Nonprostanoid prostacyclin mimetics. 2. 4,5-Diphenyloxazole derivatives.

4,5-Diphenyl-2-oxazolenonanoic acid (18b) was synthesized and found to inhibit ADP-induced aggregation of human platelets with an IC50 of 2.5 microM. Acid 18b displaced [3H]iloprost from human platelet membranes in a concentration-dependent fashion, consistent with 18b inhibiting platelet function by acting as a prostacyclin mimetic. By inserting a phenoxy ring into the side-chain moiety of 18b and systematically varying the pattern of substitution and length of the tethers, more potent inhibitors of platelet aggregation were identified. A phenoxy ring inserted centrally in the side chain proved to be the optimal arrangement but significant activity was observed when the aromatic ring was bound directly to the 2 position of the heterocycle. The meta-substituted cis-(ethenylphenoxy)acetic acid 37 is the most potent platelet aggregation inhibitor synthesized as part of this study with an IC50 of 0.18 microM. Acid 37 displaces [3H]iloprost from human platelet membranes with an IC50 of 6 nM. The trans-olefinic isomer of 37 (25p) is 72-fold weaker as an inhibitor of ADP-induced platelet aggregation, but the saturated derivative 25w (BMY 42393) is intermediate in potency. Structure-activity studies using 25w as a template focused on modification of the tethers intervening between the side-chain phenyl ring and the oxazole and carboxylate termini and substitution of the phenyl ring. These studies revealed that biological activity was sensitive to both the identity of the concatenating atoms and the pattern of ring substitution. The structure-activity relationships provide insight into the topographical relationship between the diphenylated oxazole ring and the carboxylic acid terminus that comprise the nonprostanoid prostacyclin mimetic pharmacophore.     

Octimibate, a potent non-prostanoid inhibitor of platelet aggregation, acts via the prostacyclin receptor.

1. Octimibate, 8-[(1,4,5-triphenyl-1H-imidazol-2-yl)oxy]octanoic acid, is reported to have antithrombotic properties. This is in addition to its antihyperlipidaemic effects which are due to inhibition of acylCoA:cholesterol acyltransferase (ACAT). The aim of this study was to investigate the mechanism of the antithrombotic effect of octimibate, and to determine whether the effects of octimibate are mediated through prostacyclin receptors. 2. In suspensions of washed (plasma-free) human platelets, octimibate is a potent inhibitor of aggregation; its IC50 is approx. 10 nM for inhibition of aggregation stimulated by several different agonists, including U46619 and ADP. The inhibitory effects of octimibate on aggregation are not competitive with the stimulatory agonist; the maximal response is suppressed but there is no obvious shift in potency of the agonist. In platelet-rich plasma, octimibate inhibits agonist-stimulated aggregation with an IC50 of approx. 200 nM. 3. Octimibate also inhibits agonist-stimulated rises in the cytosolic free calcium concentration, [Ca2+]i, in platelets. Both Ca2+ influx and release from intracellular stores are inhibited. The effects of octimibate on aggregation and [Ca2+]i are typical of agents that act via elevation of adenosine 3':5'-cyclic monophosphate (cyclic AMP). Similar effects are seen with forskolin, prostacyclin (PGl2) and iloprost (a stable PGl2 mimetic). 4. Octimibate increases cyclic AMP concentrations in platelets and increases the cyclic AMP-dependent protein kinase activity ratio. Octimibate stimulates adenylyl cyclase activity in human platelet membranes, with an EC50 of 200 nM. The maximal achievable activation of adenylyl cyclase by octimibate is 60% of that obtainable with iloprost.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor- and G-protein-regulated 150-kDa avian phospholipase C: inhibition of enzyme activity by isoenzyme-specific antisera and nonidentity with mammalian phospholipase C isoenzymes established by immunoreactivity and peptide sequence.

