403
J. Anat. (1976), 122, 2, pp. 403-413 With 1O figures Printed in Great Britain
On the fixation of catecholamines including adrenaline in tissue sections R. E. COUPLAND, S. KOBAYASHI AND JANET CROWE
Department of Human Morphology, University of Nottingham, and Department of Anatomy, University of Niigata Medical School, Japan
(Accepted 25 October 1975) It is well known that the mammalian adrenal medulla is a main site of synthesis, storage and secretion of catecholamines, the most abundant being adrenaline. Furthermore, the intensity of the brown-yellow chromaffin reaction when fresh adrenal tissue is exposed to solutions containing potassium dichromate to some extent reflects the concentration of adrenaline in the tissue. However, during many standard fixation procedures, including those based on glutaraldehyde (Coupland, Pyper & Hopwood, 1964; Coupland & Hopwood, 1966; Tramezzani, Chiocchio & Wasserman, 1964) and formaldehyde (Hopwood, 1968), adrenaline is lost in whole or part from storage vesicles, and it has been shown that these vesicles are particularly labile when exposed to solutions having high hydrogen ion concentrations. Aqueous solutions of dichromate salts have been known to react with the contents of the adrenal medulla since the work of Stilling (1889), but other fixatives must be included to obtain good cell preservation. For routine light microscopy the common additive is aqueous formaldehyde and optimal reactions are achieved by buffering the mixture to a pH of about 5 8 with M/5 acetate or phosphate (Coupland, 1961, 1965). Under these circumstances both adrenaline- (A) and noradrenaline-storing (NA) cells show a positive chromaffin reaction. In general, tissues fixed in formol-dichromate are unsatisfactory for electron microscopy, and hence the possibility of using mixtures of chromate salts and glutaraldehyde has been investigated. The value of glutaraldehyde in the localization of noradrenaline is now generally accepted. The independent work of Tramezzani et al. (1964), Coupland et al. (1964) and Coupland & Hopwood (1966) has shown that, during fixation of adrenal medulla in glutaraldehyde at pH 7-2-7A4, adrenaline is lost into the fixing and dehydrating fluids while noradrenaline is precipitated in situ. However, when potassium dichromate is added to the aqueous glutaraldehyde fixative, good light microscopic preservation of both amines, as judged by the chromaffin reaction, is obtained (Coupland & Hopwood, 1966). Since chromaffin granules are labile in solutions out of the range pH 6-0-7 0 (Hopwood, 1968) and the optimal pH for the glutaraldehyde reaction with noradrenaline is in the range of pH 7-0-7 4, little latitude exists with regard to the pH of the mixture. Furthermore, pilot studies have indicated widespread ultrastructural damage when potassium dichromate is added to the fixative in excess of 4 %, while an unsatisfactory light microscopic chromaffin reaction is 26
A NA I22
404
R. E. COUPLAND, S. KOBAYASHI AND JANET CROWE
V,u
Fig. 1. Autoradiograph of A cells (pale) and NA cells (dark) of mouse adrenal medulla 15 minutes after the injection of 10 ,uCi/gm body weight 3H-dopa. Note high grain density over both types of cells and lower density over a small group of cortical cells (C). Fixative glutaraldehyde. x 725.
given with concentrations of less than 1 %. However, the addition of potassium dichromate at a concentration of 1-5 % to glutaraldehyde at pH 7-2 has been found (Kobayashi & Coupland, unpublished) to give reasonable tissue fixation and so it was decided to try to assess the degree of catecholamine preservation in tissue fixed in this way using a radioisotopic method. MATERIALS AND METHODS
Male albino mice (strains CS 1 and TO) of 24-28 g body weight were injected intravenously or intraperitoneally with lO,uCi/g L-3,4-dihydroxy-(ring-2,5,6-3H) phenylalanine (3H-dopa). Animals were killed by cervical dislocation 0 25, 0 5, 1, 2, 4, 8, 24 hours and 5 days thereafter.
