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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 (EC505106mM). Pyridoxal-phosphate-6-azophenyll-29,49-disulphonic acid 4-sodium (PPADS), a P2X antago-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 (EC50534mM). The total conductance of the neurons for the kainate-induced current was significantly reduced (P,0.01) and the effect was completely reversible. BB furthermore reduced the kainate-induced current in granule and hippocampal neurons and decreased the amplitude of thea-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 100mM BB. The maximal response to kainate decreased in the presence of 20 mM BB without changing its EC , indicating a non-competitive50
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: [email protected] (C. Zona). activation of the glutamate receptor systems [2,6,29,35].
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-hydroxyethyl)piperazine-N2 9 -(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-bis(b-aminoethyl ether)-neurons in primary culture [39,40]. N,N,N9,N9-tetracetic acid (EGTA), 1; HEPES, 10; D
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 500mM 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
(100mM) in a 6-day-old cortical neuron at a holding potential of260 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 /(11(EC / [kainate]) ), with EC50 505106mM and h5 1,38. (C) Effect of CNQX (10mM) 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 10mM of CNQX. The holding potential was260 mV and the cell was the same of (A).
3. Results
Fig. 2. Effects of purinergic antagonists on the kainate-induced currents in cortical neurons in culture. (A) PPADS (100mM) did not change the amplitude of the kainate-induced currents in cortical neurons while suramine (100mM) (B) and PIT (20mM) (C) decreased the currents reversibly and irreversibly respectively. The neurons were 8, 13 and 9-day-old in culture respectively and were voltage-clamped at260 mV.
decreased the current induced by kainate (47.5613.6%, manner (n557, Fig. 3B). The EC50 of the reducing effect
n56, Fig. 2B). In addition, PIT (20mM), an antagonist of of BB was 34 mM. The minimal effective concentration P2Y receptors, decreased by 1165.7% (n58) the currents was 5mM and maximal inhibition was obtained with 1000 induced by kainate in cortical neurons and it was not mM BB (Fig. 3B).
observable the recovery of the currents from these values, To study whether the effect of reduction of the kainate-in all recorded cells, still with subsequent applications of induced current by BB was also observed in other areas of kainate (Fig. 2C). Higher concentrations than 20 mM PIT the central nervous system, we studied the current induced were not tested because in previous work it has been by kainate (100mM) in cerebellar granule (Fig. 4A) and reported to be toxic for neurons [40]. To test further if hippocampal neurons (Fig. 4B) in primary culture. In preferring P2Y receptors antagonist were involved in the granule as well as in hippocampal cells, the current effect of reduction of the kainate-induced current, we induced by kainate was comparable in amplitude and size tested BB (5–1000 mM, Fig. 3A), a preferring P2Y with that recorded in cortical neurons. In the presence of receptor antagonist, also known in literature as reactive 50 mM BB, the reduction of the current was 5365% blue 2. BB did not induce inward or outward currents (n59) and 4866% (n58) respectively.
Fig. 3. BB reduces the currents activated by 100mM kainate in a concentration-dependent manner in cultured cortical cells. (A) Currents induced by 100 mM kainate on 12, 13 and 15-day-old cortical cells in culture were recorded in the absence and in the presence of BB 10, 50 and 300mM respectively. The amplitudes of the currents were reduced during perfusion of BB at the three different concentrations and all the cells presented a full recovery. (B) Dose–response curve of the effect of BB on the amplitude of the current induced by kainate in 39 cortical cells in culture (9–15 days in culture). The EC50
of the curve was 34mM.
then decreased to a steady-state value. In all tested cortical BB by about 5566% (n56, Fig. 6A). The kainate-induced neurons (n515), the current induced by AMPA decreased currents had apparent rate of activation and deactivation or was almost abolished in the presence of BB (100–300 similar to those observed in untreated culture. Moreover,
mM). This effect was completely reversible (Fig. 5). BB when cortical neurons were intracellularly loaded with also decreased the amplitude of the current at all potentials, GTPgS (500mM), a non-hydrolysable GTP analogue, the but it did not change the reversal point of the AMPA perfusion of BB (100mM) still decreased the amplitude of current (not shown). the current induced by 100mM kainate and there were no To determine whether the effects of BB on the kainate- obvious changes in these (Fig. 6B, 7368%, n55). GTPgS induced currents involve the activation of G-proteins, we in the electrode had no observable effect on kainate-first analysed the ability of this compound to decrease induced currents.
Fig. 4. Effect of 50 mM BB on the kainate-induced currents in cerebellar granule and hippocampal neurons. (A) BB (50 mM) decreased the kainate-induced currents in cerebellar granule cell (9-day-old) and hippocampal neuron (12-day-old) (B). This effect of reduction was completely reversible. The neurons were voltage-clamped at 260 mV and the concentration of the agonist was 100mM.
membrane were significantly reduced while the reversal In n527 cortical neurons, the total conductance was potential of the kainate-induced current did not change. 10.6963.47 nS for the kainate induced current (100 mM) Using the equation: and 5.0562.35 nS in the presence of 50mM BB (P,0.01,
t-test, Fig. 7B). Gk5I /(Ek m2Erev)
In order to establish if BB acts as an antagonist of the where G is the total conductance of the membrane, I isk k AMPA / kainate receptors, different concentrations of kain-the amplitude of kain-the currents elicited by kainate, E is kain-them ate were used to elicit the current in cortical neurons in membrane potential and Erev is the reversal potential for control condition and with 20 mM BB. Concentration– currents elicited by kainate, using the experimental curren- response analysis revealed that 20 mM BB reduced the t-voltage relation induced by kainate in control condition response of kainate but did not alter the EC50 of kainate and during perfusion of 50 mM BB, we could obtain the (117mM vs. a control value of 106mM; P.0.05, n59). kainate conductance for the neurons G , where the slope ofk These data indicated a non-competitive effect of BB on the the regression fit curve indicates the total conductance. kainate action site.
Fig. 6. Effects of BB on kainate-induced currents in cortical neurons treated with ChTx and with GTPgS in the electrode solution. (A) Kainate-induced current in a 12-day-old cortical neuron treated with ChTx (250 ng / ml, 3–4 h at 378C). BB (50mM) still decreased the kainate induced current. The membrane potential was260 mV. (B) BB (100mM) reversibly decreased the current induced by 100mM kainate in an 8-day-old cortical neuron recorded with GTPgS (500 mM) in the electrode solution. The holding potential was 260 mV. The effect of reduction of the current was comparable to that observable on the neuron recorded with the standard internal solution.
4. Discussion tors antagonist, in particular BB, exerts its protecting
action from the glutamate-evoked toxicity.
The present results show that preferential P2Y It is known that purinoceptors are involved in the purinoceptor antagonists inhibit kainate-induced currents modulation of ionic channels and in the modification of the and that this effect appears to result from a direct inter- cytosolic [Ca] [7,34,37]. In rat cortical astrocytes, P2i
21 action with AMPA / kainate receptor-channels, rather than purinoceptors appear to raise also intracellular Ca and to from an inhibition of purinoceptors. stimulate mitogen-activated protein kinase, indicating a Previous work showed that only a selected number of P2 possible mechanism for controlling glutamate-mediated (but not P1) receptor modulators prevents various glu- excitatory neurotoxicity and neurotransmission [18]. In tamate-evoked actions including excitotoxicity, neurotrans- addition, BB, a P2Y preferring antagonist, prevents serum mitter release and uptake of extracellular calcium [39]. In deprivation- and low potassium-induced apoptotic cell addition, it has been shown that suramin and reactive blue death in cerebellar granule neurons [40], further indicating 2 inhibit GABA receptor channels and glutamate receptor that purinoceptors modulation is implicated in both ex-channels in rat hippocampal neurons [26], inhibit NMDA citotoxic as well as apoptotic degeneration [1,27,41,43]. receptor-channels in mouse hippocampal neurons in cul- We show in this work that BB decreases, in a dose– ture [31] and decreased the e.p.s.cs and the glutamate dependent manner, the kainate-induced currents of cortical activated currents in hippocampal slices [24]. Moreover, neurons in culture. The EC50 (34mM) is comparable with suramin but not PPADS suppressed the amplitude of the that utilised to abolish the cytotoxic action of glutamate in evoked excitatory post synaptic currents (EPSCs) and rat cerebellar granule neurons (10–20mM) [39] and with decreased the frequency and the amplitude of the miniature that for inhibition of P2 receptors that often vary depend-EPSC in rat spinal cord neurons [13]. Finally, PPADS did ing upon the experimental preparations used (1–40 mM) not affect the glutamate induced currents in rat hippocam- [12,17,25,31].
Fig. 7. BB reduces the total conductance of the cortical neuron for the kainate-induced currents and dose–response curve. (A) Kainate-induced current was recorded in a 10-day-old cortical neuron in control condition and during perfusion of 50mM BB. The holding potential of the neuron has been changed from280 mV to180 mV taking 5 s (ramp protocol). The kainate-induced current reverses around 0 mV in both experimental conditions. A net decrease of the current during perfusion of BB is also evident. (B) Histogram of conductance of the neurons (n527) for kainate-induced current in control condition and during perfusion of 50mM BB. The reduction of the total conductance is significant (P,0.01, t-test). (C) Dose–response curve of the kainate-induced current in rat cortical neurons in control condition and with 20mM of BB. The dose–response curve for the kainate-induced current was modified. The steady state of the curve decreases with BB, while the EC50 value is 106mM in control condition and 117mM with 20mM BB.
reporting the reduction of glutamate induced currents in dent manner. Within this range, the current-voltage rela-hippocampal neurons [24,26,31], therefore suggesting a tionship was approximately linear, and the degree of common mechanism of action of this compound in the inhibition produced by BB did not depend on the mem-central nervous system. brane holding potential. Our observation that BB inhibition Most of the described effects of kainate in the CNS are was not voltage-dependent is in line to a previous study mediated by its action on AMPA and kainate receptors, [31] in which BB inhibited NMDA-activated current to a because kainate is capable to activate both classes of similar extent at every tested potential.
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