(Received 5 August 1958)

Transcription

1 437 J. Physiol. (I959) I47, THE DISTRIBUTION AND FATE OF RADIOACTIVE HISTAMINE IN THE RAT BY B. N. HALPERN, TH. NEVEU AND C. W. M. WILSON* From the Clinique Medicale Propedeutique de l'h6pital Broussais, Centre de Recherches Allergiques de 1'Association Claude Bernard, and the Centre National de la Recherche Scientifique, Paris (Received 5 August 1958) It is universally accepted that histamine, whether of endogenous or exogenous origin, is disposed of by the two processes of enzymic destruction and of excretion through the renal tract. There is, however, little information about the way in which exogenous histamine is brought into contact with its specific enzymes. The rapid clearance of histamine from the blood after its intravenous injection (Rose & Browne, 1938) suggests that the uptake of histamine by the tissues may be a method for its immediate disposal from the blood, related to, and as important as, its subsequent metabolic destruction and renal excretion. The details and mechanism.s of this process are still unexplained. The purpose of the present experiments was to investigate this process by means of radioactive histamine, and to examine the fate, distribution and transfer through the tisues of exogenous histamine introduced into the circulation of the living orgamsm. Rose & Browne (1938) suggested that a powerful mechanism existed for the uptake of histamine into the tissues from the circulating blood of the rat, but their results have been criticized on account of the large doses of histamine which they used. Such doses may cause disturbances in the circulation after intravenous injection. In the present experiments, therefore, a relatively small dose of histamine was used for injections so that effects due to circulatory changes could be excluded. METHODS Albino male rats of the Wistar strain weighing g were used in all the experiments. After the intravenous injection of radioactive histamine, blood was removed at intervals of 15, 20 and 40 sec through a fine cannula inserted into the carotid artery, and at intervals from 1 to 240 min by puncturing the retro-orbital plexus, as described by Halpern & Pacaud (1951). The brain, liver, * Present address: Department of Pharmacology and General Therapeutics, University of Liverpool.

2 438 B. N. HALPERN, TH. NEVEU AND C. W. M. WILSON spleen, lungs, heart, kidneys, alimentary canal, striated muscle, and skin were removed for extraction and assay ofhistamine at intervals varying from 5 to 240 min after the injection. Urine was collected for histamine assay by puncture of the bladder after ligation of the penis to ensure that none was lost. The experiments were performed on intact animals on which an effective nephrectomy had been performed by ligation of the renal pedicles. To avoid the effect of surgical shock, silk ligatures were placed loosely round the renal pedicles in a preliminary operation, performed under ether anaesthesia, and the ends of the ligatures were brought to the surface of the skin in the back through the operation wound. Four or five days after the animals had recovered from the operation, the ligatures were tightened and secured round the renal vessels. After each experiment had been completed, post-mortem examination was performed, and it was invariably confirmed that the renal pedicles had been effectively ligated, and that the kidneys had been completely excluded from the circulatory system. Extraction and assay of radioactive 14C histamine. Histamine was used in the form of the bihydrochloride, marked with 14C in the imidazole nucleus in position 2:,NH-CH \\N-2LC{H2-CHNH2. The radioactive histamine was prepared from potassium thiocyanate K-14CNS by the method of Frazer & Raphael (1952) at the radiochemical centre, Amersham, England. The specific activities were mc/mm and 1-48 mc/mm for the two samples which were used. The first sample was diluted with non-isotopic histamine in the following proportions: crystallized histamine bihydrochloride 14C 96-2 parts parts histamine bihydrochloride (Roche). The second sample was injected undiluted. A known volume of blood plasma was spread in a thin layer on a glass slide 22 mm in diameter and the radioactivity was measured in a Geiger-Muller counter. The radioactivity of the erythrocytes was measured in the same way after the cells had been haemolysed by diluting them 1:4 with distilled water and then exposing them on the glass slide in the counter. The rats were killed by bleeding them to death. Immediately afterwards mg of each tissue was cut in a freezing microtome into sections of 1 p and placed in an aqueous solution of Tween 80 1/1000. The tissue suspension was spread in a thin uniform layer on a round glass slide of 22 mm diameter, which corresponded to the diameter of the window in the Geiger-Muller counter. The slide was mounted in an aluminium cup and was placed at the opening of the counter where the maximum exposure for measurement of the B radiation was obtained. The actual quantity of 140 in the tissues was calculated by using a layer of tissue of uniform thickness for absorption of the,b rays. The depth of the layer was established by adding known quantities of radioactive histamine to samples of the tissues of various depths. A tissue layer of 50t was finally selected as a suitable depth for giving reproducible results in all the tissues. Under these conditions, and with the apparatus which was used (Tracerlab), 10% of the radioactive 14C was recovered. In order to make comparisons between the values for the biologically active histamine and the radioactive 14C in the tissues, the latter has been expressed in terms of the chemically unmodified histamine bihydrochloride in the results, and is referred to as 'radioactive histamine'. Extraction and assay of biologically active histamine. The 1 t tissue slices were immediately placed in w-hci. After boiling, the histamine was extracted by Code's method (Code, 1937) and was assayed on the atropinized guinea-pig ileum. The results are expressed in terms of histamine bihydrochloride. RESULTS The radioactive and biologically active histamine in the blood and tissues were measured in parallel. The values for the biologically active histamine normally present in the lungs, liver, striated muscle and kidney were measured

