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Release of endogenous brain epinephrine by the calcium ionophores X537A and A23187
Brain Research, 341 (1985) 297-302 Elsevier
297
BRE 10941
Release of Endogenous Brain Epinephrine by the Calcium Ionophores X537A and A23187 NINA Y. LIANG 1, JUDY A. HOWER 1and RONALD T. BORCHARDT j.2 l Centerfor Biomedical Research, 2Departmentof Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66044 (U. S. A. ) (Accepted November 20th, 1985) Key words: endogenous brain epinephrine - - calcium ionophores - - X537A - - A23187 - - norepinephrine - - dopamine
The Ca2+ ionophores X537A and A23187 produced dose-dependent release of endogenous epinephrine (Epi) as well as norepinephrine (NE) and dopamine (DA) from chopped rat hypothalamus and brainstem. The X537A-induced release of these catecholamines (CAs) was found to be Ca2+-independent, whereas the A23187-induced release was Ca2+-dependent. X537A and A23187 were approximately equipotent in causing the release of the hypothalamic Epi, NE and DA, but X537A was much more effective than A23187 in causing the maximal release. X537A, but not A23187, reduced the total endogenous CA content and increased the total 3,4-dihydroxyphenylacetic acid (DOPAC) content in the chopped hypothalamus. CaZ+-independentrelease of Epi, NE and DA in the chopped hypothalamus was also observed with indirectly acting sympathomimetic amines (e.g. tyramine and amphetamine). Tyramine and amphetamine did not affect the total endogenous CA contents nor the total DOPAC content. These results suggest that X537A caused release of endogenous brain Epi, NE and DA by transporting the biogenic amines across vesicular or intracellular storage sites. However, A23187 caused release of these CAs by exocytosis via transport of Ca2+ into the neurons.
INTRODUCTION H6kfelt et al.llA2 reported the presences of epi-
pamine) which lower hypothalamic N E content, can also lower hypothalamic Epi 2v. In addition, Burgess and TesseP have shown that brain Epi can be released
nephrine (Epi)-containing n e u r o n s in areas of rat brain that are involved in the regulation of blood' pressure and endocrine function. Subsequently, it
by depolarizing agents such as potassium and veratridine. The present study was u n d e r t a k e n to increase our
was suggested that brain Epi may play a role as a neurotransmitterS. Efforts have been made to char-
understanding of the neurochemical properties of central Epi neurons, in particular the characteristics
acterize the neurochemical properties of central Epi
of Epi release. The release of e n d o g e n o u s Epi (as well as NE and D A ) was induced by two Ca 2+ ionophores, A23187 and X537A, and also by two indirectly acting sympathomimetic amines, amphetamine and tyramine. The brain regions studied were the hypothalamus and the brainstem, which are known to be innervated with Epi neuronslJ.lL
neurons. For example, drugs which inhibit monoamine oxidase activities (e.g. pargyline) increase brain Epi levels 24 whereas drugs which inhibit the activities of P N M T (e.g. 2-cyclooctyl-2-hydroxyethylamine is and 7,8-dichloro-l,2,3,4-tetrahydroisoquinoline 9,19) lower brain Epi levels. Some of the neurochemical properties of central Epi n e u r o n s have been shown to be quite similar to those of central norepinephrine (NE) n e u r o n s and d o p a m i n e ( D A ) neurons. For example, reserpine, which depletes the storage of brain NE and D A , can also lower brain Epi content 24 and neurotoxic agents (e.g. 6-hydroxydo-
MATERIALS AND METHODS The drugs used in these studies were obtained from the following sources: A23187 (Calbiochem, LaJolla, CA); X537A (Hoffman La Roche, Nutley, NJ);
Correspondence: R. T. Borchardt, Department of Pharmaceutical Chemistry, 3006 Malott Hall, University of Kansas, Lawrence, KS 66045, U.S.A.
29S D-amphetamine sulfate (Smith, Kline Beckman, Philadelphia, PA); and tyramine (Sigma, St, Louis, MO). Male Sprague-Dawley rats (250-300 g, Sasco) were killed by decapitation and the hypothalamus and brainstem were removed. The tissues were chopped into 0.3-ram sections with a mechanical tissue chopper and then suspended in either normal or Ca2+-free Krebs-Henseleit buffer 2. The normal medium had the following composition (mM): NaC1, 126; KCI, 240; CaCI 2, 2.70; MgC12, 0.83; Na2SO 4, 0.5; KH2PO4, 0.5; NaHCO> 27.0; glucose, 5.66. Ca2+-free medium contained all of the above with the exception of CaC12. The incubation mixture containing chopped brain tissue was then incubated at 37 °C for 30 rain. At the end of the incubation period, the medium was separated from the tissue by centrifugation. The tissue was washed with ice-cold buffer and the wash was combined with the medium. The tissue pellet was then sonicated with 0.4 N HCIO 4 and centrifuged. NE, Epi and DA and 3,4-dihydroxyphenylacetic acid (DOPAC) in both the medium and the tissue were absorbed onto alumina and then separated and quantified by high-pressure liquid chromatography using electrochemical detection as previously described 18. The amount of protein in the tissue pellet was determined by the biuret method 17. The amount of catecholamine (CA) released was expressed as pmol/mg protein.
