Receptor-Linked Hydrolysis of Phosphoinositides and Production of Prostacyclin in Cerebral Endothelial Cells

The receptor agonist-mediated hydrolysis of phosphoinositides and production of prostacyclin were studied in murine cerebral endothelial cells (MCEC). Of 11 neurotransmitters and neuromodulators examined, carbachol, noradrenaline (NE), bradykinin, and thrombin significantly increased 3H-inositol phosphate accumulation in the presence of LiCl (20 mM). The maximal stimulation of [3H]inositol monophosphate ([3H]IP1) reached approximately 11, 11, seven, and four times the basal levels for carbachol, NE, bradykinin, and thrombin, respectively. The EC50 values of IP1 accumulation for carbachol and NE were 34 and 0.16 microM, respectively. The muscarinic antagonists, atropine and pirenzepine, blocked the carbachol-induced IP1 accumulation with Ki values of 0.3 and 30 nM, respectively. The adrenergic antagonist, prazosin, blocked NE-induced IP1 accumulation with a Ki of 0.1 nM. The calcium ionophore A23187, histamine, glutamate, vasopressin, serotonin, platelet activating factor, and substance P did not stimulate IP1 accumulation. A23187, bradykinin, and thrombin stimulated prostacyclin release to approximately four, four, and two times the basal levels, respectively, whereas carbachol and NE had little effect upon prostacyclin release. These results suggest that the activation of phospholipase C and of phospholipase A2 in MCEC are regulated separately.     

Leukotriene C4-induced phosphoinositide hydrolysis in rat basophilic leukemia cell.

Leukotriene C4 (LTC4), one of the major constituents of the slow reacting substance of anaphylaxis, induced a dose-dependent hydrolysis of phosphoinositides in [3H]inositol-prelabeled rat basophilic leukemia (RBL-1) cells. The EC50 for LTC4 to elicit the half maximum accumulation of [3H]inositol phosphates (IPs) was around 20 nM. The increase in the formation of [3H]inositol bisphosphate (IP2) and [3H]inositol trisphosphate (IP3) was detectable at 2 min after the stimulation and progressed up to 30 min. Accumulation of [3H]inositol monophosphate (IP1) was observed only during the late phase of 5-30 min in the presence of LiCl. When cells were stimulated with LTC4 and LTD4 together, there was no additive accumulation in [3H]IPs. Pretreatment of cells with either LTC4 or LTD4 resulted in a decrease in production of [3H]IPs on further stimulation with the same agonist. The desensitization appeared to be heterologous since pretreatment of cells with LTC4 attenuated the responsiveness to LTD4. Conversely, pretreatment with LTD4 also diminished the responsiveness to LTC4 markedly. These results suggest that both LTC4- and LTD4-induced hydrolysis of phosphoinositides are mediated through the same effector in RBL-1 cells.     

Nerve Growth Factor Stimulation of Arachidonic Acid Release from PC12 Cells: Independence from Phosphoinositide Turnover

The effect of nerve growth factor on the metabolism of arachidonic acid and the hydrolysis of phosphatidylinositol in PC12 cells was examined. Addition of nerve growth factor to PC12 cells isotopically labeled with [3H]arachidonic acid caused an increased release of radioactivity. In a similar manner, treatment of PC12 cells prelabeled with [3H]inositol increased inositol monophosphate accumulation in the presence of LiCl. Stimulation of [3H]arachidonic acid release by nerve growth factor was concentration dependent, attaining a maximum at 0.5 nM. Concentrations of nerve growth factor above 0.5 nM caused less than maximal stimulation. In contrast, nerve growth factor-stimulated accumulation of [3H]inositol monophosphate exhibited a sigmoidal dose-response curve with an apparent maximum at 8 nM. Increased accumulation of [3H]inositol monophosphate could be detected as early as 60 s after nerve growth factor addition, whereas nerve growth factor-stimulated release of [3H]arachidonic acid was not observed until 5 min after nerve growth factor treatment. The nerve growth factor-stimulated release of [3H]arachidonic acid was independent of extracellular calcium concentration. Increased [3H]inositol monophosphate accumulation elicited by nerve growth factor was dependent on the presence of extracellular calcium. These results suggest that the increased metabolism of arachidonic acid and the enhanced hydrolysis of phosphatidylinositol are separately regulated by nerve growth factor.     

