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The postnatal development of noradrenaline uptake in the adrenergic nerves of different tissues from the rat
EUROPEAN JOURNAL OF PHARMACOLOGY 9 (1970) 67-79. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM
THE POSTNATAL DEVELOPMENT ADRENERGIC
OF NORADRENALINE
NERVES OF DIFFERENT
U P T A K E IN T H E
TISSUES FROM THE RAT
Ch, SACHS, J. de CHAMPLAIN*, T. MALMFORS and L. OLSON Department of Histology, Karolinska lnstitutet, Stockholm 60, Sweden
Received 11 July 1969
Accepted 19 September 1969
Ch. SACHS, J. de CHAMPLAIN, T. MALMFORS and L. OLSON, The postnatal development o f noradrenaline uptake in the adrenergic nerves of different tissues of the rat, European J. Pharmacol. 9 (1970) 67-79. The uptake and accumulation of 3H-NA** was studied in vitro in iris, atrium, submaxillary and sublingual glands, and the muscular and mucosal layers of the duodenum from rats at different ages (0-60 days). Most of the 3H-NA found in the tissues was located in the adrenergic nerves. The uptake of 3H-NA increased progressively with age in all the organs examined, when calculated per tissue piece. When the radioactivity was expressed per gram tissue, the concentration in the iris and atrium increased progressively as the animal grew older, but the concentration in the salivary glands and in the gut decreased. The variation in the uptake of 3H-NA per gram wet weight is probably due to differences in growth rates between neuronal and extraneuronal components of the different organs, rather than to differences in the uptake ability of the adrenergic nerves, during the time of development. Developing rat Noradrenaline
Adrenergic nerves NA uptake
1. INTRODUCTION Various tissues of the rat develop the ability t o take up and accumulate 3H-noradrenaline (3H-NA) around and shortly after birth (Glowinski et al., 1964; lversen et al., 1967), at the time when outgrowth of sympathetic adrenergic nerves takes place (Olson et al., 1970). Uptake and accumulation of NA in adult tissues are mainly located to the postganglionic sympathetic nerves (Whitby et al., 1961), since the accumulation of 3 H-NA is markedly reduced after postganglionic sympathetic denervation (Hertting et al., 1961; Jonsson et al. 1969) and in immuno* Dr. de Champlain is a Markle Scholar in Academic Medicine. Address: Dept. of Physiology, Faculty of Medicine, University of Montreal, Montreal, Canada. ** Abbreviations used: NA, noradrenaline; NM, normetanephrine; MAO, monoamine oxidase; 6-OH-DA, 6-hydroxydopamine; DMI, desmethylimipramine; dpm, disintegrations per minute.
6-OH-DA Desmethylimipramine
sympathectomized animals (Sji3qvist et al., 1965, 1967; Iversen et al., 1966). The intraneuronal uptake of NA has also been directly demonstrated at the cellular level with the histochemical fluorescence method of Falck and Hillarp (see Hamberger et al., 1964; Malmfors, 1965). The ability of tissues to take up and accumulate 3H-NA may indicate the density of the adrenergic nerves present in the tissue, provided that the uptake per "nerve unit" is constant. However, differences might exist between various organs, owing to different diffusion or circulatory conditions which can affect the uptake and retention of NA. The uptakestorage properties of the adrenergic nerves in different organs might also differ and, furthermore, nerves in the same organ might differ during various developmental stages. In an in vivo study, Iversen et al. (1967) found that in heart and spleen, the ability to accumulate 3 H-NA per weight unit developed parallel to the endogenous NA concentration, while the abil-
68
Ch.SACHS et al.
ity of the intestine and salivary glands to accumulate SH-NA was already fully developed at birth, although the NA content at this time was lower than in the adult organs. The aim of the present study was to investigate the in vitro uptake and accumulation of NA in developing tissues of the rat. The effect of certain drugs, such as reserpine, nialamide, DMI and 6-OH-DA, on this uptake were investigated, in order to obtain information on the specific neuronal uptake in relation to the outgrowth of adrenergic nerves, as demonstrated by the histochemical fluorescence method.
imately 7% less m a gland in which the lobes had been carefully loosened from each other, than in a minced gland. This difference was regarded as of minor importance for the study, and whole glands with loosened lobes were therefore used for the experiments. The wet weight of irides from very young rats was estimated indirectly by measuring the protein content (Lowry et al., 1951). The protein content of irides from 21 day old rats was 115 +- 1.9 ~g/iris, and the wet weight 1.30 -+ 0.06 mg/iris. The protein content multiplied by ten would give an approximate estimation of the mg wet weight. The wet weight of the other organs was measured directly, after incubation.
2. MATERIAL AND METHODS Rats (Sprague-Dawley) of different ages were used for the experiments. The mean body weights were: at 0 days 5 g; 4 days 12 g;8 days 17 g; 12 days 30 g; 21 days 50 g; and at around 60 days 200 g. Injections of drugs were made i.v. into a lingual vein (0.05 0.2 ml) or i.p. (0.2 1.0 ml). The animals were sacrificed by decapitation under light ether anaesthesia. The following organs were carefully dissected out under a preparation microscope: iris, atrium, submaxillary gland, sublingual gland, the external muscular coat, and the mucosal layer of the duodenum (from the pylorus to the entrance of the ductus choledocus, for details see Olson et al., 1970). Analytical determinations were made on tissues from one animal and the results were expressed per one single piece of organ, or per g wet weight. 2.1. in vitro incubations After dissection, the organs were immediately immersed in cool modified Krebs-Ringer bicarbonate buffer pH 7.4 (see Hamberger, 1967) containing 0.2 mg/ml ascorbic acid (Merck p.a.). Incubations were performed in 1.0 ml buffer containing SH-NA (10 -6 or 10-7 M) at 37°C under an atmosphere of 95% O2 - 5% CO2, using a metabolic shaker. The tissues were incubated for 30 rain and thereafter rinsed for 10min at 37°C in isotope-free buffer, unless otherwise stated (Jonsson et al., 1969). Since the submaxillary gland of 21 day old rats was the biggest organ used, some methodological studies were made to evaluate diffusion into this tissue. It was found that the uptake of radioactivity was approx-
2.2.3H-determinations The total 3H-activity taken up and accumulated in the various tissues was determined by combustion of the dried organ in an oxygen atmosphere to form CO2 and water, the ~H-water formed then being determined (Gupta, 1966: Maurer, 1968). The submaxillary glands from 12 and 21 day old rats were too large to combust and were homogenized in 1.5 ml n-butanol-0,1% HCI in a glass homogenizer. One ml of the extract was taken for counting in 5.0 ml tolueneethanol (7:3) solution. Radioactivity was determined in a Packard Model 3002 Tricarb Liquid Scintillation Spectrometer. Efficiency was determined by adding a standard amount of 3H-toluene to representative samples, and recounting the vials. Results are expressed as dpm. 3H-NA and its 3H-O-methylated and a H-deaminated metabolites were determined in tissues incubated in 10 .6 M 3 H-NA for 30 min at 37°C and reincubated in isotope-free buffer for 10 rain. The tissues were extracted with cool 0.4 N perchloric acid. The extracts were chromatographed on ion-exchange columns (Dowex 50W-X4, diameter 4.0ram, height 120 mm, at pH l) (Bertler et al., 1958). The amines were adsorbed on the resin, eluted with 1 N HC1, and measured ad rnodurn (Carlsson and Waldeck, 1063). The acid and neutral catabolites passed through in the effluent, and were determined by shaking the effluent, after acidification (pH 2), with 300 ml of ether (Rutledge and Jonasson, 1967). The volume was reduced under vacuum, and the radioactivity taken up in 1 ml ethanol/0.01% HC1 and 5 ml toluene scintillator, from which a 5 ml aliquot was taken for counting.
UPTAKE OF 3H-NORADRENALINE IN DEVELOPING ADRENERGIC NERVES
2.3. Fluorescence histochemistry
69
%
Irides, atria and duodenal tissues were prepared as whole mounts, dried i n a desiccator over phosphorous pentoxide, and thereafter exposed to formaldehyde gas of o p t i m u m humidity (Hamberger, 1967) for the histochemical demonstration o f biogenic monoamines according to the fluorescence method of Falck and Hillarp. Salivary glands were rapidly frozen in propane, cooled by liquid uitrogen, freeze-dried in a newly developed freeze-drier (Olson and Ungerstedt, 1970), treated with gaseous formaldehyde, embedded in paraffin, sectioned ( 8 / l ) and m o u n t e d in Entellan (Merck) for fluorescence microscopy (Falck et al., 1962; Falck, 1962; Norberg and Hamberger, 1964; Falck and Owman, 1965; Corrodi and Jonsson, 1967). The following substances were used: dl-7-3H-nor adrenaline-HC1, 9.0 C./mmole (New England Nuclear Corp.), dl-noradrenaline-HCl (Calbiochem), nialamide (Niamid ®, Swedish Pfizer), desmethylimipramine (Pertofran ®, Geigy), Reserpine (Serpasil ®, Ciba) and 6-hydroxydopamine-HC1 (AB H~issle, Gothenburg).
I00
i ~ AFTER PREINCUBATION IN D.M.L
u)
-~ BoJ F-
P, bJ 60 E
zE 4 0 " o~
0
I
Z 3
4
s 60
o
IRIS
t
2
3
4
8 6o AGE (DAYS)
ATRIUM
Fig. 1. Uptake of 3H-NA in iris and atrium of rat during growth. The tissues were incubated in 10-TM 3H-NA for 30 min at 37°C and rinsed for 10 rain in isotope-firee medium. Each value is the mean _+S.E.M. (2-8 observations), expressed as percentages of values from 60 day old rats (dpm/piece of tissue). The percentage of 3H-radioactivity taken up after preincubation in DMI (10 -5 M, 15 rain) is marked it, black.
3. RESULTS
the uptake of radioactivity in both tissues with increasing age of the animal. In all tissues investigated, the ability to take up and accumulate NA developed similarly between 0 and 21 days (fig. 2, table 1). In newborn rats, however, the salivary glands and the muscular coat of the intestine showed a relatively higher uptake of 3 H-NA than the iris and atrium. Irides and atria increased their weight 2 . 5 - 4 times
3.1. Uptake and accumulation of 3H-NA Irides and atria from rats of different ages ( 0 - 6 0 days) were incubated in vitro in a medium containing 3H-NA (10 -7 M). The total amount of radioactivity found per iris or atrium was calculated as a percentage o f the amount found in 60 day old animals (fig. 1). There was a progressive increase in w_ ~ 1 2 0%.