7! i
*- * A25187 • • A23187 Ca"free o ~ X537A • • X537A Ca" free
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no6 I0 5 104 Drug Concentration (M)
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Control"'
16-6 fO-5 1(3-~f Drug Concentration (M)
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RESULTS
Effects of Ca 2+ ionophores on the release of endogenous catecholamines from chopped rat hypothalamus The Ca 2+ ionophores X537A and A23187 were shown to release Epi, NE and D A from chopped hypothalamus (Fig. 1A-C). The X537A-induced release was Ca>-independent whereas the A23187-induced release was shown to be CaZ+-dependent (Fig. 1A-C). Similar results were obtained with chopped brainstem (data not shown). In chopped hypothalamus, X537A and A23187 were approximately equipotent in causing the release of Epi, NE and DA (Table I). However, X537A was more effective in causing the maximal release of the CAs. For example, the maximal release of hypothalamic Epi, NE and D A (pmol/mg protein) were 0.37 + 0.07, 19.29 +_ 4.89 and 1.81 -4- 0.50, re-
A
28 24
H •
A23187 ,t A23187 Ca'" free
t 1
[ ~1
~2o
c %
~
16
8 4
// Control
i
i
.
i
lOS i0-~ 10-4 Drug Concentration (M)
i
i(j3
Fig. l. Effectsof Ca2+ ionophoreson the release of endogenous Epi (A), NE (B) and DA (C) in the chopped hypothalamus. Data represent the mean -4-S.E.M. of 8-13 experiments. spectively, with X537A; and 0.15 + 0.04, 4.03 + 1.02 and 1.13 + 0.38, respectively, with A23187.
299 A
TABLE I
•.
The ECso values for release of endogenous hypothalamic catecholamines by A23187 and X537A
.L A M P
• . . . . • Tyromine
Catecholamine Epi NE DA
ECso (lO-6M)
free
A----A A M P Co** ~. = Tyrornine
40
Co**free..A-/"_
X3
A23187
X537A
3.2 6.0 4.7
7.1 8.9 2.7
._c 3 0 ID
B (3-
g
20
I.t.J
Effects of indirectly acting sympathomimetic amines on the release of hypothalamic Epi, NE and DA The sympathomimetic drugs tyramine and amphetamine, produced dose-dependent release of Epi, NE and D A from the chopped hypothalamus (Fig. 2 A - C ) . The release induced by these sympathomimetic amines appeared to be Ca2+-independent. Amphetamine and tyramine appeared to be equally effective in causing the release of hypothalamic Epi, NE and DA. However, the ECs0s were not determined, since the release did not plateau at high concentrations of the drugs.
E c~ I0 //
i
Control
i
1(756
i
I 0 -~
i 0 -4
Drug Concenlrotion B
18 16
/
A .L A M P A---.,L A M P Cff* free ~. = Tyramine
14
(M)
,O
f ,"
• .-~,
,/j.,/~ •
k5 c_
-~ I0 13_
Effect of Ca2+ ionophores and indirectly acting sympathomimetic amines on the total DOPAC and catecholamine content in chopped hypothalamus The ionophore, X537A, but not A23187, increased the total (medium plus tissue) hypothalamic DOPAC content (Table II). The sympathomimetic amines amphetamine and tyramine did not affect the total hypothalamic DOPAC content (Table II). Similar results were obtained in chopped brainstem (data not shown). X537A also significantly reduced the total CA content of hypothalamic Epi, NE and DA (Table III). However, this phenomenon was not observed when the tissue was pretreated with 10 .4 M pargyline and then incubated with 10 -4 M tropolone (Table lII). The CA content was not altered by A23187, amphetamine or tyramine. Similar results were observed with chopped brainstem (data not shown). DISCUSSION
o, 8
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10 -5
10 -4
Drug Concentrotion (M)
40~ E • ~SO~
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A
A AMP
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. . . . . AMP C a " f r e e -" ~ Tyramine
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* - - - * Tyramine Ca**free
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Control
One of the criteria of a neurotransmitter is release of the substance from nerve endings via exocytosis upon depolarization6. Since Ca 2+ is intimately involved in exocytosis, it was of interest to determine
t
Control
I 0 -6 10-s IO-4 Drug Concentration (M)
Fig. 2. Effects of indirectly acting sympathomimetic amines on the release of endogenous Epi (A), NE (B) and D A (C) in the chopped hypothalamus. Data represent the mean _+_S.E.M. of 8-13 experiments.
