In Vivo Electrical Stimulation of the Sympathetic Nerve of the Eye Increases Inositol Phosphate Production and Prostaglandin Release in the Rabbit Iris Muscle

The effects of in vivo electrical stimulation of the sympathetic nerve of the eye on phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis in rabbit iris and release of arachidonate and prostaglandin (PG) E2 into aqueous humor were investigated. myo-[3H]Inositol or [1-14C]arachidonate was injected intracamerally into each eye 3 h before electrical stimulation of one of the sympathetic trunks. Tissue phosphoinositides were determined by TLC, and 3H-labeled inositol phosphates were analyzed by either ion-exchange chromatography or HPLC. The aqueous humor was analyzed for 14C-labeled arachidonate and PGE2 by radiochromatography and for unlabeled PGE2 by radioimmunoassay. The results obtained from this study can be summarized as follows: (a) The rates of in vivo incorporation of myo-[3H]inositol into phosphoinositides and accumulation of 3H-labeled inositol phosphates in the iris muscle increased with time and then leveled off between 3 and 5 h. (b) Distribution of 3H radioactivity in inositol phosphates, as determined by HPLC, showed that of the total radioactivity in inositol phosphates, 53.6% was recovered in myo-inositol 1-phosphate, 36% in myo-inositol bisphosphate, 0.95% in myo-inositol 1,3,4-trisphosphate (1,3,4-IP3), and 2.6% in 1,4,5-IP3. (c) Electrical stimulation of the sympathetic nerve resulted in a significant loss of 3H radioactivity from PIP2 and a concomitant increase of that in IP3, an observation indicating that PIP2 is the physiological substrate for alpha 1-adrenergic receptors in this tissue. (d) Release of IP3 and liberation of arachidonate for PGE2 synthesis are dependent on the duration of stimulation and the intensity (voltage) of stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.     

The effects of alpha 1-adrenergic and muscarinic cholinergic stimulation on prostaglandin release by rabbit iris

We have investigated the effects of norepinephrine (NE) and acetylcholine (ACh) on prostaglandin (PGE2 and 6 keto-PGF1 alpha) production by rabbit iris, measured by radioimmunoassay (RIA), and the type of phospholipase activated by NE in irides in which phosphatidylinositol (PI) was doubly prelabeled with [3H] myo-inositol and [1-14C] arachidonic acid (14C-AA), quantitated by radiometric and chromatographic methods. PGE2 output in 60 min (3.6 micrograms/g tissue) was 2.6 times greater than 6 keto-PGF1 alpha. PG production is time-dependent and it is stimulated by NE and ACh in a dose-dependent manner. The NE- and ACh-induced release of PGE2, measured by RIA, is mediated through alpha 1-adrenergic and muscarinic cholinergic receptors, respectively, and it requires Ca2+ for maximal stimulation. Studies on the mechanism of AA release from PI in irides doubly prelabeled with 14C-AA and [3H] myo-inositol revealed the following: (a) Both NE and ACh increased the breakdown of PI, and this was accompanied by a significant increase in the release of AA and consequently PGE2. The stimulatory effects of NE and ACh are mediated through alpha 1-adrenergic and muscarinic cholinergic receptors respectively. (b) The NE-induced formation of 3H-lyso PI and the NE-induced metabolism of 14C-1,2-diacyl-glycerol (DG) are time-dependent. Two pathways for AA release from PI are probably operative in the iris: (a) An indirect release by PI-specific phospholipase C which produces DG, followed by the actions of DG- and monoacylglycerol lipases on DG to release AA. (b) A direct release by phospholipase A2. Whether lyso PI is a product of the polyphosphoinositide response remains to be established. Other phospholipids such as phosphatidylcholine and phosphatidylethanolamine could also serve as a source for AA in PG synthesis. In conclusion, the data presented provide evidence that in the iris the neuro-transmitter-stimulated release of PG and AA, from phosphoinositides, for PG synthesis is coupled to the activation of alpha 1-adrenergic and muscarinic cholinergic receptors.     

Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen.

Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.     

Release of O2- by human umbilical cord blood-derived eosinophils: role of intra- and extracellular calcium.

The aim of our study was to investigate the physiologic mechanisms involved in eosinophil activation as an essential prerequisite to disrupting the biochemical cascade that triggers inflammation, thereby attenuating the effect of this activation or, ideally, preventing it from occurring. We have, therefore, examined the nature of the fMLP- and PAF-induced [Ca2+]i rise and the relationship between the [Ca2+]i rise and O2- production in human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5. These cells responded to fMLP or PAF (1 microM each) with an increase in [Ca2+]i (217.3 +/- 22.1 and 197.8 +/- 22.1 nM respectively) which was associated with production of O2- (40.2 +/- 8.2 and 35.2 +/- 7.6 pmol/min/10(6) cells respectively). The role of Ca2+ in the induced respiratory burst was studied by changing the availability of Ca2+ in the intra- and extracellular compartments. Removal or chelation of extracellular Ca2+ induced a reduction of both the fMLP and PAF-induced [Ca2+]i rise and O2- production. Chelation of intracellular Ca2+ induced a concentration-dependent inhibition of fMLP- and PAF-induced [Ca2+]i rise and caused a decrease in O2- production. SK&F 96365 had a stimulatory effect on PAF-induced [Ca2+]i rise and on fMLP-induced O2- production, this phenomenon was not observed with extracellular Ca2+ removal or chelation. Furthermore, Ni2+ exhibited an inhibition of both fMLP and PAF-induced [Ca2+]i rise and O2- production. Finally, both fMLP and PAF induced an increase in divalent cation influx that was further augmented by thapsigargin. Our results indicate that fMLP and PAF dependent O2- production in human eosinophils require intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation.     

2-O-methyl PAF as a Ca2+ mobilizer in Madin Darby canine kidney cells

In Madin-Darby canine kidney (MDCK) cells, the effect of 2-O-methyl PAF, an inactive analogue of platelet activating factor (PAF), on intracellular Ca2+ concentration ([Ca2+]i) was measured by using the Ca2+-sensitive fluorescent dye fura-2. 2-O-methyl PAF (> or = 15 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. 2-O-methyl PAF-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. 2-O-methyl PAF-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. The 2-O-methyl PAF-induced Ca2+ influx was blocked by nifedipine, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which 2-O-methyl PAF failed to increase [Ca2+]i; also, pretreatment with 2-O-methyl PAF depleted thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not 2-O-methyl PAF)-induced [Ca2+]i rise. These findings suggest that 2-O-methyl PAF evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release.     

Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates.

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands.

1. The effect of Li+ on the agonist-dependent metabolism of [3H]inositol has been studied in rat brain, rat parotid and the insect salivary gland. 2. When brain or parotid slices were incubated in the presence of [3H]inositol, Li+ was found to amplify the ability of agonists such as carbachol, phenylephrine, histamine, 5-hydroxytryptamine and Substance P to elevate the amount of label appearing in the inositol phosphates. 3. A different approach was used with the insect salivary gland, which was prelabelled with [3H]inositol. After washing out the label, the subsequent release of [3H]inositol induced by 5-hydroxytryptamine was greatly decreased by Li+. During Li+ treatment there was a large accumulation of [3H]inositol 1-phosphate. 4. This ability of Li+ to greatly amplify the agonist-dependent accumulation of myo-inositol 1-phosphate offers a novel technique for identifying those receptors that function by hydrolysing phosphatidylinositol. 5. The therapeutic action of Li+ may be explained by this inhibition of myo-inositol 1-phosphatase, which lowers the level of myo-inositol and could lead to a decrease in the concentration of phosphatidylinositol, especially in those neurons that are being stimulated excessively. This alteration in phosphatidylinositol metabolism may serve to reset the sensitivity of those multifunctional receptors that generate second messengers such as Ca2+, cyclic GMP and the prostaglandins.     

