The effects of non-steroidal inhibitors of phospholipase A2 on leukotriene and histamine release from human and guinea-pig lung

The effects of chloroquine and mepacrine were determined on the release of slow reacting substances (leukotrienes) from lung fragments in vitro. These drugs have been shown in a variety of tissues to inhibit phospholipase A2, and thus to reduce the availability of arachidonate, which is a substrate for leukotriene biosynthesis. Leukotriene and histamine release from unsensitized human lung was stimulated by calcium ionophore A23187, and from actively sensitized guinea-pig lung, by ovalbumin. Chloroquine (10 microM and 100 microM) significantly inhibited leukotriene release in lung from both species, and at 100 microM also inhibited histamine release. Mepacrine (10 microM) inhibited leukotriene release in human lung and at 100 microM in guinea-pig lung. The effects of chloroquine (100 microM) on leukotriene release were counteracted by the presence of arachidonic acid (10 microM), which suggests that chloroquine had impaired the availability of arachidonate. It seems probable that chloroquine and mepacrine inhibit leukotriene release by inhibition of phospholipase A2 in lung.     

Phospholipase C activity in human polymorphonuclear leukocytes: partial characterization and effect of indomethacin

Several hormones act at the cellular level to increase diacylglycerol via increased catabolism of phosphatidylinositol by phospholipase C. Diacylglycerol stimulates protein kinase C, leading to protein phosphorylation and hormone action. Since phospholipase C activity has not been well studied in man, we have established an assay for phospholipase C in human neutrophils. In this assay sonicates of neutrophils were incubated with L-3-phosphatidyl-[U 14C]-inositol and the incubation mixture extracted with chloroform/methanol. Following the additions of 2 mol/l KCl and chloroform, phospholipase C activity was determined by counting [14C] in the aqueous phase. The phospholipase C activity was linear with respect to time and the quantity of added enzyme. Optimum substrate concentration and pH were 2 mmol/l and 7.0, respectively. Optimal activity was dependent on Ca2+ (2 mmol/l) and deoxycholate (2 mmol/l). Naloxone, and PGD2, which affect various aspects of leucocyte function, had no significant effects on neutrophil PLC activity. The effects of various compounds with phospholipase A2 inhibitory activity were also tested on this enzyme. Of these, mepacrine, lidocaine and indomethacin inhibited the enzyme activity. The inhibition by indomethacin was of the noncompetitive type with an apparent Km of 0.17 X 10(-6) mol/l and apparent Ki of 3.6 X 10(-6) mol/l. From these data we conclude that indomethacin is capable of inhibiting phospholipase C activity in neutrophils at clinically significant levels and that this may be relevant in the therapeutic action of this drug.     

绵羊心浦肯野纤维d-受体和β-受体激动时离子流Isi和Ix的变化

当绵羊心浦肯野纤维α-和β-受体分别激动时,用双微电极法电压箝制术研究慢内向离子流Isi和延迟整流外向离子流Ix的变化。心得安阻断β-受体时,苯肾上腺素5.0×10~(-6)mol/L使Isi的峰值由17.5增加到26nA(n=6,P<0.05).并使Isi由失活到完全恢复的时间由293±51ms延长到441±109ms(n=4,P<0.05),但对Ix无明显影响。酚妥拉明阻断α-受体时,异丙肾上腺素4×10~(-7)mol/L使Isi峰值由27.7增加到40.8nA(n=7,P<0.05),对Isi的动力过程无明显影响,却使Ix尾电流的幅值由6.7增加到14,4nA(n=6,P<0.05)。表明α-和β-受体激动剂作用的差异在于对延迟整流离子流的影响不同。     

The effects of non-steroidal inhibitors of phospholipase Az on leukotriene and histamine release from human and guinea-pig lung

