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© Copyright 2004 The British Pharmacological Society

171P GKT, University of London
Winter Meeting December 2003

Effect of sodium ions on [³H]-nemonapride dissociation from the D_2SHORTdopamine receptor

M. Vivo and P. G. Strange. School of Animal and Microbial Sciences, University of Reading, P.O. Box 228, Whiteknights, Reading, RG6 6AJ.

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Vivo M
Strange PG

It has been shown previously that the binding of substituted benzamides such as raclopride to D₂ dopamine receptors, is strongly influenced by Na⁺, while spiperone binding is less modified (Armstrong et al., 2001). Although these studies concerned effects on the affinity and occupancy, Na⁺ ions could also affect the dissociation rate of the radioligands from D₂ receptors. Thus, kinetic experiments were performed in order to determine the ability of spiperone and raclopride in dissociating [³H]-nemonapride from the short isoform of D₂ receptor (D_2S) with and without Na⁺ ions present in the assay.

Sf9 membranes were prepared from cells grown and infected to express D2 receptors as described previously (Gazi et al., 2003). The binding assays were performed incubating 15-20 mg of membranes in assay buffer (20 mM HEPES, 1mM EDTA and 1 mM EGTA; pH 7.4) with the radioligand ([³H]-nemonapride 0.75-1.0 nM), with or without the presence of 100 mM NaCl, in a final volume of 900 µl until equilibrium (3h, 25°C) and then incubated with the competing ligand, spiperone or nemonapride (1 µM) or raclopride (20 µM), in a final volume of 1 ml, for the corresponding interval of time (12 intervals from 0 to 180 min per curve). (+)-butaclamol (3µM) and assay buffer were used to determine the non-specific and total binding, respectively. Data are mean±s.e.mean for 3-6 different curves, each point being performed in triplicate and data were analysed with Prism (GraphPad Software Inc.). Statistical comparisons were performed using Student's unpaired t-test.

In the absence of ions, the radioligand was fully dissociated from the receptor after 3h of incubation with the competing ligand. In the presence of Na⁺ ions, in addition to a 3 fold slower dissociation of the radioligand compared with the control, there was also a decrease in the percentage of radioligand dissociated. That is, only ~60-70% of the radioligand was dissociated at the end of the experiment (Table 1) in the presence of Na⁺.

[³H]-nemonapride		Half life (min) (mean±s.e.mean)	% dissociated (mean±s.e.mean)
vs. nemonapride	Control	16.47 ± 4.74	100
	Na⁺	44.54 ± 6.89*	59.20 ± 3.25
vs. spiperone	Control	15.69 ± 3.07	100
	Na⁺	49.25 ±10.95*	66.56 ±12.17
vs. raclopride	Control	13.98 ± 9.35	100
	Na⁺	43.13 ± 2.49*	65.79 ± 8.67

table 1. Dissociation of [³H]-nemonapride from D_2S receptors expressed in Sf9 cells, in absence and presence of Na⁺ ions. Parameters are derived from a monoexponential fit. (*p0.0001, for effect of Na⁺).

Armstrong, D. et al. (2001). J. Biol. Chem., 276, 22621-9.
Gazi, L et al. (2003). Br. J. Pharmacol., 138, 775-86.

We thank the BBSRC for financial support.