Neuroscience Letters, 146 (1992) 231-233 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
231
NSL 09069
Activation of a metabotropic excitatory amino acid receptor potentiates Azbadenosine receptor-stimulated cyclic AMP accumulation Stephen E H . Alexander, Alistair R. Curtis, Stephen J. Hill and David A. Kendall Department of Physiology & Pharmacology, University of Nottingham Medical School, Queen's Medical Centre, Nottingham ( UK) (Received 30 June 1992; Revised version received 11 August 1992; Accepted 11 August 1992)
Key words." Excitatory amino acid receptor; A2badenosine receptor; Cyclic AMP; Receptor crosstalk Incubation of guinea-pig cerebral cortical slices with the excitatory amino acid agonists DL-~-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) or N-methyl-o-aspartate (NMDA) failed to significantly alter either basal or 5'-N-ethylcarboxamidoadenosine- (NECA-)stimulated cAMP levels. Quisqualate, L-glutamate and oL-l-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) were also without effect on basal levels of cAMP but increased NECA-stimulated levels of cAMP to approximately 190, 220 and 290% of control levels, respectively. Analysis of t-ACPD concentrationresponse data for the potentiation of NECA-stimulated cAMP elevations and the stimulation of phosphoinositide turnover gave ECs0 values of 51 and 107 pM, respectively. The enhancing effect of t-ACPD on cAMP levels was maintained for up to 40 minutes as was the stimulation of phosphoinositide turnover. We conclude that the cAMP response to A2b adenosine receptor stimulation is augmented by the rigid glutamate analogue t-ACPD, possibly through the action of products of phosphoinositide turnover.
Stimulation of A2b adenosine receptors of cerebral cortex results in raised levels of the intracellular messenger adenosine 3':5'-monophosphate (cAMP [4]). This cAMP response may be augmented through activation of a number of distinct receptor types which themselves are not directly linked to adenylyl cyclase [6]. Thus co-incubation with histamine, noradrenaline and 5-hydroxytryptamine, but not carbachol leads to an augmentation of the A2b receptor response [8]. L-Glutamate increases cAMP levels in guinea-pig cerebral cortical slices by two mechanisms. Firstly, L-glutamate stimulates the release of adenosine (presumably through ionotropic EAA receptors [7]) which then activates A2b receptors to stimulate cAMP formation [9]. Under conditions where this effect of glutamate is obviated (i.e. in the presence of adenosine deaminase), glutamate enhances the formation of cAMP by metabolically stable adenosine analogues [5]. The potentiation of A2b receptor-stimulated cAMP formation is thought to be mediated through the products of phosphoinositide turnover [6]. Since we recently observed that activation of 'metabotropic' excitatory amino acid receptors with the selective agonist DL-1aminocyclopentane-trans-l,3-dicarboxylate (t-ACPD) Correspondence: S.P.H. Alexander, Department of Physiology & Pharmacology, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. Fax: (44) (602) 709259.
stimulates phosphoinositide turnover in guinea-pig cerebral cortical slices [2] we have investigated whether tACPD potentiates A2b receptor-stimulated cAMP formation. A preliminary account of these findings has been presented to the British Pharmacological Society [1]. Guinea-pig (Dunkin-Hartley, either sex) cerebral cortical slices (350 x 350pm) were equilibrated for 60 min at 37°C with several changes of Krebs-Henseleit medium. After sedimentation under gravity, 25 i l aliquots of the slices were transferred to flat-bottomed vials containing Krebs-Henseleit medium supplemented with adenosine deaminase (1 U/ml, Sigma or Boehringer Mannheim), in the absence or presence of 10 i M 5'-N-ethylcarboxamidoadenosine (NECA, Sigma). After 10 min at 37°C, potential augmentatory agonists were added in 10 i l to give a final volume of 300 ill. After a further 10 min period, the incubation was halted by the addition of 100/11 of ice-cold perchloric acid (7.5% w/v). Following neutralisation with 0.15 M KOH or 1.2 M KHCO3, the suspension was centrifuged at 2300 x g for 10 min. An aliquot of the supernatant layer was removed and frozen for later analysis of cAMP content, and the pellet was digested in NaOH for protein estimation [3]. Cyclic AMP levels were estimated by competition with [3H]cAMP (NEN, 1646.5 GBq/mmol) binding to a soluble protein from bovine adrenal cortex (isolated as described [12]) in pH 7.4 Tris buffer (25 mM) containing EDTA (5 mM),
232 TABLE I
2500r
THE EFFECTS OF EXCITATORYAMINO ACID AGONISTS ON BASAL AND NECA-STIMULATED cAMP FORMATION
2000
Data are means _+S.E.M. determinations of cAMP levelsexpressed as a percentageof the response in the absence of EAA from quadruplicate samples in 3-9 separate experiments. Basal levelsof cAMP were 3.0 _+ 0.7 pmol/mg, raised to 181 _+ 53 pmol/mg in the presence of NECA. EAAs were present at the following concentrations: AMPA 10/aM; NMDA 100 ~tM; QA 100/aM; L-glutamate 10 raM; and t-ACPD 300 /aM.
300 25o
150@I
Control
NECA ( 10/aM)
AMPA NMDA QA L-Glutamate t-ACPD
84 69 74 105 139
70 75 187 223 289
_+ 20 _+ 11 _+ 8 + 18 _+ 27
± 18 + 9
z
2oo
¢ IoooI
£
% O'3 rTq
@/
Agent
g
x~
~
cs x>
6
,
-5 4 [t-ACPD] (log M)
3
too
*P
231
NSL 09069
Activation of a metabotropic excitatory amino acid receptor potentiates Azbadenosine receptor-stimulated cyclic AMP accumulation Stephen E H . Alexander, Alistair R. Curtis, Stephen J. Hill and David A. Kendall Department of Physiology & Pharmacology, University of Nottingham Medical School, Queen's Medical Centre, Nottingham ( UK) (Received 30 June 1992; Revised version received 11 August 1992; Accepted 11 August 1992)
Key words." Excitatory amino acid receptor; A2badenosine receptor; Cyclic AMP; Receptor crosstalk Incubation of guinea-pig cerebral cortical slices with the excitatory amino acid agonists DL-~-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) or N-methyl-o-aspartate (NMDA) failed to significantly alter either basal or 5'-N-ethylcarboxamidoadenosine- (NECA-)stimulated cAMP levels. Quisqualate, L-glutamate and oL-l-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) were also without effect on basal levels of cAMP but increased NECA-stimulated levels of cAMP to approximately 190, 220 and 290% of control levels, respectively. Analysis of t-ACPD concentrationresponse data for the potentiation of NECA-stimulated cAMP elevations and the stimulation of phosphoinositide turnover gave ECs0 values of 51 and 107 pM, respectively. The enhancing effect of t-ACPD on cAMP levels was maintained for up to 40 minutes as was the stimulation of phosphoinositide turnover. We conclude that the cAMP response to A2b adenosine receptor stimulation is augmented by the rigid glutamate analogue t-ACPD, possibly through the action of products of phosphoinositide turnover.
Stimulation of A2b adenosine receptors of cerebral cortex results in raised levels of the intracellular messenger adenosine 3':5'-monophosphate (cAMP [4]). This cAMP response may be augmented through activation of a number of distinct receptor types which themselves are not directly linked to adenylyl cyclase [6]. Thus co-incubation with histamine, noradrenaline and 5-hydroxytryptamine, but not carbachol leads to an augmentation of the A2b receptor response [8]. L-Glutamate increases cAMP levels in guinea-pig cerebral cortical slices by two mechanisms. Firstly, L-glutamate stimulates the release of adenosine (presumably through ionotropic EAA receptors [7]) which then activates A2b receptors to stimulate cAMP formation [9]. Under conditions where this effect of glutamate is obviated (i.e. in the presence of adenosine deaminase), glutamate enhances the formation of cAMP by metabolically stable adenosine analogues [5]. The potentiation of A2b receptor-stimulated cAMP formation is thought to be mediated through the products of phosphoinositide turnover [6]. Since we recently observed that activation of 'metabotropic' excitatory amino acid receptors with the selective agonist DL-1aminocyclopentane-trans-l,3-dicarboxylate (t-ACPD) Correspondence: S.P.H. Alexander, Department of Physiology & Pharmacology, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. Fax: (44) (602) 709259.
stimulates phosphoinositide turnover in guinea-pig cerebral cortical slices [2] we have investigated whether tACPD potentiates A2b receptor-stimulated cAMP formation. A preliminary account of these findings has been presented to the British Pharmacological Society [1]. Guinea-pig (Dunkin-Hartley, either sex) cerebral cortical slices (350 x 350pm) were equilibrated for 60 min at 37°C with several changes of Krebs-Henseleit medium. After sedimentation under gravity, 25 i l aliquots of the slices were transferred to flat-bottomed vials containing Krebs-Henseleit medium supplemented with adenosine deaminase (1 U/ml, Sigma or Boehringer Mannheim), in the absence or presence of 10 i M 5'-N-ethylcarboxamidoadenosine (NECA, Sigma). After 10 min at 37°C, potential augmentatory agonists were added in 10 i l to give a final volume of 300 ill. After a further 10 min period, the incubation was halted by the addition of 100/11 of ice-cold perchloric acid (7.5% w/v). Following neutralisation with 0.15 M KOH or 1.2 M KHCO3, the suspension was centrifuged at 2300 x g for 10 min. An aliquot of the supernatant layer was removed and frozen for later analysis of cAMP content, and the pellet was digested in NaOH for protein estimation [3]. Cyclic AMP levels were estimated by competition with [3H]cAMP (NEN, 1646.5 GBq/mmol) binding to a soluble protein from bovine adrenal cortex (isolated as described [12]) in pH 7.4 Tris buffer (25 mM) containing EDTA (5 mM),
232 TABLE I
2500r
THE EFFECTS OF EXCITATORYAMINO ACID AGONISTS ON BASAL AND NECA-STIMULATED cAMP FORMATION
2000
Data are means _+S.E.M. determinations of cAMP levelsexpressed as a percentageof the response in the absence of EAA from quadruplicate samples in 3-9 separate experiments. Basal levelsof cAMP were 3.0 _+ 0.7 pmol/mg, raised to 181 _+ 53 pmol/mg in the presence of NECA. EAAs were present at the following concentrations: AMPA 10/aM; NMDA 100 ~tM; QA 100/aM; L-glutamate 10 raM; and t-ACPD 300 /aM.
300 25o
150@I
Control
NECA ( 10/aM)
AMPA NMDA QA L-Glutamate t-ACPD
84 69 74 105 139
70 75 187 223 289
_+ 20 _+ 11 _+ 8 + 18 _+ 27
± 18 + 9
z
2oo
¢ IoooI
£
% O'3 rTq
@/
Agent
g
x~
~
cs x>
6
,
-5 4 [t-ACPD] (log M)
3
too
*P