The effects of chloroquine and mepacrine were determined on the release of slow reacting substances (leukotrienes) from lung fragments in vitro. These drugs have been shown in a variety of tissues to inhibit phospholipase A₂, and thus to reduce the availability of arachidonate, which is a substrate for leukotriene biosynthesis. Leukotriene and histamine release from unsensitized human lung was stimulated by calcium ionophore A23187, and from actively sensitized guinea-pig lung, by ovalbumin. Chloroquine (10 μM and 100 μM) significantly inhibited leukotriene release in lung from both species, and at 100 μM also inhibited histamine release. Mepacrine (10 μM) inhibited leukotriene release in human lung and at 100 μM in guinea-pig lung. The effects of chloroquine (100 μM) on leukotriene release were counteracted by the presence of arachidonic acid (10 μM), which suggests that chloroquine had impaired the availability of arachidonate. It seems probable that chloroquine and mepacrine inhibit leukotriene release by inhibition of phospholipase A₂ in lung.     

Effect of nonachlazine on transmembrane ionic currents in the frog atrial trabeculae

The effect of the antianginal drug nonachlazine displaying antiarrhythmic properties on transmembrane ionic currents in the frog atrial fibers was studied in experiments on isolated trabeculae of the frog atria. The transmembrane ionic currents were measured by a voltage clamp technique based on a double sucrose gap arrangement. Nonachlazine (1.03 X 10(-5) mol/l) decreased the amplitude of the fast inward current whatever the magnitude of membrane potential. The drug inhibited the slow inward current and prevented the adrenaline-increased permeability of the slow sodium-calcium channel if external sodium ions were replaced by choline chloride. Nonachlazine (1.03 X 10(-5) mol/l) diminished the amplitude of the inward ionic current in a calcium-free medium as well. The stimulatory effect of prostacycline (2 X 10(-7) mol/l) on the fast inward ionic current was inhibited by nonachlazine. The data obtained suggest that the antiarrhythmic effect of nonachlazine might be linked with the inhibition of the fast sodium inward current and the slow calcium inward current.     

Role of phospholipases in myocardial ischemia: effect of cardioprotective agents on the phospholipases A of heart cytosol and sarcoplasmic reticulum in vitro

Increased breakdown of myocardial phospholipids to fatty acids and lysophosphoglycerides is an early feature of myocardial ischemic injury and many investigators believe that enhanced phospholipase action is an important factor in the process. Several recent reports indicate that inhibitors of phospholipase A, such as mepacrine, chloroquine and chlorpromazine, can prevent heart phosphoglyceride breakdown in vivo. We isolated the phospholipases A from rat heart cytosol and sarcoplasmic reticulum and examined the effects of various cardioprotective substances on their activity. Most of the cardioprotective agents studied inhibited the heart phospholipases in vitro, providing further evidence that phospholipid degradation in ischemic myocardial injury may be modulated by pharmacologic agents.     

On the ionic mechanism underlying adrenergic-cholinergic antagonism in ventricular muscle

In atrial muscle, acetylcholine (ACh) decreases the slow inward current (Isi) and increases the time-independent outward K+ current. However, in ventricular muscle, ACh produces a marked negative inotropic effect only in the presence of positive inotropic agents that elevate cyclic adenosine monophosphate (AMP). A two-microelectrode voltage-clamp method was used on cultured reaggregates of cells from 16--20-d-old embryonic chick ventricles to determine the effects of ACh on Isi and outward current during beta-adrenergic stimulation. Only double penetrations displaying low-resistance coupling were voltage-clamped. Cultured reaggregates are advantageous because their small size (50-- 250 microns) permits better control of membrane potential and adequate space clamp. Tetrodotoxin (10(-6) M) and a holding potential of --50 to --40 mV were used to eliminate the fast Na+ current. Depolarizing voltage steps above --40 mV caused a slow inward current to flow that was sensitive to changes in [Ca]o and was depressed by verapamil (10(- 6) M). Maximal Isi was obtained at --10 mV and the reversal potential was about +25 mV. Isoproterenol (10(-6) M) increased Isi at all clamp potentials. Subsequent addition of ACh (10(-6) M) rapidly reduced Isi to control values (before isoproterenol) without a significant effect on the net outward current measured at 300 ms. The effects of ACh were reversed by muscarinic blockade with atropine (5 X 10(-6) M). We conclude that the anti-adrenergic effects of ACh in ventricular muscle are mediated by a reduction in Ca2+ influx during excitation.     

