J.R.S. Hoult

EFFECTS OF SULPHASALAZINE AND ITS METABOLITES ON PROSTAGLANDIN SYNTHESIS, INACTIVATION AND ACTIONS ON SMOOTH MUSCLE

1 We have investigated the effects of sulphasalazine and of its principal colonic metabolites (5‐aminosalicylic acid and sulphapyridine) on prostaglandin inactivation, synthesis and actions on gastrointestinal smooth muscle. 2 Sulphasalazine inhibits prostaglandin F2α breakdown in 100,000 g supernatants in all organs so far tested from 7 species with an ID50 of approx. 50 μm; it has a selective action on prostaglandin 15‐hydroxydehydrogenase and does not inhibit prostaglandin Δ‐13 reductase, prostaglandin 9‐hydroxydehydrogenase or ‘enzyme X’ at millimolar concentrations. Enzyme activities were measured radiochemically or by bioassay. 3 Sulphapyridine and 5‐aminosalicylic acid do not inhibit prostaglandin inactivation in vitro (4 species tested). A methyl analogue of sulphasalazine is a more potent inhibitor than the parent compound. Rabbit colon prostaglandin F2α metabolism in vitro was inhibited by the following drugs with ID50 values (μm) of: diphloretin phosphate 20, sulphasalazine 50, indomethacin 220, frusemide 1000 and aspirin 10,000. A similar rank order of potencies was obtained with rabbit kidney. 4 Sulphasalazine at 50 to 100 μm inhibited inactivation of prostaglandin E2 in the perfused rat and guinea‐pig lung by 3 to 40% (rat) and 32 to 100% (guinea‐pig) when measured by superfusion cascade bioassay and of prostaglandin F2α by 43.6 ± 6.5% in rat lung perfused with 50 μm sulphasalazine and assayed radiochemically. 5 Prostaglandins E1 and E2 were 97.0 ± 8.2% and 92.3 ± 6.8% inactivated in the lungs after intravenous injection in the anaesthetized rat as measured by reference to their vasodepressor potencies when injected intra‐arterially. Prostaglandin A2 was not similarly inactivated. Pulmonary inactivation was prevented in the presence of an intravenous infusion of 16.3 μg kg−1 min−1 sulphasalazine and partially inhibited at a lower infusion rate. 6 Prostaglandin biosynthesis from arachidonic acid was measured in microsomal preparations from four sources by bioassay and radiochemical methods. Indomethacin was a potent inhibitor (ID50 0.8 to 4.1 μm) but sulphasalazine and its methyl analogue were very weak inhibitors (ID50 1500 to > 5000 μm), 5‐aminosalicylic acid was weaker still and sulphapyridine inactive. 7 Sulphasalazine at 50 μm did not affect the actions of prostaglandins on five smooth muscle preparations; at 500 μm there was a rapidly reversible and probably non‐specific antagonism of responses to low doses of prostaglandins. 8 The specificity and selectivity of the interaction of sulphasalazine and its metabolites with the formation, breakdown and actions of prostaglandins are discussed.

SULPHASALAZINE IS A POTENT INHIBITOR OF PROSTAGLANDIN 15‐HYDROXYDEHYDROGENASE: POSSIBLE BASIS FOR THERAPEUTIC ACTION IN ULCERATIVE COLITIS

Sulphasalazine is a potent and selective inhibitor in vitro of prostaglandin 15‐hydroxydehydrogenase in rabbit colon (ID5() = 50 μm) and in several other organs of different species, but does not inhibit prostaglandin A‐13 reductase or microsomal prostaglandin synthesis from arachidonic acid. It is suggested that this action may underly the therapeutic usefulness of sulphasalazine in ulcerative colitis for the prevention of relapse.

