In this article, we pharmacologically characterized two naturally occurring human histamine H 3 receptor (hH 3 R) isoforms, hH 3 R(445) and hH 3 R(365). These abundantly expressed splice variants differ by a deletion of 80 amino acids in the intracellular loop 3. In this report, we show that the hH 3 R(365) is differentially expressed compared with the hH 3 R(445) and has a higher affinity and potency for H 3 R agonists and conversely a lower potency and affinity for H 3 R inverse agonists. Furthermore, we show a higher constitutive signaling of the hH 3 R(365) compared with the hH 3 R(445) in both guanosine-5Ј-O-(3-[35 S]thio)triphosphate binding and cAMP assays, likely explaining the observed differences in hH 3 R pharmacology of the two isoforms. Because H 3 R ligands are beneficial in animal models of obesity, epilepsy, and cognitive diseases such as Alzheimer's disease and attention deficit hyperactivity disorder and currently entered clinical trails, these differences in H 3 R pharmacology of these two isoforms are of great importance for a detailed understanding of the action of H 3 R ligands.The histamine H 3 receptor (H 3 R) was originally discovered in the brain on histaminergic neurons as a presynaptic autoreceptor regulating the release of histamine (Arrang et al., 1983). Subsequently, the H 3 R was found to regulate the release of other neurotransmitters, such as acetylcholine, dopamine, glutamate, noradrenalin, and serotonin (Schlicker et al., 1988(Schlicker et al., , 1989(Schlicker et al., , 1993Clapham and Kilpatrick, 1992;Brown and Reymann, 1996). The histamine-containing cell bodies, located in the tuberomammillary nucleus of the posterior hypothalamus, project to most cerebral areas in rodent and human brain (Panula et al., 1984;Watanabe et al., 1984). Brain histamine is involved in the regulation of numerous functions of the central nervous system (CNS), including arousal, cognition, locomotor activity, autonomic and vestibular functions, feeding and drinking, sexual behavior, and analgesia (Hough, 1988;Schwartz et al., 1991;Wada et al., 1991). Moreover, H 3 R-specific ligands show beneficial effects in animal models of obesity, epilepsy, and cognitive diseases such as Alzheimer's disease and attention deficit hyperactivity disorder (Hancock, 2003;Passani et al., 2004;Leurs et al., 2005). Consequently, H 3 R antagonists are con-1

Guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays were established and utilized as a reliable and high-capacity functional assay for determining antagonist and inverse agonist pharmacological parameters of novel histamine H(3) ligands, at the recombinant human H(3) receptor. [(35)S]GTPgammaS binding assays were performed with membranes prepared from human embryonic kidney 293 cells stably expressing the full-length (445 amino acids) human H(3) receptor isoform, at approximately 1 pmol/mg of protein. Utilizing robotic liquid handling, assay filtration, and scintillation counting in a 96-well format, concentration-response curves were determined for up to 40 compounds per assay. The imidazole-containing H(3) receptor antagonist ciproxifan and the non-imidazole antagonist ABT-239 inhibited (R)-alpha-methylhistamine (RAMH)-stimulated [(35)S]GTPgammaS binding in a competitive manner, and negative logarithm of the dissociation equilibrium constant (pK(b)) values determined for nearly 200 structurally diverse H(3) antagonists were very similar to the respective negative logarithm of the equilibrium inhibition constant values from N-alpha-[(3)H]methylhistamine competition binding assays. H(3) antagonists also concentration-dependently decreased basal [(35)S]GTPgammaS binding, thereby displaying inverse agonism at the constitutively active H(3) receptor. At maximally effective concentrations, non-imidazole H(3) antagonists inhibited basal [(35)S]GTPgammaS binding by approximately 20%. For over 100 of these antagonists, negative logarithm of the 50% effective concentration values for inverse agonism were very similar to the respective pK(b) values. Both H(3) receptor agonist-dependent and -independent (constitutive) [(35)S]GTPgammaS binding were sensitive to changes in assay concentrations of sodium, magnesium, and the guanine nucleotide GDP; however, the potency of ABT-239 for inhibition of RAMH-stimulated [(35)S]GTPgammaS binding was not significantly affected. These robust and reliable [(35)S]GTPgammaS binding assays have become one of the important tools in our pharmacological analysis and development of novel histamine H(3) receptor antagonists/inverse agonists.

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