic agonists stimulate hydrolysis of phosphoinositides in pancreatic islets by a phospholipase C mechanism. This effect is Ca 2+-dependent, but probably not triggered by increased Ca 2+ uptake into the islet.