Pharmacological Characterization of Homocysteine Response at the NMDA Receptor.

We first applied homocysteine plus 1 μM glycine to rat cortical neurons in culture while monitoring responses during whole-cell recording with a patch electrode or digital Ca2+ imaging with fura-2. d,l-Homocysteine (5 mM) increased [Ca2+] i by 97 ± 13 nM in rat cortical neurons (mean ± SEM, n = 7; P < 0.001 by Student’s t test). The homocysteine-mediated increases in [Ca2+] i were blocked by a variety of NMDA receptor antagonists, including d-2-amino-5-phosphonopentanoate, dizocilpine, and 7-chlorokynurenate but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (Fig. 1a). However, under these conditions, homocysteine was considerably less potent and less efficacious than NMDA (Fig. 1b): The maximal effect of homocysteine in the presence of 1 μM glycine was ≈30% that of NMDA. This lower efficacy could be secondary to a partial agonist effect of homocysteine at the NMDA binding site. Alternatively, we reasoned that homocysteine might interact with a second site on the NMDA receptor to inhibit its activity. The latter possibility was confirmed in the following experiments probing interactions of homocysteine at the glycine coagonist binding site (16, 17).

Figure 1 Homocysteine increases neuronal [Ca2+] i through NMDA receptor-channel activation. (a) Antagonism of homocysteine-mediated [Ca2+] i responses by NMDA receptor antagonists. Responses evoked by 5 mM d,l-homocysteine and 1 μM glycine were quantified in the absence and presence of NMDA- and non-NMDA-antagonists: d-2-amino-5-phosphonopentanoate (APV, 200 μM), dizocilpine (MK-801, 10 μM), 7-chlorokynurenate (7-Cl-KN, 10 μM), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 μM). Values in this and subsequent figures represent the mean ± SEM. Imaged fields contained five to seven neurons, each serving as its own control, and the results represent data from three separate experiments. Data are expressed as the percentage change compared with the control response (≈150 nM [Ca2+] i ) elicited by 5 mM homocysteine plus 1 μM glycine. Responses to homocysteine/glycine in the presence of antagonist that were statistically smaller than those obtained to homocysteine/glycine alone are indicated by asterisks (∗∗, P < 0.01, analyzed with an ANOVA followed by a Scheffé multiple comparison of means). (b) Dependence on glycine of the concentration–response relationship of NMDA- and homocysteine-mediated increases in neuronal [Ca2+] i . The increase in homocysteine responses in the presence of elevated concentrations of glycine suggests that homocysteine is not only an agonist at the NMDA binding site but also a partial antagonist of the glycine site (data from six neurons in a representative experiment from a total of eight experiments). The open boxes have been displaced slightly for clarity. (c) Partial antagonism of NMDA responses by homocysteine. NMDA (100 μM)-mediated increases in [Ca2+]i in the presence of 1 or 50 μM glycine were measured at various concentrations of homocysteine (n = 3 experiments). To exclude an effect of rundown or desensitization, high or low glycine was applied randomly during each response. The results are consistent with the notion that homocysteine is a full agonist at the NMDA binding site and that its inhibition of NMDA responses is mediated functionally by partial antagonism of the glycine site (we cannot exclude the possibility that homocysteine acts as a partial agonist at this site in the absence of glycine).

When the glycine concentration was increased, the maximal effect of homocysteine increased (with no significant change in EC 50 ) to match that of NMDA, both in digital calcium imaging experiments and in patch-clamp recordings (Figs. 1b and 2 a–d). With 10 μM glycine, the response to 10 mM d,l-homocysteine was increased 142 ± 32% (n = 4; data not shown), and with 50 μM glycine, the response was enhanced 181 ± 33% (Fig. 1b; n = 6). Moreover, in the presence of elevated glycine levels, 100–150 μM concentrations of l-homocysteine (20–300 μM d,l-homocysteine) evoked increases in neuronal [Ca2+] i or whole-cell currents (Figs. 1b and 2e). For example, approximately equivalent rises in [Ca2+] i were observed with 150 μM l-homocysteine and 10–20 μM NMDA, known neurotoxic concentrations in this preparation (11). Importantly, the dose–response curve for NMDA-induced [Ca2+] i changes was not altered by these increases in glycine concentration (Fig. 1b). Using the more sensitive technique of whole-cell recording in the presence of bicarbonate and elevated glycine, measurable NMDA receptor-mediated currents were evoked by 100 μM l-homocysteine (Fig. 2e). Taken together, these findings suggest that homocysteine competes with glycine at the glycine binding site of the NMDA receptor to decrease its activity, and in the presence of ≥50 μM glycine, homocysteine becomes a relatively high-affinity NMDA receptor agonist.

Figure 2 Whole-cell recording of homocysteine-evoked currents are increased by glycine. (a) Current evoked by 200 μM NMDA plus 1 μM glycine. (b) Current evoked by 200 μM NMDA plus 50 μM glycine. (c) The current evoked by 10 mM d,l-homocysteine (or 5 mM l-homocysteine, a maximal stimulus) with 1 μM glycine was relatively small. (d) Increasing the glycine concentration to 50 μM increased the magnitude of the current evoked by homocysteine on the same cortical neuron as in c. This action was not voltage-dependent as similar effects were seen at a holding potential of +40 mV (data not shown). Possibly due to rundown of the currents, 50 μM glycine did not always increase the maximal homocysteine-evoked current to the same level as the maximal NMDA-induced current in all neurons tested (n = 7). However, in each case the effect was qualitatively similar to that observed during the calcium imaging experiments. (e) Micromolar homocysteine evoked measurable currents in the presence of glycine and bicarbonate. When 200 μM d,l-homocysteine (equivalent to 100 μM l-homocysteine) was applied in Hanks’ balanced salt solution, a small macroscopic current was observed (left trace). To simulate physiological conditions, 24 mM bicarbonate was added, and then the same micromolar concentration of homocysteine elicited a somewhat larger whole-cell current (center trace; similar to the effect of bicarbonate on cysteine-induced currents noted previously; ref. 9). Application of 50 μM glycine in addition to the bicarbonate further increased the homocysteine-activated current to 200% (right trace). In e, the bath solution was made somewhat acidic (pH = 7.0 rather than 7.2) to more closely simulate ischemic conditions.