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Background and aims Several studies have demonstrated that both native glucagon-like peptide-1 (GLP-1) and GLP-1 receptor agonists suppress the progression of atherosclerosis in animal models. Methods We investigated whether liraglutide, a GLP-1 analogue, could prevent the development of atherosclerosis in apolipoprotein E knockout mice ( ApoE −/− ) on a high-fat diet. We also examined the influence of liraglutide on angiotensin II-induced proliferation of rat vascular smooth muscle cells (VSMCs) via enhancement of AMP-activated protein kinase (AMPK) signaling and regulation of cell cycle progression. Results Treatment of ApoE −/− mice with liraglutide (400 μg/day for 4 weeks) suppressed atherosclerotic lesions and increased AMPK phosphorylation in the aortic wall. Liraglutide also improved the endothelial function of thoracic aortas harvested from ApoE −/− mice in an ex vivo study. Furthermore, liraglutide increased AMPK phosphorylation in rat VSMCs, while liraglutide-induced activation of AMPK was abolished by exendin 9-39, a GLP-1 antagonist. Moreover, angiotensin (Ang) II-induced proliferation of VSMCs was suppressed by liraglutide in a dose-dependent manner, and flow cytometry of Ang II-stimulated VSMCs showed that liraglutide reduced the percentage of cells in G2/M phase (by arrest in G0/G1 phase). Conclusions These findings suggest that liraglutide may inhibit Ang II-induced VSMC proliferation by activating AMPK signaling and inducing cell cycle arrest, thus delaying the progression of atherosclerosis independently of its glucose-lowering effect.

In the present study, we investigated the transport of nephrotoxic mycotoxin ochratoxin A (OTxA) by a novel human organic anion transporter hNPT4 using the Xenopus oocyte expression system. hNPT4 mediated time- and concentration-dependent uptake of OTxA (Km: 802.8 μM) in a pH- and voltage-sensitive manner. Cis-inhibition experiments suggest that the substrate selectivity of hNPT4 is similar to that of hOAT4. The fact that the Km of OTxA for the efflux transporter hNPT4 was much higher than those for the uptake transporters hOAT1 and hOAT3 may favor the accumulation of OTxA in the tubular cell and lead to nephrotoxicity. Keywords:: ochratoxin A, organic anion transporter, nephrotoxicity

AIMS Several studies suggest increased mortality postcoronary angioplasty in patients on sulphonylureas. However, a theoretical reduction in cardiac risk has been suggested with the newer sulphonylurea agents, which differ from the first-generation agents. In the present study, we investigated whether a third generation of sulphonylurea, glimepiride, might stimulate nitric oxide (NO) production and thereby inhibit cytokine-induced nuclear factor (NF)-kappaB activation in endothelial cells compared with the classical sulphonylurea glibenclamide. METHODS AND RESULTS We demonstrated that glimepiride, but not glibenclamide, induces NO production in human umbilical vein endothelial cells (HUVEC). A significant increase in endothelial NO synthase (eNOS) activity, measured in terms of citrulline production, was observed with glimepiride treatment. Akt phosphorylation followed by phosphorylation of eNOS (Ser1177) was observed with glimepiride treatment in HUVEC. Moreover, two phosphoinoside 3-kinase inhibitors, wortmannin and LY294002, significantly inhibited glimepiride-induced NO production. We also demonstrated inhibition of tumour necrosis factor-alpha (TNFalpha)-induced NF-kappaB activation in HUVEC treated with glimepiride, which was attenuated by pretreatment with N(omega)-nitro-L-arginine methyl ester. We also demonstrated a marked increase in p65 in nuclear extracts from untreated HUVEC following stimulation with TNFalpha, which was dose dependently inhibited by glimepiride, but not by glibenclimide in association with NF-kappaB levels. CONCLUSION These data suggest that glimepiride might be a preferable sulphonylurea agent in the setting of type 2 diabetes and vascular disease because it may have protective effects on vascular endothelial cells.

Milrinone or olprinone, a selective phosphodiesterase (PDE) 3 inhibitor, has relaxant actions on smooth muscles in addition to positive inotropic actions. The exact mechanism on vasodilating and bronchodilating actions of milrinone or olprinone has not been elucidated. In the present experiments, relaxant responses to PDE3 inhibitors were examined on the precontracted airway or pulmonary artery smooth muscle preparations to clarify their mechanism. Both milrinone and olprinone relaxed the airway smooth muscle preparation or the pulmonary artery preparation isolated from guinea-pigs in a concentration-dependent manner. In the airway smooth muscle, these relaxations were markedly blocked by iberiotoxin (a blocker of large conductance Ca^-activated K^+ channels). On the other hand, in the main pulmonary artery, the milrinone- and olprinone-induced relaxations were significantly blocked by iberiotoxin, and were more strongly blocked by ouabain (an inhibitor of Na^+, K^+-ATPase). In the right/left (R/L) pulmonary artery, ouabain also strongly blocked relaxant responses to milrinone and olprinone, but iberiotoxin did not modify these relaxations. Similar observations were seen on the bucladesine (a cyclic AMP mimic agent)-induced relaxation. In conclusion, milrinone and olprinone cause concentration-dependent relaxations of the isolated airway and pulmonary artery smooth muscles via an increase in intracellular cyclic AMP (cAMP). In the airway smooth muscle, large conductance Ca^-activated K^+ (BK_) channels seem to play a crucial role for these relaxations. Relaxations of the main pulmonary artery induced by milrinone and olprinone are mediated predominantly by activation of Na^+, K^+-ATPase, and partly through BK_ channels. In the R/L pulmonary artery, vasorelaxant effects of milrinone and olprinone are more likely mediated by activation of Na^+, K^+-ATPase, but not BK_ channels.