GABAB Receptor-Mediated Inhibition of Histamine H1-Receptor-Induced Inositol Phosphate Formation in Slices of Rat Cerebral Cortex

Histamine-stimulated accumulation of [3H]inositol monophosphate ([3H]IP1) in lithium-treated slices of rat cerebral cortex was inhibited by gamma-aminobutyric acid (GABA) (IC50 0.30 +/- 0.03 mM). The maximum level of inhibition was 69 +/- 2%. GABA alone caused a small stimulation of basal accumulation of [3H]IP1. The inhibitory action of GABA on the response to histamine was mimicked by the GABAB agonist (-)-baclofen, IC50 0.69 +/- 0.04 microM, which was 430-fold more potent as an inhibitor than the (+)-isomer. (-)-Baclofen also inhibited histamine-induced formation of [3H]inositol bisphosphate ([3H]IP2) and [3H] inositol trisphosphate ([3H]IP3). Inhibition curves for GABA and for (-)-and and (+)-baclofen had Hill coefficients greater than unity. (-)-Baclofen, at concentrations that caused inhibition of histamine-induced [3H]IP1 accumulation, did not alter the basal level of [3H]IP1 or the incorporation of [3H]inositol into total inositol phospholipids. Isoguvacine, a GABAA agonist, had no effect on either the histamine-stimulated or basal accumulation of [3H]IP1. GABA had no effect on carbachol-stimulated [3H]IP1 formation.     

Production of 1,2-Diacylglycerol in PC12 Cells by Nerve Growth Factor and Basic Fibroblast Growth Factor

The addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) to PC12 cells prelabeled with [3H]inositol and preincubated for 15 min in the presence of 10 mM LiCl stimulated the production of inositol phosphates with maximal increases of 120-180% in inositol monophosphate (IP), 130-200% in inositol bisphosphate (IP2), and 45-50% in inositol trisphosphate (IP3) within 30 min. The majority of the overall increase (approximately 85%) was in IP; the remainder was recovered as IP2 and IP3 (approximately 10% as IP2 and 5% as IP3). Under similar conditions, carbachol (0.5 mM) stimulated about a 10-fold increase in IP, a sixfold increase in IP2, and a fourfold increase in IP3. The mass level of 1,2-diacylglycerol (DG) in PC12 cells was found to be dependent on the incubation conditions; in growth medium [Dulbecco's modified Eagle's medium (DME) plus serum], it was around 6.2 mol %, in DME without serum, 2.5 mol %, and after a 15-min incubation in Dulbecco's phosphate-buffered saline, 0.62 mol %. The addition of NGF and bFGF induced an increase in the mass level of DG of about twofold within 1-2 min, often rising to two- to threefold by 15 min, and then decreasing slightly by 30 min. This increase was dependent on the presence of extracellular Ca2+, and was inhibited by both phenylarsine oxide (25 microM) and 5'-deoxy-5'-methylthioadenosine (3 mM). Under similar conditions, 0.5 mM carbachol stimulated the production of DG to the same extent as 200 ng/ml NGF and 50 ng/ml bFGF. Because carbachol is much more effective in stimulating the production of inositol phosphates, the results suggest that both NGF and bFGF stimulate the production of DG primarily from phospholipids other than the phosphoinositides.     

Histamine stimulates inositol phosphate accumulation via the H1-receptor in cultured human endothelial cells

The effects of histamine on [3H]inositol phosphate ([3H]IP) accumulation was examined in the presence of lithium in [3H]inositol-prelabelled human umbilical vein endothelial cells. Histamine stimulated total [3H]IP formation in a dose-dependent manner with a half-maximal value (EC50) of around 1-2 X 10(-6) M. Mepyramine, but not cimetidine, completely abolished the histamine response indicating that activation of phosphoinositide hydrolysis is mediated via H1-receptors. These data are the first to suggest that activation of inositol lipid hydrolysis is the underlying transmembrane signalling mechanism histamine H1-receptors employ in mediating various endothelial cell functions.     

Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

Inositol trisphosphate (IP3), a product of the phosphoinositide cycle, mobilizes intracellular Ca2+ in many cell types. New evidence suggests that inositol tetrakisphosphate (IP4), an IP3 derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP4 are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP4 in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine (0.4 mM) stimulated an increase in [3H]IP4 and [3H]IP3 accumulation in chromaffin cells and this effect was completely blocked by atropine (0.5 mM). [3H]IP4 accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP3 and IP4 hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP4 and calcium homeostasis.     

Glycerol-3-phospho-D-myo-inositol 4-phosphate (Gro-PIP) is an inhibitor of phosphoinositide-specific phospholipase C

The deacylated forms of the phosphoinositides were used to determine whether the guinea pig uterus phosphoinositide-specific phospholipase C (PI-PLC I, Mr 60,000) required fatty acids at the sn-1 and sn-2 positions for the hydrolysis of the sn-3 phosphodiester bond. L-alpha-Glycerophospho-D-myo-inositol 4-phosphate (Gro-PIP), but not glycerol 3-phosphate (Gro-3-P), L-alpha-glycerophospho-D-myo-inositol (Gro-PI), or L-alpha-glycerophospho-D-myo-inositol 4,5-bisphosphate (Gro-PIP2), inhibited PI-PLC I in a concentration-dependent manner. Assays performed with 10 microM [3H]phosphatidylinositol ([3H]PI), 10 microM [3H]phosphatidylinositol 4-phosphate ([3H]PIP) or 10 microM [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) as substrates, with increasing [Gro-PIP] revealed an IC50 = 380 microM. Kinetic studies with increasing [3H]PI substrate concentrations in the presence of 100 microM and 300 microM Gro-PIP demonstrated that Gro-PIP exhibited competitive inhibition; Kis = 40 microM. Ca2+ concentrations over the range 1.1 microM to 1 mM did not effect inhibition, suggesting that Gro-PIP inhibition of [3H]PI hydrolysis was calcium-independent. To determine whether Gro-PIP was a substrate, 20 microM and 500 microM [3H]Gro-PIP were incubated with PI-PLC I. Anion-exchange HPLC analysis revealed no [3H]IP2 product formation, indicating that [3H]Gro-PIP was not hydrolyzed. Assays performed with [3H]PI and [3H]PIP substrates in the presence of 500 microM [3H]Gro-PIP revealed approx. 75% less [3H]inositol 1-phosphate ([3H]IP1) and [3H]inositol 1,4-bisphosphate ([3H]IP2) product formation, respectively, indicating that [3H]Gro-PIP inhibited the hydrolysis of the substrates by PI-PLC I. These data suggest that Gro-PIP does not serve as a substrate, and that it inhibits PI-PLC I by competitive inhibition in a Ca2(+)-independent fashion.     

Inositol phosphates accumulation in ovarian granulosa after stimulation by luteinizing hormone

Accumulation of inositol phosphates by granulosa cells from medium follicles of porcine ovaries was studied to determine if hydrolysis of phosphoinositides is stimulated by luteinizing hormone (LH). Although follicle-stimulating hormone (FSH), D-alanine-gonadotropin-releasing hormone (D-ala-GnRH), and dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) had no effect, LH increased accumulation of inositol phosphate (IP), -bisphosphate (IP2), and trisphosphate (IP3) by severalfold. Furthermore, 0.01 microgram LH/ml increased IP3 accumulation threefold, while 0.1 microgram/ml stimulated accumulation of all inositol phosphates. Compared to untreated cells, LH-treated granulosa cells produced approximately twice as much progesterone in 30 min. Preincubation of cells with lithium chloride (LiCl) was necessary to measure IP accumulation, but not IP2 and IP3 accumulations. However, IP2 and IP3 accumulations were higher in LH-treated granulosa after pretreatment with LiCl. Maximal increases in IP3 and IP2 accumulations occurred approximately 15 min and 30 min, respectively, after LH stimulation, whereas the effect of LH on IP accumulation continued for at least 60 min. Granulosa, made permeable to IP3 with saponin treatment, did not hydrolyze [3H]IP3 to [3H]IP2 or [3H]IP. Thus, it is hypothesized that LH stimulates phosphoinositide hydrolysis in granulosa cells, thereby generating putative second messengers.     

Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5''-[gamma-thio]triphosphate and by thrombin in the presence of GTP.

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5'-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.     

Characterization of Bradykinin-Induced Phosphoinositide Turnover in Neurohybrid NCB-20 Cells

Phosphoinositide hydrolysis was studied in neurohybrid NCB-20 cells prelabeled with myo-[3H]inositol. Among nearly 20 neurotransmitters and neuromodulators examined, only bradykinin, carbachol, and histamine significantly increased the accumulation of [3H]inositol monophosphate (IP1) in the presence of lithium. The EC50 of bradykinin was 20 nM and the saturating concentration was approximately 1 microM. The bradykinin response was robust (10-fold) and was potently and selectively blocked by a bradykinin antagonist, B 4881 [D-Arg-(Hyp3, Thi, D-Phe)-bradykinin], with a Ki of 10 nM. This effect of bradykinin appeared to be additive to that mediated by activation of muscarinic cholinergic and histamine H1 receptors. The accumulation induced by bradykinin or carbachol was dependent on the presence of calcium in the incubation medium; less than twofold stimulation was observed in the absence of exogenous calcium. Bradykinin-induced [3H]IP1 accumulation required high concentration of lithium to elicit its maximal stimulation; the concentration of lithium required for half maximal effect was about 13 mM, similar to the value reported previously for carbachol-induced accumulation in the same cell line. In contrast, using related neurohybrid NG108-15 cells, bradykinin-induced [3H]IP1 accumulation was found to require much less lithium. IN the presence of lithium, bradykinin also evoked a transient increase in the production of [3H]-inositol bis- and trisphosphate. Basal and bradykinin-induced phosphoinositide breakdown was inhibited by 4 beta-phorbol 12,13-dibutyrate, but was unaffected by the biologically inactive 4 beta-phorbol. Pretreatment of cells with pertussis toxin induced only about 30% loss of the bradykinin-induced [3H]IP1 accumulation, without affecting basal activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve growth factor promotes the activation of phosphatidylinositol 3-kinase and its association with the trk tyrosine kinase.

We investigated the involvement of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in the initiation of signal transduction by nerve growth factor (NGF) in the rat pheochromocytoma PC12 cell line. PtdIns 3-kinase catalyzes the formation of phosphoinositides with phosphate in the D-3 position of the inositol ring and previously has been found to associate with other activated protein tyrosine kinases, including growth factor receptor tyrosine kinases. Anti-phosphotyrosine immunoprecipitates had PtdIns 3-kinase activity that reached a maximum (9 times the basal activity) after a 5-min exposure of PC12 cells to NGF (100 ng/ml). Since NGF activates the tyrosine kinase activity of gp140trk, the protein product of the trk proto-oncogene, we also examined the association of PtdIns 3-kinase with gp140trk. Anti-gp140trk immunoprecipitates from NGF-stimulated PC12 cells had increased PtdIns 3-kinase activity compared to that of unstimulated cells, and larger increases were detected in cells overexpressing gp140trk, indicating that PtdIns 3-kinase associates with gp140trk. NGF produced large increases in [32P]phosphatidylinositol 3,4-bisphosphate and [32P]phosphatidylinositol 3,4,5-trisphosphate in PC12 cells labeled with [32P]orthophosphate, indicating an increase in PtdIns 3-kinase activity in intact cells. Using an anti-85-kDa PtdIns 3-kinase subunit antibody, we found that NGF promoted the tyrosine phosphorylation of an 85-kDa protein and two proteins close to 110 kDa. These studies demonstrate that NGF activates PtdIns 3-kinase and promotes its association with gp140trk and also show that NGF promotes the tyrosine phosphorylation of the 85-kDa subunit of PtdIns 3-kinase. Thus, PtdIns 3-kinase activation appears to be involved in differentiation as well as mitogenic responses.     

Stimulation of Inositol Incorporation into Lipids of PC 12 Cells by Nerve Growth Factor and Bradykinin

The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC12 cells cultured for 20 h in the presence [PC12(+)] or in the absence [PC12(-)] of nerve growth factor (NGF). BK (1 microM) induced a small stimulation of the incorporation of myo-[2-3H]inositol into the lipids of PC12(-) cells and a three- to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and greater than 10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels nine-fold in PC12 (-) and 30-fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4 degrees C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC12(+) cells. In these cells, LiCl in the presence of BK also increased several-fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.     

Increased Intracellular Calcium Stimulates 3H-Inositol Polyphosphate Accumulation in Rat Cerebral Cortical Slices

Agents that increase the intracellular Ca2+ concentration have been examined for their ability to stimulate 3H-inositol polyphosphate accumulation in rat cerebral cortex slices. Elevated extracellular K+ levels, the alkaloid sodium channel activator veratrine, the calcium ionophore ionomycin, and the marine toxin maitotoxin were all able to stimulate phosphoinositide metabolism. Certain features appear common to the agents studied. Thus, although [3H]inositol monophosphate, [3H]inositol bisphosphate ([3H]InsP2), and [3H]inositol trisphosphate were all stimulated, a proportionally greater effect was observed on [3H]InsP2 in comparison to stimulation by the muscarinic receptor agonist carbachol. However, only an elevated K+ level stimulated [3H]inositol tetrakisphosphate ([3H]InsP4) accumulation alone or produced marked synergy with carbachol on the formation of this polyphosphate. The results suggest that agents that elevate the cytoplasmic Ca2+ concentration in cerebral cells can increase the hydrolysis of membrane polyphosphoinositides. The pattern of the response differs from that produced by muscarinic receptor agonists and indicate that Ca2(+)-dependent hydrolysis may involve different pools of lipids, phosphoinositidase C enzymes, or both. However, clear differences in the ability of these agents to stimulate InsP4, alone or in the presence of muscarinic agonist, suggest that factors other than a simple elevated intracellular Ca2+ concentration are implicated.     

Plasma membrane associated phospholipase C from human platelets: synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5'-O-(3-thiotriphosphate)

The effects of thrombin and GTP gamma S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [3H]inositol-labeled membranes or with lipid vesicles containing either [3H]phosphatidylinositol or [3H]phosphatidylinositol 4,5-bisphosphate. GTP gamma S (1 microM) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP3), inositol bisphosphate (IP2), or inositol phosphate (IP) from [3H]inositol-labeled membranes. IP2 and IP3, but not IP, from [3H]inositol-labeled membranes were, however, stimulated 3-fold by GTP gamma S (1 microM) plus thrombin (1 unit/mL). A higher concentration of GTP gamma S (100 microM) alone also stimulated IP2 and IP3, but not IP, release. In the presence of 1 mM calcium, release of IP2 and IP3 was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) by platelet membrane associated PLC was also markedly enhanced by GTP gamma S (100 microM) or GTP gamma S (1 microM) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP2 was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP gamma S (100 microM) or calcium (1 mM) dependent PIP2 breakdown, while TPA inhibited GTP gamma S-dependent but not calcium-dependent phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of [3H]choline and [3H]phosphorylcholine ([3H]Pchol) from cells containing [3H]choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of [3H]phosphatidic acid ([3H]PA) in cells containing [3H]myristate-labeled PC. [3H]Diacylglycerol ([3H]DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with [3H]myristate and [14C]arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol. By analyzing the increase in 3H versus 14C in DAG, we estimate that the DAG that is formed in response to PMA arises largely from PC. Muscarinic receptor activation also causes formation of DAG from PC, but approximately 20% of carbachol-stimulated DAG appears to arise from hydrolysis of the phosphoinositides.     

Muscarinic Cholinergic Receptor-Mediated Phosphoinositide Metabolism in Peripheral Nerve

Few receptor-mediated phenomena have been detected in peripheral nerve. In this study, the ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo-[3H]inositol and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) accounting for 7-8% and 1-2% of the total, respectively. In the presence of 100 microM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 microM carbamylcholine and stimulated IP generation was abolished by 1 microM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (5.7), and 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin-rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP-ribosylated approximately 40-kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor-mediated signal transduction via the phosphoinositide cycle.     

Bradykinin-induced activation of phospholipase A2 is independent of the activation of polyphosphoinositide-hydrolyzing phospholipase C

This study evaluates the role of phosphatidylinositol 4,5-bisphosphate (PIP2) and its metabolites as possible mediators in the activation of phospholipases A2 in porcine aortic endothelial cells. We compared the time courses of bradykinin-induced turnover of phosphoinositides and the appearance of unesterified arachidonic acid (uAA) and eicosanoids. The metabolism of phosphoinositides was examined in cells prelabeled with [3H]inositol, which has a similar distribution as the endogenous inositol lipids. At 37 degrees C, bradykinin induced a rapid rise in lysophosphatidylinositol (lyso-PI) and inositol 1,4,5-trisphosphate (IP3) as well as a decrease in PIP2. Lyso-PI formation was detected at 10 s, as early as PIP2 degradation and IP3 formation. This suggests that the activation of PIP2-hydrolyzing phospholipase C and PI-hydrolyzing phospholipase A2 are simultaneous. However, at 30 degrees C, lyso-PI formation was detected in the absence of an increase in IP3 indicating that the activation of phospholipase A2 does not require the accumulation of IP3. The time course of formation of uAA and eicosanoids were examined in [3H]arachidonic acid-prelabeled cells. The 3H radioactivity was distributed among the phospholipid classes and subclasses the same as the endogenous phospholipids. Bradykinin stimulated the intracellular accumulation of uAA, detectable at 5 s, earlier than that of 1,2-diacylglycerol and phosphatidic acid. Such immediate formation of uAA further supports the notion that activation of phospholipase A2 is a very early event during the interaction of bradykinin with porcine endothelial cells, and that PIP2 hydrolysis is not prerequisite for the initial activation of phospholipase A2.     

Antigen-induced biphasic diacylglycerol formation in RBL-2H3 cells: the late sustained phase due to phosphatidylcholine hydrolysis is dependent on protein kinase C

Exposure to antigen (Ag) caused a biphasic 1,2-diacylglycerol (DG) production in [3H]myristic acid-labeled RBL-2H3 cells; the early, small transient phase and the second large sustained phase. The accumulation of phosphatidic acid (PA) or phosphatidylethanol (PEt) in the presence of ethanol was paralleled by the second-phase DG generation. Ag-induced formation of phosphocholine and choline in [3H]choline-labeled cells suggested the hydrolysis of phosphatidylcholine (PC) by phospholipases C and D. Treatment with phorbol myristate (PMA) or A23187 caused increases in [3H]DG and water-soluble [3H]choline metabolites. In protein kinase C (PKC) down-regulated cells, PEt formation was markedly reduced. In these cells DG production induced by Ag and A23187 was largely suppressed, thus indicating that PKC would play an important regulatory role for PC hydrolysis. However, because the A23187 treatment showed significant accumulation of water-soluble choline metabolites in PKC down-regulated cells, an increase in intracellular Ca2+ is another factor regulating PC hydrolysis. Taken together, these results may indicate that PC hydrolysis in response to Ag is dependent on PKC and Ca2+.     

Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.