Differential expression and signaling of the human histamine H₃ receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells

Abstract

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 H₃ receptor (hH₃R₄₄₅ and hH₃R₃₆₅) on [³⁵S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca²⁺ ions ([Ca²⁺]i) and depolarization-evoked [³H]-dopamine release. Maximal specific binding (B_max) of [³H]-N-methyl-histamine to cell membranes was 953 ± 204 and 555 ± 140 fmol/mg protein for SH-SY5Y-hH₃R₄₄₅ and SH-SY5Y-hH₃R₃₆₅ cells, respectively, with similar dissociation constants (K_d, 0.86 nM and 0.81 nM). The mRNA of the hH₃R₃₆₅ isoform was 40.9 ± 7.9% of the hH₃R₄₄₅ isoform. No differences in receptor affinity were found for the H₃R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [³⁵S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH₃R₄₄₅ cells ([³⁵S]-GTPγS binding, 158.1 ± 7.5% versus 136.5 ± 3.6% for SH-SY5Y-hH₃R₃₆₅ 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 [³H]-dopamine release evoked by 100 mM K⁺ (−18.9 ± 3.0% and −20.5 ± 3.3%, for SH-SY5Y-hH₃R₄₄₅ and SH-SY5Y-hH₃R₃₆₅ cells), or the inhibition of depolarization-induced increase in [Ca²⁺]i (S2/S1 ratios: parental cells 0.967 ± 0.069, SH-SY5Y-hH₃R₄₄₅ cells 0.639 ± 0.049, SH-SY5Y-hH₃R₃₆₅ cells 0.737 ± 0.045). These results indicate that in SH-SY5Y cells, hH₃R₄₄₅ and hH₃R₃₆₅ isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.

Keywords:

• Histamine
• H3 receptor
• receptor isoforms
• presynaptic receptors
• cAMP
• dopamine release
• calcium
• neuromodulation

Acknowledgements

We are grateful to Raúl González-Pantoja for his excellent technical assistance. G. Nieto-Alamilla and M.C. López-Méndez held Conacyt scholarships. G. Nieto-Alamilla was also supported by Mexico State Council for Science and Technology (Comecyt).

Disclosure statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funding sources were not involved at all in the study design, collection, analysis and interpretation of data, writing of the manuscript or the decision to submit this report.

Additional information

Funding

This work was supported by CINVESTAV and CONACYT [grants 220448 to J. A. A. M. and 128735 to A. G. H.].