Histological procedures Some adrenal glands were fixed by perfusion in either (a) 1-5 % glutaraldehyde in 0-2 M phosphate buffer at pH 7-3 with post-osmication in 1 % osmium tetroxide or (b) in a mixture containing 1-5 % glutaraldehyde and 1-5 % potassium dichromate at pH 7-2. In some instances one adrenal gland was bisected and fixed by immersion in glutaraldehyde and the contralateral one fixed in dichromate-glutaraldehyde. Tissues were dehydrated in graded alcohols, embedded in Araldite and sectioned on a Cambridge-Huxley microtome at 1 ,um. Sections were mounted on gelatinized slides and dipped in Ilford G5 emulsion. The slides were exposed in light tight boxes containing silica gel at 4 'C for 1-2 weeks. Autoradiographs were developed
Fixation of catecholamines
405
80 .A- Cortical rAdrenaline
70 60
ki
I
,
*Noradrenaline *
/
E
~40 30
7
IA>
20 10
15 minutes 1 hour 4 hours 1 day 30 minutes 2 hours 8 hours
5 days
Fig. 2. Grain densities over adrenal A, NA and cortical cells at various time intervals after the injection of 3H-dopa. The tissues were fixed in glutaraldehyde. Note 1 hour fall in grain counts over A cells after 8 hours as newly synthesized adrenaline (cf. Fig. 9) is lost.
at 19 °C in Kodak D 19 without agitation, rinsed briefly in 2 % acetic acid in distilled water and fixed in Hypam (1: 3 distilled water). After this they were washed in at least three changes of distilled water and stained for 6 minutes in 0-5 % toluildine blue in 0 05 phosphate buffer at pH 7-4 previously heated to 50 'C. Finally they were rinsed in three changes of distilled water, dried overnight and mounted in 'Piccolyte' resin. Grain counts were made on randomly selected fields using a x 100 oil immersion objective with normal or phase contrast microscopy. The latter aided the identification of noradrenaline-storing cells, particularly in tissues fixed in dichromate-glutaraldehyde. Areas containing only one type of chromaffin cell were delineated by means of an eyepiece graticule, and silver grains falling within the area were counted. Ten to fifteen sections were counted for each time interval. Similar estimates were made using an Olympus-MMSP.
Assay procedure Other injected animals were killed at the above time intervals and the adrenal glands removed, freed from connective tissue and homogenized in 1-5 ml N/10 HCI and 50 #1 of a mixture (1:1: 1: 3) of dopa, dopamine, adrenaline and noradrenaline (concentration 1 ,ug/ml) added as carriers. The homogenate was centrifuged immediately at 10 'C and 16000 r.p.m. for 20 minutes. The supernatant was treated with an equal volume of 40°-60° petroleum ether 'Analar' to remove lipids. The aqueous phase was then concentrated to approximately 50 ,ul under a stream of nitrogen at 4 'C. 26-2
406
R. E. COUPLAND, S. KOBAYASHI AND JANET CROWE 80 w vCortical i\A Adrenaline
70 /
60
\
*
* Noradrenaline
_
~50 m.~~
~
< 40 _
20
-
10 _ .... .A..&
30 minutes 4 hours 1 hour Time
.....
1 day
5 days
Fig. 3. Grain densities over adrenal A, NA and cortical cells at various time intervals after the injection of 3H-dopa. The tissues were fixed in dichromate-glutaraldehyde. Radioactivity persists over A cells between 8 hours and 5 days (cf. Fig. 2).
