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Interaction between catecholamines and vasoactive intestinal peptide in cultured astrocytes
Neuroplrarmocolog~ Vol. 27, No. 3, pp. 295-300, 1988 Printed in Great Britain. All rights reserved
Copyright 0
OOZS-3908jSS$3.00 + 0.00 1988 Pergamon Journals Ltd
INTERACTION BETWEEN CATEC~OLA~INES AND VASOACTIVE INTESTINAL PEPTIDE IN CULTURED ASTROCYTES ELISABETH HANSSON’
and L.
R~NNB~cK’.~
iInstitute of Neurobiology and 2Department of Neurology, University of Gteborg,
Sweden
(Accepted 21 September 1987) Summary-Receptors for vasoactive intestinal peptide (VIP) were demonstrated with cyclic AMP as the second messenger on astroglial cells cultured from the cerebral cortex, striatum, hippocampus and brain stem of the newborn rat. Vasoactive intestinal peptide produced increased accumulations of cyclic AMP in nM and pM concentrations, the former more pronounced in the cultures of brain stem, while the latter was more pronounced in the other cultures studied, indicating regional differences’ in the activation of cyclic AMP of VIP receptors. Vasoactive intestinal peptide inhibited isoproterenol-induced accumulation of cyclic AMP dose-dependently, with some regional differences, suggesting interactions between the second messenger systems of /I- and VIP receptors. On the other hand, there was no inhibition of the NA-induced accumulation of cyclic AMP in the presence of VIP, which is in agreement with an interaction between the second messenger systems for VIP and the cc-adrenoceptor. The data support interactions between the second messenger systems for VIP and a- and ~-adrenoceptors on cultured astrocytes. The functional impli~tions are at present unknown, but it might be that the peptide can modulate the response of the second messenger to catecholamines on astroglial cells through intramembrane mechanisms. Key words: astrocytes, brain stem, cerebral cortex, hippocampus, isoproterenol, noradrenaline, primary culture, striatum, vasoactive intestinal peptide.
In recent years it has become clear that several hormones exert their effects on target cells through the “second messenger”, cyclic adenosine 3’,5’ monophosphate (cyclic AMP). Interaction between such hormones and cell surface receptors results in stimulation of the membrane bound enzyme, adenylate cyclase, which catalyzes the conversion of ATP to cyclic AMP. Stimulation of astrocytes in primary cultures by c(- and /I-adrenoceptors leads to changes in cyclic AMP (McCarthy and de Vellis, 1978, 1979; Van Calker, Miiller and Hamprecht, 1980) as does stimulation by dopamine in striatal astroglial cultures (Hansson, Rijnnback and Sellstrom, 1984). The neuropeptides, vasoactive intestinal peptide (VIP), secretin and glucagon stimulate the accumulation of cyclic AMP in astrocytes (Chneiweiss, Glowinski and Premont, 1985; Evans, McCarthy and Harden, 1984; Koh, Kyritsis and Chader, 1984; Rougon, Noble and Mudge, 1983; Van Calker et al., 1980) and may also act as regulators of metabolism in brain by affecting glycogenolysis (Magistretti, Manthorpe, Bloom and Varon, 1983). The presence of a neuropeptide and a biogenic amine has been demonstrated in the same terminals of central and peripheral neurones. This has led to the hypothesis that neurotransmitters, existing together within the same neurone can interact at pre- or postsynaptic sites in a functionally coordinated manner (Hiikfelt, Johansson, Ljungdahl, Lundberg and Schultzberg, 1980).
Interactions between the cyclic AMP of VIP receptors and the adenylate cyclase system of other receptors have already been investigated on astrocytes (Chneiweiss et al., 1985; Rougon et ai., 1983). The aim of the present work was to evaluate whether or not there are differences in the VIP-induced accumulation of cyclic AMP in astrocytes, cultivated from different regions of the brain of the newborn ratcerebral cortex, striatum, hippocampus and brain stem. Furthermore, it was elucidated whether or not there are interactions between the second messenger systems of VIP and the p-agonist (ISO) or the CI-and /?-agonist noradrenaline (NA). METHODS
Tissue cultures
The primary cultures of astroglia were made from newborn rats (Sprague-Dawley strain, Alab, Sweden). Cerebral cortex, striatum, hippocampus and brain stem were extirpated as described by Hansson, Ronnblck, Persson, Lowenthal, Noppe, Alling and Karlsson (1984). In short, the cells were grown in Eagle’s minimum essential medium (MEM, Flow Laboratories, Scotland) supplied with extra substances, to make up the following final composition: double concentrations of amino acids, 2 mM glutamine, 7 mM glucose and quadruple concentrations of vitamins; 250,000 IU penicillin/l, 0.5% 295
ELISAHETH HAYSSON
296
and
L.
R&NHACK
STRlATUM
_~~_ 10 9
6
~_ 7
6
._ 5
10
9
0
7
6
5
-- -~~--_-.-_. 10
9
8
7
6
5
10
9
8
7
6
5
J
Fig. 1. The effects of increasing concentration of VIP on accumukion of cyclic AMP were estimated on primary cultures of astroglia from cerebral cortex, striatum, hippocampus and brain stem. The results are expressed as mean accumulation of cyclic AMP in relation to control values. Values are means of 5 experiments with duplicates in each. Statistical evaluation: Student’s l-test comparing largest respect values with those of controls. *P < 0.05, **P < 0.01. SEM did not exceed 10% (not indicated in the figure).
20% (v/v) fetal calf serum (Gibco were also added. The cells were grown in a humidified atmosphere with pH set at 7.3 for 2 weeks. Medium were changed three times a week. streptomycin
Bio Cult.
and
Lab.,
U.K.)
Measurement of cyclic AMP The incubation medium was changed to serum-free Eagle’s MEM 1 hr before the experiments. The cultures were exposed to VIP (Sigma Fine Chemicals, St. Louis, Missouri) in a concentration range between 10-10~10~5 M. The different cultures were further exposed to 10e6 M o,L-isoproterenol (Sigma), or to lOA M L-noradrenaline (NA) (Sigma) in the presence or absence of VIP. Antagonists, lo-’ M propranolol (Sigma) or 10. 4 M phentolamine (Sigma) were added before VIP, isoproterenol or NA. The exposure time was 1Omin and the incubations were made in the presence of the phosphodiesterase inhibitor I mM iso-butyl-I-methyl-xanthine (IBMX) (Sigma). After incubation, the medium was decanted and the cultures were immediately exposed to 2 M ice-cold perchloric acid and put on ice. The cells were scraped off, glass/glass homogenized and centrifuged at 2000 x g for 20 min, all at 4 C. The supernatants were run on anion exchange resins, Ag l-X8. 20&400 mesh (BioRad Lab., Richmond, California) at pH 6.95-7.05. The cyclic AMP was eluted using 5 ml 2 M formic acid. After freeze-drying and dissolving in 1.0 ml 0.05 M sodium acetate at pH 6.2, cyclic AMP was determined using radioimmunoassay kits (NEN, Boston, Massachusetts) as described by Steiner, Parker and Kipnis (1972a) and Steiner, Pagliara, Chase and Kipnis (1972b). Cyclic AMP
from all samples of one experiment was purified by ion exchange chromatography during one day to standardize the procedure and minimize a variable loss of the cyclic nucleotide during purification. When performed in this way, the recoveries from the columns varied less than 10%. The recovery of cyclic AMP from the columns from one day to another showed a greater variance. Therefore, the results are expressed as accumulation of cyclic AMP as a percentage of control.
Determination
of protein
were done according to Farr and Randall (1951), with bovine serum albumin as standard (Sigma). Amount of protein in one tissue culture, one sample (35 mm, in dia, Petri dish) was -0.5 mg. Determinations
Lowry,
of protein
Rosebrough,
RESl I.TS
There was an increased accumulation of cyclic AMP in the primary cultures of astrocytes from cerebral cortex, striatum, hippocampus and brain stem after incubation with different concentrations of VIP. The stimulatory effect of VIP was most pronounced in the cultures of cerebral cortex and hippocampus, reaching 5 times controls (Fig. 1). The control values were in pmol cyclic AMP,mg prot I: for cerebral cortex 39 &- 6.2; for striatum 42 & 7.1; for hippocampus 40 i 6.5 and for brain stem 43 & 7.7 (mean k SEM of 5 experiments). The detection limit of the assay was t0.2pmol cyclic AMP/ml. The increase in cyclic AMP, activated by VIP showed two peaks, one in the nM range (1 x 10 ‘-5 x 10m9 M)
8’
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298
ELISABETH
HANSON and L.
the other in the PM range (1 x 10~ ‘-1 x 10 ’ M). In the cultures of brain stem, the accumulation of cyclic AMP was more prominent in the nM range, while in the other cultures there was a more prominent accumulation of cyclic AMP in the PM range (Fig. 1). Isoproterenol (IO-” M) evoked a 2c-60 times accumulation of cyclic AMP, somewhat different in the various cultures. In the presence of 10m6 M isoproterenol, VIP evoked no further increase in cyclic AMP in the concentration interval between 10 ‘oplO ‘M in the cerebral cortex. The pattern of formation of cyclic AMP was similar to that of VIP alone, with peaks in the nM and PM ranges. However. in the presence of VIP there was a relative depression of the formation of cyclic AMP induced by isoproterenol, more pronounced at greater concentrations of VIP (Fig. 2). The stimulation of accumulation of cyclic AMP by VIP in the presence of isoproterenol and the fl-adrenergic antagonist propranolol was not different from that of VIP alone (Fig. 2). The pattern of activation of cyclic AMP after incubation with both isoproterenol and VIP was different in the various cultures, however, with the relative increase of the peaks at the same concentrations of VIP as after incubation in VIP alone. In the brain stem there was a more pronounced activation of formation of cyclic AMP in the nM range, while the other cultures showed a more prominent accumulation of cyclic AMP with VIP in the PM concentration in the presence of I PM isoproterenol. Moreover. in all cultures and especially in the striatum, there was a prominent inhibition of the isoproterenol-induced activation of cyclic AMP after incubation in large concentrations of VIP (IO-’ M). Even NA evoked a prominent activation of cyclic AMP. After incubation in IO-‘M NA there was no additive effect on accumulation of cyclic AMP in the presence of VIP in any of the cultures of astroglial primary cells studied. There were still peaks at the high- and low-affinity VIP-activated sites, but not so pronounced as with VIP alone or with VIP in the presence of isoproterenol. Neither did VIP reduce the NA-induced cyclic activation of AMP over the concentration range studied. Incubation with VIP in different concentrations, together with the fl-adrenergic antagonist propranolol and NA, showed a reduction of the formation of cyclic AMP in comparison with that caused by VIP alone. In the presence of NA and the r-adrenergic antagonist phentolamine there was a prominent accumulation of cyclic AMP with two peaks, however, never exceeding the formation of cyclic AMP for phentolamine and NA, without VIP. After incubation in phentolamine and propranolol, together with NA and VIP, the accumulation of cyclic AMP was not different from that of VIP alone (Figs 1 and 2). Phentolamine and propranolol showed no effects on the VIPinduced stimulation of cyclic AMP in the doseinterval studied.
R~~NNBACK DISCUSSION
Primary cultures of astroglia from the cerebra1 cortex, striatum, hippocampus and brain stem of the newborn rat exhibited VIP receptors that regulated the cyclic AMP system. The primary cultures used in this study were previously characterized extensively to contain immunocytochemically-defined astroglia, which express functional properties found on astroglia in ciao (Hansson, 1984, 1985, 1986; Hansson, Eriksson and Nilsson, 1985; Hansson et al., 1984). The action of VIP on the second messenger cyclic AMP system at small concentrations (nM) and at large concentrations (PM) has earlier been demonstrated on astrocytes from whole brain of mouse (Van Calker et ul.. 1980) cerebral cortex, striatum and mesencephalon (Chneiweiss rt nl., 1985). The stimulation of cyclic AMP at these two concentrations of VIP could be in accordance with the existence of two distinct VIP receptors, differing in their affinity and/or their coupling efficiency. Another possibility is the formation of a metabolite that could act as a partial agonist with high affinity for the receptor (Chneiweiss et al., 1985). The relatively low potency of VIP to increase formation of cyclic AMP (335 time of control) is in agreement with previous work (Rougon et al., 1983; Chneiweiss et al., 1985). while Van Calker et al. (1980) and Evans et al. (1984) showed a more prominent activation of cyclic AMP (20-30 times that of control). The discrepancies might be due to different sources of VIP. Another possibility might be degradation of VIP during the IO min incubation, this possibility being very unlikely, however, since Van Calker et (11.and Evans et al. performed their incubations for 10min in the presence of a phosphodiesterase inhibitor, as was done here. The VIP-induced activation of cyclic AMP differed between the various regions of the brain. Astrocyte cultures from cereral cortex, striatum and hippocampus showed a greater response in the PM-range of VIP, while cultures of brain stem showed a greater response in the nM-range. Chneiweiss et al. (1985) found a greater activation of cyclic AMP in the PM-range of VIP in astroglia from the cerebral cortex, striatum and mesencephalon of the mouse. The differences in the high-affinity receptor site might be due to differences in species and also region of the brain isolated (mesencephalon vs brain stem). The results indicate that high-affinity and low-affinity VIP receptors might differ in relative amounts in astrocytes from different regions. In view of previous work in this laboratory, demonstrating basic similarities in the cultures, as to cellular content and biochemical parameters, on one hand, and differences between the cultures from the various regions of the brain concerning functional parameters, such as uptake of neurotransmitters, activity of enzymes for catabolism of neurotransmitters, Z-. b- and DA receptors and profiles of soluble proteins, on the other hand
Interaction between ~techolamines
(Hansson, 1988), the present data with different characteristics of the VIP-activated cyclic AMP in the various regions of the brain further indicate a regional specialization of the cells. Astrocytes in primary cultures express receptors for various neurotransmitters and neuromodulators (Van Calker et al., 1980). There may be formations of mosaics of receptors on the cell membrane of astroglia and the second messengers of these receptors may “communicate” with each other through intramembrane signals. Thus, a number of interactions between second messenger systems for receptors for monoamines and neuropeptides may occur as reported on neuronal membranes in the central nervous system (Fuxe, Agnati, Benfenati, Celani, Zini, Zoli and Mutt, 1983). When VIP and isoproterenol were applied together there was an inhibition of the isoproterenol-induced activation of cyclic AMP over a broad concentration range. The inhibition was more prominent at larger concentrations of VIP. There were two peaks in the nM and p M ranges even in the presence of isoproterenol. The sum of the VIP and isoproterenol-induced accumulation of cyclic AMP did not exceed that of isoproterenol alone, which is in agreement with Chneiweiss et al. (1985). Thus, there must be an interaction between VIP and the fi -adrenoceptor-activated cyclic AMP. In the brain stem there was a partial additivity between VIP (nM range) and isoproterenol. Similar results were obtained by Chneiweiss et al. (1985) in glia from mesencephalon although they examined the low affinity site. In the presence of NA and VIP there were only very small peaks in the nM or PM ranges of VIP. Moreover, VIP exerted a much less depression of NA-induced formation of cyclic AMP than of isoproterenol-induced formation of cyclic AMP. Thus, there must be an interaction between VIP and the second messenger system of the /I-adrenoceptor, with a resulting relative increase in cyclic AMP. Rougon ef al. (1983) demonstrated a potentiation by VIP on the NA-activated formation of cyclic AMP in astrocyte cultures from cerebral cortex. The differences between the results of these two studies are at present unclear. Furthermore, NA in the presence of the P-adrenoceptor antagonist propranolol, at least partly reduced the VIP-induced stimulation of cyclic AMP. This is in agreement with the data of Van Calker er ai. (1980). It is difficult at present to draw any conclusion whether or not there is a bidirectional interaction between the second messengers coupled to VIP and the a-adrenoceptor. In cultures of astroglia from cerebral cortex and striatum, the peaks at the nM or PM ranges of VIP, in the presence of NA, were more pronounced than in cultures from hippocampus or brain stem where the peaks were hardly seen. One drawback with the culture system is that the cells are less mature than those in the intact nervous
and vasoaetive intestinal peptide
299
system (see Hansson, 1988), due to the lack of physiological contacts with other cells during cultivation. A hypersensitivity of receptors might occur in the absence of normal neurotransmission in the cultures. Thus, it can be questioned if the cells bear these receptors, and if there are interactions between the second messenger systems of the receptors in the adult nervous system, or if they represent a stage of development. Under all circumstances, the results suggest an interaction between VIP and catecholamines on cultivated astrocytes. The functional implications are at present unknown, but it might be that the peptide can modulate the response of the second messenger to catecholamines on astroglial cells through intramembrane mechanisms. Acknowledgemenrs--The author is indebted to Ulrika Sandkvist for her skilful technical assistance. The studv* was supported by grants from The Swedish Medical Research Council Cproject No. 12X-06812 and 12P-7308, from Magnus Bergvall’s Foundation, from Torsten and Ragnar SBderberg’s Foundation, from Harald and Greta Jeansson’s Foundation and from The Swedish Fund for Scientific Research without Animal Experiments.
REFERENCES
Chneiweiss H., Glowinski J. and Prkmont J. (1985) Vasoactive intestinal polypeptide receptors linked to an adenylate cyclase, and their relationship with biogenic amine- and somatostatin-sensitive adenylate cyclases on central neuronal and glial cells in primary cultures. .I. Neurochem. 44: 779-786. Evans T., McCarthy K. D. and Harden T. K. (1984) Regulation of cyclic AMP accumulation by peptide hormone receptors in immunocytochemically defined astroglial cells. /. Neurochem. 43: 131-138. Fuxe K., Agnati L. F., Benfenati F., Celani M., Zini I., Zoli M. and Mutt V. (1983) Evidence for the existence of receptor-receptor interactions in the central nervous system. Studies on the regulation of monoamine receptors bv neuropeptides. J. Ne&f Trans. Suppl. IS: 165-i79. . Hansson E. (1984) Enzyme activities of monoamine oxidase, cath~hol-O-methyl-transferal and gamma-aminobutyric acid transaminase in primary astroglial cultures and adult rat brain from different brain regions. Neurochem. Res. 9: 45-57.
Hansson E. (1985) Primary cultures from defined brain areas. Effects of seeding time on the development of beta-adrenergic and dopamine stimulated CAMP-activity _ during culti&ion. Dei Brain Res. 21: 187-192. Hansson E. (1986) Primarv cultures from defined brain areas. III effects of seeding time on (‘H) L-glutamate transport and glutamine synthetase activity. Deu. Brain Res. 21: 203-209. Hansson E. (1988) Astroglia from defined brain regions as studied with primary cultures. Prog. Neurobiol. (Ii press). Hansson E., Eriksson P. and Nilsson M. (1985) , , Amino acid and monoamine transport in primary astroglial cultures from defined brain regions. iveurochem. Res. IO: 1335-1341.
Hansson E., RiinnbPck L., Persson L. I., Lowenthal A.. Noppe M., Alling C. and Karlsson B. (1984) Cellular composition of primary cultures from cerebral cortex, striatum, hippocampus, brain stem and cerebellum. Brain Res. 300: 9-18. Hansson E., Rijnnback L. and Sellstram A. (1984) Is there a “dopaminergic glial cell”? Neurochem Res. 9: 679-689.
300
ELISABETHHANSSOK and
Hokfelt T., Johansson 0.. Ljungdahl A., Lundberg J. M. and Schultzberg M. (1980) Peptidergic neurones. Nurrcre 2% 515521. Koh S.-W. M., Kyritsis A. and Chader G. .I. (1984) Interaction of neuropeptides and cultured glial (Miiller) cells of the chick retina: elevation of intracellular cyclic AMP by vasoactive intestinal peptide and glucogen. J. ~~~r~~he~z. 43: 199-203. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent, J. hiol. Chem. 193: 265-275. Magistretti P. J., Manthorpe M.. Bloom F. E. and Varon S. (I 983) Functional receptors for vasoactive intestinal polypeptide in cultured astroglia from neonatal rat brain. R~g~~u~. Peptides 6: 7 l-80. McCarthy K. 0. and de Vellis J. (1978) Alpha-adrenergic receptor modulation of beta-adrenergic. adenosine and prosiaglandin E, increased adenosine-3’: S-cyclic monophosphate levels in primary cultures of glia. J. C.vclic Nu~lef~~ide Res. 4: 15-X.
L. RGNNB~~CK
McCarthy K. D. and de Velhs J. (1979) The regulation of adenosine 3’: 5’-cyclic monophosphate accun~u~ation m glia by alpha-adrenergic agonists. t[fi Sci. 24: 639-650. Rougon G.. Noble M. and Mudge A. W. (1983) Neuropeptides modulate the beta-adrenergic response of purified astrocytes in vitro. Nature 305: 715.-717. Steiner A. L.. Parker C. W. and Kinnis D. M. (I972a) Radioimmunoassay for cyclic nucledttdes. I, Preparation of antibodies and iodinated cyclic nucieotides. J. hiof. Chem. 247: 1106-1113. Steiner A. L., Pagliara A. S., Chase L. R. and Kipnis D. M. (1972b) Radio-immunoassay for cyclic nucleotides. II. Adenosine 3’,5’-monophosphate and guanosine 3’.5’monophosphate in mammalian tissues and body fluids. J. b&l. Chem. 247: 1 I 14-l 120. Van Calker D., Miiller M. and Hamprecht B. (1980) Regulation by secretin, vasoactive intestinal peptide, and somatostatinof cyclic AMP accumulation in cultured brain cells. Pmt. n&n. Acud. Sri. U.S.A. 71: 6907-691 I.
Copyright 0
OOZS-3908jSS$3.00 + 0.00 1988 Pergamon Journals Ltd
INTERACTION BETWEEN CATEC~OLA~INES AND VASOACTIVE INTESTINAL PEPTIDE IN CULTURED ASTROCYTES ELISABETH HANSSON’
and L.
R~NNB~cK’.~
iInstitute of Neurobiology and 2Department of Neurology, University of Gteborg,
Sweden
(Accepted 21 September 1987) Summary-Receptors for vasoactive intestinal peptide (VIP) were demonstrated with cyclic AMP as the second messenger on astroglial cells cultured from the cerebral cortex, striatum, hippocampus and brain stem of the newborn rat. Vasoactive intestinal peptide produced increased accumulations of cyclic AMP in nM and pM concentrations, the former more pronounced in the cultures of brain stem, while the latter was more pronounced in the other cultures studied, indicating regional differences’ in the activation of cyclic AMP of VIP receptors. Vasoactive intestinal peptide inhibited isoproterenol-induced accumulation of cyclic AMP dose-dependently, with some regional differences, suggesting interactions between the second messenger systems of /I- and VIP receptors. On the other hand, there was no inhibition of the NA-induced accumulation of cyclic AMP in the presence of VIP, which is in agreement with an interaction between the second messenger systems for VIP and the cc-adrenoceptor. The data support interactions between the second messenger systems for VIP and a- and ~-adrenoceptors on cultured astrocytes. The functional impli~tions are at present unknown, but it might be that the peptide can modulate the response of the second messenger to catecholamines on astroglial cells through intramembrane mechanisms. Key words: astrocytes, brain stem, cerebral cortex, hippocampus, isoproterenol, noradrenaline, primary culture, striatum, vasoactive intestinal peptide.
In recent years it has become clear that several hormones exert their effects on target cells through the “second messenger”, cyclic adenosine 3’,5’ monophosphate (cyclic AMP). Interaction between such hormones and cell surface receptors results in stimulation of the membrane bound enzyme, adenylate cyclase, which catalyzes the conversion of ATP to cyclic AMP. Stimulation of astrocytes in primary cultures by c(- and /I-adrenoceptors leads to changes in cyclic AMP (McCarthy and de Vellis, 1978, 1979; Van Calker, Miiller and Hamprecht, 1980) as does stimulation by dopamine in striatal astroglial cultures (Hansson, Rijnnback and Sellstrom, 1984). The neuropeptides, vasoactive intestinal peptide (VIP), secretin and glucagon stimulate the accumulation of cyclic AMP in astrocytes (Chneiweiss, Glowinski and Premont, 1985; Evans, McCarthy and Harden, 1984; Koh, Kyritsis and Chader, 1984; Rougon, Noble and Mudge, 1983; Van Calker et al., 1980) and may also act as regulators of metabolism in brain by affecting glycogenolysis (Magistretti, Manthorpe, Bloom and Varon, 1983). The presence of a neuropeptide and a biogenic amine has been demonstrated in the same terminals of central and peripheral neurones. This has led to the hypothesis that neurotransmitters, existing together within the same neurone can interact at pre- or postsynaptic sites in a functionally coordinated manner (Hiikfelt, Johansson, Ljungdahl, Lundberg and Schultzberg, 1980).
Interactions between the cyclic AMP of VIP receptors and the adenylate cyclase system of other receptors have already been investigated on astrocytes (Chneiweiss et al., 1985; Rougon et ai., 1983). The aim of the present work was to evaluate whether or not there are differences in the VIP-induced accumulation of cyclic AMP in astrocytes, cultivated from different regions of the brain of the newborn ratcerebral cortex, striatum, hippocampus and brain stem. Furthermore, it was elucidated whether or not there are interactions between the second messenger systems of VIP and the p-agonist (ISO) or the CI-and /?-agonist noradrenaline (NA). METHODS
Tissue cultures
The primary cultures of astroglia were made from newborn rats (Sprague-Dawley strain, Alab, Sweden). Cerebral cortex, striatum, hippocampus and brain stem were extirpated as described by Hansson, Ronnblck, Persson, Lowenthal, Noppe, Alling and Karlsson (1984). In short, the cells were grown in Eagle’s minimum essential medium (MEM, Flow Laboratories, Scotland) supplied with extra substances, to make up the following final composition: double concentrations of amino acids, 2 mM glutamine, 7 mM glucose and quadruple concentrations of vitamins; 250,000 IU penicillin/l, 0.5% 295
ELISAHETH HAYSSON
296
and
L.
R&NHACK
STRlATUM
_~~_ 10 9
6
~_ 7
6
._ 5
10
9
0
7
6
5
-- -~~--_-.-_. 10
9
8
7
6
5
10
9
8
7
6
5
J
Fig. 1. The effects of increasing concentration of VIP on accumukion of cyclic AMP were estimated on primary cultures of astroglia from cerebral cortex, striatum, hippocampus and brain stem. The results are expressed as mean accumulation of cyclic AMP in relation to control values. Values are means of 5 experiments with duplicates in each. Statistical evaluation: Student’s l-test comparing largest respect values with those of controls. *P < 0.05, **P < 0.01. SEM did not exceed 10% (not indicated in the figure).
20% (v/v) fetal calf serum (Gibco were also added. The cells were grown in a humidified atmosphere with pH set at 7.3 for 2 weeks. Medium were changed three times a week. streptomycin
Bio Cult.
and
Lab.,
U.K.)
Measurement of cyclic AMP The incubation medium was changed to serum-free Eagle’s MEM 1 hr before the experiments. The cultures were exposed to VIP (Sigma Fine Chemicals, St. Louis, Missouri) in a concentration range between 10-10~10~5 M. The different cultures were further exposed to 10e6 M o,L-isoproterenol (Sigma), or to lOA M L-noradrenaline (NA) (Sigma) in the presence or absence of VIP. Antagonists, lo-’ M propranolol (Sigma) or 10. 4 M phentolamine (Sigma) were added before VIP, isoproterenol or NA. The exposure time was 1Omin and the incubations were made in the presence of the phosphodiesterase inhibitor I mM iso-butyl-I-methyl-xanthine (IBMX) (Sigma). After incubation, the medium was decanted and the cultures were immediately exposed to 2 M ice-cold perchloric acid and put on ice. The cells were scraped off, glass/glass homogenized and centrifuged at 2000 x g for 20 min, all at 4 C. The supernatants were run on anion exchange resins, Ag l-X8. 20&400 mesh (BioRad Lab., Richmond, California) at pH 6.95-7.05. The cyclic AMP was eluted using 5 ml 2 M formic acid. After freeze-drying and dissolving in 1.0 ml 0.05 M sodium acetate at pH 6.2, cyclic AMP was determined using radioimmunoassay kits (NEN, Boston, Massachusetts) as described by Steiner, Parker and Kipnis (1972a) and Steiner, Pagliara, Chase and Kipnis (1972b). Cyclic AMP
from all samples of one experiment was purified by ion exchange chromatography during one day to standardize the procedure and minimize a variable loss of the cyclic nucleotide during purification. When performed in this way, the recoveries from the columns varied less than 10%. The recovery of cyclic AMP from the columns from one day to another showed a greater variance. Therefore, the results are expressed as accumulation of cyclic AMP as a percentage of control.
Determination
of protein
were done according to Farr and Randall (1951), with bovine serum albumin as standard (Sigma). Amount of protein in one tissue culture, one sample (35 mm, in dia, Petri dish) was -0.5 mg. Determinations
Lowry,
of protein
Rosebrough,
RESl I.TS
There was an increased accumulation of cyclic AMP in the primary cultures of astrocytes from cerebral cortex, striatum, hippocampus and brain stem after incubation with different concentrations of VIP. The stimulatory effect of VIP was most pronounced in the cultures of cerebral cortex and hippocampus, reaching 5 times controls (Fig. 1). The control values were in pmol cyclic AMP,mg prot I: for cerebral cortex 39 &- 6.2; for striatum 42 & 7.1; for hippocampus 40 i 6.5 and for brain stem 43 & 7.7 (mean k SEM of 5 experiments). The detection limit of the assay was t0.2pmol cyclic AMP/ml. The increase in cyclic AMP, activated by VIP showed two peaks, one in the nM range (1 x 10 ‘-5 x 10m9 M)
8’
VN
99 OS1
lOtiiNO9 110 S3Hlll NOIlVlIIWllS dRV,
298
ELISABETH
HANSON and L.
the other in the PM range (1 x 10~ ‘-1 x 10 ’ M). In the cultures of brain stem, the accumulation of cyclic AMP was more prominent in the nM range, while in the other cultures there was a more prominent accumulation of cyclic AMP in the PM range (Fig. 1). Isoproterenol (IO-” M) evoked a 2c-60 times accumulation of cyclic AMP, somewhat different in the various cultures. In the presence of 10m6 M isoproterenol, VIP evoked no further increase in cyclic AMP in the concentration interval between 10 ‘oplO ‘M in the cerebral cortex. The pattern of formation of cyclic AMP was similar to that of VIP alone, with peaks in the nM and PM ranges. However. in the presence of VIP there was a relative depression of the formation of cyclic AMP induced by isoproterenol, more pronounced at greater concentrations of VIP (Fig. 2). The stimulation of accumulation of cyclic AMP by VIP in the presence of isoproterenol and the fl-adrenergic antagonist propranolol was not different from that of VIP alone (Fig. 2). The pattern of activation of cyclic AMP after incubation with both isoproterenol and VIP was different in the various cultures, however, with the relative increase of the peaks at the same concentrations of VIP as after incubation in VIP alone. In the brain stem there was a more pronounced activation of formation of cyclic AMP in the nM range, while the other cultures showed a more prominent accumulation of cyclic AMP with VIP in the PM concentration in the presence of I PM isoproterenol. Moreover. in all cultures and especially in the striatum, there was a prominent inhibition of the isoproterenol-induced activation of cyclic AMP after incubation in large concentrations of VIP (IO-’ M). Even NA evoked a prominent activation of cyclic AMP. After incubation in IO-‘M NA there was no additive effect on accumulation of cyclic AMP in the presence of VIP in any of the cultures of astroglial primary cells studied. There were still peaks at the high- and low-affinity VIP-activated sites, but not so pronounced as with VIP alone or with VIP in the presence of isoproterenol. Neither did VIP reduce the NA-induced cyclic activation of AMP over the concentration range studied. Incubation with VIP in different concentrations, together with the fl-adrenergic antagonist propranolol and NA, showed a reduction of the formation of cyclic AMP in comparison with that caused by VIP alone. In the presence of NA and the r-adrenergic antagonist phentolamine there was a prominent accumulation of cyclic AMP with two peaks, however, never exceeding the formation of cyclic AMP for phentolamine and NA, without VIP. After incubation in phentolamine and propranolol, together with NA and VIP, the accumulation of cyclic AMP was not different from that of VIP alone (Figs 1 and 2). Phentolamine and propranolol showed no effects on the VIPinduced stimulation of cyclic AMP in the doseinterval studied.
R~~NNBACK DISCUSSION
Primary cultures of astroglia from the cerebra1 cortex, striatum, hippocampus and brain stem of the newborn rat exhibited VIP receptors that regulated the cyclic AMP system. The primary cultures used in this study were previously characterized extensively to contain immunocytochemically-defined astroglia, which express functional properties found on astroglia in ciao (Hansson, 1984, 1985, 1986; Hansson, Eriksson and Nilsson, 1985; Hansson et al., 1984). The action of VIP on the second messenger cyclic AMP system at small concentrations (nM) and at large concentrations (PM) has earlier been demonstrated on astrocytes from whole brain of mouse (Van Calker et ul.. 1980) cerebral cortex, striatum and mesencephalon (Chneiweiss rt nl., 1985). The stimulation of cyclic AMP at these two concentrations of VIP could be in accordance with the existence of two distinct VIP receptors, differing in their affinity and/or their coupling efficiency. Another possibility is the formation of a metabolite that could act as a partial agonist with high affinity for the receptor (Chneiweiss et al., 1985). The relatively low potency of VIP to increase formation of cyclic AMP (335 time of control) is in agreement with previous work (Rougon et al., 1983; Chneiweiss et al., 1985). while Van Calker et al. (1980) and Evans et al. (1984) showed a more prominent activation of cyclic AMP (20-30 times that of control). The discrepancies might be due to different sources of VIP. Another possibility might be degradation of VIP during the IO min incubation, this possibility being very unlikely, however, since Van Calker et (11.and Evans et al. performed their incubations for 10min in the presence of a phosphodiesterase inhibitor, as was done here. The VIP-induced activation of cyclic AMP differed between the various regions of the brain. Astrocyte cultures from cereral cortex, striatum and hippocampus showed a greater response in the PM-range of VIP, while cultures of brain stem showed a greater response in the nM-range. Chneiweiss et al. (1985) found a greater activation of cyclic AMP in the PM-range of VIP in astroglia from the cerebral cortex, striatum and mesencephalon of the mouse. The differences in the high-affinity receptor site might be due to differences in species and also region of the brain isolated (mesencephalon vs brain stem). The results indicate that high-affinity and low-affinity VIP receptors might differ in relative amounts in astrocytes from different regions. In view of previous work in this laboratory, demonstrating basic similarities in the cultures, as to cellular content and biochemical parameters, on one hand, and differences between the cultures from the various regions of the brain concerning functional parameters, such as uptake of neurotransmitters, activity of enzymes for catabolism of neurotransmitters, Z-. b- and DA receptors and profiles of soluble proteins, on the other hand
Interaction between ~techolamines
(Hansson, 1988), the present data with different characteristics of the VIP-activated cyclic AMP in the various regions of the brain further indicate a regional specialization of the cells. Astrocytes in primary cultures express receptors for various neurotransmitters and neuromodulators (Van Calker et al., 1980). There may be formations of mosaics of receptors on the cell membrane of astroglia and the second messengers of these receptors may “communicate” with each other through intramembrane signals. Thus, a number of interactions between second messenger systems for receptors for monoamines and neuropeptides may occur as reported on neuronal membranes in the central nervous system (Fuxe, Agnati, Benfenati, Celani, Zini, Zoli and Mutt, 1983). When VIP and isoproterenol were applied together there was an inhibition of the isoproterenol-induced activation of cyclic AMP over a broad concentration range. The inhibition was more prominent at larger concentrations of VIP. There were two peaks in the nM and p M ranges even in the presence of isoproterenol. The sum of the VIP and isoproterenol-induced accumulation of cyclic AMP did not exceed that of isoproterenol alone, which is in agreement with Chneiweiss et al. (1985). Thus, there must be an interaction between VIP and the fi -adrenoceptor-activated cyclic AMP. In the brain stem there was a partial additivity between VIP (nM range) and isoproterenol. Similar results were obtained by Chneiweiss et al. (1985) in glia from mesencephalon although they examined the low affinity site. In the presence of NA and VIP there were only very small peaks in the nM or PM ranges of VIP. Moreover, VIP exerted a much less depression of NA-induced formation of cyclic AMP than of isoproterenol-induced formation of cyclic AMP. Thus, there must be an interaction between VIP and the second messenger system of the /I-adrenoceptor, with a resulting relative increase in cyclic AMP. Rougon ef al. (1983) demonstrated a potentiation by VIP on the NA-activated formation of cyclic AMP in astrocyte cultures from cerebral cortex. The differences between the results of these two studies are at present unclear. Furthermore, NA in the presence of the P-adrenoceptor antagonist propranolol, at least partly reduced the VIP-induced stimulation of cyclic AMP. This is in agreement with the data of Van Calker er ai. (1980). It is difficult at present to draw any conclusion whether or not there is a bidirectional interaction between the second messengers coupled to VIP and the a-adrenoceptor. In cultures of astroglia from cerebral cortex and striatum, the peaks at the nM or PM ranges of VIP, in the presence of NA, were more pronounced than in cultures from hippocampus or brain stem where the peaks were hardly seen. One drawback with the culture system is that the cells are less mature than those in the intact nervous
and vasoaetive intestinal peptide
299
system (see Hansson, 1988), due to the lack of physiological contacts with other cells during cultivation. A hypersensitivity of receptors might occur in the absence of normal neurotransmission in the cultures. Thus, it can be questioned if the cells bear these receptors, and if there are interactions between the second messenger systems of the receptors in the adult nervous system, or if they represent a stage of development. Under all circumstances, the results suggest an interaction between VIP and catecholamines on cultivated astrocytes. The functional implications are at present unknown, but it might be that the peptide can modulate the response of the second messenger to catecholamines on astroglial cells through intramembrane mechanisms. Acknowledgemenrs--The author is indebted to Ulrika Sandkvist for her skilful technical assistance. The studv* was supported by grants from The Swedish Medical Research Council Cproject No. 12X-06812 and 12P-7308, from Magnus Bergvall’s Foundation, from Torsten and Ragnar SBderberg’s Foundation, from Harald and Greta Jeansson’s Foundation and from The Swedish Fund for Scientific Research without Animal Experiments.
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