A close relationship between oxidative stress, endothelial dysfunction, and hypoadiponectinemia has been observed. The present study was performed to investigate how glutathione depletion via buthionine sulfoximine (BSO) administration affects endothelial function and adiponectin levels in rats. Acetylcholine (Ach)-induced vasodilation was significantly enhanced in BSO-treated rats, compared with control rats. This was completely abolished by L-NAME, and Ach-induced vasodilation was not observed in the aorta without endothelium. These results suggest that Ach-induced hyper-relaxation of the aorta in BSO-treated rats is completely dependent on the presence of endothelium and mediated by changes in eNOS activity. Catalase significantly inhibited this relaxation to Ach and no effect of catalase on sodium nitroprusside-induced relaxation of the aorta without endothelium was observed in BSO-treated rats. Thus, hyper-relaxation of the aorta in BSO-treated rats is likely caused by H2O2 in addition to NO produced by the endothelium via an eNOS-dependent mechanism. Hypoadiponectinemia and decreased levels of adiponectin mRNA in adipose tissue were observed in BSO-treated rats. Protein expression of eNOS and SODs (SOD-1 and SOD-2) in the aorta was increased and plasma NOx levels were decreased in BSO-treated rats. Our results suggest that oxidative stress induced by BSO causes eNOS uncoupling and hyper-relaxation by producing H2O2, and that BSO-induced oxidative stress causes hypoadiponectinemia, probably by increasing H2O2 production in adipose tissue.

Oxidative stress induces endothelial dysfunction and hypoadiponectinemia. We previously reported that supplementation with tetrahydrobiopterin (BH4), one of the most potent naturally occurring reducing agents and an essential cofactor of enzymatic NO synthase (NOS), ameliorates endothelial dysfunction and reverses hypoadiponectinemia as a result of oxidative stress in rats. To further confirm this hypothesis, we investigated the effects of treatment with BH4 on endothelium-dependent relaxation and adiponectin levels during oxidative stress in fructose-fed rats, which provide an animal model for the metabolic syndrome. Ingestion of a fructose diet for 8 weeks significantly impaired endothelium-dependent arterial relaxation in aortic strips and decreased plasma adiponectin levels, as well as adiponectin mRNA levels within adipose tissue. However, oral supplementation with BH4 (10 mg/kg day) over the final 4 weeks leads to a significant partial reversal of impaired endothelium-dependent arterial relaxation, as well as normalization of plasma adiponectin and fat adiponectin mRNA levels. Moreover, BH4 treatment of the fructose-fed rats significantly reduced the lipid peroxidation content of aorta, heart, liver, and kidney tissues, which were increased in fructose-fed rats. This effect of BH4 treatment may be due to its function as a cofactor for eNOS, as well as its anti-oxidative effects. Thus, BH4 might show promise for the treatment of oxidative stress-induced disorders, including the metabolic syndrome.

Lamina muscularis mucosae sitting beneath mucosal surface of the digestive tract has received little attention to date compared with external smooth muscle layers. Motor activity of the muscularis mucosae shows a great regional and species difference. Autonomic innervation profile is also different from esophagus to colon or between animal species. Intracellular transduction mechanisms for motor activity of the muscularis mucosae are also different from those of external longitudinal and circular muscles or from vascular and airway smooth muscles. Since the submucosal area is a major source for eicosanoid production, abnormality of muscularis mucosae motor activity may link with abnormality of mucosal absorption and secretion functions. Inflammatory bowel diseases such as diarrhea, irritable bowel syndrome and Crohn's disease accompanied with altered motor activity of the muscularis mucosae. Much attention should be attracted to the human muscularis mucosae as a new therapeutic target for inflammatory bowel diseases.

Aldosterone is a mineralocorticoid hormone that plays an important role in regulating electrolyte balance and blood pressure and also participates in endothelial dysfunction. We evaluated the direct effect of aldosterone on human umbilical vein cells (HUVEC). Levels of eNOS phosphorylation by vascular endothelial growth factor were diminished, and the amount of NO produced in response to vascular endothelial growth factor measured as NO2+NO3 was significantly decreased in cells previously incubated with aldosterone. Incubation with aldosterone for 24 h dose-dependently increased Nox4 mRNA expression in HUVEC. Although NF-kappaB was not apparently activated by aldosterone, mRNA levels of vascular cell adhesion molecule-1, E-selectin, monocyte chemotactic protein-1, and intercellular adhesion molecule-1 in HUVEC were significantly increased after incubation with aldosterone. Thus, aldosterone directly causes the dysregulation of endothelial cell function, which may be partly responsible for high blood pressure and atherosclerosis.