A 150-kDa phospholipase C previously was purified from turkey erythrocytes and shown to be a P2Y-purinergic receptor- and guanine nucleotide-binding protein-regulated enzyme [J. Biol. Chem. 265:13508-13514 (1990)]. The relationship of this enzyme to the 150-kDa mammalian phospholipase C isoenzymes, termed phospholipase C-beta and -gamma, has been examined. Four antisera to the turkey erythrocyte phospholipase C recognized the avian enzyme in immunoblots but failed to recognize phospholipase C-gamma; one of the these weakly recognized phospholipase C-beta. Antibodies to phospholipase C-beta and -gamma failed to recognize the turkey erythrocyte phospholipase C. However, two antibodies raised against peptide sequence in regions of conserved sequence common to mammalian phospholipase C isoenzymes recognized the 150-kDa turkey erythrocyte phospholipase C. Antisera against the native form of the turkey erythrocyte phospholipase C inhibited the activity of this enzyme against phosphatidylinositol 4,5-bisphosphate presented as a component of mixed phospholipid vesicles or of mixed phospholipid and sodium cholate micelles; inhibition occurred as a decrease in Vmax, with no apparent change in Km for substrate or in the Ca2+ dependence of phospholipase C activity. Catalytic activity of phospholipase C-beta or -gamma against exogenous substrate was unaffected by antisera to the turkey erythrocyte enzyme. Antisera against the native form of the turkey erythrocyte phospholipase C also partially inhibited (50-60% inhibition) the capacity of AIF4- or adenosine 5'-O-(beta-thio) diphosphate plus guanosine 5'-O-(gamma-thio) triphosphate to stimulate phosphoinositide hydrolysis in ghosts prepared from [3H]inositol-prelabeled turkey erythrocytes. Moreover, the capacity of the purified 150-kDa enzyme to reconstitute receptor and G-protein-regulated phospholipase C activity in purified turkey erythrocyte plasma membranes devoid of this activity was completely inhibited by antisera to the turkey erythrocyte enzyme. Five peptides that were purified by high performance liquid chromatography from a tryptic digest of the turkey erythrocyte 150-kDa phospholipase C had no recognizable sequence homology with any deduced sequence of the mammalian phospholipase C isoenzymes. One turkey erythrocyte phospholipase C-derived peptide had clear homology with sequence in the first (X-domain) conserved region common to at least three of the mammalian phospholipase C isoenzymes, and another 16-amino acid peptide had partial sequence homology with the second (Y-domain) conserved region common to the mammalian enzymes. An 8-amino acid peptide from the tryptic digest had 75% homology with a sequence near the carboxyl terminus of mammalian phospholipase C-beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nonprostanoid prostacyclin mimetics. 3. Structural variations of the diphenyl heterocycle moiety.

4,5-Diphenyl-2-oxazolenonanoic acid (2) and 2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid (3) were previously identified as nonprostanoid prostacyclin (PGI2) mimetics that inhibit ADP-induced aggregation of human platelets in vitro. The effects on biological activity of substitution and structural modification of the 4- and 5-phenyl rings of 3 was examined. Potency showed a marked sensitivity to the introduction of substituents to these aromatic rings and only the bis-4-methyl derivative 9j, IC50 = 0.34 microM, demonstrated enhanced potency compared to the parent structure 3, IC50 = 1.2 microM. Substitution at the ortho or meta positions of the phenyl rings, replacement by thiopheneyl or cyclohexyl moieties, or constraining in a planar phenanthrene system resulted in compounds that were less effective inhibitors of ADP-induced platelet aggregation. In contrast, variation of the heterocycle moiety revealed a much less stringent SAR and many 5- and 6-membered heterocycles were found to effectively substitute for the oxazole ring of 2 and 3. The diphenylmethyl moiety functioned as an effective isostere for 4,5-diphenylated heterocycles since 13aad showed similar platelet inhibitory activity to 3. With the exception of the 3,4,5-triphenylpyrazole derivative 13g, compounds presenting the (m-ethylphenoxy)acetic acid side chain discovered with 3 demonstrated enhanced potency compared to the analogously substituted alkanoic acid derivative. The structure-activity findings led to a refinement of a model of the nonprostanoid PGI2 mimetic pharmacophore.     

Prostaglandin endoperoxide analogues which are both thromboxane receptor antagonists and prostacyclin mimetics.

Two prostaglandin endoperoxide analogues, EP 035 and EP 157, behave as specific thromboxane receptor antagonists on isolated smooth muscle preparations such as rabbit aorta, dog saphenous vein and guinea-pig trachea. However, in human platelet-rich plasma (PRP) they produce an unsurmountable block of aggregation induced by a wide range of agents (ADP, platelet-activating factor, thrombin); this inhibitory profile is typical of that seen with either prostaglandin I2 (PGI2) or PGD2. EP 035 and EP 157 induce large increases in cyclic AMP levels (up to 20 times basal) in human PRP. Simultaneous exposure to PGE1 markedly reduces their effect on cyclic AMP; exposure to PGD2 is much less effective in this respect. The adenylate cyclase inhibitor SQ 22,536 opposes the inhibitory action of EP 035, EP 157, iloprost (a stable PGI2 analogue) and PGD2 on platelet aggregation. However, the xanthone derivative AH 6809 blocks the inhibitory action of PGD2 but does not affect EP 035, EP 157 and PGI2 and its structural analogues. EP 035 and EP 157 displace [3H]-iloprost binding to the PGI2 receptor on human platelet membranes. Displacing ability is ranked as follows: iloprost greater than 6a-carba PGI2 greater than EP 157 greater than EP 035 greater than EP 164 (alpha-dinor derivative of EP 157). This order of potency is the same as that found for activation of adenylate cyclase in homogenates of washed human platelets and for inhibition of aggregation in washed human platelets. The activities of EP 035 and EP 157 were studied in two other systems containing PGI2 receptor-adenylate cyclase complexes, the NCB-20 cell line and human lung tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of inhibition of phospholipase activity of crotoxin B by crotoxin A

In the crotoxin complex isolated from Crotalus durissus terrificus venom, the component A inhibits the phospholipase A₂ activity of crotoxin B only when the substrate is in the aggregated form, preventing the interaction of the enzyme with lecithin-water interfaces. In contrast, with similar rates of hydrolysis of dihexanoyllecithin monomers, the activity of the crotoxin complex is lower than that of crotoxin B when the substrate is aggregated into micelles. Crotoxin B readily hydrolyses dimyristoyllecithin vesicles, the rate being modulated by the physical state of the phospholipid, suggesting that the enzyme is tightly bound to the interface. With the crotoxin complex the rate of vesicle hydrolysis is much slower (about $\text{1}\text{10}$ that of crotoxin B) and is little affected by the physical state of the lecithin. Direct binding experiments demonstrate that, in contrast to crotoxin B, the crotoxin complex is unable to interact with lecithin-water interfaces. Together with the free accessibility of the enzyme active site in the crotoxin complex, this evidence suggests that a specific area on the enzyme surface, different from the active site and shielded by crotoxin A in the complex, is responsible for the interaction of crotoxin B with lipid-water interfaces.     

Lindane stimulates neutrophils by selectively activating phospholipase C and phosphoinositide-kinase activity

The organochlorine insecticide lindane is a known activator of neutrophil responses. In this work we delineated the biochemical pathways by which lindane stimulates neutrophil oxidant production. Plasma membrane GTPase activity was not stimulated by lindane, ruling out a role for lindane-induced activation of G-proteins or G-protein coupled receptors, whereas inhibition of phospholipase C inhibited lindane-induced oxidant production. Together these data pointed to phospholipase C as the direct target of lindane activation. Type I phosphoinositide 3-kinase was not significantly activated by lindane and an inhibitor of phosphoinositide 3-kinases inhibited oxidant production by only 40%. Thus, Type I phosphoinositide 3-kinase played a minor role, if any, in lindane-induced oxidant production. Lindane stimulated an increase in phosphatidylinositol phosphate suggesting a Type II or III phosphotidylinositol 3-kinase or phosphatidylinositol 4-kinase activity was also stimulated.     