Inhibition of lipid peroxidation in muscle homogenates by phospholipase A2 inhibitors

Chlorpromazine, mepacrine, tetracaine, dibucaine, chloroquine, and procaine have been shown to inhibit the iron- and ascorbate-induced lipid peroxidation of skeletal-muscle hornogenates in vitro. These compounds are known to be inhibitors of phospholipase activity, but they were also found to be effective in blocking free-radical-mediated damage to lipids in denatured homogenates, to linoleate suspensions, and to glutamic acid solutions where phospholipase activity was not a relevant factor. The inhibitory action did not appear to be related to any iron-binding activity of the compounds.     

Factors regulating the production of prostaglandin E2 and prostacyclin (prostaglandin I2) in rat and human adipocytes.

The regulation of PGE2 (prostaglandin E2) and PGI2 (prostaglandin I2; prostacyclin) formation was investigated in isolated adipocytes. The formation of both PGs was stimulated by various lipolytic agents such as isoproterenol, adrenaline and dibutyryl cyclic AMP. During maximal stimulation the production of PGE2 and PGI2 (measured as 6-oxo-PGF1 alpha) was 0.51 +/- 0.04 and 1.21 +/- 0.09 ng/2 h per 10(6) cells respectively. Thus PGI2 was produced in excess of PGE2 in rat adipocytes. The production of the PGs was inhibited by indomethacin and acetylsalicylic acid in a concentration-dependent manner. The half-maximal effective concentration of indomethacin was 328 +/- 38 nM and that of acetylsalicylic acid was 38.5 +/- 5.3 microM. The PGs were maximally inhibited by 70-75% after incubation for 2 h. In contrast with their effect on PG production, the two agents had a small potentiating effect on the stimulated lipolysis (P less than 0.05). The phospholipase inhibitors mepacrine and chloroquine inhibited both PG production and triacylglycerol lipolysis and were therefore unable to indicate whether the PG precursor, arachidonic acid, originates from phospholipids or triacylglycerols in adipocytes. Angiotensin II significantly (P less than 0.05) stimulated both PGE2 and PGI2 production in rat adipocytes without affecting triacylglycerol lipolysis. Finally, it was shown that PGE2 and PGI2 were also produced in human adipocytes, although in smaller quantities than in rat adipocytes. It is concluded that the production of PGs in isolated adipocytes is regulated by various hormones. Moreover, at least two separate mechanisms for PG production may exist in adipocytes: (1) a mechanism that is activated concomitantly with triacylglycerol lipolysis (and cyclic AMP) and (2) an angiotensin II-sensitive, but lipolysis (and cyclic AMP)-independent mechanism.     

Inhibitors of phospholipase A2 block the stimulation of protein synthesis by insulin in L6 myoblasts.

Insulin at a concentration close to the physiological range (100 mu-units/ml) stimulated protein synthesis in L6 myoblasts by 17%. Pre-treatment with the phospholipase A2 inhibitors mepacrine or dexamethasone prevented this stimulation and decreased the release of prostaglandin F2 alpha, implicating the action of phospholipase A2 and the subsequent metabolism of arachidonic acid to prostaglandins in the stimulation of protein synthesis by physiological doses of insulin. Higher concentrations of insulin (500-1000 mu-units/ml) stimulated protein synthesis in the presence of mepacrine or dexamethasone, suggesting that an alternative pathway may become important in insulin action when phospholipase A2 is inhibited.     

Effects of mepacrine on uterine contractile responses

Mepacrine is a potent inhibitor of uterine contractile responses in vitro. Pretreatment of isolated rat uterine horns with mepacrine (1.3 X 10(-4)M) for periods of time ranging from 15 s to 5 min prior to the addition of carbachol (1.0 X 10(-4)M) showed that mepacrine could significantly reduce carbachol-induced uterine contractile responses within 15 s of exposure. The maximal inhibitory effects of mepacrine on uterine contractile responses were observed within 2 min of mepacrine treatment. A dose-response study related to the effect of increasing concentrations of mepacrine (7.5 X 10(-6) to 1.3 X 10(-4)M) on carbachol-induced (1 X 10(-4)M) uterine contractions revealed that a dose of 3.1 X 10(-5)M mepacrine reduced the carbachol-induced contraction by 50%. A dose of 7.8 X 10(-5)M mepacrine produced the maximal inhibitory effect on the carbachol-induced uterine contractions. Two doses of mepacrine (3.1 X 10(-5) and 1.3 X 10(-4)M) significantly reduced maximal contractile responses and shifted contractile dose-response curves of carbachol, oxytocin, prostaglandin F2 alpha, and BaCl2 to the right. Based on the nonselective inhibition by mepacrine of contractile responses induced by different uterotonic agents, these results suggest that mepacrine cannot be used to characterize the role of phospholipase in regulating the actions of hormones in uterine tissue.     

Effect of local anaesthetics (mesocain) on membrane properties of cat papillary muscle

In 28 experiments on cat papillary muscles the effect of mesocain (generically related to lidocaine) was studied on transmembrane currents (rapid inward INa, slow inward Isi, outward Io), on the configuration and (dV/dt)max of the action potential, on the refractory period and on isometric tension. The experiments were performed using either voltage clamp method (double sucrose gap) or microelectrode technique. All three transmembrane currents are inhibited by mesocain in a dose-dependent manner. The effect becomes measurable at 10(-6) mol/1 on INa, at 10(-5) mol/1 on Isi and at 5 X 10(-5) mol/1 on Il. INa is blocked at 10(-3) mol/1 concentration. These changes correlate with the measured alterations of action potentials: i.e. a decreased (dV/dt) max and overshoot, lower voltage and shortening of the plateau phase, slower rate of late repolarizaion. The negative inotropic effect appeared with the same treshold concentration as Isi; at higher concentrations, however, this effect was relatively much greater. The recovery of excitability measured by the interval-dependence of (dV/dt) max was prolonged 5 times on the average by mesocain at 10(-4) mol/1 and the effective refractory period was delayed beyond complete repolarization. It is concluded that the main effect of mesocain also involves a delayed kinetics of recovery from inactivation of the Na carrying system.     

Mepacrine attenuates pulmonary vasoreactivity in rabbits

The organic peroxide tert-butyl hydroperoxide (t-bu-OOH) induces pulmonary vasoconstriction by stimulating production of thromboxane in the rabbit lung, possibly by activating phospholipase A2. t-bu-OOH-induced vasoconstriction and thromboxane production is augmented by inhalational anesthetic agents, perhaps due to an effect of anesthetic agents on membrane lipids. To further investigate the mechanism of thromboxane generation, we studied the influence of the phospholipase A2 inhibitor, mepacrine, in a dose known to inhibit the enzyme in other systems, on t-bu-OOH-induced pulmonary arterial vasoconstriction. We found that 10(-4) M mepacrine completely inhibited t-bu-OOH-induced vasoconstriction. We also found that mepacrine inhibited arachidonic acid-induced pulmonary vasoconstriction but did not inhibit thromboxane productions. We also investigated the effect of mepacrine on two other pulmonary vasoconstrictors, angiotensin II (ANG II) and KCl, which do not act through arachidonic acid metabolites in the rabbit lung. Mepacrine inhibited both ANG-II and KCl-induced vasoconstriction. The inhibition by mepacrine of pulmonary vasoconstriction is reversible if the drug is washed out of the lung. This effect of mepacrine cannot be explained by phospholipase inhibition alone and is consistent with prevention of smooth muscle contraction.     

A new mechanism of action of vasopressin on the neuronal membrane]

It is shown in this work that vasopressin at the concentrations of 1 x 10(-16) to 1 x 10(-6) mol/l decreased statistically significant the amplitude of the electrosensitive sodium and calcium ionic currents of the mollusc's Lymnaea stagnalis neuronal membrane. This peptide increased the amplitude of the fast potassium current at the concentration of 1 x 10(-16) and 1 x 10(-15) mol/l. It decreased the fast potassium current and did not change the slow potassium current at the concentrations more than 1 x 10(-9) mol/l.     

