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P2 purinoceptors modulating noradrenaline release from sympathetic neurons in culture
European Journal of Pharmacology252 (1994) R7-R8
Rapid communication
P2 purinoceptors modulating noradrenaline release from sympathetic neurons in culture Clemens Allgaier *, Friedrich Pullmann, Angelika Schobert, Ivar Von Kiigelgen, Georg Hertting Institute of Pharmacology and Toxicology, Albert-Ludwigs University of Freiburg, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany
(Received 16 December 1993;accepted 28 December 1993)
Abstract
ATP (1 mM) inhibited, whereas 2-methylthio-ATP (30 /zM), a P2v-selective purinoceptor agonist, increased electrically evoked release of [3H]noradrenaline from chick sympathetic neurons. The P2x-selective purinoceptor agonist a,/3-methylene-ATP (30 ~M) had no effect. The ATP-induced inhibition of release as well as the facilitation caused by 2-methylthio-ATP was not affected by the selective adenosine (P1) receptor antagonist 8-(p-sulfophenyl)-theophylline (8-PST; 100 /zM), but completely prevented by the non-selective P2 antagonist suramin (300 /xM). The present data reveal a dual regulation of noradrenaline release from sympathetic neurons. Facilitation seems to be mediated by a P2Y purinoceptor, whereas inhibition is caused by a P2 purinoceptor which needs further subtype characterization. Key words: P2 purinoceptor; Noradrenaline release; Sympathetic neuron
A T P is a co-transmitter of noradrenaline in postganglionic sympathetic neurons (Burnstock, 1990; Von Kiigelgen and Starke, 1991). A T P released from the terminals of these neurons activates P2 purinoceptors at peripheral effector cells as well as axon terminals where they can operate as a novel kind of autoreceptors as suggested recently by Von Kiigelgen et al. (1993). Here we investigated whether depolarization-induced release of noradrenaline from sympathetic neurons in culture is modulated by P2 purinoceptors. Effects of ATP and A T P analogues on [3H]noradrenaline release from chick sympathetic neurons were measured. These neurons possess a2-adrenoceptors mediating inhibition of evoked noradrenaline release (B6hm et al., 1991). Lumbosacral paravertebral ganglia were dissected out from 12 day-old chick embryos and cultured as described previously (Greene and Rein, 1978; Edgar et al., 1981) with some modifications. Briefly, the ganglia were trypsinized and then dissociated in serum-free Dulbecco's modified Eagle medium (DMEM; Gibco BRL, Eggenstein-Leopldshafen, Germany) supple-
* Corresponding author. Tel. +761/203 5300, fax +761/203 5311. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00004-Q
mented with (in g / l ) D-glucose (1.2), N a H C O 3 (2), insulin (0.01), streptomycin (0.1), gentamicin (0.02), 100 U / I penicillin and 1 0 / z g / l nerve growth factor (Becton Dickinson, Heidelberg, Germany) by gentle trituration. The suspension of ganglionic cells was poured into a 90 mm plastic dish to which heat-inactivated horse serum was added (10%, v/v). The dish was incubated at 37°C in an atmosphere of 5% C O 2 / 9 5 % air for 60 min. The suspension was centrifuged (225 × g for 30 min) and the pellet was resuspended in medium. Approximately 30 000 cells were plated in one well of a 96-well culture plate containing a 5 mm Thermanox coverslip (Nunc, Wiesbaden-Biebrich, Germany) coated with collagen. The cultures were incubated at 37°C and fetal calf serum (5%, v / v ; Gibco BRL) was added after 60 min. The culture medium was replaced after 3 days. Seven-day-old cultures were used for release experiments. Cells were loaded with [3H]noradrenaline (0.05/zM; 42.1 C i / m m o l , NEN, Dreieich, Germany; 60 min, 37°C), and superfused at 25°C with modified KrebsRinger bicarbonate buffer (Allgaier et al., 1991) containing in addition (in mM) fumaric acid (0.5) and Na-pyruvate (5.0). (+)-Oxaprotiline (1 /~M; CibaGeigy, Basel, Switzerland) was present to prevent reuptake of noradrenaline. After 60 min of superfusion,
R8
