Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1999 Jul;127(6):1317–1326. doi: 10.1038/sj.bjp.0702679

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor

Gabriel Olmos 1, Nuria DeGregorio-Rocasolano 2, M Paz Regalado 3, Teresa Gasull 2, M Assumpció Boronat 1, Ramón Trullas 2, Alvaro Villarroel 3, Juan Lerma 3, Jesús A García-Sevilla 1,*
PMCID: PMC1760666  PMID: 10455281

Abstract

  1. This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells.

  2. Exposure (30 min) of energy deprived cells to L-glutamate (1–100 μM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 μM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine).

  3. Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 μM (EC100) L-glutamate with the rank order (EC50 in μM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole] (101)>RX821002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors.

  4. Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding.

  5. In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10–12 μM at 0 mV.

  6. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.

Keywords: Imidazoline, agmatine, neurotoxicity, neuroprotection, glutamate, N-methyl-D-aspartate receptors, cerebellar granule cells

Introduction

Several studies have demonstrated that imidazol(ine)/guanidine compounds display nanomolar affinities for various non-adrenoceptor sites, the so-called imidazoline receptors, and that the interaction with these recognition sites elicits various central and peripheral effects (for a review see Bousquet, 1995; French, 1995; Regunathan & Reis, 1996; Molderings, 1997). The amine agmatine (decarboxylated arginine) has been proposed as an endogenous agonist at imidazoline receptors (Li et al., 1994). Independently of their affinities on these receptors, imidazol(ine)/guanidine compounds also interact (in the micromolar range) with the monoamine oxidase enzymes (Ozaita et al., 1997) and various cation channels. These compounds block ATP-sensitive K+ channels (K+ATP channels) in pancreatic β-cells and rat insulinoma (RIN) cells and this leads to the stimulation of insulin release (Jonas et al., 1992; Olmos et al., 1994; Berdeu et al., 1997; Proks & Ashcroft, 1997). Imidazol(ine) drugs inhibit the acetylcholine-induced secretion of catecholamines in adrenal chromaffin cells (Ohara-Imaizumi & Kumakura, 1992) by blocking nicotinic acetylcholine receptors (Musgrave et al., 1995). These compounds also interact with 5-HT3 receptors in N1E-115 cells inhibiting the veratridine-induced influx of guanidinium to these cells (Molderings et al., 1996). Finally, an interaction with red cell Gardos channels (Coupry et al., 1996) and with rat brain N-methyl-D-aspartate (NMDA) receptors (Olmos et al., 1996) has also been demonstrated.

Glutamate, the major excitatory neurotransmitter in the brain, is also a potent excitotoxin, inasmuch as prolonged exposure of most cultured neurones to micromolar concentrations of this aminoacid leads to neurotoxicity (Choi, 1988). In this sense, glutamate exposure of primary cultures of cerebellar granule cells is a well characterized model of neurotoxicity which results from the overstimulation of the NMDA receptor (Lysko et al., 1989; Schramm et al., 1990; Berman & Murray, 1996), leading to alterations in Ca2+ homeostasis (Milani et al., 1991). In this context, imidazol(ine) drugs and agmatine have been shown to be neuroprotective in brain injuries of necrotic (Gustafson et al., 1990; Maiese et al., 1992; Gilad et al., 1996) and apoptotic neuronal cell death (Olmos et al., 1999) in which glutamate-mediated neurotoxicity is clearly involved as the mechanism determining cell death. Thus, the present study was designed (1) to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells and (2) to seek whether imidazol(ine) drugs could exert neuroprotective effects through NMDA receptor blockade. A preliminary report of a portion of this study has been previously presented in abstract form (DeGregorio-Rocasolano et al., 1999).

Methods

Cerebellar granule cell cultures

Primary cultures of granule cells were prepared from cerebella of 7-day-old Wistar rat pups as previously described (Lysko et al., 1989; Boje et al., 1993; Fossom et al., 1995). Procedures involving animals and their care were conducted in conformity with institutional guidelines that are in compliance with national and international laws and policies. Dissociated cells were plated in 24-well plastic plates (2 cm2) previously coated with poly-L-lisine hydrobromide (10 μg ml−1, MW>300,000) and the density was adjusted to give approximately 5×105 cells per cm2. Cells were cultured in Eagle's Basal Medium with the following additions: 10% heat-inactivated foetal calf serum, 2 mM L-glutamine, 0.1 mg ml−1 gentamicin, and 25 mM KCl.

The replication of non-neuronal cells was prevented by adding cytosine arabinoside (10 μM) 18–24 h after plating. The cultures were incubated at 37°C in 5% CO2 in air saturated with water vapour. Conditioned media from naive cultures were reserved for later use. The cells were used for experiments after 8–11 days in culture.

Glutamate induction of necrotic neuronal death

In cerebellar granule cells depleted of energy resources, glutamate induces a rapid necrosis, whereas development of the apoptotic programme of neuronal death requires the integrity of mitochondrial function (Dessi et al., 1993; Ankarcrona et al., 1995). Thus, cultured neurones were washed once with 0.75 ml aliquots of modified Locke-HEPES (M-LH) buffer without magnesium and glucose (in mM: NaCl 154, KCl 5.6, NaHCO3 3.6, CaCl2 1.3, and HEPES 10, (pH 7.35) and then preincubated in the same buffer for 25 min; thereafter the imidazol(ine) drugs or agmatine (see Table 1 for their chemical structures) were added for 15 min where appropriate. This preincubation time (40 min) in M-LH buffer depleted cerebellar granule cells of energy resources and rendered them susceptible to L-glutamate toxicity (Lysko et al., 1989). Neurotoxicity was then induced by a 30 min incubation with L-glutamate in M-LH buffer in the presence or absence of experimental drugs. Each experimental condition was replicated in at least three independent preparations. The cultures were washed twice with 0.75 ml aliquots of M-LH buffer containing 10 mM glucose (G-LH buffer) at the end of this incubation period. Conditioned media (0.75 ml) was added to the cultures and the cultures were returned to the incubator.

Table 1.

