419 J. Physiol. (I953) I20, 49-426 RELEASE OF HISTAMINE BY THE LIVER BY G. V. ANREP, G. S. BARSOUM AND M. TALAAT From the Physiological Laboratories, Medical Faculties of Alexandria and of Kasr-el-Aini, Cairo, Egypt (Received 10 November 1952) Following the demonstration of histamine release from skeletal muscle by curare (Alam, Anrep, Barsoum, Talaat & Wieninger, 1939) a large number of other histamine releasers have been described by Schild & Gregory (1947), Rocha e Silva & Schild (1949) and especially by MacIntosh & Paton (1949). Histamine releasers have some specificity in their action. While curare, for instance, acts on muscles and releases no histamine from lungs or the intestinal tract, snake venom releases considerable amounts from perfused lungs of the guinea-pig and of the cat (Feldberg & Kellaway, 1937). According to MacIntosh & Paton (1949), in the cat, licheniformin hydrochloride and many other substances release histamine mainly from the skin, less from the muscles and none from the digestive tract and the lungs. On the other hand, in the dog, the main site of the release is the liver. This is unlike the action of curare, which in all species so far investigated, including the dog, acts mainly on the skeletal muscle. The object of the experiments to be described was to study histamine release by the liver with special reference to the action of bile salts. METHODS All the observations were made on dogs. The experiments were performed on heart-lung-liver preparations and on anaesthetized animals, in which blood samples were collected from an artery and from the portal and hepatic veins. The livers for perfusion were taken from dogs about half the weight of those used for the heart-lung preparation. The perfusion was carried out with defibrinated blood to which heparin, 02 g/l., was added to counteract the fibrinogen which might be liberated by the liver during the course of an experiment. The livers were perfused through the portal vein and the hepatic artery or through the portal vein alone. The hepatic artery was supplied with blood directly from the arterial side of the preparation, i.e. with a pulsatile pressure of 90-110 mm Hg. The portal vein was also supplied with arterial blood, but at a pressure which was reduced to 25-40 mm Hg by means of an adjustable clamp between the heart-lung preparation and the vein. The blood 27-2
420 G. V. ANREP, G. S. BARSOUM AND M. TALAAT draining from the liver was collected through a cannula inserted into the vena cava above or below the liver. The other end of the vena cava was tied. From the liver the blood was returned to the heart-lung preparation. The lungs of the preparation were ventilated with room air or with air containing 5 % Co2. In some experiments the perfusion was carried out in situ; in others, the liver was removed from the body. Adequate precautions were always taken to prevent the perfused liver from cooling. For experiments on the whole animal the dogs were anaesthetized with chloralose. Arterial blood was collected from the femoral or carotid artery; portal blood, through a cannula inserted into a mesenteric vein pointing towards the portal vein, and hepatic blood, through a cardiac catheter introduced into the hepatic vein through the jugular vein and the inferior vena cava. The end of this catheter was so shaped as to fit one or another opening of the hepatic vein. The correct position of the cannula was verified by palpation through the abdominal cavity. With practice the position of the cannula can be ascertained by the fact that when correctly placed the outflow of the blood is markedly increased during each inspiration; this does not occur when the cannula lies in the vena cava. Since no anticoagulants were injected, the catheter had to be withdrawn between the collections of the blood from the hepatic vein. The estimation of the histamine equivalent was made by the method of Barsoum & Gaddum (1935) or its modification by Anrep, Barsoum, Talaat & Wieninger (1939); all histamine values are expressed in terms of histamine acid phosphate. The histamine-like nature of the active substance of the blood extracts was in most experiments verified by administration to the guineapig's ileum of 10-9 mepyramine maleate (neoantergan) or of 10-8 diphenhydramine