Inositol Phospholipid Metabolism Research Articles

Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: developmental changes and interaction with alpha 1-adrenoceptors.

Glutamate, aspartate, ibotenate, and quisqualate activate inositol phospholipid hydrolysis in hippocampal slices prepared from brains of 6- to 8-day-old rats. The stimulation by glutamate and aspartate progressively declines during postnatal development and is negligible after the 24th day of life. In contrast, the stimulation of inositol phospholipid hydrolysis by norepinephrine is low in hippocampal slices from newborn animals and increases during development, reaching mature values after the 35th day of life. In adult hippocampal slices, the stimulation of inositol phospholipid hydrolysis elicited by norepinephrine is inhibited by glutamate in a concentration-dependent fashion. This inhibition can also be brought about by aspartate, 2-amino-4-phosphonobutanoate, and L-phosphoserine, a product of endogenous phosphatidylserine hydrolysis.

Lithium-induced reduction in intracellular inositol supply in cholinergically stimulated parotid gland

The effects of lithium and cholinergic stimulation on inositol phospholipid metabolism have been assessed using rat parotid gland slices and isolated acinar cells labelled with 32Pi. Cholinergic stimulation using carbachol caused substantial breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and enhanced labelling of phosphatidate (PA) and phosphatidylinositol (PtdIns). Lithium alone had little effect upon 32Pi incorporation, but in combination with carbachol it greatly reduced the PtdIns labelling response to the agonist. Instead the label accumulated in a lipid identified as cytidine monophosphorylphosphatidate. There was also an enhancement of the PA labelling response to carbachol. These lithium-induced alterations in agonist-stimulated phospholipid metabolism were reversed if 10-30 mM-inositol was included in the incubation medium. Despite reduced PtdIns synthesis, lithium had relatively little effect on polyphosphoinositide labelling in stimulated cells. Resynthesis of polyphosphoinositides was monitored in acinar cells that had been stimulated with carbachol and then treated with atropine to block muscarinic receptors. Treatment with lithium during the carbachol-stimulation phase reduced the rate of phosphatidylinositol 4-phosphate synthesis, but had no significant effect upon PtdInsP2. The results suggest that an active inositol phosphatase pathway is essential to maintain intracellular inositol levels, but that PtdInsP2 synthesis is not markedly reduced by a substantial fall in intracellular inositol. This implies a close control over the rates of PtdInsP2 breakdown and resynthesis during agonist stimulation.

Antigen-stimulated metabolism of inositol phospholipids in the cloned murine mast-cell line MC9

Cells of the murine mast-cell clone MC9 grown in suspension culture were sensitized with an anti-DNP (dinitrophenol) IgE and subsequently prelabelled by incubating with [32P]Pi. Stimulation of these cells with DNP-BSA (bovine serum albumin) caused marked decreases in [32P]polyphosphoinositides (but not [32P]phosphatidylinositol) with concomitant appearance of [32P]phosphatidic acid. Whereas phosphatidylinositol monophosphate levels returned to baseline values after prolonged stimulation, phosphatidylinositol bisphosphate levels remained depressed. Stimulation of sensitized MC9 cells with DNP-BSA increased rates of incorporation of [32P]Pi into other phospholipids in the order: phosphatidylcholine greater than phosphatidylinositol greater than phosphatidylethanolamine. In sensitized cells prelabelled with [3H]inositol, release of inositol monophosphate, inositol bisphosphate and inositol trisphosphate, was observed after stimulation with DNP-BSA. When Li+ was added to inhibit the phosphatase activity that hydrolysed the phosphomonoester bonds in the sugar phosphates, greater increases were observed in all three inositol phosphates, particularly in inositol trisphosphate. The IgE-stimulated release of inositol trisphosphate was independent of the presence of extracellular Ca2+. In addition, the Ca2+ ionophore A23187 caused neither the decrease in [32P]polyphosphoinositides nor the stimulation of the release of inositol phosphates. These results demonstrate that stimulation of the MC9 cell via its receptor for IgE causes increased phospholipid turnover, with effects on polyphosphoinositides predominating. These data support the hypothesis that hapten cross-bridging of IgE receptors stimulates phospholipase C activity, which may be an early event in stimulus-secretion coupling of mast cells. The results with the Ca2+ ionophore A23187 indicate that an increase in intracellular Ca2+ alone is not sufficient for activation of this enzyme.