Potencies of various imidazol(ine) drugs and agmatine protecting against glutamate-induced neurotoxicity and affinities for NMDA receptors

graphic file with name 127-0702679t1.jpg

Open in a new tab

Low K+ induction of apoptotic neuronal death

Cerebellar granule cells maintained in serum-free medium with 25 mM K+ undergo an apoptotic death when switched to 5 mM K+ (D'Mello et al., 1993; Ishitani et al., 1997). To investigate the effects of imidazol(ine) drugs on apoptosis induced by low extracellular K+, cerebellar granule cells were washed twice and maintained in serum-free Eagle's Basal Medium containing 5 mM K+ (low [K+]out) for 24 h in the presence or absence of drugs where indicated. Other cell cultures were washed identically and maintained in serum-free Eagle's Basal Medium with 25 mM K+ (high [K+]out) in the presence or absence of drugs until neurotoxicity assays were performed.

Assessment of neurotoxicity and neuroprotection

Neurotoxicity was quantitatively determined 24 h after treatment using a tetrazolium salt colorimetric assay with 3 - (4,5 - dimethylthiazol -2-yl)-2,5-diphenyltetrazoliumbromide (MTT) (Mosmann, 1983). Mitochondrial enzymes have the capacity to transform MTT into a reduced insoluble formazan product (MTTR), thus, the MTT assay provides an indication of mitochondrial metabolic function. Decreased MTT metabolism 24 h after neurotoxic treatment indicates loss of functional mitochondria in viable neurons because results obtained with this technique correlate well with cell viability estimated by staining with fluorescein diacetate (Lysko et al., 1989) or propidium iodide (Ankarcrona et al., 1995). Cells were incubated for 30 min at 37°C with 1 ml of G-LH buffer containing 0.4 μM MTT. The buffer was aspirated and replaced with 300 μl of dimethyl sulphoxide to extract the blue formazan complex. The entire extract was transferred to a 96-well plate and the optical density of samples was measured. The MTTR formation was calculated as the difference between the optical densities measured at 490 and 630 nm. To normalize for differences in the absorbance values among different culture plates and assays, values were expressed relative to unexposed control cells (see below).

Per cent neurotoxicity was defined as the per cent decrease in the formation of MTTR in cells exposed to L-glutamate with respect to unexposed control cells and was calculated as follows:

graphic file with name 127-0702679e1.jpg

where G=MTTR in L-glutamate exposed cells/MTTR in control cells.

Per cent neuroprotection was defined as the per cent increase in the formation of MTTR in cells exposed to L-glutamate plus experimental drugs with respect to cells exposed to L-glutamate and was calculated as follows:

graphic file with name 127-0702679e2.jpg

where T=MTTR in L-glutamate plus experimental drugs exposed cells/MTTR in control cells.

Data from neurotoxicity and neuroprotection curves were fitted to a four-parameter logistic equation by computer-assisted non-linear regression using the Prism programme (GraphPAD, San Diego, CA, U.S.A.).

[3H]-(+)-MK-801 binding assays and analyses of binding data

Radioligand binding assays with [3H]-(+)-MK-801 and preparation of P2 membrane fractions from the rat parieto-occipital cortex were done as previously described (Olmos et al., 1996). Drug competition studies were performed in a total volume of 500 μl, containing 400 μl of membrane suspension and [3H]-(+)-MK-801 (4×10−9 M), in the absence or presence of various concentrations of the competing drugs (10−6–10−2 M; 11 concentrations). Non-specific binding was determined in the presence of 10−4 M ketamine. Glutamate and glycine were not included in the assays because the content of endogenous aminoacids contaminating the crude (non washed) membrane preparations is sufficient to occupy the transmitter recognition sites of the NMDA receptors and cause partial enhancement of [3H]-(+)-MK-801 binding (see Foster & Wong, 1987 for details). Furthermore, the addition of 10 μM glutamate to rat brain synaptic membrane preparations did not significantly change the affinity of agmatine for [3H]-(+)-MK-801 binding sites (Anis et al., 1990). The mixture was incubated for 45 min at 23°C and then subjected to rapid filtration through Whatman GF/C filters using a Brandel 48 R cell harvester (Biomedical Research and Development Laboratories, U.S.A.). The filters were then rinsed twice with 5 ml of ice-cold incubation buffer and counted for radioactivity by liquid scintillation spectrometry at 50% efficiency. Analysis of competition experiments as well as the fitting of data to the appropriate binding models were performed by computer-assisted non-linear regression using the EBDA-LIGAND programmes (Munson & Rodbard, 1980; McPherson, 1985).

Electrophysiology of NMDA receptor

Experiments were carried out on the cell line HEK-293. Cells were grown in Dulbecco's modified Eagle Medium supplemented with 10% foetal calf serum, 1% glutamine and 1% penicillin/streptomycin. Cell-transfection was done as previously described (Villarroel et al., 1998) by electroporating the cDNAs encoding for the NR1-1a and NR2C subunits of the NMDA receptor together with the cDNA encoding for Green Fluorescent Protein (GFP) (1 : 2 : 1 ratio) for visualization of positively transfected cells and then seeded in plastic Petri dishes containing 2 ml of medium. Non-transfected cells do not express any kind of glutamate receptor. After 3–5 h the medium was replaced with a glutamine-free medium containing the NMDA receptor glycine site antagonist 7-chlorokynurenic acid (100 μM) to prevent NMDA-receptor-mediated cell death. The cDNAs employed were a generous gift of Dr S. Nakanishi (University of Kyoto).

HEK-293 cells were recorded 1 day after transfection. Currents activated by 200 μM L-glutamate in the presence of 20 μM glycine were measured at −70 mV, unless otherwise indicated, in the whole-cell configuration of the patch-clamp technique using an EPC7 amplifier. The borosilicate glass electrodes had resistances of 3–6 MΩ and the series resistance was compensated by 30–60% when the current responses exceeded 400 pA. Solutions were delivered using a fast perfusion system (Lerma et al., 1998). The composition of the extracellular solution was (in mM): NaCl 160, KCl 2.5, CaCl2 0.5, glucose 10, HEPES 10 and glycine 0.02 (pH 8.4 with NaOH; 325 mOsM). An alkaline pH was used to evoke larger currents. The composition of the intracellular solution was (in mM): CsCH4SO3 126, CsCl 10, MgCl2 5, CaCl2 0.5, EGTA 10 and HEPES 10 (pH 7.3 with CsOH; 310 mOsM).

To evaluate channel blockade at different potentials, the current in response of a voltage ramp from −70 to +70 mV of 450 ms of duration was fitted to the Boltzman equation:

graphic file with name 127-0702679e3.jpg

where V is the membrane potential, Vrev is the reversal potential, G is the conductance, V0.5 is the voltage at which 50% of the current is blocked at a given blocker concentration, and S is the slope factor. The values for G, V0.5 and S were allowed to vary during the fitting procedure, while Vrev was fixed to a value estimated by a linear fit of the I–V relation on the control ramp. The parameters were used to generate idealized I–V relations, that in turn were used to generate idealized relations of the blocker effect at different potentials. This procedure was repeated at different concentrations of the blocker, and the concentration-response relation at a given potential was fitted to the equation:

graphic file with name 127-0702679e4.jpg

where IC50 is the concentration of the compound that produces 50% blockade, [B] is the drug concentration, and n is the Hill coefficient.

The relation of the IC50 with voltage was fitted to the Woodhull model (Woodhull, 1973) with the linear equation:

graphic file with name 127-0702679e5.jpg

where pIC50 is the negative logarithm of the IC50, IC50(0) is the IC50 value at 0 mV, RT/F=25.4 mV at 22°C and zδ is the product of the charge of the blocker and the fraction of the electrical distance within the plane of the membrane.

Statistics

Results are expressed as mean±s.e.mean. Student's unpaired t-test was used for the statistical evaluations. Correlation coefficients were calculated by the method of least squares. The level of significance was P=0.05.

Drugs

[3H]-(+)-MK-801 (23.9 Ci mmol−1) was supplied by New England Nuclear Du Pont (U.S.A.). Other drugs (and their sources) included: agmatine sulphate (Aldrich Chemical Co., U.S.A.), antazoline HCl (Sigma Chemical Co., U.S.A.); 7-chlorokynurenic acid (Tocris Cookson Ltd, U.K.); cirazoline HCl (Synthélabo Recherche, France); L-glutamic monosodium salt (Sigma); glycine (Tocris Cookson Ltd, U.K.); idazoxan HCl, LSL 60101 [2-(2-benzofuranyl)imidazole HCl], LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole HCl] and LSL 61122 [2-styryl-2-imidazoline HCl, valldemossine] (synthesized by Dr F. Geijo at S.A. Lasa Laboratorios, Barcelona, Spain); (+)-MK-801 (dizocilpine) maleate (RBI, Natick, U.S.A.); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) (Sigma) and RX821002 (2-methoxy idazoxan) HCl (synthesized by Dr F. Geijo at S.A. Lasa Laboratorios). Cell culture media were obtained from Gibco-BRL (Life technologies Ltd, U.K.). Other reagents were obtained from Sigma Chemical Co. (U.S.A.).

Results

Neuroprotective effects of imidazol(ine) drugs and agmatine against L-glutamate-induced neurotoxicity in cultured cerebellar granule cells

Thirty minutes exposure of energy deprived cerebellar granule cells to L-glutamate (1–100 μM) caused a concentration-dependent neurotoxicity, as revealed 24 h later by a decrease in the ability of these cells to metabolize MTT to formazan (Figure 1a). L-glutamate induced neurotoxicity with Emax and EC50 values of 75% and 5 μM, respectively (Figure 1a). Concurrent exposure of cells with L-glutamate and the non-competitive NMDA receptor antagonist MK-801 (1 μM) prevented (93%, P<0.001) the neurotoxicity induced by 20 μM (EC100) L-glutamate (Figure 1b); showing that NMDA receptors mediate the neurotoxic effects of L-glutamate in energy deprived cerebellar granule neurons.

Figure 1.

Figure 1

Open in a new tab

(a) Neurotoxic effect of L-glutamate on cerebellar granule cells as measured by the concentration-dependent inhibition in the formation of reduced MTT relative to unexposed control cells. Neurons were depleted of energy resources by 40 min preincubation in a medium without magnesium and glucose and then incubated for 30 min in the presence of different concentrations of L-glutamate. Neurotoxicity was determined 24 h later as described in Methods. Data shown are mean±s.e.mean of eight wells. (b) Prevention by MK-801 (dizocilpine) of the neurotoxic effect of an EC100 of L-glutamate in cerebellar granule cells. Data shown are mean±s.e.mean of eight wells. *P<0.001 as compared with cells exposed to L-glutamate (Student's t-test).

When cerebellar granule cells were exposed to 20 μM L-glutamate in the presence of various imidazol(ine) drugs and agmatine, these compounds fully prevented L-glutamate-induced neuronal death with different potencies (Figure 2 and Table 1). In the absence of L-glutamate, exposure of granule cells to imidazol(ine) drugs and agmatine (up to 500 μM) did not modify MTT reduction with respect to unexposed control cells (data not shown).

Figure 2.

Figure 2

Open in a new tab

Neuroprotective effects of (a) antazoline, cirazoline, RX821002 and idazoxan and (b) LSL 61122, LSL 60129, LSL 60101 and agmatine against the neurotoxic effect of 20 μM L-glutamate in cerebellar granule cells, as measured by the concentration-dependent increase in the formation of reduced MTT relative to L-glutamate exposed cells. Neurons were depleted of energy resources by 40 min preincubation in a medium without magnesium and glucose and then incubated for 30 min in the presence of L-glutamate. Imidazol(ine) drugs were added during the last 15 min of the preincubation time and maintained throughout the L-glutamate exposure. Neuroprotection was determined 24 h later as described in Methods. Points represent mean±s.e.mean of eight wells. See Table 1 for EC50 values.

Lack of protective effects of imidazol(ine) drugs in cerebellar granule cells exposed to low potassium concentrations

The potential neuroprotective effect of various imidazol(ine) drugs was tested in a model of apoptotic neuronal death that does not involve activation of the NMDA receptor. Switching culturing conditions of cerebellar granule cells from a medium containing high (25 mM) KCl concentrations to a medium containing low (5 mM) KCl resulted in a decrease in cell viability measured by a 64% decrease in MTT reduction in control cells (Figure 3). Exposure of cells to the imidazol(ine) drugs cirazoline, LSL 60101 and LSL 61122 did not modify significantly the loss in MTT metabolism (Figure 3).

Figure 3.

Figure 3

Open in a new tab

Effect of cirazoline, LSL 60101 and LSL 61122 on neurotoxicity induced by switching from serum-free high [K+]out (25 mM) to serum-free low [K+]out (5 mM) medium in cerebellar granule cells. Cells were maintained in serum-free Eagle's Basal Medium containing either 25 or 5 mM K+ for 24 h in the presence (100 μM) or absence (control) of drugs where indicated. Neurotoxicity was measured using the MTT reduction (mitochondrial activity) assay. Data shown are mean±s.e.mean of absorbance (optical density, O.D.) units from eight wells. *P<0.05 as compared with cells maintained in high [K+]out (25 mM) medium (Student's t-test). No significant differences were found between control cells and cells exposed to drugs and maintained in low [K+]out (5 mM) medium.

Involvement of NMDA receptor blockade in the neuroprotective effects of imidazol(ine) drugs and agmatine

The above results suggested that prevention of L-glutamate-induced necrotic neuronal death by imidazol(ine) drugs and agmatine in cerebellar granule cells could be mediated by NMDA receptor blockade. To further address this question, the ability of the imidazol(ine) drugs tested and agmatine to interact (agonist or antagonist properties) with NMDA receptors was tested.

Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to membranes from rat cerebral cortex with potencies in the micromolar range (Figure 4 and Table 1). The obtained profile of drug affinity for [3H]-(+)-MK-801 binding sites on NMDA receptors was very similar to that previously obtained for the effects of the same drugs preventing L-glutamate-induced neurotoxicity. In fact, there was a good correlation (r=0.90) between the pEC50 values for the neuroprotective effects of these drugs and the pKi values obtained in competition curves against [3H]-(+)-MK-801 binding (Table 1 and Figure 5). These results suggested that a direct interaction of imidazol(ine) drugs and agmatine with the phencyclidine/MK-801 site of the NMDA receptor could block the neurotoxic effects of L-glutamate. This question was addressed by testing if the imidazoline drug antazoline (the most potent) and agmatine (the least potent) could inhibit glutamate-induced currents.

Figure 4.

Figure 4

Open in a new tab

Inhibition of binding of [3H]-(+)-MK-801 by (a) antazoline, cirazoline, idazoxan and RX821002 and by (b) LSL 61122, LSL 60129, LSL 60101 and agmatine in the rat cerebral cortex. Membranes were incubated at 23°C for 45 min with [3H]-(+)-MK-801 (4×10−9 M) in the absence or presence of different concentrations of the competing drugs. Data shown are mean±s.e.mean of 2–4 independent experiments performed in triplicate and expressed as a percentage of total control binding (about 12,000 d.p.m.). See Table 1 for Ki values.

Figure 5.

Figure 5

Open in a new tab

Correlation between the potency (expressed as pKi=−log Ki) of several imidazol(ine) drugs and agmatine inhibiting the binding of [3H]-(+)-MK-801 to NMDA receptors in the rat brain and the potency (expressed as pEC50=−log EC50) of the same compounds protecting against L-glutamate neurotoxicity in cerebellar granule cells (data taken from Table 1). The data were best described by the equation y=0.75x+1.06 (r=0.90; P<0.01). The identification of drugs is as follows: (1) antazoline, (2) cirazoline, (3) LSL 61122, (4) LSL 60101, (5) idazoxan, (6) LSL 60129, (7) RX821002 and (8) agmatine.

Whole-cell patch clamp recordings were performed in HEK-293 cells transfected to express the NR1-1a/NR2C subunits of the NMDA receptor. In the absence of Mg2+, antazoline (3 μM) reduced the glutamate-induced current by approximately 45% at −70 mV when applied in the external solution containing 200 μM glutamate and 20 μM glycine (Figure 6a). The effect was reversible upon the washing off of antazoline (not shown). The block was most potent at hyperpolarizing membrane potentials and less effective at positive voltages (Figure 6b). Thus, the IC50 at −70 mV was 3.6 μM and increased to 215 μM at +70 mV (Figure 6c and d). The plot showing the IC50 values for antazoline (logarithmic scale) obtained at different voltages presented two linear portions, with the inflection point close to the current reversal potential. The slope values, representing the effective valences (zδ), were 0.19 and 0.55 (Figure 6d).

Figure 6.

Figure 6

Open in a new tab

NMDA receptor channel blockade by antazoline in HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor. (a) Response to 200 μM glutamate in the absence (control) or presence of the indicated concentrations of antazoline. The response returned to control levels upon removal of antazoline (not shown). The current traces were interrupted by a voltage ramp to +70 mV (shaded bar). On depolarization the channel was partially relieved of antazoline blockade. On returning to the −70 mV holding voltage the channel was blocked again, causing the observed relaxations of the traces on the right. (b) Ramp I–V curves in the absence (control) or the presence of the indicated concentrations of antazoline. Solid lines represent fits to the Boltzman equation. (c) Extent of current reduction at different voltages in the presence of the indicated concentrations of antazoline. This relationship was generated from the fitted relation in (b) (see Methods). The vertical lines represent the s.e.mean of at least four independent determinations. (d) The logarithm of the IC50 values for the inhibition of glutamate-induced currents are plotted as a function of voltage. The relation presented two linear portions that were fitted to the Woodhull model; the best fit parameters for IC50 at 0 mV and zδ are indicated on the figure.

Agmatine also reduced the glutamate-induced current in HEK-293 cells. As expected from the potency inhibiting [3H]-(+)-MK-801 binding, agmatine was less potent than antazoline and a 47% reduction of glutamate-induced current was achieved in the presence of 200 μM agmatine at −70 mV (Figure 7a). The effect was also reversible upon the washing off of agmatine (not shown) and larger at hyperpolarizing membrane potentials than at positive voltages (Figure 7b and c). A remarkable difference between the action of agmatine and antazoline was revealed upon returning to the holding potential after a voltage ramp to +70 mV (Figures 6a and 7a). The current after the voltage ramp remained at the same level when it was blocked with agmatine (Figure 7a). In contrast, in the presence of antazoline there was an increased inward current that slowly returned to the original levels after the voltage ramp. The rates of this ‘after-ramp' current relaxations were faster as the concentration of antazoline increased (Figure 6a).

Figure 7.

Figure 7

Open in a new tab

NMDA receptor channel blockade by agmatine in HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor. (a) Responses to 200 μM glutamate without (control) or with 200 μM agmatine. The response returned to control levels upon removal of agmatine (not shown). The current trace is interrupted by a voltage ramp to +70 mV (shaded bar). (b) Ramp I–V curves obtained in the absence (control) or the presence of 200 μM agmatine in the same cell as in (a). Solid lines represent fits to the Boltzman equation. These data were obtained from the same cell as in Figure 6a. (c) Extent of current reduction at different voltages in the presence of the indicated concentrations of agmatine. This relationship was generated from the fitted relation in (b) (see Methods). The vertical lines represent the s.e.mean of at least four independent determinations .

Discussion

This study demonstrates that imidazol(ine) compounds and agmatine fully protected cultured cerebellar granule cells against glutamate-induced toxicity. In the present experimental conditions, cerebellar granule cells are rendered susceptible to glutamate neurotoxicity by glucose deprivation. In energy depleted cerebellar granule cells, glutamate induces a loss of mitochondrial membrane potential (Lysko et al., 1989). Overstimulation of the NMDA receptor by glutamate leads to a Ca2+ overload; the inability to maintain osmotic integrity via ion exchange provokes cell lysis and cells die by necrosis (Ankarcrona et al., 1995). In the present study, the NMDA receptor antagonist MK-801 prevented glutamate-induced toxicity in cultured cerebellar granule cells, as previously described (Lysko et al., 1989; Berman & Murray, 1996), thus reinforcing the involvement of NMDA receptors in the neurotoxic effect of glutamate.

Recently, it has been demonstrated that after NMDA receptor stimulation there is a fast mitochondrial Ca2+ uptake, suggesting that Ca2+ influx through NMDA receptors has a privileged access to mitochondria (Peng & Greenamyre, 1998). The fact that the I2 subtype of imidazoline receptors is mainly located on mitochondrial membranes (Tesson et al., 1991), might suggest that the neuroprotective effect of the drugs tested could be mediated by activation of I2 receptors that would increase mitochondrial Ca2+ influx, and reduce excessive cytosolic Ca2+ accumulation. However, no correlation exists between the potency of the tested imidazol(ine) drugs and agmatine on rat brain I2 imidazoline receptors and the potency protecting against glutamate toxicity (r=0.17, P=0.69). These results indicate that activation of I2-imidazoline receptors is not involved in the neuroprotective effects of these drugs against glutamate-induced neurotoxicity.

Because a strong correlation (r=0.90, P<0.01) was found between the affinity of the imidazol(ine) drugs and agmatine for NMDA receptors and the potency of these compounds in protecting against glutamate-induced neuronal death, the neuroprotective effects of these drugs can be explained by a direct action at NMDA receptors. This interpretation is reinforced by two facts: (1) the lack of effect of the drugs tested in a model of apoptotic neuronal death (low K+ exposure) in which NMDA receptors are not involved and (2) the ability of antazoline and agmatine to directly block glutamate-induced currents in HEK-293 cells transfected to express the NR1-1a/NR2C subunits of the NMDA receptor. In this sense, whole cell patch-clamp experiments indicated that antazoline and agmatine behave as concentration- and voltage-dependent antagonists of the NMDA receptor. Based on the analysis of voltage dependence of the block, the action of antazoline is consistent with the mechanism of an open channel blocker (Figure 6). Also in this sense, agmatine (100 μM) has been recently shown to block NMDA currents in rat hippocampal neurones by interacting with a site located within the NMDA channel pore and the guanidino group of agmatine has been identified as the responsible moiety for blockade of the NMDA receptor channel (Yang & Reis, 1999). These results, together with the ability of imidazol(ine) drugs and agmatine to inhibit the binding of [3H]-(+)-MK-801 to the phencyclidine site of NMDA receptors (present study; Olmos et al., 1996; Boronat et al., 1998) and the similar chemical structures of antazoline, the most potent imidazoline drug tested, and phencyclidine, suggest that the binding site for imidazol(ine) drugs and agmatine could overlap with that of the phencyclidine (PCP) site. Indeed, the IC50 value of antazoline estimated at 0 mV (10–12 μM) was very close to the Ki value (13 μM) obtained in competition curves against [3H]-(+)-MK-801 binding to rat cerebral cortex membranes. Our electrophysiological analysis indicates that antazoline interacts directly with the NMDA receptor channel pore. The Woodhull model assumes that there is not interaction between a channel blocker and permeant ions (Woodhull, 1993). However, we observed a slight deviation of our data from this model (Figure 6d). The simplest explanation is that when the net current is outward (many ions flow in the outward direction) the permeating ions expel the blocker from its binding site on the pore (Antonov et al., 1998) or alternatively, that divalent cations interact with the blocker binding site within the pore (Lerma et al., 1991). The neuroprotective effect of imidazoline drugs and agmatine against glutamate-induced neurotoxicity is specific and related to NMDA receptor blockade, since neurotoxicity in cerebellar granule cells by other mechanisms not involving glutamate receptor activation cannot be prevented by glutamate receptor antagonists (Dargent et al., 1996) nor imidazol(ine) drugs.

The potencies (micromolar range) of imidazol(ine) drugs and agmatine at blocking NMDA receptors ([3H]-(+)-MK-801 binding) are in good agreement with the potencies of the same compounds at blocking other cation channels, such as K+ATP channels (Jonas et al., 1992; Shepherd et al., 1996), nicotinic acetylcholine (Musgrave et al., 1995) and 5-HT3 receptors (Molderings et al., 1996). Furthermore, as also demonstrated for these cation channels, imidazol(ine) drugs possessing two benzene rings (e.g. antazoline) are more potent on the NMDA receptor than others with only one aromatic ring (e.g. idazoxan) and these, in turn, are more potent than other molecules lacking an aromatic ring (e.g. agmatine) (Table 1). Hydrocarbon substituents on the benzene ring (e.g. cirazoline) increase the affinity of imidazol(ine) drugs for the NMDA receptor, as demonstrated for the interaction of these drugs with other cation channels (see Sakuta & Okamoto, 1994 for comparison). These findings, together with the observation that there are good correlations between the profiles of drug affinity of imidazol(ine) compounds at blocking different cation channels (K+ATP channels, NMDA and 5-HT3 receptors) (Olmos et al., 1996; Molderings, 1997), reinforce the hypothesis of the existence of a common imidazol(ine)/agmatine binding site on all these cation channels (Olmos et al., 1996).

The present results indicate that NMDA receptor blockade by imidazol(ine) drugs and agmatine is anti-excitotoxic in an in vitro model of necrotic neuronal death. The imidazoline drug idazoxan and agmatine can also exert neuroprotection after focal or global ischaemia (Gustafson et al., 1989; 1990; Maiese et al., 1992; Gilad et al., 1996), two in vivo models of necrotic neuronal death in which clear protective effects of NMDA receptor antagonists have been demonstrated (Meldrum, 1990; Holt, 1997). However, the relatively low affinity of idazoxan and agmatine for NMDA receptors and the fact that the extent of agmatine transport into the brain is unknown, make it difficult that the weak interaction of these drugs with NMDA receptors would also explain the survival-promoting effects found in vivo. In fact, it has been suggested that the neuroprotective effect of idazoxan after global ischaemia in the rat is related to the hypothermic effects of this drug (Craven & Conway, 1997). Also in this context, competitive and non-competitive NMDA receptor antagonists have been proved to attenuate the development of tolerance to morphine-induced antinociception (Trujillo & Akil, 1991; Tiseo et al., 1994). Although the same effect has been observed after concurrent chronic treatment of morphine with various imidazol(ine) drugs, no relationship between the potency of the drugs tested on NMDA receptors and their ability to attenuate morphine tolerance was found (Boronat et al., 1998).

In conclusion, the present study indicates that imidazol(ine) drugs and agmatine are neuroprotective in an in vitro model of glutamate-induced necrotic neuronal death and that this effect is mediated through NMDA receptor blockade. Further studies are needed to establish the possible in vivo functional implications of these findings.

Acknowledgments

This study was supported by DGICYT Grants PB94-0002-Mod C (J.A. García-Sevilla), PB94-0017 (R. Trullas) and PM-0008/96 (J. Lerma) from the Ministerio de Educación y Cultura. M.A. Boronat was supported by a fellowship from the Universitat de les Illes Balears. We are grateful to Consell Insular de Mallorca for support and to the pharmaceuticals firms for gifts of drugs. J.A. García-Sevilla is a member of the Institut d'Estudis Catalans.

Abbreviations

G-LH buffer

modified Locke-HEPES (M-LH) buffer containing 10 mM glucose

K+ATP

ATP-sensitive K+ channels

LSL 60101

2-(2-benzofuranyl)imidazole

LSL 60129

2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole

LSL 61122

2-styryl-2-imidazoline

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide

MTTR

reduced MTT

NMDA

N-methyl-D-aspartate

PCP

phencyclidine

RX821002

2-methoxy idazoxan

References

  1. ANIS N., SHERBY S., GOODNOW R., JR, NIWA M., KONNO K., KALLIMOPOULOS T., BUKOWNIK R., NAKANISHI K., USHERWOOD P., ELDEFRAWI A., ELDEFRAWI M. Structure-activity relationships of philanthotoxin analogs and polyamines on N-methyl-D-aspartate and nicotinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 1990;254:764–773. [PubMed] [Google Scholar]
  2. ANKARCRONA M., DYPBUKT J.M., BONFOCO E., ZHIVOTOVSKY B., ORRENIUS S., LIPTON S.A., NICOTERA P. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron. 1995;15:961–973. doi: 10.1016/0896-6273(95)90186-8. [DOI] [PubMed] [Google Scholar]
  3. ANTONOV S.M., GMIRO V.E., JOHNSON J.W. Binding sites for permeant ions in the channel of NMDA receptors and their effect on channel block. Nature Neurosci. 1998;1:451–461. doi: 10.1038/2167. [DOI] [PubMed] [Google Scholar]
  4. BERDEU D., PUECH R., RIBES G., LOUBATIÈRES-MARIANI M.-M., BERTRAND G. Antazoline increases insulin secretion and improves glucose tolerance in rats and dogs. Eur. J. Pharmacol. 1997;324:233–239. doi: 10.1016/s0014-2999(97)00126-x. [DOI] [PubMed] [Google Scholar]
  5. BERMAN F.W., MURRAY T.F. Characterization of [3H]MK-801 binding to N-methyl-D-aspartate receptors in cultured rat cerebellar granule neurons and involvement in glutamate-mediated toxicity. J. Biochem. Toxicol. 1996;11:217–226. doi: 10.1002/(SICI)1522-7146(1996)11:5<217::AID-JBT2>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
  6. BOJE K.M., WONG G., SKOLNICK P. Desensitization of the NMDA receptor complex by glycinergic ligands in cerebellar granule cell cultures. Brain Res. 1993;603:207–214. doi: 10.1016/0006-8993(93)91239-o. [DOI] [PubMed] [Google Scholar]
  7. BORONAT M.A., OLMOS G., GARCÍA-SEVILLA J.A. Attenuation of tolerance to opioid-induced antinociception and protection against morphine-induced decrease of neurofilament proteins by idazoxan and other I2-imidazoline ligands. Br. J. Pharmacol. 1998;125:175–185. doi: 10.1038/sj.bjp.0702031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. BOUSQUET P. Imidazoline receptors: from basic concepts to recent developments. J. Cardiovasc. Pharmacol. 1995;26:S1–S6. [PubMed] [Google Scholar]
  9. CHOI D.W. Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends Neurosci. 1988;11:465–469. doi: 10.1016/0166-2236(88)90200-7. [DOI] [PubMed] [Google Scholar]
  10. COUPRY I., ARMSBY C.C., ALPER S.L., BRUGNARA C., PARINI A. Clotrimazole and efaroxan inhibit red cell Gardos channel independently of imidazoline I1 and I2 binding sites. Eur. J. Pharmacol. 1996;295:109–112. doi: 10.1016/0014-2999(95)00642-7. [DOI] [PubMed] [Google Scholar]
  11. CRAVEN J.A., CONWAY E.L. Effects of α2-adrenoceptor antagonists and imidazoline2-receptor ligands on neuronal damage in global ischaemia in the rat. Clin. Exp. Pharmacol. Physiol. 1997;24:204–207. doi: 10.1111/j.1440-1681.1997.tb01808.x. [DOI] [PubMed] [Google Scholar]
  12. DARGENT B., ARSAC C., TRICAUD N., COURAUD F. Activation of voltage-dependent sodium channels in cultured cerebellar granule cells induces neurotoxicity that is not mediated by glutamate release. Neuroscience. 1996;73:209–216. doi: 10.1016/0306-4522(95)00608-7. [DOI] [PubMed] [Google Scholar]
  13. DEGREGORIO-ROCASOLANO N., OLMOS G., GASULL T., BORONAT M., TRULLAS R., GARCÍA-SEVILLA J.A.Protection by imidazol(ine) compounds of L-glutamate neurotoxicity through NMDA receptor blockade Ann. N.Y. Acad. Sci. 1999. in press [DOI] [PubMed]
  14. DESSI F., CHARRIAUT-MARLANGUE C., KHRESTCHATISKY M., BEN-ARI Y. Glutamate-induced neuronal death is not a programmed cell death in cerebellar culture. J. Neurochem. 1993;60:1953–1955. doi: 10.1111/j.1471-4159.1993.tb13427.x. [DOI] [PubMed] [Google Scholar]
  15. D'MELLO S.R., GALLI C., CIOTTI T., CALISSANO P. Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP. Proc. Natl. Acad. Sci. U.S.A. 1993;90:10989–10993. doi: 10.1073/pnas.90.23.10989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. FOSSOM L.H., BASILE A.S., SKOLNICK P. Sustained exposure to 1-aminocyclopropanecarboxylic acid, a glycine partial agonist, alters N-methyl-D-aspartate receptor function and subunit composition. Mol. Pharmacol. 1995;48:981–987. [PubMed] [Google Scholar]
  17. FOSTER A.C., WONG E.H.F. The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain. Br. J. Pharmacol. 1987;91:403–409. doi: 10.1111/j.1476-5381.1987.tb10295.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. FRENCH N. α2-adrenoceptors and I2 sites in the mammalian central nervous system. Pharmacol. Ther. 1995;68:175–208. doi: 10.1016/0163-7258(95)02005-5. [DOI] [PubMed] [Google Scholar]
  19. GILAD G.M., SALAME K., RABEY J.M., GILAD V.H. Agmatine treatment is neuroprotective in rodent brain injury models. Life Sci. 1996;58:PL41–46. doi: 10.1016/0024-3205(95)02274-0. [DOI] [PubMed] [Google Scholar]
  20. GUSTAFSON I., WESTERBORG E., WIELOCH T.W. Protection against ischemia-induced neuronal damage by α2-adrenoceptor antagonist idazoxan: Influence of time of administration and possible mechanisms of action. J. Cereb. Flow Metab. 1990;10:885–894. doi: 10.1038/jcbfm.1990.145. [DOI] [PubMed] [Google Scholar]
  21. GUSTAFSON I., YOSHITOYO M., WIELOCH T.W. Postischemic administration of idazoxan, an α2-adrenergic receptor antagonist, decreases neuronal damage in the rat brain. J. Cereb. Blood Flow Metab. 1989;9:171–174. doi: 10.1038/jcbfm.1989.25. [DOI] [PubMed] [Google Scholar]
  22. HOLT W.F.Glutamate in health and disease: the role of inhibitors Neuroprotection in CNS diseases 1997New-York: Marcel Dekker, Inc; 87–119.eds. Bär, P.R. & Beal, M.F. pp [Google Scholar]
  23. ISHITANI R., SUNAGA K., TANAKA M., AISHITA H., CHUANG D.-W. Overexpression of glyceraldehyde-3-phosphate dehydrogenase is involved in low K+-induced apoptosis but not necrosis of cultured cerebellar granule cells. Mol. Pharmacol. 1997;51:542–550. doi: 10.1124/mol.51.4.542. [DOI] [PubMed] [Google Scholar]
  24. JONAS J.C., PLANT T.D., HENQUIN J.C. Imidazoline antagonists of α2-adrenoceptors increase insulin release in vitro by inhibiting ATP-sensitive K+ channels in pancreatic β-cells. Br. J. Pharmacol. 1992;107:8–14. doi: 10.1111/j.1476-5381.1992.tb14456.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. LERMA J.L., PATERNAIN A.V., SALVADOR N., SOMOHANO F., MORALES M., CASADO M.Excitatory amino acid-activated channels Ion Channel Pharmacology 1998Oxford: Oxford University Press; 399–421.eds. Soria, B. & Ceña, V. pp [Google Scholar]
