Esophageal Smooth Muscle Cells Research Articles

Acid-induced COX-2 expression and prostaglandin E2 production via activation of ERK1/2 and p38 MAPK in cultured feline esophageal smooth muscle cells

Cyclooxygenase (COX)-2 is known to play an important role in inflammatory conditions such as reflux esophagitis resulting from acid reflux. In this study, we tested whether an acidic medium (pH 4.0) induces an increase in COX-2 expression or PGE(2) production, and explored the implication of mitogen-activated protein kinases (MAPKs) activation in these responses in cultured cat esophageal smooth muscle cells. Acidic cytotoxicity was assessed and expression changes in COXs or phosphorylated MAPKs were analyzed by Western blotting. PGE(2) production was measured by immunoassay. No significant decrease in cell viability was observed for 6 h exposure to acidic medium. COX-2 expression and PGE(2) production significantly increased to maximal levels at 6 h exposure to acidic medium. The cells also exhibited significant activation of ERK1/2 and p38 MAPK, but not JNK within 10 min under acidic medium. The increments of COX-2 expression and PGE(2) production by acidic medium were decreased by pretreatment with PD98059 or SB202190, respectively. These results suggest that acidic environments may enhance the COX-2 expression and PGE(2) production through activation of ERK1/2 and p38 MAPK in the cultured cat esophageal smooth muscle cells.

The mechanism of MAP kinase activation under acidic condition in feline esophageal smooth muscle cells

Reflux esophagitis results from repeated exposure of the esophagus to acidic gastric juice or bile-containing duodenal contents. In Barrett's adenocarcinoma, acid increases proliferation via ERK and p38 MAPK activation. This study was focused on determination of the mechanism(s) underlying MAPKs (ERK 1/2, p38 MAPK, and JNK) activation induced by acidic medium at pH 4 in normal feline primary cultured esophageal smooth muscle cells (FESMCs). We detected ERK 1/2 and p38 MAPK phosphorylation after exposure to pH 4 or neutral media in the presence or absence of several inhibitors and quantified the MAPK levels using western blotting analysis and densitometry. Acidic medium markedly increased the phosphorylation of ERK 1/2 and p38 MAPK within 10 min. Acid-induced ERK 1/2 and p38 MAPK activation was inhibited by pertussis toxin (PTX-sensitive G(i/o) protein inhibitor), DEDA (phospholipase (PL) A(2) inhibitor), ρCMB (PLD inhibitor), GF109203X (protein kinase C (PKC) inhibitor) and D609 (phosphatidylcholine-specific PLC inhibitor). But, genistein (tyrosine kinase inhibitor), forskolin (adenylate cyclase activator) and U73122 (phosphatidylinositol-specific PLC inhibitor) had no effect on acid-induced ERK1/2 and p38 MAPK activation. These findings indicate that the activation of ERK 1/2 and p38 MAPK pathways by acidic conditions, at least in part, may be mediated by activation of the G(i/o) protein coupled receptors, PC-PLC, PLD, PLA(2), and PKC in FESMCs.

Esophageal smooth muscle cells dedifferentiate with loss of α-smooth muscle actin expression after 8 weeks of explant expansion in vitro culture: Implications on esophagus tissue engineering

BACKGROUND: Esophagus tissue engineering using the hybrid-construct approach involves assembly of the esophageal components to engineer the organ. This study investigated the dedifferentiation and loss in expression of esophageal smooth muscle after explant expansion in culture. METHODS: Ovine esophagus smooth muscle cells (OESMC) were sourced from adult biopsies and expanded using the explant culture technique. The explants were maintained under static in vitro tissue culture. Flow cytometry analysis was performed for α-smooth muscle actin (α-SMA) expression at intervals of 4 weeks for up to 8 weeks. RESULTS: After 4 weeks 58.5% OESMC exhibited α-SMA which decreased to 28.5% after 6 weeks in culture. After 8 weeks in culture a mere 1.3% OESMC demonstrated α-SMA expression. CONCLUSIONS: OESMC proliferated using the explant technique exhibit a total loss of α-SMA expression after 8 weeks of static in vitro culture. This information is crucial for tissue engineering of the esophagus using the hybrid-construct approach.

Identification of GIRK2-4 subunits in human esophageal smooth muscle cells

Acetylcholine (ACh) secreted from the vagus nerve contributes to the physiological and pathological regulation of the contraction and relaxation of human esophageal smooth muscle. Expression of acetylcholine-sensitive G protein‑activated inwardly rectifying potassium channels (GIRKs) occurs widely in the heart, nervous system and gastrointestine, but the role of GIRKs in the esophagus remains unclear. In the present study, expression of the GIRK1-4 subunits in mRNA and total protein was examined in human esophageal smooth muscle cells (SMCs) by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. mRNA and protein expression of the GIRK2-4 subunits was detected in human esophageal longitudinal muscle (LM) and circular muscle (CM) cells. However, GIRK1 mRNA and protein were not observed in either the esophageal LM or CM. This study is the first to identify the expression of GIRK2-4 subunits in human esophageal SMCs.

The expression of nestin delineates skeletal muscle differentiation in the developing rat esophagus

The muscularis externa of the developing rodent esophagus is initially composed of smooth muscle, and later replaced by skeletal muscle in a craniocaudal progression. There is growing evidence of distinct developmental origins for esophageal smooth and skeletal muscles. However, the identification of skeletal muscle progenitor cells is controversial, and the detailed cell lineage of their descendants remains elusive. In the current study, we carried out multiple labeling immunofluorescence microscopy of nestin and muscle type-specific markers to characterize the dynamic process of rat esophageal myogenesis. The results showed that nestin was transiently expressed in immature esophageal smooth muscle cells in early developing stages. After nestin was downregulated in smooth muscle cells, a distinct population of nestin-positive cells emerged as skeletal muscle precursors. They were mitotically active, and subsequently co-expressed MyoD, followed by the embryonic and later the fast type of skeletal muscle myosin heavy chain. Thus, the cell lineage of esophageal skeletal muscle differentiation was established by an immunotyping approach, which revealed that skeletal myocytes arise from a distinct lineage rather than through transdifferentiation of smooth muscle cells during rat esophageal myogenesis.

Clonal overgrowth of esophageal smooth muscle cells in diffuse leiomyomatosis-Alport syndrome caused by partial deletion in COL4A5 and COL4A6 genes

This is a study of a patient who manifests all of the features of a diffuse leiomyomatosis-Alport syndrome (DL-ATS), and her two-year-old son who has already been diagnosed with Alport syndrome. Fourteen years ago, the patient underwent a partial esophageal resection followed by a replacement with jejunum. Recently, she underwent a surgical resection of the esophagus due to esophageal dysfunction. Genetic analyses of COL4A5 and COL4A6 on the X-chromosome were efficiently performed using the genomic DNA of her son. We have identified a novel deletion of 194-kb in length, encompassing COL4A5–COL4A6 promoters as well as nearly the entire large intron 1 of COL4A5 and intron 2 of COL4A6. To uncover the relationship of the esophagus-specific occurrence of the tumor and the expression of those genes, immunohistochemical analyses of type IV collagen α chains were conducted in the non-affected individuals. The esophageal smooth muscle-specific expression of α5(IV) and α6(IV) chains in the gastrointestinal tract was observed. Moreover, CAG repeat analysis of the androgen receptor gene and an immunohistochemical analysis in the leiomyoma revealed clonal overgrowth of the cells which received X-inactivation on the non-affected allele. These results may suggest that the dominant effect was caused by the partial deletion of the esophageal smooth muscle-specific genes, COL4A5 and COL4A6.

Dependence of alignment direction on magnitude of strain in esophageal smooth muscle cells

The response of cells in vitro to mechanical forces has been the subject of much research using devices to exert controlled mechanical stimulation on cultured cells or isolated tissue. In this study, esophageal smooth muscle cells were seeded on flexible polyurethane membranes to form a confluent cell layer. The cells were then subjected to uniform cyclic stretch of varying magnitudes at a frequency of approximately five cycles per minute in a custom made mechatronic bioreactor, providing similar strains experienced in the in vivo mechanical environment of the esophagus. The results show that the orientation response is dependent on the magnitude of cyclic stretch applied. Smooth muscle cells showed parallel alignment to the force direction at low cyclic strains (2%) compared to the hill-valley morphology of static controls. At higher strains (5% and 10% magnitude), the cells exhibited a consistent alignment perpendicular to the strain. To our knowledge, this is the first time that the alignment direction's dependence on strain magnitude has been demonstrated. MTS analysis indicated that cell metabolism was reduced when mechanical strain was applied, and proliferation was inhibited by mechanical strain. Protein expression indicates a decrease in smooth muscle alpha-actin, indicative of changes in cell phenotype, an increase in vimentin, which is associated with increased cell motility, and an increase in desmin, indicating differentiation in stimulated cells.