Pathways of prostaglandin F2α metabolism in mammalian kidneys

1 High‐speed cytoplasmic supernatants of rat, rabbit, pig and guinea‐pig kidneys were prepared and the metabolism of 10 μg/ml prostaglandin F2α labelled with [3H1‐9β]‐prostaglandin F2α studied by thin layer radiochromatography and bioassay. 2 The metabolism of prostaglandin F2α measured by radiochromatography parallels biological inactivation in all species except the rabbit. 3 Kidneys metabolize prostaglandin F2α by two divergent pathways, yielding a mixture of prostaglandin E and F metabolites. 4 15‐Hydroxyprostaglandin dehydrogenase and prostaglandin Δ‐13 reductase are present in all species in characteristic proportions. Thus prostaglandin F2α is metabolized sequentially to 15‐keto prostaglandin F2α and 13,14‐dihydro‐15‐keto prostaglandin F2α. The rate and profile of formation of these metabolites is species‐dependent. 5 13,14‐Dihydro‐15‐keto prostaglandin F2α is the principal prostaglandin F series metabolite in all species. 6 Pig and guinea‐pig kidney contain an unidentified enzyme which converts 13,14‐dihydro‐15‐keto prostaglandin F2α to 13,14‐dihydro prostaglandin F2α. 7 Rat kidney contains a high concentration of a prostaglandin 9‐hydroxy dehydrogenase which converts 13,14‐dihydro‐15‐keto prostaglandin F2α to 13,14‐dihydro‐15‐keto prostaglandin E2. 8 Rabbit kidney contains a novel 9‐hydroxydehydrogenase which oxidises prostaglandin F2α directly to E2, thus producing a compound with more potent renal actions. The possible implications of this enzyme for kidney homeostasis are discussed.

Inhibition of human neurophil degranulation by forskolin in the presence of phosphodiesterase inhibitors

The secretory response of cytochalasin B-treated human polymorphonuclear neutrophils to the peptide chemoattractant f-Met-Leu-Phe (FMLP), the calcium ionophore A23187 and other secretagogues was measured by assaying neutrophil supernatants for the granular enzymes beta-glucuronidase and lysozyme. The dose-dependent enzyme secretion in response to 10(-8)-10(-4) M FMLP and A23187 was unaffected by pretreatment with 10-75 microM forskolin (an activator of adenylate cyclase), but inhibited by high concentrations of prostaglandins E1 and E2. The phosphodiesterase inhibitors isobutyl-methyl-xanthine (IBMX), papaverine and Ro 20-1724 dose dependently inhibited enzyme secretion from FMLP- or A23187-treated cells, and this effect was augmented in the presence of 50-75 microM forskolin. Similar results for PGE1, forskolin and forskolin/IBMX combinations were also obtained using leukotriene B4, platelet activating factor and C5a des-Arg as secretagogues. We conclude that the adenylate cyclase system of human neutrophils is activatable by forskolin, but that the regulatory effects of adenylate cyclase stimulants in these cells are greatly attenuated unless cyclic AMP-phosphodiesterases are inhibited. Thus the phosphodiesterase activity of neutrophils may be of functional importance and is relevant to the modulation of neutrophil activity in inflammation.

Platelet activating factor is a potent colonic secretagogue with actions independent of specific PAF receptors.

Several candidate mediators of acute inflammation such as E-type prostaglandins, histamine and bradykinin are potent pro-diarrhoeal colonic secretagogues. They act to increase serosal to mucosal transport of chloride and passive water efflux. We investigated the effects of platelet activating factor (PAF) on muscle-stripped rat colon, measuring transepithelial potential difference (p.d.) and, under voltage clamp conditions, short circuit current (Isc). PAF induced dose-dependent increases in p.d. and Isc, with an approximate EC50 of 1.5 X 10(-10) M; similar concentrations of lyso-PAF had a much smaller but discernible effect. PAF and lyso-PAF both displayed sidedness with serosal application effective and mucosal application ineffective. Inhibitor studies suggest that chloride is the principal ion carrier, but the specific PAF receptor antagonists kadsurenone, L652731, CV 3988 and WEB 2086 did not block the response. Unlike bradykinin, PAF did not cause the release of PGE2 into the serosal bathing fluid, and its action was not attenuated by the cyclo-oxygenase inhibitors piroxicam, mefenamic acid or flurbiprofen. We conclude that PAF has a powerful pro-diarrhoeal secretory action on colonic epithelium which is not mediated by the previously defined PAF receptor(s) and is independent of prostanoid generation.

