Routine chest CT for staging of gastric cancer.

BACKGROUND: International guidelines on clinical staging of gastric cancer recommend the use of chest CT for the detection of pulmonary metastases. This study assessed the clinical value of routine chest CT in the staging of gastric cancer. METHODS: This retrospective study included patients identified from the gastric cancer registry of Chang Gung Memorial Hospital, Linkou, Taiwan. All patients who underwent clinical staging between 2008 and 2014 were included. The pattern, site and number of metastases at initial presentation and after surgery with curative intent were evaluated. Pulmonary metastases were defined as multiple small round pulmonary nodules with a random distribution or of variable size. RESULTS: Some 1669 patients were included, of whom 478 (28·6 per cent) had metastatic disease at clinical presentation. The majority of metastases were to the peritoneum (75·7 per cent of patients) or liver (30·5 per cent), and only 27 patients (5·6 per cent) had pulmonary metastases at presentation, none of which were isolated to the lung. Of these 27 patients, 11 had primary lesions located at the cardia/fundus. In 19 patients the lung metastases were also detected on the staging chest X-ray. After surgery there were 196 cancer recurrences. Some 15 patients (7·6 per cent) had lung metastasis and this was not the only site of metastases in any patient. The prevalence of lung metastasis at presentation of the disease and after surgery was 1·6 and 1·5 per cent respectively. CONCLUSION: This study does not support the routine use of chest CT for staging of gastric cancer as isolated pulmonary metastasis in the absence of other metastatic sites could not be detected.

Learning curve for endoscopic submucosal dissection of esophageal neoplasms.

There is a significant learning curve for endoscopic submucosal dissection of esophageal neoplasms that has not been fully characterized. This retrospective study included 33 consecutive superficial esophageal neoplasms for analysis of the learning curve for esophageal endoscopic submucosal dissection based on a single, novice endoscopist's experience. The study was divided into three periods (T1, T2, and T3) of 10 endoscopic submucosal dissection procedures in chronological order, with 13 procedures in the last period. Patient factors (age, sex, coexistent esophageal varices, or submucosal fibrosis) and tumor factors (location at upper esophagus, involving >3/4 esophageal circumference) for endoscopic submucosal dissection were not statistically different between the periods. The mean procedure time was 74.6 min/cm(2) , 23.4 min/cm(2) , and 10.5 min/cm(2) for T1, T2, and T3, respectively. The procedure time decreased over time (P = 0.02) and post hoc test revealed significant difference was only between T3 and T1 (P = 0.019). The en bloc resection rate was 50%, 100%, and 92.3% for T1, T2, and T3, respectively (P for trend = 0.015). R0 resection rate was 40%, 100%, and 84.6% for T1, T2, and T3, respectively (P for trend = 0.023). Two patients had complications: each one patient in T1 and T3 period experienced major bleeding during the procedure (P for trend = 0.875). None of the patients had esophageal perforation. The results of the study concluded that at least 30 cases of endoscopic submucosal dissection of esophageal neoplasms are needed for a novice endoscopist to gain early proficiency in this technique.

The clinical characteristics and manifestations of cytomegalovirus esophagitis.