HCI (benadryl). Heparin or Chlorazol Fast Pink were used as anticoagulants when necessary. Since injections of bile salts were made in most experiments it was important to determine whether histamine could be satisfactorily assayed in their presence. Exposure of the guinea-pig ileum to bile salts was found to reduce its sensitivity to histamine. When, however, the bile salts were added to blood and an extract prepared by a method involving hydrolysis in strong acid, the assay could be performed as accurately as with normal blood. RESULTS The removal of histamine by the liver In seventeen experiments performed on perfused livers the histamine equivalent of the hepatic venous blood was found to be lower than that of the arterial blood, indicating that the liver was removing histamine from the circulation. As can be judged from Table 1, the extent of this removal varied from one TABLE 1. Heart-lung-liver preparation. Histamine removal by liver perfused through the hepatic artery and portal vein. The blood flow was controlled in both experiments at about 200 ml./ min. Blood histamine activity expressed as /ig/ml. diphosphate Expt. 1. Blood histamine Expt. 2. Blood histamine From From Time To the the Time To the the (min) liver liver Diff. (min) liver liver Diff. 00 0-035 0 030-0 005 00 0 057 0-035 - 0-022 10 0 033 0-025 - 0-008 15 0 050 0 035-0-015 18 0-030 0-025 - 0 005 25 0 045 0 030-0-015 35 0-030 0-025 - 0 005 40 0-038 0-028 - 0.010 45 0-025 0-020 - 0 005 45 0-038 0-025 - 0-013 60 0-025 0-020 - 0 005 Mean 0 030 0-024 -0-006 Mean 0 045 0-030 - 0-015
HISTAMINE RELEASE BY LIVER 421 experiment to another. In Expt. 1 the diminution of the histamine equivalent of the blood passing through the liver was small, almost bordering on the limit of accuracy of the assay, and only the fact that the result of all the determinations pointed in the same direction justified their acceptance. A greater retention of histamine was observed in Expt. 2. When compared with the arterial blood the mean diminution in the histamine equivalent was, in the first experiment, equal to about 20 % and in the second, to 33 %. In seventy-two similar determinations the average diminution in the histamine equivalent of the blood during its passage through the liver was equal to 28% of the value for arterial blood. In six determinations no difference between the arterial and the hepatic venous blood could be detected, and in four experiments the first sample of the hepatic venous blood collected in each had a higher histamine equivalent than that of the arterial blood. As a rule, the retention of histamine was greater when the histamine equivalent of the blood supplied to the liver was high or when histamine had been added to the perfusing blood. Observations on the whole animal gave similar results. The histamine equivalent of the hepatic venous blood was found to be lower than that of the portal blood. The difference was frequently more conspicuous than in perfusion experiments, which is probably due to the fact that the portal blood in the whole animal was usually found to be richer in histamine than the blood in heart-lung preparations. As an average of fifty-six determinations made in fifteen experiments on the whole animal the histamine equivalent of the hepatic blood was 40% lower than that of the portal blood. It is obvious that with such a rate of removal no histamine should remain in the circulating blood for more than a short time during a perfusion experiment. However, in experiments lasting 1-2 hr the histamine equivalent of the perfusing blood diminished but little. In seventeen experiments on heart-lungliver preparations the average diminution was about 30 % of the initial value, ranging between 15 and 38 %. These observations indicate that the removal of histamine by the liver must to some extent be compensated by liberation of this substance by the remaining tissues of the preparation. In this respect experiments on the whole animal revealed the interesting fact that while the liver, under normal conditions, was removing histamine, the intestinal tract was continuously releasing it into the circulating blood. As a result, the histamine concentration of the portal blood was usually found to be higher than that of the arterial blood. In some experiments the portal histamine concentration was only 25 % above that of arterial blood; in others, the difference was as great as 300 %/. The maximal increase observed amongst thirty-four determinations was from 0019,ug/ml. in the arterial blood to 0-063,ug/ml. in the portal blood. As a result of the histamine removal by the liver its concentration in hepatic
422 G. V. ANREP, G. S. BARSOUM AND M. TALAAT venous blood tended to approximate to that of the arterial blood. In some experiments, like the example given in Table 2, the removal of histamine by the liver was greater than the addition by the intestine so that the blood in the hepatic vein became poorer in histamine than the arterial blood. TABLE 2. Dog weighing 8 kg, anaesthetized with chloralose. Effect of curare on the histamine retention by the liver. The blood samples were collected as nearly simultaneously as possible from the femoral artery, from the portal vein and, through a cardiac catheter, from the hepatic vein. Blood histamine activity expressed as pg/ml. diphosphate Blood histamine activity Time (min) Arterial Portal Hepatic venous 0-3 0*050 0067 0 040 15-17 0 045 0.060 0-032 20 8 ml. of 1% 'calabash' curare injected into jugular vein 25-28 0 470 0-340 0 190 38-40 0*320 0-240 0-130 Effect of curare Curare, one of the most potent histamine releasers from skeletal muscles, had no such effect on the liver. Perfusion of the liver with blood containing 'calabash' curare or D-tubocurarine neither released histamine from the liver nor diminished the rate of its withdrawal. Table 2 is given as an example of an experiment with curare performed on the whole animal. It can be seen from this experiment that before the injection of curare the intestine released and the liver withdrew histamine from the blood. After the injection, the arterial histamine concentration increased tenfold, and the intestine instead of releasing histamine as previously now removed it from the circulation. The histamine equivalent of the portal blood after the injection of curare was diminished by 25%, and that of the hepatic blood by 55%, as compared with the value for arterial blood. Effect of restriction of the blood flow Restriction of the blood flow through the liver by reducing the blood supply from the heart-lung preparation or by perfusing it only through the hepatic artery or the portal vein did not change the relative difference between the histamine equivalent of the blood before and after its passage through the liver (Table 3). Neither was the histamine concentration of the hepatic venous blood changed in the whole animal by occlusion of the hepatic artery. It follows, therefore, that so long as the histamine concentration of the inflowing blood remained unaltered the absolute amount of the histamine retained by the liver varied with the blood flow. Effect of diminished oxygen supply Livers perfused by means of heart-lung preparations are supplied with blood of a higher oxygen saturation than that of the portal blood in the intact
HISTAMINE RELEASE BY LIVER 423 animal. The possibility obviously exists that at a low oxygen tension the histamine retention by the liver might be diminished or even changed to a release. This possibility acquires a special significance since Anrep, Barsoum & Talaat (1936) showed that in an ordinary heart-lung preparation with no liver in the circulation, oxygen lack was accompanied by a conspicuous rise of the histamine equivalent of the circulating blood TABLE 3. Effect of restriction of blood supply on histamine withdrawal by the perfused liver. In Expt. 1 the liver was perfused through the hepatic artery (H) and portal vein (P), and the blood flow was changed by obstructing either one of the two blood vessels. In Expt. 2 the liver was perfused through the portal vein only, and the blood flow was regulated by restricting the blood supply. The blood samples were collected at the end of each perfusion period. Blood histamine activity expressed as ug/ml. diphosphate Expt. 1 Blood histamine Time (min) Perfusion Blood flow To the From the Expt. 1 through (ml./min) liver liver Diff. 00 H +P 220 0-036 0-028 - 0-008 12 H 90 0-040 0-030 -0.010 22 H + P 230 0-033 0-028 - 0-005 34 P 170 0 030 0-023 - 0007 45 H+P 210 0-026 0.019-0 007 55 H + P 230 0-026 0-017 - 0 009 Expt. 