In human and animal hypertension models, increased activity of G-protein-coupled receptor kinase (GRK) 2 determines a generalized decrease of ␤-adrenergic vasodilatation. We analyzed the possibility of differential changes in the expression and functionality of ␣ 1A , ␣ 1B , ␣ 1D , ␤ 1 , ␤ 2 , and ␤ 3 -ARs also being involved in the process. We combined the quantification of mRNA levels with immunoblotting and functional studies in aortas of young and adult spontaneously hypertensive rats (SHRs) and their controls (Wistar Kyoto). We found the expression and function of ␤ 1 -adrenoceptors in young prehypertensive SHRs to be higher, whereas a generalized increase in the expression of the six adrenoceptors and GRK2 was observed in aortas of adult hypertensive SHRs. ␣ 1D -and ␤ 3 -Adrenoceptors, the subtypes that are more resistant to GRK2-mediated internalization and mostly expressed in rat aorta, exhibited an increased functional role in hypertensive animals, showing two hemodynamic consequences: 1) an increased sensitivity to the vasoconstrictor stimulus accompanied by a decreased sensitivity to the vasodilator stimulus (␣ 1D -ARs are the most sensitive to agonists, and ␤ 3 -ARs are the least sensitive to agonists); and 2) a slower recovery of the basal tone after adrenergic stimulus removal because of the kinetic characteristic of the ␣ 1D subtype. These functional changes might be involved in the greater sympathetic vasoconstrictor tone observed in hypertension.Although there is growing evidence that essential hypertension is related to the overactivity of the sympathetic nervous system, the exact causes are still poorly understood. The adrenergic-dependent increase in vascular resistance could reflect an imbalance between vasoconstrictor and vasodilator mechanisms related to changes in both the expression and function of ␣ 1 -adrenoceptors (ARs), which mediate vasoconstriction, ␤-ARs, which mediate vasodilatation, and/or changes in G-protein-coupled receptor kinases (GRKs), the key regulators of the ␤-ARs (Feldman and Gros, 2006;Penela et al., 2006).

. Different expression of adrenoceptors and GRKs in the human myocardium depends on heart failure ethiology and correlates to clinical variables. Am J Physiol Heart Circ Physiol 303: H368 -H376, 2012. First published June 8, 2012; doi:10.1152/ajpheart.01061.2011.-Downregulation of ␤ 1-adrenergic receptors (␤ 1-ARs) and increased expression/function of G-protein-coupled receptor kinase 2 (GRK2) have been observed in human heart failure, but changes in expression of other ARs and GRKs have not been established. Another unresolved question is the incidence of these compensatory mechanisms depending on heart failure etiology and treatment. To analyze these questions, we quantified the mRNA/protein expressions of six ARs (␣ 1A, ␣1B, ␣1D, ␤1, ␤ 2, and ␤3) and three GRKs (GRK2, GRK3, and GRK5) in left (LV) and right ventricle (RV) from four donors, 10 patients with ischemic cardiomyopathy (IC), 14 patients with dilated cardiomyopathy (DC), and 10 patients with nonischemic, nondilated cardiopathies (NINDC). We correlated the changes in the expressions of ARs and GRKs with clinical variables such as left ventricular ejection fraction (LVEF) and left ventricular end-systolic and left ventricular end-diastolic diameter (LVESD and LVEDD, respectively). The main findings were 1) the expression of the ␣ 1A-AR in the LV positively correlates with LVEF; 2) the expression of GRK3 and GRK5 inversely correlates with LVESD and LVEDD, supporting previous observations about a protective role for both kinases in failing hearts; and 3) ␤ 1-AR expression is downregulated in the LV and RV of IC, in the LV of DC, and in the RV of NINDC. This difference, better than an increased expression of GRK2 (not observed in IC), determines the lower LVEF in IC and DC vs. NINDC. G-protein-coupled receptor kinasesHUMAN HEART FAILURE (HF) is recognized as a major public health problem arising from multiple causes that will affect one in five adults, conferring elevated mortality rates (20). Regardless of the cause, multiple organ systems attempt to compensate for the deteriorating heart and the sympathetic nervous system responds to HF with increased activity, resulting in increased levels of catecholamines and in an enhanced stimulation of adrenergic receptors (ARs; 33). Consequently, catecholamines powerfully stimulate the heart function at the expense of overproportional increases in energy consumption (13). This is a nonrigid signaling system that adapts to continuous stimulation by reducing the abundance of ␤ 1 -ARs and increasing the expression and enzymatic activity of G-proteincoupled receptor kinases (GRKs), which phosphorylate agonist-occupied receptors and facilitate their endocytosis and desensitization (26). As a result of these adaptations, cardiomyocytes in failing hearts (FH) lose their responsiveness to catecholamines over time (13).In spite of this simple scenario, many fundamental questions remain unanswered. The heart expresses different ␤-ARs (␤ 1 , ␤ 2 , and ␤ 3 ) and ␣ 1 -ARs (␣ 1A , ␣ 1B , and ␣ 1D

beta1-AR expression levels were higher in circulating lymphocytes from hypertensive patients (2-DeltaDeltaCt = 2.135 +/- 0.4252*, vs. control group), but this difference was not observed when these patients were treated with blockers of the renin-angiotensin system. beta1-AR levels directly correlated (r2 = 0.5711, P = 0.0185) with urinary albumin excretion in microalbuminuric patients, which relates alterations of this receptor to cardiovascular risk. An inverse correlation was observed between the expression levels of beta2-AR and diastolic blood pressure (r2 = 0.2078, P = 0.0031), suggesting that beta2-AR levels in lymphocytes mirror their expression in vascular cells, in which beta2-AR-mediated relaxation regulates vascular resistance. mRNA levels for GRK3 were inversely correlated with systolic and diastolic blood pressure (day, night and 24 h), which suggests a protective role for GRK3 in the regulation of human blood pressure, as supported by previous findings in transgenic mice.

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization.

No abstract