in Ringer's solution containing Ca 4~ (specific activity. 2 ~c/ml) at 6°C for 16 hours. The muscles were then removed from the isotope, rinsed in a non-radio-.
A METHOD
FOR
WATER-SOLUBLE
STUDYING ISOTOPES
INTRACELLULAR PRIOR
MOVEMENT
OF
TO RADIOAUTOGRAPHY
DONALD A. FISCHMAN and MICHAEL D. GERSHON. From the Department of Anatomy, Cornell University Medical College, New York INTRODUCTION The compartmentalization of cell structure as revealed by electron microscopy has been shown to have a striking parallelism with the segregation of enzyme systems as revealed by differential centrifugation. The biochemical anatomy of the cell is now of great interest, and a powerful technique for its investigation has been provided by the recent application of radioautography at the ultrastructural level (1-3). At present, this technique has been most successful in the localization of proteins (1) and nucleoproteins (4). However, one might also expect smaller compounds and ions to show a pattern of differential localization similar to that of the enzymes mediating the reactions with which they are concerned. T h o u g h of great interest, the intracellular localization of diffusible compounds has been a problem of exceptional difficulty, as the careful studies of D. K. Hill (5, 6) have clearly demon-
strated. While investigating such a problem, it is essential to have prior knowledge of the movement of the compound, both under the experimental conditions and during the steps of histological preparation. While studying the cellular localization of a small organic compound, serotonin, and a divalent cation, calcium, the authors found that the curves of the washout of radioactivity from the tissues of serotonin-C TM and calcium-45 provided a useful tool for investigating intracellular movement and finding a means of limiting it. MATERIALS
AND METHODS
Serotonin Mice, averaging 20 gm in weight, were injected intravenously with 5-hydroxytryptophan-3 C 14 (5HTP-C 14) 1 hour after an intraperitoneal injection of fl-phenylisopropylhydrazine (PIH), a monoamine
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oxidase inhibitor. This procedure was s h o w n to result in the production of radioactive serotonin in the g u t a n d will he dealt with in detail in a s u b s e q u e n t publication. Segments of d u o d e n u m , 25 to 40 m g in weight, were r e m o v e d from the a n i m a l 4 hours after the injection of 5 - H T P - C l t T h e l u m e n was opened by a longitudinal slit a n d the d u o d e n a l strips were
time they were m o u n t e d at resting length on Pyrex glass racks by m e a n s of cotton ties. T h e r e m a i n d e r of the procedure is described in the legend of Fig. 2.
Measurements of Radioactivity Radioactivity was assayed by m e a n s of a windowless gas flow counter. Care was taken to assure even
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Minutes FmURE I The washout of radioactive 5-OH indo]es from duodenal strips in physio]ogiea] saline and various experimental solutions. Temperature £0-¢£°C. After washout, the duodena] strips were dissoh, ed in I~ N HCI and the residual radioactivity measured. Initial radioactivity was obtained by adding that which ]eft the strips in saline or experimental solution to the residual radioactivity. The amount of radioactivity remaining in the strip, as per cent of the initial radioactivity, was then plotted sere•logarithmically against time. quickly weighed a n d t h e n transferred at various time intervals t h r o u g h a succession of tubes containing physiological saline. T h e r e m a i n d e r of the experim e n t a l procedure is described in the legend of Fig. 1.
spreading on a l u m i n u m or glass planchets, a n d s a m ples were corrected for self-absorption. T h e coefficient of variation of the counts of five pipettings of s t a n d a r d solutions containing 1000 CPM was 1.42 per cent.
