Influence of sodium substitutes on 5-HT-mediated effects at mouse 5-HT3 receptors

Abstract

1 The influence of sodium ion substitutes on the 5-hydroxytryptamine (5-HT)-induced flux of the organic cation [14C]guanidinium through the ion channel of the mouse 5-HT3 receptor and on the competition of 5-HT with the selective 5-HT3 receptor antagonist [3H]GR 65630 was studied, unless stated otherwise, in mouse neuroblastoma N1E-115 cells. 2 Under physiological conditions (135 mm sodium), 5-HT induced a concentration-dependent [14C]guanidinium influx with an EC50 (1.3 microm) similar to that in electrophysiological studies. 3 The stepwise replacement of sodium by increasing concentrations of the organic cation hydroxyethyl trimethylammonium (choline) concentration dependently caused both a rightward shift of the 5-HT concentration-response curve and an increase in the maximum effect of 5-HT. Complete replacement of sodium resulted in a 34-fold lower potency of 5-HT and an almost two times higher maximal response. A low potency of 5-HT in choline buffer was also observed in other 5-HT3 receptor-expressing rodent cell lines (NG 108-15 or NCB 20). 4 Replacement of Na+ by Li+ left the potency and maximal effects of 5-HT almost unchanged. Replacement by tris (hydroxymethyl) methylamine (Tris), tetramethylammonium (TMA) or N-methyl-d-glucamine (NMDG) caused an increase in maximal response to 5-HT similar to that caused by choline. The potency of 5-HT was only slightly reduced by Tris, to a high degree decreased by TMA (comparable to the decrease by choline), but not influenced by NMDG. 5 The potency of 5-HT in inhibiting [3H]GR65630 binding to intact cells was 35-fold lower when sodium was completely replaced by choline, but remained unchanged after replacement by NMDG. 6 The results are compatible with the suggestion that choline competes with 5-HT for the 5-HT3 receptor; the increase in maximal response may be partly due to a choline-mediated delay of the 5-HT-induced desensitization. For studies of 5-HT-evoked [14C]guanidinium flux through 5-HT3 receptor channels, NMDG appears to be an 'ideal' sodium substituent since it increases the signal-to-noise ratio without interfering with 5-HT binding.

Figure 1

Chemical structures of the organic cations used in this study to replace sodium. NMDG (pka=9.5) and Tris (pka=8.3) are positively charged at physiological pH. The ion diameters (Angstrom units, three dimensions) are as follows: sodium 1.924, 1.924 and 1.924; choline 7.487, 5.020 and 4.488; guanidinium 6.498, 4.952 and 3.103; NMDG 13.056, 5.582 and 5.332; TEA 8.431, 6.358 and 5.841; TMA 5.291, 4.824 and 4.556; and Tris 7.644, 5.712 and 4.491. The values for lithium are 1.198, 1.198 and 1.198.

Figure 2

Influence of gradual replacement of sodium by choline on the 5-HT (2-min exposure)-evoked [¹⁴C]guanidinium influx above basal influx in N1E-115 cells. The responses are expressed as percentages of the effect elicited by 100 μM 5-HT using choline (135 mM) buffer (=reference); 100 % corresponds to 51 pmol × mg protein⁻¹. Shown are means±s.e.m. of n=5–12 experiments.

Figure 3

Influence of complete replacement of sodium by different cations on the 5-HT (2-min exposure)-evoked [¹⁴C]-guanidinium influx above basal influx in N1E-115 cells. The responses are expressed as percentages of the effect elicited by 100 μM 5-HT in choline buffer (=reference effect); 100% corresponds to 51 pmol × mg protein⁻¹). Shown are means of n=5–12 experiments. Error bars were omitted for the sake of clarity; they were in the range of values shown in Figure 2.

Figure 4

Effect of preincubation with 30 μM 5-HT on the [¹⁴C]guanidinium influx above basal influx in N1E-115 cells evoked by 2-min exposure to 30 μM 5-HT. Experiments were carried out using either sodium buffer (squares) or choline buffer (triangles). The responses are expressed as absolute values of [¹⁴C]guanidinium (pmol mg⁻¹protein) and were fitted to a monoexponential (choline) and biexponential (sodium) decay. (Inset) Normalization of values to the respective maximum signal (no preincubation with 5-HT) showing that a rate difference between both conditions developed within the first minute of 5-HT preincubation. Shown are means ±s.e.m. of n=3–6 experiments.

Figure 5

Influence of complete replacement of sodium (triangles) by choline (squares) on the inhibition of [³H]GR65630 binding to intact N1E-115 cells by 5-HT. The 5-HT effects are expressed as percentages of [³H]GR65630 binding (=control) after 60 min exposure to 1 nM of the radioligand in the absence of 5-HT. Under both buffer conditions, two components with two different IC₅₀ values could be calculated. Shown are means±s.e.m. of n=4 experiments.