calcimycin and manoalide

We examined whether Ca(2+) mobilizers induce endothelium-dependent contraction and relaxation (EDC and EDR) in isolated rabbit intrapulmonary arteries. Ionomycin (10(-7) M) and A-23187 (10(-7) M), both Ca(2+) ionophores, and thapsigargin (10(-6) M), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, caused a contraction in the non-contracted preparations, and a transient relaxation followed by a transient contraction and sustained relaxation in the precontracted preparations. Endothelium-removal abolished the contraction and transient relaxation (EDC and EDR) but not sustained relaxation (endothelium-independent relaxation, EIR). In the noncontracted preparations, ionomycin-induced EDC was significantly attenuated by quinacrine (10(-5) M), manoalide (10(-6) M), both phospholipase A(2) inhibitors, indomethacin (10(-5) M) and aspirin (10(-4) M), both COX inhibitors, and ozagrel (10(-5) M), a TXA(2) synthetase inhibitor. In the precontracted arteries, EDR was markedly reduced by L-NAME (10(-4) M), a NOS inhibitor, and methylene blue (10(-6) M), a guanylate cyclase inhibitor, and was enhanced by indomethacin, aspirin and ozagrel, probably due to inhibition of EDC. ZM230487, a 5-lipoxygenase inhibitor, had no effect on EDR. EIR was not affected by L-NAME, indomethacin or ZM230487. Arachidonic acid (10(-6) M) evoked EDC sensitive to indomethacin and ozagrel. L-Arginine (10(-3) M) caused EDR sensitive to L-NAME in the ionomycin-stimulated preparations. In conclusion, Ca(2+) mobilizers cause EDC and EDR via production of TXA(2) and NO, respectively.

Topics: Animals; Aspirin; Calcimycin; Calcium; Endothelium, Vascular; Enzyme Inhibitors; Indomethacin; Ionomycin; Ionophores; Male; Methacrylates; Methylene Blue; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Pulmonary Artery; Quinacrine; Rabbits; Terpenes; Thapsigargin

The marine natural product manoalide (MLD) has been described to inactivate phospholipase A2 (PLA2) from several sources as well as to inhibit synthesis of eicosanoids in human polymorphonuclear leukocytes (HPMNL). MLD also reduces chemically-induced inflammation in vivo. In this investigation we have examined the effect of MLD on A23187-induced generation of leukotriene B4 (LTB4) and thromboxane B2 (TXB2) in HPMNL as well as 5-lipoxygenase (5-LO) activity from HPMNL sonicated preparations. In the intact cell system, MLD inhibited with similar potency biosynthesis of LTB4 and TXB2 (IC50 1.7 and 1.4 microM, respectively). In order to discern if inhibition of 5-LO is involved in the effect of MLD, we examined the action of this compound on 5-LO activity from 10,000 x g and 100,000 x g supernatants of sonicated HPMNL homogenates. The enzymatic activity was not affected at concentrations of MLD up to 50 microM. These data indicate that MLD is not a direct inhibitor of 5-LO activity from HPMNL and support the hypothesis that its anti-inflammatory action could be related with a reduction of eicosanoid biosynthesis via inhibition of PLA2.

Topics: Arachidonate 5-Lipoxygenase; Calcimycin; Dose-Response Relationship, Drug; Humans; Ionophores; Leukotriene B4; Lipoxygenase Inhibitors; Neutrophils; Terpenes; Thromboxane B2

The gastrointestinal hormone cholecystokinin and the muscarinic agonist carbamylcholine are involved in pancreatic enzyme secretion through phospholipase C activation and production of the second messengers inositol trisphosphate and diacylglycerol. However, cholecystokinin induces growth of the pancreas whereas carbamylcholine does not. This study investigated the possibility of a specific cellular signalling transduction system through which cholecystokinin may induce pancreatic growth. Rat pancreatic acini were preincubated with 3H choline or 3H myristic acid to label phosphatidylcholine. They were then stimulated by caerulein, phorbol myristate acetate, and carbamylcholine; choline and phosphocholine release as well as phosphatidic acid and phosphatidylethanol production were measured to establish phospholipase D (PLD) activation. Caerulein, phorbolester and carbamylcholine increased phosphocholine release. Choline release was induced early by caerulein and later by phorbolester but not by carbachol. PLD was activated by caerulein and phorbolester but not by carbamylcholine. Increased intracellular calcium by A23187 had no effect on PLD activation but its chelation by BAPTA prevented caerulein-induced PLD activation. In conclusion, PLD seems to be selectively activated by caerulein and phorbol ester by two different mechanisms which are insensitive to carbamylcholine. It is suggested that the PLD pathway might be the cellular signalling system leading to pancreatic growth.

