Yat-Ching Tong

Alterations in urinary bladder M2-muscarinic receptor protein and mRNA in 2-week streptozotocin-induced diabetic rats

The M2 receptor (M2-mAChR) is quantitatively the dominant muscarinic subtype in animal bladders. The alterations in its protein quantity and biosynthesis during diabetic cystopathy were investigated. Three-month-old male Wistar rats were divided into two groups: (1) 2-week-old diabetics; and (2) normoglycemic control rats. Diabetes was induced by single intravenous injection of 60 mg/kg streptozotocin. The amount of M2 receptor protein in the rat bladder body tissue was measured by Western immunoblotting using monoclonal antibodies. For determination of M2 muscarinic receptor mRNA in the bladder tissue, the method of Northern blotting was employed. The results of the Western immunoblotting showed that the amount of M2-mAChR protein in the diabetic bladder was significantly increased by 40.0 +/- 6.2% when compared with the control bladder (P < 0.05, n = 8). The Northern blotting demonstrated a 69.3 +/- 8.5% increase of the M2-mAChR mRNA in the diabetic bladder (P < 0.05, n = 8). The findings of the present study demonstrated an up-regulation of M2-mAChR biosynthesis in the diabetic urinary bladder. This phenomenon could lead to increased reactivity to acetylcholine and thus results in detrusor instability.

Transitional Cell Carcinoma in Dialysis Patients

Objective: The aim of our study was to determine whether there is an increased incidence of urothelial cancer, especially transitional cell carcinoma (TCC), in uremic patients on dialysis.Methods: Retrospective chart analyses were completed for 1,910 uremic patients undergoing maintenance dialysis between January 1987 and December 1997. The incidence of urinary tract cancer was assessed. Only the patients with cancers diagnosed after start of dialysis were enrolled in the study.Results: Of the 1,910 patients, 70 had concomitant urinary tract cancers. Nineteen patients (0.99%), including 17 patients with TCC and 2 patients with renal cell carcinoma, were diagnosed after the initiation of dialysis. The average duration from dialysis to TCC diagnosis was 38.3 (range 2–144) months. Painless gross hematuria was the cardinal symptom in 16 of the 17 patients with TCC. In the 17 patients with TCC, no distant metastases were found at the time of diagnosis. Fourteen patients (82.3%) were stage 0 or A, and 1 patient was stage B1.Conclusions: The 0.89% incidence of TCC in our dialysis patients was high as compared with that of the general population. The risks of developing urinary TCC in dialysis patients were examined, and we suggest that immunosuppressive stage, dialysis procedure, and chronic bladder irritation (decreased urinary wash effect) may play a part in the development of urinary TCC in dialysis patients. Early detection of hematuria due to regular visits and decreased exposure of urinary tract epithelium to carcinogens from urine may explain why early–stage TCC was seen in most of our patients.

Prognostic factors of primary transitional cell carcinoma of the upper urinary tract

OBJECTIVES We presented and analyzed our results in order to determine the relationship between patient survival and tumor grade and/or stage. In addition, a retrospective tumor DNA ploidy study was done to evaluate its possible role in predicting future tumor recurrence in the bladder. METHODS A total of 112 patients with upper urinary tract transitional cell carcinomas (TCCs) were recorded at our hospital. Of these, 68 patients without concurrent bladder tumors (ages ranged from 36 to 80, mean 62.4 years; male:female = 1:1.2) were treated by nephroureterectomy and bladder cuff resection. They were followed up for 14-79 months (average 38.2 months). Eight (36.4%) of the 22 patients who had stage C or D tumors had received adjuvant systemic methotrexate, vinblastine, epirubicin, cisplatin chemotherapy after surgery. DNA flow cytometry using paraffin-blocked tumor specimens was performed on the tumors of 52 patients. RESULTS Their pathologic stages and grades were 11 at stage 0, 15 at stage A, 20 at stage B, 14 at stage C, 8 at stage D; 9 of grade I, 41 of grade II, and 18 of grade III. Postoperatively, 13 patients (19.1%) subsequently developed bladder tumors with a latent period ranging from 2 to 37 months (average 14.9 months). The difference of the tumor DNA ploidy distribution pattern among tumors of high versus low stages and/or grades is not statistically significant (p > 0.05). Overall, the 5-year survival rates for patients with low- and high-stage tumors were 100 and 66.7%, respectively; for patients with grade I-II and III tumors they were 93.6 and 28.3%, respectively. CONCLUSIONS Patient survival was mainly related to both tumor stages (p = 0.0037) and grades (p = 0.0001), rather than to tumor DNA ploidy. For patients with grade II upper urinary tract tumors, tumor DNA ploidy seems to provide no additional predictive value on subsequent tumor recurrence in the bladder.