Samples were then spotted onto Whatman No. 1 chromatography paper and two dimensional chromatograms were run for 22 hours in each plane in a solvent consisting of butan-l-ol: acetic acid: water (4:1: 5). Papers were dried and sprayed with 5 % ethylenediamine and immediately dried at 100 °C for 5 minutes. Amines were identified under ultraviolet light; the dopa exhibited a yellow green fluorescence, the primary amines silver green and adrenaline yellow. Spots were circled by pencil and cut out together with two blanks of similar size taken from each side of the amine spots. The paper spots were cut into small pieces and eluted with 2 ml N/10 HCl for 1 minute, using a whirlimix (effective time previously determined), and the tubes centrifuged at 3000 r.p.m. for 5 minutes. 1 0 ml aliquots of supernatant were then added to counting vials containing 10 ml of a 'cocktail' (7 5 g 2,5-diphenyloxazole (PPO) in 1 litre of xylene plus 500 ml Triton X-100) and whirlimixed. The vials were placed in a Tracerlab Corumatic 200 liquid scintillation counter set for tritium and counted for 5 minutes. The external standard was set for 10000 counts/minute. RESULTS
Autoradiography NA cells may be distinguished by their greater cytoplasmic staining density and cytoplasmic granularity. There is a high density of reduced silver grains over both A and NA cells within 15 minutes of either an intraperitoneal or intravenous injection of 3H-dopa. The density of grains over A cells is usually slightly greater than that over NA cells at 15 minutes (Fig. 1) and this impression is borne out by grain counts on sections
Fixation of catecholamines
407
Fig. 4. Autoradiograph of mouse adrenal 1 hour after injection of 3H-dopa. Similar heavy labelling of A and NA cells. Cortical cells at the left extremity of the photomicrograph are only slightly radioactive. Fixed in glutaraldehyde. x 725. Fig. 5. Autoradiograph 4 hours after injection of 3H-dopa. Similar, but now slightly less heavy, grain concentration over A and NA cells. Fixed in glutaraldehyde. x 725.
(Figs. 2 and 3). Only a low level of radioactivity is observed over cortical cells (Figs. 1 and 4). A similar pattern and level of grain counts is observed in tissues fixed in glutaraldehyde or in dichromate-glutaraldehyde during the first 4 hours after the injection of labelled amine. At 4 hours both A and NA cells are approximately equally labelled (Figs. 2, 3 and 5). After this time, however, the grain counts over A cells fall rapidly in glutaraldehyde-fixed material and much less so in tissues fixed
408
R. E. COUPLAND, S. KOBAYASHI AND JANET CROWE
* .K;.
Fig. 6. Autoradiograph of mouse adrenal gland 24 hours after injection of 3H-dopa. Cortical cells (C) almost unlabelled. Dark staining NA cells heavily labelled, A cells (A) have only low radioactivity. Fixed in glutaraldehyde. x 725.
in dichromate-glutaraldehyde. After 24 hours only a very low level of activity exists over A cells, while NA cells are still moderately heavily labelled (Figs. 2, 3 and 6). This difference persists for up to 5 days by which time the grain counts over A cells in glutaraldehyde-fixed tissue is only slightly greater than that over cortical cells (Figs. 2, 4 and 7), while in dichromate-glutaraldehyde-fixed tissue both A and NA cells are still labelled (Fig. 8), even though counts show a slight fall in radioactivity over A cells relative to NA cells (Fig. 3). During this work it has been noted that some adrenal glands exhibit a rather patchy density of silver grains in an apparently zonal distribution. In these zones both A and NA cells are affected. There may also be a tendency to a gradient of density from the periphery of a group of chromaffin cells, where they abut well defined vascular channels, to the centre where no vascular channel is evident. No similar patchiness or gradient has been observed after the injection of L-tyrosinering 3,5-3H or L-leucine 4,5-3H: these amines also differ from L-dopa in so far as they are not selectively taken up by chromaffin cells (Kobayashi & Coupland, unpublished). Assay The radioactivity of dopa, dopamine, noradrenaline and adrenaline eluted from chromatograms (Fig. 9) was determined. Total radioactivity of the four compounds at intervals after intravenous or intraperitoneal injection of 3H-dopa is indicated in Figure 10, each reading representing the mean of three or four animals. High activity occurs during the first 4 hours and by 24 hours this has fallen to about one third of the 15 minute level. Between days 1 and 4 or 5 a slow rate of decline occurs in the amount of labelled amine. Initially (Fig. 10) the radioactivity is due primarily to
409
Fixation of catecholamines
A~~~~~~~~~~~~~~A
N
*.