Evidence for two platelet activating factor receptors on eosinophils: dissociation between PAF-induced intracellular calcium mobilization degranulation and superoxides anion generation in eosinophils

We have compared platelet activating factor (PAF)-induced eosinophil peroxidase (EPO) release and intracellular calcium mobilization with superoxide anion (.O2-) generation from guinea pig eosinophils. EPO release and Ca2+ mobilization occurred at lower concentrations of PAF (EC50 values of 1.3 nM and 11.5 nM, respectively) while .O2- production was observed at higher concentrations (EC50 of 31.7 microM). Receptor characterization with the competitive PAF antagonist, WEB 2086, gave pA2 values of 8.5 and 8.3 for EPO enzyme release and rise in [Ca2+]i, respectively, and 5.8 for the .O2- production. In addition, PAF-induced degranulation and elevation of [Ca2+]i were dependent on extracellular Ca2+ whereas PAF-stimulated .O2- generation was dependent on the presence of extracellular Mg2+ ions. These results suggest the existence either of two subtypes of the PAF receptor or a single receptor that can exist in one of two affinity states on guinea pig eosinophils.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Platelet-activating factor induces an increase in intracellular calcium and expression of regulatory genes in human B lymphoblastoid cells.

Platelet-activating factor (PAF) has recently been demonstrated to be metabolized by B lymphocytes and to cause enhancement of Ig synthesis by Ig-secreting B lymphoblastoid cell lines. We have now examined some of the early activation events triggered by PAF binding to three Ig-secreting B cell lines, LA350 (IgM secreting), HSCE- (IgG secreting), and U266 (IgE secreting). After addition of 10(-7) to 10(-11) M PAF, but not equimolar concentrations of the inactive metabolite lyso-PAF, all three cell lines demonstrated rapid dose-dependent increases in free cytosolic Ca2+ concentrations ([Ca2+]i). The increases in [Ca2+]i resulted from both the release of Ca2+ from internal stores as well as transmembrane Ca2+ uptake. Addition of PAF triggered the rapid hydrolysis of phosphatidylinositol bisphosphate and accumulation of inositol phosphates. PAF also increased expression of the cell cycle-active genes c-fos and EGR2 in a dose-dependent fashion. The stimulated increases in [Ca2+]i and phosphatidylinositol bisphosphate hydrolysis and the increases in gene expression were all inhibited by the specific PAF receptor antagonist Web 2086. The LA350 cell line (which expresses surface IgM) was also shown to increase [Ca2+]i after addition of anti-IgM antibodies. Sequential addition of PAF or anti-IgM antibody in either order failed to reveal any evidence for heterologous desensitization. Furthermore, the PAF receptor antagonist did not affect anti-IgM induced changes in [Ca2+]i. These data provide evidence for the presence of functional PAF receptors on B lymphoblastoid cells and indicate a potential role for PAF in the regulation of B cell activation.     

The CMP-stimulated production of diacylglycerol and CDPdiacylglycerol in neuronal nuclei labelled with radioactive arachidonate

A neuronal nuclear fraction (N1), isolated from immature rabbit cerebral cortex, was preincubated with [3H]arachidonate, ATP, CoA, Mg2+ and 1-acyl-sn-glycero-3-phosphocholine or 1-acyl-sn-glycero-3-phosphoinositol. Using the former lysophospholipid, a sizeable incorporation of radioactivity was seen in N1 phosphatidylcholine. In subsequent incubations in the presence of CMP and EGTA, there was a generation of radioactive diacylglycerol in N1 and a corresponding decline in phosphatidylcholine radioactivity. Both these changes could be blocked by the addition of CDPcholine. In incubations using N1 phosphatidylinositol or phosphatidylethanolamine prelabelled with [3H]arachidonate, no evidence was found to support a direct generation of diacylglycerol from these phospholipids. The back reaction of cholinephosphotransferase in N1 is likely the principal source of diacylglycerols bearing arachidonate. Using either lysophospholipid in the preincubations described in the opening sentence, more than half of the incorporated radioactivity derived from [3H]arachidonate was found in N1 phosphatidylinositol. In subsequent incubations with EGTA and CMP there was a production of radioactive CDPdiacylglycerol and a decline in radioactive phosphatidylinositol. Both events could be blocked by the presence of myo-inositol. Radioactive CDPdiacylglycerol, produced in N1 in the presence of CMP and EGTA, was converted back into phosphatidylinositol by the addition of myo-inositol. The production of CDPdiacylglycerol is likely the result of the back reaction of CDPdiacylglycerol:inositol phosphatidate transferase in N1.     