The norepinephrine tissue concentration and neuropeptide Y immunoreactivity in genitourinary organs of the spontaneously hypertensive rat

Tissue concentration of norepinephrine and neuropeptide-Y immunoreactivity (NPY-IR) were measured in the urinary bladder, urethra, prostate and corpus cavernosum of the spontaneously hypertensive rat, as well as the normotensive Wistar-Kyoto rat. The results showed significantly increased tissue norepinephrine concentrations in the urinary bladder, urethra and prostate of the spontaneously hypertensive rat when compared to those of the normotensive rat (hypertensive, n = 18: 18.3 +/- 2.1, 14.9 +/- 1.7, 22.6 +/- 2.3 vs. normotensive, n = 18: 11.2 +/- 1.9, 10.4 +/- 1.3, 16.7 +/- 2.4 nmol/g tissue, respectively, P < 0.05 in each case). No difference was noted in the cavernosal tissue (hypertensive, n = 18: 11.3 +/- 1.6 vs. normotensive, n = 18: 10.1 +/- 1.8 nmol/g tissue, P > 0.01). Correspondingly, tissue NPY-IR was significantly increased in the bladder, urethra and prostate tissue of the spontaneously hypertensive rat (hypertensive, n = 18: 39.7 +/- 5.6, 25.3 +/- 3.4, 31.5 +/- 2.8 vs. normotensive, n = 18: 27.4 +/- 3.1, 18.6 +/- 2.7, 24.2 +/- 3.2 pmol/g tissue, respectively, P < 0.05 in each case). Again, no significant difference was observed in the cavernosal tissue (hypertensive, n = 18: 15.9 +/- 2.2 vs. normotensive, n = 18: 14.8 +/- 2.6 pmol/g tissue, P > 0.01). It is therefore concluded that increased tissue concentration of norepinephrine and NPY-IR were present in the urinary bladder, urethra and prostate of the spontaneously hypertensive rat. The significance of such biochemical findings needs further investigation but may suggest increased sympathetic innervation or activity. On the contrary, no corresponding changes were observed in the corpus cavernosum of the hypertensive rat.

EVIDENCE OF ADENOSINE 5′-TRIPHOSPHATE RELEASE FROM NERVE AND P2X-PURINOCEPTOR MEDIATED CONTRACTION DURING ELECTRICAL STIMULATION OF RAT URINARY BLADDER SMOOTH MUSCLE

PURPOSE We provided direct evidence for the existence of purinergic innervation in the rat urinary bladder. MATERIALS AND METHODS The non-adrenergic non-cholinergic (NANC) innervation was studied in 4-month-old Wistar rats. Electric-field stimulation (EFS) of the detrusor muscle strips in the presence of four autonomic blockers (atropine 10(-6) M, guanethidine 10(-6) M, phentolamine 10(-6) M and propranolol 10(-6) M) showed NANC contractions accounted for about 50% of the maximum contractile response. The adenyl purines released from nerves by EFS were detected by HPLC after conversion to ethenopurines. The amount of total purine released was frequency-dependent and could be totally suppressed by tetradotoxin (10(-6) M). The amount of ATP released was significantly greater than those for ADP, AMP and adenosine (p < 0.05, n = 4). Desensitization induced by alpha, beta-MeATP (10(-6) to 10(-4) M), a P2x receptor agonist, reduced the NANC contraction. In addition, the NANC contraction was also abolished by P2 receptor blocker suramin (10(-4) to 10(-3) M) and P2x receptor blocker PPADS (10(-5) to 10(-4) M.). CONCLUSION The results of the present study give evidence to support purinergic nerve-mediated bladder smooth muscle contractions in the rat. Among the purine nucleotides, ATP is the dominant purinergic neurotransmitter released and P2x receptor activation is responsible for the NANC contractile response.

