Cell Tissue Res (1981) 221:371-383 Cell and Tissue Research �9 Springer-Verlag 1981

On the uptake of exogenous catecholamines by adrenal chromaffin cells and nerve endings

Christine Kent and R.E. Coupland

Department of Human Morphology, University of Nottingham, England

Summary. Light-microscopic autoradiography has revealed characteristic labelling patterns in adrenal medullary cells following the intravenous administration of different catecholamines. The uptake patterns for [3H] dopa, [3H] dopamine, [3H] noradrenaline and [3H] adrenaline have been compared. In all cases A cells were more active than NA cells and cells situated in the zone nearest the cortex demonstrated a markedly higher rate of uptake than central cells. It was concluded that adjacent chromaffin cells with very similar morphology may differ as much as 50 fold in their capacities to incorporate exogenous amines. The adrenergic nature of the innervation of the vessels of the adrenal cortex and capsule in the mouse was confirmed.

Key words: Catecholamines - Chromaffin cells - Nerve endings - Adrenal gland - Mice

When Coupland et al. (1976) described the distribution of radioactivity in the cells of the adrenal medulla after the injection of [3H] dopa they noted that chromaffin cells nearer to the adrenal cortex were always slightly more heavily labelled than more centrally situated cells. Similar observations were made following injections of [3H] dopamine but a much greater difference in uptake levels between the juxta- cortical zone and the central area was demonstrated (Hirano et al. 1977). In this work the wide variation in the capacities of medullary cells to incorporate exogenous dopamine was contrasted with the uniform behaviour of the same cells towards circulating amino acids such as leucine and also with the uniform way in which liver cells handle [3H] dopa. Histochemical (Bander 1950; Er~ink6 1952) and electron microscopic (Coupland and Hopwood 1966) studies established that in the rodent there are two distinct populations ofchromaffin cells in the adrenal medulla; the adrenaline-storing (A) cells constitute about 70 % of the total and the rest are noradrenaline-storing (NA) cells. Later work using immunohistochemistry (Fuxe

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372 C. Kent and R.E. Coupland

et al. 1971), silver methods (Gorgas and B6ck 1976) and autoradiography (Coupland et al. 1976; Kent unpublished) suggests that, within the two broad categories, there exists wide variation between the metabolic capacities of individual cells.

The aim of the present study was to use light microscopic autoradiography to compare the patterns of uptake of different exogenous catecholamines within the adrenal medulla. An at tempt was made to analyse the differences in the response of the two main types of adrenal medullary cells (identified in 1 ~tm plastic sections stained with toluidine blue) and to demonstrate the extent of any variation between individual cells within the two populations.

Observations were made on the uptake of catecholamines that are represented at the various stages in the synthesis of adrenaline from dopa i.e. dopa, dopamine, noradrenaline and adrenaline. The tritiated compounds were administered intravenously, a more reproducible method than intraperitoneal injection and one which more readily approaches a pulse label. Intravenous injection also mitigates the problem of the possibility of metabolism of the tritiated compound by the liver, which can be an important factor in the interpretation of results following intraperitoneal injections (Kobayashi et al 1979). Another difference between this and previous work reported by Kobayashi and coworkers is that the tissues were fixed in a fixative containing potassium dichromate, a procedure which has been shown to retain more adrenaline in the tissue than is the case when using glutaraldehyde alone (Coupland and Kent 1977).

Materials and methods

Isotopically labelled compounds were injected intravenously, via a tail vein, into male albino mice, strain CSI, body weight 23 to 28 g.

Labelled catecholamines

L-3,4-Dihydroxy (ring-2,5,6-3H) phenylalanine, specific activity 44Ci/mmol. ([3H] dopa), 3,4-dihy- droxyphenyl (1,2-3H) ethylamine hydrochloride, specific activity 10Ci/mmol. ([3H] dopamine), L(7,8-3H) noradrenaline, specific activity 24Ci/mmol. ([3H] noradrenaline) and DL-(7-3H) adrenaline hydrochloride ([3H] adrenaline) were obtained from the Radiochemical Centre, Amersham, Bucks. Table 1 gives details of how the animals were grouped and the ways in which the radioactive compounds were administered.

