Abstract
Cardiac G protein-coupled receptors that function through stimulatory G protein Galpha(s), such as beta(1)- and beta(2)-adrenergic receptors (beta(1)ARs and beta(2)ARs), play a key role in cardiac contractility. Recent data indicate that several Galpha(s)-coupled receptors in heart also activate Galpha(i), including beta(2)ARs (but not beta(1)ARs). Coupling of cardiac beta(2)ARs to Galpha(i) inhibits adenylyl cyclase and opposes beta(1)AR-mediated apoptosis. Dual coupling of beta(2)AR to both Galpha(s) and Galpha(i) is likely to alter beta(2)AR function in disease, such as congestive heart failure in which Galpha(i) levels are increased. Indeed, heart failure is characterized by reduced responsiveness of betaARs. Cardiac betaAR-responsiveness is also decreased with aging. However, whether age increases cardiac Galpha(i) has been controversial, with some studies reporting an increase and others reporting no change. The present study examines Galpha(i) in left ventricular membranes from young and old Fisher 344 rats by employing a comprehensive battery of biochemical assays. Immunoblotting reveals significant increases with age in left ventricular Galpha(i2), but no changes in Galpha(i3), Galpha(o), Galpha(s), Gbeta(1), or Gbeta(2). Aging also increases ADP-ribosylation of pertussis toxin-sensitive G proteins. Consistent with these results, basal as well as receptor-mediated incorporation of photoaffinity label [(32)P]azidoanilido-GTP indicates higher amounts of Galpha(i2) in older left ventricular membranes. Moreover, both basal and receptor-mediated adenylyl cyclase activities are lower in left ventricular membranes from older rats, and disabling of Galpha(i) with pertussis toxin increases both basal and receptor-stimulated adenylyl cyclase activity. Finally, age produces small but significant increases in muscarinic potency for the inhibition of both beta(1)AR- and beta(2)AR-stimulated adenylyl cyclase activity. The present study establishes that Galpha(i2) increases with age and provides data indicating that this increase dampens adenylyl cyclase activity.
Highlights
Cardiac contractility is controlled by several G protein-coupled receptors (GPCRs),1 such as 1- and 2-adrenergic recep
Coupling of 2AR and other GTP-binding regulatory proteins (G␣s)-coupled receptors to G␣i is relevant to cardiac function because inactivation of G␣i by pertussis toxin (PTX) increases myocyte contractility in rat heart [6] and increases both basal and receptormediated adenylyl cyclase (AC) activity in human heart [4]
We examined the expression of G␣i2, G␣i3, G␣o, G␣s, G1, and G2, all of which previously have been detected in rat heart [26, 37]
Summary
Materials—R(Ϫ)-isoproterenol-(ϩ)-bitartrate, ICI 118,551, and CGP 20712A were obtained from Research Biochemicals International (Natick, MA). Activated PTX (1 ng/l) was added to membranes (1.0 –1.5 mg/ml protein) in a buffer containing 37.5 mM Tris (pH 7.4), 6.25 mM MgCl2, 1 mM EDTA, 5 mM NAD, 2.5 mM ATP, 4 mM GTP, 10 mM thymidine, 10 mM DTT, 0.005% SDS, 0.08 mg/ml BSA, 0.5 mM benzamidine, 5 g/ml soybean trypsin inhibitor, 5 g/ml leupeptin, and 2.5 g/ml aprotinin. Activated PTX (1 ng/l) was added to membranes (1.0 –1.5 mg/ml protein) in a buffer containing 37.5 mM Tris (pH 7.4), 6.25 mM MgCl2, 1 mM EDTA, 2.5 mM ATP, 4 mM GTP, 10 mM thymidine, 10 mM DTT, 0.005% SDS, 50 M NAD, 10 Ci/mmol [32P]NAD, 0.08 mg/ml BSA, 0.5 mM benzamidine, 5 g/ml soybean trypsin inhibitor, 5 g/ml leupeptin, and 2.5 g/ml aprotinin. Each set of data was analyzed by Shapiro-Wilk tests for normality, the statistical significance of comparisons between data from young and old samples was determined by performing unpaired Student’s t tests and exact Wilcoxon Mann-Whitney tests on the mean values of each data set. p Ͻ 0.05 was considered significant for all comparisons
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