Esophagitis is the second most common gastrointestinal manifestation of cytomegalovirus (CMV) infection after colitis. CMV esophagitis has been reported in patients who have undergone transplantation, are on long-term renal dialysis, or who have the human immunodeficiency virus infection. This study aimed to investigate the clinical characteristics and manifestations of CMV esophagitis in patients who underwent diagnostic endoscopy. A total of 16 patients with histologically proven CMV infection were identified from 1539 patients with esophageal ulcers and analyzed retrospectively (January 2006 to December 2013). Patients' personal data (age, smoking, and alcohol consumption), underlying systemic diseases (diabetes mellitus, end-stage renal disease, and chronic obstructive pulmonary disease), malignancy, indication for esophagogastroduodenoscopy, endoscopic characteristics, and diagnostic methods (pathological or serological findings) were collected for further analysis. Among the patients with CMV esophagitis, the mean age was 59.94 years (range, 23-84 years). The male : female ratio was 1.67:1. Odynophagia and epigastralgia were common symptoms. Of the 16 patients, 3 (18.75%) were infected with the human immunodeficiency virus and 9 (56.25%) had an underlying malignancy, including lung cancer (6 patients), esophageal cancer (2 patients), gastric cancer (1 patient), ampulla of Vater cancer (1 patient), and lymphoma (1 patient). Six of the 9 patients (66.7%) with malignancy had been administered concurrent chemoradiotherapy (CCRT). In this study, patients with malignancy who had been administered CCRT were at increased risk for CMV esophagitis, which had not been reported before in the literature. CMV esophagitis should be considered as a potential treatment-related complication of CCRT.

Sexual genetic and simple sequence repeat (SSR) analysis for molecular marker development on the all hermaphrodite papaya.

The papaya (Carica papaya L.) is one of the most important economic tropical fruits in the world, and the hermaphrodite is the preferred type in field cultures. We analyzed the sexual ratio of offspring from the cultivar 'Taiwan Seed Station No. 7' (T7) by a self-cross and its cross with Taichung Sunrise (TS). Female progeny from the T7 self-crossing were not observed. This finding may be caused by a lethal gene that is linked to females. In this study, we selected 192 simple sequence repeats (SSRs) to analyze the polymorphism between T7 and TS. A total of 37 SSRs were identified for T7 and TS. In addition, 14 SSRs served as the molecular makers for identification of T7, TS and their hybrid offsprings. Thus, the results show that the genetic similarity between T7 and TS is rather high. This suggests that T7 may be a mutant of TS. Phylogenetic analysis from the SSR polymorphisms of the above parent strains and 15 F1 offspring revealed the genetic distance of the F1 offspring located between T7 and TS. The results of this study may provide an opportunity for elucidating the genetic characteristics of all hermaphrodites via identification of molecular makers.

Isolation and characterization of the papaya MADS-box E-class genes, CpMADS1 and CpMADS3, and a TM6 lineage gene CpMADS2.

Papaya (Carica papaya L.) plants are polygamous, with female, male, and hermaphroditic flowers. To understand the roles of MADS-box genes in flower development and sex determination, we cloned cDNAs of E-class genes CpMADS1 and CpMADS3 and a TM6 lineage of the B-class gene CpMADS2 from young flower buds of papaya. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses revealed that CpMADS1 and CpMADS3 were preferentially expressed in the carpel and also in petals and stamens. CpMADS2 was expressed in both petals and stamens early during floral development. Comparison of 10 papaya genotypes of 5 different sex phenotypes - hermaphrodite, male, female, progeny-all-hermaphrodite, and progeny-all-male - by Southern blot analysis of genomic DNAs with probes of the 3 genes revealed similar restriction patterns and copy number, suggesting a low relationship of the 3 CpMADS genes with sex expression of papaya plants at the genomic level.

Endoscopic management of suspected esophageal foreign body in adults.

Foreign bodies should not be allowed to remain in the esophagus beyond 24 hours after presentation. However, some patients with esophageal foreign body ingestion do not come to the hospital immediately and may delay medical intervention from the time of ingestion. The aim of this study was to investigate the outcomes of adults with suspected esophageal foreign body ingestion according to the time of ingestion and types of foreign bodies. A total of 326 adult patients (151 men and 175 women) were analyzed, and divided into two groups according to the time period: within or beyond 24 hours from ingestion to endoscopic intervention. A total of 172 patients (52.7%) were found to have ingested foreign bodies; 73.5% were removed smoothly, 10.3% were treated by push technique and 16.0% with failed retrieval received alternative treatments. A higher proportion of patients in the beyond-24 hours group suffered from odynophagia (25.9 vs. 12.9%, P < 0.05). Negative identification of esophageal foreign bodies was more frequent in the beyond-24 hours group (67 vs. 40.2%, P < 0.05), but these patients showed higher proportions of esophageal ulcers (21.1 vs. 7.2%, P < 0.05). The beyond-24 hours group also showed a significantly higher rate of foreign bodies in the lower esophagus (40.0 vs. 15.3%, P < 0.05). Patients with esophageal food bolus impaction had significant delayed endoscopic intervention, longer therapeutic endoscopic time, higher proportions of esophageal cancer, stricture and fewer complications. Endoscopic intervention within 24 hours from the time of ingestion should be considered early in adults, because delaying intervention may produce more symptomatic esophageal ulcerations with odynophagia.

Gout and risk of non-alcoholic fatty liver disease.

OBJECTIVES: To investigate the association between gout and non-alcoholic fatty liver disease (NAFLD). METHODS: The study subjects were participants in a health-screening programme at Chang Gung Memorial Hospital from 2000 to 2006. Subjects were classified into eight groups based on serum urate (SU) level and gout status (≤ 4.9, 5.0-6.9, 7.0-8.9, and ≥ 9.0 mg/dL, without and with gout). The association between gout and NAFLD was assessed by multiple logistic regression. RESULTS: Among a total of 54 325 subjects, 1930 (3.6%) had gout and 6169 (11.3%) had NAFLD. The prevalence of NAFLD was significantly higher in subjects with gout (23.1%, n = 445) than in those without gout (10.9%, n = 5724, p < 0.001). Among subjects with NAFLD, the severity of NAFLD was higher in gout patients. Gout was associated with an increased risk for NAFLD [odds ratio (OR) 1.42, 95% confidence interval (CI) 1.25-1.60, p < 0.001], after adjustment for age, sex, presence of metabolic syndrome, and low estimated glomerular filtration rate (eGFR). With SU ≤ 4.9 mg/dL in the absence of gout as reference, the ORs (95% CI) for NAFLD, after adjustment for age, sex, presence of metabolic syndrome, and low eGFR, were, respectively, 2.16 (1.94-2.41), 3.98 (3.55-4.46), and 5.99 (5.19-6.90) for SU levels 2-4 in those without gout and 2.61 (1.39-4.91), 2.87 (2.04-4.04), 4.53 (3.70-5.56), and 6.31 (5.12-7.77) for SU levels 1-4 in those with gout. CONCLUSIONS: There was an independent association between gout and the risk for NAFLD. In addition, there was a dose-response relationship between SU and NAFLD in subjects with and without gout.

Clinical manifestation of esophageal carcinosarcoma: a Taiwan experience.

Carcinosarcoma of the esophagus is a rare neoplasm with both carcinomatous and sarcomatous components. This study aimed to investigate its clinicopathologic features and endoscopic characteristics. The data of patients diagnosed to have esophageal carcinosarcoma pathologically in the past 30 years (January 1976-December 2007) were reviewed. Of 3318 cases of esophageal malignancy, 12 were diagnosed as esophageal carcinosarcoma, with an incidence of 0.36%. All of the cases were male with a mean age of 62.3 years. Of the 12 tumors, 8 were polypoid type, and 4 were ulcerative type. In the endoscopic ultrasonography examination, the tumors show heterogeneous hypoechoic lesions with irregular outer margins and internal multicystic components. Four patients (33.3%) had previous head and neck squamous cell carcinoma that occurred metachronously. This is the first report about the characteristics of esophageal carcinosarcoma under endoscopic ultrasonography examination. The relationship between esophageal carcinosarcomas and head and neck cancer needs further investigation.

Sonographic septation: a predictor of sequelae of tuberculous pleurisy after treatment.

BACKGROUND: Findings in the literature have been quite conflicting with respect to predicting residual pleural thickening (RPT) in tuberculous pleurisy (TP). The aim of this study was to determine which sonographic feature of TP might help in predicting the development of RPT. METHODS: Eighty-seven patients with TP were enrolled prospectively. The initial sonographic features were classified as anechoic, homogenously echogenic, complex non-septated and complex septated. The RPT level was measured 12 months after the start of antituberculosis (TB) treatment. Spirometry was performed 6 and 12 months after the start of anti-TB treatment. RESULTS: A higher odds of an RPT level >10 mm was found in patients with positive TB bacillus culture in pleural fluid (OR, 20.9; 95% CI, 2.2 to 198.0) and a complex septated sonographic pattern (OR, 145.0; 95% CI, 22.3 to 942.3). A complex septated sonographic pattern can predict RPT with a sensitivity of 80%, specificity of 96%, positive predictive value of 84% and negative predictive value of 94%. Patients with an RPT level >10 mm had a lower forced vital capacity than those without (75.4% (9.2%) predicted vs 83.2% (9.5%) predicted, p<0.01) CONCLUSION: A complex septated sonographic pattern is a useful sign to predict an RPT level >10 mm 1 year after the start of anti-TB treatment. An RPT level >10 mm is associated with a high probability of decreased lung volumes. Therefore, the initial sonographic feature is beneficial in predicting the sequelae of TP after treatment.

"Snowmelt Sign" and "Corkscrew Microvessels" Predicting Epithelium Regeneration After Acute Rejection of Small-Bowel Transplantation: A Case Report.

Intestinal failure characterized by inadequate maintenance of nutrition via normal intestinal function comprises a group of disorders with many different causes. If parenteral nutrition dependency develops, which is associated with higher mortality and complications, it is considered for intestine transplantation. However, the graft failure rate is not low, and acute cellular rejection is one of the most important reasons for graft failure. As a result, early identification of rejection and timely modification of anti-rejection medications have been considered to be associated with better graft and patient survival rates. The diagnostic gold standard for rejection is mainly based on histology, but hours of delay by pathology may occur. Some researchers investigated the association of endoscopic images with graft rejection to provide timely diagnosis. In this study, we present the first case report with characteristic features under magnifying endoscopy with a narrow-band imaging system to predict epithelial regeneration and improvement of graft rejection in a patient with small-bowel transplantation.

Up-regulation of a serine-threonine kinase proto-oncogene Pim-1 in oral squamous cell carcinoma.

The Pim-1 proto-oncogene, encoding a serine-threonine kinase, has been found to play an important role in regulating apoptosis, differentiation, proliferation and tumourigenesis. The present study was conducted to assess the importance of Pim-1 in oral tumourigenesis in vivo. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used to study the expression of Pim-1 in oral squamous cell carcinoma (OSCC) and non-cancerous match tissue (NCMT) sampled from the periphery of the tumours. Pim-1 mRNA expression in OSCC was significantly higher than that in NCMT in 36 tissue pairs (1.33+/-0.41 versus 0.97+/-0.29, P=0.03). The percentage of OSCCs exhibiting strong cytoplasmic Pim-1 immunoreactivity was significantly higher than that of NCMT (60% versus 19%, P=0.007). Pim-1 immunoreactivity is higher in the more differentiated components of a tumour. In around 10% of OSCC cases, Pim-1 immunoreactivity was found in the nucleus as well. These results show novel findings of the up-regulation of Pim-1 expression from NCMT to OSCC. The pathogenetic role of Pim-1 expression in oral tumourigenesis deserves further investigation.

Regulation of 5-hydroxytryptamine-induced signal transduction in canine cultured aorta smooth muscle cells by phorbol ester.

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT-induced IPs formation and Ca2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca2+ mobilization in ASMCs.

Forskolin inhibits 5-hydroxytryptamine-induced phosphoinositide hydrolysis and Ca+2 Mobilisation in canine cultured aorta smooth muscle cells.

The effect of forskolin on 5-hydroxytryptamine (5-HT)-induced inositol phosphate (IP) and Ca2+ mobilisation was investigated in canine cultured aorta smooth muscle cells (ASMCs). Pretreatment of ASMCs with forskolin attenuated 5-HT-induced IP accumulation and Ca2+ mobilisation in a time- and concentration-dependent manner. The half-maximal effects (pEC50) of forskolin to attenuate IP and Ca2+ responses to 5-HT occurred at concentrations of 6.28 and 6.64, respectively. Pretreatment of ASMCs with cholera toxin caused a similar inhibition on 5-HT-induced responses. Even after treatment with forskolin for 24 h, the 5-HT-induced responses were still inhibited. The inhibitory effect of forskolin resulted from both a depression of the maximal response and a shift to the right of the concentration-effect curves of 5-HT in these responses. The water-soluble forskolin analogue L-858051 [7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated IP accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on IP response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-iosquinolinesulphonamide] and HA-1004 [N-(2-guanidinoethyl)-5-iosquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IP in ASMCs. These results indicate that activation of cAMP/PKA might inhibit the 5-HT-stimulated IP accumulation and consequently reduce Ca2+ mobilisation, or inhibit both responses independently.

Uncoupling of ATP-induced inositol phosphate formation and Ca(2+) mobilization by phorbol ester in canine cultured tracheal epithelial cells.

The regulation of the increase in inositol phosphates (IPs) production and intracellular Ca(2+) concentration ([Ca(2+)](i)) by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated the ATP- and UTP-induced IPs formation and Ca(2+) mobilization. The concentrations of PMA that gave half-maximal (EC(50)) inhibition of ATP- and UTP-induced IPs accumulation and an increase in [Ca(2+)](i) were 5-10 and 4-12 nM, respectively. Prior treatment of TECs with staurosporine (1 microM), a PKC inhibitor, partially inhibited the ability of PMA to attenuate ATP- and UTP-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Furthermore, analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -theta, and -zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, and -theta from the cytosol to the membrane was seen after 5- and 30-min and 2- and 4-h treatment. However, 6-h treatment caused a partial down-regulation of these PKC isozymes. PKC-zeta was not significantly translocated and down-regulated at any of the times tested. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide (PI) hydrolysis and consequently attenuate the [Ca(2+)](i) increase or inhibit independently both responses to ATP and UTP. The translocation of PKC-alpha, -betaI, -betaII, -delta, -epsilon, -gamma, and -theta induced by PMA caused an attenuation of ATP- and UTP-induced IPs accumulation and Ca(2+) mobilization in TECs.

Effect of forskolin on bradykinin-induced calcium mobilization in cultured canine tracheal smooth muscle cells.

The effects of increases in intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) on bradykinin (BK)-induced generation of inositol phosphates (IPs) and Ca2+ mobilization were investigated in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with either forskolin or dibutyryl cyclic AMP attenuated BK-stimulated responses. The inhibitory effects of these agents produced both a depression of the maximal response and a shift to the right of the concentration-response curves of BK. The water-soluble forskolin analogue L-858051, 7-deacetyl-7 beta-(r-N-methylpiperazino)-butyryl forskolin, significantly attenuated BK-stimulated IPs accumulation, while 1,9-dideoxy forskolin, an inactive forskolin, had little effect on IPs response. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9-H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cyclic AMP-dependent protein kinase (PKA), reversed the ability of forskolin to attenuate BK-stimulated IPs accumulation. The KD and Bmax, values of the BK receptor for [3H]BK binding were not significantly changed by forskolin treatment for 30 min and 4 h. The AlF4(-)-induced IPs accumulation was attenuated by forskolin, indicating that G protein(s) are directly activated by AlF4- and uncoupled to phospholipase C by forskolin treatment. These results suggest that activation of cyclic AMP/PKA might inhibit the BK-stimulated PI breakdown and consequently reduce the [Ca2+]i increases or inhibit independently both responses, which is distal to the BK receptor in canine cultured TSMCs.

Activation of mitogen-activated protein kinase by oxidized low-density lipoprotein in canine cultured vascular smooth muscle cells.

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase inhibitor genistein for 1h, substantially reduced the [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca(2+) by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca(2+) for these responses. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.

5-Hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilisation in canine cultured aorta smooth muscle cells.

The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) changes was investigated in canine cultured aorta smooth muscle cells (ASMCs). 5-HT-stimulated inositol phosphate (IP) accumulation was time and concentration dependent with a half-maximal response (pEC50) and a maximal response at 6.4 and 10 microM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca+]i. The half-maximal response (pEC50) values of 5-HT for the peak and sustained plateau were 7.1 and 6.9, respectively. Ketanserin and mianserin (1 and 3 nM), 5-HT2A antagonists, were equipotent and had high affinity in antagonising the 5-HT-induced IP accumulation and [Ca2+]i change with pK(B) values of 8.6-9.1 and 8.6-9.4, respectively. In contrast, the concentration-effect curves of 5-HT-induced IP and [Ca2+]i responses were not shifted until the concentrations of NAN-190 and metoctopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased to as high as 1 microM with pK(B) values of 5.7-6.3 and 6.1-6.6, respectively, indicating that the 5-HT receptor-mediated responses had low affinity for these antagonists. Pre-treatment of ASMCs with pertussis toxin (100 ng/mL, 24 h) caused a significant inhibition of 5-HT-induced IP accumulation and [Ca2+]i change in ASMCs. Depletion of external Ca2+ or removal of Ca2+ by addition of EGTA led to a significant attenuation of IP accumulation and [Ca2+]i change induced by 5-HT. Influx of external Ca2+ was required for the 5-HT-induced responses, because Ca2+-channel blockers--verapamil, nifedipine and Ni2+--partly inhibited the 5-HT-induced IP accumulation and Ca2+ mobilisation. The sustained elevation of [Ca2+]i response to 5-HT was dependent on the presence of external Ca2+. Removal of external Ca2+ by addition of 5 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to lower than the resting level. The sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis and Ca2+ mobilisation, at least in part, through a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT2A receptors may be predominantly mediating IP accumulation, and subsequently IP-induced Ca2+ mobilisation may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells.

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.

Tumour necrosis factor-alpha enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells.

Inhalation of tumour necrosis factor-alpha (TNF-alpha) induced a bronchial hyperreactivity to contractile agonists. However, the mechanisms of TNF-alpha involved in the pathogenesis of bronchial hyperreactivity were not completely understood. Therefore, we investigated the effect of TNF-alpha on bradykinin (BK)-induced inositol phosphate (IP) accumulation and Ca(2+) mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with TNF-alpha potentiated BK-induced IP accumulation and Ca(2+) mobilization. However, there was no effect on the IP response induced by endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), and carbachol. Pretreatment with PDGF B-chain homodimer (PDGF-BB) also enhanced BK-induced IP response. These enhancements induced by TNF-alpha and PDGF-BB might be due to an increase in BK B(2) receptor density (B(max)), since [3H]BK binding to TSMCs was inhibited by the B(2) selective agonist and antagonist, BK and Hoe 140, but not by the B(1) selective reagents. The enhancing effects of TNF-alpha and PDGF-BB were attenuated by PD98059 (an inhibitor of activation of MAPK kinase, MEK) and cycloheximide (an inhibitor of protein synthesis), suggesting that TNF-alpha may share a common signalling pathway with PDGF-BB via protein(s) synthesis in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 mitogen-activated protein kinase (MAPK) activation induced by TNF-alpha and PDGF-BB and attenuated the effect of TNF-alpha on BK-induced IP response, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by TNF-alpha might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells.

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.