Introduction

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Aminoglycoside antibiotics (Fig. 1) are effective against many strains of aerobic Gram-negative and some aerobic Gram-positive bacteria. Despite both a rapid onset of bactericidal action and low treatment cost, the use of aminoglycosides is limited by ototoxicity that can result in permanent damage to both the cochlear and vestibular apparatus (Wersall, 1995).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Structures of aminoglycosides. Aminoglycoside antibiotics are composed of one or two aminosugars glycosidically linked to an aminocyclitol nucleus. Streptomycin and dihydrostreptomycin are composed of the aminocyclitol nucleus streptidine, which is monosubstituted on C-4 with a disaccharide composed of -L-streptose and -L-n-methylglucosamine. Both neomycin B and kanamycin A have 2-deoxystreptamine for an aminocyclitol nucleus. In neomycin B, 2-deoxystreptamine is disubstituted on C-4 and C-5 with 2,6-diaminoglucose and a disaccharide composed of 2,6-diaminoglucose and D-ribose, respectively. Kanamycin A is substituted on C-4 with 6-aminoglucose and on C-6 with kanosamine (Kirst, 1996).

Recent pharmacological evidence suggests that aminoglycoside-induced ototoxicity is, in part, an excitotoxic process mediated by an activation of N-methyl-D-aspartate (NMDA) receptors. Thus, aminoglycoside-induced hearing loss and cochlear damage in guinea pigs can be attenuated by administration of NMDA antagonists such as dizocilpine and ifenprodil (Basile et al., 1996). Furthermore, dizocilpine treatment significantly reduces the vestibular damage produced by streptomycin administration to neonatal rats (Basile et al., 1999). Neurochemical studies indicate aminoglycosides mimic the actions of polyamines such as spermine at native NMDA receptors. Like polyamines (Ransom and Stec, 1988; Sacaan and Johnson, 1990; Schoemaker et al., 1990; Hashimoto et al., 1994), aminoglycosides stimulate the binding of channel blockers such as [³H]dizocilpine (Basile et al., 1996) and [³H]1-[1-(2 thienyl)cyclohexyl]piperidine (Pullan et al., 1992) and at similar concentrations inhibit [³H]ifenprodil binding (Basile et al., 1996; Segal and Skolnick, 1998). Moreover, a positive correlation is observed between the rank order cochleotoxicity of a series of aminoglycosides and their potencies to increase [³H]dizocilpine binding in rat cortical membranes (Basile et al., 1996). Aminoglycosides enhance [³H]dizocilpine binding with EC₅₀ values of ~10 µM (Pullan et al., 1992; Basile et al., 1996; Segal and Skolnick, 1998), whereas peak concentrations in the cochlear perilymph after ototoxic doses of aminoglycosides such as gentamicin and amikacin are ~20 µM (Brummett et al., 1978). These findings led to the proposal that a polyamine-like activation of NMDA receptors is a pivotal step in the ototoxicity produced by these aminoglycosides (Skolnick, 1997).

After the initial demonstration that endogenous polyamines modulate radioligand binding to NMDA receptors, electrophysiological studies in primary neuron cultures have revealed that polyamines produce multiple actions at NMDA receptors (Benveniste and Mayer, 1993). Thus, polyamines such as spermine increase the apparent affinity of the coagonist glycine. This increase is observed at subsaturating glycine concentrations and is generally referred to as "glycine-dependent" potentiation. Polyamines also increase agonist responses in the presence of saturating glycine concentrations ("glycine-independent" potentiation), a phenomenon linked to an increase in the frequency of channel opening and a reduction in desensitization rate (Rock and Macdonald, 1995) and removal of tonic proton inhibition (Traynelis et al., 1995). Extracellular spermine also can block the NMDA receptor channel in a voltage-dependent manner, an effect more pronounced at hyperpolarized potentials (Benveniste and Mayer, 1993).

Studies in recombinant NMDA receptors demonstrate that these polyamine actions are dependent on subunit composition (Williams, 1997). Because the effects of aminoglycosides on NMDA receptors have largely been explored in native receptors with neurochemical methods, the objective of this study was to compare the electrophysiological actions of a group of aminoglycosides with spermine in recombinant NMDA receptors of defined composition (NR1A/2A, NR1A/2B, and NR1A/2C) expressed in Xenopus oocytes. We report herein that aminoglycosides generally mimic the effects of spermine on recombinant NMDA receptors. Nonetheless, several differences were evinced between these antibiotics and spermine, primarily with respect to efficacy and sensitivity to membrane voltage. In addition, significant efficacy differences were observed among these aminoglycosides that would not have been predicted from previous neurochemical studies. Finally, these data demonstrate that, unlike spermine, aminoglycosides can potentiate agonist responses in NR1A/2C receptors under glycine-dependent conditions.

	Experimental Procedures

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Materials. Xenopus laevis frogs were purchased from Xenopus-1 (Dexter, MI). Collagenase B was obtained from Boehringer Mannheim (Indianapolis, IN). All other compounds were purchased from Sigma Chemical Co. (St. Louis, MO).

cDNA Clones. The rat NMDA receptor NR1A, NR2A, and NR2C clones were gifts from Dr. S. Nakanishi (Department of Immunology, Kyoto University, Kyoto, Japan). The NR2B clone was a gift from J. Sullivan (Salk Institute, La Jolla, CA). To optimize subunit expression levels in oocytes, most of the 5' untranslated region was removed from the subunit clones using available restriction enzyme sites. The cDNAs encoding the NMDA subunits were subcloned into pcDNA3.

Injection of In Vitro Synthesized RNA into Xenopus Oocytes. Capped cRNA was synthesized from linearized template cDNA encoding the subunits with mMESSAGE mMACHINE kits (Ambion, Inc., Austin, TX). Oocytes were injected with the NR1A and NR2 subunits in a ratio of 1:5 as determined by gel electrophoresis. The NR1A subunit is the splice form lacking exon 5, and also is referred to as NR₀₁₁ (Sugihara et al., 1992; Durand et al., 1993). This subunit has been reported to form functional receptors in oocytes, perhaps as a consequence of association with an endogenous oocyte protein (Zukin and Bennett, 1995; Soloviev and Barnard, 1997). The currents resulting from injection of cRNA encoding NR1A alone were small in magnitude and, at saturating agonist concentrations, never exceeded 5% of current obtained by coinjecting cRNAs encoding NR1A with an NR2 isoform.

Electrophysiological Recordings. Oocytes were perfused at room temperature in a Warner Instruments oocyte recording chamber RC-5/18 (Hamden, CT) with perfusion solution (115 mM NaCl, 1.8 mM CaCl₂, 2.5 mM KCl, 10 mM HEPES, pH 7.2). Perfusion solution was gravity fed continuously at a rate of 15 ml/min. Compounds were diluted in perfusion solution, and applied until a steady-state current was reached, typically 60 s.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Glycine concentration-responses of recombinant NMDA NR1A/2A (), NR1A/2B (), and NR1A/2C () receptor subtypes. To comparatively evaluate glycine-independent and -dependent polyamine actions, maximal and equipotent (dotted line) glycine concentrations were determined for each subunit combination. Glutamate concentration (30 µM) is saturating. Symbols represent means ± S.D. of three or four separate oocytes and fitted to a four-parameter logistic equation. The EC50 and Hill coefficients are 1.39 ± 0.44 µM and 1.38 ± 0.22, 129 ± 26 nM and 1.21 ± 0.03, and 227 ± 97 nM and 1.06 ± 0.06 for NR1A/2A, NR1A/2B, and NR1A/2C, respectively, as determined from the fitted parameters of each individual concentration-response curve.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Aminoglycosides increase the affinity of glycine in recombinant NMDA receptors. A, representative glycine concentration response curves in the absence (, EC50 = 113 nM) and presence of 100 µM neomycin (, EC50 = 53 nM) on the same oocyte expressing NR1A/2B. B) Representative glycine concentration response curves in the absence (, EC50 =220 nM) and in the presence of 3 mM streptomycin (, EC50 =72 nM) on the same oocyte expressing NR1A/2C. Glutamate concentration is saturating (30 µM). Responses are normalized to the maximal response, and data points are fitted to a four-parameter logistic equation.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Actions of spermine (), neomycin B (), kanamycin A (), streptomycin (), and dihydrostreptomycin () on recombinant NMDA receptor subtypes NR1A/2A (A and D), NR1A/2B (B and E), and NR1A/2C (C and F) under glycine-independent (A-C) and glycine-dependent (D-F) conditions. Increasing concentrations of these compounds were perfused over the oocyte along with fixed concentrations of glutamate and glycine. The steady-state whole-cell current response in the presence of the polyamine is reported as a percentage of the response to glutamate and glycine alone. Symbols represent means ± S.D. of three or four separate oocytes, and fitted to a four-parameter logistic or a one-site competition equation where appropriate; otherwise, points were connected with a straight line.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Voltage dependence of spermine and aminoglycoside effects. The ratio of the effect at a holding potential of 85 mV to the effect at 35 mV is reported for spermine (open column), neomycin (solid column), kanamycin (stippled column), streptomycin (horizontal-striped column), and dihydrostreptomycin (hatched column). All compounds were applied at a concentration of 1 mM. Bars represent means ± S.D. for three or four separate oocytes. Significant differences from spermine are *p < .05 and **p < .01.

	Results

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Glycine-Dependent and Glycine-Independent Potentiation of NMDA Responses by Aminoglycosides: Comparison to Spermine. To evaluate the glycine-dependent (i.e., measured at subsaturating glycine concentrations) and glycine-independent (i.e., measured at saturating glycine concentrations) actions of aminoglycosides, glycine concentration-response curves were constructed for each receptor isoform. Figure 2 illustrates glycine concentration-response curves for oocytes expressing receptors composed of NR1A/2A, NR1A/2B, and NR1A/2C subunits. Based on the maximal responses obtained, a saturating glycine concentration of 10 µM was used to evaluate the glycine-independent actions of aminoglycosides at NR1A/2B and NR1A/2C receptors, and 30 µM for NR1A/2A receptors. The glycine-dependent actions of aminoglycosides were evaluated with equipotent glycine concentrations (~EC₃₅) of 30 nM, 100 nM, and 1 µM for NR1A/2B, NR1A/2C, and NR1A/2A receptors, respectively.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Aminoglycoside actions on recombinant NMDA receptor subtypes. A, voltage-clamp traces of an oocyte expressing NR1A/2A receptors during application of 30 µM glutamate and 30 µM glycine for the duration indicated by a black bar (left traces). Traces from the same oocyte subsequently applied with the coagonists along with 1 mM streptomycin for the duration indicated by a gray bar (right traces). B, voltage-clamp traces of an oocyte expressing NR1A/2C receptors during application of 30 µM glutamate and 100 nM glycine for duration indicated by a black bar (left traces). Traces from the same oocyte subsequently applied with the coagonists along with 1 mM streptomycin for duration indicated by a gray bar (right traces). Scale bar, 20 nA, 25 s.

Voltage Dependence of Aminoglycosides and Spermine at Recombinant NMDA Receptors. Based on the apparent voltage-dependent actions of polyamines on NMDA receptors (Benveniste and Mayer, 1993; Rock and Macdonald, 1992a), the influence of holding potential on aminoglycoside and spermine activities was compared under both glycine-dependent and glycine-independent conditions. The effect of each compound at a holding potential of 85 mV was compared with the effect observed at 35 mV.

Effects of Aminoglycosides on Glycine Affinity. Glycine-dependent potentiation of NMDA receptors by spermine appears to be mediated by an increase in affinity of the receptor for the coagonist glycine (Sacaan and Johnson, 1989). Glycine-dependent effects of aminoglycosides were examined by determining glycine concentration-response relationships in the presence and absence of aminoglycoside. Figure 3A illustrates representative glycine concentration-response curves measured on the same oocyte-expressing NR1A/2B receptors. Neomycin (100 µM) shifted the glycine EC₅₀ to the left an average of 2.2 ± 0.4-fold (n = 3; p < .05), a magnitude similar to that reported for spermine by Williams et al. (1994). We observed an ~4-fold left shift in glycine EC₅₀ by 1 mM spermine (data not shown). Figure 3B illustrates representative glycine concentration-response curves measured on an oocyte-expressing NR1A/2C receptors. Streptomycin (3 mM) shifted the glycine EC₅₀ to the left an average of 2.2 ± 0.8-fold (n = 3; p < .05). Consistent with previous reports (Williams, 1995), the EC₅₀ of glycine was not altered by the presence of spermine (data not shown).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The aminoglycosides examined in this study reproduced many of the electrophysiological effects of the prototypical polyamine, spermine, at recombinant NMDA receptors. Thus, like spermine, all of the aminoglycosides examined potentiated NR1A/2B receptors under both glycine-independent and glycine-dependent conditions, and potentiated NR1A/2A receptors under glycine-dependent conditions. Despite this mimicry, several significant differences were evinced between the effects of spermine and aminoglycosides with respect to subunit specificity, efficacy, and sensitivity to membrane potential. In addition, there were significant differences among aminoglycosides that would not be predicted from previous neurochemical studies (Pullan et al., 1992; Basile et al., 1996; Segal and Skolnick, 1998).

Perhaps the most striking difference between spermine and aminoglycosides such as neomycin B, kanamycin A, and streptomycin is the ability of these antibiotics to elicit a glycine-dependent potentiation in NMDA receptors composed of NR1A/2C subunits (Figs. 3B, 4F, and 6B). Consistent with Williams (1995), spermine was unable to produce a glycine-dependent augmentation in this receptor isoform, whereas under identical conditions, neomycin B and streptomycin enhanced agonist responses by >50%. The mechanism of aminoglycoside induced glycine-dependent potentiation at NR1A/2C containing receptors is via an increase in the glycine affinity of the receptor (Fig. 3B), similar to that reported for spermine on NR1A/2A and NR1A/2B receptors (Williams et al., 1994).

Although the absence of the 21-amino acid residue encoded by exon 5 of NR1 is the principal determinant of polyamine response in recombinant NMDA receptors (Durand et al., 1992), the potentiation of NR1A/2C responses by aminoglycosides is consistent with other observations that the NR2A and NR2B subunits are capable of modulating the complex actions of polyamine-like molecules in heteromeric receptors (Williams et al., 1994; Johnson, 1996). In the absence of an effect by spermine, the ability of a polyamine antagonist to block the effects of aminoglycosides in, for example, NR1A/2C receptors would provide compelling evidence that these effects are mediated through a polyamine site. However, the intrinsic actions and low potencies of previously described polyamine antagonists (e.g., arcaine, diethylenetriamine, and putrescine) confounded combination studies with aminoglycosides. For example, in a receptor isoform (NR1A/2B) where polyamine responsiveness can most readily be demonstrated, the low potency of 1,6-diaminohexane resulted in a failure to block (at 300 µM) the glycine-dependent effects of either spermine or neomycin B (at 30 µM; data not shown). Nonetheless, the failure of dihydrostreptomycin to cause potentiation at NR1A/2C receptors demonstrates that this is not a general feature of aminoglycosides and implies there is a structural specificity to this action.

Spermine produces a voltage-dependent inhibition of both native and recombinant NMDA receptors. This block is more pronounced at hyperpolarized potentials and appears to have two components. One component is a fast open-channel block, similar to that produced by Mg²⁺ (Benveniste and Mayer, 1993). Spermine also reduces the unitary conductance, an action attributed to binding to charged residues near the channel mouth, impeding cation flow (Rock and Macdonald, 1992b). The voltage-dependent inhibition by spermine and Mg²⁺ are present in the same receptor subtypes (i.e., NR1A/2A and NR1A/2B, but not NR1A/2C) (Monyer et al., 1992). Our data confirm this subtype selectivity of spermine and illustrate that aminoglycosides differ from this polyamine in two respects. Under glycine-dependent conditions, the effects of aminoglycosides are less sensitive to membrane potential than spermine on NR1A/2B receptors, and the actions of these antibiotics are sensitive to membrane potential at NR1A/2C receptors. The aminoglycosides are large, sterically bulky compounds and may not be able to enter the channel pore to block receptors composed of NR1A/2B subunits (Fig. 1). However, the rigid conformation of these molecules (relative to spermine) may actually enhance their ability to cause charge-screening on NR1A/2C subunits, resulting in voltage dependence.

Previous neurochemical studies demonstrated modest differences in potency among clinically effective aminoglycosides to enhance [³H]dizocilpine binding to rat brain membranes. Because native NMDA receptors are heterogeneous, differences in the rank order potency between those studies (Basile et al., 1996; Segal and Skolnick, 1998) and our's with recombinant NMDA receptors of defined composition are not unexpected. However, there were significant differences among aminoglycosides with respect to efficacy (Fig. 4) that would not be predicted from previous neurochemical studies. No clear structure-activity relationship emerges from these data, due perhaps to the structural diversity of the of the different aminosugar and aminocyclitol moieties on these aminoglycosides (Fig. 1). Nonetheless, the differences between streptomycin and its reduced analog dihydrostreptomycin merit further comment. Although differing from streptomycin by only the presence of a 3' CH₂OH (compared with a 3' CHO) on the -L-streptose sugar, the potentiation of glycine-dependent responses by dihydrostreptomycin in all three receptor subtypes was far more modest than that of the parent compound (Fig. 4). This finding indicates that the aldehyde group of the parent molecule may be important as a hydrogen bond-accepting site necessary for the glycine-dependent augmentation. Dihydrostreptomycin was perhaps the most cochleotoxic aminoglycoside in clinical use and was withdrawn from the market soon after its introduction (Wersall, 1995). Despite the evidence implicating NMDA receptor activation in the cochleotoxic actions of aminoglycosides (Basile et al., 1996), dihydrostreptomycin exhibits no glycine-dependent activation of NR1A/2C receptors and is less efficacious in this measure than the less ototoxic streptomycin at both NR1A/2A and NR1A/2B receptors. These data indicate that the excitotoxic component of aminoglycoside-induced ototoxicity represents a complex process. In rodents, immunocytochemistry and in situ hybridization have detected NR1, NR2A, NR2B, and NR2C subunits in both the cochlear and vestibular apparatus (Niedzielski and Wenthold, 1995). Moreover, a subpopulation of NMDA receptors is likely to be heterogeneous with respect to NR2 (Sheng et al., 1994; Luo et al., 1997), and this heterogeneity imparts a pharmacology that is unique from receptors containing a single NR2 subunit (Wafford et al., 1993). Nonetheless, the lower potentiation by dihydrostreptomycin indicates that NMDA receptor activation could, in fact, be downstream from the primary event mediating ototoxicity. Alternatively, if aminoglycoside-induced ototoxicity involves an excitotoxic component, then the relative ototoxicity of an aminoglycoside may reflect the sum total of its actions (e.g., glycine-dependent and glycine-independent efficacy, voltage-dependent block) at NMDA receptor subtypes present on target organs. If this aminoglycoside ototoxicity is, in part, an excitotoxic process, then an aminoglycoside that does not interact with NMDA receptors may have a reduced risk for ototoxicity.