Calcium
Experimental Solutions
Paired sartorius muscles, dissected from pithed frogs weighing a p p r o x i m a t e l y 20 g m , were placed in R i n g e r ' s solution containing C a 4~ (specific activity 2 ~ c / m l ) at 6°C for 16 hours. T h e muscles were t h e n r e m o v e d from the isotope, rinsed in a non-radioactive R i n g e r ' s solution for 1 hour, d u r i n g which
(1) F o r m a l i n a n d P O 4 : l 0 per cent formalin; 90 per cent 0.1 M Sorenson's p h o s p h a t e buffer, p H 7.4. (2) F o r m a l i n a n d PO4 a n d sucrose: As (1) with 9 per cent w / v sucrose. (3) F o r m a l i n a n d PO4 a n d oxalate: As (l) with s o d i u m oxalate a d d e d to give a final oxalate concentration of 0.02 M. (4) OsO4:
1400
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Palade's fixative. (5) OsO4 and sucrose: As (4) with 9 per cent w/v sucrose. (6) OsO4 and PO4: 50 per cent v/v of 2 per cent OsO4; 50 per cent v/v of 0.1 M Sorenson's phosphate buffer, pH 7.4. (7) OsO~ and PO4 and oxalate: As (6) with sodium oxalate added to give a final oxalate concentration of 0.02 M. (8) Carnoy's solution: 10 per cent glacial
increased over that in saline by the hypotonic fixatives, Os04, and formalin and P04, but was markedly increased by ethanol (70 to 100 per cent) alone and by Carnoy's solution. In contrast, the rate of washout was not increased when the O s 0 4 and formalin were m a d e hypertonic by the o : Os04 +P04 +oxalate • : Os04 +P04
o : FormaLin +PO4+oxalate • : Formalin +PO4 ]- FormaLin +oxalate
100
_
,~ OsO4+P04 +oxalate
--
T
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_
T tAIcohol ~Ringer's
"~
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t A,coho, ,~Ringer's
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• = No fixative Ringer's-',alcohol
30
20
--
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90
AIc°h°ll 120
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Minutes
Ftou~m ~ The washout of radiocaleium from frog sartorius umseles in Ringer's and experimental solutions. Temperature 6°C. After washout, the muscles were ashed at 600°C for ~4 hours and the residual radioactivity measured. The rapid phase of Ca 45release has not t)een included in the graphs. One hundred per cent has been arbitrarily set as that amount of radioactivity present in tile muscle after 1 hour in nonradioactive Ringer's solution. acetic acid; 30 per cent chloroform; 60 per cent absolute ethanol. RESULTS
addition of sucrose. All of the fixatives except Carnoy's solution prevent the subsequent extraction of radioactivity by saline, alcohol, or clearing agent.
Serotonin
Calcium
Radioactive 5-OH indoles were allowed to wash out of duodenal strips in physiological saline, and, w h e n the slow c o m p o n e n t of the washout curve had been reached, the strips were transferred to one of the experimental solutions. T h e curves of washout of radioactivity are shown in Fig. 1. T h e rate of washout of radioactivity was moderately
As in the case of the duodenal strips, Ca 45 was allowed to wash out of the muscles until the slow c o m p o n e n t of the washout curve had been reached. In both cases, it was presumed that the slow rate of washout predominantly reflected loss from an intracellular depot (see Discussion). The results are presented in Fig. 2. W h e n the muscles fixed in
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formalin or OsO4 with or without oxalate are compared, it is seen t h a t more Ca 4'~ is retained in the muscles fixed in the presence of oxalate. F u r t h e r more, the loss of isotope when the muscles are returned to the Ringer's solution is significantly decreased if oxalate h a d been included in the fixative. Carnoy's solution caused the most striking release of calcium both in the fixative a n d after r e t u r n i n g to Ringer's solution. In all cases ethanol (60 to 100 per cent) a b r u p t l y stopped calcium release. Alcohol will prevent the release of calcium even in the absence of prior fixation (1 per cent picric acid in 2-propanol also prevented calcium release). DISCUSSION By means of the relatively simple washout procedure described above, approximations of both the rate and q u a n t i t y of isotope m o v e m e n t from the cells can be made. T h e speed of fixation can be estimated from changes in the rate of isotope release, a n d an indication obtained as to not only how m u c h intracellular isotope has been retained, b u t also how quickly its m o v e m e n t has been halted. This estimation rests on the assumption t h a t m i n i m a l release from the cells indicates minimal intracellular diffusion. T h e technique gives no information, however, as to w h e t h e r a small a m o u n t of m o v e m e n t which m a y take place is selectively critical to a particular radioautographic problem. It is also possible t h a t the alterations in isotope release with fixation reflect changes in m e m b r a n e permeability to the molecules studied r a t h e r t h a n limitations to their diffusion within the cell