.~~~~~rn ~
A
Fig. 7. Autoradiogaph of mouse adrenl gland 5 days aftr injection of 3H-opa. Cortical cell (C) only slightlylabelled, NA cells (dark) heavily labelled islands of A cells (pale)showonlylo radioactivity. Fixed gn glutaraldehyde. x 725.
Fig.78 Autoadiogrph ofmouseadrena medula S ay an I. ~~~~~¼'a*
Fie radioactivity.~ 4
-
n
ngltrleyd.x75
Fig. 8. Autoradiograph of mouse adrenal meulan 5 days after injection of3H-dopa Criand follow ing fixation in 1.5 % potassium dichromate in glutaraldehyde. Both A and NA cells are labelled and A cells (pale) are more heavily labelled than in Fig. 7. x 725.
labelled dopamine. By 4 hours, however, the most abundant labelled catecholamine is noradrenaline. Adrenaline formation is evident by 1 hour and between 4 and 24 hours the normal proportions of labelled, and hence recently synthesized, adrenaline and noradrenaline are present in the glands. These are maintained during the period 1-5 days.
410
R. E. COUPLAND, S. KOBAYASHI AND JANET CROWE
20
OD ~
~
20
( .E
NA
D
1a
A
10
C
u
in
o U
, r
._N
2nd direction -v 22 hours I
ORIGIN
I
20
10 Centimetres
Fig. 9. Two-dimensional chromatogram of adrenal homogenate showing separation of dopa (D), dopamine (DA), noradrenaline (NA) and adrenaline (A). DISCUSSION
L-dopa is selectively taken up by adrenal medullary chromaffin cells as evidenced by the marked difference in radioactivity of the adrenal medulla and cortex (Figs. 2 and 3). The high grain count during the first 4 hours following the injection of 3H-dopa is in keeping with the total radioactivity of labelled amines in the adrenal medulla during this period (Fig. 10). During the first hour after the injection of 3H-dopa the most abundant labelled amine is dopamine (70-80 %) and there are also small amounts (
J. Anat. (1976), 122, 2, pp. 403-413 With 1O figures Printed in Great Britain
On the fixation of catecholamines including adrenaline in tissue sections R. E. COUPLAND, S. KOBAYASHI AND JANET CROWE
Department of Human Morphology, University of Nottingham, and Department of Anatomy, University of Niigata Medical School, Japan
(Accepted 25 October 1975) It is well known that the mammalian adrenal medulla is a main site of synthesis, storage and secretion of catecholamines, the most abundant being adrenaline. Furthermore, the intensity of the brown-yellow chromaffin reaction when fresh adrenal tissue is exposed to solutions containing potassium dichromate to some extent reflects the concentration of adrenaline in the tissue. However, during many standard fixation procedures, including those based on glutaraldehyde (Coupland, Pyper & Hopwood, 1964; Coupland & Hopwood, 1966; Tramezzani, Chiocchio & Wasserman, 1964) and formaldehyde (Hopwood, 1968), adrenaline is lost in whole or part from storage vesicles, and it has been shown that these vesicles are particularly labile when exposed to solutions having high hydrogen ion concentrations. Aqueous solutions of dichromate salts have been known to react with the contents of the adrenal medulla since the work of Stilling (1889), but other fixatives must be included to obtain good cell preservation. For routine light microscopy the common additive is aqueous formaldehyde and optimal reactions are achieved by buffering the mixture to a pH of about 5 8 with M/5 acetate or phosphate (Coupland, 1961, 1965). Under these circumstances both adrenaline- (A) and noradrenaline-storing (NA) cells show a positive chromaffin reaction. In general, tissues fixed in formol-dichromate are unsatisfactory for electron microscopy, and hence the possibility of using mixtures of chromate salts and glutaraldehyde has been investigated. The value of glutaraldehyde in the localization of noradrenaline is now generally accepted. The independent work of Tramezzani et al. (1964), Coupland et al. (1964) and Coupland & Hopwood (1966) has shown that, during fixation of adrenal medulla in glutaraldehyde at pH 7-2-7A4, adrenaline is lost into the fixing and dehydrating fluids while noradrenaline is precipitated in situ. However, when potassium dichromate is added to the aqueous glutaraldehyde fixative, good light microscopic preservation of both amines, as judged by the chromaffin reaction, is obtained (Coupland & Hopwood, 1966). Since chromaffin granules are labile in solutions out of the range pH 6-0-7 0 (Hopwood, 1968) and the optimal pH for the glutaraldehyde reaction with noradrenaline is in the range of pH 7-0-7 4, little latitude exists with regard to the pH of the mixture. Furthermore, pilot studies have indicated widespread ultrastructural damage when potassium dichromate is added to the fixative in excess of 4 %, while an unsatisfactory light microscopic chromaffin reaction is 26
A NA I22
404
R. E. COUPLAND, S. KOBAYASHI AND JANET CROWE
V,u
Fig. 1. Autoradiograph of A cells (pale) and NA cells (dark) of mouse adrenal medulla 15 minutes after the injection of 10 ,uCi/gm body weight 3H-dopa. Note high grain density over both types of cells and lower density over a small group of cortical cells (C). Fixative glutaraldehyde. x 725.
given with concentrations of less than 1 %. However, the addition of potassium dichromate at a concentration of 1-5 % to glutaraldehyde at pH 7-2 has been found (Kobayashi & Coupland, unpublished) to give reasonable tissue fixation and so it was decided to try to assess the degree of catecholamine preservation in tissue fixed in this way using a radioisotopic method. MATERIALS AND METHODS
Male albino mice (strains CS 1 and TO) of 24-28 g body weight were injected intravenously or intraperitoneally with lO,uCi/g L-3,4-dihydroxy-(ring-2,5,6-3H) phenylalanine (3H-dopa). Animals were killed by cervical dislocation 0 25, 0 5, 1, 2, 4, 8, 24 hours and 5 days thereafter.
Histological procedures Some adrenal glands were fixed by perfusion in either (a) 1-5 % glutaraldehyde in 0-2 M phosphate buffer at pH 7-3 with post-osmication in 1 % osmium tetroxide or (b) in a mixture containing 1-5 % glutaraldehyde and 1-5 % potassium dichromate at pH 7-2. In some instances one adrenal gland was bisected and fixed by immersion in glutaraldehyde and the contralateral one fixed in dichromate-glutaraldehyde. Tissues were dehydrated in graded alcohols, embedded in Araldite and sectioned on a Cambridge-Huxley microtome at 1 ,um. Sections were mounted on gelatinized slides and dipped in Ilford G5 emulsion. The slides were exposed in light tight boxes containing silica gel at 4 'C for 1-2 weeks. Autoradiographs were developed
Fixation of catecholamines
405
80 .A- Cortical rAdrenaline
70 60
ki
I
,
*Noradrenaline *
/
E
~40 30
7
IA>
20 10
15 minutes 1 hour 4 hours 1 day 30 minutes 2 hours 8 hours
5 days
Fig. 2. Grain densities over adrenal A, NA and cortical cells at various time intervals after the injection of 3H-dopa. The tissues were fixed in glutaraldehyde. Note 1 hour fall in grain counts over A cells after 8 hours as newly synthesized adrenaline (cf. Fig. 9) is lost.
at 19 °C in Kodak D 19 without agitation, rinsed briefly in 2 % acetic acid in distilled water and fixed in Hypam (1: 3 distilled water). After this they were washed in at least three changes of distilled water and stained for 6 minutes in 0-5 % toluildine blue in 0 05 phosphate buffer at pH 7-4 previously heated to 50 'C. Finally they were rinsed in three changes of distilled water, dried overnight and mounted in 'Piccolyte' resin. Grain counts were made on randomly selected fields using a x 100 oil immersion objective with normal or phase contrast microscopy. The latter aided the identification of noradrenaline-storing cells, particularly in tissues fixed in dichromate-glutaraldehyde. Areas containing only one type of chromaffin cell were delineated by means of an eyepiece graticule, and silver grains falling within the area were counted. Ten to fifteen sections were counted for each time interval. Similar estimates were made using an Olympus-MMSP.