3 DISTRIBUTION AND FATE OF HISTAMINE 439 in twelve control animals. The mean values were used in order to calculate the quantity of exogenous biologically active histamine fixed by the tissues in the experimental animals. The quantity of exogenous biologically active histamine fixed by the tissues was found by obtaining the difference between the quantity in the tissues of the experimental animals after the injection of the exogenous histamine, and the quantity present in the tissues of the control animals. By observing the changes in the values for the radioactive histamine and for the biologically active histamine at intervals after the injection of the exogenous histamine, the extent of its metabolism in different organs was calculated. The experiments were performed on normal animals and on animals whose kidneys had been excluded from the circulation, so as to assess the importance of the kidneys in the simultaneous processes of destructive oxidation and renal excretion of histamine in comparison with its destructive oxidation by the other tissues of the body. 20_ x 16 x12 C.E I% 8 - " \ I \ 4 'x Time (sec) Fig. 1. Changes in the concentration of radioactive (0) and biologically active (x ) histamine in plasma of the rat during the first 4 min after the intravenous injection of histamine 14C bihydrochloride 500 tzg/100 g. Radioactive histamine is calculated from the values for radioactive 14C, and is expressed, like the biologically active histamine, as histamine bihydrochloride pg/l. Normal anim,als Plasma histamine. Intravenously injected histamine leaves the circulating blood rapidly. The plasma histamine measured at 15, 20 and 40 sec, and at intervals from 1 to 240 min after the intravenous injection of radioactive histamine 500 p,g/100 g, is shown in Figs. 1 and 2. During the first minute the concentration of both radioactive and biologically active histamine diminished quickly, subsequently their rates of disappearance became slower, and, depending on whether the biologically active or radioactive form was being examined, the fall continued for about 15 min or several hours.

4 440 B. N. HALPERN, TH. NEVEU AND C. W. M. WILSON After the injection, when the histamine had become completely mixed with the extracellular fluid, the volume of which is about 20 ml. in rats weighing 100 g, the histamine concentration would have been 25,000 Kg/l. However, 15 sec after the injection the plasma only contained 18,300,ug/l., and after 1 min it had fallen to 3810,tg/l.; that is, about 85% of the biologically active histamine had disappeared. Its value had returned to the normal level 30 min after the injection. The concentration of radioactive histamine diminished more slowly than that of the biologically active form. This difference was already evident during the first minute, but it became more pronounced later on. Thirty minutes after the injection the plasma still contained 2010 i,g/l.; 7.5-5*0 I2-5 t X_ Time (min) Fig. 2. Changes in the concentration of radioactive (0) and biologically active (x) histamine in plasma, and of radioactive histamine (O) in the blood cells of the rat during 4 hr after the injection of histamine 14C bihydrochloride 500,tg/100 g. that is, about 8 % of the theoretical concentration at zero time. It was not until 4 hr after the injection that the radioactive histamine had almost entirely disappeared. The difference between the values for the radioactive and biologically active histamine corresponds to the quantity of histamine which contains the imidazole nucleus. By calculating the fraction biologically active histamine radioactive histamine the proportions of the active and inactive forms remaining in the circulation can be estimated, and the rate of the histamine metabolism can be measured. This has been done in Table 1, and the rate of metabolism of the biologically active histamine as expressed by its decline from unity is shown. Red blood cell histamine. Exogenous histamine penetrated rapidly into the