cytoplasm. If this were the case, then it would seem t h a t after t r e a t m e n t with alcohol the m e m b r a n e permeability changes selectively for different molecules. Ethanol a b r u p t l y halts Ca 4'~ release yet increases the rate of loss of serotonin. It would also s e e m likely t h a t if the cell m e m b r a n e were acting as a barrier to diffusion after fixation, then intracellular m e m b r a n e s would also act as similar barriers to the diffusion of molecules located within m e m b r a n e - l i m i t e d c o m p a r t m e n t s of the cell. In either case, therefore, the rate of loss of isotope from the cells would still provide useful information as to the extent of intracellular movem e n t during a n d after fixation. In order to obtain information a b o u t intracellular m o v e m e n t of the radioisotopically labeled material from the curves of washout, isotope released from within cells should be distinguishable
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from t h a t of extracellular origin. I n the case of serotonin, the serotonin-C ~4was synthesized within cells from its administered precursor, 5 - H T P - C ~4. Four hours after administration, little radioactivity was found in a muscular organ, the heart, in the connective tissue or muscles of the extremities, or free in the blood plasma. Consequently, it was assumed t h a t the slow c o m p o n e n t of the washout curves represented serotonin-C 14 from a n intracellular depot. In the case of calcium, binding to connective tissue fibers (7) a n d cell surfaces (8) complicates the analysis. However, it has been shown (7) that, while at the beginning of the washout period significant amounts of radiocalcium in the sartorius muscle are b o u n d to cell surfaces a n d connective tissue, after 90 minutes of washout 95 per cent of the r e m a i n i n g Ca 4'5 is intracellular. After 120 minutes of washout (at which time the muscles were fixed, in the present experiments) almost all of the radiocalcium leaving the muscles is from intracellular stores. T h e a m o u n t of Ca 4'~ still b o u n d to connective tissue and cell surfaces is i n a d e q u a t e to explain the m a g n i t u d e of the effects produced by immersion in fixatives. T h e authors, therefore, conclude that the alterations in the washout curves of serotonin-C t4 and Ca 4~ produced by the fixatives reflected events occurring at or inside the cells of the respective tissues. T h e curves of washout can thus be used to obtain useful information on which the radioautographic localization of these compounds can be based. SUMMARY W a s h o u t curves of serotonin-C 14 from the mouse d u o d e n u m a n d Ca a5 from frog skeletal muscle have been studied before, during, a n d after treatm e n t of the respective tissues with various fixatives. T h e release of tissue serotonin d u r i n g fixation can be markedly reduced by m a k i n g the fixative hypertonic. Alcohol causes a m a r k e d extraction of serotonin, but this extraction does not occur if fixation precedes exposure to alcohol. T h e addition of calcium precipitant, sodium oxalate, to OsO4 or formalin causes a substantial reduction in calcium release from muscle d u r i n g fixation. Alcohol a b r u p t l y stops calcium efflux w h e t h e r or not the addition of alcohol precedes or follows fixation. It is felt t h a t this washout technique m a y have wide applicability as a m e t h o d for investigating
diffusion problems prior to r a d i o a u t o g r a p h i c studies of water-soluble intracellular compounds.
3.
This research was supported in part by United States Public Health Service research grants B-1523 (C4) and AM-06290-02. Dr. Fischman is a Post-doctoral Fellow of the New York Heart Association.
4.
5.
Received for publication, October 9, 1963. REFERENCES 1. CARO, L., Electron microscopic radioautography of thin sections: The Golgi zone as a site of protein concentration in pancreatic acinar cells, J. Biophysic. and Biochem. Cytol., 1961, 10, 37. 2. REVEL, J. P., and HAY, E. D., Autoradiographic localization of DNA synthesis in a specific nltrastructural component of the interphase nucleus, Exp. Cell Research, 1961, 25, 474.
6.
7.
8.
L., and VAN TUBERGEN, R. P., High-resolution autoradiography. I. Methods, J. Cell Biol., 1962, 15, 173. HAY, E. D., and REVEL, J. P., The fine structure of the DNP component of the nucleus. An electron-microscopic study utilizing autoradiography to localize DNA synthesis, J. Cell Biol., 1963, 16, 29. HILL, D. K., The location of adenine nucleotide in frog's striated muscle, J. Physiol., 1960, 150, 347. HILL, D. K., Preferred sites of adenine nucleotide in frog's striated muscle, J. Physiol., 1960, 153,433. SHANES, A. M., and BIANCHI, C . P . , The distribution and kinetics of release of radiocalcium in tendon and skeletal muscle, J. Gen. Physiol., 1959, 42, 1123. GILBERT, D. L. and FENN, W. O., Calcium equilibrium in muscle, 3". Gen. Physiol., 1957~ 40, 393.
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