Yoko Nakahira

Blockade by 18β‐glycyrrhetinic acid of intercellular electrical coupling in guinea‐pig arterioles

1 Intercellular electrical communication between smooth muscle and endothelial cells was examined in guinea‐pig mesenteric arterioles using the whole‐cell patch‐clamp method. The time course of the current required to impose a 10 mV voltage clamp step was used to determine the extent of electrical coupling between them. Currents recorded from both smooth muscle and endothelial cells relaxed in a multi‐exponential manner, indicating the existence of electrical coupling between cells. 2 18β‐Glycyrrhetinic acid, a gap junction blocker, quickly blocked electrical communication at 40 μM, while neither heptanol nor octanol did so at concentrations of up to 1 mM. 3 In the current clamp mode, repetitive spikes, induced by 10 mM Ba2+ solutions, could be recorded from both kinds of cells. After blocking gap junctions, spikes could only be recorded from the smooth muscle cell layer, indicating that they had been conducted through myoendothelial junctions. 4 In endothelial cells, acetylcholine (ACh, 3 μM) induced hyperpolarizing responses, which had two phases (an initial fast and a second slower phase) in the current clamp condition. This ACh response persisted in the presence of 18β‐glycyrrhetinic acid, although this compound seemed to make the membrane slightly leaky. 5 After blocking gap junctions, the membrane potential of a single cell in a multicellular preparation could be well clamped. Thus, 18β‐glycyrrhetinic acid may be useful in studying the function of both arteriolar smooth muscle and endothelial cells while they remain located within a multicellular preparation.

Enhanced Antitumor Effect of Coincident Intravesical Gemcitabine Plus BCG Therapy in an Orthotopic Bladder Cancer Model

OBJECTIVES To evaluate the antitumor effect of the coincident administration of intravesical gemcitabine (Gem) plus bacillus Calmette-Guérin (BCG) in an orthotopic bladder cancer model. METHODS We evaluated the cytotoxic effect of gemcitabine against MBT-2 cells in vitro. Orthotopic tumors were established by implanting MBT-2 cells into the bladder of syngeneic female C3H mice. Intravesical Gem administration was evaluated at various doses: 0 mg (control); 1, 2, 4, and 8 mg (n = 8 for each group). Next, a comparative evaluation of tumor growth among the control, Gem-alone, BCG-alone, and combined Gem + BCG groups was performed (n = 16 for each group). Therapy was administered at 3-day intervals starting on day 5 and repeated 6 times. To evaluate the proliferative activity among the groups, Ki-67 immunostaining of the tumor was performed. RESULTS Gemcitabine exhibited a dose-dependent antitumor effect. Of the 8 mice in each group treated with a dose of 0, 1, 2, 4, or 8 mg of Gem, 1, 4, 4, 4, 5, and 4 mice failed to develop tumors and survived, respectively. The combination of Gem + BCG (54.1 ± 9.4 days) provided a significant survival advantage compared with BCG-alone (39.0 ± 16.4 days) (P = .02). Ki-67 expression, representing tumor proliferation, was significantly lower in the combined Gem + BCG group than in the BCG-alone group (P < .01). CONCLUSIONS Our results suggest that intravesical Gem + BCG treatment induces an enhanced antitumor effect against bladder tumors.

Pranidipine enhances relaxation produced by endothelium-derived relaxing factor in carotid artery.

The effects of pranidipine, a novel dihydropyridine-type Ca(2+)-channel antagonist, on acetylcholine-induced endothelium-dependent relaxation were investigated in isolated carotid artery of the guinea-pig. In arteries contracted with high-K(+) solution ([K(+)](0)=28.8 mM) containing noradrenaline, the relaxation was inhibited by N(omega)-nitro-L-arginine, indicating an involvement of endothelium-derived relaxing factor. Pranidipine (10(-9)-10(-7) M) augmented the relaxation in a concentration-dependent manner. Sodium nitroprusside produced a relaxation in arteries contracted with high-K(+) solution containing noradrenaline, in an endothelium-independent manner, and the relaxation was enhanced by pranidipine. 1H-[1,2,4] oxadiazolo [4, 3-a] quinoxalin-l-one (ODQ), an inhibitor of nitric oxide-sensitive guanylate cyclase, attenuated the relaxation produced by acetylcholine or sodium nitroprusside. In the presence of ODQ, pranidipine did not enhance the acetylcholine-induced relaxation. The relaxation produced by endothelium-derived hyperpolarizing factor was inhibited by pranidipine, with no alteration of the hyperpolarization. Thus, pranidipine augments the nitric oxide-induced relaxation, possibly by enhancing the mechanisms related to cyclic GMP.

Emphysematous pyelonephritis with successful renal preservation using open drainage surgery: A case report

Emphysematous pyelonephritis (EPN) is a rare urinary tract infection that is observed the production of gas inside and outside the kidney. Although there has been the development of antibiotics, there are some cases of EPN that have become serious. Therefore, this disease requires accurate and prompt treatment. Currently, the initial treatment for EPN consists of combination therapy with antibiotics along with percutaneous drainage (PCD). When PCD offers poor infection control, nephrectomy may be necessary. However, if the patients general health condition is poor, or if renal dysfunction is observed, nephrectomy may not be considered. Here we report on case of EPN which could be preserved renal, by open drainage surgery.

