Comp. gen. Pharmac
SUBCELLULAR D I S T R I B U T I O N OF D I A M I N E O X I D A S E * B E V E R L Y B A C K U S AND K. S. K I M Department of Pharmacology, The George Washington University School of Medicine, Washington D.C., U.S.A.
(Received 9 Dec., 1969) ABSTRACT x. Subcellular distribution of diamine oxidase (DAO) was studied in various mammalian tissues using differential centrifugation. 2. Highest DAO activity was found in the 6o0 g pellets (nuclear fraction) of the following tissues: kidney cortex (cat and dog), cat intestine, rabbit placenta, and rat uterus with deciduomata. 3. In dog and rat intestines, highest DAO activity was found in the I2o, ooog supernatant. 4. Mitochondrial and microsomal fractions contained comparatively little DAO activity. 5. In contrast, another histamine metabolizing enzyme, imidazole-N-methyltransferase (IMT), was found exclusively in the x2o, ooo g supernatant fraction of the cat kidney cortex. 6. Cat kidney was studied to determine regional distribution of DAO and IMT. Both enzymes were, for the most part, localized in the cortex, but a small amount of I M T was also found in the medulla.
THERE are at least two m a j o r h i s t a m i n e m e t a b o l i z i n g e n z y m e s in m a m m a l i a n species, n a m e l y d i a m i n e oxidase ( D A O ) a n d i m i d a z o l e - N - m e t h y l t r a n s f e r a s e ( I M T ) (Schayer, 1966). T h e r e is no a g r e e m e n t as to t h e subcellular localization o f histaminase (Cotzias a n d Dole, I952; W a t o n , I956; K o b a y a s h i a n d Ivy, 1958) a n d the reasons for disagreem e n t a m o n g these workers is n o t known. T h e m e t h o d s used for histaminase assay b y e a r l i e r workers were r a t h e r insensitive, l a c k e d specificity in some instances, a n d were used in tissues w h i c h a p p e a r to be low in histaminase activity (Cotzias a n d Dole, i952; W a t o n , 1956). F o r these reasons, s u b c e l l u l a r d i s t r i b u t i o n o f a histaminase, D A O , was e x a m i n e d in tissues w i t h h i g h D A O a c t i v i t y using a m o r e sensitive assay m e t h o d ( O k u y a m a a n d K o b a y a s h i , 196 I). * This investigation was supported by U.S.P.H.S. Research Grant GM-I3749 from the National Institutes of Health, Bethesda, Maryland, U.S.A.
Since I M T is a n o t h e r m a j o r e n z y m e conc e r n e d in h i s t a m i n e i n a c t i v a t i o n , its localization was also s t u d i e d for c o m p a r i s o n w i t h D A O in t h e cat kidney. M A T E R I A L S AND M E T H O D S Tissues from adult animals of both sexes were used, including small intestine (cat, dog, guineapig, mouse, rabbit, and rat), kidney (cat and dog), rabbit placenta, and rat uterus. Tissues were removed immediately after the animals were sacrificed by either head concussion or nembutal anaesthesia, and chilled. Small intestines were homogenized, the homogenates being prepared from either whole wall segments in the case of guinea-pigs, mice, and rats, or from the scrapings of the mucosa in the case of cats, dogs, and rabbits. The kidney was removed and rapidly sectioned to free an entire pyramid. Slices were taken from the inner medulla, inner band of the outer medulla, outer band of the outer medulla, inner cortex, and outer cortex (Fig. x). Homogenates of the kidney were prepared from these various sections and used in the study of regional localization of DAO. Only kidney cortex was used for the study of subcellular distribution since DAO and I M T are, for the most part, found to be localized in the cortex.