5 DISTRIBUTION AND FATE OF HISTAMINE 441 cellular fraction of the blood; afterwards its concentration in the red cells declined as in the plasma. The concentration in the cells was generally lower than in the plasma, but at the end of 4 hr the values for the radioactive histamine in the cells and plasma had diminished to almost the same low level (Fig. 2). Tissue histamine. Rose & Browne (1938) suggested that a powerful mechanism existed for the uptake of histamine into the tissues from the circulating blood of the rat, but their results have been criticized on account of the large doses of histamine which they used. Such doses may cause disturbances in the circulation after intravenous injection. The raised values for the tissue histamine found in the present experiments following the injection of a relatively TABLE 1. Changes in radioactive and biologically active histamine in plasma, and in the fraction biologically active histamine radioactive histamine of rats after intravenous injectionof histamine 14C bihydrochloride 500,ug/100 g. Injection at time zero Biologically active Histamine calculated from Biologically Time histamine plasma 14C active histamine after, A_, A Radioactive injection No. of animals (4gl.) No. of animals (pg/l.) histamine 15 sec * ' min * * * *560 small dose of histamine confirm their observations and show that such effects occur in the absence of any circulatory changes induced by the histamine. The disappearance of the histamine -from the circulation appeared to be too fast wholly to be explained by its enzymic destruction or urinary excretion, and so the rate of uptake of both forms of histamine by various tissues was measured. After the injection of the radioactive histamine it rapidly diffused into the different tissues, but the quantity taken up by them varied considerably. The organs which took up most histamine were the kidneys, striated muscle, liver, skin, and ileum; elsewhere the concentrations were considerably less. Biological assay was only performed in the tissues in which it was thought that the uptake or metabolism would be greatest. On account of the high normal content of the skin, the contribution made to it by the injection of exogenous histamine was relatively small and insufficiently accurate to provide figures of any value. Five minutes after the injection, 57 % of the total quantity which 28 PHYSIO. CXLVII

6 442 B. N. HALPERN, TH. NEVEU AND C. W. M. WILSON was taken up by the kidney, as indicated by the uptake ofthe radioactive form, was found to be biologically active. The biologically active histamine found in liver and striated muscles at this time was only 41 % of the total uptake by these organs. The relative quantities of both forms of histamine found in the different tissues at various periods after its administration are shown in Fig gh -c 0 d 0 eso c e hi k 0 d Cb b b~~~~~~~ CvL25 ghi- e a b d C b a a Time (min) Fig. 3. Radioactive (E) and biologically active (*) histamine found in blood, urine and different organs, 5, 30 and 240 min after the intravenous injection of histamine 14C bihydrochloride 500 ug/100 g, expressed as percentages of the injected histamine. The figures indicate the values per whole organ. The skin, striated muscle and blood have been taken as 15, 45 and 8% of the body weight, respectively. a, kidney; b, striated muscle (thigh); c, liver; d, abdominal skin; e, ileum (without contents); f, whole blood; g, lungs; h, myocardium;, colon (without contents); j, spleen; k, urine. The biologically active histamine diminished more rapidly than the radioactive histamine, and as the histamine decreased in the tissues it increased in the urine. The disappearance of the biologically active histamine in the tissues results therefore partly from urinary excretion, partly from enzymic destruction. Table 2 shows the uptake of histamine calculated not for the whole organs but per gram of tissue. It can be seen that 5 min after the injection of radioactive histamine the kidney has absorbed the largest quantity, namely 146,ug/g. The heart, liver and lung follow in descending order, each having absorbed about one tenth as much as the kidney; the striated muscle contained the lowest concentration. The concentrations of biologically active histamine in the different tissues followed the same pattern as the quantities taken up by the individual organs shown in Fig. 3. The results given in Table 2