The efficacy of anti-inflammatory agents with respect to extracellular phospholipase A2 activity

The effects of steroidal and non-steroidal anti-inflammatory agents on extracellular phospholipase A2 (PLA2) activity were investigated. Enzyme release was inhibited by 5 x 10(-8) M dexamethasone but not by indomethacin, whereas the soluble extracellular enzyme was inactivated by mepacrine but not by dexamethasone or indomethacin. PLA2, released into the interstitium by activated macrophages is both pro-inflammatory and vasoactive. The anti-inflammatory efficacy of steroidal and non-steroidal drugs may partially reside in their ability to inhibit the release of PLA2, or inactivate preformed extracellular PLA2 in chronically inflamed sites.     

Mechanism of cationic amphiphilic drug inhibition of purified lysosomal phospholipase A1

Cationic amphiphilic drugs like chlorpromazine, propranolol, and chloroquine inhibit lysosomal phospholipase A in vitro. Some workers have proposed that cationic amphiphilic drugs inhibit the activity of phospholipase A1 by forming substrate-drug complexes which cannot be degraded while others have reported competitive inhibition implying drug effects on the enzyme. To analyze the mechanism of inhibition, we examined the binding ability of these drugs to unilamellar vesicles of dioleoylphosphatidylcholine and correlated these results with a detailed kinetic analysis of phospholipase A. Chlorpromazine and propranolol bound to small unilamellar liposomes of dioleoylphosphatidylcholine substrate in a positive cooperative way consistent with two binding sites: a high-affinity site with low capacity and a low-affinity site with high capacity. The affinity of chlorpromazine for the high-affinity site was 2 times greater than that of propranolol (KA = 13 807 +/- 1722 vs. 8481 +/- 1078 M-1), and the saturation number for chlorpromazine was 3 times greater than for propranolol (N = 0.20 +/- 0.004 vs. 0.07 +/- 0.02 mol of drug/mol of phosphatidylcholine). Chloroquine did not bind to unilamellar liposomes of dioleoylphosphatidylcholine. We carried out detailed kinetic studies using purified lysosomal phospholipase A1 from rat liver. In the case of chloroquine inhibition, the Lineweaver-Burk double-reciprocal plots showed straight lines, but the slope replots were curved, indicating the formation of complexes having 2 mol of chloroquine/mol of enzyme (EI2 complexes). Thus, chloroquine is a competitive inhibitor which forms EI2 complexes with phospholipase A1. However, in the case of chlorpromazine and propranolol, the observed kinetic data do not fit to the same equilibrium used for the case of chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of antimalarial drugs on phospholipase A and lysophospholipase activities in plasma membrane, mitochondrial, microsomal and cytosolic subcellular fractions of rat liver

Activities of membrane-associated phospholipases A1 and A2, and membrane-associated as well as soluble lysophospholipases were measured in different subcellular fractions of rat liver, using suspensions of stereospecifically labelled radioactive phospholipids as substrates. Plasma membranes and endoplasmic reticulum were shown to contain phospholipase A1 and lysophospholipase activities, both of which could be stimulated by Ca2+, mitochondria Ca2+-dependent phospholipase A2 and cytosol Ca2+-independent lysophospholipase activities. Each of these lipolytic enzymes could be inhibited by antimalarial drugs (chloroquine, mepacrine, primaquine) at concentrations above 1 x 10(-4) M. Inhibition of the alkaline cytosolic lysophospholipase by these drugs was noncompetitive with respect to the substrate, and the inhibitory potency increased, when the pH was raised.     

