Cyclic AMP-Rap1A signaling activates RhoA to induce α(2c)-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells.
ABSTRACT Intracellular signaling by the second messenger cyclic AMP (cAMP) activates the Ras-related small GTPase Rap1 through the guanine exchange factor Epac. This activation leads to effector protein interactions, activation, and biological responses in the vasculature, including vasorelaxation. In vascular smooth muscle cells derived from human dermal arterioles (microVSM), Rap1 selectively regulates expression of G protein-coupled α(2C)-adrenoceptors (α(2C)-ARs) through JNK-c-jun nuclear signaling. The α(2C)-ARs are generally retained in the trans-Golgi compartment and mobilize to the cell surface and elicit vasoconstriction in response to cellular stress. The present study used human microVSM to examine the role of Rap1 in receptor localization. Complementary approaches included murine microVSM derived from tail arteries of C57BL6 mice that express functional α(2C)-ARs and mice deficient in Rap1A (Rap1A-null). In human microVSM, increasing intracellular cAMP by direct activation of adenylyl cyclase by forskolin (10 μM) or selectively activating Epac-Rap signaling by the cAMP analog 8-pCPT-2'-O-Me-cAMP (100 μM) activated RhoA, increased α(2C)-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The α(2C)-ARs mobilized from the perinuclear region to intracellular filamentous structures and to the plasma membrane. Similar results were obtained in murine wild-type microVSM, coupling Rap1-Rho-actin dynamics to receptor relocalization. This signaling was impaired in Rap1A-null murine microVSM and was rescued by delivery of constitutively active (CA) mutant of Rap1A. When tested in heterologous HEK293 cells, Rap1A-CA or Rho-kinase (ROCK-CA) caused translocation of functional α(2C)-ARs to the cell surface (~4- to 6-fold increase, respectively). Together, these studies support vascular bed-specific physiological role of Rap1 and suggest a role in vasoconstriction in microVSM.
- AJP Cell Physiology 07/2012; 303(5):C488-9. DOI:10.1152/ajpcell.00238.2012 · 3.67 Impact Factor
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ABSTRACT: Reactive oxygen species contribute to regulating the excitability of vascular smooth muscle. This study investigated the actions of the relatively stable reactive oxygen species, H(2)O(2), on nerve-evoked contractions of mouse distal tail artery. H(2)O(2) (10-100µM) increased nerve-evoked contractions of isometrically mounted segments of tail artery. Endothelium denudation increased nerve-evoked contractions and abolished the facilitatory effect of H(2)O(2). Inhibition of nitric oxide synthase with L-nitroarginine methyl ester (0.1mM) also increased nerve-evoked contractions and reduced the late phase of H(2)O(2)-induced facilitation. H(2)O(2)-induced facilitation of nerve-evoked contractions depended, in part, on synthesis of prostanoids and was reduced by the cyclooxygenase inhibitor indomethacin (1µM) and the thromboxane A(2) receptor antagonist SQ 29,548 (1µM). H(2)O(2) increased sensitivity of nerve-evoked contractions to the α(2)-adrenoceptor antagonist idazoxan (0.1µM) but not to the α(1)-adrenoceptor antagonist prazosin (10nM). Idazoxan and the α(2C)-adrenoceptor antagonist JP 1302 (0.5-1µM) reduced H(2)O(2)-induced facilitation. H(2)O(2) induced facilitation of nerve-evoked contractions was abolished by the non-selective cation channel blocker SKF-96365 (10µM), suggesting it depends on Ca(2+) influx. In conclusion, H(2)O(2)-induced increases in nerve-evoked contractions depended on an intact endothelium and were mediated by activating thromboxane A(2) receptors and by increasing the contribution of α(2)-adrenoceptors to these responses.European journal of pharmacology 11/2012; 698(1-3). DOI:10.1016/j.ejphar.2012.11.002 · 2.68 Impact Factor
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ABSTRACT: Since the discovery nearly 60 years ago, cAMP is envisioned as one of the most universal and versatile second messengers. The tremendous feature of cAMP to tightly control highly diverse physiologic processes, including calcium homeostasis, metabolism, secretion, muscle contraction, cell fate, and gene transcription, is reflected by the award of five Nobel prizes. The discovery of Epac (exchange protein directly activated by cAMP) has ignited a new surge of cAMP-related research and has depicted novel cAMP properties independent of protein kinase A and cyclic nucleotide-gated channels. The multidomain architecture of Epac determines its activity state and allows cell-type specific protein-protein and protein-lipid interactions that control fine-tuning of pivotal biologic responses through the "old" second messenger cAMP. Compartmentalization of cAMP in space and time, maintained by A-kinase anchoring proteins, phosphodiesterases, and β-arrestins, contributes to the Epac signalosome of small GTPases, phospholipases, mitogen- and lipid-activated kinases, and transcription factors. These novel cAMP sensors seem to implement certain unexpected signaling properties of cAMP and thereby to permit delicate adaptations of biologic responses. Agonists and antagonists selective for Epac are developed and will support further studies on the biologic net outcome of the activation of Epac. This will increase our current knowledge on the pathophysiology of devastating diseases, such as diabetes, cognitive impairment, renal and heart failure, (pulmonary) hypertension, asthma, and chronic obstructive pulmonary disease. Further insights into the cAMP dynamics executed by the Epac signalosome will help to optimize the pharmacological treatment of these diseases.Pharmacological reviews 02/2013; 65(2):670-709. DOI:10.1124/pr.110.003707 · 18.55 Impact Factor