Presence and passage dependent loss of biochemical M3 muscarinic receptor function in human detrusor cultured smooth muscle cells.

PURPOSE: We explored morphological and biochemical aspects of human detrusor cells in culture as a tool for investigating the physiological mechanisms underlying bladder function and disturbances. MATERIALS AND METHODS: Primary cultures of smooth muscle cells were derived from human bladder specimens of patients with an average age of 70 years undergoing cystectomy. Cultured cells were investigated by morphological, immunocytochemical and Western blot analysis. The alpha-actin content as well as the presence of muscarinic M2 and M3 receptors was determined in cell lysates and in fresh tissue homogenate for comparison. The functional response to muscarinic stimulation was assessed by measuring IP3 production induced by 1 mM. carbachol. RESULTS: Cultured smooth muscle cells showed a characteristic spindle-shaped morphology at early passages. Similarly immunocytochemical and Western blot analysis revealed an alpha-actin cell content that was unmodified up to passage 3. Conversely this marker protein sharply decreased during further passages. M3 muscarinic receptor was present in cultured cells and fresh tissue homogenates, whereas the M2 subtype was evident only in homogenates. Carbachol produced a time dependent increase in IP3 cell content, reaching maximal production after 20 minutes of exposure. This response was passage sensitive. CONCLUSIONS: Cultured human detrusor smooth muscle cells maintain their morphological and biochemical characteristics up to passage 3. With this caveat such cells can be an appropriate tool for investigating the molecular pathways underlying cholinergic activation in normal physiological and pathological bladders, and accordingly for detecting putative targets for pharmacological intervention.

Prejunctional muscarinic inhibitory control of acetylcholine release in the human isolated detrusor: involvement of the M4 receptor subtype.

1. Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response. 2. In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 microM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%). 3. Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 microM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM-1 microM), and enhanced by atropine (0.1-100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release. 4. The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (-log EC50) orders at pre- and postjunctional level were: atropine > or = 4-diphenyl-acetoxy-N-piperidine (4-DAMP) > mamba toxin 3 (MT-3) > tripitramine > para-fluorohexahydrosiladiphenidol (pF-HHSiD) > or = methoctramine > or = pirenzepine > tripinamide, and atropine > or = 4-DAMP > pF-HHSiD >> pirenzepine = tripitramine > tripinamide > methoctramine >> MT-3, respectively. 5. The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.

[Interstitial cystitis: medical treatment]. / La cistite interstiziale: terapia medica.

Interstitial cystitis is a chronic disease of the bladder and its etiology is still unknown. In this article we treat the different approaches to this disease, supported by the fact that a clear therapeutic protocol doesn't exist.

[Minimally invasive treatment of female urinary incontinence due to sphincter incompetence]. / Trattamento mini invasivo della incontinenza urinaria femminile da deficit sfinterico.

Urinary incontinence is one of the most frequent diseases in female urology. There are hundreds of therapeutic opportunities without sure outcomes, because of lack of the etiologic cause. Transurethral injection has good results in around of the 60% of the cases with minimal invasivity, good patients compliance, ripetitivity of the method. From 1993 to 1997 we treated 44 ladies with urinary incontinence due to low pressure of closure of the sphincter: 13 patients (29.55%) disease free, 17 (38.64%) meliorate.

Cisapride enhances detrusor contractility and improves micturition in a woman with lazy bladder.

A 32-year-old woman with lazy bladder was treated with cisapride (10 mg q.i.d.) for three weeks, obtaining a progressive improvement of the symptomatology and urodynamic parameters, and the return to pretreatment conditions after drug withdrawal. This case agrees with the evidence that cisapride potentiates the release of acetylcholine from postganglionic cholinergic nerves in the human isolated detrusor by activating 5-HT4 receptors, i.e. through the same mechanism responsible for its gastrointestinal prokinetic action. It is concluded that selective 5-HT4 agonists could be potentially useful to improve bladder emptying in micturition disorders associated with detrusor hypocontractility.

[Replacement bladders in Oltrepo]. / Le vesciche di sostituzione in Oltrepo.