2 00 P 260 0-048 0 035-0-013 20 P 170 0*048 0-038 - 0.010 29 P 120 0-045 0 035-0.010 37 P 70 0 040 0-032 - 0-008 46 P 50 0*040 0*030-0.010 57 P 220 0-035 0-028 - 0 007 TABLE 4. Heart-lung-liver preparation. Effect of reduced oxygen supply on histamine removal by the liver. The liver was perfused through the portal vein and the blood flow was controlled at about 190 ml./min. The preparation was first ventilated with room air and then, for 20 min, with air containing 6% oxygen. Blood histamine activity expressed as l&g/ml. diphosphate Blood histamine, ~ ~ ~~~A Time Air supplied To the From the (min) to lungs liver liver Diff. 0 Room air 0-029 0-020 - 0 009 10 Room air 0-027 0-018 - 0 009 20 6% oxygen 0.060 0-030 -0-030 30 6% oxygen 0-065 0-036 -0-029 40 Room air 0-030 0-018 -0-012 50 Room air 0-022 0-016 -0-006 It can be seen from the experiment presented in Table 4 that a similar rise occurred also in the presence of the liver, and that the liver, far from contributing to this rise, continued to withdraw histamine from the circulation. In fact, during the period of anoxia the withdrawal of histamine by the liver proceeded at a faster rate than before. In this particular experiment the oxygen saturation of the arterial blood sample collected during the exposure to low oxygen tension was reduced to 43 %. The histamine equivalent of this
424 G. V. ANREP, G. S. BARSOUM AND M. TALAAT sample was more than doubled as compared with the initial value, while the diminution of the equivalent during the passage of the blood through the liver rose from 30 to 50 %. The absolute amount of histamine removed by the liver increased from 1b7 to 5.7,ug/min. It is unlikely that this increase in the retention or destruction of histamine was directly due to oxygen deficiency since, as has been already mentioned, an increase of histamine concentration in the perfusing blood leads to a similar increase of its retention. Effect of bile salts MacIntosh & Paton (1949) found no release of histamine on intravenous injection of bile salts into the whole animal. Recently Schachter (1952) reported the liberation of small amounts of histamine from the isolated perfused cat's skin after injection of bile salts directly into the perfusion fluid. In the present experiments sodium glycotaurocholate dissolved in saline was slowly injected into the portal vein or hepatic artery in doses of 0-2-2-0 g. In perfusion experiments the blood issuing from the liver during, and for about 1 min after, the end of an injection was discarded so as to minimize the direct action of bile salts on the heart. In the whole animal this was not possible and therefore the injections were always accompanied by a considerable though transient fall in blood pressure. The results of two experiments on perfused livers are given in Table 5. It TABLE 5. Heart-lung-liver preparation. Effect of injection of bile salts into the portal vein upon the histamine equivalent of hepatic venous blood. The liver was perfused in both experiments through the portal vein. In Expt. 1 the blood flow was controlled at about 150 ml./min and in Expt. 2 at 120 ml./min. Histamine activity expressed as jug/ml. diphosphate Expt. 1. Blood histamine Expt. 2. Blood histamine From From Time To the the Time To the the (min) liver liver Diff. (min) liver liver Diff. 00 0 055 0 035-0-020 00 0 040 0-032 - 0-008 12 0 050 0-030 -0-020 10 0-040 0 030-0.010 17 2-0 g of bile salts in 10 ml. saline 12 1 0 g of bile salts in 10 ml. saline injected into portal vein injected into portal vein 20 0 110 1-460 +1-350 14 0-065 0-520 +0-455 29 0070 0-380 +0-310 20 0-060 0-210 +0-150 40 0-060 0-160 +0 100 28 0 035 0 100 +0-065 48 0-060 0-120 +0-060 38 0 030 0 055 +0-025 can be seen that on injection of bile salts the normal activity of the liver in relation to histamine was reversed. Instead of being removed by the liver, histamine was now released. In this respect it was found immaterial whether the injections were made into the portal vein or hepatic artery. The arterial histamine equivalent was in these experiments not greatly changed, probably due to the histamine-removing action of the lungs. The maximal rate of histamine release by the liver in the experiments presented in Table 5 was 200,ug/min in Expt. 1 and about 55,g/min in Expt. 2.
