Release of platelet-activating factor (PAF) and accelerated healing induced by a PAF antagonist in an animal model of chronic colitis

The role of platelet-activating factor as a mediator of inflammation was examined using a rat model of colitis. Release of platelet-activating factor by samples of colonic tissue, as measured by bioassay, was determined at various times after induction of inflammation by intracolonic administration of trinitrobenzene sulfonic acid. At the same times, colonic damage was scored and the extent of neutrophil infiltration was assessed by measuring tissue levels of myeloperoxidase activity. Platelet-activating factor release from normal colon averaged 46 +/- 17 pg/g, while not being detectable in samples taken 24 h after induction of inflammation, a time when neutrophil infiltration was maximal. However, substantial release of platelet-activating factor (12-16 times control levels; p less than 0.05) was observed in samples from rats sacrificed 1-3 weeks after induction of inflammation. In a second series of experiments, the effects of treatment with BN52021, a specific platelet-activating factor receptor antagonist, were assessed using this model. Treatment with BN52021 during either the 1st or 2nd weeks after induction of colitis resulted in significant (p less than 0.05) reductions of colonic damage scores and the wet weight of the distal colon. These results demonstrate that platelet-activating factor release is significantly elevated during the chronic phase of colitis in this animal model, and that inhibition of the action of platelet-activating factor, through receptor antagonism, leads to a significant reduction of colonic inflammation and ulceration. These observations are therefore consistent with a role for platelet-activating factor as an inflammatory mediator in chronic inflammation of the rat colon.     

Cascade Bioassay Evidence for the Existence of Urothelium-Derived Inhibitory Factor in Guinea Pig Urinary Bladder

Our aim was to investigate whether guinea pig urothelium-derived bioactivities compatible with the existence of urothelium-derived inhibitory factor could be demonstrated by in vitro serial bioassay and whether purinergic P1 receptor agonists, nitric oxide, nitrite or prostaglandins might explain observed activities. In a cascade superfusion system, urothelium-denuded guinea pig ureters were used as bioassay tissues, recording their spontaneous rhythmic contractions in presence of scopolamine. Urothelium-intact or -denuded guinea pig urinary bladders were used as donor tissues, stimulated by intermittent application of carbachol before or during the nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME), the adenosine/P1 nucleoside receptor antagonist 8-(p-sulfophenyl)theophylline (8-PST) or the cyclo-oxygenase inhibitor diclofenac infused to bath donor and bioassay tissues. The spontaneous contractions of bioassay ureters were unaltered by application of carbachol 1–5 µM in the presence of scopolamine 5–30 µM. When carbachol was applied over the urothelium-denuded bladder, the assay ureter contraction rate was unaltered. Introducing carbachol over the everted urothelium-intact bladder significantly inhibited the contraction frequency of the assay ureter, suggesting the transfer of an inhibitory activity from the bladder to the assay ureter. The transmissible inhibitory activity was not markedly antagonized by L-NAME, 8-PST or diclofenac, while L-NAME nearly abolished nitrite release from the urothelium-intact bladder preparations. We suggest that urothelium-derived inhibitory factor is a transmissible entity over a significant distance as demonstrated in this novel cascade superfusion assay and seems less likely to be nitric oxide, nitrite, an adenosine receptor agonist or subject to inhibition by administration of a cyclo-oxygenase inhibitor.     

Immunofluorescent localization of platelet-activating factor (PAF) in the rat

Summary An immunofluorescent staining method for detecting platelet-activating factor (PAF) is described. This method employs a polyclonal anti-PAF rabbit antibody. When rat brain, heart, lung, liver or kidney tissue was stained using this method, the heart, lung and kidney exhibited PAF-specific staining. Analysis of the amount of PAF in different organs, either by immunofluorescence or by bioassay, showed that kidney tissue contains the greatest amount of PAF.     

