Pseudomonas aeruginosa culture filtrates varied in their ability to slow human ciliary beat frequency (7-71%). This activity did not correlate with known virulence factors. However, a close correlation (r = 0.97) existed between ciliary slowing and pigment content. In a prolonged culture, the increase in activity correlated (r = 0.94) with pigment accumulation. Gel filtration of lyophilized filtrate yielded a single peak of activity corresponding to the pigment fraction. Pyocyanin extracted from an active strain, and 1-hydroxyphenazine were purified by high performance liquid chromatography, and characterized by ultraviolet absorbance spectra and mass spectrometry. Both slowed cilia in a dose-dependent manner, and were synthesized and shown to be indistinguishable from the biological compounds. Pyocyanin caused gradual onset of slowing and ultimate widespread ciliostasis with epithelial disruption. 1-hydroxyphenazine caused rapid onset of ciliary slowing associated with dyskinesia and ciliostasis. Pyocyanin assayed within filtrates accounted for a significant proportion of the bioactivity present.

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) -EJB 86 03401. Pyocyanin and related members of the phenazine family are produced by Pseudomonas aeruginosa and have been associated with events of pathophysiological importance.2. Pyocyanin and its base hydrolysis product 1 -hydroxyphenazine were purified to homogeneity by reversephase high-pressure liquid chromatography. Their mass spectrometric behaviour was examined with a view to evaluating the use of high-resolution chromatography/mass spectrometry in studying phenazine-mediated effects in man.3. The molecular mass of naturally derived pyocyanin was determined as 210 Da by thermospray liquid chromatography/mass spectrometry and confirmed by desorption electron-impact mass spectrometry. Mass spectrometric data could not be obtained by fast-atom bombardment or desorption chemical ionisation, techniques commonly used to determine molecular mass of polar or thermally labile species. The thermal lability of underivatised pyocyanin precluded analysis by gas chromatography/mass spectrometry.4. In contrast to pyocyanin, mass spectrometric data were readily obtained for 1-hydroxyphenazine, using direct probe analysis as well as with gas and liquid chromatography inlet systems.The phenazines have been known for over 60 years [l]; they have been synthesised and their physico-chemical properties studied [2-61. There have only been a small number of reports on their biological actions [7 -1 I], partly because of the lack of purification procedures applicable to complex biological samples, and partly due to the difficulties of analysis. Although mass spectrometric data has been reported for many of the phenazines, pyocyanin, perhaps the most interesting member of the family, yields complex mass spectrometric data only with difficulty.Procedures applicable to the high-resolution purification of pyocyanin and other phenazines are reported here in full. Definitive mass spectrometric examination (including thermospray liquid chromatography/mass spectrometry, applied for the first time in this field) has facilitated the characterisation of these species in Pseudomonas aeruginosa cultures. EXPERIMENTAL PROCEDURES Chemicals3-Methoxycatechol, o-phenylenediamine and lead dioxide were purchased from Aldrich Chemical Co. Ltd (Gillingham, UK); 3,5-bis(trifluoromethyl)benzoyl chloride was obtained from Fluorochem Ltd (Glossop, UK) and octane and diisopropylethylamine from Sigma London Chemical Co. Ltd (Poole, UK). Diisopropylethylamine was dried over calcium hydride prior to use. Ethyl acetate was Analar grade and was distilled from and stored over calcium hydride.Solvents for chromatography (acetonitrile, propan-2-01) were of high-pressure liquid chromatography (HPLC (Rathburn, Walkerburn, UK); water was of Milli-Q quality. Aristar acetic acid and trifluoroacetic acid were obtained from BDH (UK) and AR grade ammonium acetate from May & Baker, UK. Biological samplesPs. aeruginosa isolates were cultured on Kings A agar and the organisms washed from the surface. Phenazines were extracted into chloroform and crude pyocyanin obtained ...

Serotonin activates adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of NCB-20 neuroblastoma-brain hybrid cells with an activation constant of 530 nM, but has little or no effect on cellular cyclic AMP or cyclic GMP content of (4,5). Serotonin also induces contractions in ileal smooth muscle (6). However, central neuronal responses to serotonin in mammals are predominantly inhibitory and are mediated through either pre-or postsynaptic receptors, which can be distinguished by the differences in their responses to D-lysergic acid diethylamide (LSD) (5, 7). In molluscan neurons, as many as six kinds of response to serotonin have been detected (8).Serotonin-dependent activation of adenylate cyclase is preserved after electrolytic lesion of the raphe nuclei, which greatly reduces the number of serotonergic neurons innervating the colliculus. This suggests that collicular serotonin receptors that mediate activation of adenylate cyclase are postsynaptic receptors (2).In this report, effects mediated by two species of serotonin receptor in NCB-20 neuroblastoma-brain hybrid cells are described. One receptor is coupled to the activation of adenylate cyclase, and the other mediates cell depolarization and acetylcholine release. The receptors are distinguished on the basis of functional response, specificity, and desensitization.MATERIALS AND METHODS Cells. The N1E-1 15 adrenergic neuroblastoma cell line was cloned from C-1300 mouse neuroblastoma (9). The NG108-15 neuroblastoma-glioma hybrid cell line (unpublished results) was derived by Sendai virus-induced fusion of C-1300 mouse neuroblastoma clone N18TG2 resistant to 6-thioguanine (10) with rat glioma clone C6BU-1, resistant to 5-bromodeoxyuridine (11). The NCB-20 neuroblastoma-fetal Chinese hamster brain hybrid cell line (12) Adenylate Cyclase Assay. Cells were cultured as described (13). Confluent cells were detached from flasks by gentle tapping and were washed three times, each with 10 ml of Dulbecco's phosphate-buffered saline (without Ca2+ or Mg2+ ions) adjusted to 340 mOsm with NaCl. Cells were recovered by centrifugation (150 X g for 5 min) after each wash, and after the third wash were suspended in 25 mM Tris-HCl, pH 7.4/290 mM sucrose (2 ml/75-cm2 flask) and homogenized at 4°C with 50 strokes of a Dounce homogenizer. Homogenates were frozen and stored in a liquid N2 freezer. Each 100-Al reaction mixture contained 50 mM Tris-HCl (pH 7.4); 87 mM sucrose; 20 mM creatine phosphate, disodium salt (Sigma); 10 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisenment" in accordance with 18 U. S. C'. §1734 solely to indicate this fact.