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

摘要: The influence of sodium ion substitutes on the 5-hydroxytryptamine (5-HT)-induced flux organic cation [14C]guanidinium through channel mouse 5-HT3 receptor and competition 5-HT with selective antagonist [3H]GR 65630 was studied, unless stated otherwise, in neuroblastoma N1E-115 cells. Under physiological conditions (135 mM sodium), induced a concentration-dependent influx an EC50 (1.3 μM) similar to that electrophysiological studies. The stepwise replacement by increasing concentrations hydroxyethyl trimethylammonium (choline) concentration dependently caused both rightward shift concentration–response curve increase maximum effect 5-HT. Complete resulted 34-fold lower potency almost two times higher maximal response. A low choline buffer also observed other receptor-expressing rodent cell lines (NG 108-15 or NCB 20). Replacement Na+ Li+ left effects unchanged. Replacement tris (hydroxymethyl) methylamine (Tris), tetramethylammonium (TMA) N-methyl-D-glucamine (NMDG) response choline. only slightly reduced Tris, high degree decreased TMA (comparable decrease choline), but not influenced NMDG. The inhibiting [3H]GR65630 binding intact cells 35-fold when completely replaced choline, remained unchanged after results are compatible suggestion competes for receptor; may be partly due choline-mediated delay 5-HT-induced desensitization. For studies 5-HT-evoked channels, NMDG appears ‘ideal' substituent since it increases signal-to-noise ratio without interfering binding. Keywords: Mouse receptor, tracer flux, binding, substitutes, [14C]guanidinium Introduction The is (5-HT) subtype belongs superfamily ligand-gated channels (Derkach et al., 1989; Kilpatrick 1990; Maricq 1991). It permeable sodium, potassium calcium, closely related nicotinic acetylcholine receptor. characteristic feature its rapid desensitization (Jackson & Yakel, 1995; Reeves Lummis, 2002). Physiologically, receptors involved emesis pain (Aapro, 1991; Mitchelson, 1992; Karim 1996; Voog 2000; Simpson 2000). Several classes drugs, including general anaesthetics (Barann 2000), alcohols (Lovinger Zhou, 1994; Barann 1995) cannabinoids (Fan, 2002), assumed modulate directly function. Several available study native receptors, clone 1993), 20 × chinese hamster hybrid (Hellevuo 1991) NG108-15 rat glioma (Reiser Hamprecht, 1989). In addition, cells, mixture long short, mouse-specific 5-HT3A splice variants occur (Werner 1994). As alternative techniques, measurement radioactively labelled [14C]guanidinium, which behaves like (Reith, 1990), useful method investigate drugs function. Various groups have unanimously shown this suitable characterize receptor-mediated neural Emerit 1993; Bonisch 1993). However, potencies inducing were markedly different; thus, value reported al. (1993) about 10 than values groups. This discrepancy fact (1993), contrast others, used replace sodium. condition, under 5-HT-mediated sensitive ondansetron tetrodotoxin applied drug (stimulation veratridine), because larger specific fluxes, thus facilitating analysis 1993). The mechanism increased, quarternary still unknown. Therefore, main aim present elucidate underlying reduction condition. context, we examined whether such as lithium, tris(hydroxyethyl)methylamine tetramethyl ammonium differ from respect. chemical structure all cations Figure 1 (in case guanidinium, unlabelled compound shown). Figure 1 Chemical structures (pka=9.5) Tris (pka=8.3) positively charged at pH. diameters (Angstrom units, three dimensions) follows: 1.924, 1.924 ... Methods Cell culture Mouse (Amano 1972; passage numbers 40–50) grown Dulbecco's modified Eagle's medium (DMEM) HEPES (7.6 mM) bicarbonate (30 mM). growth supplemented (as described Hoyer Neijt, 1988) antibiotics penicillin (100 IU ml−1) streptomycin μg ml−1), 10% fetal calf serum (Gibco BRL) following amino-acid mix (mM): L-cysteine hydrochloride (0.3), L-alanine (0.4), L-asparagine (0.45), L-aspartic acid L-proline (0.4) L-glutamic (0.4). Cells cultured humidified atmosphere containing 5% CO2 37°C 750 ml vent flasks (Nunc) fed every 3 day. About days before starting experiments, subcultured 24-well culture cluster plates (Falcon). At beginning experiment, confluent (the protein content per well 0.2 mg). X Chinese NCB20 above except addition newborn omission mix. NG108-15, contained hypoxanthine (10 mM), thymidine (16 aminopterin (0.5 no