Stereoselectivity for the (R)-enantiomer of HA-966 (1-hydroxy-3-aminopyrrolidone-2) at the glycine site of the N-methyl-D-aspartate receptor complex.

HA-966 (1-hydroxy-3-aminopyrrolidone-2) is an antagonist at the glycine allosteric site of the N-methyl-D-aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA-966 is chiral. We report stereoselectivity for the (R)-enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)-enantiomer has an IC50 of 4.1 +/- 0.6 microM. The racemic mixture has an IC50 of 11.2 +/- 0.5 microM, whereas (S)-HA-966 has an IC50 greater than 900 microM. In glycine-stimulated [3H]1-[1-(2- thienyl)cyclohexyl]piperidine binding, the (R)-enantiomer inhibits with an IC50 of 121 +/- 61 microM, whereas the racemic mixture has an IC50 of 216 +/- 113 microM and (S)-HA-966 is inactive. The inhibition by (R)-HA-966 can be prevented by the addition of glycine. (R)-HA-966 and racemic HA-966, but not (S)-HA-966, also prevent N-methyl-D-aspartate cytotoxicity in cortical cultures. The (R)-enantiomer and, less potently, the (S)-enantiomer inhibit N-methyl-D-aspartate-evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)-HA-966 is reversed by added glycine. In glutamate-evoked contractions of the guinea pig ileum, (R)-HA-966 causes a glycine-reversible inhibition (IC50 of about 150 microM), whereas (S)-HA-966 is much less potent (IC50 of greater than 1 mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N-methyl-D-aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N-methyl-D-aspartate receptors in glutamate-evoked contractions of the guinea pig ileum, and supports their similarity to central N-methyl-D-aspartate receptors.

The polyamine spermine affects omega-conotoxin binding and function at N-type voltage-sensitive calcium channels.

1. The effects of the polyamines, spermine and spermidine on neuronal N-type voltage-sensitive calcium channels were investigated using the binding and function of the ligand omega-conotoxin GVIA (omega-CT). 2. Spermine and spermidine enhanced (EC50 approximately 0.16 and 0.45 microM) and, at higher concentrations, inhibited (IC50 of 9 and 240 microM) the binding of [125I]omega-CT to rat hippocampal synaptosomes. 3. Spermine and, less potently, spermidine inhibited the neurotransmitter-mediated, omega-CT-sensitive, electrical-field-stimulated contractile responses of the rat vas deferens. 4. The polyamines also inhibited the phenylephrine-evoked contractile responses of the vas deferens with the same rank order, consistent with a postsynaptic mechanism of inhibition. 5. However, pre-exposure to spermine prevented the irreversible inhibition of vas deferens twitch responses by omega-CT (previously found to be presynaptic). The prevention of inhibition by omega-CT demonstrates that the neuronal binding of spermine and omega-CT is mutually exclusive. Thus spermine (and presumably spermidine at higher concentrations) appears to modulate the actions of omega-CT at N-type voltage-sensitive calcium channels.

Neomycin and omega-conotoxin GVIA interact at a common neuronal site in peripheral tissues.

1. The present study examined the interaction of omega-conotoxin GVIA (omega-CT) and aminoglycoside antibiotics on electrically evoked, nerve-mediated contractile responses in the rat vas deferens, guinea-pig ileum and guinea-pig left atria. 2. omega-CT caused a time- and concentration-dependent inhibition of the electrically evoked twitch responses of the rat vas deferens and guinea-pig ileum. Aminoglycoside antibiotics inhibited the twitch responses of these preparations with a rank order of potency: neomycin greater than gentamycin greater than kanamycin. omega-CT had no effect on the postjunctional contractile responses of either noradrenaline (vas deferens) or carbachol (ileum). However, at high concentrations neomycin and gentamycin caused significant postjunctional inhibition. The results suggest that omega-CT and aminoglycosides cause prejunctional inhibition in these preparations, with the aminoglycoside antibiotics exhibiting postjunctional inhibitory effects as well at high concentrations. 3. omega-CT caused a concentration- and frequency-dependent inhibition of the neuronally mediated field stimulation enhancement of electrically paced guinea-pig left atria. omega-CT had no effect on either the electrically paced contractile response that was elicited by direct muscle stimulation or the enhancement of the paced response caused by beta-adrenoceptor agonist stimulation. Neomycin caused a concentration-dependent inhibition of the electrically paced contractile response and inhibited the field stimulation response only at concentrations which caused pronounced inhibition of the paced response. Neomycin also caused insurmountable inhibition of responses elicited by beta-adrenoceptor agonist stimulation. Thus, omega-CT caused an exclusive prejunctional inhibition in guinea-pig left atria, whereas the substantial postjunctional effects of neomycin made it difficult to discern any prejunctional activity of neomycin in these experiments. 4. In the vas deferens, ileum and atria the inhibitory effects of omega-CT were long-lasting, whereas the effects of neomycin could be reversed upon wash-out. The disparate kinetics of omega-CT and neomycin allowed for the design of receptor protection studies to determine whether neomycin acts at a prejunctional site in common with omega-CT. The pre-equilibration of a competitive antagonist (neomycin) should prevent the irreversible antagonist (omega-CT) from gaining access to receptors. Pre-exposure of tissues with neomycin prevented the irreversible inhibition of omega-CT. These receptor protection studies suggest that omega-CT and neomycin interact at common neuronal sites in the rat vas deferens, guinea-pig ileum and guinea-pig atria. Neomycin, however, exhibits activity at postjunctional sites as well.