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A dialyzable low molecular weight proinflammatory factor (X (2)) from rat spleen lymphocytes was isolated through a combination of gel filtration and high voltage paper electrophoresis (HVE) and then partially characterized. It was able to potentiate the formation of carrageenin induced edema on the rat paw. Its amino acid analysis revealed Glu, Cys and Gly (1:1:1), but gammaGlu as N-terminal residue, initially suggesting oxidized glutathione (GSSG), since it showed exactly the same HV electrophoretic mobility as GSSG at pH 6.5. However, neither GSSG nor a synthetic homologue showed any proinflammatory activity. On basis of its infrared spectrum, HVE mobility and presence of a gamma-Glu-Cys-Gly (GSH) moiety, the hypothesis of identity of X (2) with leukotriene C(4) (LTC(4)) was raised. Once again it was not confirmed, since LTC(4) did not show any proinflammatory activity too, leading us to infer that, even excluding LTC(4), our data are consistent with a structure bearing a GSH moiety conjugated with a hydroxylated insaturated fatty acid chain which contributes a -COO(-) group, thus providing a final net charge of -2 at pH 6.5 and an Mr = 600-650.

Decreased maximal responses to noradrenaline, adrenaline and phenylephrine were observed in aortas isolated from rats pretreated with triiodothyronine and thyroxine for 8 days. In addition, a potent relaxant effect occurred when supramaximal concentrations of noradrenaline and adrenaline, but not of phenylephrine, were used. The relaxant effect was not observed in preparations in which the endothelium had been removed, and it was antagonized by propranolol added to the bathing fluid. Practolol was ineffective. In vivo, adrenaline and noradrenaline evoked pressor effects which were of shorter duration and smaller magnitude in hormone-treated animals than in the controls. Furthermore, a subsequent fall in blood pressure to below normal was observed, not only with adrenaline but also with noradrenaline, in animals receiving thyroid hormones. We conclude that when large amounts of thyroid hormones circulate in the body, large arteries are more prone to distention in the presence of increased quantities of released catecholamines, and resistance vessels are less responsive to their pressor effects due to a superimposing dilator effect. These changes might represent adaptative mechanisms in hyperthyroid states, and might depend on a greater sensitivity of endothelial beta 2-receptors to noradrenaline and adrenaline.

1 The amplitudes of the first positive and the first negative waves of cortical potentials evoked by electrical stimulation of the dental pulp in the rat were decreased following electrical stimulation of the peripheral cut end of the saphenous nerve.2 This effect was greatly diminished when the stimulation of the saphenous nerve was performed with the saphenous or femoral veins ligated.3 During stimulation of the saphenous nerve of a donor rat, the subcutaneous tissue in the area supplied by the nerve was perfused; when the perfusate (in which a permeability-increasing factor was detected) was injected intravenously into a recipient animal, a decrease in the amplitude of the evoked cortical potential of the recipient rat was also observed.4 Intravenous injections of 5-hydroxytryptamine, histamine, bradykinin, prostaglandins or adenosine-triphosphate produced no effect on the evoked cortical potential, whereas large doses of acetylsalicylic acid caused a decrease.5 It is suggested that a humoral factor, released during sensory nerve stimulation, may help to modulate the processing of afferent inputs from pain receptors.

1 Subcutaneous administration of glucagon (1 and 0.5 mg/kg) 30 min before the injection of carrageenin or dextran into the rat's paw reduced oedema and the local exudation of Evans blue previously given intravenously. 2 The effect persisted after removal of the adrenal medulla but not after adrenalectomy. 3 When glucagon (1 mg/kg, s.c.) was given daily after a local reaction to Freund's adjuvant injected into the paw had developed, a decrease in the reaction was observed up to 12 days. Blood sugar levels remained within the normal range. 4 Glucagon may exert an anti-inflammatory effect through the release of adrenal corticosteroids and thus help modulate inflammatory reactions.

1 Increased vascular permeability following electric antidromic stimulation of the rat saphenous nerve was observed in the skin area supplied by the nerve, confirming previous results by other authors.2 The phenomenon was not affected by pretreatment of the rats with diphenhydramine, burimamide or their combination; atropine, methysergide, methysergide plus diphenhydramine, carboxypeptidase B, acetylsalicylic acid, indomethacin or methiazinic acid. It was partially reduced by previous injection of cellulose-sulphate, a kininogen-depleting agent.3 Perfusates from the subcutaneous tissue of the paw area supplied by the saphenous nerve contained permeability increasing activity as shown by intradermal tests in other rats. This activity was present in perfusates collected during nerve stimulation but not in those collected before stimulation. It was not destroyed by heating to 100 degrees C, or by alpha-chymotrypsin or trypsin.4 Bradykinin-like activity may appear later in the perfusates, depending on the intensity of the stimuli.5 It is concluded that following electrical antidromic stimulation of the saphenous nerve a permeability increasing factor is released, possibly from nerves. It is dialysable and can be distinguished from acetylcholine, histamine, 5-hydroxytryptamine, plasma kinins, substance P, prostaglandins and high molecular weight proteins. The increased vascular permeability induced by this factor leads to plasma exudation and activation of the kinin system.

