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.

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 of bradykinin in postprandial hypotension in humans

A transient significant decrease in mean arterial blood pressure (MAP) from 107 + ou - 3 to 98 + ou - 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 + ou - 3 to 83 + ou - 4 mmHg was also noted but it was not statistically significant. A 40 per cent 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 + ou - 1.0 to 11.0 + ou - 2.5 pg/ml plasma (P<0.05) in elderly volunteers and from 2.0 + ou - 1.0 to 10.3 + ou - 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 + ou - 0.4 to 3.0 + ou - 0.1 min (P<0.05) in young volunteers and from 5.2 + ou - 1.0 to 4.0 + ou - 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.

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 changes in human plasma following a test meal or insulin administration.

In healthy, fasted volunteers, the ingestion of a test meal produced within 30 min, a decrease of ca. 40% of high molecular weight kininogen (HK) in plasma. Return to control (fasting) levels occurred after 90-120 min. Low molecular weight kininogen (LK) remained unchanged or was slightly elevated. On another occasion, the same group of fasted volunteers received an intravenous, euglycemic-clamped infusion of insulin. This treatment reproduced the effects of the test meal on HK. Insulin had no effect on kininogen following incubation with plasma or whole blood. Slight systemic hypotension and increased leg muscle blood flow accompanied postprandial plasma kininogen change. It is suggested that such vascular changes may in part, be due to bradykinin (BK) released from HK cleaved at vascular, possibly endothelial sites activated by insulin following its release by alimentary stimulation. A decreased capacity to release BK due to absence of, or lowered sensitivity to, insulin may play a role in the origin of hypertension in diabetes.

Kininogen changes in human plasma following a test meal or insulin administration.

The effect of food intake and of insulin on plasma bradykinin (BK) reserves [total (TK), high-molecular-weight (HK), and low-molecular-weight kininogen (LK)] was followed by determining (by enzyme-linked immunosorbent assay) the amount of BK released by trypsin from plasma collected before and after a test meal or hyperinsulinemic clamping. LK was kininogen remaining in plasma after in vitro kaolin treatment, which removes all HK. HK was the difference between TK and LK. Thirty minutes after a test meal, TK and HK decreased by 8.0 +/- 0.6 and 39.7 +/- 6.6%, respectively, in six of seven subjects. Return to prealimentary levels occurred after 90-120 min. Hyperinsulinemia, comparable to that arising after the test meal, partly reproduced such treatment's effect on TK and HK but did not affect blood or plasma kininogen in vitro. Observed postprandial hypotension and increased leg blood flow could be caused, in part, by BK released from plasma HK after cleavage at vascular, possibly endothelial, sites activated by insulin.

Kininogen changes evoked in plasma by feeding, pseudo-alimentary, vagal and carbamylcholine stimulation of the rat.

Changes in plasma total (BKg), low (LKg), or high (HKg) molecular weight kininogens are indirect means of studying the participation of kinins in biological processes. To investigate kinin involvement in digestive activity, rats fasted overnight, but given access to water, were allowed to feed for one hour. BKg levels increased by 18% (p less than 0.0001); LKg by 29% (p less than 0.01); HKg remained apparently unchanged. Changes were reversed after 120 min. Decreases in circulatory HKg are probably masked by a compensatory response of the organism aimed at rapidly re-establishing kinin precursor levels in the circulation following their consumption by parasympathetic activity accompanying feeding. This conclusion is based on observations showing that fasted rats submitted to sham-feeding caused by visual/olfactory stimulation by food, present extensive (61%) reduction (p less than 0.001) of HKg, but not of LKg. It is further supported by results demonstrating that electrical stimulation of the distal stump of the cut left abdominal vagus nerve, as well as intravenous administration of carbamylcholine, a parasympathomimetic drug, also produce these changes, all of which were prevented by prior atropinization of the experimental animals. These results open the way for investigations on a possible role of kinins in the control of post-prandial vascular changes.

Parasympathetically-mediated swelling of mast cell granules during feeding.

Mesentery mast cells have been observed to swell and to spontaneously return to their original size following feeding of 12-h fasted rats. This effect may be controlled by parasympathetic efferent nerve impulses, since it is inhibited by atropine. It was reproduced in vitro in isolated rat peritoneal fluid mast cells exposed for 30s to 10(-8)-10(-11) M acetylcholine. When examined under the electron microscope, mast cell average granule diameters had increased by 29% (p less than 0.001) following treatment. Swollen granules did not leave (exocytose) acetylcholine-treated mast cells. They gradually and spontaneously returned to their original size. This recovery only differed from that occurring in the fed rat by its greater speed.

Reversible rat mesenteric mast cell swelling caused by vagal stimulation or sham-feeding.

Fragments of rat mesentery were examined using acetylthiocholine to detect cholinergic nerve fibers and toluidine blue to identify mast cells. 59.2 +/- 2.6 percent of mast cells were at less than one-half mean cell diameter (4-5 microns), from the nerve fibers. Under the electron microscope, the membrane of mast cells was within less than 50 nm from axon membranes, suggesting a synaptic type of connection. Average mast cell area in fasted rats increased following feeding, stimulation of the left abdominal vagus nerve or exposure of the animal to the smell of food. It returned to control values within 60-80 min. Granule exocytosis was not observed. Mast cell swelling was prevented by atropine and induced by intravenously administered carbamylcholine. It appears that in rat mesentery, impulses travelling via cholinergic, parasympathetic fibers innervating mast cells, cause mast cell swelling. Compound 48/80 administered to rats at doses causing little degranulation and minimum release of histamine, caused extensive, reversible swelling of mesentery mast cells.

Compound 48/80-induced secretion of histamine from rat peritoneal mast cells depends on a tryptase controlled step also leading to chymase activity.

Compound 48/80 caused activation of rat mast cell tryptase and chymase along with the release of histamine. Following low speed centrifugation, tryptase remained essentially in the supernatant while chymase sedimented. Enzyme activities as well as histamine release were inhibited by phenylmethyl-sulfonyl fluoride and tosyl-lysine chloromethyl ketone, which are respectively unspecific and specific inhibitors of trypsin-like enzymes. Chymostatin inhibited chymase, but not the release of histamine by 48/80. Tryptase activity thus seems to be essentially involved in the mechanism of histamine release, while chymase activity may be merely one of its accompanying events.

Increased levels of histamine observed in the skin of adrenaline-treated rats or mice are not the result of histamine synthesis by mast cells.

Like the skin of rats and mice injected with adrenaline (AD), rat isolated peritoneal mast cells display increased levels of perchloric acid-soluble histamine following incubation with AD. Although pre-exposure to alpha-fluoromethyl histidine (FMH), an inhibitor of histidine decarboxylase, prevented the effect of AD in vivo and in vitro, this compound was also found to inhibit mast cell granule swelling evoked by AD, a response linked to histamine changes. Absence of increased levels of isotopic histamine in mast cells incubated with labelled histidine in the presence of AD, as well as the insufficient amounts of would-be precursor histidine found in untreated mast cells, confirm the conclusion that AD does not increase mast cell histamine by stimulating its synthesis.

Atropine and hexamethonium-sensitive, Ca/K-modulated, reversible swelling of mast cells in rat mesentery, due to feeding or exposure to carbachol.

Transitory swelling of mesenteric mast cells was observed when 24 h-fasted rats were given access to food. Atropine, an anti-muscarinic drug given (1 mg/kg, i.p.) 60 min prior to feeding, prevented this response; carbachol, a cholinomimetic drug caused it to occur when given (2 micrograms/kg, i.v., 10 min) to fasted rats. Mast cells in the mesentery excised from fasted rats, presented swelling in vitro within 1 min following exposure to 10(-7) M carbachol. This response was inhibited by atropine (10(-8) M) or hexamethonium (10(-8) M), indicating that stimulation of a parasympathetic nerve pathway, reported to exist in rat mesentery, could induce mast cell swelling. Exposure to a Ca2+ free medium also led to rapid swelling of mast cells in the mesentery excised from fasted rats. This result, as well as inhibition of the mast cell response to carbachol caused by increasing the Ca2+ (but not by increasing the Mg2+) content of the incubation medium, suggests that swelling was caused by a sudden decrease of Ca at mast cell membrane sites controlling ion/water fluxes. Mast cells swollen by feeding, carbachol or Ca-lack, reverted to their original condition within 20 min when incubated in balanced salt buffer. Such reversal did not occur in a KCl-enriched medium. An equivalent (in terms of ionic strength), increase in NaCl, did not reproduce this effect, indicating that mast cells have K+-dependent means of compensating for endogenously or drug-induced volume changes. Swelling caused by cholinergic stimulation of mast cells was not accompanied by granule exocytosis.2+ carbachol-treated rat blood in vivo or in vitro, is discussed in terms of putative mast cell-controlled, localized homeostasis in the rat mesentery.

Hyposmotic swelling leads to the expression of trypsin-like activity by rat peritoneal fluid mast cells.

Rat peritoneal fluid mast cell present parallel increases in cell area (swelling), and in hydrolytic activity on the trypsin substrate p-tosyl arginine methyl ester (TAME), when placed in Tris buffers of concentrations between 0.15 and 0.03 M. Under these conditions, cells do not degranulate and preserve their trypsin-like enzyme activity after low speed centrifugation. Exposure to more dilute Tris buffers, between 0.015 and 0.003 M, leads to cell rupture accompanied by progressive degranulation and loss of activity on TAME. Protamine, a heparin antagonist prevented this loss when added to mast cells prior to hyposmotic lysis, or lysis by sonication or repeated periods of freezing and thawing. Enzyme activity released in the presence of protamine was fully recovered in supernates of cell lysates submitted to low speed centrifugation. Controlled swelling of mast cells propitiates the expression of trypsin-like activity, possibly by facilitating enzyme-substrate interaction. Cell lysis on the contrary, leads to inactivation of such activity, possibly by enzyme binding to heparin in exposed mast cell granules.

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.

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.

Stimulation of histamine synthesis in rat mast cells by compound 48/80 in vitro.

The histamine content of rat peritoneal fluid cells is doubled within 20 min by 0.5 microgram/ml of compound 48/80. Histamine catabolism inhibitors do not reproduce this effect; cells pre-incubated with alpha-fluoromethylhistidine are unresponsive to compound 48/80 which therefore activates pre-formed histidine decarboxylase rather than 'inducing' it. Non-mast cells showed no change after treatment with compound 48/80.

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.