Characteristics of the second inward current in cells isolated from rat ventricular muscle

The second inward current (Isi) in single cells isolated from ventricular muscle of adult rat hearts was measured in response to step depolarizations under voltage-clamp conditions. The major ion carrying this current was Ca, and Isi was reduced or abolished by Mn, Ni, Cd, nifedipine, nimodipine and D600. Sr and B could substitute for Ca as charge carriers, and reduced the rate of apparent inactivation of Isi. These effects of Sr and Ba, together with the relation between the steady level of apparent inactivation and membrane potential in Ca containing solution, were taken as evidence that inactivation was at least in part dependent on internal Ca. The reduction of external Na to 11% of normal caused a reduction in peak Isi when Ca was present in the external solution, but did not reduce Isi when Ca was replaced by Sr. It therefore seems unlikely that Na is a major charge carrier for Isi under the conditions of our experiments. The time-to-peak and rate of apparent inactivation of Isi were faster than in previous studies that used multicellular preparations. Both the kinetics and peak amplitude of Isi were markedly dependent on temperature (Q10 close to 3). Contraction of the cells, which was monitored optically, was initiated within 3 ms of the peak Isi, reached a maximum level after approximately 40-50 ms, and was about 100 ms in duration.     

G alpha i-3 regulates epithelial Na+ channels by activation of phospholipase A2 and lipoxygenase pathways

Polarized renal epithelial cells have pertussis toxin-sensitive Gi proteins at their apical membrane capable of modulating Na+ channel activity (Cantiello, H.F., Patenaude, C.R., and Ausiello, D.A. (1989) J. Biol. Chem. 264, 20867-20870). In this study, the patch clamp technique was used to assess if this Gi-mediated regulation of Na+ channels is a component of a phospholipid signal transduction pathway. In excised inside-out patches of apical membranes of A6 cells, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-stimulated Na+ channel activity (percent open time and channel number) was inhibited by the phospholipase inhibitor mepacrine (50 microM), which had no effect on single channel conductance. In contrast, Na+ channel activity increased in a Ca2(+)-dependent manner following the addition of 100 nM mellitin to untreated or pertussis toxin-treated patches. Addition of 10 microM arachidonic acid in the presence of mepacrine increased Na+ channel activity. Both percent open time and Na+ channel number induced by GTP gamma S, the exogenous alpha i-3 subunit, or arachidonic acid were inhibited by the addition of the 5-lipoxygenase inhibitor nordihydroguaiaretic acid. Na+ channel activity was restored with the addition of leukotriene D4 (100 nM) or the parental leukotriene substrate 5-hydroperoxyeicosatetraenoic acid (10 microM). Thus, Gi activation of apical membrane epithelial Na+ channels is mediated through the regulation of phospholipase and lipoxygenase activities. This apically located signal transduction pathway may be sensitive to, or independent of, classical second messengers generated at the basolateral membrane and known to be responsible for modulation of Na+ channel activity in epithelia.     

The role of phospholipase A activity in rat liver microsomal lipid peroxidation

The involvement of phospholipase(s) A in lipid peroxidation of rat liver microsomes was investigated by: (a) determining the effects of phospholipase A inhibitors (p-bromophenylacyl bromide, chlorpromazine, mepacrine) on the accumulation of thiobarbituric acid reactivity or on levels of oxidized phospholipids in response to selected oxidative stimuli and (b) measurement of phospholipase A activities in response to these agents. Lipid peroxidation in response to various peroxidation systems was inhibited completely by exposure of microsomes to p-bromophenylacyl bromide (250 microM). The effectiveness of p-bromophenylacyl bromide was dependent on the presence of glutathione (200 microM) in preincubation mixtures. Chlorpromazine (100 microM) and mepacrine (100 microM) also effectively inhibited peroxidation, and their potency was independent of glutathione. The accumulation of oxidized phospholipids in response to the potent peroxidation stimulus alloxan/ferrous ion was similarly inhibited by p-bromophenylacyl bromide, although the level of oxidized phospholipid in response to the initiator ADP/ferrous ion was not affected. Microsomal phospholipase A1 activity, assessed using a liposomal substrate, was substantially enhanced by promoters of lipid peroxidation. Phospholipase A2 activity was not detected using a liposomal substrate but was evident using radiolabeled microsomes as endogenous substrate and was enhanced by oxidative stimuli. We conclude that phospholipase A activity may play an integral role in the microsomal lipid peroxidation mechanism. Based on this study, we hypothesize a role for phospholipases in facilitating propagation reactions.