4 min fractions of the superfusate were collected. Electrical field stimulation was carried out after 68 min (S 1) and 96 (S 2) min with 36 pulses/3 Hz (voltage drop: 18.75 V/cm; current strength: 80 mA; pulse width 0.5 ms). ATP (disodium salt; Sigma, Deisenhofen, Germany), a,/3-methylene-ATP (lithium salt; Sigma), 2methylthio-ATP (tetrasodium salt; Biotrend, Cologne, Germany), and bromoxidine (Pfizer, Karlsruhe, Germany) were added 12 min before S 2. 8-(p-Sulfophenyl)theophylline (8-PST; Biotrend) and suramin (hexasodium salt; Bayer, Wuppertal, Germany) were present throughout superfusion as indicated. Superfusion was terminated after 112 min. Radioactivity in the samples was determined by liquid scintillation spectrometry. For determination of drug effects on evoked [3H]noradrenaline release see Allgaier et al. (1991). Electrically evoked release of noradrenaline from chick sympathetic neurons was completely Ca2÷-depen dent and sensitive to tetrodotoxin (1 /xM; Sigma) resembling action potential-induced transmitter release. The respective $2/S 1 values were 100.0 + 2.5% (n = 7) for controls, 4.3 + 0.9% (n = 7) with Ca2÷-free buffer (used from 12 min before $2), and 2.2 + 1.1% (n = 6) in the presence of 1 /~M tetrodotoxin (added from 12
with
160
min before $2). The a2-adrenoceptor agonist bromoxidine (1 tzM) inhibited transmitter release by 64.1 + 2.2% (n = 12) which was comparable to the inhibition observed previously (B6hm et al., 1991). ATP (1 mM) significantly diminished the electrically evoked release of [3H]noradrenaline (Fig. 1). In contrast, a,/3-methylene-ATP (30 ~M), a P2x-selective purinoceptor agonist, showed no effect, and 2-methylthio-ATP (30 ~M), a P2y-selective purinoceptor agonist, even enhanced evoked [3H]noradrenaline release (Fig. 1). Inhibition of [3H]noradrenaline release caused by 1 mM ATP was not affected by the selective adenosine (P~) receptor antagonist 8-PST (100 /~M), but was completely prevented by the non-selective P2 antagonist suramin (300 /zM) (Fig. 1). Likewise, the facilitatory effect of 2methylthio-ATP was not changed by 8-PST, but antagonized by suramin (Fig. 1). Taken together, it is demonstrated for the first time that evoked noradrenaline release from sympathetic neurons in culture is modulated by P2 purinoceptors. The present results reveal a dual regulation by purinoceptor ligands. They suggest that facilitation of noradrenaline release is mediated by activation of a P2v purinoceptor, whereas inhibition is caused by a P2 receptor which seems not to belong to the P2x or the P2v subtype.
B-PST
Acknowledgements 140
Supported by the Deutsche Forschungsgemeinschaft (SFB 325). o
120
with -urarnin
0
References at %,J
t~4
100
8O
60
oTI la
i
l
I IWI
I IUIV
Fig. 1. Electrically evoked release of [3H]noradrenaline from chick sympathetic neurons: effects of purinoceptor ligands. ATP (1 mM), a,/3-methylene-ATP (CH2-ATP; 30/xM), or 2-methylthio-ATP (MTATP, 30 p.M) were added 12 min before S 2. 8-PST (100 /~M) or suramin (300 p.M) were present throughout superfusion as indicated. S 2 / S 1 ratios were expressed as percentages of the corresponding drug-free control ratio (0.73 + 0.03 (n = 8), regular buffer; 0.68 + 0.02 (n = 8), with 8-PST; 0.78+0.04 (n = 6), with suramin). Means+ S.E.M. of 4-8 observations are given. Significant differences vs. respective control (Mann-Whitney test): NS, not significant; ** P < 0.01.
Allgaier, C., R. Greber and G. Hertting, 1991, Studies on the interaction between presynaptic ~2-adrenoceptors and A 1 adenosine receptors located on noradrenergic nerve terminals, Naunyn-Schmied. Arch. Pharmacol. 344, 187. B6hm, S., S. Huck, H. Drobny and E.A. Singer, 1991, Electrically evoked noradrenaline release from cultured chick sympathetic neurons: modulation via presynaptic ~2-adrenoceptors and lack of autoinhibition, Naunyn-Schmied. Arch. Pharmacol. 344, 130. Burnstock, G., 1990, Co-transmission, Arch. Int. Pharmacodyn. 304, 7. Edgar, D., Y.-A. Barde and H. Thoenen, 1981, Subpopulations of cultured chick sympathetic neurones differ in their requirements for survival factors, Nature 289, 294. Greene, L.A. and G. Rein, 1978, Release of norepinephrine from neurons in dissociated cell cultures of chick sympathetic ganglia via stimulation of nicotinic and muscarinic acetylcholine receptors, J. Neurochem. 30, 579. Von Kiigelgen, I. and K. Starke, 1991, Noradrenaline-ATP co-transmission in the sympathetic nervous system, Trends Pharmacol. Sci. 12, 319. Von Kiigelgen, I., K. Kurz and K. Starke, 1993, Axon terminal P2-purinoceptors in feedback control of sympathetic transmitter release, Neuroscience 56, 263.