Signaling mechanisms of sphingosine 1-phosphate-induced ERK1/2 activation in cultured feline esophageal smooth muscle cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid, stored and released from activated platelets, macrophages, and other mammalian cells. We previously reported that S1P induces esophageal smooth muscle contraction in freshly isolated intact cells. Here, we measured S1P-induced ERK1/2 activation and upstream signaling in cultured feline esophageal smooth muscle cells. Activation of ERK1/2 by S1P peaked at 5 min, was sustained up to 30 min, and was blocked by PTX. In contrast, S1P did not activate p38 MAPK or JNK. PTX inhibited S1P-induced ERK1/2 activation. We then used phospholipase inhibitors, DEDA for PLA(2), U73122 for PLC, and rhoCMB for PLD, to determine that ERK1/2 activation was downstream of PLC activation. The PKC inhibitors, GF109203X and chelerythrine, also suppressed ERK1/2 activation. Whereas the PTK inhibitor, genistein, partially inhibited ERK1/2 activation, the EGFR tyrosine kinase inhibitor, tyrphostin 51, had no effect. Taken together, S1P-induced ERK1/2 activation in cultured ESMCs requires a PTX-sensitive G protein, stimulation of the PLC pathway, and subsequent activation of the PKC and PTK pathways.

Relaxation Effect of Synthetic Ceramide Analogues in Cat Esophageal Smooth Muscle Cells

Ceramide has emerged as a novel second messenger for intracellular signalling. It is produced from sphingomyelin and is involved in the control of cell differntiation, proliferation, and apoptosis. C(2)-ceramide, short chain ceramide, plays a role in mediating contraction of cat esophageal smooth muscle cells. We examined the effect of synthesized ceramide analogues on the C(2)-ceramide and ACh-induced contraction in esophageal smooth muscle cells isolated with collagenase. CY3523, CY3525, or CY3723 inhibited C(2)-ceramide induced contraction, in a time dependent manne. Each analogue also inhibited the contraction in concentration dependent manners. CY 3523, CY 3525, and CY 3723 had no effect to the contraction induced by PMA. The inhibition with CY3523, CY3525 and CY3723 on the C(2)-ceramide induced contraction was recovered by PMA. These analogues decreased the density of MAPK bands (p44/42 or p38) in the western blot. These results suggest that ceramide analogues can inhibit C(2)-ceramide induced contraction via PKC and MAPK dependent pathway.

The signaling mechanism of the sphingosylphosphorylcholine-induced contraction in cat esophageal smooth muscle cells

We investigated the signaling pathway on sphingosinephosphorylcholine (SPC) -induced contraction in cat esophageal smooth muscle cells. SPC induced in a dose-dependent manner contractile effect. We have previously shown that lysophospholipid (LPL) receptor subtypes including the S1P1, S1P2, S1P3, and S1P5 receptor are present in esophageal smooth muscle. Only EDG-5 (S1P2) receptor antibody penetration into permeablilized cells inhibited the SPC-induced contraction. Pertussis toxin (PTX) and specific antibodies to G(i1), G(i2), G(i3) and G(o) inhibited the contraction, implying that SPC-induced contraction depends on PTX-sensitive G(i1), G(i2), G(i3), and G(o) protein. A phospholipase inhibitor U73122 and incubation of permeabilized cells with PLC-beta3 antibody inhibited SPC-induced contraction. The PKC-mediated contraction may be isozyme specific since only PKCepsilon antibody inhibited the contraction. Preincubation with MEK inhibitor PD98059 blocked the SPC-induced contraction, but p38 MAPK inhibitor SB202190 did not. Cotreatment with GF109203X and PD98059 did not show synergistic effects, suggesting that these two kinases are involved in the same signaling pathway in the SPC-induced contraction. The data suggest that S1P-induced contraction in feline esophageal smooth muscle cells depends on activation of the G(i1), G(i2), G(i3) and G(o) proteins and the PLCbeta3 isozyme via the S1P2 receptor, leading to stimulation of a PKCE pathway, which subsequently activates a p44/p42 MAPK pathway.

Reactive Oxygen Species Mediate ET-1-Induced Activation of ERK1/2 Signaling in Cultured Feline Esophageal Smooth Muscle Cells

Reactive oxygen species (ROS) have been shown to play a critical role in propagating the signals of several growth factors, peptide hormones, and cytokines, such as epidermal growth factor, insulin, and interleukin-1. We investigated a possible role for ROS generation in mediating the action of ET-1 on activation of ERK1/2 in cultured feline esophageal smooth muscle cells (ESMC). Confluent layers of ESMC were stimulated by 10nM ET-1; activation of ERK was examined by western blot analysis with phospho-specific antibodies of ERKs. ET-1 induced ERK1/2 phosphorylation in a dose- and time- dependent manner. ERK1/2 activation by ET-1 reached the maximal levels at 5min showing slight activation up to 20min, and then slowly declined. It was confirmed that the activation of ERK1/2 was reduced by MEK inhibitor PD98059. We observed the dose-dependent inhibitory effect of diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on the ET-1-enhanced ERK1/2 phosphorylation in ESMC. Pretreatment of ESMC with N-acetylcysteine, a ROS scavenger, also attenuated the ET-1-induced ERK1/2 activation. In addition, DPI significantly inhibited the ET-1- induced ROS production when ROS was measured as a function of DCF fluorescence. The results suggest that ROS might be critical mediators of the ET-1-induced ERK1/2 signaling events in ESMC.

Cyclic AMP dependent down regulation in the relaxation of smooth muscle cells of cat esophagitis

We investigated whether the signal mechanism for relaxation may be affected by inflammation of the cat esophagus. Acute esophagitis was induced by perfusion with 0.1N HCI at a rate of 1 mL/min for 45 min over three consecutive days. We then isolated esophageal smooth muscle cells by enzymatic digestion with collagenase. We pre-contracted the isolated smooth cells with acetylcholine (ACh) (10(-5) M) and compared the agonist-induced relaxation of pre-con tracted normal cells with those of esophagitic cells. Vasoactive intestinal polypeptide (VIP) caused a dose-dependent relaxation in normal cells, and this curve was down shifted in esophagitic cells. Sodium nitroprusside (SNP) or SIN-1 (NO donor) produced dose-dependent relaxation in normal cells, which was not affected by esophagitis. 8-Br-cGMP (a cGMP ana log) also induced dose-dependent relaxation to a similar extent in both normal and esoph agitic cells. Forskolin (a cAMP activator) or db-cAMP (a cAMP analog) produced dose-dependent relaxation in normal cells, and this relaxation curve was down shifted in esoph agitic cells. Western blotting was used to determine what subtype of adenylyl cyclase was involved in the cAMP pathway. Western blot analysis of homogenates derived from esophageal smooth muscle using antibodies against adenylyl cyclase types II, III, IV and V/VI revealed the presence of type V and/or type VI only. This result suggests that relaxation via a cAMP-dependent pathway rather than a cGMP dependent-pathway is down regulated in cat acute esophagitis. This subsensitivity of the cAMP related pathway may be related to the activ ity of adenylyl cyclase V/VI.

Density quantification of collagen grafted on biodegradable polyester: Its application to esophageal smooth muscle cell

An improved technique for quantification of collagen immobilized on polymeric substrates is needed as tissue engineering evolves. Current immobilized protein quantification methods are indirect, time-consuming, and/or inaccurate. In this study, Sirius red colorimetric microassay was shown to be feasible for quantifying the density of collagen immobilized on aminolyzed poly( l-lactic acid) (PLLA) surfaces using the specific bonding of Sirius dye to collagen. It offers a number of advantages over traditional methods, including direct staining, high sensitivity, and high stability of the dye. The detection limit is approximately 0.1 μg/cm 2, and the dynamic range is greater than 50. Sirius red dye has not been used previously for quantification of protein immobilized on polymers. The collagen densities achieved with each of the two crosslinking reagents investigated, namely glutaraldehyde (GA) and genipin, were compared. The latter is an alternative crosslinker derived from a traditional Chinese medicine. The collagen densities immobilized by the two reagents were measured to be similar. This was confirmed by the similar behaviors of esophageal primary smooth muscle cells (ESMCs) on these two modified PLLA membranes; collagen grafted with either coupler was found to greatly promote, to a similar extent, cell attachment and both short-term (4 days) and long-term (12 days) proliferation compared with unmodified PLLA. Smooth muscle cells on both modified membranes were stained to display contractile α-actin protein filaments.