Ca 2+-translocation activities of phosphatidylinositol, diacylglycerol and phosphatidic acid inferred by quin-2 in artiifical membrane systems

Ca 2+-translocating activities of phosphatidylinositol, diacylglycerol and phosphatidic acid were investigated in phosphatidylcholine liposomes. Using a fluorescent indicator of Ca 2+ concentration, quin-2, release of encapsulated Ca 2+ from egg yolk phosphatidylcholine liposomes containing 2 mol% of one of these lipids was measured at 37°C. The rate of Ca 2+ translocation across the liposomal membrane mediated by phosphatidic acid was about 3-fold larger than those mediated by phosphatidylinositol and diacylglycerol. The result implies that phosphatidic acid has Ca 2+-ionophore activity in the agonist dependent metabolism of inositol phospholipids. The ionosphoretic activity depended on the degree of unsaturation of the fatty acyl chains. The Ca 2+ translocation rate was smalles in dipalmitoylphosphatidic acid, and it increased in the order of dioleoyl-, dilinoleoyl- and dilinolenoyl-phosphatidic acid. Ca 2+ mobilization of a stimulated cell is discussed in the light of Ca 2+-ionophore activity of phosphatidic acid converted from inositol phospholipids.

Inhibitory effect of prostaglandin E2, forskolin, and dibutyryl cAMP on arachidonic acid release and inositol phospholipid metabolism in guinea pig neutrophils.

The effect of prostaglandin E2 (PGE2), forskolin, and dibutyryl cAMP on arachidonic acid release, inositol phospholipid metabolism, and Ca2+ mobilization was investigated. The chemotactic tripeptide (formylmethionyl-leucyl-phenylalanine (fMLP))-induced arachidonic acid release in neutrophils was significantly inhibited by PGE2, forskolin, and dibutyryl cAMP. Among them, PGE2 was found to be the most potent inhibitor. However, when neutrophils were stimulated by Ca2+ ionophore A23187, such inhibitory effect by these agents was less marked. PGE2 also suppressed the enhanced incorporation of [32P]Pi into phosphatidic acid (PA) and phosphatidylinositol in a dose-dependent manner in fMLP-stimulated neutrophils. Also in this case, Ca2+ ionophore-induced alterations were hardly inhibited by PGE2. As well, PGE2 inhibited the fMLP-induced decrease of [3H]arachidonic acid in phosphatidylcholine and phosphatidylinositol and the increase in PA very significantly. But the inhibitory effect by PGE2 was found to be weak in Ca2+ ionophore-stimulated neutrophils. These results suggest that a certain step from receptor activation to Ca2+ influx is mainly inhibited by PGE2. Concerning polyphosphoinositide breakdown, PGE2 did not affect the fMLP-induced decrease of [32P]phosphatidylinositol 4,5-bisphosphate which occurred within 10 s but inhibited the subsequent loss of [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol, suggesting that the compensatory resynthesis of phosphatidylinositol 4,5-bisphosphate was inhibited. On the other hand, fMLP-induced diacylglycerol formation was suppressed for the early period until 1 min, but with further incubation, diacylglycerol formation was rather accelerated by PGE2. Moreover, the inhibition of PA formation by PGE2 became evident after a 30-s time lag, suggesting that the conversion of diacylglycerol to PA is inhibited by PGE2. The formation of water-soluble products of inositol phospholipid degradation by phospholipase C, such as inositol phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, was also suppressed by PGE2 treatment. However, the inhibition was not so marked as that of arachidonic acid release and PA formation. Thus, PGE2 appeared to inhibit not only initial events such as polyphosphoinositide breakdown but also turnover of inositol phospholipids. PGE2, forskolin, and dibutyryl cAMP did not block the rapid elevation of intracellular Ca2+ which was observed within 10 s in fMLP-stimulated neutrophils. However, subsequent increase in intracellular Ca2+ which was caused from 10 s to 3 min after stimulation was inhibited by PGE2, forskolin, and dibutyryl cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Inositol 1,4,5-trisphosphate activates pharmacomechanical coupling in smooth muscle of the rabbit mesenteric artery.

To clarify the nature of the noradrenaline (NA)-induced contraction, the effects of NA on inositol phospholipid metabolism and the actions of inositol 1,4,5-trisphosphate (InsP3) on skinned muscle of the rabbit mesenteric artery were investigated. NA, in concentrations over 1 nM, reduced the amount of phosphatidylinositol 4,5-bisphosphate (PI-P2) and increased the amount of phosphatidic acid (PA). The maximum reduction in the amount of PI-P2 and the maximum increase in the amount of PA were observed in the presence of 1 microM-NA. With prolonged application of NA, the PI-P2 was gradually restored to near the control level, but with repeated applications of NA separated by rinses with Krebs solution, there was a consistent reduction of PI-P2. The NA-induced PI-P2 breakdown was inhibited by the alpha 1-adrenoceptor blocking agent, prazosin. After incubation of the tissue with radioactive inositol-containing solution, NA transiently increased the amount of radioactive InsP3 which was followed by increases in the amount of inositol 1,4-bisphosphate (InsP2) and inositol monophosphate (InsP). After accumulation of Ca by saponin-treated muscle cells of the dog mesenteric artery dispersed by collagenase, InsP3 released Ca stored in cells but InsP2 did not. A23187 (5 microM) but not InsP3 (up to 10 microM), depleted Ca accumulated in the presence of ATP. In saponin-treated skinned muscle tissues, InsP3 in concentrations over 0.3 microM, produced contraction following accumulation of Ca into the store site. InsP3 released Ca from the same source as caffeine. The release of Ca by InsP3 appeared in a concentration-dependent manner and this release also depended on the amount of Ca stored in cells (the median effective dose (ED50) was 3.0 microM in 0.1 microM-Ca and 1.0 microM in 0.3 microM-Ca). We concluded that NA activates alpha 1-adrenoceptors, thus hydrolysing PI-P2 and synthesizing InsP3. This product can release Ca stored in cells as estimated from the contraction in skinned muscle tissues, and also reduces the residual amount of Ca stored in skinned dispersed muscle cells. Contraction evoked by NA through pharmacomechanical coupling can be explained as a function of InsP3.

Inositol Phospholipid Metabolism in the Kidney

Inositol Phospholipid Metabolism in the Kidney

Metabolism and Function of myo-Inositol and Inositol Phospholipids

Metabolism and Function of myo-Inositol and Inositol Phospholipids

Determination for the Enhanced Metabolism of Inositol Phospholipids by Hormone Receptor Activation

Determination for the Enhanced Metabolism of Inositol Phospholipids by Hormone Receptor Activation

Coupling of Inositol Phospholipid Metabolism with Excitatory Amino Acid Recognition Sites in Rat Hippocampus

Ibotenate, a rigid structural analogue of glutamate, markedly enhances the hydrolysis of membrane inositol phospholipids, as reflected by the stimulation of [3H]inositol monophosphate formation in rat hippocampal slices prelabeled with [3H]inositol and treated with Li+. Quisqualate, homocysteate, L-glutamate, and L-aspartate also induce a significant (albeit weaker) increase in [3H]inositol monophosphate formation, whereas N-methyl-D-aspartate, kainate, quinolinate, and N-acetylaspartylglutamate are inactive. The increase in [3H]inositol monophosphate formation elicited by the above-mentioned excitatory amino acids is potently and selectively antagonized by DL-2-amino-4-phosphonobutyric acid, a dicarboxylic amino acid receptor antagonist. These results suggest that, in the hippocampus, a class of dicarboxylic amino acid recognition sites is coupled with phospholipase C, the enzyme that catalyzes the hydrolysis of membrane inositol phospholipids.

Phorbol ester effects on α1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells

Tumor promoting phorbol esters stimulate Ca ++ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on α 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for 125I-HEAT binding to α 1-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate α 1-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating α 1-adrenergic receptor functional properties.

Serotonergic agonists stimulate inositol lipid metabolism in rabbit platelets

The metabolism of inositol phospholipids in response to serotonergic agonists was investigated in rabbit platelets. In platelets prelabelled with [ 3H]-inositol, in a medium containing 10 mM LiCl which blocks the enzyme inositol-1-phosphatase, 5-hydroxytryptamine (5-HT) caused a dose-dependent accumulation of inositol phosphates (IP). This suggests a phospholipase-C-mediated breakdown of phosphoinositides. Ketanserin, a selective 5-HT antagonist, was a potent inhibitor of the 5-HT response, with a Ki of 28 nM, indicating that 5-HT is activating receptors of the 5-HT 2 type in the platelet. Lysergic acid diethylamide (LSD) and quipazine also caused dose-related increases in inositol phosphate levels, though these were considerably less than those produced by 5-HT. These results show that relatively small changes in phospho-inositide metabolism induced by serotonergic agonists can be investigated in the rabbit platelet, and this cell may therefore be a useful model for the study of some 5-HT receptors.

