400

Brain Research, 99 (1975) 400-404 ~77>Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Increase of tyrosine hydroxyiase activity after reserpine: evidence for the selective response of noradrenergic neurons

MASARU SORIMACHt

Department of Physiology, Ehime University Medical School, Ehime (Japan) (Accepted August 5th, 1975)

It has been well established that reserpine causes a trans-synaptic increase of some enzymes related to the synthesis of catecholamines in the peripheral sympathetic ganglia and in the adrenal glands12A7, is. Available evidence suggests that reserpine's action is also demonstrable in certain central catecholamine neurons, particularly the locus coeruleus (LC) 1,2,12,1a,1~,2°. Evidence is lacking as to whether this includes trans-synaptic events. Since the neurons o f the LC produce norepinephrine a as well as its synthesizing enzymes, tyrosine hydroxylase (TH) and dopamine-fl-hydroxylase (DBH) 4,5-s, several groups of investigators considered the noradrenergic neurons as the site for the reserpine's action~, 13. However, recent immunohistofluorescent studies on the phenytethanolamine-N-methyltransferase (PNMT) indicated that this region also includes a moderate density of adrenergic terminals whose cell bodies are located in other parts (designated as C1 and C2) of the pons-medulta 9. Since the adrenergic neurons may also include TH and DBH besides the specific enzyme PNMT, the possibility exists that these neurons are affected by reserpine, thus resulting in the increased activities of T H and DBH in their terminal regions including the LC. The present experiments were undertaken to determine the exact sites for reserpine's action by measuring these enzyme activities in the LC as well as in the regions rich in adrenergic cell bodies. Male Wistar rats, weighing 250-350 g, received subcutaneous (2.5 mg/kg, 3 successive days) or intraperitoneal injection (5-10 mg/kg, every 2 days) of reserpine (Apoplon, Daiichi-Seiyaku, Japan). Animals were killed at varying times by decapitation with or without ether anesthesia. The brain was removed and put into ice-cold isotonic saline. The regions were dissected out as described previously 18 (and in Table II, legend). Tissues were homogenized in 10-40 vol. of ice-cold Tris buffer (5 mM, pH 7.4) containing 0 . 2 ~ cutscum as described previously 16, followed by centrifugation at 10,000 rev./min for 5 min using a Beckman microfuge at 4-6 °C or at 18,000 × g for 10 min by a conventional refrigerated centrifuge. Enzyme activities measured in the supernatant obtained by 2 different centrifugation procedures produced no quantitative differences. The T H activity was measured by the COg trapping method with a 73 # M substrate concentration 19. The DBH activity was measured by

401 TABLE I E F F E C T OF R E S E R P I N E O N

TH

AND

DBH

A C T I V I T I E S I N D I F F E R E N T B R A I N R E G I O N S A N D IN A D R E N A L

Reserpine was injected intraperitoneally (5-10 mg/kg, every 2 days) unless otherwise specified and animals were killed by decapitation at varying times after the first injection. Regions were dissected as described previouslyTM. TH and DBH activities are expressed as percentages ~ S.E.M. of the mean of appropriate control groups. The numbers of animals are shown in parentheses. Regions

Day

o/ TH (,o)

o/ DBH (,o)

LC

I 2 4 5 6**

83 174 279 286 296

90±15(4) 98 5_ 4(4) 109 ~ 8 (13) 140 ± 8 (10)* 138 L- 9 (10)*

SC

3-5 6**

184 ± 10 (20)* 259 5- 27 (6)*

-115 ± 8 (4)

Pons-medulla rest

3-5

138 5_ 7 (18)*

107 ± 4 (18)

As-Alo

3-5 6**

113 ± 4 (28) 111 ~ 4(7)

--

Adrenal

1 4-5

208 ± 19 (4)* 214 - 14 (16)*

187 ± 17 (4)* 201 k 15 (20)*

± 8(4) L 14 (4)* ± 27 (13)* 5- 17 (11)* -- 23 (13)*

* Significantly different at the level of 1 ~. ** Reserpine administered subcutaneously.