Preparation of autoradiographs

Tissues were fixed by cardiac perfusion with a mixture of 1.5 % potassium dichromate and 3 % glutaraldehyde in 0.05 M phosphate buffer, adjusted to pH 7.0 with N sodium hydroxide. Thirty minutes

Table 1. Summary of experimental regime

Number of Dose Labelled Time after animals ktCi/gm b.w. catecholamine injection

Group 1 6 10 [3H]dopa 1 h Group 2 5 5 [3H]dopamine 1 h Group 3 6 5 [3H]noradrenaline 1 h Group 4 4 5 [3H]adrenaline 1 h

Catecholamine uptake by chromaffin cells and nerves 373

later glands were removed and sliced at c 1 mm thickness with a razor blade and then immersed in a fixative. After 3 h immersion in dichromate fixative the slices of adrenal gland were transferred to 3 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.3 for 2 h. Tissues were then post-osmicated, dehydrated through a graded ethanol series, embedded in Araldite and sectioned at 1 Inn on a Cambridge-Huxley microtome. Representative sections from the whole of the medulla from each adrenal were mounted on gelatinized slides: whenever possible, sections for comparison were mounted on the same glass slide.

Slides were layered with Ilford K 2 nuclear emulsion, exposed for 3 weeks, developed in D 19 and fixed with Hypam. Alternate sets of sections were stained with 0.5 % toluidine blue in 0.05 M phosphate buffer, the equivalent set being left unstained for photometric analysis.

Analysis of autoradiographs

Grain densities were measured by reflectance photometry using a Leitz MPV 2 microphotometer. All the reflectance measurements to be reported were made with a measuring diaphram 36 ~trn in diameter; this circular field delineated approximately 4 or 5 chromaffin cells. The photometer readings have been recorded in arbitrary units which are proportional to the silver grain concentration over the range used. The medulla was divided into two measuring zones; an area of c 3 cells (36 ~tm) in width, immediately adjacent to the cortex, was designated the juxta-cortical or marginal zone and the rest of the medulla was regarded as the central zone. The measuring diaphragm was moved systematically across each zone in turn and a measurement made over every group of cells encountered. Data from all the animals receiving a particular labelled catecholamine were pooled and mean photometer readings (i.e. grain concentrations) for A cells and for NA cells, in each of the zones, were computed. Corrections for background were made for each section using readings taken over areas of Araldite which contained no tissue.

Results

Zonal distribution of catecholamine uptake

A f t e r 1 h d e t e c t a b l e a m o u n t s o f all f o u r c a t e c h o l a m i n e s h a d b e e n i n c o r p o r a t e d i n t o

all t h e m e d u l l a r y cells. T a b l e 2 gives a s u m m a r y o f t h e m e a n s i lver g r a i n

c o n c e n t r a t i o n s o v e r d i f f e r e n t a r e a s o f t h e m e d u l l a a f t e r i n j e c t i o n o f t he v a r i o u s

t r i t i a t e d a m i n e s . U p t a k e p a t t e r n s we re c h a r a c t e r i z e d b y a g r a d i e n t o f g r a i n

c o n c e n t r a t i o n f r o m t he j u x t a c o r t i c a l r e g i o n i n w a r d s : dens i t i e s o v e r m e d u l l a r y cells

i m m e d i a t e l y n e x t to t h e c o r t e x ( m a r g i n a l z o n e ) w e r e a l w a y s h i g h e r t h a n t h o s e o v e r

s i m i l a r cells s i t u a t e d n e a r e r t he c e n t r e o f t he g l a n d ( c e n t r a l zone) . F o r all t h e

c a t e c h o l a m i n e s th i s p h e n o m e n o n w a s m o r e m a r k e d in t he a d r e n a l i n e - s t o r i n g cells

Table 2. Comparison of silver grain distribution over adrenal medullary cells one h after administration of labelled catecholamines

A Cells NA Cells

Marginal Central Marginal: Marginal Central Marginal: zone zone Central zone zone Central

[3H]dopa (6) 109.0+ 3.1 92.1+3.1 1.2 64.0+3.6 58.1+2.6 1.1 [3H]dopamine (5) 415.3+ 11.4 205.2+ 5.7 2.0 79.0+ 6.8 55.3+ 2.9 1.4 [3H]noradrenaline (6) 294.1+ 14.0 100.3+ 6.0 2.9 76.9+ 4.1 52.6+ 2.7 1.5 [3H]adrenaline (4) 48.7+ 3.4 25.1+ 1.2 1.9 26.5+ 1.7 19.8+ 1.4 1.3