Alterations of M2-muscarinic receptor protein and mRNA expression in the urothelium and muscle layer of the streptozotocin-induced diabetic rat urinary bladder

Diabetes associated alterations of M2-muscarinic receptors (M2-mAChR) in the urothelium and muscle layer of the urinary bladder were studied using streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into two groups; group I: normal control rats; group II: STZ-induced diabetic rats, 2 weeks after induction. The bladder was divided into urothelium and muscle layer by microdissection. Tissue M2-mAChR protein levels were measured by Western blotting. Expression of the mRNA that encoded M2-mAChR was estimated using the method of reverse transcription combined with polymerase chain reaction (RT-PCR). M2-mAChR protein and mRNA expressions were found in both the urothelium and muscle layer of the rat urinary bladder. In control rats, the M2-mAChR protein expression ratio in the urothelium and muscle layer was 1:1.66; that for mRNA was 1:0.97. Two weeks after induction of diabetes, the M2-mAChR mRNA expression in the urothelium and muscle layer were significantly increased by 44.4% (P<0.01, n=8) and 28.6% (P<0.01, n=8), respectively. Correspondently, the bladder M2-mAChR protein levels were significantly increased by 33.3% (P<0.001, n=8) in the urothelium and 25.3% (P<0.01, n=8) in the muscle layer of the diabetic rats. In conclusion, M2-mAChR mRNA and protein are expressed in both the urothelium and muscle layer of the rat urinary bladder. STZ-induced diabetes increases mRNA and protein expression of the M2-mAChR in the urothelium as well as the muscle layer.

Alteration of M(3) subtype muscarinic receptors in the diabetic rat urinary bladder.

The M3 receptor (M3-mAChR) is the major muscarinic subtype in the animal bladder responsible for detrusor contraction. The alterations in its protein quantity and biosynthesis during diabetic cystopathy were investigated. 3-month-old male Wistar rats were divided into two groups: (1) 2-week diabetic rats and (2) normoglycemic control rats. Diabetes was induced by a single intravenous injection of 60 mg/kg streptozotocin. The amount of M3 receptor protein in the rat bladder body tissue was measured by Western immunoblotting using monoclonal antibodies. For determination of M3 muscarinic receptor mRNA in the bladder tissue, the method of Northern blotting was employed. The results of the Western immunoblotting showed that the amount of M3-mAChR protein in the diabetic bladder was significantly increased by about 70.2 ± 8.5% when compared to the control bladder (p < 0.05, n = 8). Northern blotting demonstrated a 54.7 ± 6.0% increase of M3-mAChR mRNA in the diabetic bladder (p < 0.05, n = 8). The findings of the present study demonstrated an upregulation of M3-mAChR biosynthesis in the diabetic urinary bladder. This phenomenon offers an explanation of the increased contractility after muscarinic stimulation of the detrusor muscle of diabetic animals.

Pumpkin seed oil and phytosterol-F can block testosterone/prazosin-induced prostate growth in rats.