1. Inflammatory responses were induced by the injection of carrageenin into the rat paw and lymph was collected by cannulation of the thoracic duct. Cell-free lymph samples were intracutaneously injected into a second group of rats to measure vascular permeability activity by local exudation of Evans blue. 2. The vascular permeability activity in lymph samples draining inflamed tissue was consistently higher than in lymph from normal controls. Activity increased with the development of inflammation, attaining maximum values 2 to 2.5 h after the injection of carrageenin. 3. The activity was reduced by pretreatment of lymph donors with indomethacin or soybean trypsin inhibitor. Pretreatment of the animals used to measure vascular permeability activity with diphenhydramine or methysergide attenuated their responses to the permeability factors present in lymph. Thus prostaglandins, kinins, vasoactive amines or their generating systems may have contributed to the total activity detected. 4. Pretreatment of lymph donors with corticosterone or glucagon, whose anti-inflammatory action is mediated by the release of endogenous corticosteroids, did not alter the amount of activity in lymph. 5. These data suggest that the formation of the permeability factors present in lymph draining inflamed tissue was not affected by corticosteroids. We conclude that corticosteroids do not exert their anti-inflammatory activity at this level.

The response to vasoactive agents of microvessels in situ and large arteries in vitro was compared in normal and alloxan-diabetic rats. Noradrenaline was equally effective in evoking a constrictor response of mesenteric microvessels in normal and diabetic animals. The constrictor response to a standard amount of noradrenaline in such vessels was fully antagonized by acetylcholine or papaverine, the minimum effective doses being equivalent in normal and diabetic animals. In contrast, the minimum doses of histamine or bradykinin, effective in normal animals, had to be increased about 20 fold to be active in diabetic animals. Increased osmolarity of extracellular fluid caused a significant and equivalent increase in latency of the vasoconstrictor response of microvessels to noradrenaline in normal and diabetic animals. Concentration-effect curves, constructed from the response of isolated aortae to noradrenaline, were similar in normal and diabetic animals, provided the endothelium was removed. Diabetes only affected preparations in which the endothelium was left intact. In these, the median effective concentrations of noradrenaline were greatly increased in comparison with normal values. Precontracted aortae from normal and diabetic animals were equally relaxed by acetylcholine and histamine, provided the endothelium was left intact. Loss of the relaxant response of the preparations in all groups of animals was observed following removal of endothelial cells. It is suggested that different mechanisms may be involved in the effects of vasodilator agents on large arteries in vitro or small vessels in situ. Histamine and bradykinin which are potent permeability-increasing factors, may antagonize the vasoconstrictor response of microvessels to noradrenaline through an action on endothelial cells with increased vascular permeability and temporary changes in composition of extracellular fluid. The reactive process of endothelial cells to permeability factors was affected by diabetes mellitus. However, the response of microvessels to acetylcholine and papaverine which are devoid of permeability-increasing properties, was not influenced by diabetes.

1. The constrictor response of microvessels to norepinephrine and tyramine, and the dilator response to acetylcholine and papaverine was equivalent in normal and alloxan-induced diabetic rats. 2. Acetylcholine and phentolamine were equally effective as antagonists of the vasoconstrictor action of norepinephrine in both groups of animals. 3. The minimum effective doses of histamine and PAF-acether to produce maximum dilation of microvessels, or to antagonize the response to norepinephrine in normal rats had to be increased 20-fold to evoke equivalent effects in diabetic animals.

The effect of cortisol (10 and 20 mg kg-1 day-1, sc), indomethacin (2 and 4 mg kg-1 day-1, po) and piroxicam (10 and 20 mg kg-1 day-1, po) on the proliferative component of inflammation was investigated in normal, diabetic, adrenalectomized and diabetic-adrenalectomized rats using the cotton pellet test. Whereas cortisol was equally effective in preventing granulation tissue formation in all groups of animals, indomethacin and piroxicam were much less active in animals with hormonal dysfunctions. Indomethacin and piroxicam reduced thymus weight of normal and diabetic animals as much as cortisol. This was taken to be a strong indication of the effect of these non-steroidal drugs on the adrenal cortex leading to increased secretion of adrenal corticosteroids. We conclude that at least part of the anti-inflammatory effect of indomethacin and piroxicam, in the present experiments, can be ascribed to the release of endogenous corticosteroids. This would explain the decreased sensitivity of adrenalectomized animals to the non-steroidal anti-inflammatory drugs used. An additional component, however, seems to be necessary for the full expression of the anti-inflammatory effect of these drugs, since diabetic animals were also less responsive to them. When both components were absent, as in diabetic-adrenalectomized animals, indomethacin and piroxicam were practically devoid of an anti-inflammatory effect.

The response to vasoactive agents of microvessels of the rat was tested in vivo by direct microscopic observation of the exteriorized mesentery and assessment of cutaneous vascular permeability changes with Evans blue. The constrictor response to a standard amount of noradrenaline in mesenteric microvessels was fully antagonized by acetylcholine in normal, diabetic, adrenalectomized and diabetic-adrenalectomized rats. In contrast, the minimum doses of histamine or bradykinin, effective in normal or adrenalectomized animals, had to be increased about 20 fold to be active in diabetic or diabetic-adrenalectomized animals. Topical application of insulin to mesenteric microvessels of diabetic animals, in amounts not causing any increase in serum insulin levels, improved or restored the capacity of the animals to respond to histamine or bradykinin, acting as antagonists of the vasoconstrictor response to noradrenaline. Topical insulin, however, was ineffective in normal animals given 2-deoxyglucose, the acute effects of which result from cellular glucopaenia unrelated to insulin deficiency. Vascular permeability responses to intracutaneous histamine or bradykinin were decreased in animals pretreated with 2-deoxyglucose as much as in diabetic animals. Pretreatment of normal animals with indomethacin produced no effect on the responses of these animals to histamine or bradykinin, tested as antagonists of noradrenaline on mesenteric microvessels, or as vascular permeability-increasing factors in the skin. Pretreatment of normal animals with chloroquine, mepacrine or dexamethasone had no effect on the reactivity of mesenteric microvessels to histamine and bradykinin, acting as antagonists to noradrenaline. 7 It is suggested that vasoactive substances, endowed with permeability-increasing properties, evoke relaxation of microvessels through an insulin-dependent action on endothelial cells, unrelated to the release of arachidonic acid metabolites. This action would lead to increased vascular permeability, with opening of interendothelial junctions, and temporary changes in composition of extravascular fluid, which in turn, would provide the basis for vasodilatation. Diabetes mellitus apparently impairs such responses as a result of the accompanying cellular glucopaenia. Adrenal corticosteroids are not involved in the impaired responses.

1 Noradrenaline (NA) evoked a vasoconstrictor response in rat mesenteric microvessels in situ, the latency and nature of which was analogous in normal and alloxan-diabetic animals.2 Histamine and bradykinin (Bk) were capable of antagonizing the response to NA in normal but not in diabetic animals. In contrast, acetylcholine (ACh) was equally effective as an antagonist to NA in both groups of animals.3 The altered responses to histamine and Bk were not associated with hyperglycaemia since fasting rendered the diabetic animals normoglycaemic and yet did not restore the reactivity of microvessels. Previous administration of insulin to diabetic animals corrected the impaired responses to histamine and Bk.4 A similar condition of impaired responses to histamine and Bk was produced in normal animals by the intravenous injection of 2-deoxyglucose although ACh remained fully active.5 Apparently, the functional changes observed in the response to histamine or Bk, as antagonists of the vasoconstrictor reaction to NA, were not associated with a defective response of all smooth muscle. First, because ACh remained active in diabetic animals, and, second, because extravascular smooth muscles obtained from either normal or diabetic rats were equally relaxed by histamine or Bk in vitro.6 It is suggested that histamine and Bk antagonized the vasoconstrictor response of microvessels to NA through an action on lining endothelial cells resulting in increased vascular permeability and hyperosmolarity of extracellular fluids.7 The process depended on the availability of insulin, and, therefore, might be affected by intracellular glucopaenia as occurring in diabetes.8 Intracellular glucopaenia markedly affected other structures. Reduced atria rates were observed in diabetes, despite the fact that the isolated preparation responded normally to NA, ACh or tyramine. Partial substitution of glucose in the bathing fluid by 2-deoxyglucose or addition of NaF to the organ bath evoked similar changes in atria from normal animals.9 ACh which has little effect on vascular permeability must exert its vasodilator effects through mechanisms which are different from those influenced by the biochemical changes occurring in diabetes.

