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Adenosine 3′,5′ cyclic monophosphate stimulates dopamine biosynthesis in the median eminence of rat hypothalamic slices
Brain Research, 374 (1986) 37-44
37
Elsevier BRE 11712
Adenosine 3' ,5'-Cyclic Monophosphate Stimulates Dopamine Biosynthesis in the Median Eminence of Rat Hypothalamic Slices JUN ARITA and FUKUKO KIMURA
Department of Physiology, Yokohama City University School of Medicine, Yokohama 232 (Japan) (Accepted October 2nd, 1985)
Key words: tuberoinfundibular dopaminergic neuron - - dihydroxyphenylalanine - - hypothalamic slice - cyclic adenosine monophosphate - - forskolin
The regulation of dopamine biosynthesis in tuberoinfundibular dopaminergic (TIDA) neurons by adenosine 3',5'-cyclic monophosphate (cAMP) was investigated in the present study. Dopamine biosynthesis in TIDA neurons was estimated by the rate of in vitro dihydroxyphenylalanine (DOPA) accumulation in the median eminence of rat hypothalamic slices after incubation with a DOPA decarboxylase inhibitor. Addition of dibutyryl cAMP (db-cAMP) into medium caused an increase in the rate of DOPA accumulation in the median eminence in a dose- and time-dependent manner. 8-Bromo-cAMP also increased the rate of DOPA accumulation in the median eminence and cAMP was less effective than db-cAMP whereas neither adenosine nor sodium butyrate altered the rate of DOPA accumulation. An increase in the concentration of endogenous cAMP achieved by addition into medium of isobutylmethylxanthine, a phosphodiesterase inhibitor, or forskolin, an adenylate cyclase activator, was associated with an increase in the rate of DOPA accumulation in the median eminence, db-cAMP, however, had an almost negligible effect on the secretion of dopamine from the median eminence. The stimulatory effect of db-cAMP on DOPA accumulation in the median eminence was not dependent upon the presence of extracellular Ca 2+ and was not blocked by tetrodotoxin. Furthermore, the stimulation of DOPA accumulation in the median eminence induced by db-cAMP was additive with that induced by high potassium depolarization, which was Ca2+-dependent. These results suggest that dopamine biosynthesis in TIDA neurons is regulated by two distinct mechanisms, one of which involves cAMP, and another of which involves Ca 2+.
INTRODUCTION
T I D A neurons have not been known by reason of the limited availability of the tissue of the median emineu-
nence. H o w e v e r , studies, using a recently d e v e l o p e d
rons have cell bodies in the arcuate and periventricu-
Tuberoinfundibular dopaminergic ( T I D A )
method for estimation of in vitro dopamine biosyn-
lar nuclei of the hypothalamus and short axons which
thesis in the median e m i n e n c e 2, have suggested that
terminate in the vicinity of the hypophysial portal
depolarization-induced stimulation of d o p a m i n e bio-
vasculature in the median e m i n e n c e 12. D o p a m i n e re-
synthesis in T I D A neurons is mediated by Ca2+ in-
leased from the axon terminals of T I D A neurons in
flux through v o l t a g e - d e p e n d e n t Ca 2+ channels 4, and
the median e m i n e n c e exerts regulatory actions on
that Ca 2+ entering through the Ca e+ channels is ex-
the secretion of prolactin from the anterior pituitary
truded from neurons by a Na+-Ca 2+ exchange mecha-
gland via portal blood circulationS, 8.2° and possibly on
nism 3.
the release of luteinizing h o r m o n e - r e l e a s i n g hormone via axo-axonic connections 13.
The purpose of the present study was to investigate the regulatory role of adenosine 3',5'-cyclic mono-
Unlike other catecholaminergic neurons, the in-
phosphate ( c A M P ) , which is known together with
tracellular mechanisms for the regulation of dopamine biosynthesis or tyrosine hydroxylase activity in
Ca 2+ as an intracellular second messenger of various hormones and neurotransmitters, in d o p a m i n e bio-
Correspondence: J. Arita, Department of Physiology, Yokohama City University School of Medicine, 2-33 Urafune-cho. Minami-ku, Yokohama, Kanagawa 232, Japan. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
38 synthesis in T I D A neurons. The effect of exogenous and endogenous cAMP on dopamine biosynthesis in the median eminence of rat hypothalamic slices was examined. Furthermore, the relationship between Ca 2+ and cAMP in the regulation of dopamine biosynthesis in the median eminence was studied. MATERIALS AND METHODS
Hypothalamic slice preparation Female Wistar rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan), weighing 190-240 g, were used for experiments 2-3 weeks after ovariectomy. Sagittal hypothalamic slices were manually prepared using a razor blade and fine scissors, as described previously 2.
In vitro DOPA accumulation The standard medium used in the present study was composed of 116 mM NaC1, 5 mM KCI, 2.5 mM CaCI2, 1 mM MgSO 4, 1.25 mM KHzPO4, 26 mM NaHCO3, 10 mM glucose, 20 ~M tyrosine and 6 mg/1 phenol red and saturated with an atmosphere of 95% 02/5% CO 2, pH 7.4 at 37 °C. Hypothalamic slices were incubated for 3,4-dihydroxyphenylalanine (DOPA) accumulation in the median eminence according to the method described previously 2.4. Briefly, 3 or 5 hypothalamic slices were preincubated in 5 ml medium in a scintillation vial at 37 °C with continuous O2/CO 2 bubbling. After preincubation for 60-80 min, the medium was changed to 5 ml medium containing 10 mM 3-hydroxybenzylhydrazine (NSD 10t5), a D O P A decarboxylase inhibitor, to suppress the step of formation of dopamine from DOPA. The substances to be tested were added into the medium containing NSD 1015. CaZ+-free media were prepared by omission of 2.5 mM CaCI 2 and addition of 2 mM ethylene glycol bis(fl-aminoethylether)N,N,N',N'-tetraacetic acid ( E G T A ) into the medium containing NSD 1015. In the experiment using media with high K ÷ concentrations, equimolar amounts of KC1 were substituted for NaCI. Forskolin was initially dissolved in ethanol and diluted with medium so that the final concentration of ethanol was 1%. After a 1 h incubation with NSD 1015 (in the case of the experiment using media with high K + concentrations, 30 min incubation), the median eminence was
selectively dissected out from the hypothalamic slice with the aid of a microscope and fine scissors. The tissue dissected was homogenized in 100~d ol tl. 1 N perchloric acid containing 3 mM E D T A and 5 mM sodium metabisulfite. After adding 1 ng 3,4-dihydroxybenzylamine as an internal standard, the homogenate was sonified and centrifuged at IILI)01J g for 2 rain. The supernate was removed and stored at -80 °C for D O P A determination. The remaining pellet was analyzed for protein as described by Bradford 7.
