Treatment with inverse agonists enhances baseline atrial contractility in transgenic mice with chronic beta2-adrenoceptor activation

In this study, we investigate whether chronic treatment with β-adrenoceptor (βAR) ligands with inverse agonist activity enhances myocardial β2AR-mediated atrial tension more than neutral antagonists in transgenic mice (TG35). These mice exhibit chronic adrenoceptor activation because they possess a greater number of constitutively active receptors than wild type mice due to cardiac-specific overexpression of human β2ARs. TG35 and wild type mice were chronically treated for 90 h with three inverse agonists, ICI-118,551, propranolol, and carvedilol, and one neutral antagonist, alprenolol. After 96 h, we compared the basal and isoprenaline-stimulated (10 μM) increase in atrial tension in treated or untreated TG35 mice and wild type mice. In parallel, to determine the effect of chronic βAR ligand treatment on the amounts of G protein receptor kinase-2 (GRK-2) and G proteins, we performed Western blotting on myocardial cytosolic and membrane proteins. Atria from the TG35 mice treated with inverse agonists showed increases in the baseline tension compared to those from alprenolol/vehicle-treated mice. ICI-118,551 and propranolol treatment restored the elevated myocardial G-inhibitory protein (Giα) levels to that of wild type. Also, treatment with inverse agonists upregulated G-stimulatory protein (Gsα) levels and GRK2 above those levels in vehicle-treated TG35 or wild type mice. The increased baseline atrial tension was reversed by the addition of ICI-118,551. Overall, our data suggests that inverse agonists enhance baseline atrial tension more than neutral antagonists. Based on this, we propose that upregulation of the active conformation of the β2ARs, Gsα protein and restoration of Giα as three possible mechanisms to explain this enhanced receptor activity. Therefore, the favourable effects of some ligands used in pathological conditions involving chronic adrenoceptor activation may be due to the inverse agonist activity of the ligand. Keywords: β-Adrenoceptors, left atria, G proteins, G protein coupled receptor kinase, inverse agonists, isoprenaline, transgenic mice Introduction In diseases such as congestive heart failure (CHF), chronic activation of cardiac β-adrenoceptors (βARs) by endogenous agonists like adrenaline and noradrenaline can result in diminished myocardial contractility. This chronic adrenoceptor activation is accompanied by reduction in β1AR number, uncoupling of β2AR from the Gs-protein, increase in Giα protein, and increase in GRK2 protein (G protein coupled receptor kinase or β-adrenergic receptor kinase, βARK1/2) (Castellano & Bohm, 1997). Similar changes are observed during chronic activation of βARs by infusing synthetic and exogenous βAR agonists (Muller et al., 1993; Zhou et al., 1995). Recent studies show that chronic treatment with some βAR antagonists increase survival in heart failure by improving myocardial contractility (Reviewed by Krum, 1997). However, other ligands that exhibit similar βAR subtype selectivity profiles are not equally effective in heart failure suggesting mechanisms independent of antagonism of agonist actions for these ligands. Traditionally, inverse agonism has been determined by detecting changes in baseline (unstimulated) parameters. This requires enough spontaneously active receptors to reveal the inverse agonist properties of compounds. The two most extensively used techniques for increasing the number of spontaneously active receptors have been to overexpress the wild type receptor or to express a constitutively active mutant of the receptor (Milligan et al., 1995). In heart failure β-adrenoceptor numbers are decreased, and it is therefore unlikely that inverse agonists would have any effect on baseline parameters. However, recent data has suggested that even at low receptor densities, and with no detectable change in baseline, inverse agonists and neutral antagonists evoke different cellular responses (Berg et al., 1999, in press). In CHO cells transfected with relatively low levels of the 5-HT2C receptor (∼200 fm mg−1 protein), 24 h incubation with the inverse agonist for 5-HT2C receptors, SB 206553, produced different cellular effects than incubation with the neutral antagonist, 5-methoxygramine. The inverse agonist, SB206553 produced sensitization to 5-HT2C receptor agonists activating phospholipase C (PLC), and also sensitized the response to another agonist, ATP, activating purinergic receptors, which also mediates its response through PLC activation. This effect was observed even though SB 206553 had no effect on baseline IP accumulation; SB 206553 was determined to be an inverse agonist from studies in cells transfected with much higher densities (∼5–10 pm mg−1 protein) of the 5-HT2C receptor. These results suggest the cellular response of sensitization may be a more sensitive indicator of inverse agonist activity than inhibition of baseline. The neutral antagonist, 5-methoxygramine, did not produce sensitization and antagonized the sensitizing effect of SB 206553 (Berg et al., 1999, in press). Thus, cells recognize differences between antagonists and inverse agonists even when there is no change in baseline. Many βAR ligands previously classified as antagonists are now known to possess inverse agonism which imparts these ligands with negative intrinsic activity because they reduce the number of constitutively active receptors (reviewed by Milligan et al., 1995; Milligan & Bond, 1997). Constitutively active receptors can couple to G proteins and elicit biological response even in the absence of an agonist. Inverse agonists, in contrast to neutral antagonists, prevent both agonist-dependent receptor activation (like neutral antagonists do) and inhibit constitutively active receptors. We have a transgenic mouse line (TG35) which display a 50 fold increase in their total βAR density than wild type mice due to cardiac-specific overexpression of human β2ARs. This receptor overexpression increases the number of constitutively active receptors that contribute to chronic activation of the adrenoceptors (Milano et al., 1994; Bond et al, 1995). Hence, the TG35 mice provide a model to study agonist-independent chronic βAR activation. Our study is designed to assess in the TG35 hearts, whether βAR ligands exhibiting inverse agonism improve myocardial β2AR-mediated atrial tension under basal and agonist-stimulated conditions more effectively than ligands exhibiting pure antagonism. As an index of myocardial tension (contractility) in mice, we will measure basal and isoprenaline-stimulated increases in atrial tension after chronic treatment with four pharmacologically distinct βAR ligands. Also, we will examine the effect of this treatment on the amount of GRK2 and G proteins and correlate the results with the functional data.