300 TABLE II
similar to those observed for the release of NE and
Effects of Ca2÷ ionophores and sympathomimetic amines on the total DOPA C contents in the hypothalamus
DA. Our data for the release of endogenous Epi, NE and D A are similar to the data reported for X537A-
Data represent the mean + S.E.M. of 8-13 experiments.
and A23187-induced [3H]DAS,7,t0,t3,30.
Treatment
DO PA C content (pmol/mg protein)
Two features of the ionophore-induced release of hypothalamic Epi are noteworthy: (1) X537A was
Control 10-5 M A23187 10 5M X537A 3 × 10-5 M tyramine 10-5 M amphetamine
3.88 + 0.42 4.13 _+O.57 5.56 + 0.45* 3.42 + 1.49 3.26 -+ 0.42
release
of
[3H]NE
and
more effective than A23187 in causing maximal release of Epi (as well as NE and D A ) ; and (2) X537Ainduced release of Epi (as well as NE and D A ) was Ca2+-independent, while A23187-induced release was Ca2+-dependent. The effect of Ca 2+ on release
* P < 0.05 significantlyhigher than control.
of these CAs clearly indicated that the mechanism of the effect of Ca2+ ionophores on the release of central Epi. The results of our study indicate that the Ca2+ ionophores, X537A and A23187, release endo-
the release induced by X537A is different from the mechanism of the release induced by A23187. X537A appears to mediate the release of biogenic
and A23187-induced release of e n d o g e n o u s Epi are
amines by a mechanism other than one which involves carrying Ca 2÷ into the neuron. Pressman 23 has shown that X537A, unlike
TABLE III
A23187, will form complexes with amines such as N E and Epi. Other investigators have reported that
Effects of Ca2÷ ionophores and syrnpathomimetic amines on the total endogenous catecholamine contents in the normal hypothalamus and in the pargyline- and tropotone-treated hypothala-
X537A will release NE and Epi, as welt as D A , from chromaffin granules 13.14,25. Holz 14 also d e m o n s t r a t e d that X537A carried [3H]DA across bimotecular
genous Epi from both the hypothalamus and brainstem. In addition, the characteristics of the X537A-
mus
Data represent the mean + S.E.M. of 8-13 experiments. Treatment
Catecholamine content (pmol/mg protein) Normal hypothalamus
Pargyline and tropolonetreated hypothalamus
Norepinephrine Control 10-5 M A23187 10-5 M X537A 3 × 10-5 M tyramine 10 5 M amphetamine
46.57 + 5.46 55.31 _+2.79 25.28 + 0,91" 38.70 + 4.05 52.68 + 3.41
45.74 _+0.35 50.27 + 2.21 55.87 _+4.42 ---
Epinephrine Control 10-5 M A23187 10-5 M X537A 3 × 10-5 M tyramine 10-5 M amphetamine
1.15 _+_+0.10 1.18 + 0.42 0.82 -+ 0.14" 0.99 + 0.09 1.02 + 0.13
1.05 _+0.08 1.15 + 0.05 1.25 + 0.14 -
Dopamine Control 10-5 M A23187 10-5 M X537A 3 × 10-5 M tyramine 10 5 M amphetamine
9.24 + 1.10 8.53 + 0.89 2.95 + 0.25* 7.27 _ 1.36 12.50 _+ 1.01
7.66 ± 0.11 8.37 _+0.38 10.28 + 2.60 -
* P < 0.05 significantly lower than control.
