Contribution of adrenaline to the fibrinolytic activity evoked by E. coli endotoxin in the rat.

Intravenous injection of E. coli endotoxin (ETX), of adrenaline (AD) or of carbamylcholine (CBCH), caused fibrinolytic activity (FA), directly detectable on plasminogen-rich fibrin plates, to appear in the plasma of the rat. Adrenodemedullation abolished responses to ETX or CBCH, but enhanced those to AD. Rats given ETX exhibited marked hypotension, followed by a compensatory phase of normotension abolished by adrenodemedullation and significantly attenuated by phenoxybenzamine, an alpha-adrenergic blocking agent which however failed to block FA caused by either ETX or AD. Aspirin, but not indomethacin, inhibited FA evoked by ETX, AD or CBCH. These results suggest that FA evoked by ETX in the rat is caused by AD released from the adrenal gland and does involve the fatty acid cyclooxygenase system.

Role of the adrenal gland in the leucocytosis caused by bradykinin or cellulose sulphate in the rat.

Intravenous injections of bradykinin, cellulose sulphate (a kinin-releasing agent) or adrenaline cause rapid leucocytosis in the rat. The effect of the two former drugs is abolished by adrenalectomy, that of the latter is unaffected by this treatment. Bradykinin and agents capable of releasing it in plasma, may induce leucocytosis via adrenaline released from the adrenal gland.

Mechanisms of histamine release by compound 48-80.

1. Rat and guinea-pig lung tissues were incubated for 20 min at 37 degrees C in Krebs-Ringer phosphate buffer at pH 7.4, or in Tyrode-Tris buffer at pH 8.2, and the release of histamine produced by adding different concentrations of compound 48/80 to the incubation medium was determined.2. At pH 7.4, increasing concentrations of 48/80 increased the release of histamine from the rat lung, with a tendency towards a maximum. No release of histamine from guinea-pig lung was observed at this pH. At pH 8.2, histamine release occurred both from rat and guinea-pig lung, and was proportional to the logarithm of the concentration of compound 48/80.3. Histamine release from rat lung by 20 mug/ml. of 48/80 decreased when the pH was raised from 7.4 to 8.2; but the release caused by 1 mg/ml. of 48/80 increased both in rat and guinea-pig lung as the pH was raised.4. 2-4-Dinitrophenol (DNP) inhibited the release of histamine from rat lung by a concentration of 20 mug/ml. of 48/80; the inhibition was prevented by glucose. DNP did not affect histamine release from rat or guinea-pig lung by a concentration of 1 mg/ml. of 48/80 and enhanced the release when the pH was raised from 7.4 to 8.2.5. 1 mg/ml. of 48/80 did not inhibit the enhanced oxygen consumption produced by DNP in the isolated rat diaphragm.6. Iodoacetic acid (IAA) or a Ca/Mg-free medium inhibited the release of histamine by 20 mug/ml. of 48/80 from rat lung but not the release produced by 1 mg/ml. in either rat or guinea-pig lung.7. The degranulation of rat mesentery mast cells caused by 20 mug/ml. of compound 48/80 was inhibited by DNP. The degranulation evoked by 1 mg/ml. of 48/80 was also sensitive to this inhibitor; in this instance, however, the metachromatic staining reaction of the mesentery mast cells was greatly diminished.8. It is concluded that two processes of histamine release by compound 48/80 occur in rat lung. One, dependent on cell metabolism, involves, mast cell granule secretion. The other, independent of cell metabolism, seems to consist of a simple exchange reaction between histamine and compound 48/80, and this is the only one occurring in guinea-pig lung.

Lowering of kininogen in rat blood by adrenaline and its inhibition by sympatholytic agents, heparin and aspirin.

1 (-)-Adrenaline lowered the kininogen content and transitorily elevated the fibrinolytic activity of plasma following intravenous injection into the rat. Its effect on kininogen increased when administered by intravenous infusion.2 Although less effective, (-)-noradrenaline had a similar action to adrenaline; (+/-)-isoprenaline was inactive and failed to inhibit the effect of adrenaline.3 The effect of adrenaline on kininogen could be reproduced in vitro by incubation of whole blood, but not cell-free plasma, with the catecholamine for 5 min at 37 degrees C.4 Propranolol or phenoxybenzamine, as well as heparin or acetylsalicylic acid (aspirin), blocked the reduction of rat blood kininogen by adrenaline in vivo and in vitro.

Fibrinolytic activity evoked in the plasma of the normal and adrenalectomized rat by cellulose sulphate.

1. Cellulose sulphate, a kinin-releasing agent, produced fibrinolytic activity in plasma when administered intravenously to the rat but not when added to fresh rat plasma in vitro. The in vivo effect was maximal within 1 min and disappeared within 10-20 minutes. It was retained in plasma taken 1 min after the injection and kept at room temperature for 30 minutes.2. A decrease of anti-fibrinolytic potency measured against urokinase-activated bovine plasmin, was shown to occur in plasma of rats given cellulose sulphate.3. Activated rat plasma lysed heat-denatured fibrin: it probably contains free plasmin as well as plasminogen activator.4. Adrenalectomized rats did not exhibit fibrinolytic activity nor statistically significant benzoyl-arginine ethyl ester-esterase activation in plasma after cellulose sulphate treatment.5. Adrenalectomized rats had significantly increased levels of plasma kininogen, but were normally sensitive to the kininogen-depleting action of cellulose sulphate.6. The increased plasma kininogen of adrenalectomized rats seems to be a consequence of the impairment of the plasminogen activating mechanism.

Contribution of vasopressor and plasma kininogen changes towards acute adrenaline pulmonary edema in the rat.

Acute pulmonary edema, evidenced by increased lung/body weight ratios, was evoked in rats within 5 min following the intravenous injection of 16-40 mug/kg of adrenaline. This change was accompanied by a decrease of 40% of circulatory kininogen not due to generalized plasma protein loss. Rats treated 10-20 min prior to adrenaline with 10 mg/kg of acetylsalicylate (Aspirin), 1000 KIU/kg of Kunitz anti-protease (Trasylol), or 10 mg/kg of soybean trysin inhibitor (SBTI), failed to exhibit pulmonary edema or decreased plasma kininogen levels, but were as sensitive as untreated animals to the arterial hypertensive effect of adrenaline. 4.8 mug/kg of carbamylcholine administered together with 40 mug/kg of adrenaline, prevented pulmonary edema. Carbamylcholine did not reduce plasma kininogen consumption by adrenaline, but effectively decreased the raised systolic arterial blood pressure, the increased systolic-diastolic pressure interval as well as the reflex slowing of the heart presented by adrenaline-treated rats. It seems that in the adrenaline-treated rat, pulmonary edema results from the joined action of vasopressor effects leading to hydrostatically forced outflow of vascular fluid, and of kinin release leading to increased peripheral vascular permeability.

Acute pulmonary edema and plasma kininogen consumption in the adrenaline-treated rat: inhibition by acetylsalicylic acid and resistance to salicylate and indomethacin.

Pulmonary edema and plasma kininogen consumption caused by intravenously administered adrenaline, were inhibited in rats pretreated with acetylsalicylic acid, but not in rats pretreated with indomethacin or sodium salicylate. The possibility of a connection between this edema and mast cell-linked activation of kallikrein by adrenaline is discussed, as well as the possible role of acetylsalicylic acid acting as an acetylating inhibitor of these processes.

Bradykinin release from high molecular weight kininogen and increase in plasma kallikrein-like activity following sensory stimulation by food in the rat.

Cephalic stimulation by food elicits, among other responses, dilatation of mesenteric blood vessels preparatory for digestion. The possible participation of bradykinin (BK), a powerful endogenous vasodilator, in this response was studied in fasted rats prior and following stimulation by sight and scent of food (sensory stimulation, SS), actual ingestion being denied to the animals. BK content of plasma high (HK) and low molecular weight kininogen (LK) was determined by bioassay on the atropinized, antihistamine-treated isolated guinea-pig ileum following release by trypsin from heat/acid denatured plasma. BK corresponding to LK was estimated in plasma which prior to denaturation had been incubated with kaolin, a process which leads to quantitative release and inactivation of BK from HK, but does not affect LK. BK corresponding to (HK + LK) was determined in plasma not exposed to kaolin. BK contained in HK was the difference between BK of (HK + LK) and of BK of LK. Plasma and glandular kallikreins were estimated by fluorimetry, using specific synthetic substrates. A 40.6+/-4.0% decrease (P<0.001) of BK in HK occurred in rats after 90 s of SS; LK remained unaffected. Ten minutes of SS did not result in further change. Atropine inhibited the effect of SS. Return of HK to pretreatment levels occurred when, following 90s of SS, rats were allowed to rest for 60 min in the absence of food. Renewed capacity to respond to SS was then observed. Plasma kallikrein, but not glandular kallikrein, increased in plasma of rats after SS. Increased free BK was detected in the circulation of Enalapril-protected rats after SS. Electrical stimulation of the distal sector of the sectioned left abdominal vagus nerve of Nembutal-anesthetized fasted rats reproduced the effect of SS on HK. It is concluded that visuo-olfactory stimulation by food generates nerve impulses, possibly carried by the vagus nerve, which by activating plasma kallikrein lead to cleavage of circulatory HK and release of BK in the rat.

Kininogen and prekallikrein increases in the blood of streptozotocin-diabetic rats are normalized by insulin in vivo and in vitro.

Twelve days following treatment with 50 mg/kg streptozotocin (STZ), male rats were diabetic, with a three-fold increase in blood glucose (P<0.001) and increased plasma bradykinin (BK) kininogen reserves of [high-(HK)- and low- (LK)-molecular-weight kininogens,+162%, P<0.01 and +63%, P=0.05, respectively], as determined by bioassay of BK released by trypsin from these precursors under standardized conditions. Administration of a single dose (10 U/kg i.v.) of regular insulin decreased plasma HK and LK to near non-diabetic values. Within 24 h these values had returned to levels characteristic of uncorrected diabetes. Prekallikrein (PK), the precursor of plasma kallikrein, an enzyme which releases BK from HK, was increased by 63.4% (P<0.05) in STZ-diabetes, but dropped to near normal levels following insulin treatment. Incubation of whole blood of normal or diabetic rats with 0.02-0.2 mU/ml regular insulin for 10 min at 37 C, decreased HK (P<0.01) and PK (P<0.05) and led to the appearance (P<0.05) of Arg-Pro-Pro-Gly-Phe, a partially stable product of BK metabolism, detected in the incubation media by an enzyme-linked immunosorbent assay (ELISA). Incubation of cell-free plasma insulin had no effect on these parameters, suggesting that blood cells, possibly neutrophils, are required by insulin for the activation of plasma PK to kallikrein leading to BK release. Insulin may be a factor modulating BK formation; its reduction in diabetes may explain increases of plasma kininogen and PK observed in this condition.

Enhancement of histamine synthesis in mouse skin by epinephrine.

Intravenous administration of 10-40 micrograms/Kg epinephrine to mice leads to a transient 50% increase in skin histamine, followed by an increase in blood histamine. Delayed inhibition by alpha-fluorometyl histidine (alpha FMH), suggests that these changes follow stimulation of pre-formed tissue histidine decarboxylase.

Stimulation by epinephrine of histamine synthesis by rat peritoneal fluid mast cells in vitro.

In vitro exposure of mast but not of other cells of rat peritoneal fluid to epinephrine leads, within 1 min, to progressing levels of histamine in both fluid and sedimentable phases of the incubates, which present no increase in their free/total histamine ratio. Histamine increase was blocked by alpha-fluoromethyl histidine (alpha FMH), acting after a significant lag period. When compared with controls under the electron microscope, epinephrine-treated mast cells show less electron-dense, swollen intracellular granules, apparent maintenance of cell membrane continuity and an apparent decrease of peripheral finger-like projections. Histamine accumulation by epinephrine-treated mast cells may be the result of an enhanced ability of pre-formed mast cell histidine decarboxylase to attack its cell-borne substrate, consequent to an unfolding of the cell membrane during cell tumefaction evoked by epinephrine.

Endosprin, an antipyretic, antiinflammatory and analgesic preparation of lysine salicylate: reassessment of its chemical composition and effects in experimental animals in comparison with aspirin.

