Prostaglandin E2-activated Epac Promotes Neointimal Formation of the Rat Ductus Arteriosus by a Process Distinct from That of cAMP-dependent Protein Kinase A

Abstract

We have demonstrated that chronic stimulation of the prostaglandin E2-cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronan. We hypothesized that Epac, a newly identified effector of cAMP, may play a role in intimal cushion formation (ICF) in the DA distinct from that of PKA. In the present study, we found that the levels of Epac1 and Epac2 mRNAs were significantly up-regulated in the rat DA during the perinatal period. A specific EP4 agonist, ONO-AE1-329, increased Rap1 activity in the presence of a PKA inhibitor, PKI-(14-22)-amide, in DA smooth muscle cells. 8-pCPT-2′-O-Me-cAMP (O-Me-cAMP), a cAMP analog selective to Epac activator, promoted migration of DA smooth muscle cells (SMC) in a dose-dependent manner. Adenovirus-mediated Epac1 or Epac2 gene transfer further enhanced O-Me-cAMP-induced cell migration, although the effect of Epac1 overexpression on cell migration was stronger than that of Epac2. In addition, transfection of small interfering RNAs for Epac1, but not Epac2, significantly inhibited serum-mediated migration of DA SMCs. In the presence of O-Me-cAMP, actin stress fibers were well organized with enhanced focal adhesion, and cell shape was widely expanded. Adenovirus-mediated Epac1, but not Epac2 gene transfer, induced prominent ICF in the rat DA explants when compared with those with green fluorescent protein gene transfer. The thickness of intimal cushion became significantly greater (1.98-fold) in Epac1-overexpressed DA. O-Me-cAMP did not change hyaluronan production, although it decreased proliferation of DA SMCs. The present study demonstrated that Epac, especially Epac1, plays an important role in promoting SMC migration and thereby ICF in the rat DA. We have demonstrated that chronic stimulation of the prostaglandin E2-cAMP-dependent protein kinase A (PKA) signal pathway plays a critical role in intimal cushion formation in perinatal ductus arteriosus (DA) through promoting synthesis of hyaluronan. We hypothesized that Epac, a newly identified effector of cAMP, may play a role in intimal cushion formation (ICF) in the DA distinct from that of PKA. In the present study, we found that the levels of Epac1 and Epac2 mRNAs were significantly up-regulated in the rat DA during the perinatal period. A specific EP4 agonist, ONO-AE1-329, increased Rap1 activity in the presence of a PKA inhibitor, PKI-(14-22)-amide, in DA smooth muscle cells. 8-pCPT-2′-O-Me-cAMP (O-Me-cAMP), a cAMP analog selective to Epac activator, promoted migration of DA smooth muscle cells (SMC) in a dose-dependent manner. Adenovirus-mediated Epac1 or Epac2 gene transfer further enhanced O-Me-cAMP-induced cell migration, although the effect of Epac1 overexpression on cell migration was stronger than that of Epac2. In addition, transfection of small interfering RNAs for Epac1, but not Epac2, significantly inhibited serum-mediated migration of DA SMCs. In the presence of O-Me-cAMP, actin stress fibers were well organized with enhanced focal adhesion, and cell shape was widely expanded. Adenovirus-mediated Epac1, but not Epac2 gene transfer, induced prominent ICF in the rat DA explants when compared with those with green fluorescent protein gene transfer. The thickness of intimal cushion became significantly greater (1.98-fold) in Epac1-overexpressed DA. O-Me-cAMP did not change hyaluronan production, although it decreased proliferation of DA SMCs. The present study demonstrated that Epac, especially Epac1, plays an important role in promoting SMC migration and thereby ICF in the rat DA. Prostaglandin E2 (PGE2) 3The abbreviations used are: PGE2, prostaglandin E2; PKA, cAMP-dependent protein kinase A; DA, ductus arteriosus; PKI-(14-22)-amide, PKA inhibitor-(14-22)-amide; O-Me-cAMP, 8-pCPT-2′-O-Me-cAMP; pCTP-cAMP, 4-chloro-phenylthio)-adenosine 3′:5′-cyclic monophosphate cAMP; ICF, intimal cushion formation; HA, hyaluronic acid; SMC, smooth muscle cell; RT-PCR, reverse transcription-PCR; PBS, phosphate-buffered