Characteristics of oedema formation induced by N-formyl-methionyl-leucyl-phenylalanine in rabbit skin.

1. The characteristics of N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced oedema formation were investigated in vivo in rabbit skin. 2. FMLP injected intradermally alone induced a small increase in plasma leakage, but marked synergism with prostaglandin E2 (PGE2) in producing oedema responses was observed. In the presence of PGE2, FMLP was equiactive with C5a des Arg and 100-1000 times more active than histamine in terms of permeability-increasing activity. The response to FMLP was not dependent on endogenous histamine release. 3. FMLP-induced responses were of long duration (t1/2 approximately 40-50 min) when compared with bradykinin (t1/2 approximately 4-5 min). 4. The activity of a range of N-formyl peptides in increasing vascular permeability in skin correlated well with their activity as neutrophil stimulants in vitro. 5. Intravenous infusion of zymosan-activated plasma (ZAP) resulted in transient neutropenia and inhibition of oedema formation induced by FMLP and C5a des Arg in the skin. Responses to bradykinin were unaffected by the infusion of ZAP. 6. Intravenous injection of the non-steroidal antiinflammatory drug, ibuprofen, resulted in an inhibition of FMLP-induced, but not histamine-induced, oedema formation. This effect was independent of cyclo-oxygenase inhibition and the drug did not induced neutropenia. 7. Intravenous injection of the microtubule blocking agent colchicine inhibited FMLP-induced oedema formation. Responses to bradykinin were unaffected. When colchicine was administered after intradermal FMLP, subsequent plasma leakage was abolished. 8. The inference that receptors have evolved to bacterial secretions (i.e. FMLP) and products of the interaction of bacterial cell walls with tissue fluid (i.e. C5a des Arg), is consistent with the hypothesis that oedema formation is fundamentally a functional process concerned with regulating microbial lysis and opsonisation in an infected tissue.     

13-hydroxyoctadecadienoic acid attenuates oedema formation induced by leukotriene B4 in vivo in rabbit skin.

Intradermal 13-hydroxyoctadecadienoic acid (13-HODE; 10(-11)-10(-9) mol/site) inhibited oedema formation induced by the neutrophil-dependent mediator leukotriene B4 (LTB4) in the presence of calcitonin gene-related peptide (CGRP; 10(-11) mol/site) in rabbit skin. In contrast, the responses to the direct acting mediators bradykinin and histamine were unaffected by 13-HODE. 13-HODE failed to induce oedema formation in rabbit skin when injected alone or in the presence of the potent vasodilator CGRP. These results present a novel interaction between 13-HODE and LTB4 that could have important implications in the pathogenesis of inflammation.     

Steroid inhibition of oedema formation in the rat skin.

1. A model has been developed to compare the inhibitory effects of the topical steroid, betamethasone-17-valerate, to those of systemically administered betamethasone upon oedema responses induced by 5-hydroxytryptamine (5-HT), platelet activating factor (PAF) and zymosan-activated serum (ZAS) +/- prostaglandin E1 (PGE1), measured in the rat skin by use of 125I-labelled human serum albumin. 2. Systemic betamethasone had a selective, time- and dose-dependent inhibitory effect upon oedema treatment, with 1 mg kg-1 and a 3 h pretreatment having the greatest effect of the doses and times employed. 3. Topical betamethasone inhibited the oedema responses to all of the stimuli showing no apparent selectivity. 4. Topical betamethasone inhibits inflammatory stimuli in a different manner from systemic betamethasone. The broad spectrum of inhibition suggests that topical betamethasone acts by affecting a fundamental feature of the inflammatory response common to all of the stimuli.     

Effect of dexamethasone on neutrophil accumulation and oedema formation in rabbit skin: an investigation of site of action.

1. The anti-inflammatory actions of dexamethasone on vascular and leukocyte responses in rabbit skin were investigated. 2. Neutrophil accumulation and oedema formation were simultaneously measured as the local accumulation of i.v. administered 111In-labelled neutrophils and 125I-labelled albumin. Systemically administered dexamethasone (3 mg kg-1) inhibited neutrophil accumulation induced by i.d. zymosan activated plasma (ZAP), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and leukotriene B4 (LTB4) when co-injected with prostaglandin E2 (PGE2). Dexamethasone also inhibited oedema formation elicited by these stimuli and the responses induced by i.d. platelet activating factor (PAF)+PGE2 and bradykinin (BK)+PGE2. 3. Intradermal dexamethasone (2 x 10(-10) mol per site) but not indomethacin (10(-8) mol per site) inhibited oedema formation induced by i.d. ZAP+PGE2 and BK+PGE2. This inhibitory effect of dexamethasone was significant only with pretreatment periods of 4 h, shorter pretreatment periods resulting in greatly reduced effects. Intradermal dexamethasone had no effect on neutrophil accumulation induced by ZAP+PGE2. 4. Intradermal dexamethasone (2 x 10(-10) mol per site) had no effect on increase in blood flow induced by PGE2 as measured by 133Xenon clearance. 5. The accumulation of neutrophils isolated from donor rabbits pretreated with i.v. saline or dexamethasone (3 mg kg-1) was investigated in untreated recipient rabbits. The accumulation of neutrophils, induced by ZAP+PGE2, FMLP+PGE2 and LTB4+PGE2, from dexamethasone-pretreated donors was significantly smaller than the accumulation of neutrophils from saline-pretreated donors. 6. The results of this study suggest that dexamethasone can have a direct effect on vascular endothelial cells resulting in an inhibition of oedema formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ruthenium red selectively inhibits oedema formation and increased blood flow induced by capsaicin in rabbit skin.

It has been suggested that ruthenium red has a selective inhibitory effect on capsaicin-induced nociceptor stimulation. We have investigated the effect of ruthenium red on oedema formation and vasodilatation induced by intradermal (i.d.) injection of capsaicin in the rabbit in vivo. Responses induced by capsaicin were inhibited by ruthenium red, but responses induced by bradykinin, N-formyl-methionyl-leucyl-phenylalanine (FMLP), platelet activating factor (PAF), histamine and calcitonin gene-related peptide (CGRP) were not affected. These results suggest that ruthenium red selectively inhibits capsaicin-induced local plasma protein leakage and vasodilatation in the rabbit skin microvasculature.     

An investigation into the mechanism of capsaicin-induced oedema in rabbit skin.

1. Oedema formation induced by intradermal capsaicin has been studied in rabbit skin. The effect of the anti-inflammatory steroid dexamethasone and also of a range of known inhibitors of oedema formation have been investigated in order to elucidate mechanisms involved in capsaicin-induced oedema formation. 2. Oedema formation, in response to intradermally-injected test agents, was measured by the local extravascular accumulation of intravenously injected 125I-labelled albumin. In separate experiments skin blood flow was assessed by the clearance of intradermally-injected 133xenon. 3. Oedema formation induced by intradermal histamine (3 nmol) and bradykinin (1 nmol), when in the presence of vasodilator doses of calcitonin gene-related peptide (CGRP) (3 pmol) or prostaglandin E1, (PGE1) (10 pmol), was significantly inhibited (P < 0.01) in rabbits pretreated with intravenous dexamethasone (3 mg kg-1, -4 h). In contrast dexamethasone had no effect on capsaicin (3 mumol)-induced oedema formation or, on capsaicin (30-100 nmol)-induced blood flow. 4. Oedema formation observed in response to intradermal capsaicin (3 mumol) was significantly inhibited (P < 0.01) when the selective capsaicin antagonist, ruthenium red (3 nmol) was co-injected. This suggests that the mechanism of capsaicin-induced oedema involves activation of sensory nerves. However, oedema was not inhibited when capsaicin was co-injected with the neurokinin NK1 receptor antagonist, RP67580 (10 nmol), the NK2 antagonist SR48960 (10 nmol) or the CGRP antagonist CGRP8-37 (300 pmol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary adenylate cyclase activating polypeptide is a potent vasodilator and oedema potentiator in rabbit skin in vivo.

