Skip to main content
. 1997 Jan 7;94(1):137–141. doi: 10.1073/pnas.94.1.137

Figure 4.

Figure 4

Effect of in vivo treatment with adrenoreceptor ligands on CAM β2-adrenergic receptor levels and transgene expression. (A) Receptor levels in cardiac particulate preparations. The intrinsic activity of drugs at the CAM receptor was assessed in vitro at the level of adenylate cyclase in myocardial particulate preparations. Intrinsic activity (IA) was defined using untreated CAM transgenic animals (n = 2–4) as the fraction of adenylate cyclase activity evoked by the drug, compared with the reference full agonist isoproterenol (IA = 1) and the basal activity (IA = 0). Negative intrinsic activities (inverse agonists) were assessed with ICI 118,551-treated up-regulated CAM animals ([R] = 4 ± 1 pmol/mg, n = 4), with IA = 0 for basal and IA = −1 for maximal inhibition of basal, to the level of nontransgenic basal activity (see Fig. 1B). Nadolol and ICI 118,551 were used at 1 μM, and all other drugs were used at 20 × Kd (1). All reactions included 300 nM CGP 20712A, which blocks the endogenous β1-adrenergic receptor subtype selectively. 1, ICI 118,551 (0.7 mg/kg/hr); 2, nadolol (1.2 mg/kg/hr); 3, propranolol (0.4 mg/kg/hr); 4, alprenolol (0.4 mg/kg/hr); 5, pindolol (0.4 mg/kg/hr); 6, dichloroisoproterenol (1.2 mg/kg/hr); 7, ephedrine (0.3 mg/kg/hr); 8, dobutamine (1 mg/kg/hr); 9, norepinephrine (0.3 mg/kg/hr); 10, epinephrine (0.1 mg/kg/hr); 11, isoproterenol (0.1 mg/kg/hr). Crosshatched bar, untreated CAM transgenic. Shown are mean receptor densities ± SEM of 4–10 animals. (B) mRNA levels following drug treatment. NT, CAM vehicle, and CAM ICI as in Fig. 1. Total RNA was extracted from myocardial tissue (Biotecx Laboratories, Houston) and analyzed by Northern blotting. Shown are two representative lanes for each animal group (5 μg RNA per lane). The blots were probed with a simian virus 40 sequence, which is unique to the transgenic DNA construct (CAM, Upper), stripped and reprobed with the cDNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal quantity control (Lower) (12). Arrowheads, relative location of the 18S ribosomal RNA. Northern blots (3–4 animals per group) were also quantified on a PhosphorImager (Molecular Dynamics). When normalized to the amount of GAPDH mRNA, the transgene mRNA was not significantly increased by the ICI 118,551 treatment (data not shown).