saline; GFP, green fluorescent protein; siRNA, small interfering RNA. is the most potent vasodilatory lipid mediator in the ductus arteriosus (DA), a fetal arterial connection between the pulmonary artery and the descending aorta (1Smith G.C. Pharmacol. Rev. 1998; 50: 35-58PubMed Google Scholar). PGE2 increases the intracellular concentration of cAMP, which activates cAMP-dependent protein kinase A (PKA), resulting in vasodilation in the DA (1Smith G.C. Pharmacol. Rev. 1998; 50: 35-58PubMed Google Scholar, 2Waleh N. Kajino H. Marrache A.M. Ginzinger D. Roman C. Seidner S.R. Moss T.J. Fouron J.C. Vazquez-Tello A. Chemtob S. Clyman R.I. Circulation. 2004; 110: 2326-2332Crossref PubMed Scopus (54) Google Scholar). In addition to its vasodilatory effect, our recent study has identified that chronic PGE2 stimulation has another essential effect on DA development, namely intimal cushion formation (ICF) (3Yokoyama U. Minamisawa S. Quan H. Ghatak S. Akaike T. Segi-Nishida E. Iwasaki S. Iwamoto M. Misra S. Tamura K. Hori H. Yokota S. Toole B.P. Sugimoto Y. Ishikawa Y. J. Clin. Investig. 2006; 116: 3026-3034Crossref PubMed Scopus (123) Google Scholar). Briefly, via EP4, a predominant PGE2 receptor in the DA, the PGE2-cAMP-PKA stimulation up-regulates hyaluronic acid (HA) synthases, which increases HA production. Accumulation of HA then promotes smooth muscle cell (SMC) migration into the subendothelial layer to form intimal thickening. ICF then leads to luminal narrowing, helping adhesive occlusion of the vascular lumen and thus complete anatomical closure of the DA. A new target of cAMP, i.e. an exchange protein activated by cAMP, has recently been discovered; it is called Epac (4Bos J.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 733-738Crossref PubMed Scopus (414) Google Scholar). Epac has been known to exhibit a distinct cAMP signaling pathway that is independent of PKA activation (4Bos J.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 733-738Crossref PubMed Scopus (414) Google Scholar). Epac is a guanine nucleotide exchange protein that regulates the activity of small G proteins. There are two variants; Epac1 was found to be expressed in most tissues including the heart and blood vessels, whereas Epac2 is expressed in the adrenal gland and the brain (4Bos J.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 733-738Crossref PubMed Scopus (414) Google Scholar). Various roles have been proposed for Epac, such as cell proliferation or transformation, but the role of the Epac signal in cardiovascular pathophysiology remains poorly understood. Since PGE2 accumulates intracellular cAMP in the DA during late gestation, it is reasonable to assume that PGE2 activates not only PKA but also Epac pathways in the DA. Therefore, we hypothesized that Epac played a role distinct from that of PKA in vascular remodeling in the DA, especially in the ICF process. Animals and Tissue—Timed-pregnant Wistar rats were purchased from Japan SLC, Inc. (Shizuoka, Japan). The DA was obtained from rat fetuses on the 19th day of gestation (preterm) and on the 21st day of gestation (term), as well as from neonates within 6 h after delivery. On the 19th day of gestation, the DA was still immature; it became mature on the 21st day of gestation. All animals were cared for in compliance with the guiding principles of the American Physiological Society. The experiments were approved by the Ethical Committee of Animal Experiments at the Yokohama City University School of Medicine. Quantitative Reverse Transcription (RT)-PCR—Isolation of total RNA, generation of cDNA, and quantitative RT-PCR analysis were performed as described previously (3Yokoyama U. Minamisawa S. Quan H. Ghatak S. Akaike T. Segi-Nishida E. Iwasaki S. Iwamoto M. Misra S. Tamura K. Hori H. Yokota S. Toole B.P. Sugimoto Y. Ishikawa Y. J. Clin. Investig. 2006; 116: 3026-3034Crossref PubMed Scopus (123) Google Scholar, 5Yokoyama U. Minamisawa S. Adachi-Akahane S. Akaike T. Naguro I. Funakoshi K. Iwamoto M. Nakagome M. Uemura N. Hori H. Yokota S. Ishikawa Y. Am. J. Physiol. 2006; 290: H1660-1670Crossref PubMed Scopus (102) Google Scholar). Rat Epac1 primers (Rn00572463_m1) and TaqMan rodent GAPDH primers for PCR amplification were purchased from Applied Biosystems Inc. (Foster City, CA). For detection of human Epac1, the primers were used as described previously (6Ulucan C. Wang X. Baljinnyam E. Bai Y.Z. Okumura S. Sato M. Minamisawa S. Hirotani S. Ishikawa Y. Am. J. Physiol. 2007; 293: H1662-H1672Crossref PubMed Scopus (78) Google Scholar). The sequences of rat Epac2 primers were 5′-atc aga tga tgc acg gat ga-3′ (forward) and 5′-aaa ctg ctg caa aag cac ct-3 (reverse) (Invitrogen). We confirmed that the sequence of the PCR product amplified by the rat Epac2 primers was only from Epac2 cDNA. We confirmed that both Epac1 and Epac2 primers were similarly efficient to amplify each PCR product. The abundance of each gene was determined relative to GAPDH. Primary Culture of Rat DA SMCs—Vascular SMCs in primary culture were obtained from the DA as described previously (3Yokoyama U. Minamisawa S. Quan H. Ghatak S. Akaike T. Segi-Nishida E. Iwasaki S. Iwamoto M. Misra S. Tamura K. Hori H. Yokota S. Toole B.P. Sugimoto Y. Ishikawa Y. J. Clin. Investig. 2006; 116: 3026-3034Crossref PubMed Scopus (123) Google Scholar, 5Yokoyama U. Minamisawa S. Adachi-Akahane S. Akaike T. Naguro I. Funakoshi K. Iwamoto M. Nakagome M. Uemura N. Hori H. Yokota S. Ishikawa Y. Am. J. Physiol. 2006; 290: H1660-1670Crossref PubMed Scopus (102) Google Scholar). The confluent cells between passages 4 and 6 were used in the experiments. Immunohistochemistry—Paraffin-embedded blocks containing DA tissues were prepared as described previously (5Yokoyama U. Minamisawa S. Adachi-Akahane S. Akaike T. Naguro I. Funakoshi K. Iwamoto M. Nakagome M. Uemura N. Hori H. Yokota S. Ishikawa Y. Am. J. Physiol. 2006; 290: H1660-1670Crossref PubMed Scopus (102) Google Scholar); tissue staining and immunohistochemistry were performed as described previously (3Yokoyama U. Minamisawa S. Quan H. Ghatak S. Akaike T. Segi-Nishida E. Iwasaki S. Iwamoto M. Misra S. Tamura K. Hori H. Yokota S. Toole B.P. Sugimoto Y. Ishikawa Y. J. Clin. Investig. 2006; 116: 3026-3034Crossref PubMed Scopus (123) Google Scholar, 5Yokoyama U. Minamisawa S. Adachi-Akahane S. Akaike T. Naguro I. Funakoshi K. Iwamoto M. Nakagome M. Uemura N. Hori H. Yokota S. Ishikawa Y. Am. J. Physiol. 2006; 290: H1660-1670Crossref PubMed Scopus (102) Google Scholar). Antibodies of Epac1 and Epac2 were purchased from Upstate Biotechnology and Santa Cruz Biotechnology. There was little cross-reaction between Epac1 and Epac2 antibodies. Anti-αSMA antibody was obtained from Sigma-Aldrich. Anti-Ki67 and anti-von Willebrand factor antibodies were from Dako. DA SMCs cultured on 12-mm glass coverslips were serum-starved for 24 h and then stimulated for 1 h in medium alone (control) or with ONO-AE1-329 (an EP4-specific agonist, 10-8-10-6 m), O-Me-cAMP (an Epac-specific agonist, 30 μm), or pCPT-cAMP (a PKA-specific agonist, 500 μm). ONO-AE1-329 was kindly provided by Ono Pharmaceutical Inc. O-Me-cAMP and pCPT-cAMP were purchased from Sigma-Aldrich. Cells were then fixed in 10% buffered formalin for 10 min, washed twice with PBS, and permeabilized in 0.3% Triton X-100 in PBS for 10 min. DA SMCs were washed twice with (0.1% Tween) in PBS and incubated with 1% bovine serum albumin, 0.1% Tween 20 in PBS for 20 min and then with an rhodamine-conjugated phalloidin antibody (Invitrogen) and paxillin antibody (BD Biosciences) for 16 h at 4 °C. Some specimens with a primary antibody were prepared without incubation as negative controls. An additional 60-min incubation was carried out with a secondary antibody, Alexa Fluor 488 goat anti-mouse IgG (Invitrogen). Nuclear staining was achieved with 4′,6-diamidino-2-phenylindole (Invitrogen). After two washes with 0.1% Tween 20 in PBS, coverslips were mounted for microscopic imaging. All incubation and wash steps were conducted at room temperature. Morphometric analyses were performed using Nikon TE2000-E (Tokyo, Japan). Rap1 Activation Assay—DA SMCs were grown to 70% confluency, serum-starved for 24 h, and then stimulated for 15 min in medium alone (control) or with ONO-AE1-329 (10-6 m), O-Me-cAMP (30 μm), pCPT-cAMP (500 μm), or a PKA inhibitor, PKI-(14-22)-amide (10-5 m). PKI-(14-22)-amide was purchased from Calbiochem. Rap1 activity was measured using EZ-Detect RAP1 activation kit (Pierce) according to the manufacturer's protocol (6Ulucan C. Wang X. Baljinnyam E. Bai Y.Z. Okumura S. Sato M. Minamisawa S. Hirotani S. Ishikawa Y. Am. J. Physiol. 