In vitro dopamine release The standard medium containing 1 mM ascorbic acid was used for experiments of in vitro dopamine release from the median eminence. Ten median eminences dissected from rat hypothatami were placed in 2 ml medium in a scintillation vial and preincubated at 37 °C for 30 min with continuous O2/CO 2 bubbling. After the preincubation, the medium was changed sequentially at 1 h intervals by 2 ml normal medium, medium containing 5 mM dibutyryl cAMP (dbcAMP) and again normal medium. Finally, the median eminences were incubated for 15 min in medium containing 50 mM KCI. At the end of each incubation period, medium samples were centrifuged at 2000 g for 2 min. The supernatant (I).5 ml) was mixed with 0.05 ml 1 N perchloric acid containing 30 mM E D T A and 50 mM sodium metabisulfite and stored at -80 °C for dopamine determination.
Assay D O P A and dopamine concentrations were determined by high-performance liquid chromatography with electrochemical detection, as described previously2. The results of D O P A and dopamine are expressed as ng D O P A accumulated/mg protein/h and ng dopamine released/median eminence/h, respectively.
Reagents cAMP, db-cAMP, 8-bromo-cAMP, 3-isobutyl-1methylxanthine (IBMX) and tetrodotoxin were purchased from Sigma Chemicals (St. Louis; U.S.A.); NSD 1015, adenosine and sodium butyrate, from Wako Pure Chemicals (Osaka, Japan); E G T A and cycloheximide, from Nakarai Chemicals (Kyoto, Japan); forskolin, from Hoechst (Frankfurt. F. R: G. ).
39
Statistics Differences in the rate of D O P A accumulation in the median eminence between groups were statistically analyzed by one-way analysis of variance followed by Duncan's multiple range test 10. RESULTS
Effect of db-cAMP on D O P A accumulation in the median eminence The rate of in vitro D O P A accumulation in the median eminence was examined as a function of the concentration of exogenous db-cAMP. The rate of DOPA accumulation in the median eminence obtained from hypothalamic slices which were incubated for 1 h in medium containing NSD 1015 without db-cAMP was 7.5 + 0.4 ng/mg protein/h (mean + S.E.M., n = 3) (Fig. 1, left panel), db-cAMP in medium at concentrations up to 5 mM markedly raised the rate of D O P A accumulation in the median eminence in a dose-dependent manner, but no further rise in the rate of D O P A accumulation was seen at concentrations more than 5 raM. Time course for D O P A accumulation in the medi-
6
;
an eminence was examined in the presence of 5 mM db-cAMP (Fig. 1, right panel). Increasing the duration of incubation with db-cAMP within the range examined (0-120 min) produced a linear increase in D O P A accumulation in the median eminence. The amount of D O P A accumulated in the median eminence of hypothalamic slices which were incubated for 120 min with db-cAMP was approximately 5-fold greater than that without db-cAMP.
Dependence of db-cAMP-induced D O P A accumulation in the median eminence upon the c A M P moiety cAMP and its analogues had a stimulatory effect on D O P A accumulation in the median eminence (Fig. 2). 8-Bromo-cAMP at a concentration of 2 mM markedly increased the rate of D O P A accumulation in the median eminence (P < 0.01), but the rate of D O P A accumulation by 8-bromo-cAMP was significantly smaller than that by db-cAMP of the same concentration (P < 0.05). cAMP was less able to stimulate D O P A accumulation than db-cAMP or 8bromo-cAMP (P < 0.01). The stimulation of D O P A accumulation induced by db-cAMP was due to neither the butyrate moiety nor the adenosine moiety since incubation of hypothalamic slices with 2 mM sodium butyrate or adenosine had no effect on the rate of D O P A accumulation in the median eminence (P > 0.05). Effect of I B M X and forskolin on D O P A accumulation in the median eminence The presence of IBMX in medium at concentra-
UJ
o:1 i [rib-cAMP1
I;,.M
0'g'"' 30
60
90 '
120 ' rnm "
INCUBATION "rIME
Fig. 1. In vitro D O P A accumulation in the median eminence as a function of the concentration of d b - c A M P (left panel) and of incubation time (right panel). Hypothalamic slices were incubated at 37 °C in the presence of 10 m M NSD 1015 for 1 h with various concentrations of d b - c A M P (left panel) or for various incubation times with 5 m M d b - c A M P (right panel). T h e median eminence was dissected out from the hypothalamic slice after the incubation and h o m o g e n i z e d with 0.1 N perchloric acid containing 3 m M E D T A and 5 m M sodium metabisulfite. The a m o u n t of D O P A in the supernatant of the h o m o g e n a t e s was determined by high-performance liquid chromatography. Open circles and their vertical lines represent m e a n and S.E.M., respectively, based on 3 median eminences. The closed circle in right panel indicates the value obtained from hypothalamic slices which were incubated for 120 rain without db-cAMP.
~-
3C
0
~
2o x
10
0 O
CONTROL db-cAMP
8-BROMOcAMP
cAMP
SODIUM ADENOSINE BUTYRATE
Fig. 2. D e p e n d e n c e of db-cAMP-induced D O P A accumulation in the median eminence upon the c A M P moiety. Hypotbalamic slices were incubated in the presence of N S D 1015 for 1 h with one of c A M P , c A M P analogues and other substances at a concentration of 2 raM. Each column and its vertical line represent m e a n and S.E.M., respectively, based on 5 median eminences.
4O TABLE 1
ILl
rf"
z~
/
1
I/~--.I/
L
I
I-
0 c~ o[
~ _ o
i
0.0~
0.I
1~
0
[IBMX ]
I
I00 JaM
I0
[FORSKOLIN
(omparison of the eJ)ect of db-cA MP and high K -mdu~ ed ae polarization on DOPA accumulation attd dopammc t'o?lc('lllr~tion in the median eminence ~ff"hypothalamic s'/&v~ ~{/h'r incid)ation with NSD 1015 ttypothalamic slices were incubated m the plcscncc ,~1 Ht mM NSD 1015 for 30 min in either control medium, medium containing 5 mM db-cAMP or medium containing 5(~ mM K'. The supernatam of the homogenatc o! the median ;.minencc x~as analyzed for DOPA and dopaminc. Value~, are the mean .' S.E.M., based on 5 median eminences
}
Fig. 3. Effect of IBMX and forskolin on DOPA accumulation in the median eminence. Hypothalamic slices were incubated in the presence of NSD 1015 for 1 h with various concentrations of IBMX (left panel) or forskolin (right panel). Open circles and their vertical lines represent mean and S.E.M., respectively, based on 3 median eminences.
