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Experiments were performed to determine whether remodeling of the actin cytoskeleton contributes to arteriolar constriction. Mouse tail arterioles were mounted on cannulae in a myograph and superfused with buffer solution. The [[alpha].sub.1]-adrenergic agonist phenylephrine (0.1-1 [micro]mol/l) caused constriction that was unaffected by cytocbalasin D (300 nmol/l) or latrunculin A (100 nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure ([P.sub.TM]; 10--60 or 90 mmHg). Arterioles were fixed, permeabilized, and stained with Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and G-actin, respectively, using a Zeiss 510 laser scanning microscope. Elevation in [P.sub.TM], but not phenylephrine (1 [micro]mol/l), significantly increased the intensity of F-actin and significantly decreased the intensity of G-actin staining in arteriolar vascular smooth muscle cells (VSMCs). The increase in F-actin staining caused by an elevation in [P.sub.TM] was inhibited by cytochalasin D. In VSMCs at 10 mmHg, prominent F-actin staining was restricted to the cell periphery, whereas after elevation in [P.sub.TM], transcytoplasmic F-actin fibers were localized through the cell interior, running parallel to the long axis of the cells. Phenylephrine (1 [micro]mol/l) did not alter the architecture of the actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of endothelial or adventitial cells was not altered by an elevation in [P.sub.TM]. Therefore, the actin cytoskeleton of VSMCs undergoes dramatic alteration after elevation in [P.sub.TM] of arterioles and plays a selective and essential role in mechanosensitive myogenic constriction. myogenic response; laser scanning microscopy; cytochalasin D; latrunculin A; phenylephrine

The physiological role of [[alpha].sub.2C]-adrenoceptors ([[alpha].sub.2C]-ARs) in cutaneous, arteriolar, vascular smooth muscle cells (VSMs) is to mediate cold-induced constriction. In VSMs cultured from human cutaneous arterioles, there is a selective increase in [[alpha].sub.2C]-AR expression after serum stimulation. In the present study, we examined the cellular mechanisms contributing to this response. Serum induction of [[alpha].sub.2C]-ARs was paralleled by increased expression of cyclooxygenase-2 (COX-2), increased release of prostaglandins, and increased intracellular concentration of cAMP. Inhibition of COX-2 by acetyl salicylic acid (1 mM), NS-398 (5 [micro]M), or celecoxib (3 [micro]M) abolished the increase in cAMP and markedly reduced [[alpha].sub.2C]-AR induction in response to serum stimulation. The cAMP agonists, forskolin (10 [micro]M), isoproterenol (10 [micro]M), and cholera toxin (0.1 [micro]g/ml) each dramatically increased expression of [[alpha].sub.2C]-ARs in human cutaneous VSMs. The A-kinase inhibitor H-89 (2 [micro]M) inhibited phosphorylation of cAMP response element binding protein, but not the increase in [[alpha].sub.2C]-AR expression in response to these agonists. cAMP-dependent but A-kinase independent signaling can involve activation of guanine nucleotide exchange factors for the GTP-binding protein, Rap. Indeed, pull-down assays demonstrated Rap1 activation by serum and forskolin in VSM. Transient transfections using [[alpha].sub.2C]-AR promoter-luciferase reporter construct demonstrated that Rap1 increased reporter activity, whereas the A-kinase catalytic subunit decreased reporter activity. These results indicate that cAMP signaling can have dual effects in cutaneous VSMs:activation of [[alpha].sub.2C]-AR transcription mediated by Rap1 GTPase and suppression mediated by A-kinase. The former effect predominates in serum-stimulated VSMs leading to a COX-2. cAMP, and Rap 1-dependent increase in [[alpha].sub.2C]-AR expression. Such increased expression of [[alpha].sub.2C]-ARs may contribute to enhanced cold-induced vasoconstriction and Raynaud's phenomenon. microcirculation; cyclooxygenase-2; Rap GTPase; A kinase