2007; 293: H1662-H1672Crossref PubMed Scopus (78) Google Scholar). SMC Migration Assay—The migration assay was performed using 24-well Transwell culture inserts with polycarbonate membranes (8-μm pores: Corning) as described previously (3Yokoyama U. Minamisawa S. Quan H. Ghatak S. Akaike T. Segi-Nishida E. Iwasaki S. Iwamoto M. Misra S. Tamura K. Hori H. Yokota S. Toole B.P. Sugimoto Y. Ishikawa Y. J. Clin. Investig. 2006; 116: 3026-3034Crossref PubMed Scopus (123) Google Scholar) with fibronectin coating. Adenovirus Construction—For construction of adenoviral vectors, full-length cDNA-encoding human Epac1 or Epac2 was cloned into an adenoviral vector by using an AdenoX adenovirus construction kit (Clontech). Both of the cDNAs were kindly provided by Dr. J. L. Bos at University Medical Center, Utrecht, The Netherlands. Adenovirus-mediated transduction was performed as described elsewhere (7Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. PubMed Scopus Google Scholar). a study, adenovirus vector green fluorescent protein was used at the of small interfering RNAs to the of Epac1 or Epac2 were purchased from Inc. or The sequences Epac1 were and acg The sequences Epac2 were and acg negative was used as a DA SMCs were and with using the transfection (Invitrogen) according to the of the Cells were for an additional 24 h the experiments. of of HA in the cell culture was measured by a described previously (3Yokoyama U. Minamisawa S. Quan H. Ghatak S. Akaike T. Segi-Nishida E. Iwasaki S. Iwamoto M. Misra S. Tamura K. Hori H. Yokota S. Toole B.P. Sugimoto Y. Ishikawa Y. J. Clin. Investig. 2006; 116: 3026-3034Crossref PubMed Scopus (123) Google Scholar). SMC Assay—The proliferation assay was performed using a of as described previously (5Yokoyama U. Minamisawa S. Adachi-Akahane S. Akaike T. Naguro I. Funakoshi K. Iwamoto M. Nakagome M. Uemura N. Hori H. Yokota S. Ishikawa Y. Am. J. Physiol. 2006; 290: H1660-1670Crossref PubMed Scopus (102) Google Scholar). Briefly, the DA SMCs were into a 24-well culture at an of for 24 h serum with medium containing fetal Cells were then incubated with or without O-Me-cAMP for 24 h in the 1 activity for 4 h at °C. obtained from were culture was performed as described previously (3Yokoyama U. Minamisawa S. Quan H. Ghatak S. Akaike T. Segi-Nishida E. Iwasaki S. Iwamoto M. Misra S. Tamura K. Hori H. Yokota S. Toole B.P. Sugimoto Y. Ishikawa Y. J. Clin. Investig. 2006; 116: 3026-3034Crossref PubMed Scopus (123) Google Scholar). Briefly, fetal on the 19th day of gestation, including the DA, were for h with of the Epac1, Epac2, or adenovirus in fetal serum containing medium in and at °C. After the were cultured for to fixed in 10% buffered and in Morphometric analyses were performed using Japan). cushion formation was as are the of independent experiments. analysis was performed between two by or with and between by analysis of by A of was The of Epac1 and Epac2 in the Rat RT-PCR analysis that the levels of Epac1 and Epac2 mRNAs increased significantly with and on the day of A and The relative levels of Epac2 were two than those of Epac1 in a perinatal period. analysis that the of Epac1 and Epac2 in the rat DA were were expressed in the smooth muscle layer of the DA on the 19th day of gestation. The of both Epac1 and Epac2 became especially in the and subendothelial in the mature DA on the 21st day of gestation. After the DA lumen was with cells and SMCs Epac1 and Epac2 were in cells of the of the DA EP4 with PKA Rap1 activates not only but also the pathway in DA SMCs by Rap1 activity O-Me-cAMP, a cAMP analog selective to Epac M. Bos J.L. J. Biol. Chem. 2003; PubMed Scopus Google increased Rap1 whereas a cAMP analog selective to PKA, did not