1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone(AHR-5333): a selective human blood neutrophil 5-lipoxygenase inhibitor

In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 microM, respectively. Further examination of the effect of AHR-5333 on individual reactions of the 5-lipoxygenase pathway (i.e. conversion of LTA4 to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 epoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 microM) to 5-HETE was half maximally inhibited by 20 microM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformation of exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparations of sheep vesicular glands.     

Cardiorespiratory responses to systemic administration of a protein kinase C inhibitor in conscious rats

Gozal, David, Gavin R. Graff, José E. Torres, SanjayG. Khicha, Gautam S. Nayak, Narong Simakajornboon, and Evelyne Gozal. Cardiorespiratory responses to systemic administration of aprotein kinase C inhibitor in conscious rats. J. Appl.Physiol. 84(2): 641-648, 1998.Although proteinkinase C (PKC) is an essential component of multiple neurally mediatedevents, its role in respiratory control remains undefined. Theventilatory effects of a systemically active PKC inhibitor (Ro-32-0432;100 mg/kg ip) were assessed by whole body plethysmography duringnormoxia, hypoxia (10% O₂), andhyperoxia (100% O₂) inunrestrained Sprague-Dawley rats. A sustained expiratory time increaseoccurred within 8-10 min of injection in room air[mean 44.8 ± 5.2 (SE) % ], was similarto expiratory time prolongations after Ro-32-0432 administration during100% O₂ (45.5 ± 8.1%; not significant), and was associated with mildminute ventilation (E) decreases.Hypercapnic ventilatory responses (5%CO₂) remained unchanged afterRo-32-0432. During 10% O₂,E increased from 122.6 ± 15.6 to 195.7 ± 10.1 ml/min in vehicle-treated rats(P < 0.001). In contrast, markedattenuation of E hypoxic responsesoccurred after Ro-32-0432 [86.2 ± 6.2 ml/min inroom air to 104.1 ± 7.1 ml/min in 10%O₂; pre- vs. post-Ro32-0432, P < 0.001 (analysis ofvariance)]. Overall, PKC activity was reduced and increases withhypoxia were abolished in the particulate subcellular fraction of brain tissue after Ro-32-0432 treatment, indicating thatthis compound readily crosses the blood-brain barrier. We conclude thatsystemic PKC inhibition elicits significant centrally mediatedexpiratory prolongations and ventilatory reductions as well as bluntedventilatory responses to hypoxia but not to hypercapnia. Wepostulate that PKC plays an important role in signal transduction pathways within brain regions underlying respiratory control.

     

Neuropeptides in coelenterates: a review

Coelenterate neurones produce peptides containing an Arg-Phe-NH₂(RF-amide)-like carboxyterminus. RF-amide-like peptides are located in neuronal dense-cored vesicles, indicating that they are released by exocytosis and that they might function as neurotransmitters or neurohormones. Using a radioimmunoassay for the sequence RF-amide, 3 peptides were isolated from the sea anemone Anthopleura elegantissima: < Glu-Gly-Arg-Phe-NH₂(Antho-RF-amide), 2(Antho-RWamide I) and 2(Antho-RW-amide II). The general structure of these peptides can be described as 2, where X is an aromatic amino acid. From the hydromedusa Polyorchis penicillatus, the peptide 2(Pol-RF-amide I) was isolated, which also belongs to the 2 family. Using specific antisera, it was shown that all 4 peptides were located in neurones, many of which were associated with smooth muscle fibres. Application of low doses of Antho-RF-amide or of Antho-RW-amide I and II induced contractions of endodermal muscles of sea anemones. This suggests that these peptides are transmitters or modulators at neuromuscular junctions.     

Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C

We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.     

Preparation of phosphatidyl[2-3H]inositol from yeast grown in medium containing myo[2-3H]inositol

Phosphatidyl[2-3H]inositol was prepared from Saccharomyces cerevisiae (YSC-2), grown in synthetic medium containing myo[2-3H]inositol. Over 44 microCi (or 81%) of the radiolabeled inositol was taken up by the organism, with 34 microCi incorporated into phosphatidylinositol. Upon purification by silicic acid pressure liquid chromatography (MPLC), a final yield of 24 to 26 microCi of phosphatidyl[2-3H]inositol with a specific radioactivity of 40 X 10(3) dpm/nmole was obtained. The purified phosphatidyl[2-3H]inositol was found to be a suitable for phospholipase C from human platelets.     

Dispersion of Small Ceramic Particles (Al(2)O(3)) with Azotobacter vinelandii

The high surface charge of small ceramic particles such as alumina particles prevents them from dispersing evenly in aqueous suspensions and forming high-density compacts. However, suspensions of 400-nm-diameter alumina particles treated with alginate from the bacterium Azotobacter vinelandii were well dispersed. The alginate bound firmly to the particle surface and could not be removed by repeated washing with distilled water (2.82 mg of the bacterial alginate adsorbed to 1 g of the alumina particles). Furthermore, A. vinelandii grew and produced alginate in the presence of up to 15% (vol/vol) alumina particles. These results suggest that an in situ process using this bacterium to coat ceramic particles with alginate might be developed. In in situ processing experiments, the particle-packing densities were significantly increased and the viscosities of 5 and 10% (vol/vol) suspensions were reduced 4- and 60-fold, respectively, over those of controls. The bacteria were readily removed from the alumina particles by washing.