This study analyzed the regulation of [[alpha].sub.2]-adrenoceptors ([[alpha].sub.2]-ARs) in human vascular smooth muscle cells (VSMs). Saphenous veins and dermal arterioles or VSMs cultured from them expressed high levels of [[alpha].sub.2]-ARs ([[alpha].sub.2C] > [[alpha].sub.2A], via RNase protection assay) and responded to [[alpha].sub.2]-AR stimulation [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304, 1 [micro]M)] with constriction or calcium mobilization. In contrast, VSMs cultured from aorta did not express [[alpha].sub.2]-ARs and neither cultured cells nor intact aorta responded to UK-14,304. Although [[alpha].sub.2]-ARs ([[alpha].sub.2C] [much greater than] [[alpha].sub.2A]) were detected in aortas, [[alpha].sub.2C]-ARs were localized by immunohistochemistry to VSMs of adventitial arterioles and not aortic media. In contrast with aortas, aortic arterioles constricted in response to [[alpha].sub.2]-AR stimulation. Reporter constructs demonstrated higher activities for [[alpha].sub.2A]-and [[alpha].sub.2C]-AR gene promoters in arteriolar compared with aortic VSMs. In arteriolar VSMs, serum increased expression of [[alpha].sub.2C]-AR mRNA and protein but decreased expression of [[alpha].sub.2A]-ARs. Serum induction of [[alpha].sub.2C]-ARs was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) with 2 [micro]M SB-202190 or dominant-negative p38 MAPK. UK-14,304 (1 [micro]M) caused calcium mobilization in control and serum-stimulated cells: in control VSMs, the response was inhibited by the [[alpha].sub.2]A-AR antagonist BRL-44408 (100 [micro]M) but not by the [[alpha].sub.2C]-AR antagonist MK-912 (1 nM), whereas after serum stimulation, MK-912 (1 nM) but not BRL-44408 (100 nM) inhibited the response. These results demonstrate site-specific expression of [[alpha].sub.2]-ARs in human VSMs that reflects differential activity of [[alpha].sub.2]-AR gene promoters; namely, high expression and function in venous and arteriolar VSMs but no detectable expression or function in aortic VSMs. We found that [[alpha].sub.2C]-ARs can be dramatically and selectively induced via a p38 MAPK-dependent pathway. Therefore, altered expression of [[alpha].sub.2C]-ARs may contribute to pathological changes in vascular function. microcirculation; MK-912; BRL-44408; p38 mitogen-activated protein kinase

Chotani, Maqsood A., Sheila Flavahan, Srabani Mitra, David Daunt, and Nicholas A. Flavahan. Silent [[Alpha].sub.2C]-adrenergic receptors enable cold-induced vasoconstriction in cutaneous arteries. Am J Physiol Heart Circ Physiol 278: H1075-H1083, 2000.--Cold constricts cutaneous blood vessels by increasing the reactivity of smooth muscle [[Alpha].sub.2]-adrenergic receptors ([[Alpha].sub.2]-ARs). Experiments were performed to determine the role of [[Alpha].sub.2]-AR subtypes ([[Alpha].sub.2A]-, [[Alpha].sub.2B]-, [[Alpha].sub.2C]-ARs) in this response. Stimulation of [[Alpha].sub.1]-ARs by phenylephrine or [[Alpha].sub.2]-ARs by UK-14,304 caused constriction of isolated mouse tail arteries mounted in a pressurized myograph system. Compared with proximal arteries, distal arteries were more responsive to [[Alpha].sub.2]-AR activation but less responsive to activation of [[Alpha].sub.1]-ARs. Cold augmented constriction to [[Alpha].sub.2]-AR activation in distal arteries but did not affect the response to [[Alpha].sub.1]-AR stimulation or the level of myogenic tone. Western blot analysis demonstrated expression of ([[Alpha].sub.2A]- and [[Alpha].sub.2C]-ARs in tail arteries: expression of [[Alpha].sub.2C]-ARs decreased in distal compared with proximal arteries, whereas expression of the glycosylated form of the [[Alpha].sub.2A]-AR increased in distal arteries. At 37 [degrees] C, [[Alpha].sub.2]-AR-induced vasoconstriction in distal arteries was inhibited by selective blockade of [[Alpha].sub.2A]-ARs (BRL-44408) but not by selective inhibition of [[Alpha].sub.2B]-ARs (ARC-239) or [[Alpha].sub.2C]-ARs (MK-912). In contrast, during cold exposure (28 [degrees] C), the augmented response to UK-14,304 was inhibited by the [[Alpha].sub.2C]-AR antagonist MK-912, which selectively abolished cold-induced amplification of the response. These experiments indicate that cold-induced amplification of [[Alpha].sub.2]-ARs is mediated by [[Alpha].sub.2C]-ARs that are normally silent in these cutaneous arteries. Blockade of [[Alpha].sub.2C]-ARs may prove an effective treatment for Raynaud's Phenomenon. Raynaud's Phenomenon; scleroderma; microcirculation; thermoregulation; vascular smooth muscle

