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Carbachol induced release of diadenosine polyphosphates -Ap4A and Ap5A- from perfused bovine adrenal medulla and isolated chromaffin cells
Life Sciences, Vol. 48, pp. 2317-2324 Printed in the U.S.A.
CARBACHOL INDUCED R E L F ~ S E ~pSA-
FROM
PERFUSED
JesQs Pintor, Departamento
BOVINE
Magdalena
Pergamon Press
OF DIADENOSINE POLYPHOSPH~TES ADRENAL CELLS
MEDULLA
AND
ISOLATED
-AP4A
AND
CHROMAFFIN
Torres and M.Teresa M i r a s - P o r t u g a l *
de Bioqulmica, Facultad de Veterinaria, Complutense de Madrid. 28040 Madrid.
Universidad
(Received in final form April 8, 1991) Summary
The diadenosine polyphosphates - AP4A and APsA were released from perfused bovine adrenal glands and recently isolated chromaffin cells by the action of carbachol. The H.P.L.C. technique reported here allowed the quantification of pmol amounts of these compounds present in biological samples from the perfusion media after stimulation. Both compounds !AP4A and APsA ) were identified by the retention time In H.P.L.C. chromatography, co-elution with standards, re-chromatography and destruction by the phosphodiesterase action. Bovine adrenal glands s t i m u l a t e d with 100~M carbachol released 0.47 ± 0.12 n m o l / g l a n d of AP4A and i.ii ± 0.26 nmol/gland of APsA. Isolated bovine chromaffin cells after 100~M carbachol, as secretagogue, released ii.i ± 0.8 pmol/106 cells of AP4A and 15.8 ± i.i pmol/106 cells of AP5A. The ratio of these compounds with respect to the exocytotically released ATP and c a t e c h o l a m i n e s was in the same order as that found in isolated c h r o m a f f i n granules.
The presence of diadenosine polyphosphates, adenosine (5') t e t r a p h o s p h o (5') adenosine (diadenosine tetraphosphate, . . Ap 4 A) and . adenosine (5') pentaphospho (5') adenoslne (dladenoslne pentaphosphate, APsA ) has been recently described in the c h r o m a f f i n granules of bovine adrenal medulla, where they are costored with catecholamines and ATP (i). The dense granules of human p l a t e l e t s also co-stored adenosine (5') t r i p h o s p h o (5') adenosine (diadenosine triphosphate, AP3A ) and AP4A with serotonine and adenine nucleotides (2,3). The physiological actions of diadenosine polyphosphates are receiving increasing a t t e n t i o n with respect to the vascular system (4), and also in neural secretory tissues, as chromaffin cells. In the latter the d i a d e n o s i n e polyphosphates increase basal catecholamine secretion, but have an inhibitory action on nicotine evoked release (5). * To w h o m correspondence should be addressed: Prof. M.T. MirasPortugal, Dpto. Bioqu~mica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid. Spain. Telph.13943894. FAX 13943883. 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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Carbachol Released Dinucleotides
Vol. 48, No. 24, 1991
In order to know the physiological importance of these d i a d e n o s i n e polyphosphates, it is of great interest to d e t e r m i n e their r e l e a s e from stimulated neural tissues. Therefore, the e x p e r i m e n t a l work reported here tries to isolate and q u a n t i f y the AP4A and APsA from p e r f u s e d adrenal glands and isolated c h r o m a f f i n cells after carbachol stimulation.
Materials and Methods Gland perfusion. Fresh bovine adrenal glands were o b t a i n e d from the local s l a u g h t e r h o u s e 20-30 minutes after the death of animals. A d r e n a l glands were perfused with 200 ml of Locke's s o l u t i o n (mM, NaCl 140, KCl 4.4, CaCl 2 2.5, KH2PO 4 1.2, MgSO 4 1.2, HEPES i0, NaHCO 3 4.0, glucose 5.6 and ascorbic acid I, pH 7.5) at 37°C at a flow rate of 5 ml/min to eliminate the blood constituents. Then 10 ml of the perfusion media were c o l l e c t e d as control for each studied gland. Secretion was s t i m u l a t e d with 10 ml 100~M c a r b a c h o l in Locke's solution. One ml alliquot was taken for c a t e c h o l a m i n e and ATP analysis and the rest was p r o c e s s e d in order to q u a n t i f y the AP4A and APsA levels. Cell superfusion. Recently isolated c h r o m a f f i n cells were o b t a i n e d as d e s c r i b e d in (6). After the percoll g r a d i e n t step, cells were collected and 25xi06 cells were used for each experiment. Cells were superfused on a Sterivex GS filter (Millipore) with Locke's solution at a flow rate of 5 m l / m i n and 37°C temperature. 