U--t00. ~qO-
1
Ix 40"
/c. O. £3 0
4
12 2~
IRIS
0
4
12 2 I
ATRIUM
~,
12 2'
SU6MAX GLAND
O
4
12 21
SUSLING GLAND
~
4
12 9-;
MUSCULAR COAT
•
4 ,2 P, MUCOSAL
AGE (DAYS)
LAYER
Fig. 2. Uptake of 3H-NA in various organs from the growing rat. The tissues were incubated in 10-7 M 3H-NA for 30 min at 37°C and rinsed for 10 rain in isotope free buffer. Each value is the mean + S.E.M. (7-10 observations), expressed as percentages of 21 day old values (dpm/piece of tissue).
70
Ch. SACHS et al.
Table 1 Uptake of 3H-NA in various tissues of the growing r a t Tissues were incubated in 10-vM 3tl-NA for 30 rain and rinsed in isotope-free buffer for 10 min. Values expressed as dpm × 103 per piece of tissue. Organ
0
4
Iris
1.9 ± 0 , 1 8
5.0 z 0.53
17.0 ± 0.98
22.0 ± 2.49
Atrium
2.7 _+ 0,20
6.9 + 0.49
24.7 ± 1.59
49.3 +_ 4.62
46.2 _+ 5.65
68.6 ± 3.52
108.1 ± 12.22
142.7 _+ 22.54
6.0 _+0.95
12.1 _+ 1.5 l
22.8 ± 2.37
37.3 + 8.38
Duodenal muscular coat
11.8 _+ 2.23
13.9 _+ 1.19
28.3 _+2.85
61.7 _+ 10.93
Duodenal mucosal layer
7.3 ± 1.71
29.2 ± 2 85
45.4±7.13
90.2_+9.98
Submaxillary gland Sublingual gland
Each value represents the mean +_S.E.M. of 3
12
21 days
10 observations.
Table 2 Wet Weight in mg of various tissues of the growing rat Organ
0
4
12
Iris
0.25 ± 0.01
0.35 _+ 0.02
0.58 +_ 0.03
0.58 _+ 0.03
1.48 ± 0.04
Atrium
1.83 ± 0.05
2.95 + 0.00
4.92 + 0.27
6.56 _+ 0.18
21.20 ± 1.5(I
Submaxillary gland
7.85 ± 0.25
15.39 ± 0.20
40.72 _+ 0.80
75.77 +_ 2.27
198.30 ± 3.10
Sublingual gland
1.90 _+ 0.09
4.13 _+ 0.09
10.13 _+ 0.32
19.48 _+ 0.85
38.00 _t 1.60
Duodenal muscular coat
1.11 ± 0.09
3.23 +_ 0.10
5.75 _+ 0.8(I
15.53 ± 0.80
31.1 I ± 1.62
Duodenal mucosal layer
5.76 ± 0.27
13.47 + 0.36
29.09 +_ 1.58
96.81 ± 4.!18
167.40 ± 3.35
Each value represents the mean +~S.E.M. from 9
21
18 observations.
1oo. if)
80"'--"
60/
"--"
IRIS ATRIUM SUBMAX. GLAND
- - -
SUBLING. GLAND
~
MUSCULAR LAYER MUCOSAL LAYER
~ • 40-
/
ao-
,J
¢1
/
o
4
,?
2',
DAYS
Fig. 3. Increase of tissue weight of various organs, expressed as percentages of 21 day old values (cf. table 2) in the growing rat.
60 days
UPTAKE OF 3H-NORADRENALINE IN DEVELOPING ADRENERGIC NERVES
.20-
n'
METABOLITES
[]
3H-NA
FROM
71
SH-NA
UPTAKE OF ~bH-RAD(OACTIVITY AFTER PREINCUBATION IN DM.I.
% w
~:15. 0
I.-
10n~ L~
n,-
=E 5 ¸
Q£
nl nrill nNnnnn 0412
IRIS
0
4
f2 21
ATRfUM
4
12 21
SUBMAX. GLAND
0
4
12 21
SUBLING. GLAND
4
12 21
MUSCULAR COAT
0
4 12 21
AGE(DAYS)
MUCOSAL LAYER
Fig. 4. Uptake of 3H-NA in various tissues of the growing rat. The tissues were incubated in 10-7 M 3H-NA for 30 min at 37°C and rinsed in isotope-free buffer for 10 rain. Each value is the mean _+ S.E.M. (8-15 observations), expressed as dpm/g wet weight. The uptake of 3H-radioactivity after preincubation in DMI (10 -5 M, 15 rain) is shown in the dotted part of the columns. the amount of metabolites in the striped. between 0 and 21 days, and had a relatively higher weight at 0 days than the other organs, whereas the weight increase for salivary glands and duodenal tissues during the same period was in the order of ! 0 - 2 0 times (table 2 and fig. 3). There was a marked difference in uptake between the different organs when the accumulation of radioactivity was calculated per g wet weight (fig. 4). The highest uptake was then observed in iris. The concentration of radioactivity increased with age in atria and irides, whereas it decreased in the salivary glands. Of duodenal tissues, the muscular coat showed a decreased 3H-NA uptake with age, while the mucosal layer displayed no clear-cut tendency. When the total radioactivity was corrected for metabolites formed from 3H-NA (fig. 4, table 3) during the incubation period, the pattern of 3H-NA taken up was found to be the same as that for total radioactivity. Corrections were based on values obtained from incubations at 10 -6 M 3H.NA, however, which had to be used in order to obtain sufficient radioactivity for the specific chemical analysis. (The relative amount of 3H-metabolites formed is only slightly increased with higher NA concentrations in the incubation medium for adult atrial tissue (Sachs,
1969, 1970). The proportion o f total radioactivity which represented 3H-NA was high and fairly constant for iris during the age period studied, while an increase in the proportion of 3H-NA was noted in atria and duodenal tissues. A progressive relative decrease of 3 H-NA with age was noted in the salivary glands.
3.2. Extraneuronal uptake o f 3H-NA After preincubation for 15 min in DMI (10 -s M), the accumulation of radioactivity per total organ (fig. 1) or per g wet weight (fig. 4) was considerably reduced. The proportion of radioactivity, after DMI preincubation, was lowest in irides, while in the sublingual gland it accounted for about 50 percent of the value from untreated rats at 4, 12 and 21 days. However, a large proportion o f the radioactivity accumulated after DMI preincubation was found to be due to metabolites (table 4). The uptake of radioactivity when the incubation was performed at 0°C was approximately the same as after preincubation in DMI. Animals (4 days old) which had been given 6-OH-DA (20 mg/kg i.v. 4 hr) showed a reduced uptake of 3H-NA (10 -6 M) in most organs. Iris, atria and muscular coat demonstrat-
72
Ch. SACIIS et al. Table 3 Uptake of 31t-NA, :3H-NM and 3tt-acid metabolites in tissues from rats incubated in 10-6 M 3tt-NA for 30 rain and rinsed in isotope-free buffer for 10 rain, Organ
0
4
]2
21 days
,:~ 3H_NA Iris
95.0 +_ 2.7
91.1 _+ 2.5
91.5 _+ 2.8
92.0 + 3.1
Atrium
78.9 ± 3.5
86.9 ± 4.3
92.5 ± (t.6
90.4 + 2.()
Submaxillary bland
87.1 ± 2.9
82.3 ± 5.9
75.8 ± 6.3
78.3 ± 3.1
Sub/ingual gland
77.6 ± 3. I
64.0 ± 7.7
60.8 ± 7.7
42.4 ± 6.4
Duodenal muscular coat
76.3 ± 2.9
80.3 ± 2.2
85.1 _+4.5
86.7 :_~4.3
Duodenal mucosM layer
69.7 ± 6.8
87.3 ± 0.4
85.4 ± 7,7
91.5 -k 2,5
,')f 3H_NM Iris Atrium Submaxillary gland Sublingual gland
3.0 ± 2.5
3.4 ± 1.7
3.5 ± 2.5
3.7 ± 1.}
i2.7 ± 3.8
9.2 ± 3.6
3.0 + 0.6
3.3 :*- 1.2
6.6 ± 1,6
10.9 ± 4.9
20.7 _+ 6,2
15.9 _+ 2.7
"l 2.6 ± 3.2
18,3 ± 3.9
29.7 ± 8.9
43.7 ± 3.8
Duodenal muscular coat
7.7 ± 2.0
4.9 ± 2.0
1.3 ± 1.3
1.2 ± 0.7
Duodenal mucosal layer
9.3 ± 3.7
6.2 ± 1.3
12.3 _+ 7.9
3.8 ± .1.3
Iris
2.0 _+ 0.6
5.5 4_ 1.3
5.0 +_ 0.4
4.3 +_ 1.4
Atrium
8.4 ± 0,4
3.9 ± 0.8
4.5 ± 0.2
6.3 ~_- 1.3
Submaxillary gland
6.3 ± 1.5
6.9 ± 1.4
3.5 ± 1.0
5.8 ± 0.9
Sublingual gland
9.8 ± 2.7
17.7 _+ 4.5
9.5 ± 3.2
13.9 ± 2.6
Duodenal muscular coat
16.0 ± 1.0
14.8 ± 2.1
13.6 & 3.7
12.1 + 4.3
Duodenal mucosal layer
21.0 ± 7.1
6.5 _+ 1.2
2.3 ± 0,7
4.7 _+.(1.5
'?c 3H-acids
Each value is the mean _-2-S.I:2.M. obtained from 4 8 observations. The figures are e-:pressed as percentage of the sum of the 3H-radioactivity recovered in the respective fraction.
Table 4 Uptake of 3tt-NA in 4 day old rat tissues after pretreatment with wtrious drugs, expressed as percent Gf total radioactivity accumulated. The tissues were incubated in l0 -6 M 3H.NA for 30 min and rinsed in isotope-tree buffer for 10 rain.
Organ
Reserpine a + nialamide b
Reserpine a
Iris
69
28
Atrium
80
32
Submaxillary gland
36
14
80
Sublingual gland
24
8
Duodenal muscular coat
68
6
Duodenal mucosal layer
77
29
Mean from 2- 6 observations,
a Reserpine 10 mg/kg i.p. 4 hr.
DM1 preincubation (10 -s M 15 rain)
Control
72
25
9l
107
l7
87
38
82
86
18
64
98
37
80
108
45
87
Nialamide b
b Nialamide 100 mg/kg i.p. 2 hr.
UPTAKE OF 3H-NORADRENALINE IN DEVELOPING ADRENERGIC NERVES ATRIUM
IRIS
73
SuBMAX. GLAND
,oo.1n 80.
~ 6o ~
40
2O
~=
0
oc L~
4 12
SUBLING.
4
21 lAYS LAND
12
MUSCULAR
21 DAYS COAT
O
4 12
MUCOSAL
21DA'f~
LAYER
°:o°1I O 0
4
12
21 DAYS
0
4
12
21 DAYS
0
4 12
21 DAYS
RESERPINE + NIALAMIDE
[ ] NIALAMIDE
Fig. 5. Effect of nialamide (100 mg/kg i.p. 2 hr) and/or reserpine (10 mg/kg i.p. 4 hr) on the uptake of 3H-NA in various tissues from the growing rat. The tissues were incubated in 10 - 7 M 3H-NA for 30 min at 37°C and rinsed in isotope-free buffer for 10 rain. Each value is the mean + S.E.M. (2 9 observations), expressed as percentage of the value of untreated rats (dpm/g wet weight). ed the lowest uptake, about 25% o f untreated, while the uptake in the sublingual gland was the same as in untreated animals. The values were not corrected for the metabolites formed, which would reduce this percentage. 3.3. Uptake and accumulation o f 3H-NA after pretreatment with reserpine, nialamMe or reserpine + nialamide After reserpine (10 mg/kg i.p. 4 hr) + nialamide (100 mg/kg i.p. 2 hr) pretreatment, the accumulation o f 3H-NA showed a tendency to decrease with age, but the results were subjected to considerable variation (fig. 5). After nialamide pretreatment the amount o f radioactivity accumulated was about the same as in tissues from untreated rats, while after reserpine alone it was considerably lower. However, there was a high proportion o f metabolites (table 4) formed from 3H-NA after the different pretreatmerits. After subtracting the proportion of metabolites from total radioactivity the uptake of 3H-NA was somewhat higher after nialamide, than in untreated rats. After reserpine + nialamide the 3H-NA levels were close to the control values in iris and atria, while after reserpine alone only a small part o f the 3 H-activity recovered was 3 H-NA.
3.4. Retention of" 3H-NA taken up Tissues form untreated and reserpine ( 1 0 m g / k g i.p. 4 hr) + nialamide (100 mg/kg i.p. 4 hr) pretreated 4 day old rats were incubated for 30 rain in 10 -7 M 3 H-NA, and thereafter either rinsed for a few seconds and then directly dried on filter paper and combusted, or transferred to isotope-free buffer and reincubated for 10 or 30 rain at 37°C (fig. 6). There was a rapid washout o f 3H-activity in all tissues studied. The decrease o f 3 H-activity was more pronounced in tissues from reserpine + nialamide pretreated animals. However, no marked differences in the retention of 3H.activity was observed between the various tissues, except for the submaxillary gland, which showed the strongest retention. After a 10 min rinsing time, the percentage of radioactivity lost was in the same range for all organs studied.
3.5. Fluorescence histochemistry Irides, atria, salivary glands and duodenal tissues from untreated rats and treated rats ( 0 - 6 0 days old), were studied by fluorescence microscopy, either after i.v. injection of NA (0.5 mg/kg) or after incubation for 3 0 r a i n at 37°C in Krebs-Ringer bicarbonate buffer containing 10 -s M NA. There were no obvious signs of extraneuronal
74
Ch. SACHS et al. o f N A in a n y o r g a n at a n y age, after in viw) or in vitro t r e a t m e n t , as s e e n in t h e f l u o r e s c e n c e
localization I0°~)°
IRiS
8o
microscope, although the background fluorescence
\
w a s s l i g h t l y i n c r e a s e d in t h e atria o f 4 d a y old and
\
6O
\
g
20
I0
,o
30
MIN
%
older rats, after the in vitro incubation. The fluorescence intensity of the nerves and the number of terminals were slightly increased in the young animals after NA treatment (see fig. 7). In iris and atrium
\
~o
3;
MIN
'
S° L'N
MUSCULAR
GLAND
80
f r o m Very y o u n g •
IOMI N 20
~k ]j '~ ~
30
MUCOSAL
LAYER
6O
40.
"~--
~'~
\ \
-q
"'-2
20 ~ o
-
,~-
3'o
io
MIN
2'o--3o
~b MIN
MIN
Fig. 6. Retention of 3H-NA taken up in tissues from 4 day old rats. Tissues were incubated in 10 -7 M 3H-NA for 30 min at 37°C and then transferred to isotope-free buffer and incubated at 37 ° for 10 or 30 min. Each point represents the mean + S.E.M. from 4 observations, expressed as a percentage of the value from non-rinsed tissue (dpm/g wet weight). - untreated; .... pretreated with reserpine (10 mg/kg i.p. 4 hr) and nialamide (100 mg/kg i.p. 2 hr).
rats, 0 - 4
days, the varicosities and
preterminal axons seemed more pronounced after in vitro i n c u b a t i o n
in N A ,
than t i s s u e s f r o m u n t r e a t e d
rats or tissues incubated in buffer alone. In tissues from young untreated rats, the nerve fibres were coarser, with less distinct varicosities than in adults. In iris, atrium and muscular coat of 4 day old rats pretreated with reserpine (10 mg/kg i.p. 4 hr), a dose which completely abolishes NA fluorescence in the adrenergic
nerves,
no
fluorescent
nerves
could
be
observed after incubation in NA (t0 -s M). However, additional nialamide (100 mg/kg i.p. 2 hr) treatment resulted in a restitution of nerve fluorescence after incubation in NA. No fluorescent adrenergic nerves were found in organs from 6-OH-DA (20 mg/kg i.v. 4 hr) pretreated 4 day old rats, except for some single faintly fluor-
o
, ~ 2 :
r
~
r /
•
W ?
P
Fig. 7. (A) Stretch preparation of atrium from 60 day old rat. The tissue was incubated in Krebs-Ringer buffer for 30 min. Strongly fluorescent adrenergic nerves form a two-dimensional network on the endocardial surface of the atrium. X 150. (B) Atrium from 0 day old rat, treated as in (A). The adrenergic nerves are sparse and show a weak fluorescence. X 150. (C) Atrium from 0 day old rat. The tissue was incubated in NA, 10 -5 M for 30 rain. The adrenergic nerves show a stronger fluorescence than in control tissue. There is no obvious sign of extraneuronal localization of NA. X 150.
UPTAKE OF 3H-NORADRENALINEIN DEVELOPINGADRENERGIC NERVES escent nerve fibres which could occasionally be observed. The detailed morphology and course of outgrowth of the adrenergic nerves during development is described in detail elsewhere (Olson et al., 1970).
4. DISCUSSION Most of the NA taken up in adult sympathetically innervated tissues is located in adrenergic nerves, as revealed by denervation experiments (Hertting et al., 1961; Thoenen and Tranzer, 1968; Jonsson et al., 1969; Sachs, 1970). Exogenously administered NA can usually only be detected in the adrenergic nerves when studied with the histochemical fluorescence method (Hamberger et al., 1964; Malmfors, 1965). After the administration of large doses or incubation in high concentrations of NA, however, different types of extraneuronal NA uptake have been described e.g. in salivary glands (Hamberger et al., 1967), aortic smooth muscle cells (Avakian and Gillespie, 1968), rat or mouse heart (Farnebo and Malmfors, 1969; Sachs, 1970) and rat iris (Jonsson et al., 1969). Iversen et al. (1967) found in an in vivo study that the capacity of NA uptake per weight unit steadily increased in rat heart and spleen from birth to maturity, while the uptake in salivary glands and gut per weight unit which was above adult values at birth, increased until the 12th day to about 160% of the adult value, and then rapidly decreased between 12 and 21 days to adult values. They proposed three main explanations for their findings in the salivary gland and gut: 1)an increased extraneuronal binding in these organs due to their excretory function at an early age, 2)an increased ability of the developing nerves to take up and accumulate 3H-NA, or 3)a change in the distribution of blood flow. In the present study no obvious signs of extraneuronal localization of exogenous NA were observed after either injection of NA or incubation in NA. The iris from newborn rats, contains an abundant amount of yellow fluorescent mast cells, and duodenal tissue, a large number of enterochromaffin cells and mast cells (H~tkanson et al., 1968, c.f. Olson et al.. 1970). Both these cell types are considered to contain serotonin, which causes a more yellow formaldehydeinduced fluorescence than NA, and they are in all
75
probability of minor importance for the uptake of NA (Furano and Green, 1964; Ergnk/5 and Kauto, 1965; Adams-Ray et al., 1966; Er~nk0 and Jansson, 1967). In atria no fluorescent tissue cells (c.f. Farnebo and Malmfors, 1969) or cells of "chromaffin" type could be seen (c.f. Jacobowitz, 1968; Jonsson and Sachs, 1969). It has recently been shown, that 6-OH-DA can be used to obtain a chemical destruction of adrenerglc nerves (Tranzer and Thoenen, 1967; Thoenen and Tranzer, 1968; Mahnfors and Sachs, 1968). In order to determine extraneuronally located 3H.NA, 6-OH-DA pretreated 4 day old rats were investigated and the completeness of the denervation was checked by the histochemical fluorescence method. Extraneuronal uptake was also studied by inhibition of the neuronal uptake by DMI, or by incubation at 0°C. DMI specifically inhibits the uptake mechanism of the "membrane pump" of the adrenergic nerves (Hamberger, 1967). This mechanism is inactive at 0°C since it is energy dependent (see Jonsson et al., 1969; Sachs, 1970). In the present study, the marked reduction in the amount of 3H-NA taken up in the tissues after DMI preincubation, incubation at 0°C, or after 6-OH-DA pretreatment, strongly indicates that most of the 3H.NA was taken up into the adrenergic nerves in tissues from untreated rats. The sublingual gland, which is known to contain very few adrenergic nerves (Norberg and Olson, 1965), was unaffected by these pretreatments. An indication of the relative amount of nervous tissue in an organ is the amount of metabolites formed from the 3H.NA taken up, since the relative amount of 3H-NA recovered is higher in a richly innervated organ (Carlsson and Waldeck, 1963). It is known from immunosympathectomized animals that most of the 3H-NA taken up is converted to 3H-metabolites (Sj/Squist et al., 1965, 1967; Iversen et al., 1966). Table 3 shows that, in iris, the proportion of 3H.NA is high and that of metabolites low over the time of growth studied. In atria and duodenal tissues there is an increasing proportion of 3H-NA, while in the sublingual gland the proportion of 3 H-NA found gradually decreases with age. In the in vivo study of Iversen et al. (1967)and in the present in vitro study, the uptake of 3H-NA per g in salivary glands and gut from newborn animals was found to be equal to or greater than in corresponding
76
Ch. SACHS et al.