Treatment
Control 5 mM db-cAMP 50mMK ~
DO PA
DopanTine
accumulation
t onc{?/?traliO?l
(ng/mg protein/h)
(ng/mgprotein)
5.2 +_ (J.4 23.8 +_ 1.7" 59.9 _+ 4.5"
66.3 2 4.1 7{)5 :L 4,7 13.') ~: 0,4 +
* P < 0.01 vs control. tions r a n g i n g f r o m 0 to 1 m M p r o d u c e d a p r o g r e s s i v e i n c r e a s e in D O P A a c c u m u l a t i o n in the m e d i a n e m i -
directly m e a s u r e d by static incubation. T h e rate of
n e n c e (Fig. 3, left panel). F o r s k o l i n was also effec-
d o p a m i n e release f r o m the m e d i a n e m i n e n c e in nor-
tive in raising the r a t e of D O P A a c c u m u l a t i o n in the
mal m e d i u m was 0.048
m e d i a n e m i n e n c e at c o n c e n t r a t i o n s as little as 1/~M
nence/h (Fig. 4). C h a n g i n g n o r m a l m e d i u m to medi-
_+ 0,016 n g / m e d i a n emi-
(Fig. 3, right panel). A p l a t e a u in t h e r a t e of D O P A a c c u m u l a t i o n was s e e n w h e n the c o n c e n t r a t i o n of
,ol
forskolin was m o r e t h a n 1 0 # M . Effect o f d b - c A M P on d o p a m i n e release f r o m the median e m i n e n c e W h e n the effect of d b - c A M P and high K+ in m e d i -
I.t,I
"r
u m on D O P A a c c u m u l a t i o n in the m e d i a n e m i n e n c e
tl.J
x
was e x a m i n e d , it was n o t i c e d that t h e r e was a significant d i f f e r e n c e in d o p a m i n e c o n c e n t r a t i o n in the m e -
"'i
lad
~
O
x
3.5
1.0
dian e m i n e n c e after i n c u b a t i o n with N S D 1015 bet w e e n control and high K + - t r e a t e d slices ( P < 0.01) but no d i f f e r e n c e b e t w e e n c o n t r o l and d b - c A M P t r e a t e d slices ( P > 0.05) ( T a b l e I). Since the synthe-
0.5
sis of d o p a m i n e in the m e d i a n e m i n e n c e o f the hypot h a l a m i c slices was s u p p r e s s e d by N S D 1015, the decrease in the a m o u n t of d o p a m i n e c o n c e n t r a t i o n in the m e d i a n e m i n e n c e indicates the t u r n o v e r rate of d o p a m i n e in the m e d i a n e m i n e n c e . T h e result that d b - c A M P did n o t r e d u c e d o p a m i n e c o n c e n t r a t i o n in the m e d i a n e m i n e n c e s u g g e s t e d that d b - c A M P has a selective action on d o p a m i n e biosynthesis in the m e dian e m i n e n c e w h e r e a s high K + - i n d u c e d d e p o l a r i z a tion has a s t i m u l a t o r y action on b o t h biosynthesis and release of d o p a m i n e . T h e r e f o r e , the rate of in vitro release of d o p a m i n e f r o m the m e d i a n e m i n e n c e was
0 1 2 3 [CONTROL I db--cAMP [ CONTROl. IK÷t
hr
Fig. 4. Effect of db-cAMP and high K + on in vitrodopamine release from the median eminence. Ten median eminences were incubated sequentially each for 1 h in control medium (open column), medium containing 5 mM db-cAMP (dotted column) and again control medium and finally for 15 min in medium containing 50 mM K ÷ (dotted column). The media obtained at the end of each incubation period were analyzed for dopamine. Each column and its vertical line represent mean and S.E.M., respectively, based on 3 experiments,
41 um containing 5 m M d b - c A M P slightly increased the
,00[
rate of dopamine release. After an interposition of incubation with normal m e d i u m for 1 h, exposure of the median eminences to m e d i u m containing 50 mM K + caused an e n o r m o u s release of dopamine (3.762 + 0.283 ng/median eminence/h, P < 0.01).
Effect of Ca2+ removal and tetrodotoxin on dbcAMP-induced D O P A accumulation in the median eminence
la.i i,.-'T
75
z o I-.--J
'~
As shown in Fig. 5, the increase in the rate of D O P A accumulation in the median eminence by 5 mM d b - c A M P was not inhibited by Ca 2+ removal and 2 mM E G T A addition into medium (P > 0.05). Fur-
0 Q
25
thermore, 2 ~ M tetrodotoxin did not alter the effect of d b - c A M P on D O P A accumulation in the median eminence (P > 0.05).
0 CONTROL db-cAMP HIGHK+
Additive effect of db-cAMP and high K + on D O P A accumulation in the median eminence In order to examine the existence of an additive ef-
4or
db-cAMP +
HIGH K+
Fig. 6. Additive effect of db-cAMP and high K+ on DOPA accumulation in the median eminence. Hypothalamic slices were incubated in the presence of NSD 1015 for 30 min in either control medium (open column), medium containing 5 mM dbcAMP alone (dotted column), medium containing 50 mM K+ alone (shaded column) or medium containing both (closed column). Each column and its vertical line represent mean and S.E.M., respectively, based on 5 median eminences.
LLJ
fect of d b - c A M P and high K + on D O P A accumulation in the median eminence, 5 mM of d b - c A M P and 50 mM of K + were chosen, both of which were shown to be sufficient to elicit a maximal response in D O P A accumulation in the median eminence (Fig. 1 and the
rr
T
z o I--
,<
..J
"~
~" :3
K
0 O
3O
20
11]
CONTROL db-cAMP db-cAMP db-cAMP + Ca-FREE
+ TTX
Fig. 5. Effect of Ca2+ removal and tetrodotoxin (TTX) on dbcAMP-induced DOPA accumulation in the median eminence. Hypothalamic slices were incubated in the presence of NSD 1015 for 1 h in either control medium, medium containing 5 mM db-cAMP, medium containing 5 mM db-cAMP and 2 mM EGTA with CaC12 omission or medium containing db-cAMP and 2,uM tetrodotoxin. Each column and its vertical line represent mean and S.E.M., respectively, based on 5 median eminences.
previous study4). The rate of D O P A accumulation in the median eminence was raised 380 or 1100% in media containing 5 mM d b - c A M P or 50 mM K +, respectively (P < 0.01) (Fig. 6). The rate of D O P A accumulation in medium containing both 5 mM d b - c A M P and 50 m M K + was significantly greater than that in m e d i u m containing 50 m M K + alone (P < 0.01) and was raised 1790% as compared with that in control medium.