I97O , I , I 9 6 - 2 o o
In rabbit placenta studies, only the maternal decidua portion of the placenta from near-term pregnant rabbits was used. R a t uterus included deciduomata which was induced by the following regimen: starting on the day of oestrus, progesterone (2 mg. per rat per day) was injected for 4 days, on the fifth day a foreign body (silk thread suture) was introduced into the uterus, and progesterone treatment was maintained for another 6 days. The uterus was removed x day after the last progesterone injection. The freshly obtained tissues were homogenized in 9 volumes of ice-cold o'25 M sucrose solution (9 ml. per g. tissue) using a glass or a combination glass and Teflon homogenizer. Tissue homogenates were fractionated by differential centrifugation. Cell nuclei and unbroken cells were sedimented by centrifugation at 6oo g for I o minutes. Mitochondria were sedimented from the 6oo g supernatant by centrifugation at 9ooo g for 15 minutes, and microsomes were sedimented from 9ooog supernatant fraction by centrifugation at t 2o0oo g for 45 minutes. DAO and I M T were assayed by the methods described earlier (Kim, Backus, Harris, and Rourke, 1969). RESULTS REGIONAL DISTRIBUTION OF D A O AND I M T IN THE CAT KIDNEY Zeller (1938) was the first to show t h a t D A O is localized in t h e cortex b u t n o t in t h e m e d u l l a r y r e g i o n o f t h e k i d n e y o f various species. T h i s was c o n f i r m e d in cat k i d n e y , as shown in Table L D i s t r i b u t i o n o f I M T was s o m e w h a t similar b u t significantly m o r e activity was f o u n d in the m e d u l l a r y region. SUBCELLULAR DISTRIBUTION OF D A O I M T IN THE CAT KIDNEY
D A O a n d I M T activity was m e a s u r e d in various fractions o f the cat k i d n e y cortex h o m o g e n a t e w h i c h was s u b j e c t e d to t h r e e different g r a v i t a t i o n a l forces. T h e results a r e shown in Table II. M o s t o f the D A O activity was f o u n d in t h e 6o0 g pellets c o n t a i n i n g u n b r o k e n cells, nuclei, a n d v a s c u l a r a n d connective-tissue elements. D A O contents o f m i t o c h o n d r i a l a n d m i c r o s o m a l fractions were, on t h e average, 15 ~2[ld I fit p e r c e n t o f t h e t o t a l h o m o g e n a t e activity, respectively. I n contrast, I M T was f o u n d in t h e I fito,ooo g s u p e r n a t a n t (cell sap), c o n f i r m i n g t h e findings o f Brown, T o m c h i k , a n d A x e l r o d (1959).
T h e e n z y m e a c t i v i t y differences b e t w e e n h o m o g e n a t e a n d 600 g s u p e r n a t a n t , b e t w e e n 6oo g a n d 9ooo g s u p e r n a t a n t s , a n d b e t w e e n 9 o o o g a n d I2O,ooog s u p e r n a t a n t s should t h e o r e t i c a l l y be e q u a l to e n z y m e activities of 6oo g pellets (nuclear fraction), 9ooo g pellets ( m i t o c h o n d r i a l fraction), a n d 12%oo g pellets
~ [outer medulla - - F - - i n n e r band J
Fro. I . - - T h e slice of kidney used for the homogenate.
L--REGIONAL LOCALIZATION OF D A O AND I M T IN THE CAT KIDNEY ACTIVITY* AS PER CENT OF
OUTER CORTEX DAO
Outer cortex Inner cortex Outer band of outer medulla Inner band of outer medulla Inner medulla
I8+ 3 I3+ 4
* Mean ! S . E . (microsomal fraction), respectively. T h i s was f o u n d to be the ease (Table H). SUBCELLULAR DISTRIBUTION OF
D A O IN
O T H E R TISSUES
D A O a c t i v i t y was also s t u d i e d in t h e h o m o g e n a t e s a n d the s u p e r n a t a n t fractious from the small intestines (cat, dog, a n d r a t ) , d o g kidneys, r a b b i t placentas, a n d r a t uterus w i t h d e c i d u o m a t a (Table II). R a b b i t p l a c e n t a , r a t uterus w i t h d e c i d u o m a t a , d o g kidney, a n d cat intestine s h o w e d a similar
BACKUS AND KIM
pattern of D A O distribution as the cat kidney cortex shown in Table IL However, the highest D A O activity in dog and rat intestines was found in the cell sap (54 and 74 per cent, respectively) with the microsomal fraction containing the second largest fraction of D A O (24 and 23 per cent, respectively). It is assumed that the differences in D A O activities between the 9ooo g
Comp. gen. Pharmac.
homogenates. Since the present study suggested that D A O of the small intestine might not be localized in the cell sap as was reported earlier (Kobayashi and Ivy, 1958), a comparative study was made on D A O activity of the small intestines of 6 different species between tissue homogenate and its 3o,ooo g supernatant. It was found that D A O activity of the homogenates was, on the
Table ]L--SuBCELLULAR DISTRIBUTION OF D A O AND I M T IN CAT KIDNEY CORTEX ACTIVITY AS PER CENT OF HOMOGENATE
CENTRIFUGAL FRACTION OF HOMOGENATE
Enzyme activity of whole homogenate (counts per minute per Io mg.) 600 g supernatant 9oo0 g supernatant 120,0o0 g supernatant 60o g pellets 9oo0 g pellets 00,000 g pellets
13,3oo 4-°320 38 4-3 05 -4-0 II4-t 66 4-2 x54-3 104-0
37,600 +3800 95 4-7 Ioo+3 io0 4-3 3* I* I*
Values are expressed as mean ±S.E. of 3 cat kidneys. * Average of two samples.
Table IIL--DAO ACTrV~YOF 30,ooo g SUPERNATANTOF THE SMALLINTESTINEEXFaESSED AS PER CENTOF HOMOOENATEAcTIvrrY
NO. OF ANIMALS
Guinea-pig Rabbit* Mouse Rat Cat* Dog*
30,000 g SUPERNATANT
AcTivrrY (Counts per minute per IO mg.)
PER CENT OF HOMOGENATE
264 4-33 258o ± 122 546o ~ 77° 3"~o 4-438 17,700 ±2500 15,2-,0 1856o
56 ±3 7° +6 42 4"3 9o+II 25 ±3 78 +9
* Mucosal DAO activity. and 12o,ooo g supernatant fractions represent D A O activities of microsomal fractions. COMPARISON OF D A O ACTIVITY OF THE SMALL INTESTINE BETWEEN HOMOGENATE AND 3 0 , 0 0 0 g SUPERNATANT (Table III)
In our previous study on the distribution of D A O and I M T along the gastro-lntestinal tract (Kim and others, i969) , D A O activity was measured in 3o,ooog supernatant of tissue
average, higher than that of the 30,000 g supcrnatant, but there was no significant difference between the two in dog and rat intestines. DISCUSSION It was found in this study that DAO, in contrast to I M T , is not confined in the cell sap in all tissues studied. With the exception of the small intestine of rats and dogs, the
highest DAO activity in cat and dog kidneys, rabbit placenta, and rat uterus with deciduomata was found in the 6oo g pellets (Tables II, IV). Whether DAO in these tissues is localized in the nuclei or in another component of the 6oog pellets is yet to be determined. The reason for high DAO activity in the I2o,ooo g supernatant of the dog and rat intestine is not dear. DAO activity of the small intestine for the most part is localized in the mucosa (unpublished data). It is also known that the turnover rate of intestinal mucosa is rather rapid (I.4-2"8 days) (Leblond and Stevens, i948; McMinn, I954). It may be that DAO is released more readily in the aged mucosa in the process of turnover by autolytic process. This logic,
liver was localized in the mitochondria. In our series much less activity (4-I7 per cent) was found in the mitochondrial fraction of all tissues studied. It is assumed that the differenee in DAO activity between the 6oog supernatant and the 9ooog supernatant represents DAO activity of the mitochondrial fraction. T h e discrepancy between the findings of Cotzias and Dole and ours may be due to the difference in DAO assay methods. Cotzias and Dole measured DAO activity by measuring NH8 evolved during 24-hour incubation. It is now known that N H s is also given off by the action of monoamine oxidase (MAO) on 1,4-methylhistamine (Schayer, i966 ) and that rabbit liver is high in I M T activity which transforms histamine to 1,4methylhistamine (Brown and others, i959).
Table IV.--SuBCELLULAR DISTRIBUTION OF D A O ACTIVITY IN VARIOUS MAMMALIANTISSUES
N o . OF ANIMALS
HOMOGENATE ACTIVITY* (Counts per minute
SUPERNATANT ACTMTY* AS PER CENT OF HOMOGENATE
per IO mg.) Cat intestine Dog intestine Dog kidney Rabbit placenta Rat intestine Rat uterus with deciduomata
2o,2oo 4-398o 23,800 4-64oo 34,4oo 4-608o 865 4-38 4920
354-I 9or 554-IO 43 4-4 xo4
78 4-4 384-6 33 4-4 97
4-3 54 4-8 25 4-4 o6 4-3 74
'~ Mean +S.E. Average of two samples. however, does not hold in the case of the cat small intestine which showed only 21 per cent of the DAO activity in the i2o,ooo g supernatant. It is of interest to note that the mucosal turnover rate of the cat intestine is about twice that of the rat intestine. It is possible that DAO in all tissues is mostly bound in the particulate material of 6oo g pellets but in certain tissues such as dog and rat intestines DAO is readily released to cell sap during homogenization. Alternatively, the different subcellular distribution of DAO found in dog and rat intestines may be real. Cotzias and Dole (I952) found that 4o per cent of the total DAO activity of the rabbit
Monoamine oxidase is known to be localized in the mitochondria (Cotzias and Dole, I95i ). Therefore, it is possible that the method of histaminase assay used by Cotzias and Dole (i952) measured both DAO and M A O activities. Although DAO activities of the small intestine were greater in the homogenate than in 3o,ooog supernatant in all tissues, the overall conclusions of our earlier work (Kim and others, 1969) are still valid in that DAO activity of the small intestine was lower in herbivores (guinea-pigs and rabbits) than that in either omnivores (mice and rats) or carnivores (cats and dogs).
BACKUS AND KIM
Cortical localization o f D A O was confirmed in the cat kidney. I t was also shown that I M T activity o f the cat kidney for the most part is also localized in the cortex, but a significant a m o u n t was also found in other regions. Comparatively higher activity of D A O a n d I M T in the outer b a n d of outer medulla, w h e n c o m p a r e d with other regions of medulla, m a y be due to an unavoidable contamination o f cortical tissue during sectioning. W h e t h e r I M T a n d D A O are localized in the glomeruli or in the tubules is yet to be determined. Brown a n d others (1959) were able to detect only trace a m o u n t s of I M T activity in the cat kidney (one cat). T h e y also reported that guinea-pig brain showed the highest I M T activity of all tissues they studied. It was f o u n d in this laboratory that I M T activity o f the cat kidney cortex was the highest a m o n g all tissues studied, including guinea-pig brain (unpublished data). This discrepancy between Brown a n d others (1959) a n d us is not clear, but consistently high I M T activity of the kidney was noted in more t h a n 1o cats in this laboratory. O n the average, I M T activity of cat kidney cortex was more t h a n 4 times that o f the highest activity of the guinea-pig brain observed in this laboratory. SUMMARY Subcellular distribution o f D A O was studied in various tissues. After differential centrifugation of tissue homogenate, highest D A O activity was found in the 6o0 g pellets o f the kidney cortex (cat a n d dog), the cat intestine, the rabbit placenta, a n d the rat uterus with deciduomata. But in dog a n d rat intestines the highest D A O activity was f o u n d in the 120,000 g supernatant. I n contrast, I M T activity o f the cat kidney cortex was exclusively in the 120,000 g supernatant fraction of tissue homogenate. Both D A O a n d I M T were mainly localized in the cortical region of the cat kidney, b u t considerable I M T activity was also f o u n d in the medullary region.
The authors wish to thank Dr. R. A. Kenney for his kind assistance in the study of cat kidneys. Our thanks are also due to Dr. V. H. Cohn, Dr. S. Fabro, and Dr. H. G. Mandel for their helpful suggestions in the preparation of this manuscript.
REFERENCES BROWN, D. D., TOMCHICK, R., and AXELROD,J. (1959), ' T h e distribution and properties of a histamine methylating enzyme ', 07. biol. Chem., ~34~ 2948-295 o. COTZlAS, G. C., and DOLE, V. P. (I95I), ' Metabolism of amines, II. Mitochondrial localization of monoamine oxidase ', Proc. Soc. exp. Biol. Med., 78, I57-16o. (1952), ' The activity of histaminase in tissues ', 07. biol. Chem., 196, 235-242. KIM, K. S., BACKUS,B., HARMS, M., and ROURKE, P. (1969) , ' Distribution ofdiamine oxidase and imidazole-aV-methyltransferase along the gastrointestinal tract ', Comp. Biochem. Physiol., 3 x, 137-146. KOBAYASm, Y., and IvY, A. C. (1958), 'Histamine metabolizing activity of dog stomach and intestine ', Am. 07. Physiol., 195, 525527. LEmOND, C. P., and STEVENS,C. E. (1948), 'The constant renewal of the intestinal epithelium in the albino rat', Anat. Rec., too, 357378. McMaNN, R. M. H. (I954), ' The rate of renewal of intestinal epithelium in the cat ', 07. Anat., 88, 527-532. OKUYAMA, A., and KOBAYASHI, Y. (I96 Q, ' Determination of diamine oxidase activity by liquid scintillation counting ', Archs Biochem. Biophys., 95, 242-25°. SCHAYaR, R. W. (I966), 'Catabolism of histamine in vivo ', in Handbook of Experimental Pharmacology (ed. EmHLBR, O., and FARAH,A.), vol. I8, part I, pp. 672-683. Berlin: Springer Verlag. WATON, N. G. (x956), 'Studies on mammalian histidine decarboxylase ', Br. 07. Pharmac., xt, II9-127. ZELLER, E. A. (1938), ' l~ber den enzymatischen Abbau yon Histamin und Diaminen ', Helv. chim. Acta, ax, 88o-89o.
Key Word Index: Subcellular distribution of diamine oxidase and imidazole-Ar-methyltransferase, diamine oxidase, localization of diamine oxidase and imidazole-Ar-methyltransferase in cat kidney, subcellular distribution of diamine oxidase.