Brain Research 882 2000 26–35 www.elsevier.com locate bres
Research report
Effect of P2 purinoceptor antagonists on kainate-induced currents in rat cultured neurons
a,c , c
c,d b,c
` Cristina Zona
, Caterina Marchetti , Cinzia Volonte , Nicola B. Mercuri
,
b,c
Giorgio Bernardi
a
` Cattedra di Fisiologia Umana
, Dipartimento di Neuroscienze, Universita degli Studi di Roma ‘‘Tor Vergata’’, Via di Tor Vergata, 135-00133 Roma, Italy
b
` Clinica Neurologica
, Dipartimento di Neuroscienze, Universita degli Studi di Roma ‘‘Tor Vergata’’, Via di Tor Vergata, 135-00133 Roma, Italy
c
Fondazione S . Lucia, I.R.C.C.S., Via Ardeatina, 306-00179 Roma, Italy
d
Istituto di Neurobiologia , C.N.R., Viale Marx, Roma, Italy
Accepted 2 August 2000
Abstract
The action of purinergic antagonists on kainate-induced currents was studied in rat cortical neurons in primary culture using the whole-cell configuration of the patch-clamp technique. The amplitude of the currents induced by kainate in cortical neurons was
concentration-dependent EC 5106 mM. Pyridoxal-phosphate-6-azophenyll-29,49-disulphonic acid 4-sodium PPADS, a P2X antago-
50
nist, was ineffective in the reduction of the kainate-induced current in cortical neurons, while 2,29-pyridylisatogen PIT, basilen blue BB and suramin, respectively two selective P2Y and a non-selective P2 receptor antagonist, caused a reduction in the amplitude of the
current induced by kainate. BB decreased the inward current induced by kainate at all holding potentials and the reduction was dose-dependent EC 534 mM. The total conductance of the neurons for the kainate-induced current was significantly reduced P,0.01
50
and the effect was completely reversible. BB furthermore reduced the kainate-induced current in granule and hippocampal neurons and decreased the amplitude of the a-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid AMPA-evoked current in cortical neurons. Cholera
toxin ChTx did not affect the action of BB on the kainate-induced currents in cortical neurons and moreover, when guanosine 59-o-3-thiotriphosphate GTPgS was added to the electrode solution, the kainate-induced currents were still reduced by 100 mM BB.
The maximal response to kainate decreased in the presence of 20 mM BB without changing its EC , indicating a non-competitive
50
mechanism of inhibition. These results demonstrate that preferential P2Y receptor antagonists are able to modulate the kainate and AMPA-induced currents in central neurons, suggesting a potential use of these compounds as neuroprotective agents.
2000 Elsevier
Science B.V. All rights reserved.
Theme : Neurotransmitters, modulators, transporters, and receptors
Topic : Excitatory amino acids: pharmacology
Keywords : Glutamate receptor; Purinoceptor; Patch-clamp; Rat
1. Introduction are important for regulating neuronal plasticity, develop-
ment, outgrowth and survival. GluR-operated channels are Glutamate is the major excitatory neurotransmitter in the
mediators not only of normal intercellular communication, CNS. Glutamate receptor GluR-operated ion-channels
but also of neuronal injury and death. Indeed, glutamate mediate fast cellular signal information among neurons and
itself can be toxic to neurons [15]. Neurological illnesses involving strokes or epileptic seizures and many neurode-
generative diseases, such as Alzheimer’s, Huntington’s,
Corresponding author. Present address: Laboratorio di Farmacologia,
Parkinson’s and amyotrophic lateral sclerosis, are accom-
Fondazione S. Lucia, I.R.C.C.S., Via Ardeatina, 306-00179 Roma, Italy.
panied by neuronal cell death induced by excessive
Tel.: 139-6-5150-1513; fax: 139-6-5150-1384. E-mail address
: zonamed.uniroma2.it C. Zona.
activation of the glutamate receptor systems [2,6,29,35].
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 7 8 1 - 5
C . Zona et al. Brain Research 882 2000 26 –35
27
The GluR-operated ion channels have been classified by these ligands, we used the whole-cell configuration of
according to their preferred agonists as N-methyl-
D
-aspar- patch-clamp technique to study their effects on the kainate-
tate NMDA, 2-amino-3-hydroxyl-5-methyl-isoxazol-4- induced currents in rat central neurons in primary culture
propionic acid AMPA and kainate subtypes. The latter and on the AMPA-induced currents in rat cortical neurons
two types are also referred to as non-NMDA receptors. in culture.
The kainate does not act on a single type of receptor, but instead is able to activate both kainate and AMPA re-
ceptors [30,36].
2. Material and methods
Glutamate toxicity
is primarily
mediated through
NMDA-receptor activation [10,32]. However, exposure of 2.1. Cell culture
neurons to kainate can also induce dramatic cell loss [16]. Moreover, previous results indicate that kainate receptors
Experiments were performed with cortical, hippocampal can be reached by synaptically released glutamate and that
and cerebellar granule neurons in primary culture. Cortical their activation downregulates GABAergic inhibition by
neurons were isolated from 14-day-old Wistar rat embryos modulating the reliability of GABA synapses [11,33].
Morini, Reggio Emilia, Italy and grown in dissociated Thus, kainate receptors may have a role in the etiology of
cell culture for 12–16 days in basal medium Eagle’s with epilepsy and could become a target for antiepileptic drugs.
Earle’s salts BME, Gibco with 10 foetal bovine serum Several agents have been found to protect different
FBS, 6 mM glucose, 2 mM glutamine and 100 mM ml kinds of neurons from glutamate-evoked toxicity [5,9,23],
gentamicine Gibco. Hippocampal neurons were prepared among which also includes selective adenosine triphos-
from 18-day-old Wistar rat embryos and grown as cortical phate ATP receptor antagonists [39,40]. Purinoceptors
neurons. For more details, see Dichter and Zona [8]. show a wide distribution in the central nervous system
Cerebellar granule cultures from Wistar 8-day-old rat CNS and they appear to regulate important neuronal
cerebellum were prepared as previously described [22] and functions [20] and to play physiological and pathological
seeded in BME, supplemented with 25 mM KCl, 2 mM roles [18]. Purinoceptors have been classified into two
glutamine, 0.1 mg ml gentamicin, 10 heat inactivated primary classes: the P1 receptors are responsive to adeno-
foetal calf serum. sine, whereas the P2 receptors respond to a variety of
Adequate measures were taken to minimize pain or purine nucleotides, including ATP. Presently, several P2
discomfort. All the experiments were performed in accord- subtypes have been identified and assigned to two mech-
ance with the European Communities Council Directive anistically distinct subclasses of receptors [4]. The
for experimental procedures. metabotropic receptors of the P2Y subclass formerly P2U,
P2T, P2Y initiate their biological actions by G-protein- 2.2. Electrophysiology
dependent activation of phospholipase C and subsequent elevation of intracellular calcium levels, through liberation
Membrane currents from the cell-soma were recorded in of calcium from intracellular stores. The P2X purinocep-
the whole-cell configuration of the patch-clamp method tors comprise instead a distinct subclass of receptors that
[14]. Patch-clamp electrodes were obtained with capillary are ligand-gated calcium channels functionally related to
tubes pulled with a Narishige puller. The electrodes were glutamate and nicotinic acetylcholine receptors [28].
filled with the appropriate solutions see below and had a Previous works have reported different effects of
resistance of approximately 4 MV. Currents were recorded purinergic antagonists on glutamatergic transmission and
at room temperature by using a voltage-clamp amplifier on glutamate receptors in many areas of CNS. In par-
Axopatch 1D, Axon Instruments, USA. Holding poten- ticular, it has been reported that suramin and BB inhibit
tials and stimulation templates were done using a pClamp NMDA receptor-channels in mouse hippocampal neurons
6 software Axon Instruments. The current signal was [31], decrease the evoked excitatory postsynaptic currents
filtered at 2 KHz, sampled at 10 KHz and stored on a hard and the glutamate-activated currents in rat hippocampal
disk. Compensation of series resistance 50–70 was slices [24], inhibit the current activated by kainate in
routinely used. Data were digitized and analysed using the hippocampal slices [26] and suramin inhibits the gluta-
pClamp and the Origin Microcal software, USA. matergic EPSCs in spinal cord [13]. Moreover, it has been
The bathing solution contained in mM: NaCl, 130; reported that inhibition of P2 receptors, through the action
KCl, 3; CaCl , 1.5; N-2-hydroxyethylpiperazine-N9-2-
2
of selected ligands such as basilen blue BB and 2,29- ethanesulphonic acid HEPES, 10;
D
-glucose, 6; Tetra- pyridylisatogen PIT, but not the inhibition of P1 re-
ethylammonium TEA, 10; pH 7.4 with HCl. Tetrodotox- ceptors, prevents glutamate-dependent excitatory neuro-
in TTX, 1 mM was added to the extracellular medium. toxicity, neurotransmitter release and uptake of extracellu-
The solution used for filling the electrodes contained in
21
lar Ca in cerebellar granule, cortical and hippocampal
mM: CsCl, 140; Ethylene glycol-bisb-aminoethyl ether- neurons in primary culture [39,40].
N,N,N9,N9-tetracetic acid EGTA, 1; HEPES, 10;
D
-glu- In order to investigate the mechanism of action elicited
cose, 6, pH 7.4 with CsOH.
28 C
2.3. Applications of chemicals The solutions containing known concentrations of the
drugs were ejected by gravity with a multiple-barrel pipette with a total tip diameter ,2 mm placed at a distance of
less than 0.2 cm from the patched cell. Ejection of each chemical was made using a gravity perfusion system
controlled by electrically-controlled valves. The ionic currents were induced by ejection of kainate or AMPA at
the soma of the recording neurons. Other chemicals were assigned randomly to one of the multiple barrels. Their
effects on kainate or AMPA-induced currents were tested by extracellularly perfusing the cell for 20–180 s. During
the experiment, the cells were continuously perfused with the control solution at a flow-rate of 0.5–1 ml min.
Kainate and AMPA were first prepared as a concentrated stock solution and diluted in standard extracellular bath
solution prior to use at their final concentration. BB, suramin, PIT, PPADS were dissolved in water and then
diluted in a standard extracellular bath solution at different concentrations.
GTPgS was stored in aliquots at 2708C and diluted to 500 mM in a standard internal solution, immediately before
use. In experiments involving cholera toxin ChTx, the
neurons were incubated at 378C in the extracellular solu- tion containing ChTx 20 mg ml for 3–4 h. Control
preparations were obtained with the same protocol in a toxin-free extracellular solution.
All chemicals were purchased from Sigma USA except ChTx Calbiochem, USA.
2.4. Data analysis Analysis of recordings was performed with the Ax-
opatch data analysis programs. Fitting was performed with
Fig. 1. Kainate-induced currents are recorded in rat cortical neurons in culture. A Inward current were induced by bath application of kainate
Origin Microcal.
All results
are expressed
as
100 mM in a 6-day-old cortical neuron at a holding potential of 260
mean6S.E.M. n5number of neurons analysed under each
mV. For this and the following figures, bars indicate the time of drug
experimental procedure. Differences between experimen-
application. B Plot of the normalized current amplitude as a function of
tal groups were statistically analyzed by Student’s paired
the kainate concentration. The sigmoidal curve was drawn according to
h
t-test and were considered significantly different if P,
the equation: I51 11EC [kainate] , with EC 5106 mM and h5
50 50
1,38. C Effect of CNQX 10 mM on whole-cell response of cortical
0.05. Statistical analysis was performed using SPSS 6.0 for
neuron to kainate 100 mM applied with the perfusion system. The
Windows.
inward current induced by kainate was reversibly blocked by 10 mM of CNQX. The holding potential was 260 mV and the cell was the same of
A.
3. Results
