Presence Of Quinacrine Research Articles

Quinacrine Inhibits ICAM-1 Transcription by Blocking DNA Binding of the NF-κB Subunit p65 and Sensitizes Human Lung Adenocarcinoma A549 Cells to TNF-α and the Fas Ligand.

Quinacrine has been used for therapeutic drugs in some clinical settings. In the present study, we demonstrated that quinacrine decreased the expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor (TNF)-α and interleukin-1 (IL-1) α in human lung adenocarcinoma A549 cells. Quinacrine inhibited ICAM-1 mRNA expression and nuclear factor κB (NF-κB)-responsive luciferase reporter activity following a treatment with TNF-α and IL-1α. In the NF-κB signaling pathway, quinacrine did not markedly affect the TNF-α-induced degradation of the inhibitor of NF-κB or the TNF-α-induced phosphorylation of the NF-κB subunit, p65, at Ser-536 and its subsequent translocation to the nucleus. In contrast, a chromatin immunoprecipitation assay showed that quinacrine prevented the binding of p65 to the ICAM-1 promoter following TNF-α stimulation. Moreover, TNF-α and the Fas ligand effectively reduced the viability of A549 cells in the presence of quinacrine only. Quinacrine down-regulated the constitutive and TNF-α-induced expression of c-FLIP and Mcl-1 in A549 cells. These results revealed that quinacrine inhibits ICAM-1 transcription by blocking the DNA binding of p65 and sensitizes A549 cells to TNF-α and the Fas ligand.

Interaction of local anaesthetics with histamine H1 receptors in guinea-pig ileum.

The interaction of amine local anaesthetics and related compounds with histamine H1 receptors was investigated in guinea-pig ileal longitudinal muscle. Quinacrine, chloroquine, tetracaine and procaine inhibited [3H]mepyramine binding to solubilized membrane from ileal muscle with pKi values of 5.27 +/- 0.11, 5.66 +/- 0.01, 4.28 +/- 0.08 and 3.97 +/- 0.11, respectively. The pKB values obtained from the initial parallel shift of the dose-response curves for histamine in the presence of these drugs were 5.49 +/- 0.11, 6.14 +/- 0.09, 4.86 +/- 0.06 and 4.58 +/- 0.06, respectively, in reasonable agreement with the pKi values. The combined dose-ratio test with both local anaesthetics and antagonist (mepyramine) present showed that tetracaine and procaine were competitive and chloroquine was partially competitive, but that quinacrine was not competitive at histamine H1 receptors. These local anaesthetics inhibited histamine-induced desensitization in guinea-pig ileum. Receptor occupancy (%) by agonist decreased from 95.2 (without inhibitor) to 73.9, 42.8, 35.9 and 33.9 in the presence of quinacrine, chloroquine, tetracaine or procaine, respectively, under the conditions where each inhibitor drug induced half maximum inhibition of desensitization. The results suggested that most of these local anaesthetics interacted competitively at histamine H1 receptors and inhibited desensitization through their antagonizing actions, whereas quinacrine interacted allosterically and inhibited desensitization through a separate action.

Voltammetric studies of the interaction of quinacrine with DNA.

The binding interaction of the antimalarial drug quinacrine with herring sperm deoxyribonucleic acid (DNA) has been studied by square wave voltammetry. The binding parameters, the binding constant K and the binding site size s, were obtained simultaneously by the analysis of bound and free quinacrine concentration corresponding to the limits of slow and fast binding kinetics compared to the experimental timescale. The binding constant and the binding site size for the interaction of quinacrine with DNA were K = 1.59 (+/-0.18)x10(5) M(-1) and s = 7.1 (+/-0.15) base pairs and K = 7.35 (+/-0.83)x10(5) M(-1), s = 6.2 (+/-0.02) base pairs for the limiting conditions of static and mobile binding equilibrium respectively. The standard Gibbs free energy change (Delta G0 = - RT ln K) is approximately -29.67 kJ/mol at 25 degrees C, which highlights the spontaneity of the binding of quinacrine with DNA. The binding of quinacrine to herring sperm DNA results in peak potential shifts in voltammetric and a red shift in UV-absorption measurements. The ionic strength dependence of the binding constant is not large. Furthermore, the relative viscosity of DNA increases in the presence of quinacrine. These characteristics strongly support the intercalation of quinacrine into DNA. The results also show that the intercalation of quinacrine into DNA may occur at approximately every seventh base pair.

Quinacrine increases endothelial nitric oxide release: role of superoxide anion

The effect of acute quinacrine treatment on agonist-induced nitric oxide (NO) release was investigated in cultured human endothelial cells using electrochemical monitoring of the in situ NO concentration. Quinacrine dose-dependently increased NO release with an apparent EC 50 of 0.2 μM and a maximal effect at 1 μM. Quinacrine did not modify the dependence of NO release on extracellular l-arginine. Acceleration or deceleration of O 2 − dismutation, which altered NO release in control cells, did not modify it in quinacrine-treated cells. Quinacrine did not modify NO amperometric signal or reaction with O 2 − produced by xanthine oxidation. In the presence of quinacrine, agonist-induced NO release became Mg 2+-independent and could not be attributed to an inhibition of phospholipase A 2 activity. Quinacrine made NO release insensitive to Cu 2+ chelation. The present study demonstrates that acute treatment by low quinacrine concentrations increases endothelial NO release, possibly through an inhibition of O 2 − production.

The noncompetitive inhibitor quinacrine modifies the desensitization kinetics of muscle acetylcholine receptors.

Quinacrine has been shown to act as a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAChR). However, its mechanism of action is still a matter of controversy. We analyzed in detail the action of quinacrine at both the single-channel and macroscopic current levels. The main effect of quinacrine is a profound concentration-dependent decrease in both the frequency of opening events and the duration of clusters elicited by high acetylcholine concentrations. Quinacrine also significantly increases (40-fold at 30 microM) the decay rate of macroscopic currents elicited by rapid perfusion of acetylcholine to outside-out patches. This decay is still well-described by a single exponential. Quinacrine has very little effect on the peak amplitude of the response, suggesting that it acts mainly on open channels. The recovery from desensitization after removal of acetylcholine is delayed in the presence of quinacrine. Results from both single-channel and macroscopic current recordings indicate that quinacrine increases the rate of nAChR desensitization and stabilizes the desensitized state. Interestingly, in equilibrium agonist-binding assays, quinacrine does not promote the typical high-affinity desensitized state. Thus, quinacrine seems to induce an intermediate state exhibiting the permeability but not the agonist binding properties of desensitization.

Effect of quinacrine treatment on the activity of acid hydrolases (phosphatase, N-acetyl-beta-glucosaminidase, glucosidase, galactosidase and esterase) in Tetrahymena.

The effect of phospholipase A2 (PLA2) inhibitor, quinacrine, on the activity of hydrolytic enzymes in Tetrahymena pyriformis homogenate, was investigated. The activity of all of the enzymes studied (acid phosphatase, N-acetyl-beta-glusosaminidase, glucosidase, galactosidase and esterase) was significantly reduced in the presence of quinacrine. Since there are no data on the inhibitory effect of PLA2 and PLA2 influenced metabolic pathways to the hydrolytic enzymes, the direct effect of quinacrine on the hydrolytic enzymes (of Tetrahymena) can be supposed. This is supported by the fact that the other PLA2 inhibitor, 4-bromophenacyl bromide, did not influence phosphatase activity.

Binding of quinacrine to acidic phospholipids and pancreatic phospholipase A2. Effects on the catalytic activity of the enzyme.