We found that the Rap1 activity was by that PKA the effect of Epac on Rap1 A selective EP4 agonist, ONO-AE1-329 (10-6 m), did not Rap1 activity we ONO-AE1-329 did not the pathway in DA SMCs or the effect was by activation of PKA. In the presence of a PKA inhibitor, PKI-(14-22)-amide, ONO-AE1-329 increased Rap1 activity to a to O-Me-cAMP that EP4 activated not only but also pathway in DA SMCs. Epac DA SMC DA SMCs were to O-Me-cAMP for 4 h, O-Me-cAMP promoted DA SMC migration in a dose-dependent On the stimulation of pCPT-cAMP significantly inhibited DA SMC migration which is with on vascular SMCs C. E. A. D. L. A.M. I. M. Nat. PubMed Scopus Google Scholar, J. X. PubMed Scopus Google Scholar). DA SMCs were to both O-Me-cAMP and pCPT-cAMP (500 for 4 h, DA SMC migration was significantly that cell migration was by PKA Epac1 or Epac2 was by gene transfer using the of adenoviral vectors, the effect of O-Me-cAMP on DA SMC migration was further enhanced when compared with overexpression the effect of Epac1 overexpression on cell migration was stronger that that of Epac2 selective Epac is we prepared two for Epac1 or Epac2 to a in the of Epac1 or Epac2 SMC We found that transfection of both for Epac1 or Epac2 significantly decreased the of Epac1 or Epac2 A and be that the for Epac1 did not change the of Epac2 and An for Epac1 significantly decreased serum-mediated SMC migration whereas that for Epac2 did and in DA remodeling plays an important role in cell 2007; PubMed Scopus Google it is important to the effect of Epac on and focal We found that in after Epac activation were from those after PKA activation O-Me-cAMP induced a change in within min. stress fibers were well and cell shape was widely cells without On the pCPT-cAMP induced of actin stress fibers and was significantly increased with Epac activation and significantly decreased with PKA activation The of paxillin was in the of the cells. In the presence of O-Me-cAMP, paxillin was widely not only in the of the cells but also on actin as with a recent of paxillin on actin stress fibers in an cell S. 2007; PubMed Scopus Google Scholar). In the presence of paxillin staining was the cells were stimulated by O-Me-cAMP and the cell was although the staining of actin and paxillin in cells was from cells and or cells. in the presence of ONO-AE1-329, DA SMCs two distinct of which were from the of the cells stimulated by O-Me-cAMP and PGE2 stimulation a with ONO-AE1-329 not In of DA SMC in 6 as a the was to that of and in the in 6 as an the of DA SMCs cells or cells. In cells the of to cells was and at the and of ONO-AE1-329, was greater in cells than in cells. A in cell was in cells in with an in a concentration of ONO-AE1-329, but in was change in The of paxillin was widely the cells in with an in a concentration of Epac and HA in DA recent study that chronic PGE2-cAMP-PKA stimulation increased HA production, resulting in the of DA SMC migration and proliferation and thus ICF (3Yokoyama U. Minamisawa S. Quan H. Ghatak S. Akaike T. Segi-Nishida E. Iwasaki S. Iwamoto M. Misra S. Tamura K. Hori H. Yokota S. Toole B.P. Sugimoto Y. Ishikawa Y. J. Clin. Investig. 2006; 116: 3026-3034Crossref PubMed Scopus (123) Google Scholar). is important to Epac regulates proliferation and HA in DA SMCs. We found that O-Me-cAMP inhibited proliferation of rat DA SMCs We also found that O-Me-cAMP did not change HA in rat DA SMCs that migration was independent of SMC proliferation and HA production. Epac1, but Epac2, ICF in the Rat we Epac1 and Epac2 promoted ICF in the rat DA. Adenovirus-mediated Epac1 gene transfer induced prominent ICF in the rat DA when compared with those with gene transfer Epac2 gene transfer did ICF, as determined by the between and became significantly greater in the Epac1-overexpressed DA, by which that Epac1 plays an important role in promoting thickening. with the in the both Epac1 and Epac2 overexpression did not the of cells and of HA in DA explants and quantitative RT-PCR analysis confirmed that the Epac gene transfer increased the of Epac in DA The present study demonstrated that Epac promoted SMC migration and thus ICF in the rat DA. Since chronic stimulation promotes ICF in the rat DA (3Yokoyama U. Minamisawa S. Quan H. Ghatak S. Akaike T. Segi-Nishida E. Iwasaki S. Iwamoto M. Misra S. Tamura K. Hori H. Yokota S. Toole B.P. Sugimoto Y. Ishikawa Y. J. Clin. Investig. 