lipid m e m b r a n e s in the absence of Ca 2+, but similar results were not observed with A23187. In addition, it was observed that X537A-induced release of [3H]NE and [3H]DA from various brain tissues, caused a significant increase in the 3H-labelled metabolites of NE and D A , whereas A23187 did not produce a similar effect 13,15. Based on these observations, Holz 13 and Kafka and Holz 15 proposed that X537A released D A and NE by complexing with the CAs and transporting them across the vesicular membrane. This mechanism would release the CAs in the cytoplasm, exposing them to m o n o a m i n e oxidase and thereby elevating the a m o u n t of metabolites. In the present study we also observed that X537A caused an increase in catabolism of e n d o g e n o u s CAs. This conclusion was based on the observation that X537A reduced the content of Epi, NE and D A in the hypothalamus (Table III) and brainstem (data not shown). However, X537A did not effect the content of Epi, NE and D A when the hypothalamus was pretreated with pargyline ( m o n o a m i n e oxidase inhibitor) and tropolone (catechol-O-methyltransferase inhibitor). Since the total endogenous C A con-
301 tent represents a balance between synthesis and degradation, a decrease in the total content could result from: (1) an increase in the catabolism of the CA, (2) a decrease in the synthesis of the C A or (3) a concurrent decrease in synthesis and increase in the catabolism of the amine. Since treatment with drugs k n o w n to block degradation also blocked the reduction in Epi, N E and D A caused by X537A, we have concluded that the decrease in total C A content resulted at least in part from increased catabolism. The second observation which supports this conclusion is that X537A caused an increase in D O P A C content (Table II). Since the total D A content decreases while total D O P A C content increases, we can conclude that the catabolism of the D A was increased by X537A. It should be noted here that A23187 does not
[3H]DA and e n d o g e n o u s NE and D A from brain tissue 1,4,16,20,26,28,29,31. The tyramine- and amphetamine-induced release of e n d o g e n o u s hypothalamic NE and D A reported here is consistent with previous observationsl,20,zt. In addition, however, we have observed that e n d o g e n o u s hypothalamic Epi is also released by tyramine and a m p h e t a m i n e in a Ca2+-independent fashion, which confirms the results reported earlier by Burgess and Tessel 3. In summary it can be concluded that X537A caused release of e n d o g e n o u s Epi (as well as NE and DA) by transporting the biogenic amine across vesicular or intracellular storage sites. However, A23187 caused release of Epi (as well as NE and D A ) by exocytosis via transport of Ca 2+ into the Epi-containing neurons.
decrease total C A content nor does it increase total D O P A C content. All of these observations suggest that the mechanisms which have been previously put forth to explain X537A- and A23187-induced release
ACKNOWLEDGEMENTS
of [3H]NE and [3H]DA may also be applicable to explain the release of endogenous Epi by these Ca2+
the National Heart, Lung and Blood Institute (HL24093) and N.Y.L. was a recipient of a fellowship from the A m e r i c a n Heart Association - Kansas Affiliate. The authors acknowledge the sup-
ionophores. The indirectly acting sympathomimetic amines, amphetamine and tyramine, have been shown to cause Ca2+-independent release of [3H]NE or
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This work was supported by a research grant from
port provided by the staff of the Center for Biomedical Research, University of Kansas.
8 Fuller, R. W., Pharmacology of brain epinephrine neurons, Ann. Rev. Pharmacol. Toxicol., 22 (1982) 31-55. 9 Goldstein, M., Kinguasa, K., Hieble, J. P. and Pendleton, R. G., Lowering of blood pressure in hypotensive rats by SKF64139 and SKF72223, Life Sci, 30 (1982) 1951-1957. 10 Gothert, M., Ca2*-induced noradrenaline release from central noradrenergic neurons promoted by high K + concentration or ionophore A23187, Naunyn-Schmiedeberg's Arch. Pharmacol., 307 (1979) 29-37. 11 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Evidence for adrenaline neurons in the rat brain, Acta physiol, scand., 89 (1973) 286-288. 12 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., lmmunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Research, 66 (1974) 235-251. 13 Holz, R. W., The release of dopamine from synaptosomes from rat striatum by the ionophores X537A and A23187, Biochim. Biophys. Acta, 375 (1975) 138-152. 14 Johnson, R. G. and Scarpa, A., Catecholamine equilibrium gradients of isolated chromaffin granules induced by the ionophore X537A, FEBS Lett., 47 (1974) 117-121. 15 Kafka, M. S. and Holz, R. W., Ionophorcs X537A and A23187: effects on the permeability of lipid biomolecular membranes to dopamine and calcium, Biochim. Biophys. Acta, 426 (1976) 31-37. 16 Kamal, L. A., Arbilla, S. and Langer, S. Z., Presynaptic
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modulation of the release of dopamine from the rabbit caudate nucleus: differences between electrical stimulation. amphetamine and tyramine. J. Pharmacol. e,~7~. Ther.. 216 (19811 592-598. Layne, R., Spectrophotometric and turbidimetric methods for measuring proteins. In S. P. Colowick and N. O, Kaplan (Eds.), Methods in Enzymology. Vol. 3. Academic Press, New York, 1957, pp. 447-454. Liang, N. Y., Tessel, R. E., Grunewald, G. L. and Borchardt, R. T., Inhibitors of phenylethanolamine N-methyltransferase. 1. Effects of 2-cyctooctyl-2-hydroxyethanolamine on rat brain and adrenal catecholamine content and blood pressure, J. Pharmacol. exp. Ther,. 223 (1982/ 375-381. Liang, N. Y., Tessel, R. E., Grunewatd, G. L. and Borchardt, R. T., Inhibitors of phenylethanolamine-N-methyltransferase. 2. Comparison of nonaromatic analogs of phenylethanolamines, 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline (SKF64139) and 2,3-dichloro-a-methylbenzlamine: effects on rat brain and adrenal catecholamine content and blood pressure, J. Pharmacol. exp. "liter., 223 ( 19821 382-387. Lindmar, R. and Muscholl, E., Die Verst/irkung der Noradrenalinewirkung durch Tyramin, Naunyn-Schmiedeberg's Arch. exp. Pathol. Pharmacol., 252 ( t 965) 122-133. Meyerhoff, J. L. and Kant, G, J., Release of endogenous dopamine from corpus striatum, Lift' Sci., 23 (1978) 1481-1486. Myers, S. L. and Tessel, R. E., Desipramine-induced increase in efflux of endogenous dopamine from rat hypothalamus in vitro: mechanism and specificity, NeuropharmacoIogy, 22 (1983) 601-607. Pressman, B. C., Properties of ionophores with broad range cation selectivity, J. biol. Chem., 32 (19731 1698-1703.