Values are functions of silver grain concentrations over different areas of the adrenal medulla, and are expressed as means + S.E.M. (N approx. 250). Figures in brackets represent numbers of animals in each experimental group

374 C. Kent and R.E. Coupland

4 ~

300

A = A cells

NA = NA cells

m - marginal zone

c = centra l zone

mc mc mc mc mc mc mc mc

A NA A NA A NA A NA

Nooo. ooo.o,n. Fig. 1. The distribution of silver grains over adrenal medullary cells of normal animals following different labelled catecholamines. Figures represent photometer readings and are proportional to the number of silver grains per unit area: the main reading for all chromaffin cells in a particular category is presented (see Table 1). For each catecholamine the amounts of radioactivity detected in A and NA cells in the marginal zone of the medulla are compared with those in more centrally placed A and NA cells. Vertical lines represent + two SEM

than it was in the noradrenaline-storing cells (Fig. 1). The steepest gradients were found in A cells after [3H] noradrenaline, when the mean grain concentra t ion in the marginal zone was almost three times that o f the central zone. Marginal A cells incorporated about twice as much [3H] dopamine (Figs. 3, 4) and [3H] adrenaline as did more centrally placed cells. Fol lowing [3H] dopa a less steep gradient was a consistent finding, mean grain concentra t ion over marginal cells being only 1.2 times that over central cells.

Differences & uptake between A and NA cells

There were clear differences in the extent to which the A and N A cells accumulated the exogenously presented catecholamines (Figs. 3-6). All four amines gave rise to grain concentrat ions which were higher over A cells than over N A cells, especially in the marginal zone, but the ratio o f A cell uptake to NA cell uptake was different for each labelled c o m p o u n d (Table 3). The highest value for A cell to NA cell ratio (5.3)

Catecholamine uptake by chromaffin cells and nerves

Table3. Comparison of A cell uptake with NA cell uptake

Grain density ratio A:NA cells

Marginal zone Central zone

[3H]dopa 1.7 1.6 [3H]dopamine 5.3 3.7 [3H]noradrenaline 3.8 1.9 [3H]adrenaline 1.8 1.3

Values represent mean grain concentration over A cells divided by mean grain concentration over NA cells in same zone

375

was found following [3H] dopamine. A cells were 4 times more active than NA cells in taking up [all] noradrenaline and A cells accumulated [3H] dopa and [3H]adrenaline at about twice the rate of NA cells.

Range of grain densities

A consistent feature of the uptake pattern of all the catecholamines was the variation between grain concentration measurements over one type of cell within the same zone and on the same section. The degree of variation differed according to the labelled compound which had been given and, in order to illustrate and compare the distribution patterns of radioactivity, frequency distribution histograms of all the individual photometer readings were prepared. Readings were first expressed as a percentage of the mean so that different levels of uptake could be compared more easily (Fig. 2). It can be seen from Fig. 2 that the range and variability of uptake levels in cell groups were greatest in A cells following [3H] noradrenaline. The level of [3H] dopa uptake by A cells showed least variation and it was in respect of this amine that the chromaffin cells showed an uptake pattern that most nearly approached a normal distribution. The patterns for tritiated dopamine and adrenaline uptake occupied an intermediate position with [3H] adrenaline being more like that of [3H] noradrenaline.

NA cells also showed a wide variation in the amounts of isotope they incorporated during the hour following administration. The range was similar for tritiated adrenaline, noradrenaline and dopamine and, in all three cases, the readings deviated from a normal distribution, suggesting the presence of more than one population of cells.

The measurements referred to in Fig. 2 were made over 4 or 5 chromaffin cells i.e. they were the mean grain concentration of a group of cells. If single cells had been considered the range of variation would have been still greater. A frequent observation in glands labelled with [3H] noradrenaline and [3H] dopamine was the presence of cells, occurring individually or in groups of 2 or 3, with strikingly high levels of activitity compared with the surrounding cells: the grain count over these cells was often 50times that over adjacent cells which were morphologically indistinguishable (Fig. 7). The inclusion of such cells in the measuring field accounts for the occasional grain concentration of three times the mean seen in Fig. 2. These very "hot" cells occurred most frequently amongst the cells immediately next to the

376 C. Kent and R.E. Coupland

60

50 ~ 4o ~ 3O

10

H Adrenaline

o , . - ~ ~ u - r l 3H Noradrenaline

60

4O

3O

20

10

o , I h . . . . . .

