A B S T R A C T Metabolism of arachidonic acid (AA) via the cyclooxygenase pathway reduces glucose-stimulated insulin release. However, metabolism of AA by the lipoxygenase pathway and the consequent effects on insulin secretion have not been simultaneously assessed in the endocrine islet. Both dispersed endocrine cell-enriched pancreatic cells of the neonatal rat, as well as intact islets of the adult rat, metabolized [3H]AA not only to cyclooxygenase products (prostaglandins E2, F2a, and prostacyclin) but also to the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE). 12-HETE was identified by coelution with authentic tritiated or unlabeled 12-HETE using four high performance liquid chromatographic systems under eight mobile-phase conditions and its identity was confirmed by gas chromatography/mass spectrometry using selected ion monitoring. The predominant effect of exogenous AA (5 gg/ml) was to stimulate insulin release from pancreatic cells grown in monolayer. This effect was concentration-and time-dependent, and reversible. The effect of AA upon insulin release was potentiated by a cyclooxygenase inhibitor (indomethacin) and was prevented by either of two lipoxygenase inhibitors (5,8,11,14-

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) and ion trap mass spectrometry have been used to study the fragmentation behavior of native peptides and peptide derivatives prepared for de novo sequencing applications. Sulfonic acid derivatized peptides were observed to fragment more extensively and up to 28 times more efficiently than the corresponding native peptides. Tandem mass spectra of native peptides containing aspartic or glutamic acids are dominated by cleavage on the C-terminal side of the acidic residues. This significantly limits the amount of sequence information that can be derived from those compounds. The MS/MS spectra of native tryptic peptides containing oxidized Met residues show extensive loss of CH(3)SOH and little sequence-specific fragmentation. On the other hand, the tandem mass spectra of derivatized peptides containing Asp, Glu and oxidized Met show much more uniform fragmentation along the peptide backbone. The AP-MALDI tandem mass spectra of some derivatized peptides were shown to be qualitatively very similar to the corresponding vacuum MALDI postsource decay mass spectra, which were obtained on a reflector time-of-flight instrument. However, the ion trap mass spectrometer offers several advantages for peptide sequencing relative to current reflector time-of-flight instruments including improved product ion mass measurement accuracy, improved precursor ion selection and MS(n). These latter capabilities were demonstrated with solution digests of model proteins and with in-gel digests of 2D-gel separated proteins.

The three-dimensional quadrupole ion trap was first described by Paul and Steinwedel several years ago. ' The development of the mass-selective instability mode of operation, in combination with the use of a lowmolecular-weight buffer gas at pressures around lop3 Torr, has led to significant improvements in mass range, resolution and sensitivity.2 Recently, it has been demonstrated that chemical ionization (CI) and MS/MS are possible with the same basic electrode s t r u~t u r e ,~'~ without the need for specific ion sources or additional analyzers. Depending on the nature of the parent ion, collisionally activated dissociation efficiencies approaching 100% can be a~h i e v e d .~ Recent attempts to study selectivity effects in GC/MS/MS assays of prostaglandins using a reversedgeometry sector instrument (referred to as the B-E instrument) were hindered by the low efficiency of the CAD process. This limited the instrumental response obtainable in certain selected-reaction-monitoring (SRM) experiments on biological samples. Continuing evaluations of how selectivity of analysis was affected by the type of fragmentation chosen, as well as other parameters, were limited. It was therefore decided to investigate the MSIMS analysis of prostaglandins using the ion trap mass spectrometer (ITMS). The types of fragmentation observed and the daughter ion intensities were compared to results obtained on a B-E sector mass spectrometer.' Sensitivity in the GC/MS/MS mode was also evaluated using an ammonia-CI source in the first MS stage. Preliminary results are reported herein. EXPERIMENTALThe derivatization of prostaglandin E2 as the methyl ester, 0-methoxime, bis-TMS ether derivative (PGE2-MeMoxTMS2) and MS/MS analysis on the B-E instrument [ZAB-2F, Vacuum Generators, Altrincham, UK] have already been described.' A description of the ion trap mass spectrometer [ITMS, Finnigan-MAT, San Jose, CA, USA] prototype has recently been p u b l i~h e d .~ The instrument used for these studies is a modification of the prototype, and is described in detail in publications from the manufacturer. The basic methods of preparing and admitting the sample are the same for the ZAB-2F and the ITMS. In both cases, the ' Author to whom correspondence should be addressed.daughter ion spectra were obtained by direct probe analysis of standards. In the case of the B-E instrument, a 2 pg sample (based on initial PGE2 concentration) was analyzed. On the ITMS, about 100ng was used with a constant probe temperature of 60°C. The ion trap cell was kept at 105°C. G C conditions were: 15m DB-5 (0.25pm film), He carrier gas (30cm/s), 1 pL splitless injection, 60" (2 min), then 30"/min to 310" (10 min).On the ITMS, MS/MS analyses were carried out with a new scap function (Fig. 1). The stability diagram is shown in Fig. 2. It is important to remember that higher masses have lower qz values and that stable trajectories (ions stored) correspond to the cross-hatched region.Electron or ammonia-chemical ionization at relatively low-amplitude, radiofrequency (RF, 1....

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Because deformation of lung tissue stimulates prostaglandin synthesis, we wanted to investigate whether hydrodynamic forces would affect lung prostacyclin (PGI2) production. To test the hypothesis that lung prostacyclin synthesis was flow dependent, we examined lung prostacyclin production after flow alterations. Using a salt solution that contained either Ficoll or albumin as a perfusate, we changed the flow to half and to double the control flow. When flow was changed, lung prostacyclin production followed changes in flow and pressure drop. When flow was varied in lungs treated with indomethacin, prostacyclin production was too low to be measurable. Variations in pressure pulsatility at constant mean flow had no influence on lung prostacyclin production. Since vascular distension may also stimulate prostacyclin production, we increased venous pressure. An increase in venous pressure (from 2.1 to 4.8 mmHg) had no effect on prostacyclin production; a further increase in venous pressure (to 7.5 mmHg) initiated edema and caused a large increase in prostacyclin production. When we subjected monolayers of endothelial cells cultured in wells to defined shear rates, the prostacyclin concentration in the supernatant quickly increased to a maximum. The absence of further increase with greater shear may have reflected feedback control of prostacyclin synthesis. The results indicated that hydrodynamic disturbances affect endothelial cells and stimulate arachidonate metabolism. Lung prostacyclin production may be related to flow. However, this effect is small compared with the lung prostacyclin production during edema formation.