24 Scatton, B. and Bartholini, (i.. Elfects oi antih~peltcnsl~c,, and other drugs on central adrenaline utilizati,m. In K Fuxe, M. Golstein, B. H6kfelt and T, tli:,kfelt ( l:ds. }, ( e*~tral Adrenaline Neurons: Ba~ic Aspects ami */i~'~ R~h' e~,, Cardiovascular Disease, Pergamon, New '~ ork, l!~Sli. pp. 183-197. 25 Scherman, D., Roisin, M. P. and flenry. J. I'. : kmophure mediated catecholamine release from chromalfin granute, Biochem. Pharmacol., 30 ( 1981 ) 3277-3283. 26 Smith, C. B., The role of monoamme oxidasc m the intraneuronal metabolism of norepinephrine released by indirectly acting sympathomimetic amines or b). adrenergic nerve stimulation, J. Pharmac,d. e,~p. 77u¢r, 151 (1966j 2O7-22O. 27 Tessel. R, E., Kennedy, E., Burgess, S. K. and Borchardt R. T., Epinephrine in rat hypothalamus: anlagonism by desipramine of 6-hydroxydopamine-induced depletion. Brai*t Research, 153 (1978) 615-617. 28 Tessel, R. E, and Rutledge, C O., Specificity oi release ol biogenic amines from isolated rat brain tissue as a function of the meta substituent of N-ethylamphetamine derivatives, J. Pharmacol. exp, Ther.. 197 ( 19761 253-262 29 Trendelenburg, U., Release induced by phcnylethanol7 amines. In D. M. Paton ted. ). The Reh,ase ~;~"Cawchoh> mines from Adrenergic Neurons. Pergamon, New York. 1978, pp. 333-354. 30 Vargas, O,, Miranda, R. and Orrego, F., Effects ot sodium-deficient media and of calcium ionophores {A23187) on the release of -~H-noradrenalme, ~aC-(x-ammo-isobutyrate, and -~H-7-aminobutyrate from supcrfused slices of rat neocortex. Neurowience. 1 ( 19761 137 - 145. 31 Ziance, R..l., Azzaro, A. J. and Rutledge, ( . O1. Characteristics of amphetamine-induced release ol norepinephrine from rat cerebral cortex m vitro, .1. Pharmaeot. e.W). Ther.. 182 ( 19721 284-294.
297
BRE 10941
Release of Endogenous Brain Epinephrine by the Calcium Ionophores X537A and A23187 NINA Y. LIANG 1, JUDY A. HOWER 1and RONALD T. BORCHARDT j.2 l Centerfor Biomedical Research, 2Departmentof Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66044 (U. S. A. ) (Accepted November 20th, 1985) Key words: endogenous brain epinephrine - - calcium ionophores - - X537A - - A23187 - - norepinephrine - - dopamine
The Ca2+ ionophores X537A and A23187 produced dose-dependent release of endogenous epinephrine (Epi) as well as norepinephrine (NE) and dopamine (DA) from chopped rat hypothalamus and brainstem. The X537A-induced release of these catecholamines (CAs) was found to be Ca2+-independent, whereas the A23187-induced release was Ca2+-dependent. X537A and A23187 were approximately equipotent in causing the release of the hypothalamic Epi, NE and DA, but X537A was much more effective than A23187 in causing the maximal release. X537A, but not A23187, reduced the total endogenous CA content and increased the total 3,4-dihydroxyphenylacetic acid (DOPAC) content in the chopped hypothalamus. CaZ+-independentrelease of Epi, NE and DA in the chopped hypothalamus was also observed with indirectly acting sympathomimetic amines (e.g. tyramine and amphetamine). Tyramine and amphetamine did not affect the total endogenous CA contents nor the total DOPAC content. These results suggest that X537A caused release of endogenous brain Epi, NE and DA by transporting the biogenic amines across vesicular or intracellular storage sites. However, A23187 caused release of these CAs by exocytosis via transport of Ca2+ into the neurons.