10 ml of the perfused media w i t h o u t carbachol were taken for control in each done experiment. Cells were s t i m u l a t e d with 100 ~M carbachol in 10 ml of the p e r f u s i o n media. From this, 1 ml alliquot was taken for c a t e c h o l a m i n e and ATP analysis. H.P.L.C. analysis. Samples for AP4A and APsA analysis, both from i s o l a t e d c h r o m a f f i n cells and perfused adrenal glands were p r o c e s s e d as follows: samples were submitted to 100°C t e m p e r a t u r e in w a t e r bath for 10 minutes and then c e n t r i f u g e d to 12000xg for five minutes. This procedure removed the m a j o r i t y of p r o t e i n s from the samples and also destroyed part of the ATP p r e s e n t in the samples, that could interfere with the AP4A peak. Nevertheless, AP4A and APsA were not destroyed. The high c o n c e n t r a t i o n of ATP and the close r e t e n t i o n times between ATP and AP4A r e s u l t e d in an o v e r l a p p i n g of both compounds in H.P.L.C. c h r o m a t o g r a p h y in nonprocessed samples. Following this step, samples were c h r o m a t o g r a f i e d through a Sep-Pak Accell Q M A c o l u m n (Waters). All n u c l e o t i d e s were r e t a i n e d and the elution was done with 1 ml NaCl 1 M. Samples were then more concentrated and suitable for H.P.L.C. a n a l y s i s and q u a n t i f i c a t i o n of the dinucleotides. S t a n d a r d s of d i a d e n o s i n e p o l y p h o s p h a t e s added to samples of p e r f u s i o n media were r e c o v e r e d to an extent of 94-96 % of the total at the end of the c o m p l e t e treatment. The recovery of d i a d e n o s i n e p o l y ~ h o s p h a t e s from the isolated c h r o m a f f i n cells, at a density of 10 ~ cells/ml of c u l t u r e media was 90-92. % for both AP4A and APsA.. These. values were o b t a i n e d by addlng external standards of d l n u c l e o t l d e s to c e l l u l a r p r e p a r a t i o n s and then submitted to the same p r o c e d u r e as d e s c r i b e d above for the perfusion media. Routinely, 10-50~i
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Carbachol Released Dinucleotldes
2319
samples were injected for H.P.L.C. chromatography. The H.P.L.C. e q u i p m e n t was from Waters consisting of a 600 E control d e l i v e r y system, U6K injector, 4 5 1 1 max detector, a 745 data m o d u l e and a ~ b o n d a p a k C18 column (30 cm long 0.4 inner diameter). In isocratic conditions, with i0 mM KH2PO 4 buffer, pH 7.5, 2 mM t e t r a b u t y l a m m o n i u m and 15% acetonitrile as m o b i l e phase, the r e t e n t i o n times were 2.73 min for AMP, 4.60 min for ADP, 7.20 min for ATP, 11.53 min for AP4A and 19.07 min for APsA. W h e n necessary, the AP4A and AP5A peaks were c o l l e c t e d from the H.P.L.C. c o l u m n eluate and submitted to r e - c h r o m a t o g r a p h y e l u t i o n w i t h s t a n d a r d s in H.P.L.C., or p h o s p h o d i e s t e r a s e digestion. C a t e c h o l a m i n e s determination. C a t e c h o l a m i n e s were m e a s u r e d w i t h an e l e c t r o c h e m i c a l detector (Metrohm) using n o r a d r e n a l i n e as standard, as p r e v i o u s l y described (5). Chemicals. All nucleotides were from Sigma (St. Louis, Missouri) and B o e h r i n g e r (Manheim, Germany). P h o s p h o d i e s t e r a s e from C r o t a l u s durisus (EC 3.1.15.1) was from P h a r m a c i a (Upsala, Sweden). Results D i a d e n o s i n e p o l y p h o s p h a t e s were released from p e r f u s e d bovine adrenal glands and recently isolated c h r o m a f f i n cells by the action of carbachol. In absence of any secretory stimuli it was not p o s s i b l e to q u a n t i f y the AP4A and APsA in the p e r f u s i o n m e d i a of both systems. After stimulation of adrenal g l a n d s with c a r b a c h o l 100~M, both d i n u c l e o t i d e s were present at the n a n o m o l a r c o n c e n t r a t i o n in the p e r f u s i o n media. In fig. IA an H.P.L.C. c h r o m a t o g r a m is shown for a sample from p e r f u s e d adrenal glands. The total amount of d i n u c l e o t i d e s released was q u a n t i f i e d and the o b t a i n e d v a l u e s are summarized in table I. No AP3A was d e t e c t e d in our a n a l y t i c a l conditions. These values were h i g h l y v a r i a b l e due to the h e t e r o g e n i t y of bovine adrenal glands. W h e n samples of adrenal g l a n d s p e r f u s a t e were enriched with exogenous AP4A and AP5A , c o - e l u t i o n of standards with problems occurred, as shown in fig. lB. Most of the ATP released was d e s t r o y e d by the high t e m p e r a t u r e procedure, that does not affect the content of dinucleotides. I s o l a t e d bovine chromaffin cells p r o v i d e d a better model to study the e x o c y t o t i c release of AP4A and APsA than was the adrenal gland. The values obtained for both d i n u c l e o t i d e s were highly r e p r o d u c i b l e from one cellular p r e p a r a t i o n to another and are s u m m a r i z e d in table I. As occurs with adrenal glands, AP3A was not d e t e c t e d in p e r f u s a t e s from isolated chromaffin cells. In fig. iC a t y p i c a l c h r o m a t o g r a m of a sample from 100~M carbachol s t i m u l a t e d p e r f u s e d cells is shown. Due to the p r e p a r a t i o n samples, as d e s c r i b e d in methods, most of the released ATP was r e c o v e r e d as AMP. In fig. ID the c o - e l u t i o n with standards of AP4A and APsA is shown. In order to study the p h o s p h o d i e s t e r a s e action on both s u p p o s e d d i a d e n o s i n e polyphosphates, the peaks c o n t a i n i n g AP4A and AP5A were c o l l e c t e d from the H.P.L.C. column eluate. In fig. 2A
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Carbachol Released Dinucleotides
Vol. 48, No. 24, 1991
and 2C a r e - c h r o m a t o g r a p h y of AP4A and APsA is shown. The effect of p h o s p h o d i e s t e r a s e on the collected samples is shown in fig. 2B and 2D. The d i s a p p e a r a n c e of both dinucleotides was o b s e r v e d with the c o r r e s p o n d i n g appearance of adenine m o n o n u c l e o t i d e s AMP and ATP for d i a d e n o s i n e t e t r a p h o s p h a t e and the appearance of AMP and a d e n o s i n e t e t r a p h o s p h a t e for diadenosine pentaphosphate. TABLE I Adrenal Gland nmol/gland Basal Release
Chromaffin Cells pmol/106 c e l l s
Carbachol 100BM
Basal Release
Carbachol 100~M
AP4A
UD
0.47±0.14
UD
ii.i±0.8
APsA
UD
i.ii±0.30
UD
15.8±1.1
15.5±4.10
9.5±2.6
281±21
ATP
0.32±0.13
CA
ND
ND
14±3
1,039±86
The total c a t e c h o l a m i n e content was 69±10.4 nmol/106 cells m e a s u r e d after cellular lysis with Triton X-100. Total AP4A and AP5A content were 0.81±0.41 nmol/106 cells and 0.87±0.45 nmol/106 cells. The total ATP content was 42.3 ± 3.8 nmol/106 cells. The ATP was m e a s u r e d by H.P.L.C. directly, w i t h o u t processing of samples. Dinucleotides values were d e t e r m i n e d after the p r o c e s s i n g of samples as d e s c r i b e d in M a t e r i a l s and Methods. Data are expresed as means ± SD values from five d i f f e r e n t experiments in duplicate. CA m e a n s catecholamines. UD means undetectable. ND m e a n s not measured.
In c h r o m a f f i n cells AP4A and AP5A are released t o g e t h e r with ATP and catecholamines. The molar ratio ATP/AP4A and A T P / A P 5 A in the p e r f u s e d media was 25 and 18 respectively, w h i c h is in the same order to that obtained for isolated chromaffin granules. With r e s p e c t to the e x o c y t o t i c a l l y released c a t e c h o l a m i n e s the ratio CA/AP4A and CA/AP5A were 94 and 66 respectively. A ratio of 84 for both c o m p o u n d s has been found in isolated chromaffin g r a n u l e s (8). In these c e l l u l a r p r e p a r a t i o n s the catecholamines induced r e l e a s e by c a r b a c h o l r e p r e s e n t e d 1.5 % of their total cellular content. The p e r c e n t a g e values for exocytotically released AP4A and AP5A w i t h r e s p e c t to their total cellular content were 1.4 % and 1.8 % r e s p e c t i v e l y (Table I). Compared to the isolated c h r o m a f f i n
Vol. 48, No. 24, 1991
Carbachol Released Dinucleotides
2321
cells, there was a poor release of d i n u c l e o t i d e s from a d r e n a l glands (Table I), and if one bovine adrenal m e d u l l a c o n t a i n e d r o u g h l y 500x106 cells (7) the release of AP4A and AP5A w o u l d be about one order of m a g n i t u d e lower when compared to isolated c h r o m a f f i n cells. L
C
A
I
AMP
AMP
0.001 uA
"r : ADP ATP
/
~A
L ' ADP
I
~LATP
E r,a
Ap A
tn
u,I
o z <[ 0Q nO U:I m
J
D AMP
AP4A
AMP
AP5A
AP4A
AP5A
J. 1' ADP
•~ AOP
J
0
s
I0
,'s 2'0
;
~
&
;0
;s
90
Time (min)
FIG.
1
H.P.L.C. elution profile of: (A) p e r f u s i o n media from b o v i n e adrenal glands after carbachol stimulation. (B) the same as above enriched with 200 pmol each AP4A and APsA. (C) P e r f u s i o n media from isolated c h r o m a f f i n cells after stimulation. (D) The same as above e n r i c h e d with e x t e r n a l standards of AP4A and APsA, 200 pmol each. All samples were p r o c e s s e d as d e s c r i b e d in M a t e r i a l s and Methods. The procedure preserved the AP4A and APsA c o n t e n t of samples, but destroyed most of the ATP, w h i c h a p p e a r e d as AMP in the chromatogram.
Discussion The i n t r a c e l l u l a r functions of Ap4A as an a l l o s t e r i c e f f e c t o r of DNA p o l y m e r a s e u (8,9) and the p o s s i b i l i t y of it s e r v i n g as an "alarmone" in m e t a b o l i c stress (i0,ii) were the first to be reported. In addition, the presence of Ap3A and AP4A in the
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Carbachol Released Dinucleotides
Vol. 48, No. 24, 1991
secretory granules of platelets, and their release to e x t r a c e l l u l a r media, opened the p o s s i b i l i t y of their action t h r o u g h m e m b r a n e receptors, as messengers on the blood cells and t h e v a s c u l a r system (2,3,4). The presence of AP4A and APsA - but not AP3A - on the n e u r o s e c r e t o r y granules of c h r o m a f f i n cells s u g g e s t e d that these compounds could have a function on neural t i s s u e s i g n a l l i n g (i). This p o s s i b i l i t y is s t r e n g t h e n e d by the r e s u l t s r e p o r t e d here d e m o n s t r a t i n g that adrenal glands and isolated c h r o m a f f i n cells released d i a d e n o s i n e p o l y p h o s p h a t e s AP4A and A p S A - under secretaqogue stimulation. The m e t h o d d e s c r i b e d here allowed the q u a n t i f i c a t i o n of both d i n u c l e o t i d e s from the p e r f u s i o n media of n e u r o s e c r e t o r y cells.
0.001
uA
i
~ p 4A
~
E
f-
P5 A
tN .i
,¢ o ¢oo z
|
I
I
I
!
I
|
D
00
n..
AMP
AMP
ATP
L
0
5
10
15
o
20
Time
(rain)
l'S
FIG. 2 H.P.L.C. c h r o m a t o g r a p h y of recovered fractions from isolated c h r o m a f f i n cells. (A) AP4A re-chromatography. (B) The r e c o v e r e d AP4A was treated with 0.3 u.I./ml of phosphodiesterase for I0 minutes at 37°C. The d i s a p p e a r a n c e of the dinucleotide corresponds to the m o n o n u c l e o t i d e s appearance. (C) AP5A re-chromatography. (D) The same as in B but for AP5A. AT 4 means a d e n o s i n e tetraphosphate.
The d i n u c l e o t i d e s AP4A and APsA r e l e a s e d by the c a r b a c h o l action, and also the AP3A, which was not p r e s e n t in the p e r f u s e d
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Carbachol Released Dinucleotides
2323
m e d i a after stimulation, had an inhibitory action on n i c o t i n e e v o k e d r e l e a s e of c a t e c h o l a m i n e s in chromaffin cells (5). In this c e l l u l a r model the r e l e a s e d d i n u c l e o t i d e s seem to control their own s e c r e t i o n t o g e t h e r with c a t e c h o l a m i n e s and ATP. The p r e s e n c e of h i g h a f f i n i t y b i n d i n g sites with K d values under the n a n o m o l a r range, has been r e p o r t e d for AP4A in cultured c h r o m a f f i n cells (12). A n o t h e r aspect is the fate of these d i n u c l e o t i d e s at the extracellular space. In this way the p r e s e n c e of an ectop h o s p h o d i e s t e r a s e activity with high affinity for AP4A has been shown (13). The p r e s e n c e of an ecto-ATPase, e c t o - A D P a s e and ecto5' n u c l e o t i d a s e at the plasma membranes of isolated c h r o m a f f i n cells allows the complete degradation of these c o m p o u n d s to a d e n o s i n e (14), o r i g i n a t i n g a family of adenine c o m p o u n d s w i t h large m o d u l a t o r y functions on neural and non neural t i s s u e s (15). If adenosine polyphosphates are constituents of some s e c r e t o r y g r a n u l e s and released by action of secretagogues, the search for a p h y s i o l o g i c a l role in cellular s i g n a l i n g will be of some interest. The effects of d i a d e n o s i n e p o l y p h o s p h a t e s on c a t e c h o l a m i n e s s e c r e t i o n (5) could thus be a starting point to e l u c i d a t e the potential effects and relevance of these natural c o m p o n e n t s in n e u r o t r a n s m i s i o n and provide new d i r e c t i o n s in the d e v e l o p m e n t of p h a r m a c o l o g i c a l l y active drugs.
Acknowledqements
This i n v e s t i g a t i o n was supported by a research grant from the S p a n i s h M i n i s t r y of Education and Science, CICYT PB 89-00095. J. Pintor is r e c i p i e n t of a fellowship from the R e c t o r a d o of U n i v e r s i d a d Complutense. We thank Erik Lundin for his help improving this manuscript and Dr. E. Castro for the CA determinations.
References
i. 2. 3. 4. 5. 6. 7. 8. 9. i0. II.
A. R O D R I G U E Z DEL CASTILLO, M. TORRES, E.G. D E L I C A D O and M.T. M I R A S - P O R T U G A L , J.Neurochem. 5_!i 1696-1703 (1988). H. F L O D G A A R D and H. KLENOW, Biochem. J. 208 737-742 (1982). J. L U T H J E and A. OGILVIE, Biochem.Biophys.Res. Commun. 115 253-260 (1983). R. BUSSE, A. O G I L V I E and U. POHL, Am.J.Physiol. 254 H828H832 (1988). E. CASTRO, M. TORRES, M.T. M I R A S - P O R T U G A L and M.P. GONZALEZ, Br.J.Pharmacol. i 0 0 3 6 0 - 3 6 4 (1990). M.T. MIRAS-PORTUGAL, P.ROTLLAN and D. AUNIS, Neurochem. Int. 89-93 (1985). J.H. PHILLIPS, Neurosci. Z 1595-1609 (1982). E. R A P A P O R T and P.C. ZAMECNIK, Proc.Natl.Acad. S c i . u s A 733 3984-3988 (1976). F. GRUMMT, G. WALTL, H.M. JANTZEN, K . H A M P R E C H T U . H U E B S C H E R and C.C.KUENZLE, Proc.Natl.Acad. Sci.USA 76 6081-6085 (1979). P. ZAMECNIK, Anal.Biochem. 134 i-i0 (1983). P.N. GARRISON, S.A. MATHIS and L.D. BARNES, J.Bacteriol. 171 1506-1512 (1989).