  26. LERMA J., ZUKIN R.S., BENNETT M.V.L. Interaction of Mg2+ and phencyclidine in use-dependent block of NMDA channels. Neurosci. Lett. 1991;123:187–191. doi: 10.1016/0304-3940(91)90927-l. [DOI] [PubMed] [Google Scholar]
  27. LI G., REGUNATHAN S., BARROW C.J., ESHRAGHI J., COOPER R., REIS D.J. Agmatine–an endogenous clonidine-displacing substance in the brain. Science. 1994;263:966–969. doi: 10.1126/science.7906055. [DOI] [PubMed] [Google Scholar]
  28. LYSKO P.G., COX J.A., VIGANO A., HENNEBERRY R.C. Excitatory amino acid neurotoxicity at the N-methyl-D-aspartate receptor in cultured neurons: pharmacological characterization. Brain Res. 1989;499:258–266. doi: 10.1016/0006-8993(89)90773-7. [DOI] [PubMed] [Google Scholar]
  29. MAIESE K., PEK L. , BERGER S.B., REIS D.J. Reduction in focal cerebral ischemia by agents acting at imidazole receptors. J. Cereb. Blood Flow Metab. 1992;12:53–63. doi: 10.1038/jcbfm.1992.7. [DOI] [PubMed] [Google Scholar]
  30. MCPHERSON G.A. Analysis of radioligand binding experiments: a collection of computer programs for IBM PC. J. Pharmacol. Meth. 1985;14:213–228. doi: 10.1016/0160-5402(85)90034-8. [DOI] [PubMed] [Google Scholar]
  31. MELDRUM B. Protection against ischemic neuronal damage by drugs acting on excitatory neurotransmission. Cerebrovasc. Brain Metab. Rev. 1990;2:27–57. [PubMed] [Google Scholar]
  32. MILANI D., GUIDOLIN D., FACCI L., POZZAN T., BUSO M., LEON A., SKAPER S.D. Excitatory amino acid-induced alterations of cytoplasmic free Ca2+ in individual cerebellar granule neurons: role in neurotoxicity. J. Neurosci. Res. 1991;28:434–441. doi: 10.1002/jnr.490280317. [DOI] [PubMed] [Google Scholar]
  33. MOLDERINGS G.J. Imidazoline receptors: basic knowledge, recent advances and future prospects for therapy and diagnosis. Drugs Fut. 1997;22:757–772. [Google Scholar]
  34. MOLDERINGS G.J., SCHMIDT K., BÖNISCH H., GÖTHERT M. Inhibition of 5-HT3 receptor function by imidazolines in mouse neuroblastoma cells: potential involvement of σ2 binding sites. Nauynn-Schmied. Arch. Pharmacol. 1996;354:245–252. doi: 10.1007/BF00171054. [DOI] [PubMed] [Google Scholar]
  35. MOSMANN T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  36. MUNSON P.J., RODBARD D. LIGAND: a versatile computerized approach for the characterization of ligand binding systems. Anal. Biochem. 1980;107:220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  37. MUSGRAVE I.F., KRAUTWURST D., HESCHELER J., SCHULTZ G. Clonidine and cirazoline inhibit activation of nicotinic channels in PC-12 cells. Ann. N.Y. Acad. Sci. 1995;763:272–283. doi: 10.1111/j.1749-6632.1995.tb32412.x. [DOI] [PubMed] [Google Scholar]
  38. OHARA-IMAIZUMI M., KUMAKURA K. Effects of imidazole compounds on catecholamine release in adrenal chromaffin cells. Cell. Mol. Neurobiol. 1992;12:273–283. doi: 10.1007/BF00712931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. OLMOS G., CASANOVAS A., RIBERA J., BORONAT M.A., ESQUERDA J.E., GARCÍA-SEVILLA J.A.Prevention by LSL 60101 [2-(2-benzofuranyl) imidazole) of motoneuron cell death after neonatal axotomy Ann. N.Y. Acad. Sci. 1999. in press
  40. OLMOS G., KULKARNI R.N., HAQUE M., MACDERMOT J. Imidazolines stimulate release of insulin from RIN-5AH cells independently from imidazoline I1 and I2 receptors. Eur. J. Pharmacol. 1994;262:41–48. doi: 10.1016/0014-2999(94)90026-4. [DOI] [PubMed] [Google Scholar]
  41. OLMOS G., RIBERA J., GARCÍA-SEVILLA J.A. Imidazoli(di)ne compounds interact with the phencyclidine site of NMDA receptors in the rat brain. Eur. J. Pharmacol. 1996;310:273–276. doi: 10.1016/0014-2999(96)00519-5. [DOI] [PubMed] [Google Scholar]
  42. OZAITA A., OLMOS G., BORONAT M.A., LIZCANO J.M., UNZETA M., GARCÍA-SEVILLA J.A. Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat liver. Br. J. Pharmacol. 1997;121:901–912. doi: 10.1038/sj.bjp.0701214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. PENG T.-I., GREENAMYRE J.T. Privileged access to mitochondria of calcium influx through N-methyl-D-aspartate-receptors. Mol. Pharmacol. 1998;53:974–980. [PubMed] [Google Scholar]
  44. PROKS P., ASHCROFT F.M. Phentolamine block KATP channels is mediated by Kir6.2. Proc. Natl. Acad. Sci. U.S.A. 1997;94:11716–11720. doi: 10.1073/pnas.94.21.11716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. REGUNATHAN S., REIS D.J. Imidazoline receptors and their endogenous ligands. Annu. Rev. Pharmacol. Toxicol. 1996;36:511–544. doi: 10.1146/annurev.pa.36.040196.002455. [DOI] [PubMed] [Google Scholar]
  46. SAKUTA H., OKAMOTO K. Inhibition by imidazoline and imidazolidine derivatives of glibenclamide-sensitive K+ currents in Xenopus oocytes. Eur. J. Pharmacol. 1994;259:223–231. doi: 10.1016/0014-2999(94)90648-3. [DOI] [PubMed] [Google Scholar]
  47. SCHRAMM M., EIMERL S., COSTA E. Serum and depolarizing agents cause acute neurotoxicity in cultured cerebellar granule cells: role of glutamate receptor responsive to N-methyl-D-aspartate. Proc. Natl. Acad. Sci. U.S.A. 1990;87:1193–1197. doi: 10.1073/pnas.87.3.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. SHEPHERD R.M., HASHMI M.N., KANE C., SQUIRES P.E., DUNNE M.J. Elevation of cytosolic calcium by imidazolines in mouse islets of Langerhans: implications for stimulus response coupling of insulin release. Br. J. Pharmacol. 1996;119:911–916. doi: 10.1111/j.1476-5381.1996.tb15759.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. TESSON F., PRIP-BUUS C., LEMOINE A., PEGORIER J.-P., PARINI A. Subcellular distribution of imidazoline-guanidinium-receptive sites in human and rabbit liver. J. Biol. Chem. 1991;266:155–160. [PubMed] [Google Scholar]
  50. TISEO P.J., CHENG J., PASTERNAK G.W., INTURRISI C.E. Modulation of morphine tolerance by the competitive N-methyl-D-aspartate receptor antagonist LY274614: assessment of opiod receptor changes. J. Pharmacol. Exp. Ther. 1994;268:195–201. [PubMed] [Google Scholar]
  51. TRUJILLO K.A., AKIL H. Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801. Science. 1991;251:85–87. doi: 10.1126/science.1824728. [DOI] [PubMed] [Google Scholar]
  52. VILLARROEL A., REGALADO M.P., LERMA J. Glycine-dependent desensitization: localization of molecular determinants. Neuron. 1998;20:329–339. doi: 10.1016/s0896-6273(00)80460-2. [DOI] [PubMed] [Google Scholar]
  53. WOODHULL A.M. Ionic blockade of sodium channel in nerve. J. Gen. Physiol. 1973;61:687–708. doi: 10.1085/jgp.61.6.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. YANG X.C., REIS D.J. Agmatine selectively blocks the N-methyl-D-aspartate subclass of glutamate receptor channels in rat hippocampal neurons. J. Pharmacol. Exp. Ther. 1999;288:544–549. [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES