calcimycin and phenidone

Plants can acquire freezing tolerance in response to cold but non-freezing temperatures. To efficiently activate this cold acclimation, low temperature has to be sensed and processed swiftly, a process that is linked with a transient elimination of microtubules. Here, we address cold-induced microtubules elimination in a grapevine cell line stably expressing a green fluorescent protein fusion of Arabidopsis TuB6, which allows to follow their response in vivo and to quantify this response by quantitative image analysis. We use time-course studies with several specific pharmacological inhibitors and activators to dissect the signalling events acting upstream of microtubules elimination. We find that microtubules disappear within 30 min after the onset of cold stress. We provide evidence for roles of calcium influx, membrane rigidification, and activation of NAD(P)H oxidase as factors in signal susception and amplification. We further conclude that a G-protein in concert with a phospholipase D convey the signal towards microtubules, whereas calmodulin seems to be not involved. Moreover, activation of jasmonate pathway in response to cold is required for an efficient microtubule response. We summarize our findings in a working model on a complex signalling hub at the membrane-cytoskeleton interphase that assembles the susception, perception and early transduction of cold signals.

Topics: Aluminum Compounds; Benzyl Alcohol; Biphenyl Compounds; Calcimycin; Calcium; Cell Membrane; Cold Temperature; Cyclopentanes; Cytoplasm; Dimethyl Sulfoxide; Egtazic Acid; Fluorides; Gadolinium; Ionophores; Microtubules; NADPH Oxidases; Nitroprusside; Onium Compounds; Oxylipins; Pertussis Toxin; Phospholipase D; Polymerization; Pyrazoles; Signal Transduction; Stress, Physiological; Vitis

The antiinflammatory activity of rolipram, a selective inhibitor of the cyclic AMP-specific phosphodiesterase (PDE IV), was studied. Rolipram did not inhibit 5-lipoxygenase activity but did inhibit human monocyte production of leukotriene B4 (LTB4, IC50 3.5 microM). Likewise, murine mast cell release of leukotriene C4 and histamine was inhibited. In vivo, rolipram inhibited arachidonic acid-induced inflammation in the mouse, while the low Km-cyclic-GMP PDE inhibitor, zaprinast, did not inhibit. Rolipram had a modest effect on LTB4 production in the mouse, but markedly reduced LTB4-induced PMN infiltration. Beta-adrenergic receptor activation of adenylate cyclase was important for rolipram antiinflammatory activity since beta blockade abrogated arachidonic acid-induced inflammation. Thus, the antiinflammatory profile of rolipram is novel and may result from inhibition of PMN function and perhaps vasoactive amine release and leukotriene biosynthesis. These actions may be dependent upon endogenous beta-adrenergic activity and are likely mediated through inhibition of PDE IV.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Calcimycin; Cells, Cultured; Colforsin; Cyclic Nucleotide Phosphodiesterases, Type 4; Ear, External; Eicosanoids; Histamine Release; Humans; Imidazoles; Inflammation; Leukotriene B4; Male; Mast Cells; Mice; Mice, Inbred BALB C; Monocytes; Nadolol; Naproxen; Neutrophils; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Pyrazoles; Pyrrolidinones; Receptors, Adrenergic, beta; Rolipram; SRS-A; Thiazoles

The effect of ibudilast on anaphylactic bronchoconstriction was studied in guinea pigs sensitized actively with ovalbumin (OA). Animals were treated with indomethacin, tripelennamine and propranolol prior to the antigen challenge. Anaphylactic bronchoconstriction was prevented by ibudilast (1-4 mg/kg i.v. and 5-20 mg/kg p.o.) dose-dependently. FPL55712 and phenidone were also effective. Even when administered at the maximum development of bronchoconstriction, ibudilast (0.5 and 2 mg/kg i.v.) and FPL 55712 caused significant reduction of the increased airway tone, while phenidone did not. Ibudilast (1-4 mg/kg i.v.) and FPL55712 inhibited leukotriene D4-induced airway responses in nonsensitized guinea pigs pretreated with indomethacin and propranolol. Ibudilast (1.6 and 4 mg/kg i.v.) inhibited platelet-activating-factor (PAF)-induced airway responses in nonsensitized guinea pigs pretreated with indomethacin and propranolol, however, FPL 55712 inhibited PAF-induced airway responses only at a high dose such as 10 mg/kg i.v. Ibudilast (4 mg/kg i.v.) did not inhibit acetylcholine-induced airway response. Ibudilast showed inhibition of the release of slow-reacting substance of anaphylaxis (SRS-A) from guinea pig chopped lung sensitized with OA, which was significantly diminished by indomethacin. The drug little affected the activity of phospholipase A2 and 5-lipoxygenase in guinea pig polymorphonuclear leukocytes. These results indicate that ibudilast inhibits anaphylactic bronchoconstriction which is considered to be largely mediated by endogenously released SRS-A. The inhibitory effect of ibudilast on anaphylactic bronchoconstriction in the presence of indomethacin is considered to be exerted through its antagonism to SRS-A.