L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo

L-selectin has been shown to be important in mediating leukocyte recruitment during inflammatory responses. Although there are numerous in vitro studies demonstrating that engagement of L-selectin leads to the activation of several signaling pathways potentially contributing to subsequent adhesion, emigration, or even migration through the interstitium, whether this actually induces cellular events in vivo is completely unknown. Therefore, we used intravital microscopy to visualize the role of L-selectin in downstream leukocyte adhesion, emigration, and interstitial migration events in wild-type and L-selectin-deficient (L-selectin(-/-)) mice. The cremaster muscle was superfused with the chemotactic inflammatory mediators platelet-activating factor or KC. Leukocyte rolling, adhesion, and emigration in postcapillary venules were examined, and the migration of emigrated leukocytes was recorded continuously using time-lapse videomicroscopy. Platelet-activating factor increased leukocyte adhesion to a similar level in both wild-type and L-selectin(-/-) mice. In contrast, both the number of emigrated leukocytes and the distance of extravascular migration were significantly reduced in L-selectin(-/-) mice. A similar pattern was observed in response to the superfusion of KC. Because superfusion of these mediators induced chemokinesis, we developed a new in vivo chemotaxis assay using slow release of KC from an agarose gel positioned 350 microm from a postcapillary venule. These experiments showed that L-selectin(-/-) leukocytes were also severely impaired in their ability to respond to a directional cue. These findings indicate that L-selectin is important in enabling leukocytes to respond effectively to chemotactic stimuli in inflamed tissues.     

The effects of prostacyclin (PGI2) on tissues which detect prostaglandins (PG'S).

The effects of prostacyclin (PGI2) and its stable metabolite 6-oxo-PGF1alpha on various bioassay tissues are compared with those of PGE2 and PGF2alpha, using the cascade superfusion method. On vascular smooth muscle, PGI2 caused relaxation of all tissues tested except the rabbit aorta. PGE2 relaxed rabbit coeliac and mesenteric artery but contracted bovine coronary artery and had no effect on rabbit aorta. 6-oxo-PGF1alpha was ineffective at the concentrations tested. On gastro-intestinal smooth muscle, PGI2 contracted strips of rat and hamster stomach and the chick rectum. It was less potent than PGE2 or PGF2alpha. None of these substances contracted the cat terminal ileum. 6-oxo-PGF1alpha was inactive on these tissues at the doses tested. PGI2 was less active than PGE2 or PGF2alpha in contracting guinea-pig trachea and rat uterus; 6-oxo-PGF1alpha was active only on the rat uterus. Thus, PGI2 can be distinguished from the other stable prostaglandins using the cascade method of superfusion.     

Caerulein-induced desensitization of enzyme secretion fails in neonatal rat pancreas

Pancreatic segments of 1-, 3-, 5-, 10-day-old and adult female OFA (Sprague-Dowley strain) rats were superfused with graded concentrations of caerulein (10(-12)-10(-7) M) to establish concentration-response relation of amylase release. Furthermore, pancreatic segments of 3-, 5-, 10-day-old and adult rats were superfused with 10(-10) or 10(-8) M caerulein and then superfusion was repeated with 10(-10) M concentration of caerulein to show whether the phenomenon of desensitization of amylase release can be induced in the postnatal period. The 1-day-old pancreas was found practically insensitive to caerulein. The 3- and 5-day-old gland was by one order of magnitude less sensitive (EDmax = 10(-8) M) than the adult pancreas (EDmax = 10(-9) M). Repeated superfusion of the 3- and 5-day-old pancreas with 10(-10) M caerulein after the first 10(-8) M caerulein superfusion failed to cause desensitization, while the same (10(-10) M) repeated superfusion of the 10-day-old adult pancreatic segments after the first 10(-8) M caerulein superfusion evoked desensitization of enzyme release. The authors suggest that the failure of desensitization of enzyme secretion for caerulein may be due to the maturation process of newborn rat pancreatic acinar cells at receptorial and postreceptorial level.     

Prostaglandin E-2-9-ketoreductase in ovarian tissues.

The existence of the enzyme prostaglandin E-2-9-ketoreductase which can convert prostaglandin E-2 to prostaglandin F-2 alpha was indicated in experiments with pig and human ovarian tissues in vitro, using radioimmunoassay methods and a superfusion technique. Further studies involving radiotracer techniques demonstrated that the enzyme was localized in the high-speed (105 000 g) supernatant fraction of human, pig and rat luteal tissue and human stromal tissue. The enzyme was shown to be NADPH-dependent and its activity in luteal tissue increased in the order : pig less than human less than rat.     

Occurrence of platelet-activating factor (PAF) in normal rat stomach and alteration of PAF level by water immersion stress

We detected platelet-activating substance in gastrointestinal areas, which was confirmed to be platelet-activating factor (PAF) on the basis of the following findings: 1) it comigrated with authentic PAF on thin-layer chromatography; 2) it did not aggregate PAF-desensitized platelets; and 3) its activity was completely antagonized by the receptor antagonists CV3988 and L-652,731. The level of PAF was determined with a bioassay method based on the release of [3H]serotonin from washed rabbit platelets. In the normal rat stomach, the level of PAF was high in the antrum (940 +/- 200 nmol PAF/mol phosphorus of original phospholipids), especially in the antral mucosa (1801 +/- 426 nmol/mol phosphorus of original phospholipids). The stomach PAF level was significantly altered by water immersion stress. Stress for a period of 1 h was associated with a decrease in the antral PAF level to 39 +/- 7% of that of untreated controls. This low PAF level persisted during stress. On the other hand, in the corpus, stress for periods of 1 and 3 h was associated with decreases in the PAF content, and further stress (7 h) resulted in restoration of the PAF level to normal. Furthermore, 7 h of stress was associated with distinct hemorrhagic lesions, which were prevented by CV3988 infused i.v. before the stress. This is the first report of an association between a decrease of the endogenous PAF level in animal tissues and tissue damage.     

Structure-desensitization relationships of angiotensin analogues in the rat isolated uterus

The desensitizing potencies of angiotensin II (ANG II) analogues modified at positions 1, 2, 4, 7, and 8 have been examined in the rat isolated uterus assay by determining the time of recovery of the half-maximal concentration (EC50) response to angiotensin II after treatment of the tissues with a high dose (10(-5) M) of each analogue for 2 min. The magnitude of the desensitization effect was substituent dependent in the following manner: position 1, sarcosine (Sar) greater than Asp greater than des-Asp; position 2, Arg greater than Sar; position 4, Tyr greater than Tyr(Me) approximately Phe; position 7, 3,4-dehydroproline (Dpr) greater than Pro greater than thioproline (Tpr) greater than Sar; position 8, Ile greater than D-Trp greater than Ala greater than Phe. The "additivity" rule applied to these structure-desensitization relationships and the most potent desensitizer, requiring 3 h for reestablishment of the EC50 response, was [Sar1, Dpr7, Ile8]-ANG II. The desensitizing potencies of these analogues did not correlate with agonist or antagonist activities and demonstrated that the angiotensin-mediated tissue desensitization process has unique structural determinants. Methylation or elimination of the tyrosine hydroxyl group of strong desensitizers virtually eliminated the desensitization effect, implicating the phenoxyl moiety in the mechanism of desensitization. The initial phase of recovery of angiotensin responsiveness after desensitization by several analogues appeared to obey first-order kinetics. The results are discussed in the contexts of both one- and two-site receptor models.     

Endotoxin causes neutrophil-independent oxidative stress in rats

Endotoxin-induced oxidative stress is investigated in rats by measuring changes in plasma and lung tissue levels of glutathione disulfide (GSSG) using a modified enzymatic assay that allows simultaneous measurement of up to 80 samples. Salmonella enteritidis endotoxin (2 and 20 mg/kg) acutely increased both plasma reduced glutathione and GSSG with a rise in the ratio of GSSG to total glutathione. This increase in GSSG was enhanced by pretreatment with 1,3-bis(2-chloroethyl)1-nitrosourea (BCNU), an inhibitor of the glutathione reductase enzyme. However, there was no significant arteriovenous difference in plasma GSSG across the lung, and lung tissue GSSG did not increase after endotoxin treatment. The increase in plasma GSSG was not blocked by vinblastine-induced neutropenia and could not be reproduced by incubating rat blood in vitro with endotoxin. Receptor antagonists of platelet-activating factor (PAF), at a dose that previously inhibited endotoxin-induced lung injury, attenuated the endotoxin-induced increase in plasma GSSG. We conclude that endotoxin causes neutrophil-independent oxidative stress in rats, which may be enhanced by the action of platelet-activating factor.     