Introduction: This study was undertaken to investigate the effects of pumpkin seed oil alone or combined with Phytosterol-F on testosterone/prazosin-induced (T-P) prostate growth in rats. Materials and Methods: Forty adult Wistar rats were divided into five groups, including: one control group, rats treated with vehicle only, one group treated with T-P, and two groups of T-P-treated rats, one receiving orally pumpkin seed oil alone and one group receiving orally pumpkin seed oil combined with Phytosterol-F. Two weeks later, the prostatic weight-to-body weight ratio was determined after sacrifice. The total protein concentration was measured by using a protein assay. Some ventral prostatic tissues were histologically examined after hematoxylin-eosin staining. Results: Histological sections of the ventral prostate showed that the architecture of the prostate glands became hyperplastic in the T-P rats, but not in the control or vehicle-treated animals. As compared with the control or vehicle group, T-P rats had a significantly higher prostatic weight-to-body weight ratio for the ventral prostate (p = 0.05 and p = 0.007, respectively), but not for the dorsolateral prostate (p = 0.53 and p = 0.73, respectively). The T-P rats had significantly higher protein levels within both lobes (ventral lobe, p = 0.02 and p < 0.0001, respectively; dorsolateral lobe, p = 0.06 and p = 0.005, respectively). As compared with the T-P-alone rats, the TP rats treated with pumpkin seed oil alone or pumpkin seed oil combined with Phytosterol-F had a significantly lower weight ratio for the ventral prostate (p = 0.01 and p = 0.004, respectively) and significantly lower protein levels within both lobes (p = 0.03 and p = 0.003, respectively; p = 0.007 and p = 0.002, respectively). In addition, Phytosterol-F had some additive effect on the total protein synthesis within the ventral prostate (p = 0.02). Conclusion: Pumpkin seed oil alone or combined with Phytosterol-F can block the T-P-induced increases in prostatic weight-to-body weight ratio and protein synthesis.

Changes of M3-muscarinic receptor protein and mRNA expressions in the bladder urothelium and muscle layer of streptozotocin-induced diabetic rats.

Diabetes induced alterations of M3-muscarinic receptors (M3-mAChR) in the urothelium and muscle layer of the urinary bladder were studied using streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into two groups; group I: normal control rats; group II: STZ-induced diabetic rats, 2 weeks after induction. The bladder was divided into urothelium and muscle layer by microdissection. Tissue M3-mAChR protein levels were measured by Western blotting. Expression of the mRNA that encoded M3-mAChR was estimated using the method of reverse transcription combined with polymerase chain reaction (RT-PCR). M3-mAChR protein and mRNA expressions were found in both the urothelium and muscle layer of the rat urinary bladder. In control rats, the M3-mAChR protein expression ratio in the urothelium and muscle layer was 1:1.87; that for mRNA was 1:0.74. Two weeks after induction of diabetes, the M3-mAChR mRNA expression in the urothelium and muscle layer were significantly increased by 45.7% (P<0.01, n=8) and 80.8% (P<0.001, n=8), respectively. Correspondingly, the bladder M3-mAChR protein levels were significantly increased by 50.0% (P<0.01, n=8) in the urothelium and 147.1% (P<0.001, n=8) in the muscle layer of the diabetic rats. In conclusion, M3-mAChR mRNA and protein are expressed in both the urothelium and muscle layer of the rat urinary bladder. STZ-induced diabetes increases mRNA and protein expression of the M3-mAChR in the urothelium as well as the muscle layer.

Effects of Pregnancy and Progesterone on Autonomic Function in the Rat Urinary Bladder

Previous studies have shown that pregnancy is associated with a decrease in cholinergic function in the rabbit urinary bladder. The present study aimed at evaluating the effects of pregnancy on the autonomic function of the rat urinary bladder and to elucidate whether progesterone is responsible for such alterations. Female Wistar rats, 3 months old, were divided into four groups: (1) 2-week pregnant rats; (2) rats given daily intramuscular injections of progesterone 5 mg/kg for 2 weeks; (3) rats given intramuscular injections of vehicle for 2 weeks, and (4) controls. Cystometry showed a significant increase in bladder capacity in the pregnant rats. The wet weight of the pregnant rat bladder was also significantly increased. Histologic study revealed increased bladder wall thickness with interstitial edema and urothelium proliferative changes to a papillary configuration in these pregnant bladders. Bladder muscle strip study showed significantly reduced maximum contractile responses to acetylcholine and methoxamine in the pregnant and the progesterone groups. Muscarinic receptor binding study demonstrated reduced Bmax in the pregnant rats and rats receiving progesterone injections (control group Bmax = 57 +/- 11, pregnant group Bmax = 44 +/- 8, p < 0.05; progesterone group Bmax = 40 +/- 7, vehicle group Bmax = 58 +/- 9 fmol/mg protein, p < 0.05). The contractile response to lower concentrations (10(-6) mol/l to 10(-4) mol/l) of ATP was elevated in the pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS)