Urothelium dependent inhibition of rat ureter contractile activity.

PURPOSE: We investigated whether urothelium modulates isolated rat ureter contractions. MATERIALS AND METHODS: Segments of intact and urothelium-free ureters were placed in organ baths at 37C. The contractile effects of KCl and endogenous ureteral contractile agents were recorded in the absence and presence of the cyclooxygenase inhibitors indomethacin (1 microM) or ketoprofen (10 microM). The effect of the prostacyclin analogue iloprost was tested on the KCl and agonist induced responses obtained in the presence of ketoprofen. RESULTS: Without stimulation ureters were quiescent but spontaneous contractions often developed in urothelium-free ureters. Sensitivity to KCl was greater in the absence of urothelium. In intact ureters neurokinin A and vasopressin induced rhythmic contractions, whereas carbachol, norepinephrine, bradykinin and angiotensin II were inactive. In urothelium-free ureters the response to neurokinin A and vasopressin was enhanced and the other agonists, except norepinephrine, promoted contractions. In the presence of cyclooxygenase inhibitors intact ureters responded to carbachol, bradykinin and angiotensin II, and the response to neurokinin A, vasopressin and KCl increased. Responses obtained in urothelium-free ureters were not affected by the presence of cyclooxygenase inhibitors. In the presence of ketoprofen iloprost antagonized the KCl and agonist induced contractile effects in intact but not in urothelium-free ureters. CONCLUSIONS: Data suggest that the urothelium prevents spontaneous contractile activity and decreases the potential excitatory effects of endogenous contractile agents on ureteral motility. The mechanism underlying this inhibitory effect appears to involve the participation of a urothelial cyclooxygenase product such as prostacyclin, which could activate the release of urothelium derived relaxing factor(s) that are as yet unknown.

Retinoblastoma epidemiology: does the evidence matter?

It has been proposed that retinoblastoma is 'caused' by two sequential mutations affecting the RB1 gene, but this is a rather outdated view of cancer aetiology that does not take into account a large amount of new acquisitions such as chromosomal and epigenetic alterations. Retinoblastoma remains probably the only cancer in which the rather simplistic 'two hit' mutational model is still considered of value, although cancer is known to be associated with genomic and microsatellite instability, defects of the DNA mismatch repair system, alterations of DNA methylation and hystone acethylation/deacethylation, and aneuploidy. Moreover, as it is shown herein, the predictions made by the 'two hit' model, are not fulfilled by the clinical and epidemiological data reported so far. Moreover, while the role of mutational events in cancer has been largely questioned in the more recent literature, no serious effort has been done to investigate the role of epigenetic alterations and aneuploidy in retinoblastoma. Through the analysis of the specialised literature and a set of original epidemiological and biological data concerning retinoblastoma, the authors illustrate the evidences arguing against the 'two hit' hypothesis and propose that epigenetic factors and aneuploidy play central roles in the disease.

Phthisis bulbi and buphthalmos as presenting signs of retinoblastoma: a report of two cases and literature review.

PURPOSE: To report two cases of bilateral retinoblastoma (RB) with unusual presentations. METHODS: The medical records of 321 patients from the Retinoblastoma Referral Center in Siena were reviewed. A total of 111 patients had bilateral RB, 2 of them presenting with phthisis bulbi and buphthalmos. Both patients underwent bilateral enucleation. Clinical features, imaging studies, and histopathology were reviewed. RESULTS: These 2 cases represent 0.62% (2/321) in our series. Histopathology did not reveal viable tumor cells in the phthisical eyes; in both buphthalmic eyes the tumor was active, infiltrating the choroid and optic nerve. CONCLUSIONS: Phthisis bulbi and buphthalmos are unusual presenting signs of RB. This very rare combination of these two signs in different eyes of the same patient is probably due to a delay in diagnosis.

Nitric oxide production within rat urothelial cells.

PURPOSE: Recent studies have suggested that nitric oxide (NO) synthase (NOS) may be localized in the urothelium of the proximal part of the mammalian ureter. We investigated endogenous NO production in the proximal half of the rat ureter, localized its cellular source, characterized the NOS isoforms involved and assessed the impact of NO on ureteral motility. MATERIALS AND METHODS: Direct detection of NO production was performed on primary cultures of living rat ureteral cells with the fluorescent indicator diaminofluorescein. Cultures were incubated with the NO precursor L-arginine or the NOS inhibitors L-NAME (N-nitro-L-arginine-methyl ester) and 1400W. NOS expression was determined by immunofluorescence and Western blot analysis. The functional effects of NO donors were assessed on isolated ureters. RESULTS: Significant basal NO production was demonstrated by the high fluorescence level detected in diaminofluorescein treated cell cultures. NO production was strictly limited to urothelial cells since no fluorescence was seen in smooth muscle cells. Pretreatment with L-NAME or 1400W resulted in a significant decrease in fluorescence. Constitutive and inducible NOS isoforms were detected in urothelial cultured cells and in lysates of the urothelial layer. NO donors inhibited in a concentration dependent manner the agonist induced contractile activity of isolated ureters. CONCLUSIONS: These results suggest that NO production stems from the urothelium and the NO pathway inhibits contractile activity in the proximal half of the rat ureter. Hence, the nitrergic pathway may be an important target for drugs producing relaxation of the mammalian ureter.

Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation.

We have examined the role of mechanical tension in myofibroblast differentiation using two in vivo rat models. In the first model, granulation tissue was subjected to an increase in mechanical tension by splinting a full-thickness wound with a plastic frame. Myofibroblast features, such as stress fiber formation, expression of ED-A fibronectin and alpha-smooth muscle actin (alpha-SMA) appeared earlier in splinted than in unsplinted wounds. Myofibroblast marker expression decreased in control wounds starting at 10 days after wounding as expected, but persisted in splinted wounds. In the second model, granuloma pouches were induced by subcutaneous croton oil injection; pouches were either left intact or released from tension by evacuation of the exudate at 14 days. The expression of myofibroblast markers was reduced after tension release in the following sequence: F-actin (2 days), alpha-SMA (3 days), and ED-A fibronectin (5 days); cell density was not affected. In both models, isometric contraction of tissue strips was measured after stimulation with smooth muscle agonists. Contractility correlated always with the level of alpha-SMA expression, being high when granulation tissue had been subjected to tension and low when it had been relaxed. Our results support the assumption that mechanical tension is crucial for myofibroblast modulation and for the maintenance of their contractile activity.

Sepsis decreases the spontaneous and agonist-induced contractile activities in the rat portal vein.

BACKGROUND/AIMS: The portal vein has spontaneous and agonist-induced contractile activities and whether sepsis alters these two types of contractile activities is unknown. METHODS: To study the effect of sepsis on the spontaneous contractile activity and the contractile responses to norepinephrine (NE), angiotensin II (AT(II)), and neurokinin B (NKB) in the rat portal vein (RPV), we performed a cecal ligature and puncture (CLP) 24 h before RPV isolation. RESULTS: CLP decreased the spontaneous activity and induced hyporesponsiveness to AT(II) and NKB. The vascular failure was correlated to the severity of sepsis. In contrast, the reactivity to NE was not altered. Although inducible NO synthase was detected in RPV isolated from CLP rats, NO synthase inhibitors did not restore either the responsiveness to AT(II) and NKB or the spontaneous activity. Additionally, hyporesponsiveness to AT(II) and NKB was not modified by indomethacin. CONCLUSIONS: CLP decreases the spontaneous activity of the RPV as well as the contractile responses to AT(II) and NKB. The vascular failure is correlated to the severity of sepsis. The reactivity to NE is not altered in this model. Neither NO nor prostaglandins are responsible for the vascular abnormalities observed during CLP.

Metachronous tumor development in unilateral retinoblastoma.

PURPOSE: A series of 205 retinoblastoma (RB) patients referred to the Department of Ophthalmology at the University of Siena (Italy) was evaluated in order to assess the proportion of unilateral cases later developing tumors in the companion eye ("metachronous" bilateral retinobastoma) (MBRB). METHODS: The total number of unilaterally affected patients developing tumors in the fellow eye was recorded and the risk factors assessed for the development of asynchronous bilateral retinoblastoma, i.e., family history, tumor multifocality and early age at diagnosis. RESULTS: Only two out of 133 (1.5%) unilateral retinoblastoma patients in our series could be considered affected by MBRB. CONCLUSIONS: The incidence of MBRB in our series was negligible (1.5% of all unilateral cases) compared to other reports. None of the reported risk factors for the development of tumors in the fellow eye was relevant in the present series. Although close follow-up of some unilateral cases is still recommended, thorough examination of the fellow eye, to search for lesions in the peripheral retina, is essential in all cases of unilateral RB. MBRB may be a distinctive clinical entity with specific clinical, genetic and prognostic features. However, all these aspects need to be better investigated in larger series.

Diclofenac and NS-398, a selective cyclooxygenase-2 inhibitor, decrease agonist-induced contractions of the pig isolated ureter.

Non-steroidal anti-inflammatory drugs (NSAIDs) are currently considered a first-line treatment of renal colic. Their action has been ascribed to the inhibition of renal prostaglandin synthesis, which decreases renal blood flow and diuresis, and consequently lowers the pressure in the renal pelvis and ureter. However, the effects of NSAIDs on induced contractions of ureteral smooth muscle have received little attention. Also, there is a lack of clinically relevant spasmolytic drugs for the ureter. Therefore, we studied the influence of the non-selective cyclooxygenase (COX) inhibitor diclofenac, a NSAID drug customarily used in the treatment of renal colic, and of NS-398, a selective COX-2 inhibitor, on induced contractions of the pig ureter. Serotonin (0.1-30 microM), norepinephrine (0.1-30 microM) and neurokinin A (0.03-10 microM) induced reproducible concentration-dependent contractions, which were inhibited by diclofenac and NS-398 (10-300 microM) in a concentration-dependent manner. The sensitivity of neurokinin A-induced contractions to diclofenac was 3-4 times greater than that of the amines. Depending on the concentration, inhibition ranged between 25 and 96% of the initially induced contractile activity. In the presence of inhibitors, supramaximal concentrations of agonists were unable to trigger recuperation of the initially induced contractions. Prostaglandin F2alpha did not reverse the effect of diclofenac on agonist-induced contractions. Removal of diclofenac or NS-398 from the organ baths showed that the inhibition was totally reversible. Thus, the non-selective COX inhibitor diclofenac and the selective COX-2 inhibitor NS-398 are almost equipotent in reducing agonist-induced contractions in the isolated porcine ureter. Although the clinical relevance of this spasmolytic effect remains to be demonstrated, the data suggest that patients suffering from renal colic may benefit not only from the anti-diuretic and analgesic effects of diclofenac, but also from its potential spasmolytic properties. Moreover, selective COX-2 inhibitors may have clinical potential, as they may cause fewer side effects.

Hepatic vascular response to norepinephrine during endotoxemia in anesthetized pigs.

To investigate the systemic and hepatic reactivity to norepinephrine (NE) during chronic endotoxemia, we measured the reactivity of the drug in pigs infused with endotoxin (End, 160 ng/kg/min) during an 18-h period. At the end of the experiments, the hepatic vessels were removed to test the hepatic vascular reactivity in vitro. The pressive response to NE did not change over time in control (Ctrl) pigs, but endotoxin infusion decreased the response at t = 11 and 17 h. The reactivity of the portal vein blood flow did not change in Ctrl pigs but was significantly increased in End pigs at t = 5 h. Finally, endotoxin decreased the contractile response to NE only in transversal strips of portal veins isolated from End pigs. Thus, in this model, the decreased pressive response to NE develops over time, but the modifications of the hepatic vascular reactivity remain minor.

The nitric oxide pathway in pig isolated calyceal smooth muscle.

In pig and humans, whose kidneys have a multi-calyceal collecting system, the initiation of ureteral peristalsis takes place in the renal calyces. In the pig and human ureter, recent evidence suggests that nitric oxide (NO) is an inhibitory mediator that may be involved in the regulation of peristalsis. This study was designed to assess whether the NO synthase/NO/cyclic GMP pathway modulates the motility of pig isolated calyceal smooth muscle. Immunohistochemistry revealed a moderate overall innervation of the smooth muscle layer, and no neuronal or inducible NO synthase (NOS) immunoreactivities. Endothelial NOS immunoreactivities were observed in the urothelium and vascular endothelium, and numerous cyclic GMP-immunoreactive (-IR) calyceal smooth muscle cells were found. As measured by monitoring the conversion of L-arginine to L-citrulline, Ca(2+)-dependent NOS activity was moderate. Assessment of functional effects was performed in tissue baths and showed that NO and SIN-1 decreased spontaneous and induced contractions of isolated preparations in a concentration-dependent manner. In strips exposed to NO, there was a 10-fold increase of the cyclic GMP levels compared with control preparations (P < 0.01). It is concluded that a non-neuronal NOS/NO/cyclic GMP pathway is present in pig calyces, where it may influence motility. The demonstration of cyclic GMP-IR smooth muscle cells suggests that NO acts directly on these cells. This NOS/NO/cyclic GMP pathway may be a target for drugs inhibiting peristalsis of mammalian upper urinary tract. Neurourol. Urodynam. 18:673-685, 1999.

Binding of gold-protein ligand complexes to neurokinin receptors. visualization with the electron microscope on pig coronary artery and rat portal vein.

Active neurokinin (NK) receptors were visualized with 5-nm colloidal gold-protein-substance P (GPSP) and -senktide (GPSenk) complexes on vascular tissues. Electron micrographs of pig coronary strips incubated with GPSP showed gold particles either bound to the plasmalemma or inside intracytoplasmic vesicles of endothelial cells. Preincubation with SP or the NK1 receptor antagonist L-703606 prevented GPSP marking. No gold particles were seen after incubation with GPSenk. On coronary strips in vitro, which had been precontracted with U46619, GPSP induced relaxations similar to those produced by equimolar concentrations of SP, both relaxations being inhibited by L-703606. Analogous to senktide, GPSenk was totally inactive on arterial strips. Incubation of rat portal veins with GPSenk showed gold particles bound to the plasmalemma or inside intracytoplasmic vesicles of smooth muscle cells. Preincubation with senktide or the NK3 receptor antagonist R-820 prevented GPSenk marking. On portal veins in vitro GPSenk induced contractions similar to those induced by equimolar concentrations of senktide or NKB; these effects were inhibited by R-820. Our results show that colloidal gold-protein complexes present biological activity and selectivity similar to those of their respective native ligand and detect the presence of active receptors; in addition, they suggest the presence of NK-receptor-mediated endocytosis in endothelial cells of coronary artery and in smooth muscle cells of the portal vein.

Loss of heterozygosity on the long arm of chromosome 11 in orbital embryonal rhabdomyosarcoma (OERMS): a microsatellite study of seven cases.

OBJECTIVES. To investigate, by means of microsatellite analysis, regions of chromosome 11 involved in the genesis of embryonal rhabdomyosarcoma (ERMS) localized to the orbit. METHODS. Microsatellite analysis was carried out on seven cases of orbital ERMS by comparing the electrophoretic migration patterns of PCR-amplified microsatellites of chromosome 11 from both constitutional (blood) and tumor genotypes. Five of the tumors analyzed were samples frozen at the time of surgery, and two were paraffin embedded. RESULTS. Overall, microsatellites D11S1396 (11q13.1-q22.3) and D11S976 (11q) showed loss of heterozygosity (LOH) in all tumor samples, thus indicating the presence, on the long arm of chromosome 11, of one or more tumor suppressor genes with a possible role in the genesis of the disease. CONCLUSION. While the role of genes on the short arm of chromosome 11 in the genesis of ERMS is well established, much less is known of the possible involvement of tumor suppressor genes on the long arm of the same chromosome. This is the first report showing the possible involvement of tumor suppressor genes in this portion of the chromosome in ERMS localized to the orbit.

Design and implementation of a relational database used in the management of patients with retinoblastoma.

Clinical data are most useful in the management of patients with complex medical problems if they are accurate, reliable, and easily accessible by physicians and the medical community at large. Furthermore, the data are most valuable when they can be shared among cooperating institutions. We describe a computer system which exhibits a uniform taxonomy, an integrated on-line dictionary of clinical terms, a coherent temporal layout, and persistent spatial integrity with regard to the values of the variables. The system is user friendly and provides real time data access which can be retrieved by structured query language or may be programmed to be used as part of an international network in the management of patients with retinoblastoma, a malignant and potentially fatal tumor of childhood. Furthermore, because of its design flexibility, this system provides for potential application to other ophthalmic disorders, such as malignant uveal melanoma, and other areas of medicine as well.

Understanding angioid streaks.

BACKGROUND: Angioid streaks are defined as a series of linear, cracked-line dehiscences of Bruch's membrane, with secondary changes in the retinal pigment epithelium and choriocapillaris. They may be progressive or degenerative, with varied presentation, color, distribution, and retinal involvement. METHODS: The epidemiology, pathophysiology, diagnosis, and management of angioid streaks are described. In addition, the systemic diseases most commonly associated with the disease are reviewed. RESULTS: Optometrists need to be able to differentially diagnose angioid streaks and to refer for evaluation of underlying systemic disease. CONCLUSIONS: Angioid streaks are considered a rare disorder, associated with pseudoxanthoma elasticum, Paget's disease of the bone, sickle hemoglobinopathies. Marfan syndrome, and Ehlers-Danlos syndrome. Ocular complications include subretinal choroidal neovascular membrane formation. Clinicians should be aware of the disease's subtle ocular appearance, its association with systemic diseases, its potential for producing subretinal ocular complications, and correct management protocols and treatments.

The polymerase chain reaction (PCR) in the routine genetic characterization of retinoblastoma: a tool for the clinical laboratory.

The Polymerase Chain Reaction (PCR) is a highly innovative technique which allows for the generation of large amounts of DNA starting from minute quantities obtained from the blood or tissue of a patient. With the increasing knowledge concerning the structure of the human genome and the potential to amplify specific segments of DNA by the PCR technique, the molecular genetic characterization of many ocular disorders has been greatly facilitated. This is particularly true of retinoblastoma (RB) where the causative gene, RB1, gene has been identified and characterized. Using PCR technique, specific sequences of the RB1 gene can be amplified and analyzed to precisely define the genetic mutation in an affected individual. In addition, this technique can also be applied in order to characterize the genetic defect within the tumor itself. In this report we illustrate the use of the PCR technique in the genetic characterization of the RB1 gene and its application to the study of RB. These techniques are applicable even in a small clinical laboratory and can be extended to a number of ophthalmic disorders.

Effects of clonidine and alpha-methyl-p-tyrosine on the carbachol stimulation of paradoxical sleep.

Acetylcholine promotes paradoxical sleep (PS), but the role of noradrenaline in this stimulation is controversial. The relationship between cholinergic and noradrenergic systems in the production of PS was investigated in the rat implanted on a continuous basis for sleep recordings. Stimulation of PS was obtained with microinjections of carbachol (1 microgram) into the pontine reticular formation. In the presence of the alpha 2-agonist clonidine (5 micrograms/kg, IP), the carbachol activation of PS was abolished. This stimulation also disappeared when the animals were pretreated with alpha-methyl-paratyrosine (150 mg/kg, IP), an inhibitor of catecholamine synthesis. Thus, carbachol stimulation appeared inefficient when brain noradrenergic activation was decreased. This observation supports the view that the realization of PS by the cholinergic system requires a certain level of noradrenergic activity.

Concordance between parental origin of chromosome 13q loss and chromosome 6p duplication in sporadic retinoblastoma.

Two hypotheses are capable of explaining nonrandom loss of one parent's alleles at tumor suppressor loci in sporadic cases of several pediatric cancers, including retinoblastoma--namely, preferential germ-line mutation or chromosome imprinting. We have examined 74 cases of sporadic retinoblastoma for tumors in which at least two genetic events--loss of heterozygosity for chromosome 13q markers and formation of an isochromosome 6p--have occurred. Sixteen cases were found to contain both events. In 13 of 16 such tumors, the chromosomes 13q that were lost and chromosomes 6p that were duplicated are derived from the same parent. These data may be explained within the framework of the genome imprinting model but are not predicted by preferential germ-line mutation.

Italian register for retinoblastoma. Pros and cons of a retrospective statistical study.

In an attempt to verify some of the current conflicting results concerning the impact of relevant prognostic factors in the retinoblastoma therapy, the authors took into consideration, for statistical analysis, the series of 459 cases included in the Italian Registry for retrospective study of retinoblastoma. Although this series appears large enough, problems related to the continuously changing approaches to the disease and the consequent lack of standardization often make it difficult to draw significant conclusions. Hence, while historical (retrospective) analysis often allows the manipulation of a great number of data, particularly in the case of relatively rare diseases, prospective randomized controlled trials are strongly recommended to standardize definitely the relevant prognostic criteria. These and other problems related to retrospective analysis are discussed in detail.

Pathogenesis of posterior capsular opacification. Part II: Histopathological and in vitro culture findings.

The most interesting sources of information about the pathogenesis of posterior capsular opacification seem to be histopathological studies and in vitro tissue cultures. Since our surgical technique is extracapsular cataract extraction, the explants we used for tissue culture consisted of the anterior capsule epithelial sheet without the equatorial germinative zone. We successfully overcame several problems by using the autologous plasma clot culture method. This medium, considered the optimal one for this type of culture, allowed us to study the heterogeneous behavior of the epithelial cells in culture. Using the plasma clot culture method, we were able to demonstrate in vitro fibroblastic transformation of the epithelial cells. Histopathological findings of particular cases of posterior capsule opacification and immunohistochemistry of the human lens are also reported.