Leucopenic rats were injected i.v. with either allogeneic or syngeneic lymphocyte suspensions and their capacity to respond to carrageenin tested at various intervals after the injection of the cells. The carrageenin-induced inflammatory response, which is characteristically attenuated in leucopenic animals, was partially but none the less definitely restored when allogeneic cells were transfused 20 min before the irritant, and less so when they were given 1 or 2 h before. Syngeneic cells were effective in restoring this response up to 2 h after injection. Only a small fraction of the exogenous cells, either allogeneic or syngeneic, remained in the blood, and this for short intervals. One hour after administration of cell suspensions blood leucocyte counts again attained previous values. The same rate of disappearance of the cells from blood was observed in splenectomized rats. Removal of the cells from circulation was thought to reflect "homing" into lymphoid tissues and other tissues, but not selectively in the spleen. Lymphocyte constituents, obtained by cell disintegration and injected i.v. into leucopenic rats, did not cause any change in blood leucocyte counts and yet restored the previously inhibited inflammatory response to carrageenin, as much as the viable cells. It is suggested that inflammation-producing materials, either deposited in tissues or formed or transformed at a site of injury, might gain access to the lymphatics during the early stages of a non-immune, inflammatory response, and "stimulate" lymphoid cells in adjacent lymphoid tissues either to release pro-inflammatory factors or to migrate to the site of injury, where they would exert pro-inflammatory activities. Apparently, only a small number of these cells in involved in the response.

Topical application of a standard dose of noradrenaline (NA) to the anesthetized rat mesentery evoked a vasoconstrictor response the latency of which was increased in a dose-dependent way by previous addition of PAF-acether or histamine but not by Lyso-PAF. Lyso-PAF, however, blocked the antagonistic effect of PAF-acether to NA, the blockade being equivalent to that observed with PAF-antagonists (BN 52021 or WEB 2086) either injected iv, or applied topically. In contrast, the antagonistic action of histamine towards NA was not affected by Lyso-PAF or PAF-antagonists. Since platelet aggregation and leukocyte accumulation did not occur, PAF-acether appears to interact directly with microvessels to block vasoconstrictor stimuli, an effect antagonized by PAF-antagonists and the metabolite/precursor, Lyso-PAF.

1. The conditions in which the release of an active, bradykinin-like agent occurred when rat paws were heated to 46 degrees C were studied by means of the double coaxial perfusion of the subcutaneous spaces.2. The active material thus released stimulated the isolated rat uterus, produced a relaxing effect on the isolated rat duodenum, was destroyed by incubation with chymotrypsin and was potentiated by bradykinin-potentiating factor. LSD-25, in doses sufficient completely to block 5-hydroxytryptamine, did not affect the responses of the isolated uterus to the active material.3. The effects on this release of anti-proteases and hexadimethrine bromide, atropine and diphenhydramine were studied.4. Soy-bean trypsin inhibitor and hexadimethrine bromide added to the perfusion fluid produced a potent and reversible inhibition of the release of the active material; aprotinin and Kunitz inhibitor caused a temporary block.5. When administered intravenously, much larger doses of the substances were necessary to produce a similar block.6. Pretreatment of the animals with atropine plus diphenhydramine did not affect the release of the active kinin(s).7. Ligature of one iliac artery was followed by disappearance of the active material in the perfusate from the corresponding paw.8. These facts suggest that heating elicits a process leading to plasma extravasation and that the subcutaneous tissue is the chief site of release of the active material.

1. Local injection of carrageenin in the rat's paw produced oedema and leakage of dye which had been administered previously by the intravenous route. A net dissociation between both parameters was observed: while oedema developed slowly, maximal intensity being attained after 4-5 h, dye-leakage was maximum after 1 hour.2. Anti-inflammatory drugs such as acetylsalicylic acid and indomethacin were effective in reducing oedema and dye-leakage when given before the injection of carrageenin, but much less effective when given 30 or 60 min after carrageenin. Hexadimethrine bromide was effective in reducing dye-leakage also when given 1 h after the injection of carrageenin.3. Combined administration of benadryl and methysergide, before the injection of carrageenin caused only a slight reduction in oedema and dye-leakage.4. When the paws were heated (55 degrees C for 30 s) as a noxious stimulus the dissociation between maximal oedema and maximal dye-leakage was not observed, both phenomena running parallel. Pre-treatment of the animals with indomethacin did not afford any protection.5. These results suggest that the inflammatory reaction to mild stimuli (carrageenin in our experiments) develops through different phases: initially the increased vascular permeability involves extravasation of plasma proteins and that phase is followed by an increased permeability mainly to water. Stronger stimuli (heating in our experiments) produce an overlapping of both phases, probably by inflicting severe damage to the vascular bed of the affected area.6. The anti-inflammatory drugs employed affected chiefly the initial phase of the response.