7 DISTRIBUTION AND FATE OF HISTAMINE 443 show that the kidney has an extremely potent histamine-extracting capacity, which has also been observed by Lindell & Schayer (1958) for the dog's kidney. TABLE 2. Content of radioactive histamine in the organs. Values represent the mean of three animals and are expressed in ug/g of the fresh organ Tissue 5 min 30 min 240 min Kidney Heart Liver Lung 10* Ileum 9-8 6*7 3.5 Colon Spleen Stomach 5*4 2*1 19 Skin 4.2 2*9 0 7 Muscle : L r~~~~ z xx T Time (min) Fig. 4. Changes in the concentration of radioactive (-) and biologically active (x ) histamine in the plasma, and of radioactive histamine (0) in the blood cells, of the rat with kidneys excluded from the circulation during 4 hr after the injection of histamine 14C bihydrochloride 500 Pg/100 g. Animals with kidneys excluded from the circulation The predominating effect of the kidney in controlling the transfer of histamine in the living animal made it desirable to study the histamine metabolism in animals in which the renal vessels had been ligated. Blood histamine. The values for both forms of histamine in the blood of nephrectomized animals after an intravenous injection of radioactive histamine 500 plg/100 g are shown in Fig. 4 and Table 3. During the first minutes the rate of disappearance of the histamine from the circulation resembled that 28-2

8 444 B. N. HALPERN, TH. NEVEU AND C. W. M. WILSON in normal animals; but later the histamine remained about 30% higher in the nephrectomized than in the normal animals. It is clear that the values for the biologically active histamine were considerably raised in the animals whose kidneys had been excluded from the circulation, and that its rate of disappearance was reduced in comparison with normal animals. The concentration of radioactive histamine in the plasma diminished during the first 30 min, but thereafter it did not change significantly for a period of several hours. As was the case in normal animals, the changes in the concentration of radioactive histamine in the blood cells were similar to those in the plasma, although the concentration in the cells remained about 2% less during the whole experiment. TABLE 3. Changes in radioactive and biologically active histamine in plasma, and in the fraction biologically active histamine radioactive histamine of nephrectomized rats after intravenous injection of histamine 14C bihydrochloride 500 pag/ 100 g. Injection at time zero Time Biologically active Histamine calculated from Biologically after histamine plasma 14C active histamine injection, A_, _ A Radioactive (min) No. of animals (pg/l.) No. of animals (/Ag/l.) histamine * * *250 0* ' * * * ' *140 0* * *260 Tissue histamine. The levels of the biologically active and radioactive histamine in the tissues are shown in Fig. 5 at various intervals after injection of the histamine. It is clear that the radioactive histamine was distributed throughout all the remaining organs when the kidneys were excluded from the circulation, and was not selectively concentrated in any of them. Most of the tissues took up some ofthe injected histamine; the uptake varied between 2 and 5% of the total according to the organ. An interesting observation made in these experiments on nephrectomized animals was that a transfer of radioactive histamine took place between the organs. Certain tissues such as the liver, ileum and heart absorbed the histamine initially at the expense of other tissues. Later they lost this store of histamine, which passed to the muscles and skin. The quantity of biologically active histamine stored in the organs decreased progressively following the injection, and the fraction biologically active histamine radioactive histamine

9 DISTRIBUTION AND FATE OF HISTAMINE 445 correspondingly diminished. This indicates that a progressive destruction of the biologically active histamine was occurring in the tissues. Turnover of exogenous histamine in the tissues After the administration of histamine marked with 14C in the imidazole nucleus, information is obtained about the movement and fate of the radioactive histamine without knowing whether the molecule is still biologically active or has been broken down. Biological assay alone indicates whether the 100_ ff j ~~~~~f h e 75 g 2 CA 0 ee 0 d d 0.. b b Time (min) Fig. 5. Percentage of radioactive and of biologically active histamine found in the blood and different organs of nephrectomized rats after the intravenous injection of histamine 14C bihydrochloride 500 flg/100 g. Conventions as in Fig. 3. injected histamine is present in the blood and tissues in an unaltered form. The percentage of the injected histamine which has been metabolized at any instant in the tissues is indicated by the percentage of the radioactive histamine which is biologically inactive. This can be calculated from the following formula: 100- (Total biologically active - Biologically active histamine) x 100 histamine found normally present Radioactive histamine - percentage of radioactive histamine metabolized. Figures indicating the rate at which the radioactive histamine in the plasma was metabolized are shown in Table 4. These figures demonstrate that the b

10 446 B. N. HALPERN, TH. NEVEU AND C. W. M. WILSON biologically active histamine disappeared rapidly, and that 5 min after its injection 60% of the radioactive histamine had been transformed into a physiologically inactive form. In the nephrectomized animal the rate of transformation appeared to be reduced, although a larger number of observations will be necessary definitely to confirm this. TABLE 4. Changes in the value of the biologically active histamine calculated as a percentage of the radioactive histamine in the plasma of normal and nephrectomized rats after the injection of histamine 14C bihydrochloride 500 ug/100 g at zero time. Each observation represents the mean of at least three rats Percentage of radioactive histamine which was biologically inactive in Normal Nephrectomized Time (min) rats rats TABLE 5. The rate of metabolism of biologically active histamine in the different tissues: values expressed as percentage of biologically active histamine metabolized. Each observation represents the mean from at least three rats Control rats Nephrectomized rats Time (min) Kidney Liver Lung Striated muscle The metabolism of histamine by the different organs was found to be a rapid process, as can be seen from Table 5. When the metabolic rate was measured per gram of tissue the lung appeared to have considerable metabolic activity compared with the other organs. However, no definite conclusions can be drawn from this observation because of the rapid turnover and metabolism of histamine by the different tissues, which enable histamine and its metabolites to be transported from one organ to another without definite proof that the histamine was catabolized in any particular organ. It is clear that the cellular destruction of histamine in the rat is a very rapid process. Exclusion of the kidneys from the circulation did not alter this rate of degradation of biologically active histamine by the tissues, as can be seen from Table 4. The excretion of both forms of histamine in the urine was measured during a period of 4 hr after the injection of the histamine. After 30 min 27 %, and after 240 min 76 % of the injected histamine had been excreted in the form of

11 DISTRIBUTION AND FATE OF HISTAMINE 447 radioactive 14C (Fig. 3). The quantity of biologically active histamine varied between 15 and 45% of the injected histamine. From Tables 3 and 5 it can be seen that the destruction of the biologically active histamine was almost complete after 4 hr. It would therefore be expected that the renal excretion of the biologically active histamine and its metabolites would be almost complete at the end of this period also. The figures for the recovery of biologically active histamine and its metabolites in the urine confirm this, and are in close agreement with those obtained by Schayer (1952) in the rat. He recovered 95% of the injected radioactive histamine 20 hr after its injection, of which 11 % was in the form of chemically unchanged histamine. DISCUSSION Between 90 and 95% of the radioactive histamine which was injected in the present experiments was accounted for in the blood and tissues. This confirms that the method for extracting and measuring the radioactivity was sufficiently quantitative to allow valid conclusions to be drawn from the experimental results. The normal content of biologically active histamine in the different tissues of the rats showed considerable variation, but a mean value for it was obtained in a preliminary experiment on twelve animals of the same strain, so that the value for the actual uptake by the tissues was obtained by subtracting the normal value from the value obtained after injection of the exogenous histamine. The high value for the skin histamine made it impossible to obtain accurate results for the uptake of exogenous biologically active histamine in this tisue. The histamine diffused rapidly through the circulating blood after its intravenous injection. If the figures for the concentration of histamine in the plasma are used to calculate the volume ofextracellular fluid in which the histamine was distributed 1 min after its injection, it was found to have a diffusion space of about 70 ml./100 g in the rat, which is greater than the total volume of water contained in the whole animal. Therefore the histamine must have diffused out of the extracellular fluid into the cells, where it was stored in concentrations higher than those found in the blood itself. Emmelin (1951) also concluded on the basis of his calculations that histamine injected intravenously into cats passed in large quantities to the tissues, where it was subsequently inactivated. These observations do not agree with Schayer's statement (1956) concerning the fate of exogenous histamine in the rat. In the present experiments on rats the radioactive histamine was absorbed by all tissues with the exception of the central nervous system. The quantity absorbed varied from one tissue to another; it was greatest for both radioactive and biologically active histamine in the kidneys. It is possible that exogenous histamine is deposited in the cells in a biochemical form different from that in which endogenous histamine is deposited.

