CRTH2 is not involved in the anti-enteropooling effect of PGD2 in the small intestine.

The majority of prostaglandins (PGs) are known to induce intestinal fluid secretion (enteropooling). In contrast, PGD(2) has been demonstrated to inhibit fluid secretion induced by other PGs. This study was aimed to investigate, by the use of selective agonists/antagonists, which type of PGD(2) receptor mediates this inhibitory effect. The DP1 agonist BW245C dose-dependently inhibited the enteropooling effect of 16,16-dimethyl-PGE(2). This inhibition was counteracted by the DP1 antagonist BWA868C. In contrast, the CRTH2 receptor does not seem to be involved in the anti-enteropooling effect of PGD(2), since the selective agonists 13,14-dihydro-15-keto-PGD(2) and 15(R)-15-methyl-PGD(2) were without effect. Therefore, our results suggest that the inhibitory effect of PGD(2) in the small intestine is mediated via activation of the DP1 receptor.

Evidence for a direct stimulatory effect of prostacyclin on renin release in man.

THE OBJECTIVES OF THIS INVESTIGATION WERE: (a) to characterize the time and dose dependence of the effects of prostacyclin (PGI(2)) on renin release in healthy men; (b) to define whether PGI(2)-induced renin release is secondary to hemodynamic changes; (c) to determine the plasma and urine concentrations of 6-keto-PGF(1alpha) (the stable breakdown product of PGI(2)) associated with renin release induced by exogenous or pharmacologically enhanced endogenous PGI(2). Intravenous PGI(2) or 6-keto-PGF(1alpha) infusions at nominal rates of 2.5, 5.0, 10.0, and 20.0 ng/kg per min were performed in each of six normal human subjects; in three of them, PGI(2) infusion was repeated after beta-adrenergic blockade and cyclooxygenase inhibition. PGI(2), but not 6-keto-PGF(1alpha), caused a time- and dose-dependent increase of plasma renin activity, which reached statistical significance at 5.0 ng/kg per min and was still significantly elevated 30 min after discontinuing the infusion. Although combined propranolol and indomethacin treatment significantly enhanced the hypotensive effects of infused PGI(2), it did not modify the dose-related pattern of PGI(2)-induced renin release. Plasma 6-keto-PGF(1alpha) levels rose from undetectable levels (<7.5 pg/ml) in a stepwise fashion during increasingly higher infusion rates of PGI(2) or 6-keto-PGF(1alpha). The threshold concentration of plasma 6-keto-PGF(1alpha) associated with a statistically significant stimulation of renin release was approximately 200 pg/ml. Upon discontinuing PGI(2) or 6-keto-PGF(1alpha) infusion, the disappearance of 6-keto-PGF(1alpha) from blood showed an identical biphasic behavior, the initial phase having an apparent t((1/2)) of 3.2 min. The intravenous infusion of furosemide, which is known to stimulate renin release via a cyclooxygenase-dependent mechanism, caused a three-to fourfold increase of urinary 6-keto-PGF(1alpha) excretion rate, concomitant with the elevation of plasma renin activity levels, in six healthy women. 6-Keto-PGF(1alpha) remained undetectable in peripheral venous plasma throughout the study. WE CONCLUDE THAT IN HUMAN SUBJECTS: (a) PGI(2)-induced renin release occurs with a dose and time dependence similar to its reported platelet effects; (b) PGI(2)-induced renin release is not mediated by adrenergic stimuli or cyclooxygenase-dependent mechanisms secondary to hemodynamic changes; (c) furosemide-induced renin release is associated with increased renal PGI(2) formation; and (d) PGI(2) appears to act as a local modulator rather than a circulating hormone in controlling juxtaglomerular function.

On the metabolism of prostaglandins by human gastric fundus mucosa.

1. Specific radioimmunoassays for the prostaglandins E2, F2alpha and A2 and the metabolites 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2alpha and 13,14-dihydro-15-keto-prostaglandin F2alpha were used to study the metabolism of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric fundus mucosa. 2. Three prostaglandin-metabolizing enzymes were found in the 100 000 X g supernatant of human gastric fundus mucosa, 15-hydroxy-prostaglandin-dehydrogenase, delta13-reductase and delta9-reductase. The specific activity was highest for 15-hydroxy-prostaglandin-dehydrogenase and lowest for delta9-reductase. 3. Formation of prostaglandin A2 (or B2) was not observed under the same conditions. 4. None of the three enzyme activities detected in the 100 000 X g supernatant was found in the 10 000 X g and 100 000 X g pellets of human gastric fundus mucosa. 5. The results indicate that high speed supernatant derived from human gastric mucosa can rapidly metabolize prostaglandin E2 and prostaglandin F2alpha to the 15-keto and 13,14-dihydro-15-keto-derivatives. Furthermore, prostaglandin E2 can be converted to prostaglandin F2alpha, the biological activity of which, on gastric functions, differs from that of prostaglandin E2.