2006; 116: 3026-3034Crossref PubMed Scopus (123) Google both Epac and PKA, induced ICF in the DA. are PKA promotes ICF through of HA that migration and proliferation of DA SMCs. Therefore, chronic stimulation of PKA is to HA (3Yokoyama U. Minamisawa S. Quan H. Ghatak S. Akaike T. Segi-Nishida E. Iwasaki S. Iwamoto M. Misra S. Tamura K. Hori H. Yokota S. Toole B.P. Sugimoto Y. Ishikawa Y. J. Clin. Investig. 2006; 116: 3026-3034Crossref PubMed Scopus (123) Google Scholar). the present study demonstrated that stimulation of PKA inhibited DA SMC migration, which is with the on vascular SMCs C. E. A. D. L. A.M. I. M. Nat. PubMed Scopus Google Scholar, J. X. PubMed Scopus Google Scholar). Epac, on the promoted SMC migration within 4 SMC migration was inhibited by PKA an effect of Epac and PKA has been in of cells J. J.L. X. J. Biol. Chem. PubMed Scopus Google Scholar, U. N. U. S. A. PubMed Scopus Google Scholar). Since Epac did not HA in DA SMC migration was independent of Epac which is known to cell to fibronectin S. J. Bos J.L. J. Biol. 2003; PubMed Scopus Google Scholar). we found that Epac activated Rap1 and focal in DA SMCs. Since fibronectin plays a critical role in promoting migration of DA SMCs M. PubMed Scopus Google our that a in cell to fibronectin is at in for SMC the between Epac1 and Epac2 have not been understood. The present study demonstrated that Epac1 promoted SMC migration and thus ICF stronger than Epac2, although the relative levels of Epac2 were two than those of Epac1 during a perinatal when ICF Epac2 overexpression significantly increased SMC migration, it did not ICF in the rat DA We used overexpression for SMC migration and overexpression for culture as it has been demonstrated that increased and increased PGE2 in cell J. L. J. S. 2003; PubMed Scopus Google Scholar). Therefore, may assume that PGE2 promoted ICF in the DA which may in between and Epac2 overexpression in the present using the of adenoviral vectors, Epac1 overexpression promoted ICF stronger than Epac2 or study demonstrated that Epac1 and Epac2 mRNAs were up-regulated in whereas only Epac1 was increased in (6Ulucan C. Wang X. Baljinnyam E. Bai Y.Z. Okumura S. Sato M. Minamisawa S. Hirotani S. Ishikawa Y. Am. J. Physiol. 2007; 293: H1662-H1672Crossref PubMed Scopus (78) Google Scholar). In addition, we have recently found that Epac1, but not Epac2, is up-regulated in the of vascular S. T. M. and Y. Since the of ICF in the DA the of ICF by vascular or in J. PubMed Scopus Google Epac1 may play a role in promoting important in study is that DA SMCs two distinct of when stimulated with an EP4 agonist, ONO-AE1-329 or cells were cells in that of actin stress fibers and Since the of cells cells or it was to cell from be that Epac activation is to a concentration of an of a G receptor that is than the concentration of PKA (4Bos J.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 733-738Crossref PubMed Scopus (414) Google Scholar). Therefore, we that the of cells be increased in with increases in the concentration of ONO-AE1-329 in the rat DA SMCs. the cell and cell of cells were increased in the presence of a concentration of ONO-AE1-329 (10-6 in the rat DA SMCs. when cells were stimulated by both PKA and Epac the cell of cells was with and the of the cells was cells. did not or cells. stimulation of PKA and Epac was from stimulation of that when cells are activated by EP4 PKA or Epac, may be activated in each The by which cells to stimulation remains study is to of cells In the present study demonstrated that Epac, which is up-regulated during the perinatal an promoting effect on SMC migration and thus on ICF in the DA. The signal activates the cAMP both Epac and PKA, which ICF in the DA in two distinct namely and chronic further be to the of ICF in the DA, our that selective activation of Epac may as an for with DA. with