BACKGROUND Although neuroendocrine (NE) cells in prostate cancer have been speculated to accelerate the growth and progression of surrounding cancer cells, the evidence is as yet inconclusive. We investigated the effect of an NE allograft (NE-10) and its cell line, NE-CS, which were established from the prostate of the LPB-Tag 12T-10 transgenic mouse, on human prostate cancer cell line LNCaP. METHODS The proliferation and pulmonary metastasis of LNCaP xenografts in athymic mice with and without NE-10 allografts were evaluated. Boyden chamber assay and microarray analysis were performed to investigate changes in invasion/migration and mRNA of LNCaP cells under the influence of the NE cells, respectively. RESULTS NE-10 did not influence the proliferation of LNCaP. The pulmonary metastasis of LNCaP with NE-10 significantly increased compared to mice without it. The NE-CS cells accelerated the in vitro invasion/migration of adenocarcinoma cells. Increased expression of mRNA of gelsolin was observed in LNCaP cells incubated with the supernatant of NE-CS cells. CONCLUSIONS The NE-10 allograft promotes pulmonary metastasis of subcutaneously inoculated LNCaP cells by facilitating cell invasion. Secretions from NE cells upregulate the expression of gelsolin, which is an actin-binding protein, resulting in acceleration of the migration of LNCaP cells. © 2005 Wiley-Liss, Inc.

The underlying mechanism involved in the interaction between neutrophil elastase inhibitors and tachykinins has not been elucidated. In this study we have examined the effects of sivelestat, a neutrophil elastase inhibitor, on the in vitro responses of airways from lipopolysaccharide (LPS)-untreated or -treated guinea-pigs to substance P. Substance P (0.01-30 micromol/l) produced concentration-dependent contractions of both tracheal and bronchial ring preparations of LPS-untreated or -treated guinea-pigs. Responsiveness to substance P in these isolated airway preparations was augmented by either epithelium removal or LPS treatment. In epithelium-intact tracheal ring preparations isolated from LPS-untreated guinea-pigs, sivelestat (100 micromol/l) significantly inhibited substance P-induced contractions. The inhibitory action was markedly attenuated by pretreatment with L-NAME (100 micromol/l) or indomethacin (2 micromol/l), and was almost undetected following removal of the epithelium. On the other hand, in bronchial ring preparations isolated from LPS-untreated guinea-pigs, sivelestat had only a very slight effect on substance P-induced contraction of the epithelium-intact preparation, whereas sivelestat greatly inhibited contraction in epithelium-removed bronchial ring preparations. In LPS-treated guinea-pigs, whether the epithelium was intact or not, sivelestat significantly inhibited the substance P-induced contraction of bronchial ring preparations. Pretreatment with L-NAME (100 micromol/l) or indomethacin (2 micromol/l) did not affect the inhibitory effect of sivelestat in bronchial ring preparations. In conclusion, epithelium removal or LPS treatment induced hyperreactivity to substance P in the guinea-pig airway. Sivelestat caused epithelium-, nitric oxide- and prostaglandin-dependent inhibition of the substance P-induced contraction of isolated guinea-pig tracheal ring preparations. In contrast, the inhibitory effect of sivelestat on substance P-induced contraction of guinea-pig bronchial ring preparations is mediated by epithelium-, nitric oxide- and prostaglandin-independent mechanisms. Sivelestat may be effective in reducing the airway hyperresponsiveness to tachykinins induced by epithelial injury as occurs in LPS-mediated inflammatory lung diseases.

Neuroendocrine (NE) cells in prostate cancer have been shown to be associated with the progression of prostate cancer. However, little is known about the molecular basis of this association. We have previously demonstrated that NE cells promote metastasis of a human prostate cancer cell line (LNCaP) with overexpression of the gelsolin gene. The purpose of this study was to investigate the interactions between NE cells and LNCaP cells and the involvement of gelsolin in contributing to the invasive potential of LNCaP cells. In addition, we examined whether neurotensin induced gelsolin-mediated invasion. We used the NE cell line NE-CS that was established from the prostate of the LPB-Tag 12T-10 transgenic mouse. Small interfering RNA (siRNA) targeting gelsolin or not targeting it was transfected into LNCaP cells. Cell invasion was assessed by Matrigel invasion assay. The supernatant of NE-CS cells and neurotensin induced the transformation of LNCaP cells. Neurotensin was observed in the supernatant of NE-CS cells but not in LNCaP cells. The siRNA targeting of gelsolin resulted in inhibition of invasion of LNCaP cells in the culture medium with neurotensin added, and in the supernatant of NE-CS cells with epidermal growth factor. The invasive potential of LNCaP cells enhanced by neurotensin or the supernatant of NE-CS cells through neurotensin receptor 1 (NTSR1) was blocked by a phospholipase Cγ inhibitor and an intracellular calcium chelator, with concomitant gelsolin suppression. This study indicates that NE cells and neurotensin induce gelsolin-mediated invasion of LNCaP cells through NTSR1 activation.

The aim of this study was to examine the effect of exogenously applied BaCl2 on the norepinephrine-induced contraction of the rat thoracic aorta. Exogenously applied BaCl2 (0.3–1 mmol/l) slightly elevated the norepinephrine-induced sustained contraction of the rat thoracic aorta in the absence of nicardipine (1 μmol/l). In the aortic preparation pretreated with nicardipine (1 μmol/l), exogenous BaCl2 (0.1–3 mmol/l) did not elevate the norepinephrine-induced sustained contraction, but the high concentration of BaCl2 (10 mmol/l) slightly inhibited the norepinephrine-induced tone. In a Ca2+-free Krebs bi- carbonate solution (KBS) containing norepinephrine (1 μmol/l) or a Ca2+-free K+-rich (60 mmol/l) KBS, exogenously applied BaCl2 (1–30 mmol/l) caused a sustained contraction of the rat thoracic aorta, and this sustained contraction was completely inhibited by nicardipine (1 μmol/l). Exogenous CaCl2 (0.1–3 mmol/l) also caused a sustained contraction of the aortic preparation in a Ca2+- free KBS containing norephinephrine (1 μmol/l), but such a sustained contraction was partly inhibited by nicardipine (3 μmol/l). These results indicate that Ba2+ elevates the norepinephrine-induced tone of the rat isolated thoracic aorta by permeating voltage-dependent Ca2+ channels in the absence of nicardipine, but that Ba2+ has a minor modification on the norepinephrine-induced sustained contraction of the nicardipine-pretreated preparation.

1 In the oesophageal muscularis mucosae, we examined the effects of endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3) and sarafotoxin S6c (SX6c) as agonists, and FR139317, BQ-123 and RES-701-1 as endothelin receptor antagonists. 2 All of the endothelins produced tonic contractions which were frequently superimposed on rhythmic motility in a concentration-dependent manner. The order of potency (−log EC50) was ET-1 (8.61)=SX6c (8.65)>ET-2 (8.40)>ET-3 (8.18). 3 FR139317 (1–3 μM) and BQ-123 (1 μM) caused parallel rightward shifts of the concentration-response curve to ET-1, but at higher concentrations caused no further shift. RES-701-1 (3 μM) caused a rightward shift of the concentration-response curve to ET-1, while RES-701-1 (10 μM) had no additional effect. RES-701-1 (0.1–1 μM) concentration-dependently caused a rightward shift of the concentration-response curve to SX6c. The contraction to ET-1 (10 nM) in preparations desensitized to the actions of SX6c was greatly inhibited by pretreatment with FR139317 (10 μM). 4 Modulation of the Ca2+ concentration in the Krebs solution caused the concentration-response curve to ET-1 or SX6c to shift to the right and downward as external Ca2+ concentrations decreased. Verapamil (30 μM) abolished rhythmic motility induced by ET-1 or SX6c. Ni2+ (0.1 mM) weakly inhibited ET-1- or SX6c-induced tonic contraction. SK&F 96365 (60 μM) completely inhibited ET-1-induced contractions. 5 We conclude that there are two types of ET-receptors, excitatory ETA- and ETB-receptors in the oesophageal muscularis mucosae. These receptors mediate tonic contractions predominantly by opening receptor-operated Ca2+ channels (ROCs) and partly by opening T-type Ca2+ channels, and mediate rhythmic motility by opening L-type Ca2+ channels. British Journal of Pharmacology (1998) 125, 849–857; doi:10.1038/sj.bjp.0702140

We present a case of squamous cell carcinoma of the testis that metastasized from lung cancer. The patient, who had received left pneumonectomy 2 years earlier for squamous cell carcinoma (SCC) of the lung, developed pulmonary metastasis, which was treated with chemotherapy. Although the recurrence regressed after treatment, the testicular tumor progressed gradually. Left radical orchiectomy was performed. Pathological examination revealed metastatic SCC. Testicular metastasis from lung cancer is a very rare disease.

To determine the follow-up schedule in patients with non-muscle-invasive bladder cancer who had remained recurrence-free for 5 or more years, we retrospectively reviewed 258 patients with Ta and T1 bladder cancer who had been free of recurrence for at least 5 years. Of these 258 patients, subsequent recurrences developed in 100 patients. In spite of our recommendation that cystoscopic follow-up be done at 12-month intervals for patients who remained recurrence-free for more than 5 years, 45 had been followed at intervals of more than 12 months (range, 13-77 months) when the recurrences were found. Of 100 recurrent tumors, 20 (20.0%) showed bladder muscle invasion. Muscle-invasive cancer was identified more often in the patients with cytoscopic intervals of more than 12 months than in those of less than 12 months (35.6% versus 7.3%). Therefore, we recommend that cystoscopy be performed at intervals of less than 12 months in patients with non-muscle invasive bladder cancer for recurrence detection before tumors become muscle invasive, even when patients remain free of recurrence for a long period.

Leiomyosarcoma is a malignant soft-tissue cancer arising from tissues containing smooth muscle. It commonly occurs in the gastrointestinal system and retroperitoneum, but is rare in the genito-urinary system. We experienced a case of primary testicular leiomyosarcoma. A 71-year-old man presented with painless swelling of the right scrotal contents for 4 months. A high orchiectomy was performed. Histological examination revealed primary testicular leiomyosarcoma. The patient did not receive any adjuvant therapy. Seven months after the operation, there has been no recurrence. Cases of primary intratesticular leiomyosarcoma are rare. To the best of our knowledge, only sixteen cases have been reported in the literature.

The purpose of this study was to clarify the clinical relevance of carbapenem and third-generation cephalosporin treatment for febrile complicated pyelonephritis, which often leads to urosepsis. Parenteral antimicrobial treatment with a carbapenem or third-generation cephalosporin was administered to febrile patients and the treatment was switched to oral antimicrobial agents after they became afebrile. In principle, the duration of the course of antimicrobial chemotherapy was limited to a total of 14 days. Clinically, the success rates were 97.3% in the carbapenem group and 96.0% in the third-generation cephalosporin group. For microbiological efficacy, the success rates were 89.2% in the carbapenem group and 92.0% in the third-generation cephalosporin group. There were no serious adverse events in the course of the study. The treatment regimen with a carbapenem or a third-generation cephalosporin was highly effective for patients with febrile complicated pyelonephritis and was well tolerated. Either of these regimens could become one of the standard treatments for patients with febrile complicated pyelonephritis.

