neoplastic mast cells (Giarman, Potter & Day, 1960). According to Toh

Transcription

1 J. Phy8iol. (1963), 165, pp Printed in Great Britain RELEASE OF HISTAMINE FROM SPLEEN BY KIDNEY EXTRACT, RESERPINE AND COMPOUND 48/80 BY ANNIE B. ELLIOTT From the Department of Physiology, University of Singapore (Received 27 April 1962) Extracts of kidney tissue made with 0-2N-NaOH (alkali extract) release histamine and 5-hydroxytryptamine (5-HT) from separated platelets of the dog, from rabbit whole blood (Toh, 1956), and from mouse neoplastic mast cells (Giarman, Potter & Day, 1960). According to Toh (1957) the extracts also release 5-HT from the rat's spleen. It is now shown that alkali extracts of kidney tissue also release histamine from the spleen of the rat and rabbit, and this is compared with the histamine-releasing action of reserpine and of compound 48/80. Histological studies show that the effect is not located in the mast cells of the spleen; nor is it accompanied by reduction in the blood platelets, as shown by platelet counts. METHODS The experiments were carried out on rats ( g) and rabbits ( g) of both sexes. On three consecutive mornings the animals received intraperitoneal injections either of 5 ml. NaCl solution, 0 9 g/100 ml., or of 5 ml. alkali kidney extract corresponding to 2 g fresh tissue, or of 0 5 mg reserpine in 5 ml. of 0 9 % NaCl solution, or (rats only) of 0.1 mg compound 48/80 in 5 ml. 0*9 % NaCl solution. At 48 hr and 24 hr before the first, and 24 hr after the last, injections, platelet counts were made on blood drawn from the tail vein of rats or the ear vein of rabbits. The animals were then killed. Their spleens were weighed, a small portion was kept for histological examination, and the rest was extracted by the method of Douglas, Feldberg, Paton & Schachter (1951) for histamine assay. Alkali kidney extract. The extract was prepared from sheep kidneys according to the method described by Toh (1957). The ph of the different batches of extract used for injection varied between 7-5 and 9 5. HiBtamine as8ay. Histamine was assayed on the isolated guinea-pig ileum preparation suspended in atropinized Tyrode solution at 330 C against histamine acid phosphate, but all values refer to the base. After each assay the abolition of the contractions by mepyramine maleate was confirmed. Hi8tological method8. Portions of spleen were fixed in methanol and stained with (a) haematoxylin and eosin, (b) toluidine blue, (c) Leishman's stain. Pieces of mesentery were spread on cover slips, fixed in methanol and stained by a modification of the method of Steedman (1950), i.e. 10 min in Ehrlich's haematoxylin, and 10 min in an aqueous solution containing 025 % alcian blue and 0-25 % acetic acid. Platelet count. Platelets were counted by the method of Casey & Helmer (1930) as modified by Casey ( ). The diluting fluid was a sterile modified Ringer's solution containing 6-2

2 84 ANNIE B. ELLIOTT (%): NaCl 0-9, KCI 0-042, CaCl , NaHCO ; to which heparin (0-02 mg/100 ml.) was added just before use. Blood pipettes were treated with silicone, and the platelets were allowed to settle for 15 min in the haemocytometer before counting. RESULTS Experiments on rats The finding of Toh (1957) that intraperitoneal injections of alkali kidney extract increase the weight of the spleen has been confirmed. In the present experiments the mean weight of the spleen increased from to g. No such increase was observed after treatment with either reserpine or compound 48/80. The results are shown in Table 1, col. 4, and Table 2, col. 3. The effects of the various injection procedures on the histamine content of the spleen are shown in Table 2. Reserpine caused a striking reduction TABLE 1. Injection NaCl soln. (0 9 g/ 100 ml.) Alkali kidney extract Effect of intraperitoneal injections of alkali kidney extract, reserpine and compound 48/80 on the weight of rats' spleens Increase in Number Body wt. and sex (mean + s.e.; of rats g) 9f I Spleen wt. (mean +s.e.; g) spleen weight (%) and significance of difference > F> 001 Reserpine Compound 48/ > e984+0 >09P > TABLE 2. Injections Rats NaCl soln. (0-9 g/100 ml.) Alkali kidney extract Reserpine Compound 48/80 Rabbits NaCl soln. (0-9 g/100 ml.) Alkali kidney extract Reserpine Effect of intraperitoneal injections of alkali kidney extract, reserpine and compound 48/80 on spleen histamine in rats and rabbits Number Spleen wt. and sex (mean±s.e.; of rats g) 9 m) 8f J 9ml 7f S 4m 4f } 4m} 4f S 3 m) 2 27} 2f 2 m) ± ± ± ± ± ± ±0-202 Total histamine/spleen (mean± S.E.; /icg) 1-45 ± ± ± ± ± ± ±2-4 Reduction of total histamine (%), and significance of difference Histamine (,uglg spleen, mean±s.f.) 1-52 ± ± ± ±0-35 _ 92-0 ± >P> ± )-01 > P > ±1-8 Reduction of histamine (%/g spleen) and significanc( of difference > P > > P > P > > P > P

3 RELEASE OF SPLEEN HISTAMINE 85 (89 %) in the total histamine content of the spleen, whereas alkali kidney extract and compound 48/80 appeared to produce only slight reductions, 3 and 10 %, respectively. However, when calculated in terms of histamine per gram wt. of spleen the reduction following intraperitoneal injections of alkali kidney extract amounts to 33 %, because of the increase in weight of the spleen, and this decrease is probably significant. The reduction in histamine per gram wt. of spleen after compound 48/80 is 21 %, but is not significant. Histological examination of the spleen after treatment with alkali kidney extract suggested slight enlargement of the red pulp and haemosiderin inclusions. After treatment with reserpine the appearance was often suggestive of haemorrhage within the spleen. Otherwise there were no changes after treatment with any of the three histamine-releasing agents. There was no reduction in the normal rich content of all blood elements. No mast cells were seen in treated or untreated spleens. Histological examination of the mesentery showed that the normal distribution and appearance of mast cells was unchanged after reserpine or alkali kidney extract; this was in contrast to the disappearance of all mast cells after treatment with compound 48/80. The platelet count, varying between 280,000 and 1,100,000/mm.3 in the controls, was not significantly altered by treatment with any of the histamine-releasing agents. Experiments on rabbits In rabbits the effects of the different injection procedures on the histamine content of the spleen appear to be the same as in rats, although the rabbit's spleen contains about 60 times more histamine (mean value 92,g/g), most of which is located in the platelets which abound in the spleen. Rabbits from two different sources had to be used. The mean weight of the spleen in those rabbits which received intraperitoneal injections of alkali kidney extract and reserpine was smaller than that in the control rabbits. No deductions could therefore be made concerning the effect of the injections on the weight of the spleen. The histamine content was reduced by alkali kidney extract and even more by reserpine. This was evident whether the values were expressed per spleen or per gram tissue (Table 2). When expressed per gram tissue, the reduction by treatment with alkali kidney extract was 34 % as compared with 33 % in the rat; after reserpine treatment it was 90 % in both species. Histological examination of the spleen revealed no changes following the intraperitoneal injections either of alkali kidney extract or of reserpine. No mast cells were seen in the spleen of untreated or treated rabbits, and

4 86 ANNIE B. ELLIOTT within the blood vessels all the usual blood elements including numerous blood basophils and platelets were seen. The histological appearance of the mesentery was not changed after treatment with either alkali kidney extract or reserpine. Typical mast cells do not occur in the rabbit mesentery, but well stained blood basophils, which have sometimes been equated with mast cells, were visible in the mesentery of both treated and untreated animals. As in rats, the platelet count (380, ,000/mm.3) was not altered by treatment with alkali kidney extract. The effect of reserpine on the platelet count was not examined. DISCUSSION Both alkali kidney extract and reserpine cause a reduction in spleen histamine which cannot be explained by an action on mast cells, since it has been shown that these cells are absent from the spleen of rats and rabbits. Moreover, compound 48/80, which has a potent histaminereleasing action on mast cells (Fawcett, 1954; Norton, 1954; Bendit Wong, Arase & Roeper, 1955), causes only a small and statistically insignificant reduction of spleen histamine. It is also shown that the mast cells of the rat mesentery, which are disrupted and disappear after treatment with compound 48/80, are not visibly altered by either alkali kidney extract or reserpine. Both alkali kidney extract (Toh, 1956) and reserpine (Waalkes & Weissbach, 1956), however, are known to release histamine from platelets. The effect with alkali kidney extract was obtained in vitro, but with reserpine only in vivo. Although no explanation can yet be given for this difference, the mechanism by which the reduction in histamine content of the platelets is brought about may be essentially the same, since the reduction occurs in both instances without alteration in the appearance or number of platelets. Further, both alkali kidney extract and reserpine have this in common, that they reduce the 5-HT content of the platelets (Toh, 1956; Carlsson, Shore & Brodie, 1957). The observed reduction in spleen histamine is most probably also the result of an action of the alkali kidney extract and of reserpine on platelets, which abound in the spleen. This conclusion presupposes that the histamine in the spleen is, at least to a large extent, platelet histamine, and that the widely differing histamine contents of rat and rabbit spleens reflect the widely differing histamine contents of the platelets in these two species (Humphrey & Jaques, 1954). If the trapping of platelets occurs only in the spleen, this conclusion would also explain why Burkhalter, Cohn & Shore (1960) did not find any reduction in the histamine content of the rabbit's brain, lung, liver, heart and kidney after reserpine.

5 RELEASE OF SPLEEN HISTAMINE 87 A similar explanation to that given for the reduction of spleen histamine by alkali kidney extract or by reserpine, i.e. an action on the histamine content of the platelets trapped in the spleen, has been put forward by Toh (1956) for the reduction in 5-HT content of the rat spleen produced by alkali kidney extract. The release of histamine and 5-HT from platelets, either circulating in the blood or trapped in the spleen, is not analogous to the release occurring in anaphylactic shock, in which the platelets agglutinate and the platelet count falls (Waalkes, Weissbach, Bozicevich & Udenfriend, 1957). To explain the release of 5-HT by alkali kidney extract, Toh (1956) points out that the release must be considered in relation to the fact that platelets are able to absorb 5-HT against a concentration gradient and that the active processes involved in the absorption may be inhibited by the alkali kidney extract. The same explanation could apply to the action of reserpine and to the release of histamine. Whether alkali kidney extract also releases histamine from mast cells without altering their appearance has not been examined. Although such extracts release histamine from neoplastic mouse mast cells (Giarman et al. 1960), it does not necessarily follow that they would have a similar action on normal mast cells. The enlargement of the spleen produced by alkali kidney extract is not associated with visible pathological changes nor with platelet agglutination. It is not certain whether this unexplained effect of alkali kidney extract, which is not shared by reserpine, is mediated by the same substance which is responsible for the histamine release from platelets. SUMMARY 1. The effects of intraperitoneal injections of alkali kidney extract, reserpine and compound 48/80 have been examined in rats and rabbits. 2. In rats an increase in weight of the spleen is produced by alkali kidney extract, but not by reserpine or compound 48/ In rats the histamine content per gram spleen is reduced by alkali kidney extract and by reserpine. The mean reduction after alkali kidney extract is 33 %, and after reserpine 90 %. The mean reduction produced by compound 48/80 is 21 %, which is not statistically significant. 4. In rabbits the histamine content per gram spleen is reduced by alkali kidney extract and by reserpine. The mean reductions are 34 and 90 %O respectively. 5. In the mesentery of rats reserpine and alkali kidney extract, unlike compound 48/80, do not cause disruption and disappearance of the mast cells.

6 88 ANNIE B. ELLIOTT 6. It is concluded that the reduction in histamine content of spleen following treatment with alkali kidney extract or with reserpine results from a reduction of the histamine content of the platelets which abound in the spleen. The number and the appearance of circulating platelets are not affected by these treatments. REFERENCES BENDITT, E. P., WONG, R. L., ARASE, M. & ROEPER, E. (1955). 5-Hydroxytryptamine in mast cells. Proc. Soc. exp. Biol., N.Y., 90, BURKEALTER, A., CoHN, V. H. & SHORE, P. A. (1960). Effect of reserpine, serotonin and 5-hydroxytryptophan on rabbit platelet histamine in vivo and in vitro. Biochem. Pharmacol. 3, CARLSSON, A., SHORE, P. A. & BRODIE, B. B. (1957). Release of serotonin from blood platelets by reserpine in vitro. J. Pharmacol. 120, CASEY, A. E. ( ). Further note on the enumeration of blood platelets and red blood cells. Proc. Soc. exp. Biol., N.Y., 28, CASEY, A. E. & HELMER, 0. M. (1930). An accurate and practical method for blood platelet counting. Proc. Soc. exp. Biol., N.Y., 27, DO-uGLAS, W. W., FELDBERG, W., PATON, W. D. M. & SCHACHTER, M. (1951). Distribution of histamine and substance P in the wall of the dog's digestive tract. J. Physiol. 115, FAWCETT, D. W. (1954). Cytological and pharmacological observations on the release of histamine by mast cells. J. exp. Med. 100, GIARmAN, N. J., POTTER, L. T. & DAY, M. (1960). Release of 5-hydroxytryptamine and histamine from neoplastic mast cells by alkaline tissue extracts. Experientia, 16, HUMPHREY, J. H. & JAQUEmS, R. (1954). The histamine and serotonin content of the platelets and polymorphonuclear leucocytes of various species. J. Physiol. 124, NORTON, S. (1954). Quantitative determination of mast cell fragmentation by compound 48/80. Brit. J. Pharmacol. 9, STEEDMAN, H. F. (1950). Alcian blue 8GS: a new stain for mucin. Quart. J. micr. Sci. 91, TOH, C. C. (1956). Release of 5-hydroxytryptamine (serotonin) and histamine from platelets by tissue extracts. J. Phy8iol. 133, TOH, C. C. (1957). The presence of a 5-hydroxytryptamine (serotonin) liberator in the gastro-intestinal tract. J. Phy8iol. 138, WIAATxS, T. P. & WEISSBACH, H. (1956). In vivo release of histamine from rabbit blood by reserpine. Proc. Soc. exp. Biol., N.Y., 93, WAAIxES, T. P., WEISSBACH, H., BOZICEVICH, J. & UDENFRIEND, S. (1957). Serotonin and histamine release during anaphylaxis in the rabbit. J. clin. Invest. 36,