(Received 17 June 1952)

Transcription

1 20 J. Physiol. (I953) I20, THE EFFECT OF QUININE ON THE HISTOCHEMICAL DEPHOSPHORYLATION OF RIBONUCLEIC ACID IN THE TISSUES OF THE RAT AND RABBIT BY A. F. BARADI AND G. H. BOURNE From the Departwent of Histology, London Hospital Medical College, London, E.1 (Received 17 June 1952) Some details of the histochemical dephosphorylation of ribonucleic acid have been given by previous authors; Gomori (1948), Newman, Fiegin, Wolf & Kabat (1950), Baradi & Bourne (1951). The enzyme responsible in both rat and rabbit has been described as being localized mainly in the nuclei of the cells of some organs and in the cytoplasm of the cells of some others. Some of the descriptions (e.g. localization of the reaction in brush borders) suggest that the reaction is not completely specific. The present work deals with the distribution of the histochemical reaction for this enzyme in different organs and tissues of the rabbit and rat, and gives details of the unexpected effects of adding a small quantity of quinine to the substrate mixture. METHODS Nineteen different organs of rabbit and rat were used. Immediately after death 2 mm thick slices of these organs were fixed for 17 hr in 85% (v/v) ethyl alcohol in a refrigerator. Dehydration was effected by three changes of absolute alcohol at room temperature over a period of 24 hr. Clearing was in two changes of methyl benzoate for about 12 hr, each followed by two 15 min changes of benzene. Embedding in wax was carried out.at 580 C for the first 30 min in a mixture of 50% benzene and 50% paraffin wax followed by three changes of 560 C melting-point paraffin wax of 30 min each. Sections were cut at 7 u and were dried on to slides for 3 hr in an incubator at 370 C. After bringing to water in the usual way the sections were incubated at 370 C for 17 hr in the following substrate solution: 2% (w/v) sodium barbitone, 10 ml.; 2% (w/v) yeast nucleic acid, 10 ml. (ribonucleic acid); 2% (w/v) calcium chloride, 5 ml.; 2% (w/v) magnesium sulphate, 2 ml.; distilled water to make 50 ml. The ph of this mixture was 9-2. Following incubation the slides were treated with cobalt chloride and ammonium sulphide as in the Gomori method for alkaline phosphatase. Sites of enzyme activity were demonstrated black by this technique. A second substrate solution was also used. In this quinine h 'rr@joride, dissolved in a few drops of alcohol! had been added, to make a concentration o f sues incubated in this substrate were treated in exactly the same way as those passing through the control substrate. In all cases in the text instead of' quinine hydrochloride ' read 'quinine base '.

2 QUININE AND RIBONUCLEIC DEPHOSPHORYLATION 21 RESULTS During an investigation into the localization of various enzymes in the papillae foliatae of the rabbit (Baradi & Bourne, 1951), it was found that the addition of quinine hydrochloride to the substrate used for demonstrating the dephosphorylation of ribonucleic acid caused a remarkable increase in the reaction. This led us to study the effects of quinine on this reaction in other organs of both rabbit and rat. Papinla foliata. The only regions giving a positive reaction for this enzyme were the small areas of epithelium immediately overlying the various taste buds (P1. 1, fig. 1). The addition of quinine to the substrate caused a great increase in the reaction. It was found to be present in all the cells of the epithelium lining the gutters between the papillae, the nerve plexuses underlying the epithelium, and Ebner's glands lying beneath the papiliae (P1. 1, fig. 2). Liver. Quinine caused, in the normally negative liver of the rabbit and rat, a positive reaction in the nuclei and cytoplasm of the hepatic cells and the endothelium of the blood vessels and sinusoids (P1. 2, figs. 7, 8). Kidney. There was a slight general reaction by the whole kidney in the rabbit, but it was concentrated mainly in the cortex. The reaction was diffused through the whole of the cell in the different regions of the kidney, the exception being those of the proximal convoluted tubules in which the nuclei were strongly positive. Other workers have recorded a concentration of the reaction in the brush borders of the proximal convoluted tubule cells, but we have found only a slight concentration, not comparable with that found with glycerophosphatase. With quinine an enormous increase of enzyme activity in all sites was obtained. The medulla remained unchanged, but there was a great increase in the cells lining the pelvis and the ureter. In the kidney of the rat there was a similar distribution of enzyme activity which was only slightly increased by quinine. In this animal the quinine caused the nuclei of the medullary cells to become positive. Lung. A very faint reaction was given by nuclei and cytoplasm of most of the cells present in the rabbit lung. With quinine there was a considerable increase in the staining of all nuclei, the endothelium of the blood vessels and the epithelium of the bronchi. A similar result was obtained for the rat. Heart muscle. This gave, in the rabbit, a very faint general reaction (P1. 1, fig. 5). Quinine caused the nuclei of the fibres and the endothelium of the blood vessels to become much more stained (P1. 1, fig. 6). In the rat the heart muscle appeared almost unstained but quinine produced a reaction in the nuclei. Skeletal muscle. This was negative in both the rabbit and rat, and quinine had no effect.