Binding of quinacrine to phospholipids and porcine pancreatic phospholipase A2 (PLA2) was investigated using fluorescence resonance energy transfer, Langmuir films, assay for the enzymatic activity, and molecular modeling. No significant binding of this drug to the zwitterionic phosphatidylcholine was observed whereas a high affinity for acidic phospholipids was revealed by quenching of pyrene-labeled phospholipid analogues. Partial reversal of this binding was observed due to the addition of 4 mM CaCl2. Quinacrine efficiently and independently of the lipid surface pressure penetrated into monolayers of phosphatidylglycerol while only a weak penetration into phosphatidylcholine films was evident. Quinacrine also bound to eosin-labeled PLA2, and the addition of 4 mM CaCl2 reversed this interaction almost completely. In the presence of acidic phospholipids both the drug and the enzyme were attached to the lipid surface. Studies on the influence of quinacrine on the activity of PLA2 toward pyrene-labeled phospholipid analogues revealed that the hydrolysis of phosphatidylcholine was progressively reduced as a function of increasing [quinacrine]. At low [CaCl2] and low quinacrine:lipid molar ratios (<1:5) quinacrine enhanced slightly the rate of hydrolysis of acidic phospholipids whereas at higher drug:lipid molar ratios (>1:2) an inhibition was observed. In the presence of 1 mM CaCl2 quinacrine inhibited PLA2-catalyzed hydrolysis of phosphatidylglycerol only when the drug:lipid molar ratio exceeded 1:1. The presence of 4 mM CaCl2 abolished nearly completely the inhibition with all the substrate analogues used. Our data suggest that the inhibition of PLA2 by quinacrine is due to its binding to the enzyme. This is supported also by molecular modeling which suggested a binding site for quinacrine close to the active site and Ca2+ binding site of the enzyme. Importantly, our data indicate that quinacrine binds avidly to acidic phospholipids and their presence may influence the drug-enzyme interaction and the inhibition of the enzyme action. Accordingly, presence of quinacrine may interfere also with other processes that require the presence of acidic lipids and/or Ca2+, such as the function of the nicotinic acetylcholine receptor.

Evidence against a role of cytochrome P450-derived arachidonic acid metabolites in endothelium-dependent hyperpolarization by acetylcholine in rat isolated mesenteric artery.

1. In rat mesenteric artery, acetylcholine (ACh) causes endothelium-dependent hyperpolarization by releasing endothelium-derived hyperpolarizing factor (EDHF). Recent evidence suggests that EDHF may be a cytochrome P450-derived arachidonic acid metabolite. The aim of the present study was to investigate whether such a metabolite is indeed contributing to ACh-induced hyperpolarization observed in rat mesenteric artery. 2. The phospholipase A2 inhibitor quinacrine (30 microM) nearly completely eliminated ACh-induced hyperpolarization. However, the hyperpolarizing effect of pinacidil was also abolished in the presence of quinacrine. 3. The imidazole antimycotic agents ketoconazole (50 microM), clotrimazole (30 microM) and miconazole (10 microM), which bind to the heme moiety of cytochrome P450, eliminated not only ACh-induced hyperpolarizations but also those induced by pinacidil. SKF525A (30 microM), a prototype inhibitor of the enzyme, also abolished the hyperpolarizing responses to both agents. In contrast, neither 17-octadecynoic acid (10 microM), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, nor eicosatetraynoic acid (20 microM), an inhibitor of all arachidonic acid metabolic pathways, altered ACh-induced hyperpolarization. Furthermore, the hyperpolarization was unaffected by the preferential inhibitors of specific cytochrome P450 isozymes, alpha-naphtoflavone (1 microM), diedthyldithiocarbamate (50 microM), metyrapone (20 microM) and troleandomycin (10 microM). 4. Pretreatment of rats with lipopolysaccharide (2 mg kg-1) and exposure to nitroprusside (10 microM), both of which are expected to inhibit cytochrome P450 activity due to nitric oxide overproduction, were without effect on ACh-induced hyperpolarization. Pretreatment of rats for 3 days with pentobarbitone (80 mg kg-1 day-1), a cytochrome P450 inducer, also did not affect the hyperpolarizing response to ACh. 5. Arachidonic acid in concentrations up to 100 microM had no detectable effect on smooth muscle membrane potential. 11, 12-Epoxyeicosatrienoic acid (EET, 10 microM), one of cytochrome P450-derived epoxygenase metabolites of arachidonic acid, elicited a small endothelium-independent membrane hyperpolarization. The hyperpolarizing response to EET was blocked by glibenclamide (30 microM), in contrast to the response to ACh. 6. These results suggest that the contribution of a cytochrome P450-derived metabolite of arachidonic acid to ACh-induced hyperpolarization via EDHF release is minimal or absent in rat mesenteric artery.

Ca(2+)-independent fusion of synaptic vesicles with phospholipase A2-treated presynaptic membranes in vitro.

To clarify the mechanism of exocytosis in neurotransmitter release, the fusion of synaptic vesicles with presynaptic membranes prepared from rat brain synaptosomes and concomitant acetylcholine (ACh) release induced by fusion of them were studied in vitro. Fusion of the synaptic vesicles with presynaptic membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B. Synaptic vesicles fused with presynaptic membranes which had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ and released ACh, whereas synaptic vesicles did not interact with non-pretreated membranes. The fusion followed by ACh release depended (i) on the activity of PLA2 during the membrane pretreatment, (ii) on the amount of pretreated membrane and (iii) on the duration of the pretreatment. The presence of Ca2+ ions during the pretreatment was essential for inducing a fusogenic activity of the membranes, but Ca2+ ions were not required for the fusion itself because the fusion experiment was carried out in the presence of 5mM EGTA without added Ca2+. The presence of quinacrine, an antagonist of PLA2, during the membrane pretreatment inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Presence of albumin during the pretreatment, which is an adsorbent of free fatty acids, also inhibited the fusogenic activity. Arachidonic acid, when added during the pretreatment, potentiated the fusogenic activity of the membrane. These findings suggest that the conformational change in the presynaptic membrane phospholipids induced by PLA2 and the presence of arachidonic acid produced by PLA2 are important in the process of fusion of synaptic vesicles with the presynaptic membranes of rat brain, and that the fusion process itself is independent of Ca2+.

Enhancement of stress-induced synthesis of stress proteins by mastoparan in C6 rat glioma cells.

The levels of two small stress proteins, hsp27 and alpha B crystallin, were low in C6 glioma cells confluency. However, the levels of the two proteins increased after exposure of cells to heat (42 degrees C for 30 min) or arsenite (50-100 microM for 1 h) stress. When cells were exposed to arsenite or heat in the presence of mastoparan, a peptide toxin from wasp venom, the induction of hsp27 and alpha B crystallin was markedly stimulated, as detected by means of specific immunoassays, Western blot analysis, and Northern blot analysis. The response of hsp70 to each stress was also enhanced in the presence of mastoparan. Treatment of cells with 40 microM mastoparan alone barely induced the accumulation of hsp27 and alpha B crystallin. The stimulatory effect of mastoparan was little affected in cells that had been treated with pertussis toxin, but it was strongly suppressed in the presence of quinacrine, an inhibitor of phospholipase A2. These results suggest that mastoparan, which is an activator of phospholipase A2, enhances the responses to stress of hsp27, alpha B crystallin and hsp70 by increasing the metabolic activity of the arachidonic acid cascade.

The Role of Plasma Membrane Oxidoreductase Activity in Proton Transport

Summary Inside-out plasma membrane vesicles were obtained after partition of the freeze/thawed right-side out vesicles from cucumber roots in a two phase system. A sidedness of the plasma membrane vesicles was determined by the latency of vanadate-sensitive ATPase and trypsin digestion of the enzyme. In experiments with ferricyanide-loaded vesicles a low transplasma membrane electron transport with NADH as an electron donor was generated. However, introduction of Triton X-100 into the reaction medium increased the rate of NADH oxidation several times. During incubation with ATP of both unloaded vesicles and vesicles loaded with ferricyanide, the decrease in acridine orange absorbance was observed. DCCD and sodium orthovanadate added to the reaction mixture diminished distinctly the changes of acridine absorbance. These results indicate that in vesicles the ATP-dependent H + pump is active. Incubation of the ferricyanide-loaded vesicles with NADH caused a small increase of acridine absorbance that was deteriorated in the presence of quinacrine. The contribution of the plasma membrane NADH-ferricyanide oxidoreductase in the H + transport is discussed.

Modulation of NMDA receptor-mediated release of [3H]arachidonate in hippocampal slices of immature rats.

Using superfusion with albumin-containing medium of hippocampal and striatal slices of adult and developing rats at postnatal days (PND) 7-10, prelabelled with [3H]arachidonic acid ([3H]AA), we detected N-methyl-D-aspartate (NMDA) evoked release to the superfusion medium of radiolabelled material, 70% of which was associated with arachidonic acid (AA) and its metabolites. [3H]AA release was much more pronounced in PND 7-10 rats than in adults, and the response to NMDA in the hippocampal slices exceeded the reactions in the striatal slices. The subsequent experiments, employing only hippocampal slices of PND 7-10 rats, demonstrated that NMDA-stimulated [3H]AA release was dose-dependent in the micromolar range, was sensitive to NMDA receptor antagonists, and was inhibited in calcium-free medium and in the presence of quinacrine. [3H]AA release induced by 100 microM NMDA was not significantly inhibited by magnesium but was completely blocked by 7 Cl-kynurenic acid and ifenprodil (both antagonists 100 microM). The sulfhydryl reducing reagent dithiothreitol induced [3H]AA release; this response was sensitive to NMDA receptor antagonists. These data indicate that the NMDA induced, calcium triggered, and phospholipase A2 dependent AA release is highly pronounced in the developing rat hippocampus. NMDA receptors mediating AA release in the hippocampus of PND 7-10 rats are subject to glycine, polyamine and redox modulation, but they show low sensitivity to Mg2+ inhibition.

Differential involvement of phospholipase A 2 in phorbol ester-induced luteinizing hormone and growth hormone release from rat anterior pituitary tissue

The protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu) induced the release of both luteinizing hormone (LH) and growth hormone (GH) from proestrous rat anterior pituitary pieces in vitro. Phorbol 12,13-di-butyrate-induced LH, but not GH release was readily inhibited by the phospholipase A 2 (PLA 2) inhibitors, quinacrine, aristolochic acid, ONO-RS-082 and chloracysine. Furthermore, PDBu induced release of [ 3H]arachidonic acid ([ 3H]AA) from pre-labelled anterior pituitary tissue that was prevented in the presence of quinacrine, aristolochic acid and ONO-RS-082 but not the diglyceride lipase inhibitor RHC 80267. The effect of PDBu was completely inhibited by staurosporine and the selective PKC inhibitor Ro 31-8220 but only partially by low concentrations of H7; consistent with the involvement of both H7-sensitive and H7-resistant forms of PKC in the activation of PLA 2 by PDBu. The protein synthesis inhibitor cycloheximide inhibited the release of both [ 3H]AA and LH that had been induced by PDBu, whereas LH release induced by the PLA 2 activator mellitin was cycloheximide-insensitive. These results suggest that PKC activators may induce LH but not GH release from anterior pituitary tissue by a mechanism involving activation of a PLA 2, brought about by a process which is reliant on protein synthesis.

A nuclear magnetic resonance study of the interactions of the antimalarials chloroquine, quinacrine, quinine and mefloquine with lipids extracted from normal human erythrocytes

The interaction of four antimalarials (chloroquine, quinacrine, mefloquine and quinine) with lipid membranes re-formed from lipid extracts of normal human erythrocytes was studied using 2H- and lipid extracts of normal human erythrocytes was studied using 2H- and 31P-nuclear magnetic resonance (NMR). Inclusion of small amounts of chain-perdeuterated dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylethanolamine (DPPE) as an 2H-NMR probe allowed us to study separately the effects of drugs on PC or PE components of the membranes. Only a very small decrease in the order parameters of the DPPE, but not the DPPC probe, was observed in the presence of chloroquine at a molar ratio of 1:5 of drug to lipid. Addition of quinacrine at the same molar ratio resulted in a small but significant decrease in the order parameters of the lipid side chains; identical effects were obtained with DPPC or DPPE perdeuterated probes. The presence of quinacrine did not induce non-bilayer lipid phases. In contrast, mefloquine and quinine produced a significant disordering of the lipid side chains; the effect was considerably larger with the DPPE probe. In addition, both mefloquine and quinine induced non-bilayer phases of the lipids; mefloquine induced formation of hexagonal and micellar lipid conformation, whereas addition of quinine resulted in the formation of lipid micelles only. The lipid polymorphism induced by either of these drugs was more pronounced when the DPPE component was observed, indicating that the non-bilayer phases were enriched in PE. The results suggest the presence of strong interactions between mefloquine and quinine with lipid bilayers, especially with the PE component.(ABSTRACT TRUNCATED AT 250 WORDS)

Human cytomegalovirus stimulates arachidonic acid metabolism through pathways that are affected by inhibitors of phospholipase A2 and protein kinase C

Inhibitors of phospholipase A 2, tetracaine and quinacrine, inhibitors of protein kinases, H-7 and H-8, and a diacylglycerol lipase inhibitor reduced the level of CMV-induced [ 3H]AA release. A combination of H-7 and quinacrine inhibited stimulation of [ 3H]AA by about 80%. LU cells chronically treated with TPA and infected with CMV, had a reduced level of CMV-induced [ 3H]AA release and in the presence of quinacrine it was completely inhibited. These results suggest that CMV-induced stimulation of AA metabolism is mediated by pathways which are associated with activation of PLA 2 and protein kinase C.

Mechanism of calcium ionophore stimulated Cl secretion from frog skin glands

The aim of the present study was to investigate the mechanism by which the calcium ionophore A23187 stimulates Cl and water secretion from exocrine glands in the frog skin. The Cl secretion was visualized as changes in short-circuit current (SCC) in skins where the Na absorption was blocked by amiloride applied to the apical membrane. Measurements of A23187 stimulated ion fluxes showed that the ionophore induced a net secretion of Cl, Na and K. The active Cl secretion was enhanced more than the Na and K secretion, resulting in a net secretion of negative ions which closely resembled the A23187-stimulated SCC. The effect of A23187 was abolished in skins pretreated with indomethacin, implying the involvement of prostaglandins in the response. Furthermore, the effect of A23187 was inhibited in the presence of quinacrine, indicating that the activation of the cyclooxygenase pathway is dependent on phospholipase A2 activity. In addition, the A23187, but not the arachidonic acid stimulated Cl secretion was abolished in the presence of trifluoperazine, suggesting that the effect of the ionophore may be mediated via a Ca2+ -calmodulin-dependent step located before the activation of the cyclooxygenase. The net water flow and the Cl secretion were measured simultaneously under the conditions outlined above. The stimulation, inhibition, and time-course of the water secretion were similar to the changes observed for the Cl secretion. The A23187 stimulated Cl secretion was enhanced by the phosphodiesterase inhibitor, theophyllin, indicating that the effect of A23187 was caused by an increase in the intracellular cAMP level in the gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Preliminary observations on the pharmacology of petaline chloride, a quaternary alkaloid from Leontice leontopetalum

1. 1. Low concentrations of petaline chloride (1–300 μg/ml) caused relaxation of the epinephrine-contracted aorta, contraction of the ileum, and no effect on the trachea. 2. 2. Larger concentrations (up to 3 mg/ml) caused nonsustained large contractions of the aorta and the trachea and increased the amplitude of the phasic contractions of the ileum. 3. 3. The above effects were not inhibited in the presence of atropine. 4. 4. Petaline chloride increased, in a concentration-dependent manner, the contractions of the spontaneously-beating atrium and the isolated perfused heart. 5. 5. The effects of petaline chloride on the atrium and on the perfused heart were not affected by propranolol but were significantly reduced in the presence of quinacrine, suggesting the participation of arachidonic acid metabolism to this effect. 6. 6. In anesthetized rats, petaline chloride (0.3–3 mg/100 g body weight; i.p.) increased both the systolic and diastolic blood pressure and increased the heart rate.

Mechanism for leukotriene C4 stimulation of chloride transport in cornea.

The leukotriene, LTC4, exerts a stimulatory effect on chloride transport in the frog cornea. In the work described here, the mechanism of action of LTC4 to stimulate chloride transport was studied. In corneas pretreated with indomethacin, the effect of LTC4 was abolished, suggesting the involvement of cyclo-oxygenase products in the response. Incubation of corneas with LTC4 resulted in a significant stimulation in PGE2 synthesis, as determined by TLC-autoradiography and radioimmunoassay. In addition, LTC4 was found to stimulate cAMP synthesis in the cornea, and this stimulation was blocked with indomethacin. PGE2 was previously shown by us to be the dominant cyclo-oxygenase product formed in the frog cornea, and is capable of stimulating cAMP and chloride transport. We suggest that LTC4 stimulation of chloride transport is mediated via activation of the cyclo-oxygenase pathway, resulting in enhanced PGE2 synthesis. Elevated PGE2 levels induce cAMP synthesis, and ultimately, the stimulation of chloride transport. Further, the activation of cyclo-oxygenase was found to be dependent on phospholipase A2 activity. This was shown by the inhibition of the LTC4 effect in the presence of quinacrine. Similarly, inhibition of the LTC4 effect in the presence of trifluoperazine suggests that cyclo-oxygenase activation by LTC4 may be mediated via calmodulin. We have previously demonstrated that the frog cornea has the biosynthetic capacity to produce LTC4. Therefore LTC4 may function as an endogenous regulator of chloride transport in this tissue.

Is an acetylcholine transport system responsible for nonquantal release of acetylcholine at the rodent myoneural junction?

Experiments were performed to investigate the effects on the spontaneous, nonquantal release of acetylcholine (AcCho) from motor nerve terminals of substances known to inhibit the AcCho transport system present in cholinergic synaptic vesicles. In mouse diaphragms, the hyperpolarization normally produced by d-tubocurarine in the endplate area of muscle fibers that had been treated by an anticholinesterase was partly or completely blocked by 2-(4-phenylpiperidino)cyclohexanol (AH5183, 0.1-1 microM), quinacrine (0.1 microM), and tetraphenylborate (1 microM). Since the sensitivity of the subsynaptic area to AcCho was not changed, the block of the hyperpolarizing action of d-tubocurarine indicated in inhibition of the spontaneous, nonquantal release of AcCho. This was confirmed in experiments on rat diaphragm using direct radioenzymatic measurement of the AcCho released into the incubation medium. The release of AcCho from the innervated diaphragm was decreased by about 50% in the presence of AH5183 (0.01-1 microM) and by 42% in the presence of quinacrine (0.1-1 microM). The AcCho released was presumably neural, since the release of AcCho from 4-day denervated diaphragms was not diminished by either AH5183 or quinacrine. The results indicate that the spontaneous release of AcCho from the motor nerve terminals is highly sensitive to low concentrations of specific inhibitors and is probably mediated by a carrier. It is proposed that spontaneous release is due to the incorporation into the membrane of the nerve terminal during exocytosis of the vesicular transport system responsible for moving AcCho into the vesicle.

Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine platelets.

Phosphatidylinositol-specific phospholipase C was purified to homogeneity from soluble fraction of bovine platelets by ammonium sulfate fractionation, hydrophobic chromatography, DEAE ion exchange chromatography and gel filtration. The purified enzyme has a narrow pH optimum ranging from 6.5 to 7.5 and the molecular weight of the enzyme was estimated to be 143,000 by sodium dodecyl sulfate slab gel electrophoresis. The purified enzyme requires Ca2+ strictly for activity, which was markedly enhanced in the presence of arachidonate. No enhancement of the activity was observed in the presence of purified calmodulin. The activity was markedly inhibited in the presence of quinacrine but no inhibition by indomethacin was observed.