The diastereomeric mixture of d/l-2,7-diaminooctanedioyl-bis(YRLRY-NH2) (BVD-74D, 2) was described in the literature as a high affinity Y4 receptor agonist. Here we report on the synthesis and pharmacological characterization of the pure diastereomers (2R,7R)- and (2S,7S)-2 and a series of homo- and heterodimeric analogues in which octanedioic acid was used as an achiral linker. To investigate the role of the Arg residues, one or two arginines were replaced by Ala. Moreover, N(ω)-(6-aminohexylaminocarbonyl)Arg was introduced as an arginine replacement (17). (2R,7R)-2 was superior to (2S,7S)-2 in binding and functional cellular assays and equipotent with 17. [(3)H]Propionylation of one amino group in the linker of (2R,7R)-2 or at the primary amino group in 17 resulted in high affinity Y4R radioligands ([(3)H]-(2R,7R)-10, [(3)H]18) with subnanomolar Kd values.

In an integrative approach, we studied the role of histamine H 2 receptors in the mouse heart. We noted that histamine, added cumulatively to the organ bath, failed to affect the force of contraction in left atrial preparations and did not change spontaneous heart rate in right atrial preparations from wildtype mice. By contrast, in the same preparations from mice that overexpressed the human H 2 receptor in a cardiac-specific way, histamine exerted concentration-and time-dependent positive inotropic and positive chronotropic effects. Messenger RNA of the human H 2 receptor was only detected in transgenic mice. Likewise, immunohistology and autoradiography only gave signals in transgenic but not in wild-type cardiac preparations. Similarly, a positive inotropic and positive chronotropic effect was observed with histamine in echocardiography of living transgenic mice and isolated perfused hearts (Langendorff preparation). Phosphorylation of phospholamban was increased in atrial and ventricular preparations from transgenic mice, but not in wild-type animals. The effects of histamine were mimicked by dimaprit and amthamine and antagonized by cimetidine. In summary, we generated a new model to study the physiologic and pathophysiologic cardiac role of the human H 2 receptor.