the P N M T c o u p l e d m e t h o d using an o p t i m a l c o n c e n t r a t i o n o f CHSO411. The P N M T activity was assayed a c c o r d i n g to the highly sensitive m e t h o d by S a a v e d r a et al. 14. The c o n c e n t r a t i o n o f substrate a n d [3H]S-adenosyl-e-methionine were 0.2 m M a n d 3.6 # M (0.92 m C i / # m o l e ) , respectively. O r g a n i c solvent extracts were e v a p o r a t e d at a r o u n d 50 °C with a stream o f air and this step was repeated after the a d d i t i o n o f 3 ml o f fresh organic solvent. Consistent with the earlier findings, reserpine m a r k e d l y increased T H activity in the L C (Table 1). The earliest increase o f T H activity was f o u n d 2 days after a singre i n t r a p e r i t o n e a l injection, while the D B H activity r e m a i n e d u n c h a n g e d at this time. The increase o f T H activity was also f o u n d in o t h e r parts o f the p o n s - m e d u l l a . The activity increased by a p p r o x i m a t e l y 80~o in the subcoeruleus regions (SC) rich in n o r a d r e n e r g i c cell bodies. The increase was smallest ( a p p r o x i m a t e l y 40 ~o) in the rest o f the p o n s - m e d u l l a . O n the o t h e r hand, little if any increase in the T H activity was f o u n d after reserpine in the A8-10 regions where m o s t o f the d o p a m i n e r g i c cell bodies in the b r a i n are localized. These results suggest t h a t all o f the n o r a d r e n e r g i c cell bodies located in different p a r t s o f the p o n s - m e d u l l a r e s p o n d to reserpine by increasing T H activity a n d t h a t the smaller increases in the relatively larger areas dissected are due to the diluted density o f n o r a d r e n e r g i c cell bodies. Indeed, the D B H activity in the L C increased significantly after reserpine. H o w e v e r , this response c o u l d be differentiated f r o m t h a t o f T H in two respects; the increase in D B H activity needed much

402 TABLE 11 TH, DBH

AND

PNMT ACTIVITIESIN VARIOUS

REGIONS OF THE PONS-MEDULLA

Rats were killed by decapitation under ether anesthesia. "l-he values of 3 enzyme activities were not different from those without anesthesia. The most caudal coronal section was obtained by placing cuts at the level 1 mm caudal to and 1 mm rostral to the area postrema. Ventro-lateral and dorsomedial portions of this section are referred to as C1 and C2 (caudal half), respectively. The rest region of the same section is referred to as R1. The rostral half of Cz was isolated from the next section~ the rostral cut of which was made at the level 1 mm caudal to the indentations of sulci limitans (i.e., 2 mm rostrat to the caudal cut). The rest is referred to as R2. Ra is designated as the whole coronal section placed between the second and the fourth section which includes LC and SC. TH, DBH and PNMT activities are expressed as nmole of ~4CO2 evolved, nmole phenylethanolamine formed and nmole N-methylphenylethanolamine formed/g tissue/h, respectively. The number of samples is shown in parentheses. Region

TH

C1 C2 R1 R2 R3 LC SC As-Aao

32 33 11 I1 6 78 10 329

:!: 4 (t9) ~: 2 (21) :~: 2 (9) !: 2(10) ::I_ 1 (4) ± 6 (13) :_:z 1 (7) ~: 13 (13)

D BH

P NMT

190 :Jz 18 (12) 222 ± 16 (12) 91 :~: 27 (4) 103_~26(5)

1.76 5_ 0.27 (10) 5.05 j_~0.41 (10) 0.47 ~: 0.08 (7) 1.91±0.12(7) 0.49 :~: 0.04 (7) 0.88 :~: 0.15 (6) 0.27 -i 0.01 (31)

....

866 ± 46 (14) 104 :Jz 11 (8) . . . . .

