Differential expression and signaling of the human histamine H-3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells

摘要

In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H-3 receptor (hH(3)R(445) and hH(3)R(365)) on [S-35]-GTP gamma S binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca2+ ions ([Ca2+]i) and depolarization-evoked [H-3]-dopamine release. Maximal specific binding (B-max) of [H-3]-N-methyl-histamine to cell membranes was 953 +/- 204 and 555 +/- 140 fmol/mg protein for SH-SY5Y-hH(3)R(445) and SH-SY5Y-hH(3)R(365) cells, respectively, with similar dissociation constants (K-d, 0.86 nM and 0.81 nM). The mRNA of the hH(3)R(365) isoform was 40.9 +/- 7.9% of the hH(3)R(445) isoform. No differences in receptor affinity were found for the H3R ligands histamine, immepip, (R)(-)-alpha-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [S-35]-GTP gamma S binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH(3)R(445) cells ([S-35]-GTP gamma S binding, 158.1 +/- 7.5% versus 136.5 +/- 3.6% for SH-SY5Y-hH(3)R(365) cells; cAMP accumulation, -74.0 +/- 4.9% versus -43.5 +/- 5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [H-3]-dopamine release evoked by 100 mM K+ (-18.9 +/- 3.0% and -20.5 +/- 3.3%, for SH-SY5Y-hH(3)R(445) and SH-SY5Y-hH(3)R(365) cells), or the inhibition of depolarization-induced increase in [Ca2+]i (S2/S1 ratios: parental cells 0.967 +/- 0.069, SH-SY5Y-hH(3)R(445) cells 0.639 +/- 0.049, SH-SY5Y-hH(3)R(365) cells 0.737 +/- 0.045). These results indicate that in SH-SY5Y cells, hH(3)R(445) and hH(3)R(365) isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.