3 22 2 A. F. BARADI AND G. H. BOURNE Oesophagus and trachea. In the rabbit and rat both these structures were faintly positive and the reaction was slightly increased by the addition of quinine. Stomach (fundus). The only positive reaction in the rabbit stomach was given by the capillary endothelium and a few connective tissue nuclei. Quinine had little effect on the reaction in this tissue, only the surface epithelium and some of the nuclei of the gastric glands becoming slightly positive. The rat stomach was negative but with quinine all nuclei in the section became positive. A reaction was also found in the cytoplasm of the deeper cells of the stomach glands and in the smooth muscle layers and Auerbach's nerve plexus. Duodenum. The epithelium, including the brush border, and the distal part of the cytoplasm of the cells, the smooth muscle and some nuclei of the cells in the lamina propria gave a positive reaction in the rabbit and rat duodenum. Brunner's glands were negative (Pl. 2, fig. 13). With quinine there was a general increase of the reaction in all the sites where it normally occurred except in the nuclei of Brunner's glands (P1. 2, fig. 14). Jejunum. The reaction in the jejunum was similar to that in the duodenum. With quinine there was a general intensification of enzyme activity. The nuclei of the epithelial cells and the connective tissue cells of the lamina propria showed intense activity. The distal half of each villus was more positive than the proximal half. In the rat without quinine the villi showed signs of enzyme activity. In the epithelial cells of the villus the reaction was distributed in the nuclei, in the brush borders and in the distal third of the cytoplasm. Fine black granules were present in other parts of the cells. After quinine everything became positive including nuclei and cytoplasm of all cells, the smooth muscle layers and Auerbach's nerve plexus. Rectum. A very faint general reaction was present in the rectum of the rabbit (negative in the rat). With quinine the superficial epithelial cells in both species showed signs of considerable enzyme activity, and in some cells this could be seen particularly well in the brush border and distal part of the cytoplasm. Deep positive staining of the nuclei of the connective tissue cells of the lamina propria was also present. Spleen. The whole organ in both rabbit and rat showed slight general enzyme activity with the centres of the Malpighian corpuscles particularly active, both the nuclei and cytoplasm of the constituent cells in this region staining intensely. The fibroblast cells in the capsule and the media of the small vessels and the nuclei of isolated cells in the white pulp were also positive. With quinine the enzyme activity of the whole Malpighian corpuscle was greatly increased.