.

l o n g e r reserpine t r e a t m e n t (more t h a n 5 days) as was also f o u n d by B r o o k e a n d F i b i g e r 2 b u t n o t by others ta, a n d the extent o f increase was m u c h smaller. I n contrast, c o m p a r a b l e increases in b o t h e n z y m e activities are f o u n d in the adrenals. Since b o t h increases are a l r e a d y m a x i m a l 1 d a y after reserpine, the possibility c a n n o t be neglected t h a t there exists a differential rate o f increase earlier t h a n 1 day. However, the differential increases are m o r e r e m a r k a b l e in the LC, a n d therefore this w o u l d m a k e one feature in the central n o r a d r e n e r g i c neurons. A p a r t from these results, it s h o u l d be recalled t h a t 2 enzymes m a y be involved n o t only in the n o r a d r e n e r g i c b u t a l s o in the adrenergic neurons, whose cell bodies have been recently identified in p a r t i c u l a r regions o f the p o n s - m e d u l l a 9. I next s o u g h t to see if the increases o f T H a n d D B H activities in the L C reflect j u s t the a c c u m u l a t i o n o f these in the adrenergic terminals, whose existence in the L C a r e a was i m m u n o h i s t o c h e m i c a U y d e m o n s t r a t e d 9. I f this is the case, reserpine w o u l d increase these enzyme activities in the adrenergic cell b o d i e s , thus allowing differential increases o f T H a n d D B H activities in the L C by the differences in the rate o f a x o n a l t r a n s p o r t o f the 2 enzymes. Before starting this e x p e r i m e n t , I m e a s u r e d the T H , the D B H a n d the P N M T activities in several regions o f the p o n s - m e d u l l a (Table II). In the LC, the highest T H a n d D B H activities were f o u n d c o m p a r e d to a relatively low P N M T activity, a fact confirming t h a t this i e g i o n includes a high density o f n o r a d r e n e r g i c neurons. I n contrast, the highest activity o f P N M T was f o u n d in the C2 region, followed by the (21 reg i o n a n d the rest o f the regions (R~) o f the c o r o n a l section f r o m which the rostral

403 TABLE IlI EFFECT OF RESERPINEON TH AND PNMT ACTIVITIESIN SEVERALREGIONSOF THE PONS-MEDULLA Rats received subcutaneous injection of reserpine (2.5 mg/kg, for 3 days) and were killed by decapitation 6 days after the first injection. Values are expressed as percentages ± S.E.M. of the mean of control group in each experiment. Number of animals is shown in parenthesis.

Region

TH (°o)

P N M T (%)

LC C1 C2 R1 ÷ R 2

310 ± 30 (10)* 108 ± 6 (15) 1 1 4 ± 5(21) 1 4 5 ± 5(7)*

93 ± 10 (11) -102 ± 6(14) 89i 6(7)

* Significantly different at the level of 1 ~ .

half of the C2 is obtained. These regions have relatively low T H and DBH activities, facts indicating that the adrenergic neurons include higher PNMT, but much lower T H and DBH activities than the dopaminergic and the noradrenergic neurons. The increase of T H activity in the C1 and the C2 regions was statistically insignificant even after the longest treatment of reserpine (Table III). On the other hand, small but significant increases were found in the rest of the regions of C1 and C2. The increase in these regions may therefore be responsible for the previously observed increase of T H in the rest of the pons-medulla (Table I). Furthermore, no alterations of P N M T activity by reserpine in the C2 region as well as in the LC seem to add further evidence that the adrenergic cell bodies are little influenced by this drug as was recently found in the superior cervical ganglia 10 where no depletion of adrenaline was observed. It should be noted here that the immunohistofluorescent studies on T H in the LC would provide quite valuable information on these problems. In conclusion, the results in this experiment suggest that the noradrenergic cell bodies are the targets among central catecholaminergic ones for reserpine's action. The control mechanisms for T H and DBH in these neurons might be somewhat different from those in the peripheral tissues such that T H behaves as a more important rate-limiting enzyme than DBH for noradrenaline synthesis.

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