Assay procedure Other injected animals were killed at the above time intervals and the adrenal glands removed, freed from connective tissue and homogenized in 1-5 ml N/10 HCI and 50 #1 of a mixture (1:1: 1: 3) of dopa, dopamine, adrenaline and noradrenaline (concentration 1 ,ug/ml) added as carriers. The homogenate was centrifuged immediately at 10 'C and 16000 r.p.m. for 20 minutes. The supernatant was treated with an equal volume of 40°-60° petroleum ether 'Analar' to remove lipids. The aqueous phase was then concentrated to approximately 50 ,ul under a stream of nitrogen at 4 'C. 26-2
406
R. E. COUPLAND, S. KOBAYASHI AND JANET CROWE 80 w vCortical i\A Adrenaline
70 /
60
\
*
* Noradrenaline
_
~50 m.~~
~
< 40 _
20
-
10 _ .... .A..&
30 minutes 4 hours 1 hour Time
.....
1 day
5 days
Fig. 3. Grain densities over adrenal A, NA and cortical cells at various time intervals after the injection of 3H-dopa. The tissues were fixed in dichromate-glutaraldehyde. Radioactivity persists over A cells between 8 hours and 5 days (cf. Fig. 2).
Samples were then spotted onto Whatman No. 1 chromatography paper and two dimensional chromatograms were run for 22 hours in each plane in a solvent consisting of butan-l-ol: acetic acid: water (4:1: 5). Papers were dried and sprayed with 5 % ethylenediamine and immediately dried at 100 °C for 5 minutes. Amines were identified under ultraviolet light; the dopa exhibited a yellow green fluorescence, the primary amines silver green and adrenaline yellow. Spots were circled by pencil and cut out together with two blanks of similar size taken from each side of the amine spots. The paper spots were cut into small pieces and eluted with 2 ml N/10 HCl for 1 minute, using a whirlimix (effective time previously determined), and the tubes centrifuged at 3000 r.p.m. for 5 minutes. 1 0 ml aliquots of supernatant were then added to counting vials containing 10 ml of a 'cocktail' (7 5 g 2,5-diphenyloxazole (PPO) in 1 litre of xylene plus 500 ml Triton X-100) and whirlimixed. The vials were placed in a Tracerlab Corumatic 200 liquid scintillation counter set for tritium and counted for 5 minutes. The external standard was set for 10000 counts/minute. RESULTS
Autoradiography NA cells may be distinguished by their greater cytoplasmic staining density and cytoplasmic granularity. There is a high density of reduced silver grains over both A and NA cells within 15 minutes of either an intraperitoneal or intravenous injection of 3H-dopa. The density of grains over A cells is usually slightly greater than that over NA cells at 15 minutes (Fig. 1) and this impression is borne out by grain counts on sections
Fixation of catecholamines
407
Fig. 4. Autoradiograph of mouse adrenal 1 hour after injection of 3H-dopa. Similar heavy labelling of A and NA cells. Cortical cells at the left extremity of the photomicrograph are only slightly radioactive. Fixed in glutaraldehyde. x 725. Fig. 5. Autoradiograph 4 hours after injection of 3H-dopa. Similar, but now slightly less heavy, grain concentration over A and NA cells. Fixed in glutaraldehyde. x 725.
(Figs. 2 and 3). Only a low level of radioactivity is observed over cortical cells (Figs. 1 and 4). A similar pattern and level of grain counts is observed in tissues fixed in glutaraldehyde or in dichromate-glutaraldehyde during the first 4 hours after the injection of labelled amine. At 4 hours both A and NA cells are approximately equally labelled (Figs. 2, 3 and 5). After this time, however, the grain counts over A cells fall rapidly in glutaraldehyde-fixed material and much less so in tissues fixed
408
R. E. COUPLAND, S. KOBAYASHI AND JANET CROWE
* .K;.