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser2113. Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function. [ABSTRACT FROM AUTHOR]

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 (micro-VSM), Rap1 selectively regulates expression of G protein-coupled α2C-adrenoceptors (α2C-ARs) through JNK-c-jun nuclear signaling. The α2C-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 α2C-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 α2C-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The α2C-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 α2C-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. [ABSTRACT FROM AUTHOR]

The physiological role of α2-adrenoceptors (α2-ARs) in cutaneous, arteriolar, vascular smooth muscle cells (VSMs) is to mediate cold-induced constriction. In VSMs cultured from human cutaneous arterioles, there is a selective increase in α2C-AR expression after serum stimulation. In the present study, we examined the cellular mechanisms contributing to this response Serum induction of α2C-ARs was paralleled by increased expression of cyclooxygenase-2 (COX-2), increased release of prostaglandins, and increased intracellular concentration of cAMP. Inhibition of COX-2 by acetyl salicylic acid (1 mM). NS-398 (5 μM), or celecoxib (3 μM) abolished the increase in cAMP and markedly reduced α2C-AR induction in response to serum stimulation. The cAMP agonists, forskolin (10 μM), isoproterenol (10 μM), and cholera toxin (0.1 μg/ml) each dramatically increased expression of α2C-ARs in human cutaneous VSMs. The A-kinase inhibitor H-89 (2 μM) inhibited phosphorylation of cAMP response element binding protein, but not the increase in α2C-AR expression in response to these agonists, cAMP dependent but A-kinase independent signaling can involve activation of guanine nucleotide exchange factors liar the GTP-binding protein, Rap. Indeed, pull-down assays demonstrated Rapl activation by serum and forskolin in VSM. Transient transfections using α2C-AR promoter-luciferase reporter construct demonstrated that Rap1 increased reporter activity, whereas the A-kinase catalytic subunit decreased reporter activity These resells indicate that cAMP signaling can have dual effects in cutaneous VSMs:activation of α2C-AR transcription mediated by Rapl GTPase and suppression mediated by A kinase. The former effect predominates in serumstimulated VSMs leading to a COX-2, cAMP, and Rap 1-dependent increase in α2C-AR expression. Such increased expression of α2C-Rs may contribute to enhanced cold-induced vasoconstriction and Raynaud's phenomenon. [ABSTRACT FROM AUTHOR]

This study analyzed the regulation of α[sub 2]-adrenoceptors (α[sub 2]-ARs) in human vascular smooth muscle cells (VSMs). Saphenous veins and dermal arterioles or VSMs cultured from them expressed high levels of α[sub 2]-ARs (α[sub 2C] > α[sub 2A], via RNase protection assay) and responded to α[sub 2]-AR stimulation [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304, 1 µM)] with constriction or calcium mobilization. In contrast, VSMs cultured from aorta did not express α[sub 2]-ARs and neither cultured cells nor intact aorta responded to UK-14,304. Although α[sub 2]-ARs (α[sub 2C] » α[sub 2A]) were detected in aortas, α[sub 2C]-ARs were localized by immunohistochemistry to VSMs of adventitial arterioles and not aortic media. In contrast with aortas, aortic arterioles constricted in response to α[sub 2]-AR stimulation. Reporter constructs demonstrated higher activities for α[sub 2A]and α[sub 2C]-AR gene promoters in arteriolar compared with aortic VSMs. In arteriolar VSMs, serum increased expression of α[sub 2C]-AR mRNA and protein but decreased expression of α[sub 2A]-ARs. Serum induction of α[sub 2C]ARs was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) with 2 µM SB-202190 or dominant-negative p38 MAPK. UK-14,304 (l µM) caused calcium mobilization in control and serum-stimulated cells: in control VSMs, the response was inhibited by the α[sub 2A]-AR antagonist BRL-44408 (100 nM) but not by the α[sub 2C]-AR antagonist MK-912 (1 nM), whereas after serum stimulation, MK-912 (1 nM) but not BRL-44408 (100 nM) inhibited the response. These results demonstrate site-specific expression of α[sub 2]-ARs in human VSMs that reflects differential activity of α[sub 2]-AR gene promoters; namely, high expression and function in venous and arteriolar VSMs but no detectable expression or function in aortic VSMs. We found that α[sub 2C]-ARs can be dramatically and selectively induced via a p38 MAPK-dependent pathway. Therefore, altered expression of α[sub 2C]-ARs may contribute to pathological changes in vascular function. [ABSTRACT FROM AUTHOR]