Leukotriene synthesis by human gastrointestinal tissues.

The prostaglandin and leukotriene synthesizing capacity of human gastrointestinal tissues obtained at surgery was investigated using radioimmunoassay for prostaglandin E2, leukotriene B4 and sulfidopeptide leukotrienes. The leukotriene immunoassay data were validated by high-pressure liquid chromatography (HPLC). During incubation at 37 degrees C, fragments of human gastric, jejuno-ileal and colonic mucosa released considerably larger amounts of prostaglandin E2 than of leukotriene B4 and sulfidopeptide leukotrienes. Gastrointestinal smooth muscle tissues released even larger amounts of prostaglandin E2, but smaller amounts of leukotrienes than the corresponding mucosal tissues. Adenocarcinoma tissue released larger amounts of leukotriene B4, sulfidopeptide leukotrienes and prostaglandin E2 than normal colonic mucosa. Ionophore A23187 (5 micrograms/ml) did not stimulate release of prostaglandin E2 from any of the tissues investigated, but enhanced release of leukotriene B4 and sulfidopeptide leukotrienes. HPLC analysis demonstrated that immunoreactive leukotriene B4 co-chromatographed almost exclusively with standard leukotriene B4, while immunoreactive sulfidopeptide leukotrienes consisted of a mixture of leukotrienes C4, D4 and E4. Leukotriene synthesis by human gastrointestinal tissues was inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the dual enzyme inhibitor BW755C (3-amino-1-(trifluoromethylphenyl)-2-pyrazoline hydrochloride). Synthesis of prostaglandin E2 was inhibited by the cyclooxygenase inhibitor indomethacin as well as by BW755C. Incubation of gastrointestinal tissues in the presence of glutathione decreased the amounts of leukotrienes D4 and E4, while release of leukotriene C4 was simultaneously increased. On the other hand, incubation of tritiated leukotriene C4 with incubation media from human gastric or colonic mucosa resulted in conversion of the substrate to [3H]leukotriene D4 and [3H]leukotriene E4. The results indicate the capacity of human gastrointestinal tissues to synthesize the 5-lipoxygenase-derived products of arachidonate metabolism, leukotriene B4 and sulfidopeptide leukotrienes, in addition to larger amounts of prostaglandin E2. Furthermore, considerable activities of the sulfidopeptide leukotriene-metabolizing enzymes gamma-glutamyl transpeptidase and dipeptidase were detected in human gastrointestinal tissues. These enzymes might play an important role in biological inactivation and/or change of biological profile of sulfidopeptide leukotrienes generated in the human gastrointestinal tract.

Skin levels of arachidonic acid-derived inflammatory mediators and histamine in atopic dermatitis and psoriasis.

Since the biochemical events leading to cutaneous inflammation in atopic dermatitis and psoriasis are unknown, we studied the levels of arachidonic acid-derived mediators of inflammation as well as histamine in the suction blister fluid obtained from lesional and nonlesional skin of patients with these dermatoses. Mediator levels were determined radioimmunologically. Skin from healthy controls and uninvolved skin from patients contained very low or unmeasurable levels of the 5-lipoxygenase metabolite of arachidonic acid, leukotriene (LT) B4. In contrast, higher levels of LTB4-like immunoreactivity were detected in suction blister fluid from lesional atopic dermatitis skin, and even higher concentrations occurred in psoriasis lesions. LTB4-like immunoreactivity from atopic dermatitis suction blister fluid cochromatographed on reverse-phase high-pressure liquid chromatography with authentic LTB4, thus excluding cross-reaction of the LTB4-antibody with arachidonic acid or monohydroxyeicosatetraenoic acids. In contrast, suction blister concentrations of the cyclooxygenase metabolite of arachidonic acid prostaglandin (PG) E2 showed no significant differences between lesional and nonlesional patient skin and healthy control skin. PGD2 determined as a stable metabolite could not be detected in these samples. Histamine concentrations in lesional skin were within normal range. The elevated levels of the potent proinflammatory and immunomodulating mediator LTB4 could be involved in the pathogenesis of cutaneous inflammation in atopic dermatitis and psoriasis. In addition, they might explain the therapeutic efficiency of glucocorticosteroids, which among other actions inhibit the release of arachidonic acid from phospholipid stores by blocking the enzyme phospholipase A2. However, the specificity of disease expression in atopic dermatitis and psoriasis must be due to factors other than cutaneous LTB4 elevation.