Background Endothelial dysfunction plays a key role in atherogenesis. We investigated whether AMP-activated protein kinase (AMPK) activity is a downstream mediator of the beneficial effects of cilostazol on vascular endothelial cells and whether cilostazol might reverse endothelial dysfunction in diabetic rats. Methods and results Treatment of human umbilical vein endothelial cells (HUVECs) with cilostazol resulted in time-dependent activation of AMPK, as monitored by phosphorylation of AMPK and its down-stream target, acetyl-CoA carboxylase (ACC). Activation of AMPK by cilostazol was through signaling pathway independent of cyclic AMP and caused phosphorylation of endothelial nitric oxide synthase (eNOS), leading to increased production of nitric oxide (NO), while inhibiting cytokine-induced nuclear factor-kappaB (NF-kappaB) activation, leading to suppression of VCAM-1 gene expression. Significantly reduced eNOS activity and NO production in response to cilostazol and attenuation of cilostazol-induced inhibition of NF-kappaB activation were observed in cells treated with AMPK siRNA. We also demonstrated that administration of cilostazol to diabetic rats significantly restored endothelium-dependent vasodilation. Furthermore, treatment of diabetic rats with cilostazol increased tetrahydrobiopterin (BH4) levels in the aorta. Thus, recovery of BH4 following administration of cilostazol might also contribute to restoration of endothelial function in diabetic rats. Conclusions Our findings suggest that the beneficial effects of cilostazol on endothelial function may be due to AMPK activation. Restoration of endothelial dysfunction in diabetic rats by cilostazol is at least partly attributed to amelioration of biopterin metabolism in the aorta.

We focused on the establishment of a trial procedure for the collection and distribution of Japanese liver tissues obtained from waste surgical resections for drug development and research use. The following procedures were prepared for this project: the pretreatment of liver tissues before storage, their storage at 4 degrees C, the transport of liver samples, the setting up of a communication network among the participating hospitals and laboratories and the approval of each ethics committee. Thirteen liver samples (1.6-7.6 g) obtained from patients whose livers were excised due to cirrhosis, hepatocellular carcinoma, cholangiocarcinoma, or metastasis from colorectal carcinoma and were donated for research. Informed consent was obtained from every patient. Freshly isolated human hepatocytes were prepared from nine liver samples (viability 34.3-86.1%). Four samples were unsuitable to prepare hepatocytes. The profile of testosterone metabolism as 6beta-, 2beta-, 16beta-, 16alpha- and 2alpha-hydroxytestosterone and androstenedione in freshly isolated hepatocytes was shown to be specific for human liver. The 6beta-hydroxylation activity catalyzed by CYP3A4/5 indicated a high level of metabolism (139-996 pmol/min/million cells). Levels of 7-ethoxycoumarin O-deethylation and glucronidation activities were sufficient for analysis in freshly isolated human hepatocytes. We conclude that liver tissues from waste surgical resections supplied from a participating hospital can constitute a valuable source of freshly isolated human hepatocytes for drug development and safety evaluation.

Objective The pteridine cofactor tetrahydrobiopterin (BH4) has emerged as a critical determinant of endothelial nitric oxide synthase (eNOS) activity. When BH4 availability is limited, eNOS does not produce nitric oxide (NO) but instead generates superoxide. BH4 may reverse endothelial dysfunction due to cardiovascular disease, including atherosclerosis, coronary artery disease and hypertension. In this study, the influence of BH4 on cardiovascular parameters and the production of free radicals following angiotensin II (Ang II) infusion was assessed. Methods BH4 (20 mg/kg per day in drinking water) was administered with Ang II (300 ng/kg per min subcutaneously, osmotic pump) for 7 days in Sprague-Dawley rats. In addition, BH4 was also given in vehicle-infused rats. Results Treatment with BH4 significantly prevented some of the effects of Ang II, such as impaired vascular responses to acetylcholine, hypertension and increases in heart weight index values. Treatment with BH4 also significantly reduced Ang II-induced increases in inducible NO synthase expression, nitrotyrosine immunostaining, NO production and superoxide anion formation in rats. Conclusion These results indicate that BH4 might prevent the development of hypertension and myocardial hypertrophy, as well as the Ang II-induced production of superoxide and NO, thereby reducing the production of peroxynitrite. Therefore, BH4 may protect against the cardiovascular manifestations of oxidative and nitrosative stress in this experimental model of Ang II-mediated hypertension.

We have compared the reactivity to carbachol and high potassium of circular smooth muscle isolated from segments of human colon which was freeze-stored in different preservative solutions for more than one month following surgical resection. Concentration-dependent contractions in response to carbachol were reduced in terms of both their sensitivity (pEC50) and reactivity (Emax), depending on the preservative solutions used. Similar reduction of reactivity to 100 mM KCl was also observed. The best responsiveness was shown when the tissue was freeze-stored in SFM101. It is concluded that the freeze-storage of surgically excised human colon in SFM101 or phosphate buffer solution for more than one month provided the best preservation of smooth muscle function for in vitro pharmacological examination.