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12. 13. 14. 15.
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Vol. 48, No. 24, 1991
J. PINTOR, M. TORRES, E. C A S T R O & M.T. M I R A S - P O R T U G A L , Br. J. P h a r m a c o l . (in press). (1991). M.T. M I R A S - P O R T U G A L , J. PINTOR, P. R O T L L A N a n d M. T O R R E S , N . Y . A c a d . Sci. 603 5 2 3 - 5 2 6 (1990). M. T O R R E S , J. P I N T O R a n d M.T. M I R A S - P O R T U G A L , A r c h . B i o c h e m . B i o p h y s . 279 37-44 (1990). M. W I L L I A M S , A n n . R e v . Pharmacol. Toxicol. 2_/7 3 1 4 - 3 4 5 (1987).
CARBACHOL INDUCED R E L F ~ S E ~pSA-
FROM
PERFUSED
JesQs Pintor, Departamento
BOVINE
Magdalena
Pergamon Press
OF DIADENOSINE POLYPHOSPH~TES ADRENAL CELLS
MEDULLA
AND
ISOLATED
-AP4A
AND
CHROMAFFIN
Torres and M.Teresa M i r a s - P o r t u g a l *
de Bioqulmica, Facultad de Veterinaria, Complutense de Madrid. 28040 Madrid.
Universidad
(Received in final form April 8, 1991) Summary
The diadenosine polyphosphates - AP4A and APsA were released from perfused bovine adrenal glands and recently isolated chromaffin cells by the action of carbachol. The H.P.L.C. technique reported here allowed the quantification of pmol amounts of these compounds present in biological samples from the perfusion media after stimulation. Both compounds !AP4A and APsA ) were identified by the retention time In H.P.L.C. chromatography, co-elution with standards, re-chromatography and destruction by the phosphodiesterase action. Bovine adrenal glands s t i m u l a t e d with 100~M carbachol released 0.47 ± 0.12 n m o l / g l a n d of AP4A and i.ii ± 0.26 nmol/gland of APsA. Isolated bovine chromaffin cells after 100~M carbachol, as secretagogue, released ii.i ± 0.8 pmol/106 cells of AP4A and 15.8 ± i.i pmol/106 cells of AP5A. The ratio of these compounds with respect to the exocytotically released ATP and c a t e c h o l a m i n e s was in the same order as that found in isolated c h r o m a f f i n granules.
The presence of diadenosine polyphosphates, adenosine (5') t e t r a p h o s p h o (5') adenosine (diadenosine tetraphosphate, . . Ap 4 A) and . adenosine (5') pentaphospho (5') adenoslne (dladenoslne pentaphosphate, APsA ) has been recently described in the c h r o m a f f i n granules of bovine adrenal medulla, where they are costored with catecholamines and ATP (i). The dense granules of human p l a t e l e t s also co-stored adenosine (5') t r i p h o s p h o (5') adenosine (diadenosine triphosphate, AP3A ) and AP4A with serotonine and adenine nucleotides (2,3). The physiological actions of diadenosine polyphosphates are receiving increasing a t t e n t i o n with respect to the vascular system (4), and also in neural secretory tissues, as chromaffin cells. In the latter the d i a d e n o s i n e polyphosphates increase basal catecholamine secretion, but have an inhibitory action on nicotine evoked release (5). * To w h o m correspondence should be addressed: Prof. M.T. MirasPortugal, Dpto. Bioqu~mica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid. Spain. Telph.13943894. FAX 13943883. 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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In order to know the physiological importance of these d i a d e n o s i n e polyphosphates, it is of great interest to d e t e r m i n e their r e l e a s e from stimulated neural tissues. Therefore, the e x p e r i m e n t a l work reported here tries to isolate and q u a n t i f y the AP4A and APsA from p e r f u s e d adrenal glands and isolated c h r o m a f f i n cells after carbachol stimulation.
Materials and Methods Gland perfusion. Fresh bovine adrenal glands were o b t a i n e d from the local s l a u g h t e r h o u s e 20-30 minutes after the death of animals. A d r e n a l glands were perfused with 200 ml of Locke's s o l u t i o n (mM, NaCl 140, KCl 4.4, CaCl 2 2.5, KH2PO 4 1.2, MgSO 4 1.2, HEPES i0, NaHCO 3 4.0, glucose 5.6 and ascorbic acid I, pH 7.5) at 37°C at a flow rate of 5 ml/min to eliminate the blood constituents. Then 10 ml of the perfusion media were c o l l e c t e d as control for each studied gland. Secretion was s t i m u l a t e d with 10 ml 100~M c a r b a c h o l in Locke's solution. One ml alliquot was taken for c a t e c h o l a m i n e and ATP analysis and the rest was p r o c e s s e d in order to q u a n t i f y the AP4A and APsA levels. Cell superfusion. Recently isolated c h r o m a f f i n cells were o b t a i n e d as d e s c r i b e d in (6). After the percoll g r a d i e n t step, cells were collected and 25xi06 cells were used for each experiment. Cells were superfused on a Sterivex GS filter (Millipore) with Locke's solution at a flow rate of 5 m l / m i n and 37°C temperature. 10 ml of the perfused media w i t h o u t carbachol were taken for control in each done experiment. Cells were s t i m u l a t e d with 100 ~M carbachol in 10 ml of the p e r f u s i o n media. From this, 1 ml alliquot was taken for c a t e c h o l a m i n e and ATP analysis. H.P.L.C. analysis. Samples for AP4A and APsA analysis, both from i s o l a t e d c h r o m a f f i n cells and perfused adrenal glands were p r o c e s s e d as follows: samples were submitted to 100°C t e m p e r a t u r e in w a t e r bath for 10 minutes and then c e n t r i f u g e d to 12000xg for five minutes. This procedure removed the m a j o r i t y of p r o t e i n s from the samples and also destroyed part of the ATP p r e s e n t in the samples, that could interfere with the AP4A peak. Nevertheless, AP4A and APsA were not destroyed. The high c o n c e n t r a t i o n of ATP and the close r e t e n t i o n times between ATP and AP4A r e s u l t e d in an o v e r l a p p i n g of both compounds in H.P.L.C. c h r o m a t o g r a p h y in nonprocessed samples. Following this step, samples were c h r o m a t o g r a f i e d through a Sep-Pak Accell Q M A c o l u m n (Waters). All n u c l e o t i d e s were r e t a i n e d and the elution was done with 1 ml NaCl 1 M. Samples were then more concentrated and suitable for H.P.L.C. a n a l y s i s and q u a n t i f i c a t i o n of the dinucleotides. S t a n d a r d s of d i a d e n o s i n e p o l y p h o s p h a t e s added to samples of p e r f u s i o n media were r e c o v e r e d to an extent of 94-96 % of the total at the end of the c o m p l e t e treatment. The recovery of d i a d e n o s i n e p o l y ~ h o s p h a t e s from the isolated c h r o m a f f i n cells, at a density of 10 ~ cells/ml of c u l t u r e media was 90-92. % for both AP4A and APsA.. These. values were o b t a i n e d by addlng external standards of d l n u c l e o t l d e s to c e l l u l a r p r e p a r a t i o n s and then submitted to the same p r o c e d u r e as d e s c r i b e d above for the perfusion media. Routinely, 10-50~i
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samples were injected for H.P.L.C. chromatography. The H.P.L.C. e q u i p m e n t was from Waters consisting of a 600 E control d e l i v e r y system, U6K injector, 4 5 1 1 max detector, a 745 data m o d u l e and a ~ b o n d a p a k C18 column (30 cm long 0.4 inner diameter). In isocratic conditions, with i0 mM KH2PO 4 buffer, pH 7.5, 2 mM t e t r a b u t y l a m m o n i u m and 15% acetonitrile as m o b i l e phase, the r e t e n t i o n times were 2.73 min for AMP, 4.60 min for ADP, 7.20 min for ATP, 11.53 min for AP4A and 19.07 min for APsA. W h e n necessary, the AP4A and AP5A peaks were c o l l e c t e d from the H.P.L.C. c o l u m n eluate and submitted to r e - c h r o m a t o g r a p h y e l u t i o n w i t h s t a n d a r d s in H.P.L.C., or p h o s p h o d i e s t e r a s e digestion. C a t e c h o l a m i n e s determination. C a t e c h o l a m i n e s were m e a s u r e d w i t h an e l e c t r o c h e m i c a l detector (Metrohm) using n o r a d r e n a l i n e as standard, as p r e v i o u s l y described (5). Chemicals. All nucleotides were from Sigma (St. Louis, Missouri) and B o e h r i n g e r (Manheim, Germany). P h o s p h o d i e s t e r a s e from C r o t a l u s durisus (EC 3.1.15.1) was from P h a r m a c i a (Upsala, Sweden). Results D i a d e n o s i n e p o l y p h o s p h a t e s were released from p e r f u s e d bovine adrenal glands and recently isolated c h r o m a f f i n cells by the action of carbachol. In absence of any secretory stimuli it was not p o s s i b l e to q u a n t i f y the AP4A and APsA in the p e r f u s i o n m e d i a of both systems. After stimulation of adrenal g l a n d s with c a r b a c h o l 100~M, both d i n u c l e o t i d e s were present at the n a n o m o l a r c o n c e n t r a t i o n in the p e r f u s i o n media. In fig. IA an H.P.L.C. c h r o m a t o g r a m is shown for a sample from p e r f u s e d adrenal glands. The total amount of d i n u c l e o t i d e s released was q u a n t i f i e d and the o b t a i n e d v a l u e s are summarized in table I. No AP3A was d e t e c t e d in our a n a l y t i c a l conditions. These values were h i g h l y v a r i a b l e due to the h e t e r o g e n i t y of bovine adrenal glands. W h e n samples of adrenal g l a n d s p e r f u s a t e were enriched with exogenous AP4A and AP5A , c o - e l u t i o n of standards with problems occurred, as shown in fig. lB. Most of the ATP released was d e s t r o y e d by the high t e m p e r a t u r e procedure, that does not affect the content of dinucleotides. I s o l a t e d bovine chromaffin cells p r o v i d e d a better model to study the e x o c y t o t i c release of AP4A and APsA than was the adrenal gland. The values obtained for both d i n u c l e o t i d e s were highly r e p r o d u c i b l e from one cellular p r e p a r a t i o n to another and are s u m m a r i z e d in table I. As occurs with adrenal glands, AP3A was not d e t e c t e d in p e r f u s a t e s from isolated chromaffin cells. In fig. iC a t y p i c a l c h r o m a t o g r a m of a sample from 100~M carbachol s t i m u l a t e d p e r f u s e d cells is shown. Due to the p r e p a r a t i o n samples, as d e s c r i b e d in methods, most of the released ATP was r e c o v e r e d as AMP. In fig. ID the c o - e l u t i o n with standards of AP4A and APsA is shown. In order to study the p h o s p h o d i e s t e r a s e action on both s u p p o s e d d i a d e n o s i n e polyphosphates, the peaks c o n t a i n i n g AP4A and AP5A were c o l l e c t e d from the H.P.L.C. column eluate. In fig. 2A
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and 2C a r e - c h r o m a t o g r a p h y of AP4A and APsA is shown. The effect of p h o s p h o d i e s t e r a s e on the collected samples is shown in fig. 2B and 2D. The d i s a p p e a r a n c e of both dinucleotides was o b s e r v e d with the c o r r e s p o n d i n g appearance of adenine m o n o n u c l e o t i d e s AMP and ATP for d i a d e n o s i n e t e t r a p h o s p h a t e and the appearance of AMP and a d e n o s i n e t e t r a p h o s p h a t e for diadenosine pentaphosphate. TABLE I Adrenal Gland nmol/gland Basal Release
Chromaffin Cells pmol/106 c e l l s
Carbachol 100BM
Basal Release
Carbachol 100~M
AP4A
UD
0.47±0.14
UD
ii.i±0.8
APsA
UD
i.ii±0.30
UD
15.8±1.1
15.5±4.10
9.5±2.6
281±21
ATP
0.32±0.13
CA
ND
ND
14±3
1,039±86
The total c a t e c h o l a m i n e content was 69±10.4 nmol/106 cells m e a s u r e d after cellular lysis with Triton X-100. Total AP4A and AP5A content were 0.81±0.41 nmol/106 cells and 0.87±0.45 nmol/106 cells. The total ATP content was 42.3 ± 3.8 nmol/106 cells. The ATP was m e a s u r e d by H.P.L.C. directly, w i t h o u t processing of samples. Dinucleotides values were d e t e r m i n e d after the p r o c e s s i n g of samples as d e s c r i b e d in M a t e r i a l s and Methods. Data are expresed as means ± SD values from five d i f f e r e n t experiments in duplicate. CA m e a n s catecholamines. UD means undetectable. ND m e a n s not measured.
In c h r o m a f f i n cells AP4A and AP5A are released t o g e t h e r with ATP and catecholamines. The molar ratio ATP/AP4A and A T P / A P 5 A in the p e r f u s e d media was 25 and 18 respectively, w h i c h is in the same order to that obtained for isolated chromaffin granules. With r e s p e c t to the e x o c y t o t i c a l l y released c a t e c h o l a m i n e s the ratio CA/AP4A and CA/AP5A were 94 and 66 respectively. A ratio of 84 for both c o m p o u n d s has been found in isolated chromaffin g r a n u l e s (8). In these c e l l u l a r p r e p a r a t i o n s the catecholamines induced r e l e a s e by c a r b a c h o l r e p r e s e n t e d 1.5 % of their total cellular content. The p e r c e n t a g e values for exocytotically released AP4A and AP5A w i t h r e s p e c t to their total cellular content were 1.4 % and 1.8 % r e s p e c t i v e l y (Table I). Compared to the isolated c h r o m a f f i n
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cells, there was a poor release of d i n u c l e o t i d e s from a d r e n a l glands (Table I), and if one bovine adrenal m e d u l l a c o n t a i n e d r o u g h l y 500x106 cells (7) the release of AP4A and AP5A w o u l d be about one order of m a g n i t u d e lower when compared to isolated c h r o m a f f i n cells. L
C
A
I
AMP
AMP
0.001 uA
"r : ADP ATP
/
~A
L ' ADP
I
~LATP
E r,a
Ap A
tn
u,I
o z <[ 0Q nO U:I m
J
D AMP
AP4A
AMP
AP5A
AP4A
AP5A
J. 1' ADP
•~ AOP
J
0
s
I0
,'s 2'0
;
~
&
;0
;s
90
Time (min)
FIG.
1
H.P.L.C. elution profile of: (A) p e r f u s i o n media from b o v i n e adrenal glands after carbachol stimulation. (B) the same as above enriched with 200 pmol each AP4A and APsA. (C) P e r f u s i o n media from isolated c h r o m a f f i n cells after stimulation. (D) The same as above e n r i c h e d with e x t e r n a l standards of AP4A and APsA, 200 pmol each. All samples were p r o c e s s e d as d e s c r i b e d in M a t e r i a l s and Methods. The procedure preserved the AP4A and APsA c o n t e n t of samples, but destroyed most of the ATP, w h i c h a p p e a r e d as AMP in the chromatogram.
Discussion The i n t r a c e l l u l a r functions of Ap4A as an a l l o s t e r i c e f f e c t o r of DNA p o l y m e r a s e u (8,9) and the p o s s i b i l i t y of it s e r v i n g as an "alarmone" in m e t a b o l i c stress (i0,ii) were the first to be reported. In addition, the presence of Ap3A and AP4A in the
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secretory granules of platelets, and their release to e x t r a c e l l u l a r media, opened the p o s s i b i l i t y of their action t h r o u g h m e m b r a n e receptors, as messengers on the blood cells and t h e v a s c u l a r system (2,3,4). The presence of AP4A and APsA - but not AP3A - on the n e u r o s e c r e t o r y granules of c h r o m a f f i n cells s u g g e s t e d that these compounds could have a function on neural t i s s u e s i g n a l l i n g (i). This p o s s i b i l i t y is s t r e n g t h e n e d by the r e s u l t s r e p o r t e d here d e m o n s t r a t i n g that adrenal glands and isolated c h r o m a f f i n cells released d i a d e n o s i n e p o l y p h o s p h a t e s AP4A and A p S A - under secretaqogue stimulation. The m e t h o d d e s c r i b e d here allowed the q u a n t i f i c a t i o n of both d i n u c l e o t i d e s from the p e r f u s i o n media of n e u r o s e c r e t o r y cells.
0.001
uA
i
~ p 4A
~
E
f-
P5 A
tN .i
,¢ o ¢oo z
|
I
I
I
!
I
|
D
00
n..
AMP
AMP
ATP
L
0
5
10
15
o
20
Time
(rain)
l'S
FIG. 2 H.P.L.C. c h r o m a t o g r a p h y of recovered fractions from isolated c h r o m a f f i n cells. (A) AP4A re-chromatography. (B) The r e c o v e r e d AP4A was treated with 0.3 u.I./ml of phosphodiesterase for I0 minutes at 37°C. The d i s a p p e a r a n c e of the dinucleotide corresponds to the m o n o n u c l e o t i d e s appearance. (C) AP5A re-chromatography. (D) The same as in B but for AP5A. AT 4 means a d e n o s i n e tetraphosphate.
The d i n u c l e o t i d e s AP4A and APsA r e l e a s e d by the c a r b a c h o l action, and also the AP3A, which was not p r e s e n t in the p e r f u s e d
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Carbachol Released Dinucleotides
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m e d i a after stimulation, had an inhibitory action on n i c o t i n e e v o k e d r e l e a s e of c a t e c h o l a m i n e s in chromaffin cells (5). In this c e l l u l a r model the r e l e a s e d d i n u c l e o t i d e s seem to control their own s e c r e t i o n t o g e t h e r with c a t e c h o l a m i n e s and ATP. The p r e s e n c e of h i g h a f f i n i t y b i n d i n g sites with K d values under the n a n o m o l a r range, has been r e p o r t e d for AP4A in cultured c h r o m a f f i n cells (12). A n o t h e r aspect is the fate of these d i n u c l e o t i d e s at the extracellular space. In this way the p r e s e n c e of an ectop h o s p h o d i e s t e r a s e activity with high affinity for AP4A has been shown (13). The p r e s e n c e of an ecto-ATPase, e c t o - A D P a s e and ecto5' n u c l e o t i d a s e at the plasma membranes of isolated c h r o m a f f i n cells allows the complete degradation of these c o m p o u n d s to a d e n o s i n e (14), o r i g i n a t i n g a family of adenine c o m p o u n d s w i t h large m o d u l a t o r y functions on neural and non neural t i s s u e s (15). If adenosine polyphosphates are constituents of some s e c r e t o r y g r a n u l e s and released by action of secretagogues, the search for a p h y s i o l o g i c a l role in cellular s i g n a l i n g will be of some interest. The effects of d i a d e n o s i n e p o l y p h o s p h a t e s on c a t e c h o l a m i n e s s e c r e t i o n (5) could thus be a starting point to e l u c i d a t e the potential effects and relevance of these natural c o m p o n e n t s in n e u r o t r a n s m i s i o n and provide new d i r e c t i o n s in the d e v e l o p m e n t of p h a r m a c o l o g i c a l l y active drugs.
Acknowledqements
This i n v e s t i g a t i o n was supported by a research grant from the S p a n i s h M i n i s t r y of Education and Science, CICYT PB 89-00095. J. Pintor is r e c i p i e n t of a fellowship from the R e c t o r a d o of U n i v e r s i d a d Complutense. We thank Erik Lundin for his help improving this manuscript and Dr. E. Castro for the CA determinations.
References
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12. 13. 14. 15.
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J. PINTOR, M. TORRES, E. C A S T R O & M.T. M I R A S - P O R T U G A L , Br. J. P h a r m a c o l . (in press). (1991). M.T. M I R A S - P O R T U G A L , J. PINTOR, P. R O T L L A N a n d M. T O R R E S , N . Y . A c a d . Sci. 603 5 2 3 - 5 2 6 (1990). M. T O R R E S , J. P I N T O R a n d M.T. M I R A S - P O R T U G A L , A r c h . B i o c h e m . B i o p h y s . 279 37-44 (1990). M. W I L L I A M S , A n n . R e v . Pharmacol. Toxicol. 2_/7 3 1 4 - 3 4 5 (1987).