organs from 21 day old animals. The present in vitro study gives no support for the view that this increased uptake might be due to an increased excretory function of these tissues at an early age, since no marked differences in extraneuronal uptake over age was observed, which could account for this. The effect of differences in regional blood flow, which might affect the uptake in vivo in these tissues, is eliminated in the in vitro system. This study thus rules out the possibility of differences in regional blood flow, since the findings in vitro and in vivo were similar, ls it due, then, to an increased ability of the developing adrenergic nerves of the salivary glands and gut to take up exogenous NA? This is somewhat more difficult to elucidate, since the ~aumber of nerves per g wet weight cannot be estimated in the different organs, lf, however, it is assumed that the NA content of all adrenergic nerves remains constant during development, then the endogenous NA of the organs can be taken as a measure of the number of adrenergic nerves. The uptake o f 3 H-NA per "unit" nerve could therefore be estimated by calculating the specific activity of 3'H-NA in the tissue. When the data from lversen et al. (1967) are expressed in terms of specific activity, it appears that the uptake per nerve " u n i t " is highest in the youngest animals and decreases with age in a similar pattern for all organs (de Champlain, unpublished observations). In heart, at any age, the specific activity was 3 4 times higher than in any other of the organs investigated. If the above assumption holds true, it seems that the uptake ability is greatest at birth and then decreases; from histochemical fluorescence data, however, it can be assumed that the endogenous concentration o f NA as interpreted from fluorescence intensity increases with age. It is therefore uncertain whether or not there is any change in uptake ability with age. The adrenergic terminals in young tissues, such as iris, atrium and submaxillary gland, show a weaker fluorescence and a less varicose appearance than adult tissues (c.f. Olson et al., 1970). The main axons are more strongly fluorescent in young tissue than in adult. However, the subnmcosal and myenteric nerve plexa of the duodenum differ having a rich adrenergic innervation of the adult type at birth. Friedman et al. (1968) found a good correspondance between the endogenous NA content and the outgrowth of adrenergic nerves as studied by the
monoamine fluorescence technique in the rabbit heart. The NA level was about 0.18 #g/g m a two day old aminal, rising to about adult level, 0.93/~g/g, at 4 wk. It was not stated whether any weakly fluorescent terminals were present in, the newborn rabbit, although strongly fluorescent nerves were found within large nerve trunks. Schiebler and Heene (1968) found, in their study of the outgrowth of adrenergic nerves in the rat heart, that the specific NA fluorescence of the adrenergic nerves was weak m the newborn animal. They also noticed the strongly fluorescent axons growing into the heart muscles from the periphery. On the 12th and 16th day the terminals had the same weak fluorescence, and strongly fluorescent axons were present, while by the 22nd day the innervation of the heart had become of the adult type. This would mean that the number of nerves is higher at birth than indicated by endogenous NA levels, since it appears that these new nerves contain less NA than nerves from adult animals. A close correlation has been shown to exist between the number of adrenergic nerves estimated front fluorescence microscopy and the radioactivity accumulaled in iris and salivary glands of adult animals (Olson et al., 1968). Owing to the rather long incubation time (30 rain) and the following rinsing (10 min) used in the present study, differences in the retention ability of the nerves might have accounted for the differences m radioactivity found. However, after incubation in 3H-NA (fig. 6), the young nerves (4 days) showed about the same capacity to retain NA as adult irides or atria, either before or after pretreatment with reserpine and nialamide (c.f. Jonsson et al., 1969: Sachs, 1970). The percentage of retained 3H-NA in untreated and reserpine + nialamide pretreated rats after rinsing for 10 min was about the same for all tissue~ studied, which makes a comparison between organs possible. It has been shown that, in a reserpine pretreated animal, NA is rapidly taken up by the axonal "membrane p u m p " and then immediately deaminated by MAO, since reserpine efficiently blocks the granular uptake-storage mechanism. MAO-inhibition by e.g. nialamide, causes an extragranular accumulation of NA within the adrenergic nerves (Stitze! and Lundborg, 1967; Jonsson and Sachs, 1969). Furthermore, MAO inhibition alone leads to an increased accumula-
UPTAKE OF 3H-NORADRENALINE IN DEVELOPING ADRENERG1C NERVES tion of NA (Pletscher et al.. 1966). Both the MAO activity and the uptake-storage properties of the amine granules are of great importance for the accumulation of NA (Jonsson et al., 1969; Sachs, 1970). If MAO activity was lower in the adrenergic nerves shortly after birth, then the amount of 3H.NA taken up at that time would be proportionally higher than in organs from 21 day old animals. This difference could be eliminated by giving an MAO-inhibitor. The results (fig. 5), however, show no clear tendency for nialamide to cause an increase or a decrease in the uptake of radioactivity with increasing age, with the exception of iris, in which there is some increase in the radioactivity with age, indicating a more active enzyme at 21 days than at 0 days. However, these values were not corrected for the metabolites formed, which might obscure minor changes in the 3H-NA uptake. Furthermore, no restitution of the fluorescent nerves could be obtained by the administration o f NA in 4 day old animals pretreated with reserpine, which suggests the presence of MAO-activity (see Malmfors, 1965). An increased storage capacity o f the amine granules at birth would likewise result in a proportionally higher uptake and accumulation in young tissues than in tissues from 21 day old animals. If the uptake and storage of the amine granules is blocked by reserpine (Carlsson, 1966) and the MAO-activity inhibited, the uptake and accumulation is dependent only on the uptake at the axonal membrane (Malmfors, 1965) and any difference due to a different storage capacity of the amine storage granules is eliminated. In iris, atrium, submaxillary gland and duodenal mucosa, there is a tendency for a decrease in the amount of radioactivity with age after reserpine + nialamide pretreatment (fig. 5). This suggests a smaller proportion of NA to be taken up and stored in the amine granules o f animals at 0 days than at 21 days, or fewer amine granules present at this time, or that the membrane mechanism is more efficient at 0 days. Although immature nerves seem to contain less endogenous NA than mature nerves, their capacity for NA uptake and storage appears to be the same as that of adult nerves. The continuous increase with age o f the radioactivity accumulated per whole organ thus demonstrates a continuous growth of the adrenergic nerves in all organs studied (fig. 2). In irides
77
and atria the outgrowth of nerves was probably more rapid than the growth of the extraneuronal tissue, resulting in an increased density of nerves and in an increased uptake per g with age. In the submaxillary gland and in the gut, the amount o f nerves at birth was relatively high and the following nerve outgrowth was probably relatively slower than the growth of the extraneuronal tissue, resulting in a decreased uptake per g tissue with age.
ACKNOWLEDGEMENTS The investigation has been supported by research grants from the Swedish Medical Research Council (B68-12X-71103-04 and B70-14X-2295-03), "Stiftelsen Therese och Johan Anderssons Minne", "Ollie och Elof Ericssons Stit'telse", and "Svenska LivfOrs~ikringsbolags N/imnd fOr Medicinsk Forskning". Mrs Eva Lindquist and Miss Ulla Enberg are thanked for their skillful technical assistance. For a generous supply of drugs we thank Doc. Hans Corrodi, AB. H~issle, Goteborg (6-OH-DA), Swedish Ciba, Stockholm (Serpasil@) and Swedish Pfizer, Stockholm (NiamidQL
REFERENCES Adams-Ray, J., A. Dahlstr6m and Ch. Sachs, 1966, Uptake of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan by catecholamine forming mast ceils in the hamster, Acta Physiol. Scand. 67,259. Avakian, O.V. and J.S. GiUespie, 1968, Uptake of noradrenaline by adrenergic nerves, smooth muscle and connective tissue in isolated perfused arteries and its correlation with vasoconstrictor response, Brit. J. Pharmacol. 32, 168. Bertler, /~., A. Carlsson and E. Rosengren, 1958, A method for the fluorimetric determination of adrenaline and noradrenaline in tissues, Acta Physiol. Scand. 44,273. Carlsson, A. and B. Waldeck, 1963, On the role of the liver catechol-O-methyl transferase in the metabolism of circulating catecholamines, Acta Pharmacol. (Kbh) 20, 47. Carlsson, A., 1966, Drugs which block the storage of 5-hydroxy-tryptamine and related amines, in; Handbuch der Experimentellen Pharmakologie, Vol. 19, eds, O. Eichler and A. Farah (Springer, Berlin, Heidelberg, New York) p. 529. Corrodi, H. and G. Jonsson, 1967, The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines: A review on the methodology, J. Histochem. Cytochem. 15, 65. Erank0, O. and L. Kauko, 1965, Uptake of monoamines by
78
Ch. SACHS et al.
mesenteric mast cells of the mouse, Acta Physiol. Scand. 64,283. Er~inkt~, O. and S.-E. Jansson, 1967, Uptake of monoamines by mouse peritoneal mast cells in vitro, Acta Physiol. Scand. 70, 449. Falck, B., 1962, Observations on the possibilities of the cellular localization of monoamines by a fluorescence method, Acta Physiol. Scand. 56, Suppl. 197. Falck, B., N.-,~. Hillarp, G. Thieme and A. Torp, 1962, Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. ttistochem. Cytochem. 10, 348. Falck, B. and C. Owman, 1965, A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic monoamines, Acta Univ. Lund, Section lI, No. 7, 1. Farnebo, L.-O. and T. Malmfors, 1969, Histochemicai studies on the uptake of noradrenaline and O~-methyl-noradrenaline in the perfused rat heart, European J. Pharmacol. 5. 313. Friedman, W.F., P.E. Pool, D. Jacobowitz and S.C. Seagren, 1968, Sympathetic innervation of the developing rabbit heart. Biochemical and histochemical comparisons of fetal, neonatal and adult myocardium, Circulation Res. 23, 25. Furano, A.V. and J.P.Green, 1964, The uptake of biogenic amines by mast cells of the rat, J. Physiol. (London) 170, 263. Glowinski, J., J.Axelrod, l.J. Kopin and R. J. Wurtman, 1964, Physiological disposition of 3H-norepinephrine in the developing rat, J. Pharmacol. Exptl. Therap. 146, 48. Gupta, G.N., 1966, A simple in vial combustion method for assay of hydrogen-3, carbon-14 and sulfur-35 in biological, biochemical and organic materials, Anal. Chem. 38. 1355. Hamberger, B., 1967, Reserpine-resistant uptake of catecholamines in isolated tissues of the rat, Acta Physiol. Stand. Suppl. 295. Hamberger, B., T. Malmfors, K.-A. Norberg and Ch. Sachs. 1964, Uptake and accumulation of catecholamines in peripheral adrenergic neurons of reserpinized animals, studied with a histochemical method, Biochem. Pharmacol. 13, 841. Hamberger, B., K.-A. Norberg and L. Olson, 1967, Extraneuronal binding of catecholamines and 3,4-dihydroxyphenylalanine (dopa) in salivary glands, Acta Physiol. Scand. 69, 1. Hertting, G., J. Axelrod, J. Kopin and L.G. Whitby, 1961, Lack of uptake of catecholamines after chronic denervation of sympathetic nerves, Biochem. Pharmacol. 8, 246. H~kansson, R., Ch. Owman and N.-O. Sji3berg, 1969, Three different systems of monoamine-storing cells in the gastrointestinal tract of fetal and neonatal rats, Acta Physiol. Scand. 75,213. lversen, L.L., J. Glowinski and J. Axelrod, 1966, The physiological disposition and metabolism of norepinephrine in immunosympathectomized animals, J. Pharm. Exptl. Therap. 151,273.
Iversen, L.L., J. de Champlain, J. Glowinski and J. Axe[rod, 1967, Uptake, storage and metabolism of norepinephrine in tissues of the developing rat. J. Pharm. Exptl. Therap. 157,509. Jacobowitz, D., 1967. ttistochemical studies of the relationship of chromaffin cells and adrenevgic nerve fibres to the cardiac ganglia of several species, J. Pharmacol. Exptl. Therap. 158,227. Jonsson, G. and Ch. Sachs, 1969, Subcellular distribution of 3H-noradrenaline in adrenergic nerves of mouse atriumeffect of reserpine, nlonoamine oxidase and tyrosine hydroxylase inhibition, Acta Physiol. Scand. 77,344. Jonsson, G., B. Hamberger, T. Malmfors and Ch. Sachs, 1969, Uptake and accumulation of 3tl-noradrenaline in adrenergic nerves of rat iris: Effect of reserpine, monoamine oxidase and tyrosine hydroxylase inhibition. F,uropean 1. Pharmacol. 8, 58. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall. 1951, Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265. Malmfors, T., 1965, Studies on adrenergic nerves. Acta Physiol. Scand. 64, Suppl. 248. Malmfors, 1". and Ch. Sachs, 1968, Degeneration of adrenergic nerves produced by 6-hydroxydopamine, l:;uropean. J. Pharmacol. 3, 89. Maurer, H.R., 1968, Eine modifizierte MikroverbrennungsMethode zur Bestimmung yon Tritium in organischem Material, Z. Physiol. (?hem. 349, 115. Norberg, K.-A. and B. llamberger. 1964, The sympathetic adrenergic neuron. Some characteristtcs revealed by histochemical studies on the intraneuronal distribution of the transmitter, Acta Physiol. Stand. 63, Suppl. 238. Norberg, K.-A. and L. Olson, 1965, Adrenergic innervation :~I" the salivary glands in the rat, Z. Zellforsch. 68, 183. Olson, L., B. Hamberger, G. Jonsson and 1'. Malmfors, 1968, Combined fluorescence histochelnistry and 3tt-noradrenaliue measurenlents of adrenergic nerves, ltistochemie 15, 38. Olson, I;., J. de Champlain, T. Mahnfors and Ch. Sachs, 1970, Fluorescence morphology of developing peripheral adrenergic nerves in the rat, Acta Physiol. Stand. Olson, L. and U. Ungerstedt, 1970, A simple high capacity freeze-dryer for histochemical use, to be published. Pletscher, A., K.F. Gey and W.P. Burkard, /966, lnhibitors of monoamine oxidase and decarboxylase of aromatic amino acids, in: Handbuch der Experimentellen Pharmakologie, Vol. 19, eds, O. Eichler and A. l:arah (Springer Berlin, lfeidelberg, New York) p. 593. Rutledge, Ch. and J. Jonasson, 1967, Metabolic pathways of dopamine and norepinephrine in rabbit brain m vitro. J. Pharmacol. Exptl. Therap. 157,493. Sachs, Ch., 1969, Uptake and accunmlation in vitro of 3H-noradrenaline in adrenergic nerves of human atrium, ttistochemie, 12, 189. Sachs, Ch., 1970, Studies on the uptake and accumulation of 3H-noradrenaline in adrenergic nerves of mouse atrium, in preparation. Schiebler, T.H. and R. Heene, 1968, Nachweis yon Katechol-
UPTAKE OF 3H-NORADRENALINE IN DEVELOPING ADRENERGIC NERVES aminen im Rattenherzen w~ihrend der Entwicklung, Histochemie 14,328. Sj6qvist, F., E. Titus, 1.A. Michaelson, P. Taylor, Jr. and K.C. Richardson, 1965, Uptake and metabolism of dl-norepinephrine-7-3tt in tissues of irnmunosympathectomized mice and rats, Life Sci. 4, 1125. Sj~iqvist, F., W.T. Palmer, Jr. and E. Titus, 1967, The effect of immunosympathectomy on the retention and metabolism of noradrenaline, Acta Physiol. Scand. 69, 13. Stitzel, R. and P. Lundborg, 1967, Effect of reserpine and monoamine oxidase inhibition on the uptake and subcellular distribution of 3H-noradrenaline, Brit. J. Pharmacol. 29, 99.
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Thoenen, H. and J.P. Tranzer, 1968, Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hyroxydopamine, Arch. Exptl. Pathol. Pharmakol. 261,271. Tranzer, J.P. and H. Thoenen, 1967, Ultramorphologische Ver~inderungen der sympatischen Nervendigungen der Katze nach Vorbehandlung mit 5- und 6-HydroxyDopamin, Arch. Exptl. Pathol. Pharmakol. 257,343. Whitby, L.G., J. Axelrod and H. Weil-Malherbe, 1961, The fate of 3H-norepinephrine in animals, J. Pharmacol. Exptl. Therap. 132, 193.
THE POSTNATAL DEVELOPMENT ADRENERGIC
OF NORADRENALINE
NERVES OF DIFFERENT
U P T A K E IN T H E
TISSUES FROM THE RAT
Ch, SACHS, J. de CHAMPLAIN*, T. MALMFORS and L. OLSON Department of Histology, Karolinska lnstitutet, Stockholm 60, Sweden
Received 11 July 1969
Accepted 19 September 1969
Ch. SACHS, J. de CHAMPLAIN, T. MALMFORS and L. OLSON, The postnatal development o f noradrenaline uptake in the adrenergic nerves of different tissues of the rat, European J. Pharmacol. 9 (1970) 67-79. The uptake and accumulation of 3H-NA** was studied in vitro in iris, atrium, submaxillary and sublingual glands, and the muscular and mucosal layers of the duodenum from rats at different ages (0-60 days). Most of the 3H-NA found in the tissues was located in the adrenergic nerves. The uptake of 3H-NA increased progressively with age in all the organs examined, when calculated per tissue piece. When the radioactivity was expressed per gram tissue, the concentration in the iris and atrium increased progressively as the animal grew older, but the concentration in the salivary glands and in the gut decreased. The variation in the uptake of 3H-NA per gram wet weight is probably due to differences in growth rates between neuronal and extraneuronal components of the different organs, rather than to differences in the uptake ability of the adrenergic nerves, during the time of development. Developing rat Noradrenaline
Adrenergic nerves NA uptake
1. INTRODUCTION Various tissues of the rat develop the ability t o take up and accumulate 3H-noradrenaline (3H-NA) around and shortly after birth (Glowinski et al., 1964; lversen et al., 1967), at the time when outgrowth of sympathetic adrenergic nerves takes place (Olson et al., 1970). Uptake and accumulation of NA in adult tissues are mainly located to the postganglionic sympathetic nerves (Whitby et al., 1961), since the accumulation of 3 H-NA is markedly reduced after postganglionic sympathetic denervation (Hertting et al., 1961; Jonsson et al. 1969) and in immuno* Dr. de Champlain is a Markle Scholar in Academic Medicine. Address: Dept. of Physiology, Faculty of Medicine, University of Montreal, Montreal, Canada. ** Abbreviations used: NA, noradrenaline; NM, normetanephrine; MAO, monoamine oxidase; 6-OH-DA, 6-hydroxydopamine; DMI, desmethylimipramine; dpm, disintegrations per minute.
6-OH-DA Desmethylimipramine
sympathectomized animals (Sji3qvist et al., 1965, 1967; Iversen et al., 1966). The intraneuronal uptake of NA has also been directly demonstrated at the cellular level with the histochemical fluorescence method of Falck and Hillarp (see Hamberger et al., 1964; Malmfors, 1965). The ability of tissues to take up and accumulate 3H-NA may indicate the density of the adrenergic nerves present in the tissue, provided that the uptake per "nerve unit" is constant. However, differences might exist between various organs, owing to different diffusion or circulatory conditions which can affect the uptake and retention of NA. The uptakestorage properties of the adrenergic nerves in different organs might also differ and, furthermore, nerves in the same organ might differ during various developmental stages. In an in vivo study, Iversen et al. (1967) found that in heart and spleen, the ability to accumulate 3 H-NA per weight unit developed parallel to the endogenous NA concentration, while the abil-
68
Ch.SACHS et al.
ity of the intestine and salivary glands to accumulate SH-NA was already fully developed at birth, although the NA content at this time was lower than in the adult organs. The aim of the present study was to investigate the in vitro uptake and accumulation of NA in developing tissues of the rat. The effect of certain drugs, such as reserpine, nialamide, DMI and 6-OH-DA, on this uptake were investigated, in order to obtain information on the specific neuronal uptake in relation to the outgrowth of adrenergic nerves, as demonstrated by the histochemical fluorescence method.
imately 7% less m a gland in which the lobes had been carefully loosened from each other, than in a minced gland. This difference was regarded as of minor importance for the study, and whole glands with loosened lobes were therefore used for the experiments. The wet weight of irides from very young rats was estimated indirectly by measuring the protein content (Lowry et al., 1951). The protein content of irides from 21 day old rats was 115 +- 1.9 ~g/iris, and the wet weight 1.30 -+ 0.06 mg/iris. The protein content multiplied by ten would give an approximate estimation of the mg wet weight. The wet weight of the other organs was measured directly, after incubation.
2. MATERIAL AND METHODS Rats (Sprague-Dawley) of different ages were used for the experiments. The mean body weights were: at 0 days 5 g; 4 days 12 g;8 days 17 g; 12 days 30 g; 21 days 50 g; and at around 60 days 200 g. Injections of drugs were made i.v. into a lingual vein (0.05 0.2 ml) or i.p. (0.2 1.0 ml). The animals were sacrificed by decapitation under light ether anaesthesia. The following organs were carefully dissected out under a preparation microscope: iris, atrium, submaxillary gland, sublingual gland, the external muscular coat, and the mucosal layer of the duodenum (from the pylorus to the entrance of the ductus choledocus, for details see Olson et al., 1970). Analytical determinations were made on tissues from one animal and the results were expressed per one single piece of organ, or per g wet weight. 2.1. in vitro incubations After dissection, the organs were immediately immersed in cool modified Krebs-Ringer bicarbonate buffer pH 7.4 (see Hamberger, 1967) containing 0.2 mg/ml ascorbic acid (Merck p.a.). Incubations were performed in 1.0 ml buffer containing SH-NA (10 -6 or 10-7 M) at 37°C under an atmosphere of 95% O2 - 5% CO2, using a metabolic shaker. The tissues were incubated for 30 rain and thereafter rinsed for 10min at 37°C in isotope-free buffer, unless otherwise stated (Jonsson et al., 1969). Since the submaxillary gland of 21 day old rats was the biggest organ used, some methodological studies were made to evaluate diffusion into this tissue. It was found that the uptake of radioactivity was approx-
2.2.3H-determinations The total 3H-activity taken up and accumulated in the various tissues was determined by combustion of the dried organ in an oxygen atmosphere to form CO2 and water, the ~H-water formed then being determined (Gupta, 1966: Maurer, 1968). The submaxillary glands from 12 and 21 day old rats were too large to combust and were homogenized in 1.5 ml n-butanol-0,1% HCI in a glass homogenizer. One ml of the extract was taken for counting in 5.0 ml tolueneethanol (7:3) solution. Radioactivity was determined in a Packard Model 3002 Tricarb Liquid Scintillation Spectrometer. Efficiency was determined by adding a standard amount of 3H-toluene to representative samples, and recounting the vials. Results are expressed as dpm. 3H-NA and its 3H-O-methylated and a H-deaminated metabolites were determined in tissues incubated in 10 .6 M 3 H-NA for 30 min at 37°C and reincubated in isotope-free buffer for 10 rain. The tissues were extracted with cool 0.4 N perchloric acid. The extracts were chromatographed on ion-exchange columns (Dowex 50W-X4, diameter 4.0ram, height 120 mm, at pH l) (Bertler et al., 1958). The amines were adsorbed on the resin, eluted with 1 N HC1, and measured ad rnodurn (Carlsson and Waldeck, 1063). The acid and neutral catabolites passed through in the effluent, and were determined by shaking the effluent, after acidification (pH 2), with 300 ml of ether (Rutledge and Jonasson, 1967). The volume was reduced under vacuum, and the radioactivity taken up in 1 ml ethanol/0.01% HC1 and 5 ml toluene scintillator, from which a 5 ml aliquot was taken for counting.
UPTAKE OF 3H-NORADRENALINE IN DEVELOPING ADRENERGIC NERVES
2.3. Fluorescence histochemistry
69
%
Irides, atria and duodenal tissues were prepared as whole mounts, dried i n a desiccator over phosphorous pentoxide, and thereafter exposed to formaldehyde gas of o p t i m u m humidity (Hamberger, 1967) for the histochemical demonstration o f biogenic monoamines according to the fluorescence method of Falck and Hillarp. Salivary glands were rapidly frozen in propane, cooled by liquid uitrogen, freeze-dried in a newly developed freeze-drier (Olson and Ungerstedt, 1970), treated with gaseous formaldehyde, embedded in paraffin, sectioned ( 8 / l ) and m o u n t e d in Entellan (Merck) for fluorescence microscopy (Falck et al., 1962; Falck, 1962; Norberg and Hamberger, 1964; Falck and Owman, 1965; Corrodi and Jonsson, 1967). The following substances were used: dl-7-3H-nor adrenaline-HC1, 9.0 C./mmole (New England Nuclear Corp.), dl-noradrenaline-HCl (Calbiochem), nialamide (Niamid ®, Swedish Pfizer), desmethylimipramine (Pertofran ®, Geigy), Reserpine (Serpasil ®, Ciba) and 6-hydroxydopamine-HC1 (AB H~issle, Gothenburg).
I00
i ~ AFTER PREINCUBATION IN D.M.L
u)
-~ BoJ F-
P, bJ 60 E
zE 4 0 " o~
0
I
Z 3
4
s 60
o
IRIS
t
2
3
4
8 6o AGE (DAYS)
ATRIUM
Fig. 1. Uptake of 3H-NA in iris and atrium of rat during growth. The tissues were incubated in 10-TM 3H-NA for 30 min at 37°C and rinsed for 10 rain in isotope-firee medium. Each value is the mean _+S.E.M. (2-8 observations), expressed as percentages of values from 60 day old rats (dpm/piece of tissue). The percentage of 3H-radioactivity taken up after preincubation in DMI (10 -5 M, 15 rain) is marked it, black.
3. RESULTS
the uptake of radioactivity in both tissues with increasing age of the animal. In all tissues investigated, the ability to take up and accumulate NA developed similarly between 0 and 21 days (fig. 2, table 1). In newborn rats, however, the salivary glands and the muscular coat of the intestine showed a relatively higher uptake of 3 H-NA than the iris and atrium. Irides and atria increased their weight 2 . 5 - 4 times
3.1. Uptake and accumulation of 3H-NA Irides and atria from rats of different ages ( 0 - 6 0 days) were incubated in vitro in a medium containing 3H-NA (10 -7 M). The total amount of radioactivity found per iris or atrium was calculated as a percentage o f the amount found in 60 day old animals (fig. 1). There was a progressive increase in w_ ~ 1 2 0%.
U--t00. ~qO-
1
Ix 40"
/c. O. £3 0
4
12 2~
IRIS
0
4
12 2 I
ATRIUM
~,
12 2'
SU6MAX GLAND
O
4
12 21
SUSLING GLAND
~
4
12 9-;
MUSCULAR COAT
•
4 ,2 P, MUCOSAL
AGE (DAYS)
LAYER
Fig. 2. Uptake of 3H-NA in various organs from the growing rat. The tissues were incubated in 10-7 M 3H-NA for 30 min at 37°C and rinsed for 10 rain in isotope free buffer. Each value is the mean + S.E.M. (7-10 observations), expressed as percentages of 21 day old values (dpm/piece of tissue).
70
Ch. SACHS et al.
Table 1 Uptake of 3H-NA in various tissues of the growing r a t Tissues were incubated in 10-vM 3tl-NA for 30 rain and rinsed in isotope-free buffer for 10 min. Values expressed as dpm × 103 per piece of tissue. Organ
0
4
Iris
1.9 ± 0 , 1 8
5.0 z 0.53
17.0 ± 0.98
22.0 ± 2.49
Atrium
2.7 _+ 0,20
6.9 + 0.49
24.7 ± 1.59
49.3 +_ 4.62
46.2 _+ 5.65
68.6 ± 3.52
108.1 ± 12.22
142.7 _+ 22.54
6.0 _+0.95
12.1 _+ 1.5 l
22.8 ± 2.37
37.3 + 8.38
Duodenal muscular coat
11.8 _+ 2.23
13.9 _+ 1.19
28.3 _+2.85
61.7 _+ 10.93
Duodenal mucosal layer
7.3 ± 1.71
29.2 ± 2 85
45.4±7.13
90.2_+9.98
Submaxillary gland Sublingual gland
Each value represents the mean +_S.E.M. of 3
12
21 days
10 observations.
Table 2 Wet Weight in mg of various tissues of the growing rat Organ
0
4
12
Iris
0.25 ± 0.01
0.35 _+ 0.02
0.58 +_ 0.03
0.58 _+ 0.03
1.48 ± 0.04
Atrium
1.83 ± 0.05
2.95 + 0.00
4.92 + 0.27
6.56 _+ 0.18
21.20 ± 1.5(I
Submaxillary gland
7.85 ± 0.25
15.39 ± 0.20
40.72 _+ 0.80
75.77 +_ 2.27
198.30 ± 3.10
Sublingual gland
1.90 _+ 0.09
4.13 _+ 0.09
10.13 _+ 0.32
19.48 _+ 0.85
38.00 _t 1.60
Duodenal muscular coat
1.11 ± 0.09
3.23 +_ 0.10
5.75 _+ 0.8(I
15.53 ± 0.80
31.1 I ± 1.62
Duodenal mucosal layer
5.76 ± 0.27
13.47 + 0.36
29.09 +_ 1.58
96.81 ± 4.!18
167.40 ± 3.35
Each value represents the mean +~S.E.M. from 9
21
18 observations.
1oo. if)
80"'--"
60/
"--"
IRIS ATRIUM SUBMAX. GLAND
- - -
SUBLING. GLAND
~
MUSCULAR LAYER MUCOSAL LAYER
~ • 40-
/
ao-
,J
¢1
/
o
4
,?
2',
DAYS
Fig. 3. Increase of tissue weight of various organs, expressed as percentages of 21 day old values (cf. table 2) in the growing rat.
60 days
UPTAKE OF 3H-NORADRENALINE IN DEVELOPING ADRENERGIC NERVES
.20-
n'
METABOLITES
[]
3H-NA
FROM
71
SH-NA
UPTAKE OF ~bH-RAD(OACTIVITY AFTER PREINCUBATION IN DM.I.
% w
~:15. 0
I.-
10n~ L~
n,-
=E 5 ¸
Q£
nl nrill nNnnnn 0412
IRIS
0
4
f2 21
ATRfUM
4
12 21
SUBMAX. GLAND
0
4
12 21
SUBLING. GLAND
4
12 21
MUSCULAR COAT
0
4 12 21
AGE(DAYS)
MUCOSAL LAYER
Fig. 4. Uptake of 3H-NA in various tissues of the growing rat. The tissues were incubated in 10-7 M 3H-NA for 30 min at 37°C and rinsed in isotope-free buffer for 10 rain. Each value is the mean _+ S.E.M. (8-15 observations), expressed as dpm/g wet weight. The uptake of 3H-radioactivity after preincubation in DMI (10 -5 M, 15 rain) is shown in the dotted part of the columns. the amount of metabolites in the striped. between 0 and 21 days, and had a relatively higher weight at 0 days than the other organs, whereas the weight increase for salivary glands and duodenal tissues during the same period was in the order of ! 0 - 2 0 times (table 2 and fig. 3). There was a marked difference in uptake between the different organs when the accumulation of radioactivity was calculated per g wet weight (fig. 4). The highest uptake was then observed in iris. The concentration of radioactivity increased with age in atria and irides, whereas it decreased in the salivary glands. Of duodenal tissues, the muscular coat showed a decreased 3H-NA uptake with age, while the mucosal layer displayed no clear-cut tendency. When the total radioactivity was corrected for metabolites formed from 3H-NA (fig. 4, table 3) during the incubation period, the pattern of 3H-NA taken up was found to be the same as that for total radioactivity. Corrections were based on values obtained from incubations at 10 -6 M 3H.NA, however, which had to be used in order to obtain sufficient radioactivity for the specific chemical analysis. (The relative amount of 3H-metabolites formed is only slightly increased with higher NA concentrations in the incubation medium for adult atrial tissue (Sachs,
1969, 1970). The proportion o f total radioactivity which represented 3H-NA was high and fairly constant for iris during the age period studied, while an increase in the proportion of 3H-NA was noted in atria and duodenal tissues. A progressive relative decrease of 3 H-NA with age was noted in the salivary glands.
3.2. Extraneuronal uptake o f 3H-NA After preincubation for 15 min in DMI (10 -s M), the accumulation of radioactivity per total organ (fig. 1) or per g wet weight (fig. 4) was considerably reduced. The proportion of radioactivity, after DMI preincubation, was lowest in irides, while in the sublingual gland it accounted for about 50 percent of the value from untreated rats at 4, 12 and 21 days. However, a large proportion o f the radioactivity accumulated after DMI preincubation was found to be due to metabolites (table 4). The uptake of radioactivity when the incubation was performed at 0°C was approximately the same as after preincubation in DMI. Animals (4 days old) which had been given 6-OH-DA (20 mg/kg i.v. 4 hr) showed a reduced uptake of 3H-NA (10 -6 M) in most organs. Iris, atria and muscular coat demonstrat-
72
Ch. SACIIS et al. Table 3 Uptake of 31t-NA, :3H-NM and 3tt-acid metabolites in tissues from rats incubated in 10-6 M 3tt-NA for 30 rain and rinsed in isotope-free buffer for 10 rain, Organ
0
4
]2
21 days
,:~ 3H_NA Iris
95.0 +_ 2.7
91.1 _+ 2.5
91.5 _+ 2.8
92.0 + 3.1
Atrium
78.9 ± 3.5
86.9 ± 4.3
92.5 ± (t.6
90.4 + 2.()
Submaxillary bland
87.1 ± 2.9
82.3 ± 5.9
75.8 ± 6.3
78.3 ± 3.1
Sub/ingual gland
77.6 ± 3. I
64.0 ± 7.7
60.8 ± 7.7
42.4 ± 6.4
Duodenal muscular coat
76.3 ± 2.9
80.3 ± 2.2
85.1 _+4.5
86.7 :_~4.3
Duodenal mucosM layer
69.7 ± 6.8
87.3 ± 0.4
85.4 ± 7,7
91.5 -k 2,5
,')f 3H_NM Iris Atrium Submaxillary gland Sublingual gland
3.0 ± 2.5
3.4 ± 1.7
3.5 ± 2.5
3.7 ± 1.}
i2.7 ± 3.8
9.2 ± 3.6
3.0 + 0.6
3.3 :*- 1.2
6.6 ± 1,6
10.9 ± 4.9
20.7 _+ 6,2
15.9 _+ 2.7
"l 2.6 ± 3.2
18,3 ± 3.9
29.7 ± 8.9
43.7 ± 3.8
Duodenal muscular coat
7.7 ± 2.0
4.9 ± 2.0
1.3 ± 1.3
1.2 ± 0.7
Duodenal mucosal layer
9.3 ± 3.7
6.2 ± 1.3
12.3 _+ 7.9
3.8 ± .1.3
Iris
2.0 _+ 0.6
5.5 4_ 1.3
5.0 +_ 0.4
4.3 +_ 1.4
Atrium
8.4 ± 0,4
3.9 ± 0.8
4.5 ± 0.2
6.3 ~_- 1.3
Submaxillary gland
6.3 ± 1.5
6.9 ± 1.4
3.5 ± 1.0
5.8 ± 0.9
Sublingual gland
9.8 ± 2.7
17.7 _+ 4.5
9.5 ± 3.2
13.9 ± 2.6
Duodenal muscular coat
16.0 ± 1.0
14.8 ± 2.1
13.6 & 3.7
12.1 + 4.3
Duodenal mucosal layer
21.0 ± 7.1
6.5 _+ 1.2
2.3 ± 0,7
4.7 _+.(1.5
'?c 3H-acids
Each value is the mean _-2-S.I:2.M. obtained from 4 8 observations. The figures are e-:pressed as percentage of the sum of the 3H-radioactivity recovered in the respective fraction.
Table 4 Uptake of 3tt-NA in 4 day old rat tissues after pretreatment with wtrious drugs, expressed as percent Gf total radioactivity accumulated. The tissues were incubated in l0 -6 M 3H.NA for 30 min and rinsed in isotope-tree buffer for 10 rain.
Organ
Reserpine a + nialamide b
Reserpine a
Iris
69
28
Atrium
80
32
Submaxillary gland
36
14
80
Sublingual gland
24
8
Duodenal muscular coat
68
6
Duodenal mucosal layer
77
29
Mean from 2- 6 observations,
a Reserpine 10 mg/kg i.p. 4 hr.
DM1 preincubation (10 -s M 15 rain)
Control
72
25
9l
107
l7
87
38
82
86
18
64
98
37
80
108
45
87
Nialamide b
b Nialamide 100 mg/kg i.p. 2 hr.
UPTAKE OF 3H-NORADRENALINE IN DEVELOPING ADRENERGIC NERVES ATRIUM
IRIS
73
SuBMAX. GLAND
,oo.1n 80.
~ 6o ~
40
2O
~=
0
oc L~
4 12
SUBLING.
4
21 lAYS LAND
12
MUSCULAR
21 DAYS COAT
O
4 12
MUCOSAL
21DA'f~
LAYER
°:o°1I O 0
4
12
21 DAYS
0
4
12
21 DAYS
0
4 12
21 DAYS
RESERPINE + NIALAMIDE
[ ] NIALAMIDE
Fig. 5. Effect of nialamide (100 mg/kg i.p. 2 hr) and/or reserpine (10 mg/kg i.p. 4 hr) on the uptake of 3H-NA in various tissues from the growing rat. The tissues were incubated in 10 - 7 M 3H-NA for 30 min at 37°C and rinsed in isotope-free buffer for 10 rain. Each value is the mean + S.E.M. (2 9 observations), expressed as percentage of the value of untreated rats (dpm/g wet weight). ed the lowest uptake, about 25% o f untreated, while the uptake in the sublingual gland was the same as in untreated animals. The values were not corrected for the metabolites formed, which would reduce this percentage. 3.3. Uptake and accumulation o f 3H-NA after pretreatment with reserpine, nialamMe or reserpine + nialamide After reserpine (10 mg/kg i.p. 4 hr) + nialamide (100 mg/kg i.p. 2 hr) pretreatment, the accumulation o f 3H-NA showed a tendency to decrease with age, but the results were subjected to considerable variation (fig. 5). After nialamide pretreatment the amount o f radioactivity accumulated was about the same as in tissues from untreated rats, while after reserpine alone it was considerably lower. However, there was a high proportion o f metabolites (table 4) formed from 3H-NA after the different pretreatmerits. After subtracting the proportion of metabolites from total radioactivity the uptake of 3H-NA was somewhat higher after nialamide, than in untreated rats. After reserpine + nialamide the 3H-NA levels were close to the control values in iris and atria, while after reserpine alone only a small part o f the 3 H-activity recovered was 3 H-NA.
3.4. Retention of" 3H-NA taken up Tissues form untreated and reserpine ( 1 0 m g / k g i.p. 4 hr) + nialamide (100 mg/kg i.p. 4 hr) pretreated 4 day old rats were incubated for 30 rain in 10 -7 M 3 H-NA, and thereafter either rinsed for a few seconds and then directly dried on filter paper and combusted, or transferred to isotope-free buffer and reincubated for 10 or 30 rain at 37°C (fig. 6). There was a rapid washout o f 3H-activity in all tissues studied. The decrease o f 3 H-activity was more pronounced in tissues from reserpine + nialamide pretreated animals. However, no marked differences in the retention of 3H.activity was observed between the various tissues, except for the submaxillary gland, which showed the strongest retention. After a 10 min rinsing time, the percentage of radioactivity lost was in the same range for all organs studied.
3.5. Fluorescence histochemistry Irides, atria, salivary glands and duodenal tissues from untreated rats and treated rats ( 0 - 6 0 days old), were studied by fluorescence microscopy, either after i.v. injection of NA (0.5 mg/kg) or after incubation for 3 0 r a i n at 37°C in Krebs-Ringer bicarbonate buffer containing 10 -s M NA. There were no obvious signs of extraneuronal
74
Ch. SACHS et al. o f N A in a n y o r g a n at a n y age, after in viw) or in vitro t r e a t m e n t , as s e e n in t h e f l u o r e s c e n c e
localization I0°~)°
IRiS
8o
microscope, although the background fluorescence
\
w a s s l i g h t l y i n c r e a s e d in t h e atria o f 4 d a y old and
\
6O
\
g
20
I0
,o
30
MIN
%
older rats, after the in vitro incubation. The fluorescence intensity of the nerves and the number of terminals were slightly increased in the young animals after NA treatment (see fig. 7). In iris and atrium
\
~o
3;
MIN
'
S° L'N
MUSCULAR
GLAND
80
f r o m Very y o u n g •
IOMI N 20
~k ]j '~ ~
30
MUCOSAL
LAYER
6O
40.
"~--
~'~
\ \
-q
"'-2
20 ~ o
-
,~-
3'o
io
MIN
2'o--3o
~b MIN
MIN
Fig. 6. Retention of 3H-NA taken up in tissues from 4 day old rats. Tissues were incubated in 10 -7 M 3H-NA for 30 min at 37°C and then transferred to isotope-free buffer and incubated at 37 ° for 10 or 30 min. Each point represents the mean + S.E.M. from 4 observations, expressed as a percentage of the value from non-rinsed tissue (dpm/g wet weight). - untreated; .... pretreated with reserpine (10 mg/kg i.p. 4 hr) and nialamide (100 mg/kg i.p. 2 hr).
rats, 0 - 4
days, the varicosities and
preterminal axons seemed more pronounced after in vitro i n c u b a t i o n
in N A ,
than t i s s u e s f r o m u n t r e a t e d
rats or tissues incubated in buffer alone. In tissues from young untreated rats, the nerve fibres were coarser, with less distinct varicosities than in adults. In iris, atrium and muscular coat of 4 day old rats pretreated with reserpine (10 mg/kg i.p. 4 hr), a dose which completely abolishes NA fluorescence in the adrenergic
nerves,
no
fluorescent
nerves
could
be
observed after incubation in NA (t0 -s M). However, additional nialamide (100 mg/kg i.p. 2 hr) treatment resulted in a restitution of nerve fluorescence after incubation in NA. No fluorescent adrenergic nerves were found in organs from 6-OH-DA (20 mg/kg i.v. 4 hr) pretreated 4 day old rats, except for some single faintly fluor-
o
, ~ 2 :
r
~
r /
•
W ?
P
Fig. 7. (A) Stretch preparation of atrium from 60 day old rat. The tissue was incubated in Krebs-Ringer buffer for 30 min. Strongly fluorescent adrenergic nerves form a two-dimensional network on the endocardial surface of the atrium. X 150. (B) Atrium from 0 day old rat, treated as in (A). The adrenergic nerves are sparse and show a weak fluorescence. X 150. (C) Atrium from 0 day old rat. The tissue was incubated in NA, 10 -5 M for 30 rain. The adrenergic nerves show a stronger fluorescence than in control tissue. There is no obvious sign of extraneuronal localization of NA. X 150.
UPTAKE OF 3H-NORADRENALINEIN DEVELOPINGADRENERGIC NERVES escent nerve fibres which could occasionally be observed. The detailed morphology and course of outgrowth of the adrenergic nerves during development is described in detail elsewhere (Olson et al., 1970).
4. DISCUSSION Most of the NA taken up in adult sympathetically innervated tissues is located in adrenergic nerves, as revealed by denervation experiments (Hertting et al., 1961; Thoenen and Tranzer, 1968; Jonsson et al., 1969; Sachs, 1970). Exogenously administered NA can usually only be detected in the adrenergic nerves when studied with the histochemical fluorescence method (Hamberger et al., 1964; Malmfors, 1965). After the administration of large doses or incubation in high concentrations of NA, however, different types of extraneuronal NA uptake have been described e.g. in salivary glands (Hamberger et al., 1967), aortic smooth muscle cells (Avakian and Gillespie, 1968), rat or mouse heart (Farnebo and Malmfors, 1969; Sachs, 1970) and rat iris (Jonsson et al., 1969). Iversen et al. (1967) found in an in vivo study that the capacity of NA uptake per weight unit steadily increased in rat heart and spleen from birth to maturity, while the uptake in salivary glands and gut per weight unit which was above adult values at birth, increased until the 12th day to about 160% of the adult value, and then rapidly decreased between 12 and 21 days to adult values. They proposed three main explanations for their findings in the salivary gland and gut: 1)an increased extraneuronal binding in these organs due to their excretory function at an early age, 2)an increased ability of the developing nerves to take up and accumulate 3H-NA, or 3)a change in the distribution of blood flow. In the present study no obvious signs of extraneuronal localization of exogenous NA were observed after either injection of NA or incubation in NA. The iris from newborn rats, contains an abundant amount of yellow fluorescent mast cells, and duodenal tissue, a large number of enterochromaffin cells and mast cells (H~tkanson et al., 1968, c.f. Olson et al.. 1970). Both these cell types are considered to contain serotonin, which causes a more yellow formaldehydeinduced fluorescence than NA, and they are in all
75
probability of minor importance for the uptake of NA (Furano and Green, 1964; Ergnk/5 and Kauto, 1965; Adams-Ray et al., 1966; Er~nk0 and Jansson, 1967). In atria no fluorescent tissue cells (c.f. Farnebo and Malmfors, 1969) or cells of "chromaffin" type could be seen (c.f. Jacobowitz, 1968; Jonsson and Sachs, 1969). It has recently been shown, that 6-OH-DA can be used to obtain a chemical destruction of adrenerglc nerves (Tranzer and Thoenen, 1967; Thoenen and Tranzer, 1968; Mahnfors and Sachs, 1968). In order to determine extraneuronally located 3H.NA, 6-OH-DA pretreated 4 day old rats were investigated and the completeness of the denervation was checked by the histochemical fluorescence method. Extraneuronal uptake was also studied by inhibition of the neuronal uptake by DMI, or by incubation at 0°C. DMI specifically inhibits the uptake mechanism of the "membrane pump" of the adrenergic nerves (Hamberger, 1967). This mechanism is inactive at 0°C since it is energy dependent (see Jonsson et al., 1969; Sachs, 1970). In the present study, the marked reduction in the amount of 3H-NA taken up in the tissues after DMI preincubation, incubation at 0°C, or after 6-OH-DA pretreatment, strongly indicates that most of the 3H.NA was taken up into the adrenergic nerves in tissues from untreated rats. The sublingual gland, which is known to contain very few adrenergic nerves (Norberg and Olson, 1965), was unaffected by these pretreatments. An indication of the relative amount of nervous tissue in an organ is the amount of metabolites formed from the 3H.NA taken up, since the relative amount of 3H-NA recovered is higher in a richly innervated organ (Carlsson and Waldeck, 1963). It is known from immunosympathectomized animals that most of the 3H-NA taken up is converted to 3H-metabolites (Sj/Squist et al., 1965, 1967; Iversen et al., 1966). Table 3 shows that, in iris, the proportion of 3H.NA is high and that of metabolites low over the time of growth studied. In atria and duodenal tissues there is an increasing proportion of 3H-NA, while in the sublingual gland the proportion of 3 H-NA found gradually decreases with age. In the in vivo study of Iversen et al. (1967)and in the present in vitro study, the uptake of 3H-NA per g in salivary glands and gut from newborn animals was found to be equal to or greater than in corresponding
76
Ch. SACHS et al.
organs from 21 day old animals. The present in vitro study gives no support for the view that this increased uptake might be due to an increased excretory function of these tissues at an early age, since no marked differences in extraneuronal uptake over age was observed, which could account for this. The effect of differences in regional blood flow, which might affect the uptake in vivo in these tissues, is eliminated in the in vitro system. This study thus rules out the possibility of differences in regional blood flow, since the findings in vitro and in vivo were similar, ls it due, then, to an increased ability of the developing adrenergic nerves of the salivary glands and gut to take up exogenous NA? This is somewhat more difficult to elucidate, since the ~aumber of nerves per g wet weight cannot be estimated in the different organs, lf, however, it is assumed that the NA content of all adrenergic nerves remains constant during development, then the endogenous NA of the organs can be taken as a measure of the number of adrenergic nerves. The uptake o f 3 H-NA per "unit" nerve could therefore be estimated by calculating the specific activity of 3'H-NA in the tissue. When the data from lversen et al. (1967) are expressed in terms of specific activity, it appears that the uptake per nerve " u n i t " is highest in the youngest animals and decreases with age in a similar pattern for all organs (de Champlain, unpublished observations). In heart, at any age, the specific activity was 3 4 times higher than in any other of the organs investigated. If the above assumption holds true, it seems that the uptake ability is greatest at birth and then decreases; from histochemical fluorescence data, however, it can be assumed that the endogenous concentration o f NA as interpreted from fluorescence intensity increases with age. It is therefore uncertain whether or not there is any change in uptake ability with age. The adrenergic terminals in young tissues, such as iris, atrium and submaxillary gland, show a weaker fluorescence and a less varicose appearance than adult tissues (c.f. Olson et al., 1970). The main axons are more strongly fluorescent in young tissue than in adult. However, the subnmcosal and myenteric nerve plexa of the duodenum differ having a rich adrenergic innervation of the adult type at birth. Friedman et al. (1968) found a good correspondance between the endogenous NA content and the outgrowth of adrenergic nerves as studied by the
monoamine fluorescence technique in the rabbit heart. The NA level was about 0.18 #g/g m a two day old aminal, rising to about adult level, 0.93/~g/g, at 4 wk. It was not stated whether any weakly fluorescent terminals were present in, the newborn rabbit, although strongly fluorescent nerves were found within large nerve trunks. Schiebler and Heene (1968) found, in their study of the outgrowth of adrenergic nerves in the rat heart, that the specific NA fluorescence of the adrenergic nerves was weak m the newborn animal. They also noticed the strongly fluorescent axons growing into the heart muscles from the periphery. On the 12th and 16th day the terminals had the same weak fluorescence, and strongly fluorescent axons were present, while by the 22nd day the innervation of the heart had become of the adult type. This would mean that the number of nerves is higher at birth than indicated by endogenous NA levels, since it appears that these new nerves contain less NA than nerves from adult animals. A close correlation has been shown to exist between the number of adrenergic nerves estimated front fluorescence microscopy and the radioactivity accumulaled in iris and salivary glands of adult animals (Olson et al., 1968). Owing to the rather long incubation time (30 rain) and the following rinsing (10 min) used in the present study, differences in the retention ability of the nerves might have accounted for the differences m radioactivity found. However, after incubation in 3H-NA (fig. 6), the young nerves (4 days) showed about the same capacity to retain NA as adult irides or atria, either before or after pretreatment with reserpine and nialamide (c.f. Jonsson et al., 1969: Sachs, 1970). The percentage of retained 3H-NA in untreated and reserpine + nialamide pretreated rats after rinsing for 10 min was about the same for all tissue~ studied, which makes a comparison between organs possible. It has been shown that, in a reserpine pretreated animal, NA is rapidly taken up by the axonal "membrane p u m p " and then immediately deaminated by MAO, since reserpine efficiently blocks the granular uptake-storage mechanism. MAO-inhibition by e.g. nialamide, causes an extragranular accumulation of NA within the adrenergic nerves (Stitze! and Lundborg, 1967; Jonsson and Sachs, 1969). Furthermore, MAO inhibition alone leads to an increased accumula-
UPTAKE OF 3H-NORADRENALINE IN DEVELOPING ADRENERG1C NERVES tion of NA (Pletscher et al.. 1966). Both the MAO activity and the uptake-storage properties of the amine granules are of great importance for the accumulation of NA (Jonsson et al., 1969; Sachs, 1970). If MAO activity was lower in the adrenergic nerves shortly after birth, then the amount of 3H.NA taken up at that time would be proportionally higher than in organs from 21 day old animals. This difference could be eliminated by giving an MAO-inhibitor. The results (fig. 5), however, show no clear tendency for nialamide to cause an increase or a decrease in the uptake of radioactivity with increasing age, with the exception of iris, in which there is some increase in the radioactivity with age, indicating a more active enzyme at 21 days than at 0 days. However, these values were not corrected for the metabolites formed, which might obscure minor changes in the 3H-NA uptake. Furthermore, no restitution of the fluorescent nerves could be obtained by the administration o f NA in 4 day old animals pretreated with reserpine, which suggests the presence of MAO-activity (see Malmfors, 1965). An increased storage capacity o f the amine granules at birth would likewise result in a proportionally higher uptake and accumulation in young tissues than in tissues from 21 day old animals. If the uptake and storage of the amine granules is blocked by reserpine (Carlsson, 1966) and the MAO-activity inhibited, the uptake and accumulation is dependent only on the uptake at the axonal membrane (Malmfors, 1965) and any difference due to a different storage capacity of the amine storage granules is eliminated. In iris, atrium, submaxillary gland and duodenal mucosa, there is a tendency for a decrease in the amount of radioactivity with age after reserpine + nialamide pretreatment (fig. 5). This suggests a smaller proportion of NA to be taken up and stored in the amine granules o f animals at 0 days than at 21 days, or fewer amine granules present at this time, or that the membrane mechanism is more efficient at 0 days. Although immature nerves seem to contain less endogenous NA than mature nerves, their capacity for NA uptake and storage appears to be the same as that of adult nerves. The continuous increase with age o f the radioactivity accumulated per whole organ thus demonstrates a continuous growth of the adrenergic nerves in all organs studied (fig. 2). In irides
77
and atria the outgrowth of nerves was probably more rapid than the growth of the extraneuronal tissue, resulting in an increased density of nerves and in an increased uptake per g with age. In the submaxillary gland and in the gut, the amount o f nerves at birth was relatively high and the following nerve outgrowth was probably relatively slower than the growth of the extraneuronal tissue, resulting in a decreased uptake per g tissue with age.
ACKNOWLEDGEMENTS The investigation has been supported by research grants from the Swedish Medical Research Council (B68-12X-71103-04 and B70-14X-2295-03), "Stiftelsen Therese och Johan Anderssons Minne", "Ollie och Elof Ericssons Stit'telse", and "Svenska LivfOrs~ikringsbolags N/imnd fOr Medicinsk Forskning". Mrs Eva Lindquist and Miss Ulla Enberg are thanked for their skillful technical assistance. For a generous supply of drugs we thank Doc. Hans Corrodi, AB. H~issle, Goteborg (6-OH-DA), Swedish Ciba, Stockholm (Serpasil@) and Swedish Pfizer, Stockholm (NiamidQL
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