br Discussion This study dedicated to

Discussion This study, dedicated to ciprofloxacin H3 and H4 receptors, was carried out on superfused tissues preincubated with 3H-choline or 3H-noradrenaline; electrical stimulation was used to elicit quasi-physiological exocytotic release of the respective transmitter. Since inhibition of transmitter release by Gi/o protein-coupled receptors (to which H3 and H4 receptors belong) can be improved by the simultaneous blockade of the muscarinic (Hiramatsu et al., 1998, Nishiwaki et al., 1998, Giaroni et al., 1999, Shakirzyanova et al., 2006) or α2-autoreceptor (for review, see Schlicker and Göthert, 1998) AF-DX 384 and rauwolscine were used in our experiments, respectively. Since it was unclear whether H3 and H4 receptors can be detected in each of the five experimental models, the muscarinic receptor agonist oxotremorine was studied as a positive control. This drug elicits its maximum inhibitory effect at 10 μM on sympathetically innervated tissues (Kurz et al., 2008). In hippocampal slices, oxotremorine had to be administered at a tenfold higher concentration since those experiments were routinely carried out in the presence of AF-DX 384 (Schulte et al., 2012). As expected, oxotremorine led to a marked inhibition of transmitter release in each of the five experimental models, clearly demonstrating that modulation of transmitter release by presynaptic inhibitory receptors is possible under the experimental conditions of the present study. The H4 receptor agonist 4-methylhistamine (Lim et al., 2005) was used at a concentration (10 μM) that exceeds its Ki value at the gpH4 receptor by a factor of 200 (Lim et al., 2010) and was close to the concentration (20 μM) used on guinea-pig cardiac synaptosomes by Chan et al. (2012). 4-Methylhistamine did not affect transmitter release in the release models of our study, including the heart. In the latter preparation, the H3 agonist R-α-methylhistamine (Arrang et al., 1987), used at a concentration (1 μM) that elicits the maximum effect at functional H3 receptors of the guinea-pig (Luo and Tan, 1994, Timm et al., 1998), did inhibit noradrenaline release. The reason why the cardiac synaptosome preparation allowed the identification of H3and H4 receptors whereas only the H3 receptor could be identified on the isolated atrium is unclear. Presynaptic cardiac H3 receptors, first shown by Luo et al. (1991) on electrically stimulated atrial strips of the guinea-pig, have also been showed on the human (Imamura et al., 1995) and rat atrium (Malinowska and Schlicker, 1993). R-α-Methylhistamine inhibited noradrenaline release also in the aorta, renal cortex and vas deferens. The presynaptic H3 receptor on the guinea-pig vas deferens has been shown for the first time by Luo and Tan (1994), who examined the electrically induced contractile response instead of directly quantifying noradrenaline release. The occurrence of H3 receptors has also been suggested for the guinea-pig aorta in vitro (Schultheiß et al., 2005) and the dog kidney in vivo (Yamasaki et al., 2001). Since the latter two studies are, however, based on single concentrations or doses of H3 receptor ligands only, additional experiments based on whole concentration–response curves were carried out in the aorta and renal cortex. Thioperamide, which possesses a high preference for H3 over H1 or H2 receptors (Arrang et al., 1987, Hough, 2001), served as the antagonist. Although it does not discriminate well between H3 and H4 receptors (Hough, 2001) it was used anyway since affinity data for each of the four histamine receptor subtypes in guinea-pig tissues are available. Its apparent pA2 value of 8.0–8.1 in our interaction experiments agreed well with its pA2 in other functional H3 receptor models of the guinea-pig (8.1–8.2; Timm et al., 1998, Feliszek et al., 2015) but exceeded its affinity (pKi) at H4 receptors (7.1; Lim et al., 2010) markedly and its affinity at H1 (pKB < 4; Arrang et al., 1987) and H2 receptors (pKB < 5; Arrang et al., 1987) extremely. These data allow us to conclude that the inhibitory presynaptic histamine receptor in the aorta and renal cortex is indeed an H3 receptor.