Topics: Acetylcholine; Anaphylaxis; Animals; Arachidonate 5-Lipoxygenase; Asthma; Bronchoconstriction; Bronchodilator Agents; Calcimycin; Chromones; Guinea Pigs; Male; Neutrophils; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Pyrazoles; Pyridines; SRS-A

Intraperitoneal and intra-articular (knee joint) injection of zymosan in the rat caused two phases of increased vascular permeability, a rapid increase (0.25-0.5 h) and a secondary increase (2-3 h) which was temporally associated with the onset of leukocyte infiltration. Intraperitoneal injection of zymosan led to a single peak of eicosanoid production (LTB4, C4, D4, E4 and 6-oxo-PGF1 alpha) which was maximal at 0.125-0.25 h. Intra-articular injection led to an initial peak of LTB4 production (maximal at 0.25 h) and a secondary peak of LTB4 and PGE2 production (maximal at 3 h). Oral administration of the 5-lipoxygenase (5-LO) inhibitors phenidone, BW A4C (N-hydroxy-N-[3-(3-phenoxyphenyl)-2-propenyl] acetamide), A63162 (N-hydroxy-N-[1-(4-(phenylmethoxy) phenyl)ethyl] acetamide and ICI 207 968 (2-[3-pyridylmethyl]-indazolinone inhibited LTB4 production in A23187 stimulation blood ex vivo. The glucocorticosteroid dexamethasone had no effect in this model. The initial phase of increased vascular permeability in the peritoneal cavity and LTB4 production was dose dependently inhibited by the 5-LO inhibitors phenidone, BW A4C, A63162, and ICI 207 968 but not by dexamethasone or colchicine. The initial phase of increased permeability in the joint was unaffected by phenidone, BW A4C, dexamethasone or colchicine. However the latter two drugs inhibited the later phase of increased permeability and leukocyte infiltration in the joint and peritoneal cavity. These results demonstrate that zymosan induces eicosanoid production in vivo but the relative importance of these mediators varies depending on the inflammatory site.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arthritis; Benzeneacetamides; Calcimycin; Colchicine; Dexamethasone; Dinoprostone; Disease Models, Animal; Hydroxamic Acids; Inflammation; Kinetics; Knee Joint; Leukocytes; Leukotriene B4; Leukotrienes; Lipoxygenase Inhibitors; Male; Peritonitis; Pyrazoles; Rats; Zymosan

We compared the effects of the leukotriene (LT) D4 receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 microM), and by arachidonic acid (AA), melittin and LTD4. FPL55712 (0.1 and 1 microM) inhibited contractions induced by AA (100 microM) and the phospholipase A2 activator melittin (3 micrograms/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM) was a more effective inhibitor of the melittin response than the AA response. FPL55712 inhibited contractions induced by OA (100 micrograms/ml) more than by A23187 (1 microgram/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC4 conversion to LTD4. NDGA (10 microM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 microM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 microM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD4-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD4-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Animals; Antigens; Arachidonate Lipoxygenases; Calcimycin; Chromones; Guinea Pigs; In Vitro Techniques; Male; Masoprocol; Muscle Contraction; Pyrazoles; Receptors, Immunologic; Receptors, Leukotriene; SRS-A; Trachea

The role of arachidonic acid metabolism in the efflux of intracellular enzymes from damaged skeletal muscle has been examined in vitro using inhibitors of cyclo-oxygenase and lipoxygenase enzymes. Damage to skeletal muscle induced by either calcium ionophore A23187 (25 microM) or dinitrophenol (1 mM) caused an increase in the efflux of prostaglandins E2 and F2 alpha together with a large efflux of intracellular creatine kinase. Use of a cyclo-oxygenase inhibitor completely prevented the efflux of prostaglandins, but had no effect on creatine kinase efflux. However, several agents having the ability to inhibit lipoxygenase enzymes dramatically reduced creatine kinase efflux following damage. These data suggest that a product or products of lipoxygenase enzymes may be mediators of the changes in plasma membrane integrity which permit efflux of intracellular enzymes as a consequence of skeletal muscle damage.

Topics: 2,4-Dinitrophenol; 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Creatine Kinase; Cyclooxygenase Inhibitors; Dinitrophenols; Female; In Vitro Techniques; Masoprocol; Muscles; Muscular Diseases; Phenylacetates; Prostaglandins; Pyrazoles; Rats; Rats, Inbred Strains