Profile of gastrointestinal damage induced by platelet-activating factor

The ulcerogenic actions of an intravenous infusion of platelet-activating factor (100 ng/kg/min) was studied in the rat. Damage to the stomach, duodenum, jejunum, ileum and colon were assessed histologically and using intraluminal acid phosphatase release as a marker of cellular damage. A 10-min infusion of platelet-activating factor caused extensive haemorrhagic damage to each of the regions examined, with the exception of the colon. Acid phosphatase release was significantly elevated in the stomach, jejunum, ileum (p less than 0.001) and duodenum (p less than 0.01), but not in the colon. These studies demonstrate that platelet-activating factor is a potent ulcerogen in the stomach and small intestine, and support a role for this endogenous phospholipid as a mediator of the ulceration associated with endotoxin-induced shock.     

Human spermatozoa produce C16-platelet-activating factor.

Recent data indicate that human spermatozoa produce platelet-activating factor as determined by the rabbit platelet [3H]serotonin release bioassay. In this report, we examined by fast atom bombardment/mass spectrometry the molecular species of platelet-activating factor generated by these germ cells. Extracted spermatozoal samples that contained platelet-activating factor bioactivity underwent straight-phase high-performance liquid chromatography, and fractions which coeluted with authentic C16- and C18-platelet-activating factor standards were subjected to fast atom bombardment/mass spectrometry. Our mass spectral data indicate that human spermatozoa synthesize C16-platelet-activating factor but not C18-platelet-activating factor.     

Platelet-activating factor contracts human myometrium in vitro

The myometrial contractile responses to synthetic 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet-activating factor, PAF) and to oxytocin were evaluated in vitro on uterine (lower segment) strips obtained from pregnant women at term (39th week), undergoing elective cesarean section. Contractility was measured isometrically in an isolated organ bath using a superfusion technique. PAF in a concentration range between 5 and 100 nM as well as oxytocin (0.1-10 mU/ml) induced a dose-dependent contraction which could be categorized in two patterns, depending on whether spontaneous activity was present. In resting strips, oxytocin induced a prompt (0.5-1 min) development of active tension, followed by a prolonged (6-18 min), slow contraction and a final relaxation. However, at variance with oxytocin, PAF-induced contractions were rhythmic (3-8/hr), and characterized by a prompt (0.5-2 min) development of tension, followed by a brief (0.5-2 min) plateau, and a final, rapid relaxation. In spontaneously active strips, both stimuli induced a marked potentiation of the contractile activity. PAF response was dependent on both cyclooxygenase- and lipoxygenase-derived products as inferred from the abrogating effects of indomethacin and FPL 55712. A receptor-mediated mechanism of action was inferred from the occurrence of specific desensitization to PAF (but not to oxytocin), and from the blocking effect of CV 3988, a specific PAF receptor antagonist. The present study indicates that PAF stimulates the contraction of human myometrium in vitro and suggests that this mediator may have a role in labor.     

Quantitative analysis of platelet-activating factor in rat brain.

Age-related decrease of the platelet-activating factor (PAF) content in rat brain was shown by a convenient method consisting of solid extraction of lipids with a Sep-Pak C-18 cartridge, lipid separation by HPLC and bioassay on rabbit platelets. This method was sufficiently sensitive to allow measurement of PAF in a single brain, and the recovery of PAF was quite high throughout the procedure.     

Characterization of Nicotine-Induced Desensitization of Evoked Dopamine Release from Rat Striatal Synaptosomes

Abstract: Nicotine has been shown to stimulate neurotransmitter release from brain tissue by acting on presynaptic receptors. In this study, the ability of nicotine pretreatment to produce functional desensitization was investigated in rat striatal synaptosomes in which the release of [³H]dopamine was measured with an in vitro superfusion system. Pretreatment of synaptosomes with low concentrations of l -nicotine resulted in a decrease in the ability of a subsequent nicotine challenge to evoke [³H]dopamine release. The IC₅₀ for nicotine-induced desensitization was found to be 12 n M with a maximum inhibition of >90% at 300 n M . Nicotine pretreatment did not affect the release evoked by amphetamine, veratridine, or 15 m M K⁺. The onset of nicotine-induced desensitization occurred with a t _1/2 of 43 s at 30 n M nicotine. The temperature dependence of onset yielded a Q10 of 1.2.Recovery from desensitization was slower ( t _1/2 = 4.33 min), and both the onset and recovery appeared to follow a single first-order process. Several intermittent schedules of nicotine treatment were found to be effective at inducing and maintaining desensitization. The results of this study show that nonstimulating concentrations of nicotine can produce a complete functional desensitization of subsequent nicotine-induced neurotransmitter release.     

Structural analogs of alkylacetylglycerophosphocholine inhibitory behavior on platelet activation

Several analogs of alkylacetylglycerophosphocholine (AGEPC; platelet-activating factor) were investigated as potential selective inhibitors of AGEPC-induced activation of washed rabbit platelets. Two particular compounds, CV-3988 (rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl-2-thiazolioethyl++ + phosphate) and U66985 (1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-6'-trimethylammonium-hexyl ester) emerged as particularly active and effective inhibitors. Aggregation and secretion profiles, as well as the degradation of inositol phospholipids and production of phosphatidic acid, were used as monitors of their inhibitory capabilities. U66985 was the most effective inhibitor, giving an IC50 value of 4.1 +/- 1.5 X 10(-8) M against a challenge of 1 X 10(-10) M AGEPC in the secretion assay. Phospholipid turnover was blocked completely at this inhibitor concentration. On the other hand, while CV-3988 was an effective inhibitor, a higher concentration was required and a more restricted range of activity was noted with an IC50 value of 5.9 +/- 1.3 X 10(-7) M against a challenge of 1 X 10(-10) M AGEPC in the secretion assay. While CV-3988 did indeed completely block the turnover of inositol phospholipids and phosphatidic acid formation, these effects were noted at a higher concentration than with U66985. On the basis of data obtained in desensitization experiments with AGEPC and U66985, it appears that each inhibitor occupies the same receptor site as the agonist, AGEPC. These results illustrate the usefulness of these AGEPC analogs in exploring the biochemical characteristics of the interaction of AGEPC with a cell.     

Endothelin, a potent peptide agonist in the liver

Endothelin, a peptide mediator produced by vascular endothelial cells, caused sustained vasoconstriction of the portal vasculature in the perfused rat liver. The vasoactive effect of endothelin was accompanied by increased glycogenolysis and alterations in hepatic oxygen consumption. The endothelin-induced increase in the portal pressure was concentration-dependent with an EC50 of 1 nM. Endothelin-induced hepatic glycogenolysis was dose-dependent but exhibited a different EC50 than for the vasoconstrictive effects of endothelin. Hepatic vasoconstriction and glycogenolysis following endothelin infusion were inhibited when Ca2+ was removed from the perfusion medium. The endothelin-induced responses in the liver were not altered by prior infusion of phenylephrine (alpha-adrenergic agonist), isoproterenol (beta-adrenergic agonist), angiotensin II, glucagon, platelet-activating factor, or the platelet-activating factor antagonist, BN52021. However, repeated infusion of endothelin resulted in desensitization of the glycogenolytic response but was without a significant effect on hepatic vasoconstriction. Endothelin also stimulated metabolism of inositol phospholipids in isolated hepatocytes and Kupffer cells in primary culture. The present experiments demonstrate, for the first time, that endothelin is a very potent agonist in the liver eliciting both a sustained vasoconstriction of the hepatic vasculature and a significant increase in hepatic glucose output.     

A limited role for leukotrienes and platelet-activating factor in food protein induced jejunal smooth muscle contraction in sensitized rats.

To determine whether the release of newly formed mediators such as the peptidoleukotrienes and platelet-activating factor might modulate the food protein induced jejunal smooth muscle contraction observed in sensitized rats, Hooded-Lister rats were sensitized by injection of ovalbumin (10 micrograms i.p.) and controls were sham sensitized with saline. Fourteen days later the contractility of longitudinally (n = 9) and circularly (n = 9) oriented jejunal segments (mucosa intact) were examined in standard tissue baths in response to antigen, leukotrienes, and platelet-activating factor alone and in the presence of a specific leukotriene receptor antagonist (MK-571), a 5-lipoxygenase inhibitor (L651,392), and a platelet-activating factor receptor antagonist (WEB 2086). Although the responses of control and sensitized tissues to stretch and 10(-4) M bethanechol were similar, only sensitized tissues contracted in response to antigen (1 mg/mL). MK-571 (10(-5) M) reduced or significantly inhibited the contractile response of sensitized longitudinally and circularly oriented tissues to 10(-7) M leukotrienes C4, D4, or E4, but neither L651,392 (10(-4) M) nor MK-571 (10(-5) M) significantly reduced the contractile response of sensitized tissues to antigen challenge. WEB 2086 (10(-4) M) significantly (p less than 0.01) reduced the contractile response of sensitized longitudinally and circularly oriented tissues to 10(-7) M platelet-activating factor but did not significantly alter the response to antigen in longitudinally (45% of control, p = 0.14) or circularly (118% of control, ns) oriented jejunal smooth muscle. In this model leukotrienes and platelet-activating factor play an insignificant role in modulating food protein induced jejunal smooth muscle contraction in intestinal anaphylaxis.     

The priming of neutrophils by lipopolysaccharide for production of intracellular platelet-activating factor. Potential role in mediation of enhanced superoxide secretion

LPS priming of the neutrophil results in enhanced release of superoxide upon subsequent stimulation, but the mechanism of this effect remains obscure. The recent recognition that neutrophils synthesize and retain platelet-activating factor within the cell led us to hypothesize that enhanced synthesis of platelet-activating factor in the LPS-primed cell might account for the observed effects of lipopolysaccharide. Using human neutrophils isolated on plasma-Percoll gradients, we found that incubation with 100 ng/ml LPS for 60 min resulted in a small but significant increase in intracellular platelet-activating factor assessed after lipid extraction, TLC, and bioassay. The further stimulation of primed neutrophils with FMLP resulted in a marked increase in neutrophil platelet-activating factor compared with non-LPS-treated controls. The priming effect of LPS was time dependent (30 to 60 min), dose dependent, and inhibited at 0 degree C and did not require protein synthesis. Platelet-activating factor so generated was not released but rather retained within the neutrophil, and the molecular species of platelet-activating factor produced was predominantly 1-O-hexadecyl-2-acetyl-sn-3-phosphorylcholine. Platelet-activating factor production in LPS-treated neutrophils was also enhanced by PMA, suggesting that receptor-mediated events could not account exclusively for the enhancement. Considering the ability of nanomolar concentrations of exogenously added platelet-activating factor to prime the neutrophil for enhanced release of superoxide, the rapid intracellular accumulation of platelet-activating factor that accompanies stimulation of an LPS-primed cell by FMLP may modulate the secretory events that accompany such stimulation.     

Modification of the polar head group of platelet-activating factor: influence on the biological activity

Racemic analogues of platelet-activating factor and its lyso derivatives have been prepared in which one methyl of the trimethylammonium group has been replaced by ethyl, propyl, allyl, or carboxymethylene. The influence of chemical modification on the biological activity was assessed by measuring platelet aggregation and desensitization. The results point to a crucial role of a positively charged polar head group for the expression of biological activity of platelet-activating factor. There are also some indications of a more non-specific interaction of the polar head group of platelet-activating factor with its platelet binding sites.