Radioimmunoassay for leukotriene E4. Use for determination of total sulfidopeptide-leukotriene release from rat gastric mucosa.

A conjugate of leukotriene (LT) E4 and bovine serum albumin (BSA) was prepared by covalently linking the free amino group of the hapten to the protein using dimethyl pimelindiimidate (DMP) as coupling reagent. Anti-LTE4 antibodies were raised in rabbits immunized with the conjugate. Binding of [3H]LTE4 to the antibodies is inhibited by 50% with 0.63 ng LTE4, while the relative cross-reaction of LTC4 and LTD4 is 46.3% and 12.6%, respectively. Using the radioimmunoassay release of sulfidopeptide-LT (SP-LT) from rat gastric mucosa incubated in vitro was determined after quantitative enzymatic conversion of SP-LT to LTE4. It could be demonstrated that this method is suitable for determination of SP-LT in biological material.

Monoclonal antibodies against E- and F-type prostaglandins. High specificity and sensitivity in conventional radioimmunoassays.

Polyclonal antisera against prostaglandins (PGs) are widely used for the assessment of the biological role of these mediators, but even the most specific contain antibodies against the major metabolites and degradation products of the haptens employed. To overcome this inherent problem we produced monoclonal antibodies (mAs) against PGE2, PGF2 alpha and 6-keto-PGF1 alpha using the somatic cell hybridization technique. The mAs against 6-keto-PGF1 alpha and PGF2 alpha proved to be highly specific, but allowed only for moderate detection limits (1-2 ng) in conventional fluid phase radioimmunoassays (RIAs). One of the mAs against PGE2 permitted a 100-fold improvement in the detection limit while being almost devoid of cross-reactivity with metabolites and other structurally related PGs. These results show that highly specific mAs against PGs can be produced to improve the available RIA technique for PG quantification.

Release of leukotriene C4 from human polymorphonuclear leucocytes as determined by radioimmunoassay.

Rabbits were immunized with a conjugate of leukotriene (LT) C4 and bovine serum albumin prepared by coupling the single free amino group of the hapten to the protein using gluteraldehyde. Binding of [3H]LTC4 to the antibodies obtained is inhibited by 50% with 1.5 ng LTC4. The relative cross-reaction of LTD4 is 16% and of LTC4-methyl ester 3.6%. The validity of the radioimmunoassay was demonstrated by comparison with bioassay using the isolated guinea pig ileum. Using the radioimmunoassay it could be shown that endogenous LTC4 is released in a dose-dependent manner by human polymorphonuclear leucocytes stimulated with the divalent cation ionophore A23187.

Effect of endotoxin treatment on the expression of cyclooxygenase-2 and prostaglandin synthases in spinal cord, dorsal root ganglia, and skin of rats.

Peripheral inflammation causes upregulation of cyclooxygenase in the spinal cord and subsequent increase in prostaglandin biosynthesis. However, prostaglandin synthases, which are downstream of cyclooxygenase control the type of prostaglandin that is formed predominantly. Since there is little known about the regulation of prostaglandin synthases, the present study was conducted in order to determine the effect of endotoxin treatment on the expression of messenger RNA encoding interleukin 1beta, cyclooxygenase-2, and prostaglandin synthases mediating the formation of prostaglandin E(2) (membrane bound prostaglandin E synthase) and prostaglandin D(2) (lipocalin prostaglandin D synthase) in spinal cord, dorsal root ganglia and skin of rats. Endotoxin (2 mg/kg i.p.) induced the expression of interleukin-1beta, cyclooxygenase-2, and membrane bound prostaglandin E synthase messenger RNA in spinal cord, dorsal root ganglia, and skin as determined by reverse transcription polymerase chain reaction. In contrast, basal expression of lipocalin prostaglandin D synthase messenger RNA in spinal cord and dorsal root ganglia was not significantly altered by endotoxin. Dexamethasone (1 mg/kg s.c. at -18 h and -1 h) attenuated the effect endotoxin on the expression of interleukin-1beta, cyclooxygenase-2, and membrane bound prostaglandin E synthase messenger RNA in all tissues investigated, but did not significantly influence expression of lipocalin prostaglandin D synthase mRNA in spinal cord and dorsal root ganglia. In situ hybridisation histochemistry showed endotoxin-induced expression of cyclooxygenase-2 and membrane bound prostaglandin E synthase messenger RNA throughout gray and white matter of spinal cord sections. In dorsal root ganglia, expression of membrane bound prostaglandin E synthase seemed primarily located to non-neuronal cells, while cyclooxygenase-2 messenger RNA was not detectable. The results show that the immune response elicited by endotoxin induced cyclooxygenase-2 and membrane bound prostaglandin E synthase, but not lipocalin prostaglandin D synthase messenger RNA in spinal cord and dorsal root ganglia of rats. The distribution of cyclooxygenase-2 and membrane bound prostaglandin E synthase messenger RNA expressing cells suggests major involvement of non-neuronal cells in spinal prostaglandin biosynthesis. Determination of the regulation of enzymes downstream of cyclooxygenase at the messenger RNA level may represent a valuable tool to investigate effects of analgesic/anti-inflammatory drugs on the regulation of spinal prostaglandin biosynthesis.