1757 CYTOKERATIN 18 AND EAU SCORE PREDICT TUMOR RECURRENCE IN PATIENTS WITH NON-MUSCLE-INVASIVE BLADDER CANCER FOLLOWING SINGLE POSTOPERATIVE IMMEDIATE INTRAVESICAL CHEMOTHERAPY INSTILLATION

269 Background: We examined the prognostic factors for recurrence after TURBT using molecular markers as well as the scoring system of the EAU. METHODS Eighty-eight patients with primary or recurrent bladder tumors who underwent TURBT followed by the single postoperative immediate instillation of pirarubicin and no further instillations were enrolled between 2003 and 2006; the median follow-up period was 46 months. The time to first recurrence was the primary end point of this study. Patients were divided into EAU recurrence risk groups as follows: low-risk group (total score, 0), intermediate-risk group (total score, 1-9) and high-risk group (total score, 9-17). The intermediate-risk group patients were subdivided into a total score of 1-4 and a total score of 5-9. Immunostaining using Ki-67, pHH3, CK18 and Survivin were performed on the TURBT specimens. RESULTS According to the risk stratification, 5, 82, and 1 were assigned to the low-, intermediate-, and high-risk recurrence groups, respectively. During the follow-up, recurrences were observed in 0% of the low-risk group, 45% (37 out of 82) in the intermediate-risk group and 100% in the high-risk group. We evaluated various predictors of a recurrence-free outcome among the 82 intermediate-risk patients. In univariate analyses, EAU score (1-4, 32.1% vs 5-9, 62.1%; p = 0.0011), high CK18 expression (negative, 31.4% vs positive 88.8%; p < 0.0001), high Ki-67 index (< 5%, 35.4% vs > 5%, 52.5%; p = 0.017) and high Survivin nuclear staining (< 5%, 35.9% vs > 5%, 62.5%; p = 0.004) were associated with recurrence. In a multivariate analysis, EAU score (HR 2.95, p = 0.003) and a high CK18 immunostaining (HR 6.70, p < 0.0001) were independent predictors of disease recurrence. CONCLUSIONS A single immediate chemotherapy instillation is, by itself, insufficient for the treatment of patients in the intermediate- or high-risk recurrence groups defined by the EAU guidelines. Strong immunohistochemical expression of CK18 and the EAU scoring system appeared to be independent predictors of clinical outcome among patients with urothelial carcinoma of the bladder. No significant financial relationships to disclose.

ENHANCED ANTITUMOR EFFECT OF INTRAVESICAL GEMICITABINE THERAPY PLUS BCG IN AN ORTHOTOPIC MURINE BLADDER CANCER MODEL

INTRODUCTION AND OBJECTIVES: Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) is the most successful adjuvant agent for superficial bladder tumors. However, tumors recur in 60 to 70% of the cases, and 30 % of these recurrent tumors present with a higher grade with invasive properties.Gemicitabine is a strong and specific deoxycytidine analog with activity in bladder tumors. To treat superficial bladder tumors that are resistant, we evaluated the combination treatment strategies with intravesical BCG plus Gemicitabine for bladder tumors in a model of orthotopic murine bladder cancer(MBT-2). METHODS: Superficial murine bladder cancer was established by simple instillation of 1 x 106 MBT-2 cells into the lumen of the bladder of female C3H mouse. To assess the antitumoral effect of gemicitabine, intravesical gemicitabine therapy was administered at various doseescalating concentrations; 0(control), 1mg, 2mg, 4 mg and 8mg (n = 8 for each group). Intravesical therapy was administered at 3-days intervals starting on day 5 and repeated 6 times. Next, comparative evaluation of tumor growth among the control group, Gemicitabine-alone group, Gemicabine plus BCG group and BCG-alone group were performed (n= 16 for each group). On day 60 after the initial implantation of the MBT-2 cells, all the surviving mice were sacrificed and necropsied. To evaluate the proliferative activity among the group, Ki67 staining of the tumor cells was performed following corresponding single intavesical administration in an orthotopic bladder tumor model. RESULTS: In the Gemicitabine-only groups of 8 mice each at the dose of 0(control), 1mg, 2mg, 4 mg and 8mg, one, four, four, four, five and four of the mice, respectively, survived. In the 8mg group, three of the eight mice were died due to adverse events. There was no significant difference in the tumor growth rate between any doses of Gemicitabine groups. There was a significant survival advantage in the Gemecitabine plus BCG group (54.1 ± 9.4 days) as compared with that in the BCG-alone group (39.0 ± 16.4 days) (p = 0.02). The Ki67 expression level as the proliferation index was significantly decreased in the Gemecitabine plus BCG group as compared with that in the BCG-alone group (p <0.05). CONCLUSIONS: Our results suggest that intravesical BCG plus Gemicitabine exhibited augmented antitumor effect against bladder tumors. Combined BCG plus Gemicitabine therapy was associated with a reduced proliferation rate as compared to BCG-alone therapy in the orthotopic bladder tumor model.