Influence of cell membrane integrity on the activity of α 1- adrenoceptor subtypes

E Carceller, M Pérez-Aso, V Segura, MA Noguera, MP D\'Ocon. Universitat de València, Pharmacology, 46100, Spain

Background: The three subtypes of α1-adrenoceptors (ARs), α1A, α1B and α1D, have different pharmacological properties and signaling mechanisms that modulate their trafficking (1). Recent studies have shown that α1-ARs can themselves initiate ERK1/2 (extracellular regulated kinase) cascade by both G-protein and ß-arrestin-dependent process, with a subtype different regulation of ERK1/2 activation over time (2). Among the three subtypes, the α1D exhibit a peculiar behavior. When expressed in cells and tissues, it is mainly intracellularly located and shows constitutive activity through p-ERK1/2 signaling and contractile activity in vessels.

Aims: To analyze the influence of cell membrane integrity and caveolae on the activation of the α1A- and the α1D-AR subtype and their functional consequences in regulating vascular tone.

Material and Methods: HEK293 cell lines stably transfected with each α1-AR subtype were stimulated with phenylephrine (PHE, 100μM) in the presence or absence of the cholesterol-depleting agent methyl-β-cyclodextrin (mβcd, 10mM), which alters membrane integrity. ERK1/2 activation was quantified by Western blot. Functional studies (isometric recordings) were performed in isolated aorta and tail artery from male Wistar rats (250-300g) where the α1D- and the α1A-subtype, respectively, are the main responsible of the adrenergic contraction. Contractile response to PHE (1μM in aorta and 10μM in tail artery) was analyzed after a preincubation time (30/60 min) with mβcd 10mM. Electron micrographs (80000x) corroborate the membrane disruption in both vessels.

Statistical analysis: Students´ t test.

Results: PHE induced an early (1-5 min) and transient ERK1/2 phosphorylation in α1A- and α1B-AR transfected cells which is PKC-mediated (2) and was inhibited by mβcd, indicating its dependence on cellular membrane integrity. The presence of mβcd did not modify the later (10-15min) sustained ERK1/2 activation only observed in α1A-transfected cells. Interestingly, stimulation of α1D-AR by PHE activates ERK1/2 in a PKC independent manner (2) and preincubation with mβcd causes a slower and sustained pERK1/2 signal, indicating that destabilization of the cell membrane delays, but does not inhibit ERK1/2 phosphorylation. Functional results obtained in rat aorta, shows that destabilization of membrane by mβcd significantly increases the magnitude of the contractile response to PHE (8.25±0.81mN, n=4 vs. 5.09±0.61mN, n=5, p<0.05) and modifies the profile and kinetic of the contraction, since it developed more slowly (25.69±0.89min, n=4) vs. control (12.42±0.20min, n=5; p<0.001). By the contrary, pretreatment with mβcd completely inhibits PHE-induced contractile response in tail artery, where the α1A subtype exhibits the main functional role.

Conclusions: Adrenergic responses mediated by α1A-ARs were dependent on the membrane integrity, corroborating the classical activating pathway through G-proteins located in the cell membrane. α1D-ARs, exhibit a peculiar mechanism of activation, which did not require the membrane integrity, suggesting an internal location and a different activating pathway not exclusively mediated by G-protein activation at the membrane, but related to ERK1/2 phosphorylation.

References:

(1) Chalothorn, D. et al. (2002) Differences in the Cellular Localization and Agonist-Mediated Internalization Properties of the Alpha(1)-Adrenoceptor Subtypes. Mol Pharmacol 61: pp 1008-1016.

(2) Perez-Aso, M. et al. The three α1-drenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation. J. Mol. Pharmacol. (in review)

Acknowledgements: Study supported by a grant from Universitat de València.