4 QUININE AND RIBONUCLEIC DEPHOSPHORYLATION 23 Pancreas. No reaction was found in the rabbit pancreas either with or without quinine. The acinar cells of the rat pancreas gave a slight positive cytoplasmic reaction, and with quinine this was increased, and was diffused in both cytoplasm and nuclei. Thyroid. There was a slight reaction in cytoplasm and nuclei of all cells in rabbit and rat thyroid. The colloid was negative. With quinine there was a great increase in the cellular reaction and the colloid became strongly positive. Adrenal. The capsule and the zona glomerulosa of the rabbit adrenal were negative. A faint positive reaction was given by the outer zona fasciculata cells and this reaction became progressively greater towards the medulla, the zona reticularis showing intense activity. The medulla gave an intermediate positive reaction. In the cortical cells the reaction was diffuse and was present in both nuclei and cytoplasm and it increased progressively towards the meduula. In the zona reticularis, however, the nuclear reaction was much greater than that of the cytoplasm. With quinine the capsule and the zona glomerulosa became positive, equal in intensity to that of most of the zona fasciculata. Patches of more deeply staining cells were present in the latter zone and there seemed to be an increase in the reaction between and around the cells, in the walls of the sinusoids. The reaction in both the zona reticularis and the medulla had become even stronger, particularly in the former, between and around the cells. In the zona reticularis the nuclei and cytoplasm contained numerous fine positive granules and nuclear membranes. In the rat the adrenal was almost negative, but with quinine a considerable positive reaction was given by the zona glomerulosa and capsule and a slight one in the medulla. Moderate activity was also present in the other cortical zones. The reaction was mainly diffuse but, in addition, fine granules were present in most cells. Vas deferens. Only very faint reaction could be detected in the epithelium of this organ in the rabbit (P1. 1, fig. 3), but after quinine the epithelium became intensely positive (P1. 1, fig. 4), particularly in the distal portions of the cells. The nuclei of the cells of the lamina propria also showed considerable activity. A similar result was given by the vas deferens of the rat with the addition that quinine also caused a positive reaction in all nuclei and in the endothelium of capillary vessels. Epididymis. The rabbit epididymis showed strong reactions in the epithelium and lamina propria. The distal part of the cytoplasm of the epithelial cells was more stained than the cilia. The stroma also gave a good reaction and so did the spermatozoa in the tubules. After quinine there was a great increase in the reaction of all positive areas. Testis. All parts of the testis, including the Leydig cells, in the rabbit and rat were intensely positive. The reaction was mainly diffuse but was partly

5 24 A. F. BARADI AND G. H. BOURNE granular as well. The nuclei of the sperm heads gave an extremely positive reaction. The reaction in all areas was greatly increased by the addition of quinine. The sex organs of some female rats were examined. Uterus. This gave practically no reaction, but with quinine all the nuclei in the section became positive and so did the distal margins of the epithelial cells. Leucocytes present in the epithelium were negative. Fallopian tube. There was very little reaction in the Fallopian tube (P1. 2, fig. 11), but with quinine all nuclei became positive and all the tissues which make up the wall of the tube gave a diffuse reaction (P1. 2, fig. 12). Ovary. Practically all varieties of cells in the ovary showed a moderate degree of activity which was mainly diffuse, but fine positive granules were present as well in both cytoplasm and nuclei. With quinine the nuclear reaction was increased both in intensity and in the numbers of nuclei giving a positive reaction. This was particularly so in the case of the theca cells. In addition the walls of large blood vessels and the stroma also showed activity. Salivary glands. Serous and mixed salivary glands of the rat were negative (P1. 2, fig. 9), but with quinine the connective tissue around the ducts and acini became positive. The nuclei and, to a lesser extent, the basal cytoplasm of the acinar cells as well as the nuclei and cytoplasm of the duct cells also showed activity (P1. 2, fig. 10). Thymus. A faint reaction was given by the nuclei of the rabbit thymus. Hassall's corpuscles were negative. Quinine had no effect on the reaction in this organ. Lymph node. A lymph node from a rabbit showed a positive reaction in a few nuclei and with quinine there was some intensification. Skin. A positive reaction was found in the cornified layer of the epithelium and in the hair follicles of the rabbit skin and in the nuclei of connective tissue cells. With quinine there was a slight intensification of the reaction in the first and last sites and a great increase in that present in the hair follicles. DISCUSSION Generally speaking, the distribution of the reaction in untreated tissues was similar to that recorded in earlier literature (Gomori, 1948; Newman et al. 1950). The distribution is similar in comparable rat and rabbit organs, although more rat organs appear completely negative, whereas the corresponding rabbit organs show a slight positive reaction. The outstanding exception is that of the adrenal. In the rat the adrenal is almost negative, whereas in the rabbit only the outer zones are negative, the inner zones giving an intense reaction. Quinine either has no effect on these reactions or else it intensifies the positive reaction and causes it to become more widespread. It has no inhibitory effect in any organ. Only in three organs is there much difference in the two

6 QUININE AND RIBONUCLEIC DEPHOSPHORYLATION 25 species in the degree of intensification which quinine exerts upon this reaction. These are the kidney, the stomach and the rectum. In the first of these quinine causes a considerable increase in the reaction in the rabbit kidney but only a slight increase in that of the rat kidney. In the second, quinine causes only a slight increase in the fundus of the rabbit stomach but greater increase in that of the rat. In the third there is a slight increase in the reaction in the rabbit rectum but a moderate increase in that of the rat. Knowing, however, how capricious histochemical tests can be, we feel that even these differences may be attributed to technical variation. It is of interest that although in many organs the increase of positive reaction due to quinine occurs in both cytoplasm and nucleus, in other organs the increase in the nuclear reaction is the sole or the main effect of the quinine. In some organs quinine has little or no effect on the reaction; these are: skeletal muscle, oesophagus, pancreas, lymph node and thymus. In general the greatest intensification is apparent in those organs which already give a strong reaction. The exception to this statement is the papilla foliata of the rabbit where the initial reaction is very small and the increase, due to the influence of the quinine, is phenomenal. The reaction of quinine on the dephosphorylating reaction differs from its well-known biochemical effect on esterases and also from the histochemical effects we have observed on simple esterase and on enzymes which split other phosphate esters. In the case of all other histochemical reactions where it has any action at all, this is inhibitory. It becomes necessary at this point to consider whether this intensifying effect is due to a real interference with enzyme/substrate reaction or whether it is due to some technical freak. Factors which influence histochemical tests for phosphatase enzymes have been discussed by Gomori (1950), Danielli (1946), Doyle (1953), Cleland (1950), and others. It has been shown, for example, that method and time of fixation, dehydration and time and temperature of embedding all affect the reaction. These, however, were standardized in the present work, and in any case it would be an impossible coincidence for the reaction to be virtually inactivated by any of these means in all control slides, and not affected in all slides treated with quinine. The most likely technical effect therefore is some direct interference with the reaction during incubation. The fundamental process which takes place during incubation is the splitting off of phosphate, its combination with calcium and deposition as a calcium salt at the site of enzyme activity. This process can be affected by altering the ph which not only moderates enzyme activity but also affects the precipitation of calcium phosphate. The ph was, however, quite unaffected by the amount of quinine used in this work. The temperature and time of incubation affect the reaction, but these were

7 26 A. F. BARADI AND G. H. BOURNE standardized in this work. Doyle (1953) has pointed out that random currents in the substrate during incubation can affect the result, but in this case the consistency of the activity of the quinine makes it unlikely that the results obtained would be due to such a cause. The most important argument against the activity of the quinine being due to its effect on the formation or deposition of calcium phosphate, is the fact that in the histochemical demonstration of other phosphatases, the addition of quinine to the substrate either has no effect at all (yeast adenylic acid and adenosinetriphosphate) or has an inhibitory action (muscle adenylic acid and fl-glycerophosphate). There is a possibility that quinine may facilitate the penetration of the substrate into the cells, but we feel that the most likely explanation of the results offered in this paper is that it affects the enzyme/ substrate reaction. Whether it does this by affecting the enzyme or co-enzyme, or by combining with the substrate so that it is more easily split by the enzyme we do not know, although the latter seems the more acceptable explanation. Nor is it known whether quinine would affect the splitting of ribonucleic acid by purified extracts of the enzyme. This has not been tried because, although our histochemical preparations can be regarded as a type of artifact, we feel that a purified enzyme extract is also an artifact. A result with one artifact would not necessarily be of any significance as far as any other artifact is concerned. We do not know whether quinine would accentuate the activity of the enzyme in living tissue or not, but we believe that our results nevertheless may prove of some interest. SUMMARY 1. The addition of quinine hydrochloride to the substrate mixture has been found to intensify the histochemical reaction for the dephosphorylation of ribonucleic acid in the papilla foliata, liver, kidney, lung, heart, alimentary tract, spleen, trachea, thyroid, adrenal and male sex organs of the rabbit, and in the liver, kidney, lung, heart, alimentary tract, adrenal, salivary glands and female sex organs of the rat. 2. In general the rabbit tissues were more affected than the rat tissues by the addition of quinine. 3. In many tissues of both animals, nuclei which had been previously negative gave a strong positive reaction in the presence of quinine. REFERENCES BAXADI, A. F. & BouNE, G. H. (1951). Localization of gustatory and olfactory enzymes in the rabbit, and the problems of taste and smell. Nature, Lond., 68, 977. CLELAND, K. W. (1950). A study of the allkaline phosphatase reaction in tissue sections. Part II. The histological and cytological validity. Proc. Linn. Soc. N.S.W. 76, DAxImia, J. F. (1946). A critical study of techniques for determining the cytological position of alkaline phosphatase. J. exp. Biol. 22,

8 THE JOURNAL OF PHYSIOLOGY, VOL. 120, Nos. 1 & 2 PLATE 1 r Z :0~~~~~~~~~~~~~~~~4 l;s2 t=f W+f C~~~~~M..b,.... o... _~~~~W _0s;.uS.+. ~~~~~~~~ 3~~~~~~~~~~~~I0 To face p. 26

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10 QUININE AND RIBONUCLEIC DEPHOSPHORYLATION 27 Doyimz, W. (1953). Quantitative histochemistry of phosphatase. International Review of Cytolgy, 2, (in the Press). Ed. BoUENE, G. H. & DANizTu, J. F. New York: Academic Press. GOmo1RI, G. (1948). Histochemical differentiation between esterases. Proc. Soc. exp. Biol., N.Y., 67, 4-. GoMoRI, G. (1950). Sources of error in enzymatic histochemistry. J. Lab. din. Med. 85, NEWMN, W., FIEGiN, I., WoLF, A. & KABAT, E. A. (1950). Histochemical studies on tissue enzymes. Part n; Distribution of some enzyme systems which liberate phosphate at ph 9*2 as determined with various substrates and inhibitors, demonstration of three groups of enzymes. Amer. J. Path. 26, EXPLANATION OF PLATES PLATE 1 Fig. 1. Reaction in papilla foliata of the rabbit's tongue. There is a strong positive reaction in epithelium just overlying the taste buds. Fig. 2. Reaction in papilla foliata of the rabbit's tongue after adding quinine to substrate mixture. All the cells of the epithelium lining the gutters between the papillae are positive; so are the nerve plexuses underlying the epithelium and the glands of Ebner. Fig. 3. Reaction in vas deferens of the rabbit. A faint positive reaction is given by the epithelium of the mucosa. Fig. 4. Reaction in vas deferens of rabbit after adding quinine to substrate mixture. The epithelium becomes more intensely positive than in Fig. 3. Fig. 5. Reaction in cardiac muscle fibres of the rat. Negative reaction. Fig. 6. Reaction in cardiac muscle fibres of rat after adding quinine to substrate mixture. There is a strong positive reaction in the nuclei and a diffuse positive reaction in the fibres. PLATE 2 Fig. 7. Reaction in liver of the rabbit. Almost negative. Fig. 8. Reaction in liver of the rabbit after adding quinine to substrate mixture. A positive reaction is given by the nuclei and cytoplasm of hepatic cells and by the endothelium of blood vessels. Fig. 9. Reaction in mixed salivary gland of the rat. There is a faint positive reaction in connective tissue surrounding the acini and in nuclei and cytoplasm of acini and ducts. Fig. 10. Reaction in mixed salivary gland of the rat after adding quinine to substrate mixture. The reaction is in the same sites as in fig. 9 but more dense. Fig. 11. Reaction in Fallopian tube of the rat. A faint reaction is given by the epithelium of the mucosa and the nuclei of the cells of the lamina propria. Fig. 12. Reaction in Fallopian tube of the rat after adding quinine to substrate mixture. Reaction is in the same sites as fig. 11 but much intensified. Fig. 13. Reaction in duodenum of the rabbit. Faint positive reaction is given by brush border of the epithelium, by the distal portion of epithelium in some cases and by nuclei of lamina propria and Brunner's glands. Fig. 14. Reaction in duodenum of the rabbit after adding quinine to substrate mixture. The reaction is increased in all sites shown in fig. 13 except in nuclei of Brunner's glands.