Rat Peritoneal Fluid Research Articles

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 α-fluoromethyl histidine (αFMH), acting after a significant lag period. When compared with controls under the electron microscope, epinephrine-treated mast cells show less electron-dense, swollen intercellular 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.

A peritoneal fluid activator for histamine release from isolated peritoneal mast cells of the rat.

A factor present in rat peritoneal fluid has been found to be necessary for optimal release of histamine from peritoneal mast cells by dextran and antigen, but not by concanavalin A. Washed peritoneal mast cells suspended in a physiological medium containing bovine serum albumin require ten-fold higher concentrations of phosphatidyl serine for optimal release by concanavalin A. The peritoneal fluid activator is probably a protein of molecular weight about 180,000 Daltons.

Hydrolysis of arginine and tyrosine esters in mast cells exposed to epinephrine

Acting in a dose-dependent fashion in vitro, l-epinephrine caused rat peritoneal fluid cells to hydrolyze substituted arginine (BAEE, TAME) and tyrosine (BTEE) esters. Cell activation was complete within 5 min; repeated washing of activated cells did not result in loss of their BAEE-hydrolyzing capacity. BTEE-hydrolyzing capacity fell to pre-activation levels within 10 min, even in the absence of washing. This loss may be the result of an interaction of BTEE-esterase with cell-borne inhibitory factors. 8-Br-cGMP or noradrenaline acted like epinephrine. diBu-cAMP was inactive but inhibited cell activation by epinephrine or cGMP. Neither epinephrine, 8-Br-cGMP nor diBu-cAMP released histamine. Exposure of peritoneal cells to a hypotonie salt medium caused histamine release but not activation of esterase. However, hypotonically pretreated cells responded to epinephrine in the same way as normal cells. Diisopropyl-fluorophosphate prevented esterase activity on TAME and BTEE. Following differential centrifugation of peritoneal fluid cells, epinephrine-sensitive esterase was found only in mast cells; eosinophils, lymphocytes and monocytes were inactive. Compound 48 80 , although less active than epinephrine, was also able to activate mast cells. The concentrations required were ten times higher than those needed to cause substantial release of histamine. The appearance of esterase activity in epinephrine-treated rat mast cells seems to be the outcome of cGMP-dependent changes, associated with reversible morphological alterations, but not with the release of histamine,

Release of prostaglandins E and F in an algogenic reaction and its inhibition

E and F prostaglandins were measured by radioimmunoassay in the peritoneal fluid of rats which had been injected with an irritant, acetic acid. The considerable increase recorded 5 min after the injection virtually disappeared in 90 min. For the first 15 min the PGE 2 level was twice that of PGF 2α, the levels then equalized and after 90 min the PGE 2 level was less than that of PGF 2α. This balance between PGEs, which are hyperalgesic, and PGF 2α, which has often been shown to be a PGE antagonist, could regulate defence mechanisms. An examination of cells collected by washing the peritoneum revealed a large decrease between 15 and 30 min after injection of the irritant and suggested that the prostaglandins could be produced by neutrophil polynuclear cells but also by destruction of macrophages. Various types of prostaglandin biosynthesis inhibitors (non-steroid anti-inflammatory agents, non-narcotic analgesics and some monoamino-oxidase inhibitors and antioxidants) prevented prostaglandin release. Their activity on release paralleled their activity on acetic acid-induced writhing.

The separation of mast cells with a modified coil planet centrifuge

Mast cells of peritoneal and pleural fluid of albino rats were separated by a modified coil planet centrifuge. This apparatus was composed of a double rotating system and utilized the steady flow of medium (acid-citrate-dextrose solution with 15% rat serum) through a coiled Teflon tube (0.66 mm × 10 m) in a weak centrifugal field (40 g). This system prevented cell adhesion and aggregation during centrifugation. Mast cells were separated from the other cells by densities and size. A final purity of 97.2 ± 1.0% was achieved reproducibly with reasonable yield. Morphological and chemical examination indicated little change in the mast cells after separation; separated mast cells were able to undergo granule discharge and histamine release.

IDENTIFICATION OF MAST CELLS IN THE SCANNING ELECTRON MICROSCOPE BY MEANS OF X-RAY SPECTROMETRY

In mixed populations of rat peritoneal fluid cells, the mast cells can be differentiated from other cell types in the scanning electron microscope by virtue of the X-ray emission referable to their sulfur content. Both stationary probe and X-ray mapping are feasible; the amount of sulfur eliciting a signal is estimated to be about 8 x 10(-18) g.

Consumption of kininogen, formation of kinin and activation of arginine ester hydrolase in rat plasma by rat peritoneal fluid cells in the presence of l-adrenaline

Cells harvested from the peritoneal cavity of the rat, were able to lower kininogen, release kinin and activate benzoylarginine ester (BAEE) hydrolase following brief incubation with rat plasma in the presence of l-adrenaline. These effects were not reproduced when either cells or the catecholamine were incubated with plasma. Peritoneal cells incubated with l-adrenaline did not acquire detectable BAEE-esterase activity, but became able to induce this activity as well as lowering of kininogen, in adrenaline-free plasma. Phenoxybenzamine and, to a lesser extent, propranolol, blocked the action of adrenaline, which was also sensitive to Trasylol, the anti-kallikrein inhibitor from bovine tissues. l-Noradrenaline was less effective than adrenaline, isoprenaline, ineffective. Peritoneal cavity washings which contained no mast cells, but were normal in their lymphocyte and eosinophil content, were unable to lower plasma kininogen in the presence of l-adrenaline. Pretreatment with compound 48/80, leading to substantial release of histami did not decrease the cells' ability to lower plasma kininogen in the presence of adrenaline. The catecholamine failed to release histamine from peritoneal fluid cells. Acetylsalicylic acid (Aspirin®) blocked cell-linked lowering of kininogen and the activation of BAEE-esterase by l-adrenaline. This effect was obtained at a dose (2–10 μg/ml) of the analgesic, 200–500 times lower than that required to block histamine release by compound 48/80. While the action of acetylsalicyclic acid on this release was counteracted by glucose, its effect on kininogen breakdown and esterase activation occurred in the presence of physiological levels of the sugar. While peritoneal fluid cells pretreated with acetylsalicylic acid could not be activated by adrenaline, the action of adrenaline-activated cells on plasma kininogen and BAEE-esterase was not inhibited by the presence of acetylsalicylic acid in the incubation medium. Acetylsalicylic acid prevented the appearance of the discrete morphological alterations normally evoked by adrenaline in rat mesentery mast cells, but failed to affect the degranulation evoked in these cells by compound 48/80.

Selective secretion of lysosomal enzymes of the Yoshida hepatoma AH 130 in the peritoneal fluid.

We measured the lysosomal enzymes in the peritoneal fluid of rats bearing Yoshida hepatoma AH 130 and related our results to tumor growth. Between the 6-th and 21-th day after inoculation the concentration of the lysosomal enzymes increased by the following multiples; cathepsin 16.8; β-glucuronidase 14.0; acid phosphatase 5.3; β-acetyl-glucosaminidase 3.19; hyaluronidase 1.66; alkaline phosphatase 3.7. No significant change was observed in the mitochondrial enzyme glutamic pyruvic transaminase. Activities in the tumor cells increased in the same period to multiples: cathepsin 2.2; acid phosphatase-1.7; and β-glucuronidase-1.1. Treatment of the tumor bearing rats with the cytostatic agent podophyllinic acid ethylhydracid produced no significant change in the lysosomal enzymes. The results are discussed in relation to the enzyme release hypothesis and tumor growth.

Mast cell count in the peritoneal fluid of rats with adjuvant-induced polyarthritis.

Adjuvant-arthritis was induced in rats by means of a subplantar injection (left hind paw) of M. butyricum in paraffine oil and the severity of the disease determined volumetrically on the right, uninjected hind paw when the secondary lesions had reached near-maximal extent. One group of the rats was treated on days 18 to 21 post-adjuvant with hydrocortisone at a daily oral dose of 30 mg/kg and the therapeutic effect assessed in terms of gross paw-oedema changes and of certain parameters mentioned hereafter Using a variety of ‘clinical parameters’ (ESR, albumin/globulin ratio, fibrinogen, fibrinolysis, serum iron and serum copper) as well as other parameters still under experimental evaluation (peritoneal mast cells and ‘inflammatory’ rest cells) it was found that: (a) the ‘clinical parameters’ undergo much the same changes in adjuvant-induced arthritis in the rat as they do in human rheumatoid arthritis; (b) steroid therapy reverses such changes to normal in the rat in a fashion similar to that obtained in man suffering from rheumatoid arthritis and the like; (c) peritoneal mast cell count is low and peritoneal ‘inflammatory’ rest cell count high in full-blown adjuvant-arthritis; (d) a short-term steroid therapy tends to normalise the changes mentioned under (c).

Turnover of lymphocytes in rat peritoneal fluid

Zellkinetische Untersuchungen an Ratten nach Injektion von H3-Thymidin ergaben eine rasche Markierung der grossen mononukleären Zellen innerhalb von 20 Min und eine langsaniere Markierung der kleinen Lymphozyten, für welche eine Turnover-Zeit von 23,4 Tagen errechnet wurde. Stündlich gelangen ca. 105 kleine Lymphozyten in die Bauchhöhlenflüssigkeit.

Histamine release inhibition in anti-inflammatory mechanism.

Rats were depleted of skin histamine by more than 80 % by intraperitoneal injections of sinomenine with daily increasing doses for 6 days. In these rats, egg-white edema induced in the hind paws was inhibited by 68 % of control. The weight of the wall of granuloma pouch made by croton oil was also evidently smaller in the rat treated similarly with sinomenine than that of control. This suggests an important role of histamine participating in the inflammation. It has been observed that a variety of non-steroidal anti-inflammatory drugs inhibited both degranulation and histamine release induced by compound 48/80 of mast cells isolated from rat peritoneal fluid. The degranulation inhibiting actions of anti-inflammatory drugs were markedly decreased in the presence of glucose as in cases of dinitrophenol, dicumarol and warfarin which are known uncouplers of oxidative phosphorylation. Also, prevention of edema provoked by anti-rat serum is roughly correlated to a potency of degranulation inhibiting effect of anti-inflammatory agents. These observations suggest that there is a common mechanism between these two phenomena, and the prevention of mast cell degranulation by the anti-inflammatory agents is, at least, partially due to their uncoupling effects. A working hypothesis explaining the process of edema formation at the inflammatory site has. been made based on the data of the present experiment and other ob3ervations: a leakage of plasma into the tissue space from the gap between two adjacent endothelial cells which are contracted by released histamine may activate a kinin-forming system in the plasma, and kinin(s) may further aggravate a leakage. The mechanism of action of anti-inflammatory agents, which interfere with the histamine effect in inflammation, should be understood in twofold: one is prevention of histamine release from the tissue, mainly by inhibiting mast-cell degranulation, and the other is prevention of the contraction of endothial cells by their uncoupling activities.

Cytological studies on normal and surviving mast cells in vitro.

AbstractCytological features of rat peritoneal fluid mast cells are studied to advantage with differential interference contrast optics (Nomarski System). These cells are particularly sensitive to handling; they rapidly undergo a variety of cytological changes which, in the literature, repeatedly have been interpreted as reflecting physiological activity related to mast cell secretory function. These interpretations are hereby questioned and, alternately, it is suggested that the changes are agonal ones and that an absorptive function must at least be considered.Some cytological features not detectable with conventional optics are described. The special optics were combined with time‐lapse cinematography and dynamic records were obtained. These support the concept, advanced elsewhere, that sets of mast cell granules are confined within a common membranous sac and that each such set may behave as an independent physiological unit.

Untersuchungen über die Phagozytoseaktivität von Peritonealflüssigkeitszellen während der Neonatalperiode der Ratte

Phagocytic activity of rat peritoneal fluid cells was investigated after the injection of foreign erythrocytes into the peritoneal cavity of 1 to 30 days old rats. Peritoneal mononuclears as well as neutrophilic granulocytes phagocytised the erythrocytes. Age related differences in the phagocytic activity were not observed. It is concluded that single cells are mature for phagocytosis from the first day of extrauterine live.

Untersuchungen zum Ablauf der Phagozytose in der Peritonealflüssigkeit der Ratte

Phagocytic activity of mononuclear cells of the rat peritoneal fluid has been investigated at different times, after the addition of foreign red blood cells (sheep and chicken red blood cells). The mononuclear cells phagocytized both types of erythrocytes, but preferentially chicken red blood cells. To a lesser extent the neutrophilic granulocytes phagocytized chicken red blood cells. The investigations showed that in all instances a number of the mononuclear cells exhibited no phagocytic activity even in the presence of free foreign red blood cells.

Comparative peritoneal cellular content as related to species and sex.

AbstractCytologic fluid specimens were obtained by aspiration from the abdominal peritoneal cavities of young adult male and female mice, hamsters, guinea pigs, gerbils and rats. Two hundred cells were randomly counted and grouped as mesothelial cells, lymphocytes, polymorphonuclear leukocytes, histiocytes, mast cells, bare nuclei, monocytes and daisy cells. In all mammals studied, regardless of sex, mesothelial cells were the predominate cells in abdominal serous fluid. Relatively high distributions of mast cells were observed in rats and hamsters. Bare nuclei without cytoplasm were found in all animals except male mice. Special attention was given to the daisy cell which occurred in rat peritoneal fluid. It is concluded that species and sex difference markedly influence the cytologic content of abdominal peritoneal fluid.

EFFECTS OF COMPOUND 48/80, CHYMOTRYPSIN AND ANTISERUM ON ISOLATED MAST CELLS UNDER AEROBIC AND ANAEROBIC CONDITIONS

The anaphylactic histamine release from guinea-pig and rat lung tissues was completely or largely depressed by oxygen lack, respiratory inhibitors and uncouplers of oxidative phosphorylation when the incubation medium was devoid of glucose (1-7). Similar inhibition has been found of compound 48/80-induced histamine release from rat lung tissue and of degranulation of rat mesentery mast cells (6, 8, 9). Since these inhibitions were largely overcome by the presence of glucose (4-9), and phlorizin (10) and 2-deoxyglucose (7) inhibited these glucose-dependent release of histamine, glycolysis should be considered to take part in supplying the energy required for these processes. Chymotrypsin is also known to cause histamine release (11) and degranulation of mast cells (11, 12), and the latter action is also blocked by anoxia and glucose has a preventive effect on this block. There are a number of works demonstrating the histamine-releasing or degranulating effect of compound 48/80 and some other agents including anti-serum (13) on isolated mast cells or mixed cell suspension taken from rat peritoneal fluid. However, the observations relevant to glucose dependency are all based on the experiments with excised tissues but not with isolated cells, because an anaerobic experiment with isolated mast cells seems to have not yet been successful. Thus, Keller and Beeger (14) reported that the introduction of nitrogen gas into the cell suspension resulted in a complete destruction of mast cells; Moran et al. (15) were unable to demonstrate an inhibition of 48/80induced amine release from mixed peritoneal cells by incubating in their supposedly oxygen-free atmosphere in the absence of glucose. In the present experiments, with isolated mast cells, the glucose-dependent degranulation by compound 48/80, α-chymotrypsin and anti-serum is demonstrated under nitrogen anaerobiosis with success, and the effects of some inhibitors including phlorizin and 2-deoxyglucose are studied. Observations in aerobiosis are also described.

ISOLATION OF GRANULES FROM EOSINOPHIL LEUCOCYTES AND STUDY OF THEIR ENZYME CONTENT.

Eosinophils were separated from other types of cells in horse blood or rat peritoneal fluid by centrifugation in concentrated albumin solutions. Eosinophils did not appear to be damaged by this separation procedure. A technique was also devised for isolation of cytoplasmic granules from eosinophils, thus allowing studies on enzyme content of the granules. Granules from both horse and rat eosinophils contained a number of hydrolytic enzymes, similar in variety and in concentration to those previously found in granules of rabbit polymorphonuclear leucocytes. Eosinophil granules differed from those of the rabbit granulocyte in their high content of peroxidase and the absence of lysozyme and phagocytin. On disruption of eosinophil granules by repeated freezing and thawing in saline, cathepsin, ribonuclease, arylsulfatase and beta glucuronidase were released into solution, but phosphatases were partially and peroxidase completely bound to the insoluble granule residue. Peroxidase could be extracted from the granule residue with weak acid. Eosinophil granules thus are lysosome-like structures.

Effect of catecholamines and their chloro-analogs on the in vitro release of histamine front cells of rat peritoneal fluid

Effect of catecholamines and their chloro-analogs on the in vitro release of histamine front cells of rat peritoneal fluid

The effect of hydrocortisone on the mast cells in the peritoneal fluid and mesentery of the rat.

SummaryMast cells were studied in the mesentery and peritoneal fluid of rats after local (intraperitoneal) and general (subcutaneous) administration. Hydrocortisone did not disrupt or degranulate the mast cells. There were no significant changes in the number of mast cells in the mesentery although there was a tendency to lower values after a daily intraperitoneal injection for fourteen days. In the peritoneal fluid the mast cells were more resistant than the eosinophils and the mononuclears to the action of hydrocortisone and were relatively increased after daily intraperitoneal injections for two to three days and subcutaneous injections for fourteen days although their total number was unaffected. Their size and morphology was unaltered. After fourteen days intraperitoneal treatment they had disappeared from the peritoneal fluid, however. The eosinophil leucocytes were reduced in number after two to three days and had practically disappeared after fourteen days. Prednisolone produced changes similar to...