The effects on platelet aggregation and prostanoid biosynthesis of two parenteral analgesics: ketorolac tromethamine and dipyrone.

The pharmacokinetics and effects on platelet function of dipyrone (1.0 g; 2.5 g; i.v.) and ketorolac tromethamine (30 mg; i.m.) were studied in a three-way crossover study in twelve healthy subjects. The biosynthesis of thromboxane A2 in clotting whole blood ex vivo as well as collagen-induced platelet aggregation were determined before and up to 48 h after administration. Both prostanoid biosynthesis and platelet aggregation were inhibited by ketorolac tromethamine for a significantly longer period of time than by both doses of dipyrone. The changes in platelet functions correlated well with the serum concentrations of ketorolac or 4-methylaminoantipyrine and 4-aminoantipyrine. Using the sigmoidal Emax model the mean serum concentration (SD) of ketorolac, 4-methylaminoantipyrine and 4-aminoantipyrine inhibiting platelet TXB2 generation by 50% (EC50) in vitro was found to be 0.088 +/- 0.031, 1.2 +/- 0.3 and 10.2 +/- 3.4 micrograms ml-1, respectively. In conclusion the recovery of platelet function after dipyrone administration is faster as compared to ketorolac tromethamine. This is in line with clinical observations and may be an advantage when these drugs are given as postoperative analgesics at the doses tested.

Effect of exogenous 5,8,11,14,17-eicosapentaenoic acid on cardiac anaphylaxis.

The effects of infusions of eicosapentaenoic acid (EPA) (6 X 10(-8) mol min-1 and 15 X 10(-8) mol min-1) on the coronary constriction and the release of immunoreactive sulphidopeptide-leukotrienes (SP-LT), thromboxane B2(TXB2) and 6-keto-prostaglandin F1 alpha (PGF1 alpha) from perfused anaphylactic guinea-pig hearts were investigated. EPA dose-dependently inhibited the profound early coronary flow reduction after antigen injection. The less pronounced late phase of anaphylactic coronary flow reduction was, however, not significantly affected. EPA (15 X 10(-8) mol min-1) significantly shortened the average duration of antigen-induced arrhythmias. EPA dose-dependently decreased release of immunoreactive TXB2 and 6-keto-PGF1 alpha from anaphylactic guinea-pig hearts. Release of immunoreactive SP-LT was dose-dependently increased after antigen challenge in the presence of EPA. Inhibiton of the release of SP-LT by the lipoxygenase inhibitor esculetin (1 X 10(-7) mol min-1) was accompanied by a significant attenuation of flow reduction during the late phase of anaphylactic vasoconstriction. Reversed phase h.p.l.c. of perfusates from anaphylactic guinea-pig hearts revealed immunoreactivity comigrating with authentic leukotriene C4 (LTC4), LTD4, and LTE4. In perfusates from hearts treated with EPA infusions, additional immunoreactivity was detected comigrating with LTC5, LTD5 and LTE5. In addition to immunoreactivity migrating with LTB4, as observed in control heart perfusates, in perfusates from EPA-treated hearts, a second peak was observed, which coincides with the retention time described for LTB5. Exogenous LTC5 (1 X 10(-12) mol min-1 and 20 X 10(-12) mol min-1) induced dose-dependent reductions of coronary flow and was found to be a slightly weaker constrictor than LTC4, but no significant differences were observed. Coronary vasoconstriction elicited by infusion of exogenous LTC4 (20 X 10(-12) mol min-1) was dose-dependently inhibited by infusions of EPA. However, the negative inotropic effect of LTC4 remained unaffected. Thus, in the isolated anaphylactic heart of the guinea-pig exogenous EPA was effectively metabolized via the 5-lipoxygenase pathway whereas the cyclo-oxygenase pathway of polyunsaturated fatty acid metabolism was found to be inhibited. The results are in agreement with the suggestion that cyclo-oxygenase products are mediators of the early phase of the anaphylactic coronary constriction, while vasoconstrictor SP-LT are involved in the later phase. However, in spite of enhanced release of SP-LT, EPA infusion did not result in increased coronary constriction. Considering the fact that EPA antagonizes LTC4-induced coronary constriction, it seems possible, that EPA might act as a functional antagonist of vasoconstrictor eicosanoids including EPA-derived SP-LT.

Effects of terbutaline on NGF formation in allergic inflammation of the rat.

1. The aim of this study was to determine the effects of the beta adrenergic agonist terbutaline on NGF increase caused by allergic inflammation in rats. 2. Intraplantar antigen injection in sensitized rats increased paw volume and stimulated NGF biosynthesis in the skin of the injected paw as determined 3 and 6 h after injection. Treatment of rats with terbutaline (0.1 - 0.3 mg kg(-1), s.c.) had no significant effect on the NGF concentration in non-inflamed skin, but reduced oedema, and at 0.3 mg kg(-1) also NGF mRNA and immunoreactive NGF in the skin of the inflamed paw in a propranolol-reversible manner. In carrageenan-induced inflammation, terbutaline did not significantly reduce the inflammation-induced increase of NGF in paw skin. 3. Exposure of sensitized rats to aerosolized antigen (twice, 24 h interval) increased protein content, eosinophil leukocytes, and immunoreactive NGF in the bronchoalveolar lavage fluid (BAL, obtained 16 h after the second antigen exposure). Treatment of rats with terbutaline (0.3 mg kg(-1), s.c. 30 min before the second antigen challenge) suppressed antigen-induced elevation of protein and eosinophil leukocytes, and reduced the concentration of NGF in BAL to values similar to those found in non-sensitized rats. 4. The present results demonstrate anti-allergic properties of terbutaline in rats that were accompanied by a marked reduction of antigen-induced NGF increase in skin and BAL, respectively. These results are compatible with the assumption that terbutaline primarily suppressed the immune response to antigen thereby attenuating the release of vasoactive mediators and the stimulation of NGF biosynthesis.

Mediation by prostaglandins of the nitric oxide-induced neurogenic vasodilatation in rat skin.

1. Intraplantar administration of the nitric oxide (NO) donor, sodium nitroprusside (SNP), induces hyperaemia in the rat paw skin, which is in part due to release of calcitonin gene-related peptide (CGRP) from afferent nerve fibres. The present study examined whether prostaglandins or other inflammatory mediators participate in the neurogenic vasodilatation caused by SNP. Blood flow in the plantar hindpaw skin of urethane-anaesthetized rats was measured by laser Doppler flowmetry. 2. The hyperaemic responses to intraplantar administration of the NO donors SNP (150 pmol) and 3-morpholino-sydnonimine (SIN-1, 15 nmol) were attenuated by 45% and 61%, respectively, after injection of the CGRP antagonist, CGRP8-37 (50 nmol kg-1, i.v.) which did not significantly change baseline blood flow. 3. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 15 mg kg-1, i.v.), the bradykinin antagonist Hoc-140 (100 nmol kg-1, i.v.) and the histamine antagonists, pyrilamine (2 mg kg-1, i.v.) plus cimetidine (10 mg kg-1, i.p.) were without effect on baseline blood flow and the vasodilatation caused by SNP. 4. The cyclo-oxygenase inhibitors, indomethacin (10 mg kg-1, i.p.) and flurbiprofen (5 mg kg-1, i.p.) depressed the SNP-induced hyperaemia by 65% and 42%, respectively, without altering baseline blood flow. The ability of CGRP8-37 to inhibit the vasodilator response to SNP was lost in indomethacin-treated rats. 5. Intraplantar administration of prostaglandin E2 (PGE2, 15 pmol) evoked cutaneous vasodilatation which was attenuated by 66% after administration of CGRP8-37 but remained unaltered by indomethacin or L-NAME. 6. These data indicate that the neurogenic hyperaemia which in rat skin is induced by intraplantar administration of NO donors involves the formation of prostaglandins which in turn cause release of the vasodilator peptide, CGRP, from perivascular afferent nerve fibres.

Effects of non-steroidal anti-inflammatory drugs on rat gastric mucosal leukotriene C4 and prostanoid release: relation to ethanol-induced injury.

1. The effects of oral and subcutaneous administration of the non-steroidal anti-inflammatory drugs sodium salicylate, aspirin and indomethacin on ex vivo gastric mucosal release of leukotriene C4 (LTC4) prostaglandin E2 (PGE2), 6-oxo-PGF1 alpha and thromboxane B2 (TXB2) were investigated in rats under basal conditions as well as after challenge with ethanol. 2. Basal release of PGE2, 6-oxo-PGF1 alpha and TXB2 was inhibited by oral administration of aspirin (0.6-400 mgkg-1) and indomethacin (4 or 20 mgkg-1), but not by sodium salicylate (up to 400 mgkg-1), in a dose-dependent manner. Oral administration of aspirin in the dose range 3.2-400 mgkg-1 and of indomethacin (20 mgkg-1) additionally inhibited release of LTC4, while sodium salicylate (up to 400 mgkg-1) had no effect. Indomethacin (20 mgkg-1) and aspirin (400 mgkg-1) administered subcutaneously inhibited generation of cyclo-oxygenase products of arachidonate metabolism, but did not significantly affect LTC4 synthesis. 3. Oral instillation of ethanol caused gastric mucosal damage and simultaneously induced a selective increase in the ex vivo release of LTC4 from rat gastric mucosa, while release of cyclo-oxygenase products of arachidonate metabolism was not significantly affected. Oral pretreatment of rats with sodium salicylate protected the gastric mucosa and simultaneously inhibited the ethanol-stimulated gastric mucosal LTC4 release in a dose-dependent manner. Sodium salicylate had no effects on the release of PGE2 and TXB2, while that of 6-oxo-PGF1 alpha was slightly increased. 4. Pretreatment with indomethacin (4 or 20mg kg- p.o.) or aspirin in doses up to 25mg kg-1 p.o. prior to oral instillation of ethanol did not inhibit gastric mucosal damage and had no effect on the stimulatory action of ethanol on LTC4 release. Higher doses of aspirin (100mgkg-1 or 400mgkg-1 p.o.) reduced the mucosal damaging effect of ethanol and simultaneously inhibited LTC4 release. 5. The results suggest that aspirin and indomethacin in concentrations higher than those necessary to inhibit the cyclo-oxygenase pathway of arachidonate metabolism additionally inhibit gastric mucosal LTC4 synthesis under basal conditions, while sodium salicylate has no such effect. On the other hand, sodium salicylate, but not indomethacin or low doses of aspirin (up to 25mg kg 1), by an unknown mechanism inhibits stimulation of LTC4 biosynthesis by ethanol and simultaneously protects the gastric mucosa against ethanol-induced damage. Similar effects of high oral doses (> 100mgkg- 1) of aspirin might be due to significant formation of salicylate. These results suggest that there is a causal relationship between enhanced LTC4 biosynthesis and the development of ethanol-induced gastric injury.

Disturbance of peristalsis in the guinea-pig isolated small intestine by indomethacin, but not cyclo-oxygenase isoform-selective inhibitors.

1. Since the cyclo-oxygenase (COX) isoform-nonselective inhibitor indomethacin is known to modify intestinal motility, we analysed the effects of COX-1 and COX-2 inhibition on intestinal peristalsis. 2. Peristalsis in isolated segments of the guinea-pig small intestine was triggered by a rise of the intraluminal pressure and recorded via the pressure changes associated with peristalsis. 3. The COX-1 inhibitor SC-560, the COX-2 inhibitor NS-398 (both at 0.1 -- 1 microM) and the isoform-nonselective inhibitors flurbiprofen (0.01 - 10 microM) and piroxicam (0.1 - 50 microM) were without major influence on peristalsis, whereas indomethacin and etodolac (0.1 -- 10 microM) disturbed the regularity of peristalsis by causing nonpropulsive circular muscle contractions. 4. Radioimmunoassay measurements showed that SC-560, NS-398, indomethacin and etodolac (each at 1 microM) suppressed the release of 6-keto-prostaglandin F(1 alpha) (6-keto-PGF(1 alpha)) from the intestinal segments. 5. Reverse transcription - polymerase chain reaction tests revealed that, relative to glyceraldehyde-3 phosphate dehydrogenase ribonucleic acid, the expression of COX-1 mRNA increased by a factor of 2.0 whereas that of COX-2 mRNA rose by a factor of 7.9 during the 2 h experimental period. 6. Pharmacological experiments indicated that the action of indomethacin to disturb intestinal peristalsis was unrelated to inhibition of L-type calcium channels, adenosine triphosphate-sensitive potassium channels or phosphodiesterase type IV. 7. These results show that selective inhibition of COX-1 and COX-2 does not grossly alter peristaltic motor activity in the guinea-pig isolated small intestine and that the effect of indomethacin to disturb the regular pattern of propulsive motility in this species is unrelated to COX inhibition.

Dilatation by angiotensin II of the rat femoral arterial bed in vivo via pressure/flow-induced release of nitric oxide and prostaglandins.

1. The haemodynamic effects of angiotensin II (AII) and, for comparison, arginine vasopressin (AVP) in the femoral and superior mesenteric artery of urethane-anaesthetized rats were analysed with the ultrasonic transit time shift technique. 2. I.v. bolus injection of AII (0.1-3 nmol kg-1) and AVP (0.03-1 nmol kg-1) increased blood pressure which was accompanied by a decrease in blood flow through the superior mesenteric artery and an increase in femoral blood flow. The femoral hyperaemia was in part due to vasodilatation as indicated by a rise of femoral vascular conductance up to 200% relative to baseline. The femoral vasodilatation caused by AVP, but not AII, was followed by vasoconstriction. 3. Blockade of angiotensin AT1 receptors by telmisartan (0.2-20 mumol kg-1) prevented all haemodynamic responses to AII. 4. The femoral dilator responses to AII and AVP depended on the increase in vascular perfusion pressure since vasodilatation was reversed to vasoconstriction when blood pressure was maintained constant by means of a gravity reservoir. However, the AII-evoked femoral vasodilatation was not due to an autonomic or neuroendocrine reflex because it was not depressed by hexamethonium (75 mumol kg-1), prazosin (0.25 mumol kg-1) or propranolol (3 mumol kg-1). 5. The AII-induced femoral vasodilatation was suppressed by blockade of nitric oxide (NO) synthesis with NG-nitro-L-arginine methyl ester (L-NAME, 40 mumol kg-1) and reversed to vasoconstriction when L-NAME was combined with indomethacin (30 mumol kg-1), but was left unaltered by antagonism of endothelin ETA/B receptors with bosentan (37 mumol kg-1). 6. These results demonstrate that the effect of AII to increase systemic blood pressure and the resulting rise of perfusion pressure in the femoral artery stimulates the formation of NO and prostaglandins and thereby dilates the femoral arterial bed. This local vasodilator mechanism is sufficient to mask the direct vasoconstrictor response to AII.

Effect of inhibition of synthesis and receptor antagonism of SRS-A in cardiac anaphylaxis.

The effects of infusions of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 1.1 X 10(-7) mol min-1) and the antagonist of slow-reacting substance of anaphylaxis (SRS-A) FPL 55712 (1.2 X 10(-7) mol min-1) on the coronary constriction and the release of SRS-A, leukotreine C4-like immunoreactivity, thromboxane B2 and 6-keto-prostaglandin F1 alpha from perfused anaphylactic guinea-pig hearts were investigated. Both NDGA and FPL 55712 in the concentrations used induced an increase in basal coronary flow, but did not prevent the coronary flow reduction in the early phase (0-4 min) after antigen injection. On the other hand, NDGA and FPL 55712 inhibited the less pronounced long-lasting coronary flow reduction in the later phase of cardiac anaphylaxis. NDGA decreased the release of SRS-A from the anaphylactic guinea-pig hearts below or close to the detection limit of the bioassay and simultaneously diminished the release of leukotriene C4-like immunoreactivity. On the other hand, FPL 55712 did not influence the amounts of leukotriene C4-like immunoreactivity released in cardiac anaphylaxis. Neither NDGA nor FPL 55712 affected the release of immunoreactive thromboxane B2 (TXB2) from anaphylactic guinea-pig hearts. Release of 6-keto-prostaglandin F1 alpha after challenge, however, was decreased by NDGA, while FPL 55712 had no significant effect. These results suggest, that SRS-A may be a relatively more important mediator in the late phase of coronary constriction occurring during cardiac anaphylaxis, while the effects of other mediators, particularly vasoconstrictor cyclo-oxygenase products, seem to prevail in the early phase.

Prostaglandin D2 is the prevailing prostaglandin in the acute inflammatory exudate of urate arthritis in the chicken.

An acute inflammation was elicited in intertarsal joints of chicken by injection of urate crystals. Inflammatory exudates recovered at different times were assayed for prostaglandin D2(PGD2)E2 and F2 alpha and thromboxane B2 content by specific radioimmunoassays. We found that PGD2 was the prevailing prostaglandin reaching concentrations up to 10 times in excess of PGE2. This finding was confirmed by gas chromatography-mass spectrometry. It is concluded that PGD2 should be considered as a possible mediator of acute inflammation.

Bradykinin-evoked sensitization of neuropeptide release from afferent neurons in the guinea-pig lung.

1. It has been shown that bradykinin (BK) causes sensitization of airway sensory neurons and an enhancement of the cough reflex in guinea-pigs. In the present study, the guinea-pig isolated perfused lung was used to investigate the possible enhancement by BK of histamine-evoked neuropeptide release from peripheral terminals of primary afferent neurons, and to determine the contribution of cyclooxygenase products of arachidonate metabolism to this effect. 2. The lung was perfused with oxygenated physiological salt solution containing peptidase inhibitors (thiorphan, bestatin and captopril, 1 microM each). BK and histamine were added to the perfusate for 10 and 5 min, respectively. 3. BK alone (0.1 microM) evoked the release of 10.35+/-2.4 fmol immunoreactive calcitonin gene-related peptide (CGRP), histamine alone (100 microM) evoked the release of 12.7+/-1.6 fmol CGRP. Stimulation with 100 microM histamine in the presence of 0.1 microM BK (added 5 min before histamine and present during histamine) evoked the release of 67.1+/-5.3 fmol CGRP. 4. Prostaglandin (PG) release was stimulated by BK (418+/-71 pmol 15-keto-13,14-dihydro-PGF2alpha and 345+/-59 pmol 6-keto-PGF1alpha), and, to a lesser extent, by histamine (36.1+/-7.4 pmol 15-keto-13,14-dihydro-PGF2alpha, and 24.6+/-3.9 pmol 6-keto-PGF1alpha). Prostaglandin release induced by histamine in the presence of BK was not significantly higher than with BK alone. 5. Indomethacin (5 microM) as well as the bradykinin B2 receptor antagonist HOE140 (icatibant, 1 microM) inhibited prostaglandin release following stimulation with histamine in combination with BK. CGRP release evoked by histamine in combination with BK was attenuated by indomethacin and HOE140 to 22.1+/-7.8 fmol and 16.4+/-3.8 fmol, respectively, significantly less than the value obtained in control experiments (67.1+/-5.3 fmol). 6. The results suggest that BK-induced stimulation of prostaglandin synthesis results in facilitation of histamine-evoked release of pro-inflammatory neuropeptides from afferent neurons, a mechanism that probably becomes relevant during inflammation, and that can be blocked by a bradykinin B2 receptor antagonist.

Effect of carbenoxolone on the biological activity of nitric oxide: relation to gastroprotection.

1. The interactions between carbenoxolone and nitric oxide (NO) were examined by investigating their effects on human platelet aggregation, on rat aortic strips precontracted by phenylephrine and on protection of rat gastric mucosa against ethanol-induced injury. 2. Carbenoxolone (100-300 microM) caused a significant and concentration-dependent potentiation of rat peritoneal neutrophil (RPN)- 3-morpholino-syndnonimine (SIN-1)- or iloprost-induced inhibition of platelet aggregation. Higher concentrations (500 microM) of carbenoxolone alone markedly inhibited platelet aggregation. Pretreatment with carbenoxolone (100-300 microM) antagonized the reversal of the RPN- or SIN-1-induced antiaggregatory effect by oxyhaemoglobin (10 microM). 3. Rat aortic strips with intact endothelium precontracted by phenylephrine (0.1-0.3 microM) were relaxed by carbenoxolone (100-300 microM) in a concentration-dependent manner. Relaxations were abolished by mechanical removal of the endothelium or by incubation with methylene blue (10 microM) or NG-nitro-L-arginine (L-NNA, 100 microM). Sodium nitroprusside (10 nM)-induced relaxations of endothelium-denuded rat aortic strips were potentiated by carbenoxolone (100 microM). . The carbenoxolone (200 mg kg-1, p.o.)-induced gastroprotection against ethanol was antagonized by L-NNA (5-40 mg kg-1) in a dose-dependent manner. Pretreatment of rats with indomethacin (10 mg kg-1, s.c.) increased the effect of L-NNA. 5. The results suggest that the activity of carbenoxolone in the experimental systems tested is due to phosphodiesterase inhibition, although radical scavenging properties of the drug could contribute to some of the effects observed. In the rat gastric mucosa both increased prostaglandin levels and effects on the NO system could contribute to the protective action of carbenoxolone.