Inositol Phospholipid Metabolism Research Articles

Muscarinic Receptor Stimulation Increases Inositol‐Phospholipid Metabolism and Inhibits Cyclic AMP Accumulation in PC 12 Cells

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate receptor subtypes may be classified into two major categories: A study on Xenopus oocytes injected with rat brain mRNA

Three major subtypes of glutamate receptors that are coupled to cation channels are known. Recently an additional subtype that is coupled to G proteins and stimulates inositol phospholipid metabolism (the metabotropic glutamate receptor) has been proposed. The pharmacological characteristics of this receptor have now been examined. Although it shares some agonists with N-methyl- d-aspartate- and quisqualate-subtype receptors, it shares virtually no antagonists with any of the three cation channel-coupled receptor subtypes. Thus the metabotropic glutamate receptor belongs to a receptor category that is completely different from that of the other three receptor subtypes, not only functionally, but also pharmacologically.

Phorbol ester effects on coupling mechanisms during cholinergic contraction of swine tracheal smooth muscle.

1. We studied effects of the phorbol ester, phorbol 12,13-dibutyrate (PDB), on carbachol-induced contractions of swine trachealis muscle. PDB (1-10 microM) markedly inhibited 5.5 microM-carbachol-induced inositol phosphate synthesis allowing us to study (a) whether the membrane potential-independent component of force (pharmacomechanical coupling component) developed in carbachol-stimulated trachealis muscle is dependent on activation of inositol phospholipid metabolism, and (b) whether carbachol-induced membrane depolarization and contraction are altered in muscle where second messenger signals generated by inositol phospholipid metabolism are inhibited and activation of protein kinase C (PKC) is already maximal. 2. Application of PDB (10 microM) to unstimulated trachealis muscle resulted in a small slowly developing contraction associated with a 10 m V membrane depolarization. PDB-evoked contractions were not influenced by Na+ or Cl- ion substitutions, or administration of amiloride, all of which inhibited PDB-evoked membrane depolarization. 3. Pre-treatment with PDB had no effect on [K+]-force, or [K+]-membrane potential relationships, over a range of extracellular [K+] from 40 to 70 mM. Pretreatment with PDB had no effect on extracellular [Ca2+]-force relationships during 40 mM-K+. 4. Carbachol-evoked contractions of muscle treated with PDB became similar to K+ contractions in regard to effects of organic Ca2+ antagonist drugs or decrease in bathing solution [Ca2+]. At low carbachol concentrations, verapamil plus PDB completely inhibited force development. With 5.5 microM-carbachol, over 90% of total carbachol-induced force was inhibited by verapamil, or nifedipine, plus PDB. 5. Control carbachol-evoked contractions were associated with 20-25 mV membrane depolarizations. In PDB-treated muscle, carbachol-evoked contraction occurred with a blunted depolarization, i.e. about 5 mV. 6. Force controlled by pharmacomechanical coupling mechanisms operating during maintained carbachol-evoked contractions was inhibited by treatment with PDB. Carbachol-induced force dependent on pharmacomechanical coupling mechanisms could be explained by signals generated via inositol phospholipid metabolism. 7. Electromechanical coupling mechanisms were augmented during carbachol in PDB-treated muscle. This appears to be due primarily to changes in the properties or number of surface membrane voltage-gated Ca2+ channels. 8. Data suggest an important role of PKC-mediated phosphorylations for control of both pharmacomechanical coupling mechanisms mediated by activation of inositol phospholipid metabolism and electromechanical coupling mechanisms mediated by effects on operation of surface membrane ion channels.

Modulation of Kupffer cell membrane phospholipid function by n-3 polyunsaturated fatty acids