INTRODUCTION H6kfelt et al.llA2 reported the presences of epi-
pamine) which lower hypothalamic N E content, can also lower hypothalamic Epi 2v. In addition, Burgess and TesseP have shown that brain Epi can be released
nephrine (Epi)-containing n e u r o n s in areas of rat brain that are involved in the regulation of blood' pressure and endocrine function. Subsequently, it
by depolarizing agents such as potassium and veratridine. The present study was u n d e r t a k e n to increase our
was suggested that brain Epi may play a role as a neurotransmitterS. Efforts have been made to char-
understanding of the neurochemical properties of central Epi neurons, in particular the characteristics
acterize the neurochemical properties of central Epi
of Epi release. The release of e n d o g e n o u s Epi (as well as NE and D A ) was induced by two Ca 2+ ionophores, A23187 and X537A, and also by two indirectly acting sympathomimetic amines, amphetamine and tyramine. The brain regions studied were the hypothalamus and the brainstem, which are known to be innervated with Epi neuronslJ.lL
neurons. For example, drugs which inhibit monoamine oxidase activities (e.g. pargyline) increase brain Epi levels 24 whereas drugs which inhibit the activities of P N M T (e.g. 2-cyclooctyl-2-hydroxyethylamine is and 7,8-dichloro-l,2,3,4-tetrahydroisoquinoline 9,19) lower brain Epi levels. Some of the neurochemical properties of central Epi n e u r o n s have been shown to be quite similar to those of central norepinephrine (NE) n e u r o n s and d o p a m i n e ( D A ) neurons. For example, reserpine, which depletes the storage of brain NE and D A , can also lower brain Epi content 24 and neurotoxic agents (e.g. 6-hydroxydo-
MATERIALS AND METHODS The drugs used in these studies were obtained from the following sources: A23187 (Calbiochem, LaJolla, CA); X537A (Hoffman La Roche, Nutley, NJ);
Correspondence: R. T. Borchardt, Department of Pharmaceutical Chemistry, 3006 Malott Hall, University of Kansas, Lawrence, KS 66045, U.S.A.
29S D-amphetamine sulfate (Smith, Kline Beckman, Philadelphia, PA); and tyramine (Sigma, St, Louis, MO). Male Sprague-Dawley rats (250-300 g, Sasco) were killed by decapitation and the hypothalamus and brainstem were removed. The tissues were chopped into 0.3-ram sections with a mechanical tissue chopper and then suspended in either normal or Ca2+-free Krebs-Henseleit buffer 2. The normal medium had the following composition (mM): NaC1, 126; KCI, 240; CaCI 2, 2.70; MgC12, 0.83; Na2SO 4, 0.5; KH2PO4, 0.5; NaHCO> 27.0; glucose, 5.66. Ca2+-free medium contained all of the above with the exception of CaC12. The incubation mixture containing chopped brain tissue was then incubated at 37 °C for 30 rain. At the end of the incubation period, the medium was separated from the tissue by centrifugation. The tissue was washed with ice-cold buffer and the wash was combined with the medium. The tissue pellet was then sonicated with 0.4 N HCIO 4 and centrifuged. NE, Epi and DA and 3,4-dihydroxyphenylacetic acid (DOPAC) in both the medium and the tissue were absorbed onto alumina and then separated and quantified by high-pressure liquid chromatography using electrochemical detection as previously described 18. The amount of protein in the tissue pellet was determined by the biuret method 17. The amount of catecholamine (CA) released was expressed as pmol/mg protein.
