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Research Article

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

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Pages 141-150 | Received 06 Nov 2017, Accepted 26 Feb 2018, Published online: 20 Mar 2018
 

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 [3 H]-dopamine release. Maximal specific binding (Bmax) of [3 H]-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 (Kd, 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 [3 H]-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.

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.].

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