Dietary n-3 polyunsaturated fatty acids (PUFAs) have been reported to improve clinical outcome in a number of inflammatory diseases including burns and sepsis. One mechanism contributing to the anti-inflammatory effect is the incorporation of n-3 PUFAs into membrane phospholipids which decreases macrophage eicosanoid production. We hypothesize that an additional mechanism for their effects is an alteration of membrane signal transduction that decreases macrophage responsiveness to inflammatory stimuli. Kupffer cells, the fixed macrophages of the liver, were obtained from rats pair fed diets for 6 weeks with 15% of calories supplied as menhaden (high n-3), corn (control), or safflower (high n-6) oils. The effects of the dietary oils on Kupffer cell membrane signal transduction and eicosanoid production were assessed by measuring inositol phospholipid (PI) metabolism, intracellular calcium responses, and prostaglandin E 2 (PGE 2) production to the inflammatory signals endotoxin (LPS) and platelet activating factor (PAF). The menhaden oil diet resulted in significant incorporation of n-3 PUFAs into total cellular PUFAs compared to corn and safflower oil. (total n-3 PUFAs, 28.1% menhaden vs 2.1% corn vs 1.2% safflower, P < 0.03) This incorporation altered signal transduction of PAF as both PI turnover (65% ± 10% of corn oil) and calcium response (0.6-fold vs 5.0-fold for corn oil) were significantly reduced in the menhaden oil group. ( P < 0.05) The menhaden oil diet also reduced significantly PGE 2 production in response to PAF and LPS (corn, 348 ± 23 pg/ml; menhaden, 48 ± 6 pg/ml, P < 0.01). We conclude that, in addition to modulating eicosanoid production, n-3 PUFAs can also alter macrophage membrane signal transduction. These mechanisms may contribute to observed anti-inflammatory effects and offer the potential ability to down-regulate macrophage-mediated hypermetabolism in critically ill surgical patients.

Role of Intracellular pH in the Axolemma‐ and Myelin‐Induced Proliferation of Schwann Cells

In order to provide additional information on the biochemical events that interact to cause Schwann cells to proliferate, we have monitored the intracellular pH of Schwann cells that have been stimulated to divide with myelin-enriched fractions (MEF) or axolemma-enriched fractions (AEF). The intracellular pH of Schwann cells was monitored using 2',7'-bis(carboxymethyl)-5(6)-carboxyfluorescein (BCECF), which displays an increase in fluorescence upon alkalinization. Both AEF and MEF caused dose-dependent increases in the intracellular fluorescence of the Schwann cell cultures. At their maximum doses, AEF and MEF stimulation resulted in a 260 and 300% increase in intracellular fluorescence, respectively. The increase in intracellular fluorescence was abolished when cells were stimulated in Na+-free media, suggesting a role for the Na+/H+ exchanger. Mitotic stimulation required integrity of the Na+/H+ exchanger, as inhibition of the Na+/H+ exchanger for periods up to 1 h after addition of mitogen caused a significant inhibition of subsequent mitosis. Phorbol esters, which can potentiate AEF- and MEF-induced Schwann cell proliferation, increased intracellular fluorescence fivefold, an effect which was also dependent upon the presence of Na+ in the culture media. The specificity of the increase in intracellular pH for AEF and MEF was tested by incubating Schwann cells with liver microsomes and a biologically inactive phorbol alcohol, neither of which is significantly mitogenic for Schwann cells. Neither liver microsomes nor phorbol alcohol had a significant effect on intracellular pH. The implications of the increase in intracellular pH in Schwann cells with respect to inositol phospholipid metabolism, protein kinase C activation, and cellular proliferation are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of cholera toxin on the inhibition of vasopressin-stimulated inositol phospholipid hydrolysis is a cyclic AMP-mediated event at the level of receptor binding

Incubation of L6 skeletal myoblasts for 16 h with cholera toxin but not with pertussis toxin, led to the inhibition of inositol phosphate generation induced by subsequent exposure to vasopressin. The effects of the toxin on inositol lipid metabolism were accompanied by the total ADP-ribosylation of the available cholera-toxin substrates within the cells. Immunological analysis demonstrated that the two polypeptides modified in vivo by cholera toxin were different forms of Gs alpha (alpha subunit of Gs). No novel cholera-toxin substrate(s) were detected. The cholera-toxin-mediated inhibition of vasopressin-stimulated inositol phosphate generation could be mimicked by both forskolin and dibutyryl cyclic AMP, but not by the separated subunits of the toxin. Receptor-binding studies demonstrated that the inhibition of agonist-stimulated inositol phosphate generation was accompanied by a decrease in cell-surface vasopressin-binding sites, with no effect on the affinity of these for the hormone. We suggest that the effect of cholera toxin and agents which increase intracellular cyclic AMP on vasopressin-stimulated inositol lipid hydrolysis is an effect on receptor number, and that there is no requirement to postulate a role for a novel G-protein, which is a substrate for cholera toxin, in the regulation of inositol phospholipid metabolism.

Retinoic acid inhibits phosphatidylinositol turnover only in RA-sensitive while not in RA-resistant human neuroblastoma cells

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line which can be induced to differentiate along the neuronal pathway by retinoic acid (RA), and a derivated RA-resistant subline of it (LAN-1-res). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2- 3H] inositol or [1,(3)- 3H] glycerol. Analysis of labelled phosphatidylinositol metabolites from prelabelled LAN-1 cells indicated a rapid decrease of inositol (1,4,5)-trisphosphate and (1,2) diacylglycerol within 1 min. of induction of differentiation by RA, while no changes were observed in RA-treated LAN-1-res cells. These findings indicate that phosphoinositides-derived metabolites may be directly implicated in the induction processes of RA-triggered NB cell differentiation.

Glucagon desensitization of adenylate cyclase and stimulation of inositol phospholipid metabolism does not involve the inhibitory guanine nucleotide regulatory protein Gi, which is inactivated upon challenge of hepatocytes with glucagon

Brief exposure of hepatocytes to glucagon, angiotensin or the protein kinase C activator TPA (12-O-tetradecanoylphorbol 13-acetate) caused the inactivation of the inhibitory guanine nucleotide regulatory protein Gi. Glucagon-mediated desensitization of glucagon-stimulated adenylate cyclase activity was seen in hepatocytes from both normal rats and those made diabetic with streptozotocin, where Gi is not functionally expressed. Normal glucagon desensitization was seen in hepatocytes from young animals, 6 weeks of age, which had amounts of Gi in their hepatocyte membranes which were some 45% of that seen in mature animals (3.4 pmol/mg of plasma-membrane protein). Streptozotocin-induced diabetes in young animals abolished the appearance of functional Gi in hepatocyte plasma membranes. Pertussis-toxin treatment of hepatocytes from both normal mature animals and those made diabetic, with streptozotocin, blocked the ability of glucagon or angiotensin or TPA to elicit desensitization of adenylate cyclase. The isolated B (binding)-subunit of pertussis toxin was ineffective in blocking desensitization. Neither induction of diabetes nor treatment of hepatocytes with pertussis toxin inhibited the ability of glucagon and angiotensin to stimulate the production of inositol phosphates in intact hepatocytes. Thus (i) Gi does not appear to play a role in the molecular mechanism of glucagon desensitization in hepatocytes, (ii) the G-protein concerned with receptor-stimulated inositol phospholipid metabolism in hepatocytes appears not to be a substrate for the action of pertussis toxin, (iii) in intact hepatocytes, treatment with glucagon and/or angiotensin can elicit the inactivation of the inhibitory G-protein Gi, and (iv) pertussis toxin blocks desensitization by a process which does not involve Gi.

Potentiation by lithium of CMP-phosphatidate formation in carbachol-stimulated rat cerebral-cortical slices and its reversal by myo-inositol.

This paper describes a rapid and simple method for measuring CMP-phosphatidate (CMP-PA; CDP-diacylglycerol), providing a novel assay for inositol phospholipid metabolism. Rat cerebral-cortical slices labelled with [14C]cytidine were incubated with the muscarinic cholinergic agonist carbachol in the presence of various concentrations of LiCl; 10 mM-LiCl greatly enhanced the carbachol-stimulated formation of [14C]CMP-PA over a 60 min incubation period. The potentiation by Li+ was concentration-dependent, with a maximal enhancement at 3 mM and half-maximal enhancement at 0.6 mM-LiCl. The enhancement by Li+ could be reversed by incubation with myo-inositol; a maximal effect was observed with 10 mM-inositol. A similar, though smaller, enhancement of CMP-PA concentrations in the presence of LiCl was observed in slices stimulated with noradrenaline, 5-hydroxytryptamine and K+. The results are discussed in relation to previously observed effects of Li+ on inositol phospholipid metabolism.

Inositol phospholipid metabolism during and following synaptic activation: role of adenosine.

The metabolic pathway of inositol phospholipids represents a series of synthetic and hydrolytic reactions with inositol as a by-product. Hence, the rate of [3H]inositol release from prelabeled phospholipids can be used as a reflection of activity of this pathway. In the frog sympathetic ganglion prelabeled with [3H]inositol, we studied the effect of synaptic activity (orthodromic stimulation) on release of 3H-label into the medium. This release was interpreted as [3H]inositol release. The value was low at rest and increased significantly by 32% during orthodromic stimulation (20 Hz for 5 min). However, on cessation of the stimulation, [3H]inositol release increased rapidly by 148% and remained elevated for at least 45 min. This increase in [3H]inositol release during and after the stimulation period was reduced by suffusion of the ganglia with adenosine. We hypothesized that synaptic activation releases a long-lasting stimulatory agonist and a short-lasting inhibitory (adenosine) agonist or agonists affecting [3H]inositol release. To demonstrate the presence of a stimulatory agonist, two sympathetic ganglia were used. One was prelabeled with [3H]inositol, and the other was not. The two ganglia were placed together in a 5-microliter droplet of Ringer's solution containing atropine. Orthodromic stimuli applied to the nonlabeled ganglion elicited release of [3H]inositol from the nonstimulated ganglion. To test whether the adenosine formed during orthodromic stimulation inhibits [3H]inositol release, we destroyed endogenous adenosine by suffusion of the ganglia with adenosine deaminase during the stimulation period. We found that adenosine deaminase induced large increases in [3H]inositol release during the stimulation period, in contrast to an increase seen only during the poststimulation period when adenosine deaminase was omitted. Because [3H]inositol release is assumed to parallel changes in content of inositol phosphates, we anticipated no changes of the levels of these compounds during orthodromic stimulation. However, measurements showed that levels of inositol phosphates and inositol phospholipids were all elevated except for phosphatidylinositol 4-phosphate. On termination of the stimulus, they remained elevated, with a further increase in levels of inositol trisphosphate and phosphatidylinositol 4-phosphate. We conclude that endogenous adenosine inhibits [3H]inositol release, possibly by modulating several of the steps of the inositol phospholipid pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Subacute and Chronic In Vivo Lithium Treatment Inhibits Agonist‐ and Sodium Fluoride‐Stimulated Inositol Phosphate Production in Rat Cortex

We have investigated the effects of in vivo lithium treatment on cerebral inositol phospholipid metabolism. Twice-daily treatment of rats with LiCl (3 mEq/kg) for 3 or 16 days resulted in a 25-40% reduction in agonist-stimulated inositol phosphate production, compared with NaCl-treated controls, in cortical slices prelabelled with [3H]inositol. A small effect was also seen with 5-hydroxytryptamine (5-HT) 24 h after a single dose of LiCl (10 mEq/kg). Dose-response curves to carbachol and 5-HT showed that lithium treatment reduced the maximal agonist response without altering the EC50 value. This inhibition was not affected by the concentration of LiCl in the assay buffer. Stimulation of inositol phosphate formation by 10 mM NaF in membranes prepared from cortex of 3-day lithium-treated rats was also inhibited, by 35% compared with NaCl-treated controls. Lithium treatment did not alter the kinetic profile of inositol polyphosphate formation in cortical slices stimulated with carbachol. Muscarinic cholinergic and 5-HT2 bindings were unaltered by lithium, as was cortical phospholipase C activity and isoproterenol-stimulated cyclic AMP formation. [3H]Inositol labelling of phosphatidylinositol 4,5-bisphosphate was significantly enhanced by 3-day lithium treatment. The results, therefore, indicate that subacute or chronic in vivo lithium treatment reduces agonist-stimulated inositol phospholipid metabolism in cerebral cortex; this persistent inhibition appears to be at the level of G-protein-phospholipase C coupling.

Inositol lipid turnover and compartmentation in canine trachealis smooth muscle.

We established conditions for the study of metabolism and compartmentation of inositol phospholipids in canine trachealis muscle. Unstimulated muscle was incubated with myo-[3H]inositol for 30 min at 37 degrees C which resulted in labeling of the tissue free myo-inositol pool, whereas only a small amount of radioactivity was incorporated into inositol phospholipids or inositol phosphates. After addition of 5.5 microM carbachol, phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2), specific radioactivities increased exponentially, reaching apparent constant values in 180-240 min. Initial rates of increases in PI, PIP, and PIP2 specific radioactivities were 39, 32, and 66 times that measured in unstimulated muscle. Metabolic flux rates (nmol.100 nmol total lipid Pi-1.min-1) during development of force averaged 0.42 +/- 0.09 and during force maintenance averaged 0.14 +/- 0.01. Fractions of total PI, PIP, and PIP2 pools that were linked to muscarinic cholinergic activation were estimated to be 0.97, 0.85, and 0.65, respectively. Initial rates of increase in specific radioactivities and specific radioactivities during carbachol activation were similar in PI, PIP, and PIP2 fast active compartments, suggesting metabolic flux from PI to PIP to PIP2 was in near chemical equilibrium. Turnover times for PI, PIP, and PIP2 fast active compartments were estimated to be 21, 1.6, and 4.0 min, respectively.

Activation of Phosphatidylinositol Kinase and Phosphatidylinositol-4-phosphate Kinase by cAMP in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, cAMP-dependent phosphorylation plays an essential role at the start of the cell cycle. It has also recently been demonstrated that the breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is a requisite process for cell proliferation (Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) Nature 333, 188-190). To clarify the relationship between the cAMP- and inositol phospholipid-mediated signal transduction systems, alterations in the inositol phospholipid metabolism of cAMP mutants were examined. The incorporation of [32P]Pi into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was markedly reduced in ras2, which produces low levels of cAMP, and increased in bcy1, which produces cAMP-independent protein kinase. The incorporation of [32P]Pi into ATP and phosphatidylinositol (PI) was almost the same in wild type, ras1, ras2, and bcy1 yeast strains. The addition of exogenous cAMP to cyr1-2 caused a tremendous increase in [32P]Pi incorporation into PIP and PIP2 without any effect on incorporation into ATP and PI, suggesting that cAMP plays an important role in polyphosphoinositide synthesis. We therefore examined the activities of PI and PIP kinases, the enzymes that catalyze the sequential steps from PI to PIP2 via PIP. The activities of both kinases were found to be very low in the membranes of cry1-2 and ras2 but very high in the membranes of bcy1 and ras1 ras2 bcy1 strain cells. The addition of cAMP to cyr1-2 cells caused the activation of PI and PIP kinases. Furthermore, the treatment of membranes with cAMP or dibutyryl cAMP caused the activation of PI kinase in wild type, ras1, cry1-2, and ras2 strains, but not in bcy1 strain cells. The effect was most prominent in membranes from cyr1-2 and ras2 cells. These results show that cAMP-dependent phosphorylation enhances polyphosphoinositide synthesis through activation of PI and PIP kinase, an effect which may lead to the enhanced production of inositol 1,4,5-trisphosphate and diacylglycerol.

Attenuation of phosphoinositidase activity and phosphatidylinositol bisphosphate level of bovine retinal capillary pericytes in high glucose

Both phosphoinositidase (PIase) and individual species of inositol phospholipid (IPL) of bovine retinal capillary pericytes (BRCP) were quantitatively determined. When glucose in growth medium was increased from 5- to 15- or 30 m m, PIase activity was attenuated to 82% or 55%, respecitively. In contrast, when glucose (5-, 15-, 30 m m) was added to an enzyme extract from cells grown in the standard growth medium (5 m m glucose, 0·04 m m myo-inositol) the PIase activity was not changed, indicating that the reduced PIase activity was not due to the direct effect of glucose. When IPLs from BRCP were analysed by HPLC and TLC, we observed reduction of the total and newly formed IPLs including the substrate of PIase, Phosphatidylinositol bisphosphate (PIP 2). Reduced levels of IPLs were associated with a decrease in myo-inositol and an increase in sorbitol. The changes in IPL metabolism were reversed by adding either free myo-inositol or AL1576, an aldose reductase inhibitor (ARI), to the high-glucose medium. However, the addition of myo-inositol to the growth medium with a standard concentration of glucose only caused a marked increase in phosphatidylinositol, but not in PIP or PIP 2, while the supplement of AL1576 in the standard medium did not cause any changes in IPL formation. These findings suggest that the alteration in IPL metabolism in BRCP may be related to insufficient myo-inositol or activated sorbitol pathway under high-glucose conditions. Further explanation of the role of the altered hydrolysis of PIP 2 triggered by PIase may provide clues to understanding of the mechanism of decreased pericyte viability in the presence of high glucose concentrations.

Epinephrine stimulates inositol phospholipid metabolism by activating alpha-2 adrenergic receptors in human platelets

The metabolism of inositol phospholipids in response to epinephrine was investigated in intact human platelets. In platelets prelabelled with [ 3H]-myo-inositol in Ca 2+-free HEPES buffer containing 10 mM LiCl, epinephrine caused an accumulation of inositol-1-phosphate in a concentration-dependent manner. The EC 50 value for epinephrine was 5 uM. Yohimbine (1 uM), a selective alpha-2 adrenergic receptor antagonist, inhibited 88% of the epinephrine (10 uM) response, whereas prazosin (1 uM), a selective alpha-1 adrenergic receptor antagonist, failed to inhibit the response. Yohimbine inhibited the epinephrine (10 uM) response in a concentration-dependent manner. The inhibition constant (Ki) value for yohimbine was 60.3 nM. These data indicate that epinephrine stimulates phosphoinositide (PI) turnover by activating adrenergic receptors of the alpha-2 type in human platelets. In addition, this PI response elicited by epinephrine was found to be inhibited in a concentration-dependent manner by treatment of platelets with dibutyryl cyclic AMP and 8-bromo-cyclic GMP which are knowm as potent inhibitors for platelet activation, and may therefore be a useful biochemical index for the study of the function of human alpha-2 adrenergic receptors.

走化性因子刺激モルモット腹腔しん出マクロファージにおけるイノシトールりん脂質代謝とＧＴＰ結合蛋白質の連関性

The involvement of GTP-binding protein in inositol phospholipid metabolism in guinea pig peritoneal exudate macrophages stimulated with the chemoattractant N-formyl-Methionyl-Leucyl-Phenylalanine (fMLP) was examined. The GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) caused a dose-dependent increase in the formation of inositol triphosphate in membranes of macrophages. This effect was specific for GTP and its analog. fMLP-induced inositol phospholipid turnover was markedly inhibited by the prior exposure of macrophages to 100 ng/ml of pertussis toxin (PT). Likewise, the pretreatment of macrophages with 100 ng/ml of PT evoked the inhibition of the increase in the intracellular free Ca2+ concentration and the spreading of macrophages induced by fMLP. These actions of PT were not associated with an alteration in the cellular concentration of cyclic AMP. Incubation of the membranes of macrophages with [32P]NAD and PT resulted in the ADP-ribosylation of a 41,000 Da protein. This ADP-ribosylation was diminished by the prior incubation of the membranes with 100 microM GTP gamma S plus 1 mM MgCl2, indicating that the 41,000 Da protein may be the alpha subunit of a GTP-binding protein. Moreover, there was a parallel between the time course of the ADP-ribosylation of intact macrophages by PT and the inhibition of the increase in intracellular free Ca2+ concentration as well as of the enhancement of the spreading of macrophages. These results suggest that the 41,000 Da protein, a GTP-binding protein, mediates the fMLP-stimulated inositol phospholipid metabolism.

In vivo stimulation of phosphoinositide metabolism in the brainstem of rats following osmotic stress

We have investigated the effects of osmotic stress on the activity of inositol phospholipid turnover in the central nervous system of rats. Intracerebroventricular injection of [ 3H]myo-inositol was used for metabolic labelling of brain phosphoinositides in vivo. The levels of radiolabelled inositol lipids increased in a time-dependent manner in several areas of the brain, reaching a maximum 24 h after the injection. Treatment with LiCl 20 h after the administration of tritiated myo-inositol did not modify the levels of inositol lipids, but resulted in a dose-dependent accumulation of inositol phosphates. In rats treated with 10mEq/kg LiCl, intraperitoneal injections of hypertonic saline (1.50 M NaCl) resulted in the stimulation of phosphoinositide turnover in the brainstem, but not in any of the other regions of the brain studied. This response was not prevented by unilateral cervical vagotomy, but was significantly lower in vasopressin-deficient Brattleboro rats. Brain phosphoinositide metabolism was not stimulated by acute blood volume depletion. We conclude that osmotic stress, but not acute hypovolemia, results in vasopressin-dependent activation of brainstem neurons by stimulating inositol phospholipid metabolism. In addition, metabolic labelling in vivo followed by treatment with LiCl provides a useful approach for assessing the physiological significance of the activation of polyphosphoinositide metabolism in the central nervous system in vivo. Our study provides evidence for a functional role of this second messenger system in the brainstem.

Regulatory Role of Retinoic Acid on Cultured Mouse Keratinocyte Inositol Phospholipid Metabolism: Dose-dependent Release of Inositol Triphosphate

The incorporation of precursor 14C-myoinositol into the three cellular inositol phospholipids (PtdIns, PtdInsP, and PtdInsP2) of cultured, rapidly proliferating keratinocytes is significantly enhanced by the exogenous addition of a high concentration (1 X 10(-7) M) of all-trans retinoic acid or its analog 13-Cis analog, whereas a similar incubation with a low concentration (1 X 10(-10) M) of the same retinoid resulted in an insignificant incorporation of the radio-precursor into the three inositol phospholipids. Incorporation was most marked into the more phosphorylated PtdIns4P and PtdIns4,5P2. These results indicate that retinoic acid affects the biosynthesis of the inositol phospholipids at high concentrations. In contrast, the hydrolysis of 14C-PtdIns4,5P2 and release of 14C-InsP3 from the prelabeled keratinocytes is markedly enhanced by a low physiologic concentration (1 X 10(-10) M) of retinoic acid or its 13-Cis analog. The hydrolysis is rapid, with an accompanying elevated transient release of 14C-InsP3. High concentration (1 X 10(-5) M), on the other hand, supresses 14C-InsP3 release. These results taken together underscore a bifunctional, dose-dependent effect of both the all-trans-RA and its 13-Cis analog on the synthesis and hydrolysis of keratinocyte PtdIns4,5P2. Furthermore, the results suggest that at low physiologic concentrations, these retinoids may function as agonists to perturb the membrane resulting in induced rapid hydrolysis of cellular PtdIns4,5P2, which is coupled to a "transient" generation of InsP3 (an intracellular second messenger). The rapid formation of this putative "second messenger" may in turn play a role in the cellular proliferative or differentiating biochemical events in the murine keratinocytes.

Cyclic AMP Inhibits Chemotactic-Peptide-Induced but not Ca&lt;sup&gt;2+&lt;/sup&gt;-Ionophore- or Tetradecanoylphorbol-Acetate-Induced Enzyme Secretion in Guinea Pig Neutrophils

cAMP-increasing agents such as prostaglandin El, dibutyryl cAMP and 8-bromo-cAMP inhibited N-acetyl-beta-D-glucosaminidase secretion induced by the chemotactic peptide formylmethionyl-leucyl-phenylalanine in guinea pig neutrophils. However, they could not inhibit but rather potentiated the secretion significantly when secretion was induced by the Ca2+ ionophore A23187. Similarly, these agents did not inhibit tetradecanoylphorbol acetate (TPA)-induced secretion. Moreover, the secretion induced by combined treatment with Ca2+ ionophore A23187 and TPA was hardly blocked by cAMP-increasing agents. These results strongly suggest that cAMP inhibits the secretion through suppression of a signal transduction from receptor activation to events such as Ca2+ mobilization and protein kinase C activation, probably of the inositol phospholipid metabolism.

Enhancement of inositol phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts.

The inositol phospholipid metabolism is one of the main pathways of signal transduction in cells. We measured the activities of its key enzymes in v-Ha-ras-transformed 208F rat fibroblasts. In the ras-transformed clones, incorporation of [32P]Pi into intermediates of the inositol phospholipid metabolism was stimulated. The activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in the transformed clones were about 35-50% more than in untransformed cells, indicating increased inositol phospholipid metabolism. However, the activity of diacylglycerol kinase in their membrane fraction was 25-35% less than that of untransformed cells, although the total diacylglycerol kinase activity did not change. The imbalance of these kinases could constitute one of the main reasons leading to the increased level of inositol phosphates and the accumulation of diacylglycerol to 2-2.2 times that in control 208F cells. Phosphatidylinositol-4,5-bisphosphate-phospholipase C activity did not change on the transformation when assayed under various conditions. The increased level of diacylglycerol caused intracellular translocation, activation, and down-regulation of protein kinase C changes which may be one of the essential events in transformation by the v-Ha-ras gene.