Inhibition of inositol phospholipid breakdown by D 2 dopamine receptors in dissociated bovine anterior pituitary cells

Inositol phospholipids have been labelled with [ 3H]inositol in a lactotroph-enriched preparation of dissociated bovine anterior pituitary cells. Stimulation of cells with thyrotropin-releasing hormone receptor agonists leads to accumulation of [ 3H]inositol phosphates, and this effect may be inhibited by dopamine (DA) agonists. The DA agonist effect may be prevented by D 2 DA receptor selective antagonists. Thus the D 2 receptors on these cells are linked to inhibition of inositol phospholipid metabolism, and this provides a functional assay for the receptor.

Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.

DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of alpha 1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular alpha 1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same rapid time course with a t1/2 less than 2 min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.

Purification and properties of myo-inositol-1-phosphatase from rat brain.

myo-Inositol-1-phosphatase [EC 3.1.3.25] was purified from a cytosolic fraction of rat brain. The purified enzyme appeared homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 29,000. The molecular weight of the native enzyme was 55,000 as determined by molecular sieve chromatography. These values indicated that the native enzyme was composed of two identical subunits. The isoelectric point of the enzyme was 4.6. The enzyme hydrolyzed inositol-1-phosphate, 2'-AMP, 2'-GMP, beta-glycerophosphate, and alpha-glycerophosphate; the ratio of the reaction rates was 100 : 84 : 73 : 64 : 32. The Km values for inositol-1-phosphate, 2'-AMP, and beta-glycerophosphate were 1.2 X 10(-4) M, 1.9 X 10(-4) M, and 7.7 X 10(-4) M, respectively. Mn2+ and Ca2+ were strong competitive inhibitors against Mg2+, with Ki values of 3 microM and 20 microM, respectively. This result suggests that myo-inositol-1-phosphatase might be regulated by intracellular Ca2+ and/or Mn2+. Li+, which is known to show a therapeutic effect on manic-depressive disease and also to prolong the intrinsic periods of circadian rhythms in various organisms, was a potent uncompetitive inhibitor and inhibited 50% of the activity at 1 mM. The possibility that myo-inositol-1-phosphatase and inositol phospholipid metabolism are involved in circadian rhythm oscillation is discussed in terms of Li actions.

α1-adrenoceptor-mediated inositol phospholipid hydrolysis in rat cerebral cortex: Relationship between receptor occupancy and response and effects of denervation

The characteristics of catecholamine-mediated breakdown of inositol phospholipids in rat cerebral cortex slices have been examined using a direct assay involving prelabeling with [ 3H]inositol and examining the production of labelled inositol phosphates in the presence of lithium. Noradrenaline produced a marked stimulation of inositol phosphate accumulation and this response could be potently and competitively antagonised by the α 1-adrenoceptor antagonist prazosin. The α 2-antagonist yohimbine was almost 1000-fold less potent at antagonising noradrenaline inositol phospholipid response. Noradrenaline and adrenaline were full agonists at α 1-adrenoceptors but phenylephrine and methoxamine were only partial agonists in their ability to stimulate inositol phospholipid metabolism. There was a significant correlation between the ability of a variety of agonists and antagonists to activate or inhibit [ 3H]inositol phosphate accumulation and their ability to displace the α 1-adrenoceptor selective ligand [ 3H]prazosin from specific binding sites when assays were performed on rat cerebral cortical slices under identical conditions. The similarity of EC 50 values of agonists stimulating inositol phosphate accumulation and their IC 50 values in [ 3H]prazosin binding experiments suggested a close relationship between receptor occupancy and α 1-mediated inositol phosphate accumulation. Further experiments were performed to examine this directly by inactivating α 1-adrenoceptors with the alkylating antagonist phenoxybenzamine. After washing out unbound antagonist, [ 3H]prazosin binding was reduced to a very similar proportion to that observed on the maximal noradrenaline-stimulated accumulation of [ 3H]inositol phosphates in the slices. The EC 50 values for noradrenaline-stimulated inositol phosphate accumulation was unaltered and the affinity of [ 3H]prazosin for the remaining sites was equally unaffected. In rats treated 14 days previously with i.c.v. 6-hydroxydopamine (2 × 250 μg) there was a small increase in α 1-adrenoceptor binding sites but a parallel shift to the left in the noradrenaline [ 3H]inositol phosphate accumulation dose-response curve. On the other hand, the partial agonist phenylephrine induced a larger maximal response in denervated animals without a change in the EC 50 values. When slices from 6-hydroxydopamine treated animals were preincubated with phenoxybenzamine, the loss in α 1-adrenoceptor binding sites was greater than the reduction in the maximal response to noradrenaline. This may indicate the development of a small receptor reserve after denervation. The results presented provide evidence that noradrenaline stimulates inositol phospholipid hydrolysis via a closely coupled α 1-adrenoceptor in the cerebral cortex and, furthermore, that this coupling may be altered by noradrenergic denervation.