Rapid communication
P2 purinoceptors modulating noradrenaline release from sympathetic neurons in culture Clemens Allgaier *, Friedrich Pullmann, Angelika Schobert, Ivar Von Kiigelgen, Georg Hertting Institute of Pharmacology and Toxicology, Albert-Ludwigs University of Freiburg, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany
(Received 16 December 1993;accepted 28 December 1993)
Abstract
ATP (1 mM) inhibited, whereas 2-methylthio-ATP (30 /zM), a P2v-selective purinoceptor agonist, increased electrically evoked release of [3H]noradrenaline from chick sympathetic neurons. The P2x-selective purinoceptor agonist a,/3-methylene-ATP (30 ~M) had no effect. The ATP-induced inhibition of release as well as the facilitation caused by 2-methylthio-ATP was not affected by the selective adenosine (P1) receptor antagonist 8-(p-sulfophenyl)-theophylline (8-PST; 100 /zM), but completely prevented by the non-selective P2 antagonist suramin (300 /xM). The present data reveal a dual regulation of noradrenaline release from sympathetic neurons. Facilitation seems to be mediated by a P2Y purinoceptor, whereas inhibition is caused by a P2 purinoceptor which needs further subtype characterization. Key words: P2 purinoceptor; Noradrenaline release; Sympathetic neuron
A T P is a co-transmitter of noradrenaline in postganglionic sympathetic neurons (Burnstock, 1990; Von Kiigelgen and Starke, 1991). A T P released from the terminals of these neurons activates P2 purinoceptors at peripheral effector cells as well as axon terminals where they can operate as a novel kind of autoreceptors as suggested recently by Von Kiigelgen et al. (1993). Here we investigated whether depolarization-induced release of noradrenaline from sympathetic neurons in culture is modulated by P2 purinoceptors. Effects of ATP and A T P analogues on [3H]noradrenaline release from chick sympathetic neurons were measured. These neurons possess a2-adrenoceptors mediating inhibition of evoked noradrenaline release (B6hm et al., 1991). Lumbosacral paravertebral ganglia were dissected out from 12 day-old chick embryos and cultured as described previously (Greene and Rein, 1978; Edgar et al., 1981) with some modifications. Briefly, the ganglia were trypsinized and then dissociated in serum-free Dulbecco's modified Eagle medium (DMEM; Gibco BRL, Eggenstein-Leopldshafen, Germany) supple-
* Corresponding author. Tel. +761/203 5300, fax +761/203 5311. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00004-Q
mented with (in g / l ) D-glucose (1.2), N a H C O 3 (2), insulin (0.01), streptomycin (0.1), gentamicin (0.02), 100 U / I penicillin and 1 0 / z g / l nerve growth factor (Becton Dickinson, Heidelberg, Germany) by gentle trituration. The suspension of ganglionic cells was poured into a 90 mm plastic dish to which heat-inactivated horse serum was added (10%, v/v). The dish was incubated at 37°C in an atmosphere of 5% C O 2 / 9 5 % air for 60 min. The suspension was centrifuged (225 × g for 30 min) and the pellet was resuspended in medium. Approximately 30 000 cells were plated in one well of a 96-well culture plate containing a 5 mm Thermanox coverslip (Nunc, Wiesbaden-Biebrich, Germany) coated with collagen. The cultures were incubated at 37°C and fetal calf serum (5%, v / v ; Gibco BRL) was added after 60 min. The culture medium was replaced after 3 days. Seven-day-old cultures were used for release experiments. Cells were loaded with [3H]noradrenaline (0.05/zM; 42.1 C i / m m o l , NEN, Dreieich, Germany; 60 min, 37°C), and superfused at 25°C with modified KrebsRinger bicarbonate buffer (Allgaier et al., 1991) containing in addition (in mM) fumaric acid (0.5) and Na-pyruvate (5.0). (+)-Oxaprotiline (1 /~M; CibaGeigy, Basel, Switzerland) was present to prevent reuptake of noradrenaline. After 60 min of superfusion,
R8
4 min fractions of the superfusate were collected. Electrical field stimulation was carried out after 68 min (S 1) and 96 (S 2) min with 36 pulses/3 Hz (voltage drop: 18.75 V/cm; current strength: 80 mA; pulse width 0.5 ms). ATP (disodium salt; Sigma, Deisenhofen, Germany), a,/3-methylene-ATP (lithium salt; Sigma), 2methylthio-ATP (tetrasodium salt; Biotrend, Cologne, Germany), and bromoxidine (Pfizer, Karlsruhe, Germany) were added 12 min before S 2. 8-(p-Sulfophenyl)theophylline (8-PST; Biotrend) and suramin (hexasodium salt; Bayer, Wuppertal, Germany) were present throughout superfusion as indicated. Superfusion was terminated after 112 min. Radioactivity in the samples was determined by liquid scintillation spectrometry. For determination of drug effects on evoked [3H]noradrenaline release see Allgaier et al. (1991). Electrically evoked release of noradrenaline from chick sympathetic neurons was completely Ca2÷-depen dent and sensitive to tetrodotoxin (1 /xM; Sigma) resembling action potential-induced transmitter release. The respective $2/S 1 values were 100.0 + 2.5% (n = 7) for controls, 4.3 + 0.9% (n = 7) with Ca2÷-free buffer (used from 12 min before $2), and 2.2 + 1.1% (n = 6) in the presence of 1 /~M tetrodotoxin (added from 12
with
160
min before $2). The a2-adrenoceptor agonist bromoxidine (1 tzM) inhibited transmitter release by 64.1 + 2.2% (n = 12) which was comparable to the inhibition observed previously (B6hm et al., 1991). ATP (1 mM) significantly diminished the electrically evoked release of [3H]noradrenaline (Fig. 1). In contrast, a,/3-methylene-ATP (30 ~M), a P2x-selective purinoceptor agonist, showed no effect, and 2-methylthio-ATP (30 ~M), a P2y-selective purinoceptor agonist, even enhanced evoked [3H]noradrenaline release (Fig. 1). Inhibition of [3H]noradrenaline release caused by 1 mM ATP was not affected by the selective adenosine (P~) receptor antagonist 8-PST (100 /~M), but was completely prevented by the non-selective P2 antagonist suramin (300 /zM) (Fig. 1). Likewise, the facilitatory effect of 2methylthio-ATP was not changed by 8-PST, but antagonized by suramin (Fig. 1). Taken together, it is demonstrated for the first time that evoked noradrenaline release from sympathetic neurons in culture is modulated by P2 purinoceptors. The present results reveal a dual regulation by purinoceptor ligands. They suggest that facilitation of noradrenaline release is mediated by activation of a P2v purinoceptor, whereas inhibition is caused by a P2 receptor which seems not to belong to the P2x or the P2v subtype.
B-PST
Acknowledgements 140
Supported by the Deutsche Forschungsgemeinschaft (SFB 325). o
120
with -urarnin
0
References at %,J
t~4
100
8O
60
oTI la
i
l
I IWI
I IUIV
Fig. 1. Electrically evoked release of [3H]noradrenaline from chick sympathetic neurons: effects of purinoceptor ligands. ATP (1 mM), a,/3-methylene-ATP (CH2-ATP; 30/xM), or 2-methylthio-ATP (MTATP, 30 p.M) were added 12 min before S 2. 8-PST (100 /~M) or suramin (300 p.M) were present throughout superfusion as indicated. S 2 / S 1 ratios were expressed as percentages of the corresponding drug-free control ratio (0.73 + 0.03 (n = 8), regular buffer; 0.68 + 0.02 (n = 8), with 8-PST; 0.78+0.04 (n = 6), with suramin). Means+ S.E.M. of 4-8 observations are given. Significant differences vs. respective control (Mann-Whitney test): NS, not significant; ** P < 0.01.
Allgaier, C., R. Greber and G. Hertting, 1991, Studies on the interaction between presynaptic ~2-adrenoceptors and A 1 adenosine receptors located on noradrenergic nerve terminals, Naunyn-Schmied. Arch. Pharmacol. 344, 187. B6hm, S., S. Huck, H. Drobny and E.A. Singer, 1991, Electrically evoked noradrenaline release from cultured chick sympathetic neurons: modulation via presynaptic ~2-adrenoceptors and lack of autoinhibition, Naunyn-Schmied. Arch. Pharmacol. 344, 130. Burnstock, G., 1990, Co-transmission, Arch. Int. Pharmacodyn. 304, 7. Edgar, D., Y.-A. Barde and H. Thoenen, 1981, Subpopulations of cultured chick sympathetic neurones differ in their requirements for survival factors, Nature 289, 294. Greene, L.A. and G. Rein, 1978, Release of norepinephrine from neurons in dissociated cell cultures of chick sympathetic ganglia via stimulation of nicotinic and muscarinic acetylcholine receptors, J. Neurochem. 30, 579. Von Kiigelgen, I. and K. Starke, 1991, Noradrenaline-ATP co-transmission in the sympathetic nervous system, Trends Pharmacol. Sci. 12, 319. Von Kiigelgen, I., K. Kurz and K. Starke, 1993, Axon terminal P2-purinoceptors in feedback control of sympathetic transmitter release, Neuroscience 56, 263.