Platelet-activating factor stimulates production of prostaglandin E2 in murine osteoblast-like cell line MC3T3-E1.

We found that platelet-activating factor (PAF) stimulated the production of prostaglandin (PG) E2 in MC3T3-E1 cells in a time- and dose-dependent manner. 1.0 microM PAF gave a maximal stimulation of PGE2 production by MC3T3-E1 cells after a 4 hr PAF-treatment. Furthermore, the PAF-induced PGE2 production was abolished by the pre-treatment of the cells with a PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine, which occupied the same receptor site as PAF. These results suggest that PAF stimulates the PGE2 synthesis through a PAF receptor mediated pathway. Possibly PAF modulates bone metabolism by stimulating PGE2 synthesis.     

Gonadotropin-releasing hormone activates a rapid Ca2+-independent phosphodiester hydrolysis of polyphosphoinositides in pituitary gonadotrophs

Addition of gonadotropin releasing hormone (GnRH) to pituitary cells prelabeled with [32P]Pi or with myo-[2-3H]inositol, resulted in a rapid decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate (approximately 10 s), and in [32P]phosphatidylinositol 4-phosphate (approximately 1 min), followed by increased labeling of [32P]phosphatidylinositol and [32P]phosphatidic acid (1 min). GnRH stimulated the appearance of [3H]myo-inositol 1,4,5-trisphosphate (10 s), [3H]myo-inositol 1,4-bisphosphate (15 s), and [3H]myo-inositol 1-phosphate (1 min) in the presence of Li+ (10 mM). Li+ alone stimulated the accumulation of [3H]myo-inositol 1-phosphate and [3H]myo-inositol 1,4-bisphosphate but not [3H]myo-inositol 1,4,5-trisphosphate, but had no effect on luteinizing hormone release. The effect of GnRH on inositol phosphates (Ins-P) production was dose-related (ED50 = 1-5 nM), and was blocked by a potent antagonist [D-pGlu,pClPhe,D-Trp]GnRH. Elevation of cytosolic free Ca2+ levels ([Ca2+]i), by ionomycin and A23187 from intracellular or extracellular Ca2+ pools, respectively, had no significant effect on [3H]Ins-P production. GnRH-induced [3H]Ins-P production was not dependent on extracellular Ca2+ and was noticed also after extracellular or intracellular Ca2+ mobilization by A23187 or ionomycin, respectively. The effect of GnRH on [3H]Ins-P accumulation was not affected by prior treatment of the cells with the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with islet-activating protein pertussis toxin. These results indicate that GnRH stimulates a rapid phosphodiester hydrolysis of polyphosphoinositides. The stimulatory effect is not mediated via an islet-activating protein-substrate, is not dependent on elevation of [Ca2+]i, neither is it negatively regulated by 12-O-tetradecanoylphorbol-13-acetate which activates Ca2+/phospholipid-dependent protein C kinase. The results are consistent with the hypothesis that GnRH-induced phosphoinositide turnover is responsible for Ca2+ mobilization followed by gonadotropin release.     

Bacterial lipopolysaccharide up-regulates platelet-activating factor-stimulated Ca2+ mobilization and eicosanoid release in human Mono Mac 6 cells.

When human monocytic Mono Mac 6 cells were treated with bacterial LPS (10 ng/ml, 72 h), they showed an increase in phagocytic activity, superoxide anion production, and expression of monocyte/macrophage-associated cell surface Ag. In these more mature (LPS-treated) cells but not in untreated cells, platelet-activating factor (PAF) (100 nM) produced a three- to fourfold increase in cytosolic free Ca2+ concentration. The cytosolic free Ca2+ concentration increase was inhibited by the PAF receptor antagonist L-659,989 (10 microM) and by EGTA (2 mM), indicating receptor-dependent Ca2+ influx. Furthermore, L-659,989 (10 microM), as well as PAF (1 microM), inhibited specific [3H]PAF binding in LPS-treated but not in untreated cells. Consistent with these results, PAF (100 nM) stimulated release of arachidonic acid and thromboxane B2 only in LPS-treated cells, and this could be inhibited by L-659,989 (10 microM) and EGTA (2 mM). Our data indicate that LPS up-regulates PAF-induced Ca2+ influx, resulting in arachidonic acid and eicosanoid release in Mono Mac 6 cells.     

Mechanisms of interaction of a plasmalogenic analog of platelet-activating factor with human platelets.

The interaction of a plasmalogenic analog of platelet-activating factor (1-O-alk-1;-enyl-2-acetyl-sn-glycero-3-phosphocholine; 1-alkenyl-PAF) with human platelets was studied. 1-Alkenyl-PAF induced an increase in intracellular Ca2+ concentration and inhibition of adenylate cyclase at significantly higher concentrations than PAF. 1-Alkenyl-PAF inhibits PAF-induced platelet aggregation but has no effect on ADP- or thrombin-induced aggregation of human platelets. In contrast to PAF, 1-alkenyl-PAF increases [3H]PGE1 binding with human platelets. The properties of 1-alkenyl-PAF as an agonist or antagonist of PAF receptors apparently depend on its concentration in the cell medium. Under physiological conditions 1-alkenyl-PAF might be a natural PAF antagonist acting in the human cardiovascular system.     

Platelet-activating factor stimulates multiple signaling pathways in cultured rat mesangial cells.

We have previously reported that platelet-activating factor (PAF) elevates cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded glomerular mesangial cells. To confirm that this increase in [Ca2+]i is a result of receptor-mediated activation of phospholipase C, we investigated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) in PAF-treated mesangial cells. PAF (10(-7) M) stimulated a rapid and transient formation of inositol trisphosphate. In concomitant experiments, PAF stimulated a biphasic accumulation of 3H-arachidonate-labeled 1,2-diacylglycerol (DAG). The secondary elevation in DAG was coincident with a rise in 3H-phosphorylcholine (PC) and 3H-phosphorylethanolamine (PE) suggesting that PAF stimulates delayed phospholipase activities which hydrolyze alternate phospholipids besides the polyphosphoinositides. This PAF-stimulated elevation in 3H-water soluble phosphorylbases was seen at 5 min but not at 15 sec suggesting that the initial rise in DAG as well as the initial elevation in [Ca2+]i are due primarily to PtdIns-4,5-P2 hydrolysis. PAF also stimulated PGE2 as well as 3H-arachidonic acid and 3H-lyso phosphatidylcholine (PtdCho) formation. We suggest that arachidonate released specifically from PtdCho via phospholipase A2 is a source of this PAF-elevated PGE2. It has been postulated that anti-inflammatory prostaglandins may antagonize the contractile and proinflammatory effects of PAF via activation of adenylate cyclase. Surprisingly, exogenous PAF reduced basal and receptor-mediated cAMP concentration indicating that PAF-stimulated transmembrane signaling pathways may oppose receptor-mediated activation of adenylyl cyclase. We have taken advantage of the different sensitivities of phospholipases A2 and C(s) to PMA, EGTA, and pertussis toxin to dissociate phospholipase A2 and C activities. Acute PMA-treatment enhanced PAF-stimulated PGE2 formation, reduced PAF-induced elevations in [Ca2+]i and had no effect upon PAF-stimulated 3H-PE. We have also demonstrated that phospholipase A2, but not PtdIns-specific phospholipase C, was sensitive to external calcium concentration. The role of a GTP-binding protein to couple PAF-receptors to the PtdIns-specific phospholipase C was confirmed as GTP gamma S synergistically elevated PAF-stimulated inositol phosphate formation. We also demonstrated that pertussis toxin ADP-ribosylates a single protein of an apparent 42 kD mass and that PAF pretreatment reduced subsequent ADP-ribosylation in a time-dependent manner. However, pertussis toxin had no effect upon phospholipase C-generated water soluble phosphorylbases or inositol phosphates. In contrast, PAF-stimulated phospholipase A2 and PAF-inhibited adenylyl cyclase activities were sensitive to pertussis toxin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endothelin-induced mobilization of Ca2+ and the possible involvement of platelet activating factor and thromboxane A2

