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[3H]5'-N-Ethylcarboxamidoadenosine (NECA) labels both Al and A2 adenosine receptors in mammalian brain tissue. In the presence of 50 nM N'-cyclopentyladenosine (CPA), however, to block selectively the Al component of binding, [3H]NECA labels two binding components in striatal tissue. Such binding is dependent on the presence of Mg2+ ions and the removal of endogenous adenosine, using the catabolic enzyme, adenosine deaminase. The two binding components were observed in six mammalian species-rabbit, man, rat, mouse, guinea pig, and calf-with dissociation constants varying between 3.2 and 9.6 nM for the higher-affinity site and 54 and 232 nM for the lower-affinity site. In rat brain tissue, specific binding was reduced by the presence of 5'guanylyimidodiphosphate Gpp(NH)p, indicating the involvement of a G-protein. Gpp(NH)p effects were attributable to a conversion of a proportion of high-affinity to low-affinity components, as determined by saturation experiments. These results, together with the observation that Gpp(NH)p shifted the inhibition curve for the agonist, 2-chloro-adenosine(2-CADO), to the right indicate that the sites labeled by [3H]NECA in the presence of 50 nM CPA represent multiple affinity states rather than multiple receptor subtypes.Pharmacological examination of the higher-aff inity binding component using 2 nM [3H]NECA showed that binding was consistent with the labeling of an A2 adenosine receptor in all species. The order of activity for adenosine agonists was NECA > 2-CAD0 > R-phenylisopropyladenosine (R-PIA) > N'cyclohexyladenosine 2 CPA > > S-PIA. The activity ratios of the diastereomers of PIA (SIR) varied between 7.9 (mouse) and 14.5 (rat). The interaction of a series of 8-phenyl-substituted xanthines showed marked species differences with 1,3-dipropyl-8-(2-amin0-4-chloro) phenylxanthine (PACPX) being the most active at 14-15 nM in rabbit and man, 3-fold less active in calf, and 24-40-fold less active in guinea pig, mouse, and rat. A similar trend was seen for 8-phenyltheophylline (8-PT), 8-para-sulfo-phenyltheophylline (8-PST), and 1,3-diethyl-8-phenylxanthine (DPX), the relative order of activity of these adenosine antagonists being PACPX > 8-PT 2 DPX 2 8-PST. Caffeine and theophylline were much less active (1Cs0 > 10,000 nM), while the phosphodiesterase inhibitor 3-isobutyl-1 -methyl xanthine (IBMX I C5, , 2,257-4,410 nM) was 2-3 times more potent in man and calf than in the other species studied.

The binding of the selective ligand [3H]cyclohexyladenosine (CHA) to adenosine A1 receptors was studied in brain membrane from six species; cow, rabbit, rat, mouse, human, and guinea pig. Saturation analysis gave evidence for a single binding site in each species. The number of binding sites (2.6-4.7 pmollmg protein), based on saturation analysis, was virtually identical in each species. The Kd values were, however, different; whereas rat, rabbit, and mouse A1 receptors had similar affinities (Kd = 1-2 nM), the binding site in bovine tissue had three to four times greater affinity (Kd = 0.59 nM). Adenosine binding sites in human and guinea pig brain had two to three times less affinity, with Kd values 3.7 and 6.6 nM, respectively, than those in rat, rabbit, and mouse brain. Examination of the binding of five agonists, the R and S diastereomers of N6-phenylisopropyladenosine (PIA), CHA, 5"-ethylcarboxamido adenosine (NECA), and 2-chloroadenosine, and of five xanthine antagonists, PACPX [ 1,3,dipropyl-8-(2-amino-4-chloro)phenylxanthine], 1,3 diethyl-8-phenylxanthine (DPX), 8-phenyltheophylline (8PT), 8-parasulfophenyl-theophylline (8PST), and theophylline, showed that across the species N'-substituted adenosine analogs showed consistent differences in rank order activity. R-PIA 2 CHA > S-PIA, with the compounds being most active in bovine tissue and leas: active in guinea pig, showing an 18-fold difference. In contrast, the 2-substituted analog, 2-chloroadenosine was most active in guinea pig and human brain membranes, and least active in rat and bovine brain. The 5"-substituted analog NECA had similar activity in all species studied. PACPX was the most potent of the xanthines studied, being most active in bovine brain tissue and least active in guinea pig; the difference in activities was 393-fold. The interaction of the remainder of the xanthines studied was complex. In general, the rank order of potency was PACPX > DPX = 8 phenyltheophylline > 8PST > > theophylline. However, in human and rabbit brain 8PT was more active than DPX, and in guinea pig 8PT was twofold less active than DPX. In addition, in human and guinea pig brain, 8PST had comparable activities. These differences in pharmacological profiles may be attributable to differences in the adenosine systems studied as previously noted by Murphy and Snyder [Mol. Pharmacol. 22:250-257, 19821, which may in turn reflect distinct differences in A1 receptors or the presence of different subtypes of adenosine receptor in the vacious species. While these interspecies studies need to be extended to both A2 receptors and to coupled, i.e., adenylate cyclase, systems, they underline the need for care in choosing the mammalian species in which structure activity relationships are generated. Some caution is necessary in the use of bovine brain tissue in development programs targeted towards therapeutic entities in the adenosine A1 antagonist area.

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