Protein kinase C-promoted inhibition of Galpha(11)-stimulated phospholipase C-beta activity.

The effects of protein kinase C (PKC) activation on inositol lipid signaling were examined. Using the turkey erythrocyte model of receptor-regulated phosphoinositide hydrolysis, we developed a membrane reconstitution assay to study directly the effects of activation of PKC on the activities of Galpha(11), independent of potential effects on the receptor or on PLC-beta. Membranes isolated from erythrocytes pretreated with 4beta-phorbol-12beta-myristate-13alpha-acetate (PMA) exhibited a decreased capacity for Galpha(11)-mediated activation of purified, reconstituted PLC-beta1. This inhibitory effect was dependent on both the time and concentration of PMA incubation and occurred as a decrease in the efficacy of GTPgammaS for activation of PLC-beta1, both in the presence and absence of agonist; no change in the apparent affinity for the guanine nucleotide occurred. Similar inhibitory effects were observed after treatment with the PKC activator phorbol-12,13-dibutyrate but not after treatment with an inactive phorbol ester. The inhibitory effects of PMA were prevented by coaddition of the PKC inhibitor bisindolylmaleimide. Although the effects of PKC could be localized to the membrane, no phosphorylation of Galpha(11) occurred either in vitro in the presence of purified PKC or in intact erythrocytes after PMA treatment. These results support the hypothesis that a signaling protein other than Galpha(11) is the target for PKC and that PKC-promoted phosphorylation of this protein results in a phosphorylation-dependent suppression of Galpha(11)-mediated PLC-beta1 activation.     

Characterisation of human recombinant somatostatin receptors. 4. Modulation of phospholipase C activity

Total [3H]phosphoinositide (IPx) accumulation, a measure of phospholipase C (PLC) activity, induced by somatostatin (somatotropin release-inhibiting factor, SRIF) and cortistatin (CST) analogues was studied at human somatostatin receptor subtypes 1-5 (hsst1-5) recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. SRIF14 (10 microM) stimulated total [3H]-IPx production 200% and 1070% over basal levels, and increased intracellular Ca2+ ([Ca2+]i) 1600% and 2790%, in cells expressing hsst3 and hsst5 receptors, respectively. The SRIF14-stimulated IPx production was partly blocked by 100 ng/ml pertussis toxin (PTX) (30% and 15% inhibition, respectively). At hsst1, hsst2, and hsst4 receptors, only weak or no stimulation of PLC activity was found (Emax = 114%, 122%, and 102%, respectively). Consequently, hsst3 and hsst5 receptors were subjected to more detailed studies to establish pharmacological profiles of PLC stimulation. At hsst3 receptors, the relative efficacies of most ligands were in the same range (maximum response Emax = 218-267%). At hsst5 receptors Emax varied over a broad range, seglitide, CST17, SRIF28 displaying almost full agonism compared to SRIF14, whereas octreotide and BIM 23052 showed very low partial agonism. BIM 23056 behaved as an antagonist on SRIF14-induced total [3H]-IPx accumulation with a pKB (negative logarithm of antagonist binding constant) of 6.74 at hsst3 receptors, and of 6.94 at hsst5 receptors. The putative cycloantagonist SA showed weak antagonist activity on SRIF14-induced total [3H]-IPx levels at hsst3 (pKB = 5.85), but not at hsst5 receptors. The [3H]-IPx accumulation profiles at sst3/sst5 receptors were compared to their respective radioligand binding ([125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide binding), to [35S]GTPgammaS binding, and to forskolin-stimulated adenylate cyclase (FSAC) inhibition profiles determined previously in CCL39 cells. The different affinity profiles correlated relatively well at both receptor subtypes with PLC activation (sst3: r = 0.90-0.97; sst5: r = 0.80-0.87). However, [35S]GTPgammaS binding correlated only minimally with stimulation of [3H]-IPx levels at sst5 receptors (r = 0.59), but rather well at sst3 receptors (r = 0.80). A moderate correlation was also observed between inhibition of FSAC activity and stimulation of PLC activity for hsst3 and hsst5 receptors with correlation coefficients of 0.85 and 0.70, respectively. In summary, most SRIF analogues behave as full agonists at hsst3 receptors and agonist-induced phosphoinositide turnover correlates well with radioligand binding, [35S]GTPgammaS binding and inhibition of adenylate cyclase activity, all measured in CCL39 cells. By contrast, at hsst5 receptors, most SRIF analogues behave as intermediate or very low partial agonists (although receptor levels are comparatively high, 7000 vs. 400 fmol/mg), and the agonist-induced phosphoinositide turnover correlates rather poorly with radioligand binding, [35S]GTPgammaS binding or inhibition of adenylate cyclase activity, all measured in the same cell line. Agonist-induced phosphoinositide turnover, [35S]GTPgammaS binding and inhibition of adenylate cyclase activity, show differences both in the rank orders of potency and relative efficacy at hsst3 and markedly at hsst5 receptors, suggesting either that PLC activity is functionally irrelevant or, more probably, that agonist-dependent receptor trafficking is taking place in CCL39 cells.     

The antiplatelet activity of Geiji-Bokryung-Hwan,Korean traditional formulation,is mediated through inhibition of phospholipase C and inhibition of TxB(2) synthetase activity

Geiji-Bokryung-Hwan (GBH), consisting of herbes of Cinnamomi ramulus (Geiji), Poria cocos (Bokryun), Mountan cortex radicis (Mokdanpi), Paeoniae radix (Jakyak), and Persicae semen (Doin), on antiplatelet activity in human platelet suspensions was studied. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes and GBH (15 and 30 microg/ml) significantly inhibited [3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. GBH did not significantly affect nitrate production in collagen (10 microg/ml)-induced human platelet aggregation. Various concentrations of GBH (0, 5, 10, 15, and 30 microg/ml) dose-dependently inhibited [3H]inositol monophosphate formation stimulated by collagen (10 microg/ml) in [3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 min). These results indicated that the antiplatelet activity of GBH may possibly be due to the inhibition of phospholipase C (PLC) activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A(2) formation, and then inhibition of [Ca(2+)](i) mobilization of platelet aggregation stimulated by agonists. In conclusion, GBH suppressed PLC in a dose-dependent manner, and may have pharmaceutical applications. These data suggest that GBH extracts merit investigation as a potential anti-atherosclerogenic agent in humans.     

Selective inhibition by minoxidil of prostacyclin production by cells in culture

The effect of minoxidil on arachidonic acid metabolism by cells in culture was studied. In bovine aorta endothelial cells, treatment with minoxidil in the presence of various stimulators of arachidonic acid metabolism was accompanied by a dose-dependent inhibition of prostacyclin production (measured as 6-keto-prostaglandin F1 alpha). Synthesis of the other cyclooxygenase products (prostaglandins E2, F2 alpha and thromboxane) was not inhibited. When the bovine aorta endothelial cells were stimulated by the Ca2+ ionophore A-23187, the inhibition was seen as early as 2 min. Minoxidil also inhibited prostacyclin production by a second cell line of bovine aorta endothelial cells (the established CPAE cell line), bovine aorta smooth muscle cells, porcine aorta endothelial cells, and rat liver cells (the C-9 cell line)--the latter, less effectively. Again, formation of all the other cyclooxygenase products studied was not inhibited. Minoxidil did not affect significantly prostaglandin E2 and F2 alpha production by newborn rat keratinocytes (the NBR cell line)--a cell that does not produce PGI2. The clinical, biochemical, and pharmacologic implications are discussed.     

Recovery of prostacyclin synthesis by rabbit aortic endothelium and other tissues after inhibition by aspirin.

The effect of aspirin on prostacyclin (PGI2) and thromboxane B2 (TXB2) synthesis was studied in rabbits. Tissues were removed from animals killed at intervals after injection of aspirin, and incubated with Hanks' solution. PGI2 synthesis was monitored by radioimmunoassay of its hydrolysis product, 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha). TXB2 production in clotted blood, also measured by radioimmunoassay, was determined as an index of platelet cyclo-oxygenase activity. 6-oxo-PGF1 alpha and TXB2 production 0.5 h after aspirin were similarly inhibited to less than 5% of control in all incubations. Subsequent recovery of PGI2 synthesis occurred more rapidly in aortic endothelium than in other tissues, including aorta denuded of endothelium. Recovery of TXB2 production was slower than that of PGI2. Intravenous cycloheximide prevented the partial recovery of PGI2 synthesis that otherwise occurred 6 h after aspirin, while intravenous epidermal growth factor increased recovery. It is concluded that in the rabbit, cyclo-oxygenase is synthesized more rapidly in aortic endothelium than in deep layers of aorta, or in the other tissues studied.     

Reactive blue 2 inhibition of cyclic AMP-dependent differentiation of rat C6 glioma cells by purinergic receptor-independent inactivation of phosphatidylinositol 3-kinase

Cyclic AMP-dependent differentiation of rat C6 glioma cells into an astrocyte type II is characterized by inhibition of cell growth and induction of glial fibrillary acidic protein (GFAP) synthesis. Activation of the P2Y(12) receptor with 2-methylthioadenosine-5'-diphosphate inhibited beta-adrenergic receptor-induced differentiation. The selective P2Y(12) receptor antagonist N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene ATP abolished the receptor-mediated effect on differentiation. In contrast non-selective antagonists of P2Y receptors did not revert the inhibiting effect of the P2Y(12) receptor on differentiation. Reactive blue 2 (RB2), a potent P2Y(12) receptor antagonist, completely inhibited the synthesis of GFAP, while the P2Y receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid were less efficient. However, although P2Y receptor antagonists inhibited GFAP synthesis to a different extent they were unable to relieve the growth inhibition that accompanied induction of differentiation, whereas stimulation of the P2Y(12) receptor with 2-methylthioadenosine-5'-diphosphate inhibited GFAP expression and restored cell proliferation. Assay of the activity of phosphatidylinositol 3-kinase (PI 3-K), an enzyme required for GFAP expression [J. Neurochem. 76 (2001) 610], showed that RB2 inhibited this enzyme after cellular uptake, while suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid inhibited PI 3-K to a lesser extent. The intracellular concentration of RB2 increased in time and attained the ic(50) for PI 3-K inhibition (4microM) after 40-min incubation with 50microM RB2. In conclusion, cAMP-induced differentiation in C6 cells is inhibited by activation of the P2Y(12) receptor. In addition, synthesis of GFAP is also inhibited by cellular uptake of non-selective nucleotide receptor antagonists that inhibit PI 3-K, a kinase required for the cAMP-dependent induction of differentiation.     

Identification of a compound that directly stimulates phospholipase C activity

Phosphoinositide-specific phospholipase C (PLC) plays a pivotal role in the signal transduction of various cellular responses. However, although it is undeniably important that modulators of PLC activity be identified, no direct PLC activity modulator has been identified until now. In this study, by screening more than 10,000 different compounds in human neutrophils, we identified a compound that strongly enhances superoxide-generating activity, which is well known to be PLC-dependent. The active compound 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS) stimulated a transient intracellular calcium concentration ([Ca(2+)](i)) increase in neutrophils. Moreover, m-3M3FBS stimulated the formation of inositol phosphates in U937 cells, indicating that it stimulates PLC activity. The compound showed no cell-type specificity in terms of [Ca(2+)](i) increase in the various cell lines including leukocytes, fibroblasts, and neuronal cells. We also ruled out the possible involvement of heterotrimeric G proteins in m-3M3FBS-stimulated signaling by confirming the following: 1) pertussis toxin does not inhibit m-3M3FBS-induced [Ca(2+)](i) increase; 2) m-3M3FBS does not stimulate cyclic AMP generation; and 3) the inhibition of G(q) by the regulator of G protein-signaling 2 does not affect the m-3M3FBS-induced [Ca(2+)](i) increase. We also observed that m-3M3FBS stimulated PLC activity in vitro. The purified isoforms of PLC that were tested (i.e., beta2, beta3, gamma1, gamma2, and delta1) were activated by m-3M3FBS and showed no isoform specificity. Taken together, these results demonstrate that m-3M3FBS modulates neutrophil functions by directly activating PLC. Because m-3M3FBS is the first compound known to directly activate PLC, it should prove useful in the study of the basic molecular mechanisms of PLC activation and PLC-mediated cell signaling.     