In our Department we performed 269 radical cystectomies from January 1985 to April 1996. As regard the urinary diversion 64 patients were undergone to a cutaneous ureterostomy; in 137 we performed a Wallace uretero-intestinal anastomosis; in 6 a Mauclaire rectal diversion; in 12 patients a Mainz Pouch II and 50 selected patients (44 man and 6 ladies) we performed a Vesica Ileale Padovana (VIP). In these cases the stage was lowly, good renal function, no are hydronephrosis. The patients were motivated for a long follow-up and preoperatively undergone to urethral biopsies. We have had the following complications: an early bowel obstruction, 4 wound infections and for the long term follow-up 2 inguinal hernias, 1 laparocele, 5 patients with bilateral hydronephrosis. At the moment we cannot state a correct oncological report for the short follow-up: only 1 patient died after 18 months for a pelvic relapse. We can suggest that VIP is a good solution for the ladies: its mandatory to take care of the urethra and the pelvic floor for a good continence limiting the lymphadenectomy. We haven't any suggestions about the deep of the colpectomy and the opportunity of the vaginal suspension.

Neural 5-HT4 receptors in the human isolated detrusor muscle: effects of indole, benzimidazolone and substituted benzamide agonists and antagonists.

1. In strips of human isolated detrusor muscle, the 5-hydroxytryptamine (5-HT) receptor (5-HT4) that mediates facilitation of neuromuscular cholinergic transmission was further characterized by using 5-HT and a series of ligands known for their 5-HT4 agonist (5-methoxytryptamine: 5-MeOT, cisapride, (R,S)-zacopride, BIMU 8) or antagonist (RS 23597, GR 125487, DAU 6285) properties. 2. In the presence of methysergide (1 microM) and ondansetron (3 microM) to isolate pharmacologically the 5-HT4 receptors, 5-HT (0.3 nM-1 microM), 5-MeOT (10 nM -30 microM), BIMU 8 (10 nM-3 microM), cisapride (0.1-10 microM) and (R,S)-zacopride (0.1-30 microM) potentiated cholinergic contractions to electrical field stimulation in a concentration-dependent manner. RS 23597 (10 nM-10 microM), a competitive 5-HT4 receptor antagonist in other systems, also showed agonist properties. The following rank order of potency as an agonist was obtained: 5-HT (pEC50 = 8.0) > RS 23597 (7.0) = BIMU 8 (6.9) > or = cisapride (6.6) > 5-MeOT (6.0) > or = (R,S)-zacopride (5.7). Relative to 5-HT (intrinsic activity = 1), 5-MeOT acted as a full agonist (1.03), while BIMU 8 (0.76), (R,S)-zacopride (0.61), RS 23597 (0.60) and cisapride (0.41) behaved as partial agonists. 3. The potentiation by 5-HT was competitively antagonized by the selective 5-HT4 receptor antagonist GR 125487 (0.3-3 nM) with a pA2 estimate of 9.75 (Schild slope of 1.09), and by DAU 6285 (1 microM; pK3 = 6.45). Additionally, GR 125487 (3 nM) antagonized the responses to 5-MeOT (pKB = 9.72) and reversed the potentiation induced by RS 23597. As an antagonist, RS 23597 (10, 30 and 100 nM) inhibited the response to 5-HT. In addition, 30 and 100 nM RS 23597 reduced the 5-HT response maximum by 30 and 50%, respectively. The pKB value calculated at 10 nM was 8.0. 4. Thus, in the human isolated detrusor muscle, the 5-HT4 receptors mediating facilitation of cholinergic neuromuscular transmission are activated by indoleamines (5-HT, 5-MeOT), substituted benzamide (cisapride, (R,S)-zacopride), benzoate (RS 23597) and benzimidazolone (BIMU 8) derivatives. The activities (in terms of both potency and efficacy) of most agonists, as well as the affinity estimates of the antagonists GR 125487 and DAU 6285, are comparable to those found in other peripheral tissues. Exceptions are RS 23597, which acted either as a partial agonist or as an antagonist of the response to 5-HT1 and 5-MeOT that showed an unusually low potency. The latter findings may be ascribed to differences in the efficiency of receptor coupling mechanisms and/or in the molecular structure (i.e. splice variants) of the 5-HT4 receptor.

[pT1G3 tumor of the bladder]. / Il tumore della vescica pT1 G3.

The management of the superficial bladder tumor is commonly performed by transurethral resection (TUR) and a following chemotherapy or immunotherapy with exception of Tis. A lot of doubts and questions raise in the management of pT1G3 bladder tumor (BT) for the high grade of recurrencies and progressions. Our experience from 1984 to 1994 about 109 patients (pts) with pT1G3 BT undergone to radical cystectomy (47 pts) and TUR (62 pts) allows us to state that when Tis is present a radical cystectomy is mandatory, but the TUR option must be followed to frequent and accurate random biopsies in order to discovery foci of Tis. In the case of pT1G3 monofocal there is always a therapeutic disconcerting.

Quantitative and qualitative AgNORs rates of prostate cancer on needle core biopsies: a multicentric study.

From 1992 to 1993, the Scarpa Foundation Center of Pavia (SFCP) with 12 associated Italian Urological Units selected 40 cases of prostate diseases discovered on needle core biopsies, 5 of which were benign hyperplasias (BPH) in patients without clinical and morphological evidence of cancer and 35 prostate cancers (PRC) classified according to Gleason's histological grades (GLG) of PRC malignancy. Serum prostatic specific antigen (PSA) values were tested before clinical urological examination or biopsy or surgery. In all groups, AgNORs scores/nucleus were obtained by semi- and -automatic computerized image analysis and also by qualitative subjective counts of three observers on light microscopy. Our results pointed out a good correlation between PSA levels, GLG of PRC malignancy and AgNORs scores. The quantitative method showed an average number of AgNORS dots per nucleus between 2 and 3 in well differentiated PRC and higher than 3 in moderately differentiated and undifferentiated PRC, and exhibited more sensitivity over GLG3 than the qualitative investigation. The qualitative subjective count of AgNORs dots/nucleus seemed to be more reliable in differentiating the AgNORs scores of BPH (average of 1.81 dots/nucleus) from very well differentiated PRC with GLG1 + 2(average of 2.25 dots/nucleus) than quantitative analysis, which showed the same average value in both groups (2.11 dots/nucleus). For these reasons, also on needle biopsies of benign and malignant prostate diseases the subjective AgNORs count may aid the histological diagnostic judgement of malignancy, by avoiding misleading diagnoses of microscopic pictures of BPH cancer look likes and a predictive histologic malignant factor, in identifying PRC with low or high progression.

[Incision of the bladder neck]. / L'incisione del collo vescicale.

Transurethral incision of the bladder neck (TUIBN) is a simple method of relieving bladder outflow obstruction producing similar results to transurethral resection of the prostate (TURP) when obstruction is caused by small prostate (< 40 gr). The complication rate is low and retrograde ejaculation has a far less degree than after TURP, so less bleeding. The major advantage of TUIBN are: short hospitalization, less bladder neck contractures. The drawback of the absence of prostate tissue for an earlier diagnosis of cancer can be balanced performing a biopsy before treatment.

[Intraurethral devices in the treatment of prostatic obstruction]. / I dispositivi intrauretrali nella terapia dell'ostruzione prostatica.

We describe our experience (started in 1988) about the use of urethral stents implanted into patients with prostatic outflow obstruction, unit for prostatic surgery. This technique has been applied successfully with few complications. The use of different stents (Fabian's spiral, mesh stent) allowed us to get the right choices in the different shapes of prostatic urethra and in the different expectancies of the patients. Lastly we can offer an useful recommendation about the patients evaluation and the stents selection.

[Immunotherapy with low-dose subcutaneous interleukin-2 in metastatic renal carcinoma]. / Immunoterapia con basse dosi di interleuchina-2 per via sottocutanea nel carcinoma renale metastatico.

The intravenous injection of interleukin-2 (IL-2) has appeared to induce tumor regression in metastatic renal cell carcinoma (RCC). IL-2 given subcutaneously has also appeared to be effective when it is administered in association with interferon-alpha (INF), but with a lower toxicity in comparison to the intravenous route of administration. The present study was carried out to evaluate the efficacy of a subcutaneous immunotherapy with IL-2 alone in metastatic RCC. The study included 30 consecutive patients affected by metastatic RCC, 14 of whom had been pretreated with INF plus vinblastine, while the other 16 patients received IL-2 as a first line therapy of their metastatic disease. IL-2 was given subcutaneously at a dose of 3 million IU twice/day for 5 days/week for 6 consecutive weeks, corresponding to one cycle of immunotherapy. No complete response was obtained. A partial response (PR) was achieved in 10/30 (33%) patients (median duration: 7 months, range 5-25), without any significant difference between patients pretreated with IFN and nonpretreated patients (4/14 vs 6/16). Response rate was significantly higher in nephrectomized patients than in those who did not undergo nephrectomy (10/25 vs 0/5; P < 0.01). Moreover, response rate was significantly higher in patients with performance status (PS) greater than 40% than in those with PS lower than 40% (10/23 vs 0/7; P < 0.01). A stable disease (SD) was obtained in 12/30 (40%) patients (median duration 5 months, range 3-13), while the remaining 8/30 (27%) progressed. The increase in lymphocyte and eosinophil mean number was significantly higher in patients with PR or SD than in the progressed ones.(ABSTRACT TRUNCATED AT 250 WORDS)

[Experience with rIL2 in the treatment of metastatic renal carcinoma]. / Esperienze con rIL2 nel trattamento del carcinoma renale metastatizzato.