Secretory effects of kinins on colonic epithelium in relation to prostaglandins released from cells of the lamina propria

1 Sheets of muscle‐stripped rat and rabbit colon with epithelium intact or removed were mounted in Ussing‐type chambers for recording of transepithelial p.d., resistance and short circuit current (Isc), and measurement by radioimmunoassay (RIA) of the release of prostaglandins into serosal and mucosal bathing solutions. 2 In epithelial‐intact preparations prostaglandin E2 (PGE2), PGE1, PGF2α, U46619 and prostacyclin (10−7‐10−6m) caused increases in Isc and transepithelial p.d., in (approximate) descending order of potency. Epithelial‐removed preparations did not exhibit any transepithelial p.d. 3 In epithelial‐intact preparations, lysyl‐bradykinin (LBk) applied serosally but not mucosally caused increased p.d. and release of PGE2 (and to a lesser extent other prostaglandins) into serosal but not mucosal bathing solutions. In epithelial‐removed tissues, responsiveness to LBk was maintained, but it did not exhibit ‘sidedness’, i.e. LBk was effective when applied on either side and PGE2 release occurred into both compartments. 4 Indomethacin and other non steroidal anti‐inflammatory drugs (NSAIDs) abolished the LBk‐induced p.d. and reduced PGE2 release if applied serosally but not mucosally in epithelial‐intact preparations. In epithelial‐removed tissues, indomethacin added to either side abolished prostaglandin release into both compartments. 5 Calcium removal from serosal but not mucosal bathing solution (Ca2+‐free EGTA Krebs) abolished p.d. generation by LBk in epithelial‐intact preparations, and reduced PGE2 release in rabbit but not rat colon. Similarly, in epithelial‐removed preparations, calcium removal did not affect kinin‐induced PGE2 generation in rat but strongly attenuated it in rabbit colon. 6 We conclude that (i) kinins activate the arachidonate cascade principally by interactions with cells in the subepithelial (lamina propria) layer, rather than with the epithelial cells themselves, (ii) PGE2 contributes substantially to the kinin‐induced increase of transepithelial p.d. as a messenger released from kinin‐responsive subepithelial cells and acting on the basolateral pole of the epithelial cells, (iii) the apparent sidedness of colonic epithelium in terms of responses to kinins, NSAIDs and calcium removal is due to the barrier properties of the epithelial cell layer, and (iv) there are differences in calcium sequestration and apparent calcium dependence of prostaglandin biosynthesis between rat and rabbit colonic subepithelial cells.

Divergent effects of co-carcinogenic phorbol esters and a synthetic diacylglycerol on human neutrophil chemokinesis and granular enzyme secretion.

1 The effects of two co‐carcinogenic phorbol esters (phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu)) and a synthetic diacylglycerol (OAG, 1‐oleoyl‐2‐acetyl‐glycerol), which all stimulate protein kinase C, were compared with two inactive phorbol compounds (4α‐phorbol and 4α‐phorbol didecanoate (4α‐PDD)) on three functional properties of stimulated human polymorphonuclear leukocytes (PMNs): release of granular enzymes lysozyme and β‐glucuronidase, chemokinesis, and changes in cytoplasmic free calcium [Ca2+]i. 2 PMA, PDBu and the diacylglycerol, OAG, all caused a dose‐dependent and slow (max by 15 min) release of small amounts of lysozyme with much less β‐glucuronidase and no release of cytoplasmic lactate dehydrogenase. Release was unaffected by removal of extracellular Ca2+. 3 PMA, PDBu and OAG inhibited random movement of the cells, did not cause chemokinesis and induced a slow reduction in the basal [Ca2+]i, as measured by the quin‐2 method. 4 PMA, PDBu and OAG increased the capacity of five independently‐acting stimulants (N‐formyl‐Met‐Leu‐Phe, leukotriene B4, C5a des‐Arg, platelet activating factor and A23187) to cause release of lysozyme and β‐glucuronidase but strongly inhibited PMN chemokinesis induced by the same five agents and reduced the stimulant‐induced increases in [Ca2+]i. 5 PMA was always more potent than PDBu and much more potent than OAG in eliciting these stimulatory or inhibitory effects on human PMNs. In all tests, 4α‐phorbol and 4α‐PDD were inactive. 6 The results confirm that stimulation of the diacylglycerol/protein kinase C system in human PMN, either by active phorbol esters or the synthetic diacylglycerol, causes bidirectional effects on human PMN function. In particular, activation of the C‐kinase causes inhibition of stimulated neutrophil motility, whereas the secretory functions of the cells are enhanced.

Phorbol myristate acetate enhances human polymorphonuclear neutrophil release of granular enzymes but inhibits chemokinesis

1 The effects of the co‐carcinogenic phorbol ester, phorbol myristate acetate (PMA), on N‐formyl‐Met‐Leu‐Phe (FMLP)‐induced human polymorphonuclear leukocyte chemokinesis and release of granular lysozyme and β‐glucuronidase were compared with those of the inactive phorbol didecanoate (PDD). 2 Release of the enzymes was enhanced by PMA but was unaffected by PDD which also had no effect on chemokinesis. 3 In contrast, FMLP‐induced chemokinesis was completely suppressed by PMA in a dose‐dependent fashion (ID50 = 3.5 nM). 4 PMA also inhibited the FMLP‐induced increase in cytoplasmic calcium level, measured by the fluorescent indicator quin‐2. 5 These and other results suggest that although the diacylglycerol/protein kinase C system is involved in the positive regulation of certain neutrophil functions (degranulation and superoxide generation), if it is very powerfully stimulated, as with PMA, it has inhibitory actions on other neutrophil properties such as motility.

Identification of 6‐oxo‐prostaglandin E1 as a naturally occurring prostanoid generated by rat lung

1 The spontaneous release of prostanoids from rat isolated perfused lungs was studied after acid/organic extraction of perfusates by bioassay, radioimmunoassay, thin layer and high performance liquid chromatographic methods and by gas chromatography‐negative ion mass spectroscopy (g.c.‐n.i.m.s.). 2 An acid/organic extractable anti‐aggregatory vasodilator prostaglandin which inhibited the twitch response of the field‐stimulated guinea‐pig vas deferens was released from the Krebs‐perfused rat lung in nanogram amounts similar to those of other detected prostanoids. Parallel biological assay suggested that this prostaglandin had very closely similar pharmacological activity to authentic 6‐oxo‐prostaglandin E1 (6‐oxo‐PGE1), a metabolite of prostacyclin (PGI2) generated by the action of the enzyme 9‐hydroxyprostaglandin dehydrogenase (9‐PGDH). 3 6‐oxo‐PGE1 was identified conclusively in extracts of rat lung perfusate by thin layer chromatography, high performance liquid chromatography and g.c./m.s. combined with bioassay (inhibition of platelet aggregation), and its covalent structure was defined by g.c. negative ion chemical ionization mass spectroscopy. 4 The rank order of spontaneous release of prostanoids (measured by radioimmunoassay) from the perfused rat lung was 6‐oxo‐PGF1α > thromboxane B2 (TXB2) > PGE2 > 6‐oxo‐PGE1 (measured biologically) > PGF2α. Release of all five prostanoids was inhibited by indomethacin, but only that of 6‐oxo‐PGE1 was inhibited by naringenin. 5 Rat lung 100,000 g cytosolic supernatants contained 9‐PGDH activity capable of removing 9β‐tritium from labelled prostacyclin and forming an acid/organic extractable 6‐oxo‐PGE1‐like anti‐aggregatory substance. This 9‐PGDH activity was inhibited by naringenin (IC50 10.3 μM). 6 The relevance of these findings to the possible physiological role of 6‐OXO‐PGE1 in the lung is discussed, and we propose that 6‐oxo‐PGE1 should be accorded the status of a physiologically relevant, naturally occurring metabolite of arachidonic acid.

Kinin-induced prostaglandin release in rat colon does not display serosal/mucosal ‘sidedness' after epithelial removal

Release of prostaglandin E2 (PGE2) from rat colon in response to 1 μM lysylbradykinin (LBk) displayed ‘sidedness’ in preparations with an intact epithelial cell layer (PGE2 release, sensitivity to LBk and inhibition by indomethacin all occurred on the serosal side only). Preparations with histologically‐verified removal of the epithelial layer and which were impermeable to prostaglandins (i.e. intact) continued to demonstrate LBk‐induced PGE2 generation, but this and indomethacin inhibition did not display sidedness. The results show that kinin‐induced PGE2 derives principally from cells in the lamina propria and not from the epithelial cells as previously supposed, and that the apparent sidedness of LBk responsiveness, PGE2 generation and its inhibition by indomethacin results from the barrier property of the epithelial cells and is not indicative of an asymmetric response.