Hormone and growth factor receptor-mediated regulation of phospholipase C activity

The broad importance of receptor-activated phosphoinositide hydrolysis in the physiological action of hormones, neurotransmitters and growth factors has sparked interest in the study of transmembrane signalling events responsible for activation of phospholipase C. As with receptors involved in regulation of adenylyl cyclase, ion channels and phototransmission, it is clear that a guanine nucleotide regulatory protein (G protein) is a necessary component of the hormone- and neurotransmitter-regulated phosphoinositide signalling mechanism. Recent evidence to support a possible second mode of regulation of phospholipase C by growth factor receptors is emerging in the form of realization that at least one isozyme of phospholipase C serves as a substrate for the tyrosine kinase activity of growth factor receptors known to stimulate phosphoinositide hydrolysis. In this review, José Boyer and colleagues summarize progress towards delineating the properties and identity of the G protein(s) involved in this pathway, recent advances in purification and molecular cloning of phospholipase C isozymes, and the current understanding of growth factor receptor-mediated regulation of phosphoinositide hydrolysis.     

Competitive regulation of phospholipase C responses by cAMP and calcium.

DDT1-MF2 smooth muscle cells demonstrated a robust phospholipase C response to norepinephrine, as detected by inositol phosphate accumulation. A selective A1-adenosine receptor agonist, cyclopentyladenosine, caused only a minor stimulation of phospholipase C, which was eliminated in the absence of added extracellular calcium. The simultaneous addition of norepinephrine and cyclopentyladenosine resulted in a synergistic increase in phosphoinositide hydrolysis either in the absence or in the presence of external calcium. In the presence of external calcium and a calcium ionophore, and adenosine agonist caused a significant stimulation of phosphoinositide hydrolysis without the addition of norepinephrine. Influx of extracellular calcium through voltage-sensitive calcium channels did not appear to be required to observe an effect of cyclopentyladenosine, because neither calcium channel antagonists (nifedipine, verapamil, and LaCl3) nor a chelator of extracellular calcium (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) were able to alter the degree of potentiation of norepinephrine-stimulated phosphoinositide hydrolysis due to the adenosine agonist. On the other hand, buffering of intracellular calcium concentration with the membrane-permeant calcium chelator quin2 blocked the potentiation. This blockade of potentiation by quin2 was reversed by the addition of extracellular calcium. Agents that stimulated cAMP production or membrane-permeable analogues of cAMP also blocked the action of the adenosine agonist to potentiate norepinephrine-stimulated phosphoinositide hydrolysis. This effect of cAMP was less pronounced in the presence of elevated extracellular calcium and was abolished in the presence of a calcium ionophore. When norepinephrine-stimulated calcium transients were quantitated using fura-2 fluorescence, a reduction in the amplitude of the calcium response was observed in the presence of forskolin. Conversely, both the amplitude and the duration of the calcium response were enhanced by the addition of the adenosine agonist. The results of these studies suggest that the mechanism by which adenosine receptors enhance the stimulation of phosphoinositide hydrolysis is dependent upon a rise in intracellular Ca2+ concentration resulting from the simultaneous activation of alpha 1-adrenergic receptors. The results further suggest that cAMP inhibits this mechanism by decreasing the norepinephrine-stimulated rise in intracellular Ca2+ concentration.