3H Doparnine

50

4O

311

20

10

0 r , , , o r a l , �9

50 I60 3H Dopa ~' 4o ~ 3o o

10

0 , , ,= I I

M(IO(~)

, l l l l l , , , l l l l l l l l l l l l l l l l l l , ,

% 0 20 40 60 80 100 140 180 200 240 280 %

Fig. 2. Frequency distribution of silver grain densities over A and NA cells in the marginal zone of the medulla. The mean (M) photometer reading for each medulla was taken as 100 % and the frequencies of readings (expressed as a percentage) at either side of the mean were plotted at 10 ~o intervals. Clear bars represent A cells and black bars represent NA ceils

cortex but could occasionally be seen deeper in the gland. Most could be identified as A cells but many NA cell nests also contained at least one very heavily labelled cell; the latter could well have been S.G.C. cells which can only be confidently identified at the electron microscopic level (Kobayashi and Coupland 1977). The overall level of labelling was much lower with [3H] adrenaline but isolated A and NA cells with grain counts several times greater than the surrounding cells were seen in similar locations. [3H] dopa labelling revealed none of these "hot" cells.

Labelling of adrenal vessels

[3H] noradrenaline, [3H] dopamine and [3H] adrenaline consistently labelled structures lying adjacent to the majority of capsular vessels (Fig. 8, 10). A similar uptake was also seen, in most sections, deep in the cortex and in the juxtamedullary zone associated with blood vessels including medullary arteries in this situation

Fig.3. Autoradiograph showing distribution of radioactivity in mouse adrenal medulla 1 h after injection of [3HI dopamine. C cortex. Darker medullary cells are NA-storing (N). x 500

Fig. 4. Heavier labelling of chromaffin cells adjacent to cortex (C) 1 h after injection of [3H] dopamine. x 1,260

Fig. 5. Autoradiograph showing distribution of radioactivity in mouse adrenal medulla 1 h after injection of [3HI noradrenaline. Note heavier labelling of juxta-cortical (marginal zone) cells. NA- storing cells have dark staining cytoplasm. NA cells (N), cortex (C). x 500

Fig. 6. Heavier labelling ofchromaffin cells adjacent to cortex (C) with [3H] noradrenaline affects both A cells (A) and NA cells (N). • 1,260

Fig. 7. Autoradiograph showing marked variation in intensity of labelling of A and NA cells 1 h after injection of [3HI noradrenaline, x 1,260

Fig.& Perivascular labelling in subcapsular zone due to uptake of [3H] noradrenaline. Heavy concentration of silver grains overlies perivascular nerve fibres. Capsule (K). • 1,260

Fig. 9. Labelling of perivascular nerve fibres associated with a medullary artery in the inner part of the cortex (labelled medullary cells can be seen to the lower left) 1 h after injection of [3H] dopamine, x 1,260

Fig. 10. Labelling of perivascular nerve fibres in the subcapsular zone of mouse adrenal by [3HI adrenaline. Capsule (K). • 1,260

Catecholamine uptake by chromaffin cells and nerves 379

(Fig. 9). Some of the vessels associated with the label had very thin walls; many were without smooth muscle and had the appearance of capillaries with pericytes. Silver grains were arranged in dense clusters at intervals around the vessels, consistent with uptake by nerve endings, a suggestion confirmed by other studies including catecholamine fluorescent microscopy and electron microscopy (Coupland unpublished). The concentration of silver grains associated with nerve endings after [3H] adrenaline labelling was only slightly below that after [3H] noradrenaline and was always greater than grain concentrations measured over medullary chromaffin cells.

In several instances in which the grain density over the medulla was unusually low after injection of [3H] adrenaline, but in which the cortical and capsular nerve endings had accumulated a high concentration of labelled adrenaline, it was possible to determine that the medullary vessels including medullary arteries were not associated with labelled nerve fibres.

Uptake of [all] dopa by nerve endings was not detected in any of the glands processed.

Discussion

An active transport system which accumulates exogenous catecholamines against a considerable concentration gradient is known to exist in the postganglionic neurons of the sympathetic nervous system. Evidence from studies on a variety of preparations rich in sympathetic endings suggests that the uptake occurs where there are already stores of endogenous amines (Iversen 1970; Budd and Salpeter 1969). Amines cross the cell membrane and then enter, and are bound by, the amine-storing granules found in nerve terminals and chromaffin cells (Stj~irne 1964; Potter 1967).

Although it is possible, indeed likely that some of the radioactivity in the A and NA cells revealed by autoradiography is due to the presence of catecholamine metabolites, the proportion of metabolites bound by glutaraldehyde fixation is low (Descarries and Dupin 1974) and in addition, from the work ofAxelrod et al. (1959) and Whitby et al. (1961), only a relatively small proportion of labelled adrenaline and noradrenaline would be converted to the metanephrine metabolite in the adrenal gland following the intravenous injection during the time period of the present experiment. Hence it may safely be assumed that the observed radioactivity represents adrenaline and noradrenaline fixed in situ and from previous work (Coupland and Kobayashi 1976; Coupland et al. 1976) in chromaffin granules in association with endogenous amines.

The experiments were designed primarily for relative measurements within the adrenal medulla but, since the labelled catecholamines had similar specific activities, and they were given in the same radioactive dose and all samples were autoradiographed simultaneously, comparisons can be made between uptake levels of different amines. Dopamine and noradrenaline were accumulated in the highest concentrations, especially by A cells. A cells contain chromaffin granules which are currently thought to be almost exclusively adrenaline-storing, but the current results indicate that there must also exist binding sites which are available for the trapping of exogenous dopamine and noradrenaline and in consequence it is likely

380 C. Kent and R.E. Coupland

that, if available, endogenous amines would be similarly bound. The catecholamine uptake system in adrenergic neurons is also capable of transporting a variety of structurally related amines (Iversen 1970) and noradrenaline-storing areas of rat brain have been shown to accumulate high concentrations of dopamine whilst conversely, dopamine-containing neurons take up noradrenaline (Glowinski and Iversen 1966).

The present work shows that adrenaline is also incorporated into adrenal medullary cells but to a much lesser extent than dopamine and noradrenaline. The lower levels of adrenaline uptake may be due to the active transport system across the cell membrane having a lower affinity for adrenaline, as is known to be the case in sympathetic axons (Iversen 1963) or to greater loss during fixation. However, loss during fixation is unlikely to account for a six fold difference between adrenaline and noradrenaline uptake by chromaffin cells because fixatives containing dichromate are known to reduce adrenaline loss (Coupland and Kent 1977) and the fact that the concentration of radioactivity in perivascular nerve endings following [3H] adrenaline administration was only slightly below that after [3H] noradrenaline indicates significant fixation of adrenaline.

Although medullary A cells are known to store adrenaline, their capacity to accumulate and bind exogenous adrenaline appears to be very low compared with their ability to concentrate other amines. The characteristics of [3H] dopa uptake by adrenal medullary cells have been reported and discussed elsewhere (Elfvin et al. 1966; Coupland et al. 1976) but it is interesting to note here that, although the specific activity of [all] dopa was comparable and the dose twice that of the other catecholamines, the amounts of radioactivity accumulated were relatively low. From previous studies (Coupland and Kobayashi 1976; Coupland et al. 1976). 1 h after intravenous injection of [3H] dopa the labelled amine would be mainly [3H] dopamine - a catecholamine difficult to fix in situ even in the presence of dichromate (Coupland and Kent 1977), as is dopa, and this may account for the lower radioactivity. However, Glowinsky and Iversen (1966), using assay methods, reported a similar finding in various areas of rat brain and hence it is possible that the observation reflects a less active uptake mechanism for dopa than for noradrenaline and that its movement into the cell is closely dependent upon the rate of entry of [3H] dopa into the metabolic sequence dopa ~ dopamine noradrenaline --* adrenaline. It should be noted, however, that from previous work (Coupland and Kobayashi 1976) it is apparent that dopa is selectively taken up by the chromaffin cells in preference to the natural precursor of catecholamine synthesis tyrosine.

Zonation of radioactivity after [3H] dopa was slight and may either be due to the uptake of the dopamine synthesized in the 1 h period (Hirano et al. 1977) or from more rapid in situ synthesis of catecholamines in the marginal zone, as well as slight selective uptake. There was also far less variation in labelling of individual cells after [3H] dopa and the variation which did exist followed a normal distribution, suggesting that the chromaffin cells form a single population in respect of dopa uptake and metabolism.

The zonation in [3H] dopamine uptake has been previously reported by Hirano et al. (1977) and Hirano and Kobayashi (1978) have shown that the effect is dependent upon pituitary ACTH. It is not known whether the uptake of adrenaline

Catecholamine uptake by chromaffin cells and nerves 381

and noradrenaline are affected by blood levels ofACTH. Other factors which might affect uptake will be considered in a following paper.

Irrespective of the nature of the catecholamines injected the mean uptake levels for NA cells were always considerably lower than that for A cells. If it is accepted that there is normally a correlation between the presence of endogenous amines and the ability to accumulate exogenous amine, the variation in uptake is an unexpected finding. Either the capacity of the storage granules to bind amine is much lower in NA cells or the mechanism for transporting amines into the cell is much less effective. Soluble compound autoradiography might be helpful in elucidating this problem. However, some individual NA cells did incorporate very high levels of radioactivity and this variability may reflect the presence of different types of NA cell, as demonstrated by Gorgas and B6ck (1976). Individual A cells were also variable in their ability to accumulate amines, especially noradrenaline. It is difficult to relate this finding to morphological characteristics as A cells appear to be more homogenous than NA cells. There are "light" and "dark" A cells and other intermediate types can be distinguished in sections fixed with glyoxylic acid- containing fixatives (Kent unpublished) but their distribution does not correlate with the patterns of amine uptake. Ultra-structural studies do not show any changes from cell to cell which could account for the very large differences in uptake. It would appear, therefore, from this work and from the results of electronmicroscopic autoradiography (Budd and Salpeter 1969), that the ability to take up catecholamines may not necessarily parallel the amine stores as they are visualized by electron microscopy i.e. the presence of dense cores of the storage- granules in chromaffin cells and nerve endings.

Comparing the uptake patterns for [3H] dopamine and [3H] noradrenaline, some differences emerge; the zonal gradient is steeper for [3H] noradrenaline and the ratio of A to NA cell uptake is greater for [3H] dopamine than for [3H] noradrenaline. These observations alone are not sufficient to postulate different uptake mechanisms but they may indicate that the kinetics for the two amines are different. The details of the kinetics for dopamine and noradrenaline uptake into brain slices and synaptosomes have also been shown to be different (Snyder and Coyle 1968; Coyle and Snyder 1969).

In view of the findings that both A and NA cells have the ability to take up the full spectrum of adrenal catecholamines it seems likely that in vivo although chromaffin cells undoubtedly store mainly adrenaline or noradrenaline both types probably take up and store fractional amounts of amines released by adjacent cells so that trace quantitites of adrenaline will be present in predominantly NA cells and vice versa.

In contrast to chromaffin cells, which had a low affinity for exogenous adrenaline, nerve endings associated with cortical vessels demonstrated a marked capacity to accumulate adrenaline. The physiological significance of this observation is currently unknown but the phenomenon may be important in generalized sympathoadrenal activity. The uptake of noradrenaline and dopamine confirmed the partially adrenergic nature of cortical innerveration in the mouse. Densely labelled structures seen in the cortex which in some cases appeared not to be associated with vessels and in others were close to capillaries are probably the equivalent of the adrenergic endings described by Unsicker (1973) in birds and

382 C. Kent and R.E. Coupland

similar adrenergic endings have been observed in the mouse, rat and cat adrenal cortex (Coupland unpublished). Currently there is some doubt about whether these endings are functionally related only to blood vessels or to both cortical cells and blood vessels.

It is also interesting to note that we have obtained no evidence of adrenergic nerve endings in the vicinity of blood vessels of the adrenal medulla; this suggests a different mechanism of control of cortical and medullary vessels.

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Catecholamine uptake by chromaffin cells and nerves 383

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Accepted August 31, 1981