Eight patients with metastatic renal cell carcinoma, all nephrectomized, were treated with rIL2 continuous i.v. infusion or subcutaneous administering (+/- INF). Six patients alive. Continuous infusion is possible event out of Intensive Care Unity. Adverse effects, sometimes serious, rapidly decrease with suspension. Subcutaneous infusion is more easy and with lower adverse effects. We don't know methodic of which can give more security.

[Interferon and vinblastine in advanced renal carcinoma]. / Interferone e vinblastina nel carcinoma renale avanzato.

19 patients with renal adenocarcinoma and mets were treated with INF alfa 2b (10 x 10(6) U/m square s.c. three times weekly) plus VBL (0.1 mg/kg every 3 weeks). We followed up the patients for 12 weeks with monitoring of major parameters. The outcomes have been positive in 16.6% of the cases with mean survival of 20.7 months. We point out the efficacy in lung mets. The outcomes of therapy with INF alfa 2b plus VBL are unsuccessful and we state that an alternative solution could be interleukin 2.

Measurement of estrogen receptors in normal and neoplastic lymph node tissue from dogs.

Estrogen receptors were measured in normal canine lymph nodes and neoplastic tissue from dogs with lymphoma, using a commercially available [3H]estradiol dextran-coated charcoal assay. Using the same assay, estrogen receptors were detected in the positive-control tissues--dog uterus, rat uterus, and lyophilized bovine uterus. Specific binding of [3H]estradiol was not detected in rat skeletal muscle or in any of the canine lymphoid tissues, indicating that the specimens did not contain estrogen receptors.

Recombinant alpha interferon in metastatic renal cell carcinoma. A cooperative phase II study.

A total of 61 evaluable patients with advanced renal cell carcinoma have been treated with 3 x 10(6) IU per square meter of body surface with recombinant alpha 2b interferon three times a week within a Cooperative Phase II Study. Toxic death for terminal renal failure occurred in 1 patient (1.63%), and toxicities greater than WHO grade 2 were present in 10 cases (16%). The overall response rate after six months of treatment was 13.1% (partial response 4, minor response 4). Two complete responses were obtained at nine and fifteen months (3.3%). Long-lasting stabilization of disease was 13.1 percent.

[Carcinoma of the urethra in patients having undergone cystectomy for neoplasm of the bladder]. / Il carcinoma dell'uretra nei pazienti cistectomizzati per neoplasia vescicale.

The multifocality of the cancer of the urethra is a concept already known so for time, therefore urethral lesions are expected in patients with carcinoma of the bladder with an incidence which varies from 4% to 18%. A retrospective study of incidence of transitional cell carcinoma of the urethra of personal sample of patients having cystectomy is reported. Of 259 patients who underwent cystectomy 13 developed a urethral tumour; 7.7% according to the international literature. The paper regards the characteristics of the urethral tumour by defining patients liable to risk where the carcinoma is plurifocal, or situated at the neck of the bladder and where frequent endoscopic operations have been performed or when there is a urethral "ca in situ". In these cases cystourethrectomy is suggested while the urethra is removed in presence of positive cytology in other forms. According to the authors' experience the survival of patients who have undergone a urethrectomy after cystectomy is 46%.

Peripheral nerve biopsy for diagnosis of globoid cell leukodystrophy in a dog.

Saphenous nerve biopsy was performed to diagnose globoid cell leukodystrophy in a 3-month-old West Highland White Terrier. The dog had progressive neurologic disease that appeared to involve the cerebellum, spinal cord, and lower motor neurons to the hind limbs. Light and transmission electron microscopic findings of the nerve biopsy specimen established the diagnosis. Peripheral nerve biopsy may provide a reliable antemortem method for diagnosis of globoid cell leukodystrophy.