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.

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 [ ³⁵ S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca ²⁺ ions ([Ca ²⁺ ]i) and depolarization-evoked [ ³ H]-dopamine release. Maximal specific binding (B _max ) of [ ³ H]-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 H ₃ R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [ ³⁵ S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH ₃ R ₄₄₅ cells ([ ³⁵ S]-GTPγS 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 [ ³ H]-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 [Ca ²⁺ ]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.