Effect of cycloheximide on db-cAMP-induced DOPA accumulation in the median eminence Cycloheximide at a concentration of 10/~M had no effect on 5 mM d b - c A M P - i n d u c e d stimulation of D O P A accumulation in the median eminence (dbc A M P alone, 51.9 + 4.9 (n = 5); d b - c A M P plus cy-
42 cloheximide, 5 0 . 7 + 2.1 (n = 5) ng/mg protein&, P > 0.05). DISCUSSION The present study demonstrates that exogenous db-cAMP increased the rate of in vitro D O P A accumulation in the median eminence in a dose- and timedependent manner. The stimulatory effect of dbc A M P on D O P A accumulation has been shown to be due to the c A M P moiety of the analogue since neither adenosine nor sodium butyrate altered the rate of D O P A accumulation whereas the other c A M P analogue 8-bromo-cAMP and c A M P itself were effective in stimulating D O P A accumulation. These results are suggestive of the view that c A M P plays a stimulatory role in the regulation of dopamine biosynthesis in T I D A neurons. The stimulatory role of c A M P is further supported by the result that an increase in the concentration of endogenous c A M P by treatment with I B M X or forskolin produced an increase in the rate of D O P A accumulation in the median eminence. I B M X probably inhibits the phosphodiesterase which catalyzes degradation of cAMP~S, resulting in an increase in intracellular c A M P concentration. On the other hand, forskolin is considered to activate the catalytic subunit of adenylate cyclase 23 or act at the guanine nucteotide regulatory protein of adenylate cyclase ~,1~. Since there are many examples which show the close causal relationship between Ca > system and cAMP system in the regulation of cell functions (for a review, see refs. 6 and 22), the relationship between Ca ",+ and c A M P actions on dopamine biosynthesis in T I D A neurons was studied. The result of the failure of Ca 2÷ omission and E G T A addition into medium to block db-cAMP-induced D O P A accumulation in the median eminence indicates that the stimulation of dopamine biosynthesis in T I D A neurons by c A M P does not require extracellular Ca 2÷. There still remains the possibility that the action of c A M P may be mediated by an increase in the concentration of intracellular Ca2+ resulting from Ca2+ release into the cytoplasm from intracellular reservoirs 6.22. If this is the case, db-cAMP should also stimulate the release of dopamine from the median eminence since the dopamine release is triggered by the increase in intracellular C a > concentration. The present study, however,
demonstrates that d b - c A M P had ahnosl negligible effect on m vitro dopamine release from the median eminence at the concentration sufficient to cause :~ prominent increase in in vitro D O P A accumulation m the median eminence, r h u s . Ca : ' mediation ot cAMP action on dopamine biosynthesis in T I D A neurons seems unlikely. On the other hand, it might be possible that a rise in intraccllular ¢'aZ- concentration induces either the activanon of adenylate cyclase or the inhibition of phosphodiesterasc, both of which, in turn, result in a rise in the intracellulm cAMP concentration. If the stimulatory action of Ca 2+ on dopamine biosynthesis in the median eminence is mediated by cAMP, there should be no summation of their actions. However, in fact, the effect of c A M P on D O P A accumulation in the median eminence has been shown to be additive with that of K -~. Taken together with the previous finding that high K +-induced dopamine biosynthesis in T I D A neurons was highly dependent upon Ca z~ influx through voltage-dependent Ca 2+ channels 4, this result is inconsistent with the possibility of c A M P mediation of Ca:+-dependent dopamine biosynthesis in T l I ) A neurons. Collectively, regarding the regulation of dopamine biosynthesis in T I D A neurons, there seems an independence between the mechanisms by which Ca > and c A M P exert their effects On the basis of the results that the stimulatory action of db-cAMP on D O P A accumulation in the median eminence was blocked by neither the absence of extraceilular Ca > nor tetrodotoxin and was additive with the action of high K+-induced depolarization, it seems likely that db-cAMP acts directly on T I D A neurons, more specifically, on the axon terminals of T I D A neurons in the median eminence, 1o stimulate dopamine biosynthesis. As for nigrostriatal dopaminergic neurons, it has been shown that c A M P increased tyrosine hydroxylase activity m slices and cell-free systems prepared from the striatum with an alteration in the kinetics of either decreased K,, of tyrosine hydroxylase for cofactor N,~f or increased Vmax1,21. Although there are no data on the effect of c A M P on the kinetics of tyrosine hydroxylase in T I D A neurons, it is reasonable to speculate, by analogy with nigrostriatal dopaminergic neurons, that,. also in T I D A neurons, c A M P stimulates dopamine biosynthesis by increasing tyrosine hydroxylase activity. The result that the protein synthesis inhibitor.
43 cycloheximide failed to block the stimulatory action of db-cAMP on D O P A accumulation in the median eminence indicates that cAMP-induced dopamine biosynthesis in T I D A neurons is not dependent upon protein synthesis. Therefore, it is suggested that cAMP-induced dopamine biosynthesis in T I D A neurons is not due to induction but is due to activation of tyrosine hydroxylase. In conclusion, cAMP stimulates dopamine biosynthesis in TIDA neurons independently from Ca 2+ action. cAMP may activate cAMP-dependent protein kinase or suppress protein phosphatase, the substrate of which is the phosphoprotein tyrosine hydroxylase, as suggested in other catecholaminergic neurons t 1,17,19.24. Although the stimulus for Ca2+-in-
duced dopamine biosynthesis in TIDA neurons is indicated to be membrane depolarization, it is unknown what neurotransmitter(s) and hormone(s) activate or inhibit adenylate cyclase in axon terminals of TIDA neurons, and this question remains to be investigated in future studies.
REFERENCES
11 Edelman, A.M., Raese, J.D., Lazar, M.A. and Barchas, J.D., Tyrosine hydroxylase: studies on the phosphorylation of a purified preparation of the brain enzyme by the cyclic AMP-dependent protein kinase, J. Pharmacol. Exp. Ther.. 216 (1981) 647-653. 12 Fuxe, K., Cellular localization of monoamines in the median eminence and in the infundibular stein of some mammals, Acta Physiol. Scand., 58 (1963) 383-384. 13 Fuxe, K., H6kfelt, T., L6fstr6m, A., Johansson, O., Agna+ ti, L., Everin, B., Goldstein, M., Jeffcoatc, S., White, N., Eneroth, P., Gustafson, J.-A. and Skett, P., On the role of neurotransmitters and hypothalamic hormones and their interactions in hypothalamic and extrahypothalamic control of pituitary function and sexual behavior. In F. Naftotin, K.J. Ryan and I.J. Davies (Eds.), Subcelhdar Mechanisms in Reproductive Neuroendocrinology+ Elsevier, Amsterdam, 1976, pp. 193-246. 14 Goldstein, M., Bronaugh, R.L., Ebstein, B. and Roberge, C., Stimulation of tyrosine hydroxylase activity by cyclic AMP in synaptosomes and in soluble striatal enzyme preparation, Brain Research, 109 (1976) 563-574. 15 Harris, J.E., Morgenroth IlI. V.H.. Roth. R.H. and Baldessarini, R.J., Regulation of catecholamine synthesis in the rat brain in vitro by cyclic AMP, Nature (London), 252 (1974) 156-158. 16 Insel+ P.A., Stengel, D., Ferry, N. and Hanoune, J., Regulation of adenylate cyclase of human platelet membranes by forskolin+ J. Biol. Chem. 257 (1982)7485-7490. 17 Joh, T.H., Park, D.H. and Reis, D.J.+ Direct phosphorylation of brain tyrosine hydroxylase by cyclic AMP-dependent protein kinase: mechanism of enzyme activation, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 4744-4748. 18 Kramer, G.L., Garst, J.E., Mitchel. S.S. and Wells, J.N., Selective inhibition of cyclic nucleotide phosphodiesterases by analogues of 1-methyl-3-isobutylxanthine, BiochemistO,, 16 (1977) 3316-3321. 19 Lovenberg, W., Bruckwick+ E.A. and Hanbauer, I.. ATP, cyclic AMP, and magnesium increase the affinity of rat striatal tyrosine hydroxylase for its cofactor. Proc. Natl. Acad. Sci. U.S.A.. 72 (1975) 2955 2958.
1 Anagnoste, B., Shirron, C., Friedman, E. and Goldstein, M., Effect of dibutyryl cyclic adenosine monophosphate on a~C-dopamine biosynthesis in rat brain striatal slices, J. Pharmacol. Exp. Ther., 191 (1974)370-376. 2 Arita, J. and Kimura, F., Estimation of in vitro activity of tuberoinfundibular dopaminergic neurons by measurement of DOPA synthesis in the median eminence of hypothalamic slices, Neuroendocrinology, 39 (1984) 524-529. 3 Arita, J. and Kimura, F., In vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices: possible involvement of a Na+-Ca 2+ exchange mechanism, Brain Research, 338 (1985) 384-386. 4 Arita, J. and Kimura, F., In vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices: involvement of voltage-dependent Ca 2+ channels, Brain Research, 347 (1985) 299-305. 5 Ben-Jonathan, N., Oliver, C., Weiner, H.J., Mical, R.S. and Porter, J.C., Dopamine in hypophysial portal plasma of the rat during the estrous cycle and throughout pregnancy, Endocrinology, 100 (1977) 452-458. 6 Berridge, M.J., Modulation of nervous activity by cyclic nucleotides and calcium. In F.O. Schmitt and F.G. Worden (Eds.), The Neurosciences, Fourth Study Program, MIT Press, Cambridge, 1979, pp. 873-889. 7 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 8 Carom M.G., Beaulieu, M., Raymond, V., Gagn6, B., Drouin, J., Lefkowitz, R.J. and Labrie, F., Dopaminergic receptors in the anterior pituitary gland, J. Biol. Chem., 253 (1978) 2244-2253. 9 Darfler, F.J., Mahan, L.C., Koachman, A.M. and Insel+ P.A., Stimulation by forskolin of intact $49 lymphoma cells involves the nucleotide regulatory protein of adenylate cyclase, J. Biol. Chem., 257 (1982) 11901-11905. 10 Duncan, D.B., Multiple range and multiple Ftests, Biometrics, 11 (1955) 1-25.
ACKNOWLEDGEMENTS
The authors thank Nippon Kayaku (Tokyo, Japan) and Hoechst AG for the generous gift of forskolin. This work was supported by a Grant-in-Aid for Encouragement of Young Scientist of the Ministry of Education, Science and Culture, Japan.
44 20 MacLeod, R.M., Fontham, E.H. and Lehmeyer, J.E., Prolactin and growth hormone production as influenced by catecholamines and agents that affect brain catecholamines, Neuroendocrinology, 6 (1970) 283-294. 21 Pollock, R.J., Kapatos, G. and Kaufman, S., Effect of cyclic AMP-dependent protein phosphorylating conditions on the pH-dependent activity of tyrosine hydroxylase from beef and rat striata, J. Neurochem., 37 (1981) 855-860. 22 Rasmussen, H. and Goodman, D.B.P., Relationships between calcium and cyclic nucleotides in cell activation, Physiol. Rev., 57 (1977) 421-.5(19.
23 Seamon, K. and Daly, J.W., Activation ol adcnylatc c~. clase by the diterpene forskolin does not require the gm~nine nucleotide regulatory protein, J. Bio!. ~'hem., 256 ( 1981) 9799 - 9801. 24 Yamauchi, T. and Fujisawa, 1t.. Regulation ~f bovine adrenal tyrosine 3-monooxygenase by phosphorylation-dc~ phosphorylation reaction, catalyzed by adenosine 3':5'. monophosphate-dependent protein kinasc and phospho.. protein phosphatase, J. Biol. ('hem., "54 (107~)) 6408-6413.
37
Elsevier BRE 11712
Adenosine 3' ,5'-Cyclic Monophosphate Stimulates Dopamine Biosynthesis in the Median Eminence of Rat Hypothalamic Slices JUN ARITA and FUKUKO KIMURA
Department of Physiology, Yokohama City University School of Medicine, Yokohama 232 (Japan) (Accepted October 2nd, 1985)
Key words: tuberoinfundibular dopaminergic neuron - - dihydroxyphenylalanine - - hypothalamic slice - cyclic adenosine monophosphate - - forskolin
The regulation of dopamine biosynthesis in tuberoinfundibular dopaminergic (TIDA) neurons by adenosine 3',5'-cyclic monophosphate (cAMP) was investigated in the present study. Dopamine biosynthesis in TIDA neurons was estimated by the rate of in vitro dihydroxyphenylalanine (DOPA) accumulation in the median eminence of rat hypothalamic slices after incubation with a DOPA decarboxylase inhibitor. Addition of dibutyryl cAMP (db-cAMP) into medium caused an increase in the rate of DOPA accumulation in the median eminence in a dose- and time-dependent manner. 8-Bromo-cAMP also increased the rate of DOPA accumulation in the median eminence and cAMP was less effective than db-cAMP whereas neither adenosine nor sodium butyrate altered the rate of DOPA accumulation. An increase in the concentration of endogenous cAMP achieved by addition into medium of isobutylmethylxanthine, a phosphodiesterase inhibitor, or forskolin, an adenylate cyclase activator, was associated with an increase in the rate of DOPA accumulation in the median eminence, db-cAMP, however, had an almost negligible effect on the secretion of dopamine from the median eminence. The stimulatory effect of db-cAMP on DOPA accumulation in the median eminence was not dependent upon the presence of extracellular Ca 2+ and was not blocked by tetrodotoxin. Furthermore, the stimulation of DOPA accumulation in the median eminence induced by db-cAMP was additive with that induced by high potassium depolarization, which was Ca2+-dependent. These results suggest that dopamine biosynthesis in TIDA neurons is regulated by two distinct mechanisms, one of which involves cAMP, and another of which involves Ca 2+.
INTRODUCTION
T I D A neurons have not been known by reason of the limited availability of the tissue of the median emineu-
nence. H o w e v e r , studies, using a recently d e v e l o p e d
rons have cell bodies in the arcuate and periventricu-
Tuberoinfundibular dopaminergic ( T I D A )
method for estimation of in vitro dopamine biosyn-
lar nuclei of the hypothalamus and short axons which
thesis in the median e m i n e n c e 2, have suggested that
terminate in the vicinity of the hypophysial portal
depolarization-induced stimulation of d o p a m i n e bio-
vasculature in the median e m i n e n c e 12. D o p a m i n e re-
synthesis in T I D A neurons is mediated by Ca2+ in-
leased from the axon terminals of T I D A neurons in
flux through v o l t a g e - d e p e n d e n t Ca 2+ channels 4, and
the median e m i n e n c e exerts regulatory actions on
that Ca 2+ entering through the Ca e+ channels is ex-
the secretion of prolactin from the anterior pituitary
truded from neurons by a Na+-Ca 2+ exchange mecha-
gland via portal blood circulationS, 8.2° and possibly on
nism 3.
the release of luteinizing h o r m o n e - r e l e a s i n g hormone via axo-axonic connections 13.
The purpose of the present study was to investigate the regulatory role of adenosine 3',5'-cyclic mono-
Unlike other catecholaminergic neurons, the in-
phosphate ( c A M P ) , which is known together with
tracellular mechanisms for the regulation of dopamine biosynthesis or tyrosine hydroxylase activity in
Ca 2+ as an intracellular second messenger of various hormones and neurotransmitters, in d o p a m i n e bio-
Correspondence: J. Arita, Department of Physiology, Yokohama City University School of Medicine, 2-33 Urafune-cho. Minami-ku, Yokohama, Kanagawa 232, Japan. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
38 synthesis in T I D A neurons. The effect of exogenous and endogenous cAMP on dopamine biosynthesis in the median eminence of rat hypothalamic slices was examined. Furthermore, the relationship between Ca 2+ and cAMP in the regulation of dopamine biosynthesis in the median eminence was studied. MATERIALS AND METHODS
Hypothalamic slice preparation Female Wistar rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan), weighing 190-240 g, were used for experiments 2-3 weeks after ovariectomy. Sagittal hypothalamic slices were manually prepared using a razor blade and fine scissors, as described previously 2.