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RESULTS
Effects of Ca 2+ ionophores on the release of endogenous catecholamines from chopped rat hypothalamus The Ca 2+ ionophores X537A and A23187 were shown to release Epi, NE and D A from chopped hypothalamus (Fig. 1A-C). The X537A-induced release was Ca>-independent whereas the A23187-induced release was shown to be CaZ+-dependent (Fig. 1A-C). Similar results were obtained with chopped brainstem (data not shown). In chopped hypothalamus, X537A and A23187 were approximately equipotent in causing the release of Epi, NE and DA (Table I). However, X537A was more effective in causing the maximal release of the CAs. For example, the maximal release of hypothalamic Epi, NE and D A (pmol/mg protein) were 0.37 + 0.07, 19.29 +_ 4.89 and 1.81 -4- 0.50, re-
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Fig. l. Effectsof Ca2+ ionophoreson the release of endogenous Epi (A), NE (B) and DA (C) in the chopped hypothalamus. Data represent the mean -4-S.E.M. of 8-13 experiments. spectively, with X537A; and 0.15 + 0.04, 4.03 + 1.02 and 1.13 + 0.38, respectively, with A23187.
299 A
TABLE I
•.
The ECso values for release of endogenous hypothalamic catecholamines by A23187 and X537A
.L A M P
• . . . . • Tyromine
Catecholamine Epi NE DA
ECso (lO-6M)
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X537A
3.2 6.0 4.7
7.1 8.9 2.7
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20
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Effects of indirectly acting sympathomimetic amines on the release of hypothalamic Epi, NE and DA The sympathomimetic drugs tyramine and amphetamine, produced dose-dependent release of Epi, NE and D A from the chopped hypothalamus (Fig. 2 A - C ) . The release induced by these sympathomimetic amines appeared to be Ca2+-independent. Amphetamine and tyramine appeared to be equally effective in causing the release of hypothalamic Epi, NE and DA. However, the ECs0s were not determined, since the release did not plateau at high concentrations of the drugs.
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Effect of Ca2+ ionophores and indirectly acting sympathomimetic amines on the total DOPAC and catecholamine content in chopped hypothalamus The ionophore, X537A, but not A23187, increased the total (medium plus tissue) hypothalamic DOPAC content (Table II). The sympathomimetic amines amphetamine and tyramine did not affect the total hypothalamic DOPAC content (Table II). Similar results were obtained in chopped brainstem (data not shown). X537A also significantly reduced the total CA content of hypothalamic Epi, NE and DA (Table III). However, this phenomenon was not observed when the tissue was pretreated with 10 .4 M pargyline and then incubated with 10 -4 M tropolone (Table lII). The CA content was not altered by A23187, amphetamine or tyramine. Similar results were observed with chopped brainstem (data not shown). DISCUSSION
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One of the criteria of a neurotransmitter is release of the substance from nerve endings via exocytosis upon depolarization6. Since Ca 2+ is intimately involved in exocytosis, it was of interest to determine
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Fig. 2. Effects of indirectly acting sympathomimetic amines on the release of endogenous Epi (A), NE (B) and D A (C) in the chopped hypothalamus. Data represent the mean _+_S.E.M. of 8-13 experiments.
300 TABLE II
similar to those observed for the release of NE and
Effects of Ca2÷ ionophores and sympathomimetic amines on the total DOPA C contents in the hypothalamus
DA. Our data for the release of endogenous Epi, NE and D A are similar to the data reported for X537A-
Data represent the mean + S.E.M. of 8-13 experiments.
and A23187-induced [3H]DAS,7,t0,t3,30.
Treatment
DO PA C content (pmol/mg protein)
Two features of the ionophore-induced release of hypothalamic Epi are noteworthy: (1) X537A was
Control 10-5 M A23187 10 5M X537A 3 × 10-5 M tyramine 10-5 M amphetamine
3.88 + 0.42 4.13 _+O.57 5.56 + 0.45* 3.42 + 1.49 3.26 -+ 0.42
release
of
[3H]NE
and
more effective than A23187 in causing maximal release of Epi (as well as NE and D A ) ; and (2) X537Ainduced release of Epi (as well as NE and D A ) was Ca2+-independent, while A23187-induced release was Ca2+-dependent. The effect of Ca 2+ on release
* P < 0.05 significantlyhigher than control.