α2C-adrenoceptors (α2C-ARs) are sequestered in the transGolgi whereas α2A-ARs are expressed on the plasma membrane (PM) of microvascular smooth muscle (VSM). Experiments were performed to identify mechanisms regulating cell-surface delivery of α2C-ARs, which occurs on cooling or serum stimulation. In human cultured VSMs, Rap1 stimulation by forskolin (10µM) or 8-pCPT-2′-O-Me-cAMP (100 µM) activated RhoA (4.1±1.2 fold, P<0.01, n=3), increased F-actin, and caused translocation of perinuclear α2C-ARs to intracellular filamentous structures and PM. A yeast 2-hybrid screen with VSM cDNA library and the α2C-AR C-terminus identified a novel interaction with the actin cross-linker, filamin-2. In Rap1-stimulated VSMs, α2C-ARs co-localized with filamin or PO4Ser2113-filamin on intracellular filaments and at the PM. In HEK293 cells transfected with α2C-ARs, constitutively-active (CA) mutants of Rap1 or ROCK caused translocation of α2C-ARs to the cell surface (6 ± 1 fold increase, P<0.05, n=3) and increased PO4Ser2113-filamin. The effects of Rap1CA were reduced by the ROCK inhibitor fasudil, siRNA mediated knockdown of filamin2 inhibited Rap1-induced redistribution of α2C-ARs. Rap1CA did not affect α2A-ARs. Co-immunoprecipitation experiments demonstrated that α2C-ARs, but not α2A-ARs, interacted with filamin. Unlike α2A-ARs, α2C-AR cell surface delivery is mediated by Rap1-ROCK-filamin signaling. [ABSTRACT FROM AUTHOR]

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser(2113). Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.

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.

The aim of this study was to determine whether prolonged loss of NO activity, in endothelial NO synthase knockout (eNOS(-/-)) mice, influences endothelin (ET) ETA receptor-mediated smooth muscle contraction and, if so, to define the underlying mechanism(s). In isolated endothelium-denuded abdominal aortas, contractions to the selective ETA receptor agonist ET-1(1-31) were significantly increased in aortas from eNOS(-/-) compared with wild-type (WT) mice. In contrast, contractions to the alpha1-adrenergic agonist phenylephrine or the thromboxane (TX) A2 analog U-46619 were similar between eNOS(-/-) and WT mice. Immunofluorescent and Western blot analysis demonstrated that the aortic expression of ETA receptors was decreased in eNOS(-/-) compared with WT mice. Contractions evoked by ET-1(1-31), but not phenylephrine, were reduced by inhibition of cyclooxygenase-2 (COX-2) (indomethacin or celecoxib) or of TXA2/prostaglandin H2 receptors (SQ-29548). After COX inhibition, contractions to ET-1(1-31) were no longer increased and were actually decreased in eNOS(-/-) compared with WT aortas. Western blot analysis revealed that endothelium-denuded abdominal aortas express COX-2, but not COX-1, and that expression of COX-2 was significantly increased in eNOS(-/-) compared with WT mice. Contractions to the COX substrate arachidonic acid were also increased in eNOS(-/-) aortas. Furthermore, ET-1(1-31) but not phenylephrine stimulated production of the TXA2 metabolite TXB2, which was increased in eNOS(-/-) compared with WT aortas. Therefore, COX-2 plays a crucial and selective role in ETA-mediated smooth muscle contraction. Furthermore, COX-2 expression is increased in eNOS(-/-) mice, which overcomes a reduced expression of ETA receptors and enables a selective increase in contraction to ETA receptor stimulation.