Topics: Alkaloids; Animals; Calcimycin; Carbachol; Ceruletide; Cholecystokinin; Choline; Egtazic Acid; Enzyme Activation; Glycerophospholipids; In Vitro Techniques; Male; Pancreas; Phosphatidic Acids; Phospholipase D; Phosphorylcholine; Protein Kinase C; Rats; Rats, Sprague-Dawley; Signal Transduction; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Type C Phospholipases

The effects of the stable analogue of TxA2, U46619, on polymorphonuclear leukocyte (PMN) function were investigated. U46619, at micromolar concentrations, inhibited fMLP-stimulated aggregation, beta-glucuronidase release, and superoxide production. fMLP-induced LTB4 synthesis was also inhibited. U46619 did not modify intracellular Ca2+ increase induced by fMLP in Fura-2-loaded PMN, suggesting that early events of cell activation were not involved. In fact, U46619 also inhibited aggregation, beta-glucuronidase release, superoxide anion and LTB4 production induced by the calcium ionophore A23187. By comparison with the specific 5-lipoxygenase inhibitor, L-663,536, which prevented LTB4 synthesis without affecting degranulation, we excluded the impairment of PMN function by U46619 as a consequence of the reduction of this endogenous agonist. TLC separation of lipid extracts from [3H]-AA-loaded PMN, stimulated by A23187, showed significant reduction of the radioactivity associated with authentic free AA, suggesting that U46619 could interfere with mechanisms regulating AA release from membrane phospholipids. This suggestion is also supported by the observation that manoalide, a standard inhibitor of phospholipase A2, similarly to U46619, inhibits beta-glucuronidase release from stimulated PMN. Prostaglandin endoperoxides, produced by cells participating in inflammatory reactions, might therefore play a role in modulating PMN activities.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Calcimycin; Cell Adhesion; Fura-2; Humans; Indoles; Leukotriene Antagonists; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases A; Phospholipases A2; Prostaglandin Endoperoxides, Synthetic; Superoxides; Terpenes; Thromboxane A2; Vasoconstrictor Agents

AGN 190383 is a 5-hydroxy-2(5H)-furanone ring analog of the marine natural product manoalide. When applied topically, AGN 190383 inhibits phorbol ester induced mouse ear edema. It is a potent inhibitor of bee venom phospholipase A2 and blocks the release of arachidonic acid from calcium ionophore A23187 stimulated human neutrophils. AGN 190383 also inhibits both hormone-operated and depolarization-dependent calcium mobilization in GH3 cells, as well as fMLP stimulated increases in free cytosolic calcium in human PMNs. Furthermore, it is also able to block the release of the neutral protease elastase from stimulated neutrophils. The effects of AGN 190383 on arachidonic acid metabolism and leukocyte function may account, in part, for its anti-inflammatory activity in vivo.

Topics: Administration, Topical; Amino Acid Sequence; Animals; Anti-Inflammatory Agents; Arachidonic Acid; Calcimycin; Furans; Mice; Molecular Sequence Data; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pancreatic Elastase; Phospholipases; Phospholipases A; Phospholipases A2; Terpenes

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostagl

Topics: Acetophenones; Calcimycin; Cell Line; Dinoprostone; Fatty Acids, Unsaturated; Kinetics; Macrophages; Melitten; Phospholipases; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Substrate Specificity; Terpenes

The marine natural produce manoalide has been reported to inactivate venom phospholipase A2 from several sources and phospholipase A2 from polymorphonuclear leukocytes. In this investigation, the regulation of eicosanoid production was studied both in an in vitro and in an in vivo model. The release of arachidonic acid and prostaglandin E2 was inhibited by manoalide when cultured mouse peritoneal macrophages were stimulated with phorbol myristate acetate (apparent IC50 = 0.23 microM), calcium ionophore A23187 (apparent IC50 = 0.23 microM) and zymosan (apparent IC50 = 0.18 microM). Leukotriene C4 production was inhibited by manoalide when macrophages were stimulated by A23187 (IC50 = 0.35 microM) but was enhanced when the cells were stimulated with zymosan. In an in vivo model, manoalide antagonized zymosan-induced peritoneal writhing in the mouse (ED50 = 0.71 mg/kg) and inhibited the i.p. release of 6-keto-prostaglandin F1 alpha (ED50 = 0.2 mg/kg) and leukotriene C4 (ED50 = 0.24 mg/kg). Our results suggest that: 1) manoalide modifies arachidonic acid release and metabolism to prostaglandins and leukotrienes in mouse cultured peritoneal macrophages stimulated by phorbol myristate acetate, calcium ionophore A23187 and zymosan and 2) the analgesic properties of manoalide seem to be partially correlated with reduced eicosanoid production in zymosan-stimulated mouse peritoneal exudates.

Topics: 6-Ketoprostaglandin F1 alpha; Analgesics; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Dinoprostone; In Vitro Techniques; Macrophages; Male; Mice; Phospholipases; Phospholipases A; Phospholipases A2; SRS-A; Terpenes; Tetradecanoylphorbol Acetate; Zymosan