BACKGROUND The role of neuroendocrine (NE) cells in prostate cancer remains unclear. A useful model is necessary to study the biology of NE cells. We herein describe the establishment and characterization of an immortalized cell line from an NE-10 allograft of murine prostatic NE carcinoma. METHODS A novel cell line, designated NE-CS, was developed from an NE-10 allograft that was established from the ventral prostate of the LPB-T-antigen (Tag) transgenic mouse, line 10 (12T-10). We investigated the growth, karyotype, electron microscopic findings, expression of Tag and androgen receptor (AR), and tumorigenesis of the cells in athymic mice. RESULTS The immortal cell line NE-CS was maintained in vitro for more than 2 years. The NE-CS cells had dendritic-like extensions with dense core granules in the cytoplasm and produced serotonin and somatostatin in conditioned medium. The cells expressed neither Tag nor AR. They showed androgen-independent growth in vitro and a hypotetraploid karyotype similar to the original NE-10 allograft. The NE-CS cells, which were subcutaneously inoculated into athymic mice, formed tumors with the NE phenotype. The tumors exhibited accelerated growth compared to the original NE-10 allograft. CONCLUSIONS The established cell line has characteristics of NE differentiation and tumorigenic ability. This cell line may be a promising model to understand the molecular mechanisms associated with the acquisition of hormone refractory prostate cancer. © 2004 Wiley-Liss, Inc.

We retrospectively reviewed the records of 54 patients with RCC who underwent partial nephrectomy for the primary lesion between 1992 and 2001. The indications for partial nephrectomy were elective in 43 and imperative in 11 patients. We selected 51 patients with clinical stage T1a who underwent open radical nephrectomy for localized RCC for comparison during the same period. We evaluated the peri- and postoperative complications, disease-free survival rates and changes of renal function in the partial nephrectomy (PN) group, compared to the radical nephrectomy (RN) group. There was no significant difference with regard to pathological findings and clinical outcomes between two groups, except for the amount of intraoperative bleeding. Three patients in the PN group developed postoperative complications, consisting of urine leakage in 2 patients and renal hypertension in 1 patient. The 5-year disease-free survival rates in the PN and RN groups were 90% and 97%, respectively. Local recurrence from the resected area of the renal parenchyma was not found in patients in the PN group. All patients in the PN group maintained satisfactory and stable renal function. In the RN group, renal function slowly deteriorated in 2 patients. Therefore, partial nephrectomy offers cancer control and an acceptable low mortality rate, comparable to those of radical nephrectomy.

Objectives To examine whether peroxisome proliferator-activated receptor gamma (PPARγ) is expressed in human renal cell carcinoma (RCC) cells, and whether activation of PPARγ by its ligands can have multiple antitumor effects on human RCC cells in vitro. Methods We examined the expression of PPARγ in four human RCC cell lines by reverse transcriptase-polymerase chain reaction and immunocytochemical staining. The effects of two PPARγ ligands, pioglitazone and 15-deoxy-Δ12,14-prostaglandin J2, on cell proliferation were investigated by 3-[4,5-dimethylthiazol-2-thiazoly]-2,5-diphenyltetrazolium bromide assay. The induction of apoptosis by the ligands was examined using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method and Annexin V assay. Furthermore, we investigated whether these ligands suppressed the production of angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, by enzyme-linked immunosorbent assay. Results PPARγ and retinoid X receptor, which forms a heterodimer with PPARγ, were expressed in all RCC cell lines. In addition, immunocytochemical studies showed expression of PPARγ protein in the RCC cells. PPARγ ligands inhibited the cell growth in all cells in a dose-dependent manner. These ligands also induced apoptosis. Furthermore, secretion of both vascular endothelial growth factor and basic fibroblast growth factor was inhibited by these ligands in a dose-dependent and time-dependent manner. Conclusions Ligands for PPARγ have multiple antitumor effects in human RCC cells in vitro. Activation of the PPARγ pathway may be a new strategy for treatment of patients with RCC.

We present a case of squamous cell carcinoma of the testis that metastasized from lung cancer. The patient, who had received left pneumonectomy 2 years earlier for squamous cell carcinoma (SCC) of the lung, developed pulmonary metastasis, which was treated with chemotherapy. Although the recurrence regressed after treatment, the testicular tumor progressed gradually. Left radical orchiectomy was performed. Pathological examination revealed metastatic SCC. Testicular metastasis from lung cancer is a very rare disease.

We have compared the reactivity to spasmogens of longitudinal muscularis mucosae isolated from the human, guinea pig and rat colon in vitro. The muscularis mucosae isolated from the human distal colon responded with a sustained contractions to carbachol (10 nM-30 microM), in a concentration-dependent manner, and the maximum contraction was comparable to that with high potassium concentration (100 mM). Among several spasmogens, neurokinin A was the most potent with the following order of potency: carbachol, prostaglandin F2alpha and acetylcholine. Histamine, 5-hydroxytryptamine and bradykinin did not produce a recognizable contraction of this tissue. The muscularis mucosae isolated from the guinea pig distal colon demonstrated a concentration-dependent contraction in response to neurokinin A, carbachol, histamine and acetylcholine, but not to prostaglandin F2alpha or 5-hydroxytryptamine, and the maximum contraction was obtained with histamine. The muscularis mucosae from the rat distal colon was very sensitive to neurokinin A and bradykinin, less to carbachol and acetylcholine, and not at all sensitive to histamine, 5-hydroxytryptamine and prostaglandin F2alpha. It is concluded that the colonic muscularis mucosae respond to pharmacological agents in a species-different manner.

Objectives: We evaluated clinical features and predictive factors for the recurrence of renal cell carcinoma (RCC) developing more than 5 years after nephrectomy. Methods: We retrospectively reviewed 239 patients with RCC who underwent surgery for the primary lesion. To identify factors that affected recurrence more than 5 years after nephrectomy (delayed recurrence) and its clinical outcomes, we performed a multivariate analysis using Cox’s proportional hazards model and a survival study. Results: Recurrence developing within 5 years after nephrectomy (early recurrence) was found in 57 patients and delayed recurrence in 11 patients. The multivariate analysis revealed no clinical and pathologic features influencing delayed recurrence in 114 patients who survived more than 5 years after nephrectomy without having early recurrence. The patients with delayed recurrence showed better clinical outcomes than those with early recurrence when the rate was determined from the time of recurrence. Conclusions: Although delayed recurrence is not a rare event for patients with RCC, no clinical and pathologic factors at the time of the initial treatment can predict the recurrence. Patients who are free of recurrence for more than 5 years after surgery for a primary lesion should be carefully followed up for delayed recurrence.

The effects of Ba2+ on receptor-mediated sustained contraction of rat pulmonary artery and guinea-pig oesophageal muscularis mucosae were studied in-vitro. In rat isolated pulmonary artery, sustained contraction induced by noradrenaline (1 μm) was resistant to nicardipine (1 μm), but this same sustained contraction was completely inhibited by SK&F 96365 (30 μm), a blocker of voltage-dependent L-type Ca2+ channels and receptor-activated Ca2+ influx. The SK&F 96365-sensitive sustained contraction induced by noradrenaline (1 μM) may be due primarily to Ca2+ influx through receptor-activated Ca2+ channels resistant to nicardipine. Cumulatively applied BaCl2 (0.1 — 10 mM) increased the noradrenaline (1 μM)-induced sustained contraction of the pulmonary arterial preparation in the absence of nicardipine, but in the presence of nicardipine (1 μM), BaCl2 (0.1 — 3 mM) did not affect this contraction. A higher concentration of BaCl2 (10 mM), however, weakly inhibited the noradrenaline (1 μM)-induced tone. In addition, BaCl2 (3–10 mM) increased the tone induced by KCl (60 mM), and the BaCl2-elevated KCl tone was markedly inhibited by nicardipine (1 μM) treatment. In the guinea-pig isolated oesophageal muscularis mucosae, sustained contraction induced by acetylcholine (3 μM) was resistant to nicardipine (1 μM) but was returned to its basal level by SK&F 96365 (30–60 μM). The SK&F 96365-sensitive, acetylcholine-induced sustained contraction of the oesophageal muscularis mucosae is also likely to link with receptor-activated Ca2+ channels resistant to nicardipine. In contrast to the rat pulmonary artery, cumulatively applied BaCl2 (0.3–10 mM) inhibited the acetylcholine (3 μM)-induced sustained contraction of the oesophageal muscularis mucosae in a concentration-dependent manner in the presence of nicardipine (1 μM). In conclusion, Ba2+ presumably activates voltage-dependent Ca2+ channels by depolarizing plasma membrane and also passes through voltage-dependent Ca2+ channels to contract the pulmonary artery in the absence of nicardipine, and Ba2+ also has a minor effect on the nicardipine-resistant, SK&F 96365-sensitive sustained contraction induced by noradrenaline in rat isolated pulmonary artery.

The aim of the present study was to examine the effect of Ba2+ on acetylcholine- and KCl-induced contractions of the guinea-pig esophageal muscularis mucosae. When the muscularis mucosae was pretreated with nicardipine (1 microM), Ba2+ (0.1-30 mM) markedly inhibited the acetylcholine (3 microM)-induced tone, and at 10-30 mM the tone returned to its basal level. In contrast, Ba2+ (0.1-30 mM) slightly increased the KCl (60 mM)-induced tone. Moreover, the Ba2+ (30 mM)-increased KCl tone was completely inhibited by treatment with nicardipine (0.3-1 microM). In conclusion, Ba2+ both selectively inhibits receptor-mediated contraction of the muscularis mucosae and itself permeates through voltage-dependent Ca2+ channels.

We examined the role of receptor-operated Ca 2+ influx in endothelin-1 (ET-1)- or sarafotoxin S6c (S6c)-induced contraction of the muscularis mucosae. Responses of the esophageal muscularis mucosae isolated from guinea-pigs were recorded by an isotonic transducer and a polygraph. ET-1 and S6c produced contraction of the esophageal muscularis mucosae in a concentration-dependent manner. The contractile responses to ETs were abolished in a Ca 2+ -free EGTA-containing medium, weakly inhibited by nicardipine. and markedly inhibited by SK&F96365. In addition, both H-7 and U-73122 strongly inhibited the ET-induced contractions. but U-73343 weakly inhibited these responses. These results indicate that the esophageal muscularis mucosae of guinea pigs has ET receptors that are coupled mainly to receptor-operated Ca 2+ influx and linked with the phospholipase C-protein kinase C pathway.

We present a case of juvenile hemangioma of the testis associated with hemangiomas of the liver and skin. The testicular hemangioma appeared to progress after therapy with interferon alpha-2a (IFN alpha-2a), whereas the liver and skin hemangiomas regressed. Analysis of growth factors responsible for proliferation of hemangioma revealed that the tumor expressed vascular endothelial growth factor but not basic fibroblast growth factor (bFGF), which is reported to be inhibited by IFN alpha-2a. This finding suggests that a possible explanation for the inadequate efficacy of IFN alpha-2a for testicular hemangioma is a lack of bFGF expression.

A 59-year-old woman presented elsewhere with genital bleeding, pollakiuria and urgency. The gynecologist noted a raised tumor next to the external meatus and the patient was referred to us. On physical examination the tumor was erythematous, protrusive, inflamed and surrounding the urethral meatus. The lesion was 2.5 cm. in diameter, solid and partly rubbery in consistency. It did not appear to be a urethral caruncle, which is typically a benign, red, raspberrylike, friable tumor involving the posterior lip of the external meatus. There were no lesions on the body surface. The uterus and adnexa were unremarkable. Laboratory values were normal except for urinalysis, which revealed numerous red blood cells. Urinary cytology demonstrated no malignancy. Cystourethroscopy revealed reddish mucosa at the distal urethra but not in the bladder. The lesion was not obstructive. Pelvic magnetic resonance imaging (MRI) in the sagittal plane showed a protrusive mass with low intensity on T1-weighted images, which was clear with high intensity on T2-weighted images (fig. 1). There was clinical suspicion for a malignant tumor. After obtaining informed consent, we performed a biopsy with the patient under lumbar anesthesia. Microscopically, the specimens consisted of clusters of dilated venous-like vessels with thrombi, which were composed of thin endothelial lined spaces. Blood containing spaces were large sinusoids lined by flattened endothelial cells (fig. 2). The specimen was pathologically diagnosed as cavernous hemangioma of the urethra. The tumor was resected completely. The patient was symptom-free at 6-month followup with no evidence of tumor recurrence.

1 The submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus was used to study the actions of catecholamines on the twitch responses to electrical stimulation.2 When the preparation was stimulated coaxially (0.1 Hz, 0.5 ms, supramaximal voltage), stable twitch-like contractions were obtained. These were abolished by tetrodotoxin (0.1 muM) and atropine (0.1 muM), potentiated by physostigmine (0.1 muM), and were mediated presumably by stimulation of intramural cholinergic nerves.3 The twitch contractions of the muscularis mucosae were inhibited by catecholamines, in a concentration-dependent manner. The order of potency was isoprenaline > adrenaline > noradrenaline > dopamine.4 The inhibitory actions of noradrenaline (1 muM) and adrenaline (1 muM) were partly reversed by phentolamine (1 muM) or by propranolol (1 muM), and completely abolished by both antagonists together. The inhibitory effect of dopamine (300 muM) was largely reversed by phentolamine (1 muM), but not by propranolol (1 muM), while the inhibitory action of isoprenaline was competitively antagonized only by propranolol (pA(2) of 7.6).5 The contraction of the muscularis mucosae to exogenously applied acetylcholine (ACh, 20 nM) which was comparable in magnitude with that to electrical stimulation was also inhibited by isoprenaline (0.1 muM), adrenaline (1 muM) and noradrenaline (1 muM), but not by dopamine (300 muM). In the presence of propranolol (1 muM), noradrenaline, adrenaline and dopamine potentiated the ACh-induced contraction, while the effect of isoprenaline was mainly antagonized. The potentiating effects were antagonized by further treatment with phentolamine (1 muM).6 Adrenaline, noradrenaline and dopamine but not isoprenaline, produced a weak contraction of the longitudinal muscularis mucosae in the presence of propranolol (3 muM). The contractile responses were completely inhibited by phentolamine (3 muM). Tone in the muscularis mucosae induced by carbachol (3 muM) in the presence of phentolamine (10 muM) was inhibited by catecholamines, in a concentration-dependent manner, an effect that was competitively antagonized by propranolol.7 In the submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus there are three types of adrenoceptor, inhibitory prejunctional alpha-adrenoceptors, excitatory postjunctional alpha-adrenoceptors and inhibitory postjunctional beta-adrenoceptors, and cholinergic neurotransmission is inhibited by catecholamines acting at both prejunctional alpha- and postjunctional beta-adrenoceptors.

The effects of long-term incubation and of indomethacin on contractile responses to adrenaline and noradrenaline of the guinea-pig esophageal muscularis mucosae were studied in vitro.

Pharmacologic characteristics of muscarinic receptors in the muscularis mucosae of the guinea pig esophagus were examined in vitro and compared with those of the longitudinal muscle of the guinea pig ileum. All cholinomimetics tested produced a sustained contraction of the muscularis mucosae, in a concentration-dependent manner, which was associated with a small biphasic change in membrane potential, initially a hyperpolarization followed by a depolarization. The contractile response was hardly modified by verapamil, but was depressed by calcium removal from the bathing medium. Both atropine and pirenzepine antagonized the contractile response in a competitive manner, with higher pA2 values than those in the ileum. These results indicate that the muscarinic receptors of the muscularis mucosae of the guinea pig esophagus mainly link with calcium ion channels which are independent of changes in membrane potential and that their subtype populations are probably pharmacologically distinct from those in the ileal longitudinal muscle.

The effects of histamine were studied on the isolated circular muscle strip and longitudinal muscle strip of the dog portal vein. Histamine-induced contractions of the circular muscle were inhibited by H1-receptor antagonist pyrilamine but increased by H2-receptor antagonist ranitidine. When the tissues were contracted with PGF2α in the presence of pyrilamine, histamine produced the relaxation of the circular muscle but not longitudinal muscle. The relaxation of the circular muscle was inhibited by ranitidine in a concentration dependent manner, the pA2 value for ranitidine being 6·97 (6·55–7·39). It is concluded that the response of the circular muscle to histamine is the sum of two components, H1-receptor mediated contraction and H2-receptor mediated relaxation.