Activation of p38 MAPK Is Involved in Endothelin-1-stimulated COX-2 Expression in Cultured Feline Esophageal Smooth Muscle Cells

Activation of p38 MAPK Is Involved in Endothelin-1-stimulated COX-2 Expression in Cultured Feline Esophageal Smooth Muscle Cells

IL-1β activates p44/42 and p38 mitogen-activated protein kinases via different pathways in cat esophageal smooth muscle cells

To examine the pathway related to the IL-1beta-induced activation of mitogen-activated protein (MAP) kinases in cat esophageal smooth muscle cells. Culture of the esophageal smooth muscle cells from cat was prepared. Specific inhibitors were treated before applying the IL-1beta. Western blot analysis was performed to detect the expressions of COX, iNOS and MAP kinases. In the primary cultured cells, although IL-1beta failed to upregulate the COX and iNOS levels, the levels of the phosphorylated forms of p44/42 MAP kinase and p38 MAP kinase increased in both concentration- and time-dependent manner, of which the level of activation reached a maximum within 3 and 18 h, respectively. The pertussis toxin reduced the level of p44/42 MAP kinase phosphorylation. Tyrphostin 51 and genistein also inhibited this activation. Neomycin decreased the density of the p44/42 MAP kinase band to the basal level. Phosphokinase C (PKC) was found to play a mediating role in the IL-1beta-induced p44/42 MAP kinase activity. In contrast, the activation of p38 MAP kinase was inhibited only by a pretreatment with forskolin, and was unaffected by the other compounds. Based on these results, IL-1beta-induced p44/42 MAP kinase activation is mediated by the Gi protein, tyrosine kinase, phospholipase C (PLC) and PKC. The pathway for p38 MAP kinase phosphorylation is different from that of p44/42 MAP kinase, suggesting that it plays a different role in the cellular response to IL-1beta.

The growth improvement of porcine esophageal smooth muscle cells on collagen‐grafted poly(DL‐lactide‐co‐glycolide) membrane

Synthetic polyester and the extracellular matrix component collagen are among the most widely used materials in tissue engineering. However, the integration of collagen into polyester scaffolds without loss of its biological function is a problem that has not been fully solved. This article investigates the covalent immobilization of collagen onto poly(DL-Lactide-co-Glycolide) (PLGA) membrane surfaces via a bridge of 1,8-diaminooctane and with glutaraldehyde as crosslinking agent. X-ray photoelectron spectroscopy (XPS) and fluorescence measurements confirmed the presence of bonded collagen. The effect of collagen grafting on cell behavior was investigated by comparing collagen-PLGA with unmodified PLGA sample and tissue culture polystyrene (TCPS) plates by using porcine esophageal smooth muscle cells (ESMC). DNA analysis showed that collagen-modified PLGA improved the overall proliferation of the ESMCs compared with unmodified PLGA and TCPS plates. Cells seeded on collagen-modified PLGA also showed a more extended morphology. Thus, we believe that collagen-modified PLGA shows good potential to be used as a scaffold material for tissue engineering of the esophagus.

Functional and molecular analysis of L-type calcium channels in human esophagus and lower esophageal sphincter smooth muscle

Excitation of human esophageal smooth muscle involves the release of Ca(2+) from intracellular stores and influx. The lower esophageal sphincter (LES) shows the distinctive property of tonic contraction; however, the mechanisms by which this is maintained are incompletely understood. We examined Ca(2+) channels in human esophageal muscle and investigated their contribution to LES tone. Functional effects were examined with tension recordings, currents were recorded with patch-clamp electrophysiology, channel expression was explored by RT-PCR, and intracellular Ca(2+) concentration was monitored by fura-2 fluorescence. LES muscle strips developed tone that was abolished by the removal of extracellular Ca(2+) and reduced by the application of the L-type Ca(2+) channel blocker nifedipine (to 13 +/- 6% of control) but was unaffected by the inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). Carbachol increased tension above basal tone, and this effect was attenuated by treatment with CPA and nifedipine. Voltage-dependent inward currents were studied using patch-clamp techniques and dissociated cells. Similar inward currents were observed in esophageal body (EB) and LES smooth muscle cells. The inward currents in both tissues were blocked by nifedipine, enhanced by Bay K8644, and transiently suppressed by acetylcholine. The molecular form of the Ca(2+) channel was explored using RT-PCR, and similar splice variant combinations of the pore-forming alpha(1C)-subunit were identified in EB and LES. This is the first characterization of Ca(2+) channels in human esophageal smooth muscle, and we establish that L-type Ca(2+) channels play a critical role in maintaining LES tone.