Inositol phospholipid and phosphatidic acid metabolism in response to membrane receptor activation.

Inositol phospholipid and phosphatidic acid metabolism in response to membrane receptor activation.

Inositol phospholipid levels of rat forebrain obtained by freeze-blowing method

Four different techniques of handling rat brain prior to lipid extraction and assay were tested to investigate the levels of inositol phospholipids in the brain. In these four techniques, the rat forebrains were either 1. (1) freeze-blown followed by being preserved in liquid N 2, 2. (2) subjected to microwave irradiation prior to decapitation, 3. (3) removed and frozen in liquid N 2, or 4. (4) removed at room temperature and subjected to lipid extraction as rapidly as possible. There was little change in phosphatidylinositol levels under any of these conditions; however, higher levels of phosphatidylinositol 4-phosphate were observed in freeze-blown and microwave-irradiated samples compared to the other samples. Even more striking differences were seen in phosphatidylinositol 4,5-bisphosphate fractions. The highest level of this lipid, 763 ± 39 nmol/g tissue, which was obtained from freeze-blown samples, was more than 2-fold higher than that of the lowest values which were obtained by extraction without prior inactivation. These results indicate that the values of phosphatidylinositol 4,5-bisphosphate in brain in situ are higher than those generally reported, and that the freeze-blowing method has an advantage for further investigation of inositol phospholipid metabolism in brain due to the rapid breakdown of these compounds.

Pirenzepine indicates heterogeneity of muscarinic receptors linked to cerebral inositol phospholipid metabolism

Muscarinic receptor-mediated inositol phospholipid metabolism was examined in rat cerebral cortex and medulla-pons slices prelabelled with 3H-inositol. Carbachol stimulated accumulation of 3H-inositol phosphate in both regions with identical EC 50 values though the maximal stimulation was considerably greater in cortex. The carbachol response was potently suppressed by atropine with very similar affinities in both regions. However, the M 1 antagonist pirenzepine displayed a 10-fold greater affinity for the cortical carbachol response. The data supports the concept of heterogeneity of cerebral muscarinic receptors and suggests that both M 1 and M 2 sites may be linked to inositol phospholipid metabolism in brain.

Role of enhanced inositol phospholipid metabolism in neutrophil activation

When guinea pig neutrophils were stimulated with chemotactic peptide [formylmethionyl-leucyl-phenylalanine (fMLP)], a marked release of lysosomal enzyme and production of Superoxide anion were detected. The breakdown of phosphatidylinositol 4,5-bisphosphate (TPI) and the subsequent formation of diacylglycerol, phosphatidic acid and free arachidonic acid also occurred during the processes. Ca 2+ ionophore A23187 caused an evident secretion of lysosomal enzyme but no Superoxide anion production. Ca 2+ ionophore also caused TPI breakdown to diacylglycerol although this breakdown was not as significant as that detected by fMLP. The tumor promotor tetradecanoylphorbol acetate (TPA), which is a strong activator of Superoxide anion production but not a good stimulator of lysosomal enzyme secretion, did not cause a significant decrease of TPI or arachidonic acid release. Since TPA is known not to increase the intracellular Ca 2+ level, these results suggest that lysosomal enzyme secretion is correlated closely with enhanced inositol phospholipid metabolism and Ca 2+-dependent processes. On the other hand, Superoxide anion production seemed to be caused mainly by Ca 2+-independent processes, perhaps by protein kinase-C activation through newly formed diacylglycerol, when neutrophils were activated by chemotactic peptide.