1. The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on microvascular blood flow and plasma protein leakage were investigated in rabbit skin in vivo. 2. Intradermal injection of PACAP38, the 38 amino acid form of the peptide, caused a dose-dependent increase in blood flow measured by a 133Xe clearance technique. An equivalent increase in blood flow was induced by 10(-12) mol per site of PACAP38, 10(-12) mol per site of human alpha-calcitonin gene-related peptide (CGRP) and 10(-10) mol per site of vasoactive intestinal polypeptide (VIP). 3. The vasodilator activity of PACAP38 was not significantly different from that of the 27 amino acid form of the peptide, PACAP27, when measured with a laser Doppler flow meter, causing a 104 +/- 14% compared with 110 +/- 18% increase above basal blood flow at 10(-12) mol per site respectively. 4. At 10(-12) mol per site the effect of PACAP38 was longer lasting than that of CGRP. Blood flow remained significantly increased above control at 2 h with PACAP38 (P less than 0.05) whereas blood flow after intradermal CGRP had returned to control values by this time. 5. PACAP38 injected alone had no significant effect on microvascular leakage of 125I-labelled albumin. However, PACAP38 significantly potentiated bradykinin-induced oedema where it was approximately 100 fold more potent than VIP. 6. Oedema potentiation induced by PACAP38 was not inhibited by indomethacin at a dose which did inhibit potentiation of bradykinin-induced oedema by arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoactive intestinal polypeptide is more potent than prostaglandin E2 as a vasodilator and oedema potentiator in rabbit skin.

1 The 28 amino acid polypeptide, vasoactive intestinal polypeptide (VIP) induced increased local blood flow when injected intradermally in the rabbit. 2 VIP was found to be even more potent than prostaglandin E2 (PGE2) in increasing blood flow; VIP induced a significant effect at a 1 pmol dose. 3 VIP was shown to be poor in increasing microvascular permeability but very potent in enhancing local oedema induced by two substances which increase permeability, bradykinin and C5a des Arg. VIP was more potent than PGE2 as an oedema potentiator. 4 Indomethacin had no effect on oedema potentiation induced by VIP, suggesting that a release of endogenous prostaglandins was not involved. 5 These results support our hypothesis for the regulation of oedema formation by arteriolar vasodilators, although the observations do not exclude the possibility of additional regulation by agonist interaction in the region of the venule. 6 VIP may be involved in the physiological control of normal blood flow and in hyperaemia in some types of inflammatory reactions.     

Antagonism of acetylcholine by adrenaline antagonists

Phenoxybenzamine antagonized the inhibitory action of acetylcholine on the guinea-pig isolated atrium. The antagonism was slow in onset, very slowly reversible, and could be overcome by increased concentrations of acetylcholine. In contrast, atropine inhibited the action of acetylcholine quickly, and the effect disappeared soon after withdrawal. The pA₁₀ of phenoxybenzamine (2 hr of contact) was 6.8, and that of atropine (30 min of contact) was 8.4. In the presence of atropine phenoxybenzamine did not exert a slowly reversible antagonism, and the dose-ratio of acetylcholine returned to normal soon after withdrawal of both drugs. Phenoxybenzamine also antagonized acetylcholine in the guinea-pig isolated ileum, but with higher concentrations acetylcholine did not overcome the antagonism. The pA₁₀ (60 min of contact) was 6.6. The pA₁₀ of chlorpromazine in the atrium (2 hr of contact) and ileum (60 min of contact) was 5.9. Phentolamine, 2-diethylaminomethylbenzo-1,4-dioxan hydrochloride (883 F), and yohimbine antagonized acetylcholine in the atrium and ileum but required higher concentrations than chlorpromazine.     

Transmitter release modulated by alpha-adrenoceptor antagonists in the rabbit mesenteric artery: a comparison between noradrenaline outflow and electrical activity.

Effects of alpha-adrenoceptor blockers (prazosin, yohimbine, phentolamine and phenoxybenzamine) on the outflow of noradrenaline (NA) and 3,4-dihydroxyphenylglycol (DOPEG) during perivascular nerve stimulation were observed in relation to electrical events in the rabbit mesenteric artery. Cocaine or imipramine increased the NA outflow and reduced the DOPEG outflow induced by nerve stimulation. In the absence of stimulation, cocaine and imipramine did not significantly modify the NA and DOPEG outflows. The alpha-adrenoceptor blockers we used enhanced the NA and DOPEG outflow during nerve stimulation, in a dose-dependent manner; the potency of the enhancement was higher for phentolamine and phenoxybenzamine than for prazosin and yohimbine. Higher concentrations (10(-5) M) of yohimbine reduced the NA and DOPEG outflows induced by nerve stimulation. Prazosin increased the DOPEG outflow in the absence of stimulation, and this effect was not inhibited by pretreatment with cocaine. Guanethidine increased the NA and DOPEG outflow in the absence of stimulation, and the NA outflow was reduced during nerve stimulation. These effects of guanethidine were prevented by pretreatment with cocaine or imipramine. Perivascular nerve stimulation evoked excitatory junction potentials (e.j.ps) and with high frequency stimulation, slow depolarization and spike potentials. Application of phentolamine, phenoxybenzamine or yohimbine enhanced, and of prazosin had no effect, on the amplitude of the e.j.p. Spike potentials were not affected by these alpha-adrenoceptor blockers. Slow depolarization ceased in the presence of prazosin, phentolamine or phenoxybenzamine, and was slightly inhibited by yohimbine. Guanethidine blocked all of these electrical responses induced by perivascular nerve stimulation. Application of prazosin, phentolamine or phenoxybenzamine did not alter the resting membrane potential of the smooth muscle cells. Depolarizations of smooth muscle membrane produced by exogenously applied NA were inhibited by prazosin, phentolamine or phenoxybenzamine. Yohimbine itself depolarized the membrane and the inhibitory effects on the NA-induced depolarization were weaker. We conclude that the smooth muscle membrane of the rabbit mesenteric artery possesses alpha 1-adrenoceptors. Increase in NA outflow by alpha-adrenoceptor antagonists during nerve stimulation was not always consistent with increase in e.j.p. amplitude, presumably due to involvement of actions other than alpha-adrenoceptor blockade with each of these antagonists.     

E-type prostaglandins enhance local oedema formation and neutrophil accumulation but suppress eosinophil accumulation in guinea-pig skin.

1. Prostaglandins possess both pro- and anti-inflammatory actions depending on their route of administration and the experimental model used. In this study, we have investigated the effect of locally injected prostaglandins on oedema formation, neutrophil accumulation and eosinophil accumulation in inflammatory responses in guinea-pig skin. 2. Prostaglandin E1 (PGE1) significantly enhanced local oedema formation induced by zymosan-activated plasma (ZAP), bradykinin and in a passive cutaneous anaphylactic (PCA) reaction. The accumulation of ZAP-induced 111In-labelled neutrophils was also significantly enhanced by PGE1. In addition, the prostacyclin analogue, iloprost, enhanced ZAP-induced responses. 3. In contrast PGE1 decreased the accumulation of 111In-labelled eosinophils in skin sites. This was demonstrated on eosinophil accumulation and local oedema formation induced by PAF, compound 48/80 and in the PCA reaction. PGE2 also suppressed eosinophil accumulation while iloprost had no detectable effect. 4. Isoprenaline inhibited eosinophil accumulation in a dose-dependent manner with no effect on local oedema formation, except in the case of responses to ZAP where suppression was observed. 5. The vasodilator neuropeptide, calcitonin gene-related peptide (CGRP), enhanced local oedema formation but had no detectable effect on eosinophil accumulation. 6. In conclusion, the magnitude of a given response to an inflammatory mediator in vivo depends on the net effect of stimulation of several cell types e.g. arteriolar smooth muscle cells, microvascular endothelial cells, mast cells and accumulating leukocytes. In this study, we have demonstrated that different components of the inflammatory response in guinea-pig skin can be differentially modulated by E-type prostaglandins and isoprenaline, suggesting that cyclic AMP has an important regulatory role.     

Measuring side-effects of beta-adrenoceptor antagonists: a comparison of two methods.

The prevalence of side-effects of beta-adrenoceptor antagonists among hypertensive patients was assessed by two methods. Using visual analogue scales, scores for tired legs, cold digits and vivid dreaming were significantly higher in patients taking beta-adrenoceptor blockers than in patients not taking beta-adrenoceptor blockers. When measured by numerical scales, from 1 to 10, these symptoms showed no relation to beta-adrenoceptor blocker treatment. The visual analogue scales were more sensitive than the numerical scales because the scores were distributed more evenly over the analogue scales.     

Suppression by intradermal administration of heparin of eosinophil accumulation but not oedema formation in inflammatory reactions in guinea-pig skin.

1. Heparin is widely used in the treatment of thrombotic disorders and as an aid in surgery. Anti-inflammatory effects of heparin have also been described. In this study, we have investigated the effects of locally-injected heparin on the oedema formation and eosinophil accumulation induced by various inflammatory stimuli in guinea-pig skin. 2. Heparin dose-dependently suppressed the accumulation of 111In-labelled eosinophils induced by i.d. injection of zymosan-activated plasma (ZAP). The 111In-eosinophil accumulation induced by other inflammatory stimuli (compound 48/80, platelet activating factor, interleukin-8 and in a passive cutaneous anaphylaxis reaction) was also suppressed by locally-injected heparin. 3. Oedema formation in response to these same stimuli was not altered by the local injection of heparin. 4. Fucoidin, a negatively-charged sulphated algal polymer, had no effect on the 111In-eosinophil accumulation or oedema formation induced by ZAP. Nevertheless, fucoidin significantly suppressed the oedema formation induced by i.d. injection of cationic protein-containing extracts of Schistosoma mansoni larvae. Heparin also inhibited oedema induced by the extracts, suggesting that both fucoidin and heparin were effectively neutralizing the cationic protein of the extracts to inhibit their oedema-inducing activity. 5. Thus, heparin significantly inhibited the accumulation of 111In-eosinophils, but not oedema formation, induced by various inflammatory stimuli. This, taken together with the lack of effect of fucoidin, suggests that heparin interferes with the process of eosinophil trafficking by a mechanism that does not depend on neutralisation of the charge of the stimulatory molecules.     

Antagonism of morphine by long acting narcotic antagonists

Thirty-six female albino rats, trained to run for a chocolate reward in a circular runway, were treated according to 6×6 Latin square schemes with five doses of 3-quinuclidinylbenzilate, ranging from 0.1 to 10 mg/kg, or the vehicle. On experimental days there were 6 consecutive trials. Intraperitoneal injections were administered 20 min before the first trial. The apparatus was automated as far as the opening of sliding doors and the recording of the duration of running, subdivided into latency and running-time, were concerned. Along with the duration of running, the behaviour shown in the various parts of the maze was analysed. The drug caused a marked, dose-dependent increase of the latency, whereas the effect on running-time was comparatively small. During the latency the frequency of ambivalent behaviours, shown at the transition of the start-goal compartment and the runway, increased under the influence of 1 mg/kg or more. Concomitant increases were noted in the frequency of displacement activities, which were absent in control animals. The results were interpreted as a drug-induced intensification of a conflict, existing in normal animals between the tendency to stay in the vicinity of the reward and the tendency to run for a subsequent reward.     

Profile of capsaicin-induced mouse ear oedema as neurogenic inflammatory model: comparison with arachidonic acid-induced ear oedema.

1. We have investigated the mechanism of capsaicin-induced mouse ear oedema compared with that of arachidonic acid (AA)-induced ear oedema, and evaluated the possible involvement of neuropeptides in the development of capsaicin-induced oedema. 2. Topical application of capsaicin (0.1-1.0 mg per ear) to the ear of mice produced immediate vasodilatation and erythema followed by the development of oedema which was maximal at 30 min after the treatment. This oedema was of shorter duration with less swelling than AA-induced oedema (2.0 mg per ear). 3. Capsaicin-induced ear oedema was unaffected when inhibitors of arachidonate metabolites including platelet activating factor (PAF) were administered before capsaicin (250 micrograms per ear) application, while these agents significantly prevented AA-induced oedema. Dexamethasone, histamine H1 and/or 5-hydroxytryptamine (5-HT) antagonists, and substance P (SP) antagonists were effective in inhibiting both models. Furthermore, a Ca(2+)-channel blocker and the capsaicin inhibitor, ruthenium red, were effective inhibitors of capsaicin oedema but had no effect on AA-induced oedema. 4. Phosphoramidon (50 micrograms kg-1, i.v.), an endopeptidase inhibitor, markedly (P < 0.001) enhanced only capsaicin-induced ear oedema, but bestatin (0.5 mg kg-1, i.v.), an aminopeptidase, failed to enhance oedema formation. 5. Neuropeptides (1-100 pmol per site) such as rat calcitonin gene-related peptide (CGRP), SP, neurokinin A (NKA), and vasoactive intestinal peptide (VIP), which are released from capsaicin-sensitive neurones, caused ear oedema by intradermal injection. Furthermore, a synergistic effect of CGRP (10 fmol per site) and SP (10pmol per site) on oedema formation was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative dermatotoxicology: I. Direct comparison of rabbit and human primary skin irritation responses to isopropylmyristate

The primary skin irritation properties of isopropylmyristate (IPM) have been determined for rabbit and human skin. In rabbit studies, an occluded patch procedure was used in which irritation due to a vehicle was eliminated. Known amounts of IPM were applied to patches with ethanol as a vehicle. A radiotracer study showed the ethanol could be evaporated to a nonirritating level prior to use of the patch. This procedure was used with an experimental design that improved the efficiency of the irritation test by allowing removal of animal-to-animal variability and permitting four different treatments on each animal at one time. Four consecutive 23-hr occluded exposures of rabbit skin to 6.3 mg IPM/cm² of patch produced moderate erythema according to the Draize scale while four exposures to 23 or 85 mg IPM/cm² of patch produced moderate to severe erythema with very slight edema. A single exposure at doses ranging from 1.7 to 85 mg IPM/cm² of patch produced responses ranging from no erythema to well-defined erythema. The responses to a single exposure were less with 1 or 4 hr of occlusion than with 23 hr. A statistical analysis showed substantial variation in irritation scores from skin site-to-skin site but no tendency for one site on the rabbits' backs to have systematically higher scores than another. Human studies were conducted with experimental conditions the same as for the rabbit. Four consecutive 23-hr occluded exposures of human back skin to 85 mg IPM/cm² of patch produced only very slight erythema with no edema. The results of these studies support the use of IPM as a model species-variable skin irritant to study the biological basis for the variability of skin irritation among species.     

Role of CD18 in the accumulation of eosinophils and neutrophils and local oedema formation in inflammatory reactions in guinea-pig skin.

1. Intradermal injection of the complement fragment C5a des Arg induces local oedema formation that, in rabbits and man, is dependent on circulating neutrophils. Monoclonal antibodies to the leukocyte adhesion molecule CD11/CD18 block neutrophil accumulation and prevent neutrophil-dependent oedema formation. The role of CD11/CD18 in mediating eosinophil accumulation in vivo is less established. In this study we have used an anti-human CD18 monoclonal antibody, 6.5E, to investigate the neutrophil-dependency of oedema formation induced by C5a des Arg in guinea-pig skin. We also studied the role of CD18 in mediating eosinophil accumulation in the same model. 2. Stimulated adhesion of 111In-labelled guinea-pig neutrophils and eosinophils to serum-coated plastic was inhibited in a dose-dependent manner by 6.5E suggesting that the monoclonal antibody recognizes and blocks the guinea-pig CD18 adhesion molecule. 3. The accumulation of 111In-labelled neutrophils induced by zymosan-activated plasma (ZAP, as a source of C5a des Arg) in skin sites was reduced by up to 89% in animals treated intravenously with F(ab')2 fragments of 6.5E. ZAP-induced accumulation of 111In-labelled eosinophils was also greatly reduced (by up to 78%) by treatment with 6.5E. 4. Despite the inhibition of ZAP-induced neutrophil accumulation by 6.5E, local oedema formation in the same skin sites was unaffected, except at the top dose of ZAP, by treatment with the anti-CD18 monoclonal antibody, suggesting that the oedema response was largely neutrophil-independent. Indeed, ZAP-induced oedema formation was reduced by up to 81% by the H1 receptor antagonist, mepyramine.(ABSTRACT TRUNCATED AT 250 WORDS)