In vitro DOPA accumulation The standard medium used in the present study was composed of 116 mM NaC1, 5 mM KCI, 2.5 mM CaCI2, 1 mM MgSO 4, 1.25 mM KHzPO4, 26 mM NaHCO3, 10 mM glucose, 20 ~M tyrosine and 6 mg/1 phenol red and saturated with an atmosphere of 95% 02/5% CO 2, pH 7.4 at 37 °C. Hypothalamic slices were incubated for 3,4-dihydroxyphenylalanine (DOPA) accumulation in the median eminence according to the method described previously 2.4. Briefly, 3 or 5 hypothalamic slices were preincubated in 5 ml medium in a scintillation vial at 37 °C with continuous O2/CO 2 bubbling. After preincubation for 60-80 min, the medium was changed to 5 ml medium containing 10 mM 3-hydroxybenzylhydrazine (NSD 10t5), a D O P A decarboxylase inhibitor, to suppress the step of formation of dopamine from DOPA. The substances to be tested were added into the medium containing NSD 1015. CaZ+-free media were prepared by omission of 2.5 mM CaCI 2 and addition of 2 mM ethylene glycol bis(fl-aminoethylether)N,N,N',N'-tetraacetic acid ( E G T A ) into the medium containing NSD 1015. In the experiment using media with high K ÷ concentrations, equimolar amounts of KC1 were substituted for NaCI. Forskolin was initially dissolved in ethanol and diluted with medium so that the final concentration of ethanol was 1%. After a 1 h incubation with NSD 1015 (in the case of the experiment using media with high K + concentrations, 30 min incubation), the median eminence was
selectively dissected out from the hypothalamic slice with the aid of a microscope and fine scissors. The tissue dissected was homogenized in 100~d ol tl. 1 N perchloric acid containing 3 mM E D T A and 5 mM sodium metabisulfite. After adding 1 ng 3,4-dihydroxybenzylamine as an internal standard, the homogenate was sonified and centrifuged at IILI)01J g for 2 rain. The supernate was removed and stored at -80 °C for D O P A determination. The remaining pellet was analyzed for protein as described by Bradford 7.
In vitro dopamine release The standard medium containing 1 mM ascorbic acid was used for experiments of in vitro dopamine release from the median eminence. Ten median eminences dissected from rat hypothatami were placed in 2 ml medium in a scintillation vial and preincubated at 37 °C for 30 min with continuous O2/CO 2 bubbling. After the preincubation, the medium was changed sequentially at 1 h intervals by 2 ml normal medium, medium containing 5 mM dibutyryl cAMP (dbcAMP) and again normal medium. Finally, the median eminences were incubated for 15 min in medium containing 50 mM KCI. At the end of each incubation period, medium samples were centrifuged at 2000 g for 2 min. The supernatant (I).5 ml) was mixed with 0.05 ml 1 N perchloric acid containing 30 mM E D T A and 50 mM sodium metabisulfite and stored at -80 °C for dopamine determination.
Assay D O P A and dopamine concentrations were determined by high-performance liquid chromatography with electrochemical detection, as described previously2. The results of D O P A and dopamine are expressed as ng D O P A accumulated/mg protein/h and ng dopamine released/median eminence/h, respectively.
Reagents cAMP, db-cAMP, 8-bromo-cAMP, 3-isobutyl-1methylxanthine (IBMX) and tetrodotoxin were purchased from Sigma Chemicals (St. Louis; U.S.A.); NSD 1015, adenosine and sodium butyrate, from Wako Pure Chemicals (Osaka, Japan); E G T A and cycloheximide, from Nakarai Chemicals (Kyoto, Japan); forskolin, from Hoechst (Frankfurt. F. R: G. ).
39
Statistics Differences in the rate of D O P A accumulation in the median eminence between groups were statistically analyzed by one-way analysis of variance followed by Duncan's multiple range test 10. RESULTS
Effect of db-cAMP on D O P A accumulation in the median eminence The rate of in vitro D O P A accumulation in the median eminence was examined as a function of the concentration of exogenous db-cAMP. The rate of DOPA accumulation in the median eminence obtained from hypothalamic slices which were incubated for 1 h in medium containing NSD 1015 without db-cAMP was 7.5 + 0.4 ng/mg protein/h (mean + S.E.M., n = 3) (Fig. 1, left panel), db-cAMP in medium at concentrations up to 5 mM markedly raised the rate of D O P A accumulation in the median eminence in a dose-dependent manner, but no further rise in the rate of D O P A accumulation was seen at concentrations more than 5 raM. Time course for D O P A accumulation in the medi-
6
;
an eminence was examined in the presence of 5 mM db-cAMP (Fig. 1, right panel). Increasing the duration of incubation with db-cAMP within the range examined (0-120 min) produced a linear increase in D O P A accumulation in the median eminence. The amount of D O P A accumulated in the median eminence of hypothalamic slices which were incubated for 120 min with db-cAMP was approximately 5-fold greater than that without db-cAMP.
Dependence of db-cAMP-induced D O P A accumulation in the median eminence upon the c A M P moiety cAMP and its analogues had a stimulatory effect on D O P A accumulation in the median eminence (Fig. 2). 8-Bromo-cAMP at a concentration of 2 mM markedly increased the rate of D O P A accumulation in the median eminence (P < 0.01), but the rate of D O P A accumulation by 8-bromo-cAMP was significantly smaller than that by db-cAMP of the same concentration (P < 0.05). cAMP was less able to stimulate D O P A accumulation than db-cAMP or 8bromo-cAMP (P < 0.01). The stimulation of D O P A accumulation induced by db-cAMP was due to neither the butyrate moiety nor the adenosine moiety since incubation of hypothalamic slices with 2 mM sodium butyrate or adenosine had no effect on the rate of D O P A accumulation in the median eminence (P > 0.05). Effect of I B M X and forskolin on D O P A accumulation in the median eminence The presence of IBMX in medium at concentra-
UJ
o:1 i [rib-cAMP1
I;,.M
0'g'"' 30
60
90 '
120 ' rnm "
INCUBATION "rIME
Fig. 1. In vitro D O P A accumulation in the median eminence as a function of the concentration of d b - c A M P (left panel) and of incubation time (right panel). Hypothalamic slices were incubated at 37 °C in the presence of 10 m M NSD 1015 for 1 h with various concentrations of d b - c A M P (left panel) or for various incubation times with 5 m M d b - c A M P (right panel). T h e median eminence was dissected out from the hypothalamic slice after the incubation and h o m o g e n i z e d with 0.1 N perchloric acid containing 3 m M E D T A and 5 m M sodium metabisulfite. The a m o u n t of D O P A in the supernatant of the h o m o g e n a t e s was determined by high-performance liquid chromatography. Open circles and their vertical lines represent m e a n and S.E.M., respectively, based on 3 median eminences. The closed circle in right panel indicates the value obtained from hypothalamic slices which were incubated for 120 rain without db-cAMP.
~-
3C
0
~
2o x
10
0 O
CONTROL db-cAMP
8-BROMOcAMP
cAMP
SODIUM ADENOSINE BUTYRATE
Fig. 2. D e p e n d e n c e of db-cAMP-induced D O P A accumulation in the median eminence upon the c A M P moiety. Hypotbalamic slices were incubated in the presence of N S D 1015 for 1 h with one of c A M P , c A M P analogues and other substances at a concentration of 2 raM. Each column and its vertical line represent m e a n and S.E.M., respectively, based on 5 median eminences.
4O TABLE 1
ILl
rf"
z~
/
1
I/~--.I/
L
I
I-
0 c~ o[
~ _ o
i
0.0~
0.I
1~
0
[IBMX ]
I
I00 JaM
I0
[FORSKOLIN
(omparison of the eJ)ect of db-cA MP and high K -mdu~ ed ae polarization on DOPA accumulation attd dopammc t'o?lc('lllr~tion in the median eminence ~ff"hypothalamic s'/&v~ ~{/h'r incid)ation with NSD 1015 ttypothalamic slices were incubated m the plcscncc ,~1 Ht mM NSD 1015 for 30 min in either control medium, medium containing 5 mM db-cAMP or medium containing 5(~ mM K'. The supernatam of the homogenatc o! the median ;.minencc x~as analyzed for DOPA and dopaminc. Value~, are the mean .' S.E.M., based on 5 median eminences
}
Fig. 3. Effect of IBMX and forskolin on DOPA accumulation in the median eminence. Hypothalamic slices were incubated in the presence of NSD 1015 for 1 h with various concentrations of IBMX (left panel) or forskolin (right panel). Open circles and their vertical lines represent mean and S.E.M., respectively, based on 3 median eminences.
Treatment
Control 5 mM db-cAMP 50mMK ~
DO PA
DopanTine
accumulation
t onc{?/?traliO?l
(ng/mg protein/h)
(ng/mgprotein)
5.2 +_ (J.4 23.8 +_ 1.7" 59.9 _+ 4.5"
66.3 2 4.1 7{)5 :L 4,7 13.') ~: 0,4 +
* P < 0.01 vs control. tions r a n g i n g f r o m 0 to 1 m M p r o d u c e d a p r o g r e s s i v e i n c r e a s e in D O P A a c c u m u l a t i o n in the m e d i a n e m i -
directly m e a s u r e d by static incubation. T h e rate of
n e n c e (Fig. 3, left panel). F o r s k o l i n was also effec-
d o p a m i n e release f r o m the m e d i a n e m i n e n c e in nor-
tive in raising the r a t e of D O P A a c c u m u l a t i o n in the
mal m e d i u m was 0.048
m e d i a n e m i n e n c e at c o n c e n t r a t i o n s as little as 1/~M
nence/h (Fig. 4). C h a n g i n g n o r m a l m e d i u m to medi-
_+ 0,016 n g / m e d i a n emi-
(Fig. 3, right panel). A p l a t e a u in t h e r a t e of D O P A a c c u m u l a t i o n was s e e n w h e n the c o n c e n t r a t i o n of
,ol
forskolin was m o r e t h a n 1 0 # M . Effect o f d b - c A M P on d o p a m i n e release f r o m the median e m i n e n c e W h e n the effect of d b - c A M P and high K+ in m e d i -
I.t,I
"r
u m on D O P A a c c u m u l a t i o n in the m e d i a n e m i n e n c e
tl.J
x
was e x a m i n e d , it was n o t i c e d that t h e r e was a significant d i f f e r e n c e in d o p a m i n e c o n c e n t r a t i o n in the m e -
"'i
lad
~
O
x
3.5
1.0
dian e m i n e n c e after i n c u b a t i o n with N S D 1015 bet w e e n control and high K + - t r e a t e d slices ( P < 0.01) but no d i f f e r e n c e b e t w e e n c o n t r o l and d b - c A M P t r e a t e d slices ( P > 0.05) ( T a b l e I). Since the synthe-
0.5
sis of d o p a m i n e in the m e d i a n e m i n e n c e o f the hypot h a l a m i c slices was s u p p r e s s e d by N S D 1015, the decrease in the a m o u n t of d o p a m i n e c o n c e n t r a t i o n in the m e d i a n e m i n e n c e indicates the t u r n o v e r rate of d o p a m i n e in the m e d i a n e m i n e n c e . T h e result that d b - c A M P did n o t r e d u c e d o p a m i n e c o n c e n t r a t i o n in the m e d i a n e m i n e n c e s u g g e s t e d that d b - c A M P has a selective action on d o p a m i n e biosynthesis in the m e dian e m i n e n c e w h e r e a s high K + - i n d u c e d d e p o l a r i z a tion has a s t i m u l a t o r y action on b o t h biosynthesis and release of d o p a m i n e . T h e r e f o r e , the rate of in vitro release of d o p a m i n e f r o m the m e d i a n e m i n e n c e was
0 1 2 3 [CONTROL I db--cAMP [ CONTROl. IK÷t
hr
Fig. 4. Effect of db-cAMP and high K + on in vitrodopamine release from the median eminence. Ten median eminences were incubated sequentially each for 1 h in control medium (open column), medium containing 5 mM db-cAMP (dotted column) and again control medium and finally for 15 min in medium containing 50 mM K ÷ (dotted column). The media obtained at the end of each incubation period were analyzed for dopamine. Each column and its vertical line represent mean and S.E.M., respectively, based on 3 experiments,
41 um containing 5 m M d b - c A M P slightly increased the
,00[
rate of dopamine release. After an interposition of incubation with normal m e d i u m for 1 h, exposure of the median eminences to m e d i u m containing 50 mM K + caused an e n o r m o u s release of dopamine (3.762 + 0.283 ng/median eminence/h, P < 0.01).
Effect of Ca2+ removal and tetrodotoxin on dbcAMP-induced D O P A accumulation in the median eminence
la.i i,.-'T
75
z o I-.--J
'~
As shown in Fig. 5, the increase in the rate of D O P A accumulation in the median eminence by 5 mM d b - c A M P was not inhibited by Ca 2+ removal and 2 mM E G T A addition into medium (P > 0.05). Fur-
0 Q
25
thermore, 2 ~ M tetrodotoxin did not alter the effect of d b - c A M P on D O P A accumulation in the median eminence (P > 0.05).
0 CONTROL db-cAMP HIGHK+
Additive effect of db-cAMP and high K + on D O P A accumulation in the median eminence In order to examine the existence of an additive ef-
4or
db-cAMP +
HIGH K+
Fig. 6. Additive effect of db-cAMP and high K+ on DOPA accumulation in the median eminence. Hypothalamic slices were incubated in the presence of NSD 1015 for 30 min in either control medium (open column), medium containing 5 mM dbcAMP alone (dotted column), medium containing 50 mM K+ alone (shaded column) or medium containing both (closed column). Each column and its vertical line represent mean and S.E.M., respectively, based on 5 median eminences.