HISTAMINE RELEASE BY LIVER 425 In fifteen heart-lung-liver preparations in which bile salts had been administered, the maximal increase of the histamine equivalent of the blood emerging from the liver bore a relation to the amount of bile salts injected. On injection in different preparations of 0f25, 05, 1.0 and 2.0 g the average maximal increase of the histamine equivalent per ml. was 007, 0X28, 0 49 and 1-2 jug respectively. Observations on the whole animal confirmed the results obtained with liver perfusion; injections of bile salts into the portal vein or into the hepatic artery caused a considerable increase of the histamine equivalent of the blood in the hepatic vein (Table 6). With injections of 0*5-1-0 g of bile salts the histamine TABLE 6. Dog weighing 11 kg, anaesthetized with chloralose. Effect of injection of bile salts into the portal vein upon the histamine concentration in the hepatic vein. Blood histamine activity expressed as Fg/ml. diphosphate Blood histamine Time (min) Arterial Portal Hepatic venous 0-3 0*008 0-016 0*012 7-8 1*5 g of bile salts in 15 ml. saline injected into the portal vein 10-12 0 033 0*034 0 370 20-22 0020 0019 04046 30-32.0010 0-018 0-030 equivalent of arterial blood was usually not affected, but with larger doses it increased in most experiments, never, however, to the same extent as in the hepatic vein. Experiments on the whole animal were complicated by a considerable fall in arterial blood pressure which followed the injection of bile salts. This was due to a direct action and not to the release of histamine since it was not modified by administration of various antihistamines. The possibility that the fall in blood pressure might be the cause of the histamine liberation by the liver is excluded by the fact that a similar liberation was observed also in perfused livers, where the blood pressure is not changed by administration of the bile salts, and also by the fact that injections of bile salts into the general circulation through the jugular vein caused a much smaller liberation of histamine from the liver than injections into the portal vein, although the effect on the arterial blood pressure was in both cases approximately the same. No evidence could be found to show that histamine is released by bile salts from other organs besides the liver. There was no increase in the blood histamine equivalent of the general circuit or coronary sinus blood when bile salts were injected into a heart-lung preparation in the absence of the liver; and, in the intact animal, injections of bile salts into the femoral artery werenot followed by an increase of histamine concentration in the femoral vein.
426 G. V. ANREP, G. S. BARSOUM AND M. TALAAT SUMMARY AND CONCLUSIONS 1. The removal and release of histamine by the liver were studied in heartlung-liver preparations and in the whole animal by comparison of the histamine equivalents of arterial, portal and hepatic venous blood samples. 2. In most determinations portal blood was found to be richer in histamine than arterial blood, indicating that the intestine continuously released histamine into the circulation. 3. The histamine equivalent of hepatic venous blood was usually lower than that of the portal blood, indicating that histamine was withdrawn from the blood during its passage through the liver. As a result, the histamine equivalent of hepatic venous blood was reduced to the level of the arterial blood or less. 4. On increasing the histamine equivalent of the arterial blood by injection of histamine or of a strong histamine releaser, such as curare, both the intestine and the liver were found to remove histamine from the blood. 5. Administration of curare, restriction of the blood supply to the liver or severe anoxia caused no release of histamine from the liver. 6. Injections of bile salts into the hepatic artery or into the portal vein caused a conspicuous release of histamine by the liver. No such release by bile salts could be observed from other organs such as the lungs, the heart, the intestine or the limbs. REFERENCES ALAm, M., ANREP, G. V., BRAsoum, G. S., TATrAAT, M. & WIENINGER, E. (1939). Liberation of histamine from skeletal muscle by curare. J. Physiol. 95, 148-158. ANREP, G. V., BAsoum, G. S. & TATAAT, M. (1936). Liberation of histamine by the heart muscle. J. Physiol. 86, 431-451. ANREP, G. V., BARsoum, G. S., TALAAT, M. & WIENINGER, E. (1939). Determination of histamine in blood. J. Physiol. 95, 476-484. BARsourM, G. S. & GADDUM, J. H. (1935). The pharmacological estimation of adenosine and histamine in blood. J. Physiol. 85, 1-14. FELDBERG, W. & KETrrAwAY, C. H. (1937). Histamine liberation from perfused lungs by snake venoms. J. Phy8iol. 90, 257-279. MACINTOsH, F. C. & PATON, W. D. M. (1949). The liberation of histamine by certain organic bases. J. Physiol. 109, 190-219. RoCHA E SnLVA, M. & SCHMD, H. 0. (1949). The release of histamine by D-tubocurarine from the isolated diaphragm. J. Phy8iol. 109, 448-458. ScHAcHTER, M. (1952). Release of histamine from the skin by neoarsphenamine and bile salts. J. Phy8iol. 116, lop. SCHULD, H. 0. & GREGORY, R. A. (1947). Liberation of histamine from striated muscle by curarine, strychnine and related substances. Abdtr. XVII int. physiol. Congr. p. 288.