of these CAs clearly indicated that the mechanism of the effect of Ca2+ ionophores on the release of central Epi. The results of our study indicate that the Ca2+ ionophores, X537A and A23187, release endo-
the release induced by X537A is different from the mechanism of the release induced by A23187. X537A appears to mediate the release of biogenic
and A23187-induced release of e n d o g e n o u s Epi are
amines by a mechanism other than one which involves carrying Ca 2÷ into the neuron. Pressman 23 has shown that X537A, unlike
TABLE III
A23187, will form complexes with amines such as N E and Epi. Other investigators have reported that
Effects of Ca2÷ ionophores and syrnpathomimetic amines on the total endogenous catecholamine contents in the normal hypothalamus and in the pargyline- and tropotone-treated hypothala-
X537A will release NE and Epi, as welt as D A , from chromaffin granules 13.14,25. Holz 14 also d e m o n s t r a t e d that X537A carried [3H]DA across bimotecular
genous Epi from both the hypothalamus and brainstem. In addition, the characteristics of the X537A-
mus
Data represent the mean + S.E.M. of 8-13 experiments. Treatment
Catecholamine content (pmol/mg protein) Normal hypothalamus
Pargyline and tropolonetreated hypothalamus
Norepinephrine Control 10-5 M A23187 10-5 M X537A 3 × 10-5 M tyramine 10 5 M amphetamine
46.57 + 5.46 55.31 _+2.79 25.28 + 0,91" 38.70 + 4.05 52.68 + 3.41
45.74 _+0.35 50.27 + 2.21 55.87 _+4.42 ---
Epinephrine Control 10-5 M A23187 10-5 M X537A 3 × 10-5 M tyramine 10-5 M amphetamine
1.15 _+_+0.10 1.18 + 0.42 0.82 -+ 0.14" 0.99 + 0.09 1.02 + 0.13
1.05 _+0.08 1.15 + 0.05 1.25 + 0.14 -
Dopamine Control 10-5 M A23187 10-5 M X537A 3 × 10-5 M tyramine 10 5 M amphetamine
9.24 + 1.10 8.53 + 0.89 2.95 + 0.25* 7.27 _ 1.36 12.50 _+ 1.01
7.66 ± 0.11 8.37 _+0.38 10.28 + 2.60 -
* P < 0.05 significantly lower than control.
lipid m e m b r a n e s in the absence of Ca 2+, but similar results were not observed with A23187. In addition, it was observed that X537A-induced release of [3H]NE and [3H]DA from various brain tissues, caused a significant increase in the 3H-labelled metabolites of NE and D A , whereas A23187 did not produce a similar effect 13,15. Based on these observations, Holz 13 and Kafka and Holz 15 proposed that X537A released D A and NE by complexing with the CAs and transporting them across the vesicular membrane. This mechanism would release the CAs in the cytoplasm, exposing them to m o n o a m i n e oxidase and thereby elevating the a m o u n t of metabolites. In the present study we also observed that X537A caused an increase in catabolism of e n d o g e n o u s CAs. This conclusion was based on the observation that X537A reduced the content of Epi, NE and D A in the hypothalamus (Table III) and brainstem (data not shown). However, X537A did not effect the content of Epi, NE and D A when the hypothalamus was pretreated with pargyline ( m o n o a m i n e oxidase inhibitor) and tropolone (catechol-O-methyltransferase inhibitor). Since the total endogenous C A con-
301 tent represents a balance between synthesis and degradation, a decrease in the total content could result from: (1) an increase in the catabolism of the CA, (2) a decrease in the synthesis of the C A or (3) a concurrent decrease in synthesis and increase in the catabolism of the amine. Since treatment with drugs k n o w n to block degradation also blocked the reduction in Epi, N E and D A caused by X537A, we have concluded that the decrease in total C A content resulted at least in part from increased catabolism. The second observation which supports this conclusion is that X537A caused an increase in D O P A C content (Table II). Since the total D A content decreases while total D O P A C content increases, we can conclude that the catabolism of the D A was increased by X537A. It should be noted here that A23187 does not
[3H]DA and e n d o g e n o u s NE and D A from brain tissue 1,4,16,20,26,28,29,31. The tyramine- and amphetamine-induced release of e n d o g e n o u s hypothalamic NE and D A reported here is consistent with previous observationsl,20,zt. In addition, however, we have observed that e n d o g e n o u s hypothalamic Epi is also released by tyramine and a m p h e t a m i n e in a Ca2+-independent fashion, which confirms the results reported earlier by Burgess and Tessel 3. In summary it can be concluded that X537A caused release of e n d o g e n o u s Epi (as well as NE and DA) by transporting the biogenic amine across vesicular or intracellular storage sites. However, A23187 caused release of Epi (as well as NE and D A ) by exocytosis via transport of Ca 2+ into the Epi-containing neurons.
decrease total C A content nor does it increase total D O P A C content. All of these observations suggest that the mechanisms which have been previously put forth to explain X537A- and A23187-induced release
ACKNOWLEDGEMENTS
of [3H]NE and [3H]DA may also be applicable to explain the release of endogenous Epi by these Ca2+
the National Heart, Lung and Blood Institute (HL24093) and N.Y.L. was a recipient of a fellowship from the A m e r i c a n Heart Association - Kansas Affiliate. The authors acknowledge the sup-
ionophores. The indirectly acting sympathomimetic amines, amphetamine and tyramine, have been shown to cause Ca2+-independent release of [3H]NE or
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