Endothelin (ET) caused transient and sustained elevations of cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat and rabbit vascular smooth muscle cells (VSMC). Specific platelet activating factor (PAF) antagonists (CV-6209 and WEB-2086) and arachidonic acid (AA) cascade blockers (chlorpromazine, indomethacin, CV-4151 and AA-2414) potently inhibited the ET-induced increase in [Ca2+]i. Additionally, these compounds inhibited the PAF-induced increase in [Ca2+]i. These results suggest that PAF and thromboxane A2 (TXA2) may be involved in the mechanism of ET-induced mobilization of Ca2+ in cultured rat and rabbit VSMC.     

Platelet-activating factor induces matrix metalloproteinase-9 expression through Ca(2+)- or PI3K-dependent signaling pathway in a human vascular endothelial cell line

Platelet-activating factor (PAF) augments angiogenesis by promoting the synthesis of a variety of angiogenic factors, via the nuclear factor (NF)-kappaB activation. Recently, we reported that PAF upregulates MMP-9 expression in a NF-kappaB-dependent manner. In this study, we investigated the signaling pathway involved in PAF-induced MMP-9 expression in ECV304 cells. Our current data indicate that the Ca(2+)- or phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway is necessary for PAF-induced MMP-9 expression. Furthermore, PAF-induced NF-kappaB activation was blocked by selective inhibitors of Ca(2+), PI3K, or extracellular signal-regulated kinase (ERK). Our results suggest that PAF-induced MMP-9 expression, in a NF-kappaB-dependent manner, is regulated by Ca(2+), PI3K and ERK signaling pathways.     

Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells

The effect of prostaglandins (PG) on free cytosolic calcium concentrations [( Ca2+]i) and cAMP levels was studied in the osteosarcoma cell line UMR-106. PGF2 alpha and PGE2, but not 6-keto-PGF1 alpha, induced an increase in [Ca2+]i which was mainly due to Ca2+ release from intracellular stores. The EC50 for PGF2 alpha was approximately 7 nM, whereas that for PGE2 was approximately 1.8 microM. Maximal doses of PGF2 alpha increased [Ca2+]i to higher levels than PGE2. Both active PGs also stimulated phosphatidylinositol turnover in UMR-106 cells. The effects of the two PGs were independent of each other and appear to involve separate receptors for each PG. PGE2 was a very potent stimulator of cAMP production and increased cAMP by approximately 80-fold with an EC50 of 0.073 microM. PGF2 alpha was a very poor stimulator of cAMP production; 25 microM PGF2 alpha increased cAMP by 5-fold. The increase in cellular cAMP levels activated a plasma membrane Ca2+ channel which resulted in a secondary, slow increase in [Ca2+]i. High concentrations of both PGs (10-50 microM) inhibited this channel independent of their effect on cAMP levels. Pretreatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited the PG-mediated increase in phosphatidylinositol turnover and the increase in [Ca2+]i. However, pretreatment with 12-O-tetradecanoyl-13-acetate had no effect on the PGE2-mediated increase in cAMP. The latter finding, together with the dose responses for PGE2-mediated increases in [Ca2+]i and cAMP levels, suggests the presence of two subclasses of PGE2 receptors: one coupled to adenylate cyclase and the other to phospholipase C. With respect to osteoblast function, the cAMP signaling system is antiproliferative, whereas the Ca2+ messenger system, although having no proliferative effect by itself, tempers cAMP's antiproliferative effect.