Endosprin, an antiinflammatory-antipyretic-analgesic salicylate drug marketed in Brazil since 1970, has been shown by NMR-1H spectral analysis to contain salicylic acid and not, as formerly believed, acetylsalicylic acid (aspirin). Its analgesic and antiinflammatory activity in mice and rats was similar to that of aspirin but its antipyretic activity against bacterial pyrogen fever in rabbits was significantly lower than that of aspirin. This result stands in contrast to the large body of clinical evidence indicating a rather high degree of effectiveness of Endosprin in lowering fever of various origin in children.

In vitro reduction of human blood kininogen by catecholamines.

Plasma kininogen levels were significantly reduced in normal human blood, but not in cell-free human plasma, following 10 min in vitro exposure to, in order of decreasing effectiveness, 6 microM adrenaline, noradrenaline or isopropyl-noradrenaline. Phenoxybenzamine (0.1 mM), an alpha-receptor blocking drug, and 0.5 mM aspirin, an inhibitor of prostaglandin (PG) synthesis, inhibited the action of adrenaline, whereas 0.1 mM propranolol, a beta-receptor blocker, and 0.5 mM indomethacin, another inhibitor of the formation of PG, failed to do so. The results suggest that catecholamines are able to activate cell-mediated activation of the kallikrein system in human blood and that this process can be inhibited by aspirin.

Role of bradykinin in postprandial hypotension in humans.

A transient significant decrease in mean arterial blood pressure (MAP) from 107 +/- 3 to 98 +/- 3 mmHg (P < 0.05) was observed in elderly (59-69 years of age), healthy volunteers 25-30 min following ingestion of a test meal. In young volunteers (22-34 years of age), a postprandial decrease of MAP from 88 +/- 3 to 83 +/- 4 mmHg was also noted but it was not statistically significant. A 40% decrease in bradykinin (BK) content of circulatory high molecular weight kininogen had previously been observed in human subjects given the same test meal. We presently demonstrate by specific ELISA that the stable pentapeptide metabolite (1-5 BK) of BK increases from 2.5 +/- 1.0 to 11.0 +/- 2.5 pg/ml plasma (P < 0.05) in elderly volunteers and from 2.0 +/- 1.0 to 10.3 +/- 3.2 pg/ml plasma (P < 0.05) in young volunteers 3 h following food intake. This result suggests that ingestion of food stimulates BK release from kininogen in normal man. Postprandial splanchnic vasodilatation, demonstrated by a decrease of plasma half-life of intravenously administered indocyanine green (ICG), a marker of mesenteric blood flow to the liver, from 4.4 +/- 0.4 to 3.0 +/- 0.1 min (P < 0.05) in young volunteers and from 5.2 +/- 1.0 to 4.0 +/- 0.5 min (P < 0.05) in elderly volunteers, accompanied BK release. The participation of BK in this response was investigated in subjects given the BK-potentiating drug captopril prior to food intake. Postprandial decreases of ICG half-lives were not changed by this treatment in either young or elderly subjects, a result which may indicate that BK released following food intake plays no role in postprandial splanchnic vasodilatation in normal man.

Role in kinin in rat epinephrine pulmonary edema (REPE).

High doses (10-40 micrograms/Kg, i.v.), of epinephrine evoke conspicuous consumption of circulatory rat kininogen (Kg), an effect not observed in animals pre-treated with either soybean trypsin inhibitor (SBTI, 10 mg/Kg, i.v.), Trasylol (1000 KIU/Kg, i.v.) or Aspirin (10 mg/Kg). Kg consumption by epinephrine is accompanied by a raise in rat plasma TAME-esterase attributed to the activation of plasma kallikrein by pro-kininogenase generated in circulatory basophils or mast cells exposed to epinephrine. The severe pulmonary edema observed in rats given epinephrine, is very markedly reduced in animals pre-treated with either SBTI, Trasylol or Aspirin at doses which inhibit Kg consumption by the catecholamine. Indomethacin (1-10 mg/kg) did not reduce REPE nor inhibit Kg loss. These results indicate that while kinin released via the action of epinephrine-activated basophils and/or mast cells, could play a major role in REPE, the same cannot be suggested for prostaglandins, whose eventual formation in the epinephrine-treated lung, would be expected to be fully prevented by Indomethacin. Since Aspirin, known as a less effective inhibitor of PG formation than Indomethacin, was nevertheless a highly effective inhibitor of both REPE and Kg consumption, an explanation for the action of Aspirin not involving the lung PG system, is clearly called for.