LLJ
fect of d b - c A M P and high K + on D O P A accumulation in the median eminence, 5 mM of d b - c A M P and 50 mM of K + were chosen, both of which were shown to be sufficient to elicit a maximal response in D O P A accumulation in the median eminence (Fig. 1 and the
rr
T
z o I--
,<
..J
"~
~" :3
K
0 O
3O
20
11]
CONTROL db-cAMP db-cAMP db-cAMP + Ca-FREE
+ TTX
Fig. 5. Effect of Ca2+ removal and tetrodotoxin (TTX) on dbcAMP-induced DOPA accumulation in the median eminence. Hypothalamic slices were incubated in the presence of NSD 1015 for 1 h in either control medium, medium containing 5 mM db-cAMP, medium containing 5 mM db-cAMP and 2 mM EGTA with CaC12 omission or medium containing db-cAMP and 2,uM tetrodotoxin. Each column and its vertical line represent mean and S.E.M., respectively, based on 5 median eminences.
previous study4). The rate of D O P A accumulation in the median eminence was raised 380 or 1100% in media containing 5 mM d b - c A M P or 50 mM K +, respectively (P < 0.01) (Fig. 6). The rate of D O P A accumulation in medium containing both 5 mM d b - c A M P and 50 m M K + was significantly greater than that in m e d i u m containing 50 m M K + alone (P < 0.01) and was raised 1790% as compared with that in control medium.
Effect of cycloheximide on db-cAMP-induced DOPA accumulation in the median eminence Cycloheximide at a concentration of 10/~M had no effect on 5 mM d b - c A M P - i n d u c e d stimulation of D O P A accumulation in the median eminence (dbc A M P alone, 51.9 + 4.9 (n = 5); d b - c A M P plus cy-
42 cloheximide, 5 0 . 7 + 2.1 (n = 5) ng/mg protein&, P > 0.05). DISCUSSION The present study demonstrates that exogenous db-cAMP increased the rate of in vitro D O P A accumulation in the median eminence in a dose- and timedependent manner. The stimulatory effect of dbc A M P on D O P A accumulation has been shown to be due to the c A M P moiety of the analogue since neither adenosine nor sodium butyrate altered the rate of D O P A accumulation whereas the other c A M P analogue 8-bromo-cAMP and c A M P itself were effective in stimulating D O P A accumulation. These results are suggestive of the view that c A M P plays a stimulatory role in the regulation of dopamine biosynthesis in T I D A neurons. The stimulatory role of c A M P is further supported by the result that an increase in the concentration of endogenous c A M P by treatment with I B M X or forskolin produced an increase in the rate of D O P A accumulation in the median eminence. I B M X probably inhibits the phosphodiesterase which catalyzes degradation of cAMP~S, resulting in an increase in intracellular c A M P concentration. On the other hand, forskolin is considered to activate the catalytic subunit of adenylate cyclase 23 or act at the guanine nucteotide regulatory protein of adenylate cyclase ~,1~. Since there are many examples which show the close causal relationship between Ca > system and cAMP system in the regulation of cell functions (for a review, see refs. 6 and 22), the relationship between Ca ",+ and c A M P actions on dopamine biosynthesis in T I D A neurons was studied. The result of the failure of Ca 2÷ omission and E G T A addition into medium to block db-cAMP-induced D O P A accumulation in the median eminence indicates that the stimulation of dopamine biosynthesis in T I D A neurons by c A M P does not require extracellular Ca 2÷. There still remains the possibility that the action of c A M P may be mediated by an increase in the concentration of intracellular Ca2+ resulting from Ca2+ release into the cytoplasm from intracellular reservoirs 6.22. If this is the case, db-cAMP should also stimulate the release of dopamine from the median eminence since the dopamine release is triggered by the increase in intracellular C a > concentration. The present study, however,
demonstrates that d b - c A M P had ahnosl negligible effect on m vitro dopamine release from the median eminence at the concentration sufficient to cause :~ prominent increase in in vitro D O P A accumulation m the median eminence, r h u s . Ca : ' mediation ot cAMP action on dopamine biosynthesis in T I D A neurons seems unlikely. On the other hand, it might be possible that a rise in intraccllular ¢'aZ- concentration induces either the activanon of adenylate cyclase or the inhibition of phosphodiesterasc, both of which, in turn, result in a rise in the intracellulm cAMP concentration. If the stimulatory action of Ca 2+ on dopamine biosynthesis in the median eminence is mediated by cAMP, there should be no summation of their actions. However, in fact, the effect of c A M P on D O P A accumulation in the median eminence has been shown to be additive with that of K -~. Taken together with the previous finding that high K +-induced dopamine biosynthesis in T I D A neurons was highly dependent upon Ca z~ influx through voltage-dependent Ca 2+ channels 4, this result is inconsistent with the possibility of c A M P mediation of Ca:+-dependent dopamine biosynthesis in T l I ) A neurons. Collectively, regarding the regulation of dopamine biosynthesis in T I D A neurons, there seems an independence between the mechanisms by which Ca > and c A M P exert their effects On the basis of the results that the stimulatory action of db-cAMP on D O P A accumulation in the median eminence was blocked by neither the absence of extraceilular Ca > nor tetrodotoxin and was additive with the action of high K+-induced depolarization, it seems likely that db-cAMP acts directly on T I D A neurons, more specifically, on the axon terminals of T I D A neurons in the median eminence, 1o stimulate dopamine biosynthesis. As for nigrostriatal dopaminergic neurons, it has been shown that c A M P increased tyrosine hydroxylase activity m slices and cell-free systems prepared from the striatum with an alteration in the kinetics of either decreased K,, of tyrosine hydroxylase for cofactor N,~f or increased Vmax1,21. Although there are no data on the effect of c A M P on the kinetics of tyrosine hydroxylase in T I D A neurons, it is reasonable to speculate, by analogy with nigrostriatal dopaminergic neurons, that,. also in T I D A neurons, c A M P stimulates dopamine biosynthesis by increasing tyrosine hydroxylase activity. The result that the protein synthesis inhibitor.
43 cycloheximide failed to block the stimulatory action of db-cAMP on D O P A accumulation in the median eminence indicates that cAMP-induced dopamine biosynthesis in T I D A neurons is not dependent upon protein synthesis. Therefore, it is suggested that cAMP-induced dopamine biosynthesis in T I D A neurons is not due to induction but is due to activation of tyrosine hydroxylase. In conclusion, cAMP stimulates dopamine biosynthesis in TIDA neurons independently from Ca 2+ action. cAMP may activate cAMP-dependent protein kinase or suppress protein phosphatase, the substrate of which is the phosphoprotein tyrosine hydroxylase, as suggested in other catecholaminergic neurons t 1,17,19.24. Although the stimulus for Ca2+-in-
duced dopamine biosynthesis in TIDA neurons is indicated to be membrane depolarization, it is unknown what neurotransmitter(s) and hormone(s) activate or inhibit adenylate cyclase in axon terminals of TIDA neurons, and this question remains to be investigated in future studies.
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ACKNOWLEDGEMENTS
The authors thank Nippon Kayaku (Tokyo, Japan) and Hoechst AG for the generous gift of forskolin. This work was supported by a Grant-in-Aid for Encouragement of Young Scientist of the Ministry of Education, Science and Culture, Japan.
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