The subcutaneous sponge implant model of acute inflammation in the rat has been evaluated as a suitable test system for evaluating the potential anti-inflammatory efficacy of 5-lipoxygenase inhibitors. The inflammatory parameters measured were exudate volume and leukocyte recruitment. Specific radioimmunoassays were used to measure (1) 5-lipoxygenase (LPO) and cyclo-oxygenase (CO) activity in exudate leukocytes stimulated ex vivo with A23187, and (2) the LTB4 and PGE2 content of inflammatory exudate. The NSAIDs flurbiprofen and indomethacin inhibited cell recruitment, exudate volume and CO activity with ED50S of approximately 1 mg per kg p.o. but failed to inhibit LPO activity at 10 mg per kg p.o. Nafazatrom (Bayer 6575), quercetin and NDGA, which inhibit LPO activity in vitro, were inactive against all parameters when dosed at 100 mg per kg p.o. The "mixed inhibitors" BW755C and phenidone were approximately equipotent inhibitors of LPO activity but BW755C was 10 times more potent than phenidone against CO activity. BW755C was also greater than 10 times more potent at inhibiting cell recruitment and exudate volume than phenidone suggesting that the anti-inflammatory efficacy of the mixed inhibitors reflect their potency against CO rather than LPO activity. Time course studies demonstrated that the inhibitor effects of BW755C and phenidone on leukocyte recruitment reflected a reduction in the PGE2 but not the LTB4 content of the inflammatory exudate. Polyester sponges soaked in high concentrations of LTB4 caused only a modest (2-fold) increase in leukocyte recruitment whilst physiological levels were inactive. The results taken together suggest that CO products make a major contribution to leukocyte recruitment in this model whilst the LPO product LTB4 has little role. This model therefore is of little value for evaluating the anti-inflammatory efficacy of 5-lipoxygenase inhibitors. Moreover, the rat would appear to be unsuitable for evaluating the role of LTB4 in acute inflammation.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Acute Disease; Animals; Blood Proteins; Calcimycin; Chemotaxis, Leukocyte; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Flurbiprofen; Indomethacin; Inflammation; Leukocytes; Leukotriene B4; Lipoxygenase Inhibitors; Male; Prostaglandins E; Pyrazoles; Pyrazolones; Rats; Rats, Inbred Strains; Skin

The effects of inhibitors of the cyclooxygenase and lipoxygenase pathways of arachidonic acid (AA) metabolism were studied on the contractions of guinea pig tracheal spirals and lung parenchymal strips induced by antigen and calcium ionophore A23187. Inhibition of the cyclooxygenase pathway with indomethacin results in enhancement of antigen- and A23187-induced contraction of trachea which is a result of diversion of AA into the lipoxygenase pathway and inhibition of modulatory cyclooxygenase products. Indomethacin does not enhance parenchymal contractions but partly inhibits contraction induced by a strong stimulus (5.7 microM A23187 or 100 micrograms/ml ovalbumin). Parenchymal contraction is inhibited by agents that block the lipoxygenase pathway of AA metabolism, phenidone and nordihydroguaiaretic acid. These agents inhibit the prolonged phase of antigen-induced tracheal contraction but in some cases enhance the early phase of tracheal contraction induced by antigen or A23187. The results suggest that contraction of parenchyma induced by antigen or A23187 are the result of a bronchoconstrictor lipoxygenase product and only have a cyclooxygenase bronchoconstrictor component following a strong stimulus. In contrast, contraction of trachea also appears to be the result of a bronchoconstrictor lipoxygenase product which may not be identical to that contracting parenchyma, and is modulated by cyclooxygenase products. The results emphasize the importance of comparing small and large airways to further our understanding of asthmatic broncho-constriction.

Topics: Airway Resistance; Animals; Anti-Bacterial Agents; Antigens; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cyclooxygenase Inhibitors; Guinea Pigs; In Vitro Techniques; Lipoxygenase Inhibitors; Male; Ovalbumin; Pyrazoles; Trachea

Ovalbumin (OA) and the calcium ionophore A23187 induced a dose-dependent contraction of guinea pig tracheal strips. The OA-induced contraction (of sensitized trachea) consisted of an initial peak concentration, maximal between 5 and 10 min, followed by a very gradual decline from the peak. On the other hand, A23187 induced a sustained contraction of the trachea with a more gradual onset. Both antigen- and A23187-induced contractions required the presence of extracellular calcium. The response was not reduced by delaying (up to 10 min) the addition of calcium, suggesting that the mechanism of antigen-induced contraction differs from that of antigen-induced histamine secretion from rat mast cells and human basophils. The 1st min of the OA-induced contraction was inhibited significantly by mepyramine (10(-5) M) suggesting that histamine contributed to the contraction at this time point. In contrast, A23187-induced contraction was unaffected by mepyramine. On the other hand, both the A23187-induced contraction and the prolonged phase of the OA-induced contraction were enhanced by indomethacin, a cyclooxygenase inhibitor, and inhibited by phenidone, a cyclooxygenase-lipoxygenase inhibitor. This suggests that a product of the lipoxygenase pathway of arachidonic acid metabolism contributes to OA- and A23187-induced contraction of the guinea pig trachea.

Topics: Animals; Anti-Bacterial Agents; Antigens; Calcimycin; Calcium; Contracture; Guinea Pigs; Indomethacin; Male; Ovalbumin; Pyrazoles; Pyrilamine; Time Factors; Trachea