Fig. 6. Autoradiograph of mouse adrenal gland 24 hours after injection of 3H-dopa. Cortical cells (C) almost unlabelled. Dark staining NA cells heavily labelled, A cells (A) have only low radioactivity. Fixed in glutaraldehyde. x 725.
in dichromate-glutaraldehyde. After 24 hours only a very low level of activity exists over A cells, while NA cells are still moderately heavily labelled (Figs. 2, 3 and 6). This difference persists for up to 5 days by which time the grain counts over A cells in glutaraldehyde-fixed tissue is only slightly greater than that over cortical cells (Figs. 2, 4 and 7), while in dichromate-glutaraldehyde-fixed tissue both A and NA cells are still labelled (Fig. 8), even though counts show a slight fall in radioactivity over A cells relative to NA cells (Fig. 3). During this work it has been noted that some adrenal glands exhibit a rather patchy density of silver grains in an apparently zonal distribution. In these zones both A and NA cells are affected. There may also be a tendency to a gradient of density from the periphery of a group of chromaffin cells, where they abut well defined vascular channels, to the centre where no vascular channel is evident. No similar patchiness or gradient has been observed after the injection of L-tyrosinering 3,5-3H or L-leucine 4,5-3H: these amines also differ from L-dopa in so far as they are not selectively taken up by chromaffin cells (Kobayashi & Coupland, unpublished). Assay The radioactivity of dopa, dopamine, noradrenaline and adrenaline eluted from chromatograms (Fig. 9) was determined. Total radioactivity of the four compounds at intervals after intravenous or intraperitoneal injection of 3H-dopa is indicated in Figure 10, each reading representing the mean of three or four animals. High activity occurs during the first 4 hours and by 24 hours this has fallen to about one third of the 15 minute level. Between days 1 and 4 or 5 a slow rate of decline occurs in the amount of labelled amine. Initially (Fig. 10) the radioactivity is due primarily to
409
Fixation of catecholamines
A~~~~~~~~~~~~~~A
N
*.
.~~~~~rn ~
A
Fig. 7. Autoradiogaph of mouse adrenl gland 5 days aftr injection of 3H-opa. Cortical cell (C) only slightlylabelled, NA cells (dark) heavily labelled islands of A cells (pale)showonlylo radioactivity. Fixed gn glutaraldehyde. x 725.
Fig.78 Autoadiogrph ofmouseadrena medula S ay an I. ~~~~~¼'a*
Fie radioactivity.~ 4
-
n
ngltrleyd.x75
Fig. 8. Autoradiograph of mouse adrenal meulan 5 days after injection of3H-dopa Criand follow ing fixation in 1.5 % potassium dichromate in glutaraldehyde. Both A and NA cells are labelled and A cells (pale) are more heavily labelled than in Fig. 7. x 725.
labelled dopamine. By 4 hours, however, the most abundant labelled catecholamine is noradrenaline. Adrenaline formation is evident by 1 hour and between 4 and 24 hours the normal proportions of labelled, and hence recently synthesized, adrenaline and noradrenaline are present in the glands. These are maintained during the period 1-5 days.
410
R. E. COUPLAND, S. KOBAYASHI AND JANET CROWE
20
OD ~
~
20
( .E
NA
D
1a
A
10
C
u
in
o U
, r
._N
2nd direction -v 22 hours I
ORIGIN
I
20
10 Centimetres
Fig. 9. Two-dimensional chromatogram of adrenal homogenate showing separation of dopa (D), dopamine (DA), noradrenaline (NA) and adrenaline (A). DISCUSSION
L-dopa is selectively taken up by adrenal medullary chromaffin cells as evidenced by the marked difference in radioactivity of the adrenal medulla and cortex (Figs. 2 and 3). The high grain count during the first 4 hours following the injection of 3H-dopa is in keeping with the total radioactivity of labelled amines in the adrenal medulla during this period (Fig. 10). During the first hour after the injection of 3H-dopa the most abundant labelled amine is dopamine (70-80 %) and there are also small amounts (