Potassium channel diversity within the muscular components of the feline lower esophageal sphincter

We hypothesized that regional differences in electrophysiological properties exist within the musculature of the feline lower esophageal sphincter (LES) and that they may potentially contribute to functional asymmetry within the LES. Freshly isolated esophageal smooth muscle cells (SMCs) from the circular muscle and sling regions within the LES were studied under a patch clamp. The resting membrane potential (RMP) of the circular SMCs was significantly more depolarized than was the RMP of the sling SMCs, resulting from a higher Na+ and Cl- permeability in circular muscle than in sling muscle. Large conductance Ca2+-activated K+ (BKCa) set the RMP at both levels, since specific BKCa inhibitors caused depolarization; however, BKCa density was greatest in the circular region. A significant portion of the outward current was due to non-BKCa, especially in sling muscle, and likely delayed rectifier K+ channels (KDR). There was a large reduction in outward current with 4-aminopyridine (4-AP) in sling muscle, while BKCa blockers had a limited effect on the voltage-activated outward current in sling muscle. Differences in BKCa:KDR channel ratios were also manifest by a leftward shift in the voltage-dependent activation curve in circular cells compared to sling cells. The electrophysiological differences seen between the circular and sling muscles provide a basis for their different contributions to LES activities such as resting tone and neurotransmitter responsiveness, and in turn could impart asymmetric drug responses and provide specific therapeutic targets.

A neo-esophagus reconstructed by cultured human esophageal epithelial cells, smooth muscle cells, fibroblasts, and collagen.

The scientists involved in this study attempted to develop an artificial esophagus constructed of autologous cells grown by cell culture methods on an extracellular matrix. An artificial esophagus consisting of human esophageal epithelial cells, dermal fibroblasts, and smooth muscle cells isolated from the aortic media, was attempted. The purpose of this study was to examine whether smooth muscle cells could be used in the transforming matrix. Human fibroblasts were embedded in Type I collagen superimposed on the collagen layer of smooth muscle cells. Next, human esophageal epithelial cells were cultured on the collagen layer of the fibroblasts. The resulting collagen sheets were cultured in vitro for 1 week, then transplanted on the latissimus dorsi muscles of athymic rats. The sheets were examined histologically at 1 and 2 weeks using hematoxylin eosin and immunologic stain methods (antiactin antibody). At the end of 2 weeks after transplantation, on microscopic observation of the collagen sheets, it appeared that the epithelial layer, the submucosal tissue layer, and the proper muscle layer had been reconstructed. Additionally, the authors successfully isolated smooth muscle cells from the media of the left gastric artery as a surgical specimen by explant cell culture. The ability to transform collagen sheets consisting of esophageal epithelial cells, fibroblasts, and smooth muscle cells from a surgical specimen into a luminal structure may enable clinical application of the artificial esophagus.

Construction of a recombinant adenovirus carrying endothelial nitric oxide synthase gene and its expression and control in esophageal smooth muscle cells

Nitric oxide (NO) is a major inhibitory neurotransmitter, and its deficiency plays an important role in the pathogenesis of motility disorders of the gastrointestinal tract. The present study was designed to generate a recombinant adenovirus containing the tetracycline (Tet)-regulated endothelial nitric oxide synthase (eNOS) gene and to detect the controllable expression of the gene in esophageal smooth muscle cells (ESMC). The construction of the recombinant adenovirus was completed in three steps: (1) a Tet-responsible expression cassette was made by cloning the full-length cDNA encoding eNOS into a pTRE-Shuttle Vector, which can be regulated by tetracycline or its analogs, such as doxycycline (Dox); (2) the expression cassette was transferred to Adeno-X viral DNA to form a recombinant adenoviral plasmid (pAd-eNOS) by means of an in vitro ligation reaction; and (3) the Ad-eNOS was packaged into infectious adenoviral particles (Adeno-X-TRE-eNOS) by transfecting human embryonic kidney (HEK) 293 cells. Cultured ESMC were coinfected by Adeno-X-TRE-eNOS and regulation virus (Adeno-X Tet-off virus), and the Dox-regulated eNOS expression was detected by RT-PCR and western blot. The recombinant adenovirus (Adeno-X-TRE-eNOS) was generated successfully by an in vitro ligation reaction. The expression of the eNOS gene in the coinfected ESMC was confirmed by RT-PCR and western blot. Furthermore, the transcription could be precisely regulated in a dose-dependent manner in a series of concentrations of Dox, and it was completely turned off when the concentration reached 0.01 microg/mL. A Tet- (or Dox-) regulated recombinant adenovirus carrying eNOS was successfully generated and controllable expression of eNOS in ESMC was achieved, which provides some material for conducting further gene therapy studies with eNOS.