12 448 B. N. HALPERN, TH. NEVEU AND C. W. M. WILSON Study of the distribution of histamine and its metabolites in the normal animal is made difficult by their rapid excretion through the kidneys. Exclusion of the kidneys from the circulation overcame this difficulty, and the two-stage character of the operation prevented undesirable effects produced by complete bilateral nephrectomy on the day of the experiment. In the nephrectomized animals the biologicallyactive histamine in the blood decreased progressively while the radioactivity persisted at a constant level from 15 min until several hours after the injection. The biologically active histamine was being progressively metabolized, and a diffusion equilibrium between the histamine and its inactive metabolites became established across the cell membranes. During this period there was also a transfer of radioactive 14C from liver and other abdominal viscera to striated muscle and lungs. The present results show that the metabolism of histamine by the tissues is extremely rapid, almost 100% of the histamine taken up by the tissues being metabolized in 30 min. It is clear that exogenous histamine in normal animals can be metabolized in large quantities and this metabolic process appears to occur especially in the kidney. It was found that the kidney had considerable power of extracting histamine from the blood and that a small proportion of this histamine was excreted in a chemically unchanged form. SUMMARY 1. By the intravenous injection of radioactive 14C histamine, the turnover, tissue distribution and fate of histamine has been investigated in the rat. The value of this method of investigation lay in the possibility of making comparisons between the concentrations for the radioactive 14C and the biologically active histamine introduced into the body. The radioactive 14C measured the total histamine concentration both in its biologically active form and after its metabolic transformation in the tissues. 2. Diffusion of the histamine out of the blood occurred very rapidly during the first few seconds after its injection. The factors which modified its rate of diffusion were investigated in normal rats, and in rats whose kidneys were excluded from the circulation. 3. Histamine penetrated rapidly into the cells of all the tissues except the brain. The extent of the uptake varied in the different organs. The kidney had the greatest power of extracting histamine from the blood. 4. Histamine taken up by the tissues was rapidly metabolized and the value for the fractionbiologically active for thefraction radioactive histamine 14C correspondingly diminished. 5. The rate at which histamine was metabolized in the extracellular fluid and in the different organs was determined. In the rats whose kidneys were excluded from the circulation, the metabolic products of histamine were gradually transferred between the tissues.

13 DISTRIBUTION AND FATE OF HISTAMINE 449 This work was done during the tenure by one of us (C.W.M.W.) of a Wellcome Research Travel Grant. The authors wish to acknowledge the technical assistance of M'elle Annie Brannelec, aide technique a l'association Claude Bernard. REFERENCES CODE, C. F. (1937). The quantitative estimation of histamine in the blood. J. Physiol. 89, EMMELw, N. (1951). The disappearance ofinjected histamine from the blood stream. Actaphysiol. scand. 22, FRAZER, M. M. & RAPHAEL, R. A. (1952). A synthesis of histamine from but. 2-ync-1:4-diol. J. Chem. Soc., Lond., 1, HALPERN, B. N. & PACAUD, A. (1951). Technique de pr6k6vement d'echantillons de sang chez les petits animaux de laboratoire par ponction du plexus ophthalnique. C.R. Soc. Biol., Paris, 143, LiNDELL, S. E. & ScHAYER, R. W. (1958). The renal removal of injected (14C) histamine from blood in dogs. Brit. J. Pharmacol. 13, ROSE, B. & BROWNE, J. S. L. (1938). The distribution and rate of disappearance of intravenously injected histamine in the rat. Amer. J. Physiol. 124, ScHAYER, R. W. (1952). The metabolism of ring-labelled histamine. J. biol. Chem. 196, ScHAYER, R. W. (1956). The origin of histamine in the body. Ciba Foundation Symposium on Histamine. London: J. and A. Churchill.