Effects of heat shock on the Mre11/Rad50/Nbs1 complex in irradiated or unirradiated cells.

The mechanism by which hyperthermia sensitizes mammalian cells to ionizing radiation remains to be elucidated, but an overwhelming amount of circumstantial evidence suggests that heat radiosensitization might be mediated by inhibition of double-strand break repair, particularly after exposure of irradiated cells to heat treatments in excess of about 43 degrees C. In mammalian cells, double-strand break repair usually occurs via two pathways, non-homologous end-joining and homologous recombination. Several reports suggest a role for non-homologous end-joining in heat radiosensitization, while others implicate homologous recombination as a target. However, cell lines that are compromised in either the non-homologous end-joining or homologous recombination pathway are still capable of being radiosensitized, suggesting that heat affects both pathways. Indeed, several of the proteins involved in one or both of these pathways have been observed to undergo alterations or translocation after unirradiated or irradiated cells are exposed to heat shock. The work summarized in this review implicates proteins of the Mre11/Rad50/Nbs1 complex as targets for heat radiosensitization.

Mice overexpressing genes from the 22q11 region deleted in velo-cardio-facial syndrome/DiGeorge syndrome have middle and inner ear defects.

Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is a congenital anomaly disorder associated with hemizygous 22q11 deletions. We previously showed that bacterial artificial chromosome (BAC) transgenic mice overexpressing four transgenes, PNUTL1, (CDCrel-1), GP1B beta, TBX1 and WDR14, had reduced viability, cardiovascular malformations and thymus gland hypoplasia. Since these are hallmark features of VCFS/DGS, we analyzed the mice for additional anomalies. We found that the mice have important defects in the middle and inner ear that are directly relevant to the disorder. The most striking defect was the presence of chronic otitis media, a common finding in VCFS/DGS patients. In addition, the mice had a hyperactive circling behavior and sensorineural hearing loss. This was associated with middle and inner ear malformations, analogous to Mondini dysplasia in humans reported to occur in VCFS/DGS patients. We propose that overexpression of one or more of the transgenes is responsible for the etiology of the ear defects in the mice. Based upon its pattern of expression in the ear and functional studies of the gene, TbX1 likely plays a central role. Haploinsufficiency of TBX1 may be responsible for ear disorders in VCFS/DGS patients.

Characterization of ataxia telangiectasia fibroblasts with extended life-span through telomerase expression.

Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by progressive cerebellar degeneration, immunodeficiencies, genomic instability and gonadal atrophy. A-T patients are hypersensitive to ionizing radiation and have an elevated cancer risk. Cells derived from A-T patients require higher levels of serum factors, exhibit cytoskeletal defects and undergo premature senescence in culture. We show here that expression of the catalytic subunit of telomerase (hTERT) in primary A-T patient fibroblasts can rescue the premature senescence phenotype. Ectopic expression of hTERT does not rescue the radiosensitivity or the telomere fusions in A-T fibroblasts. The hTERT+AT cells also retain the characteristic defects in cell-cycle checkpoints, and show increased chromosome damage before and after ionizing radiation. Although A-T patients have an increased susceptibility to cancer, the expression of hTERT in A-T fibroblasts does not stimulate malignant transformation. These immortalized A-T cells provide a more stable cell system to investigate the molecular mechanisms underlying the cellular phenotypes of Ataxia-telangiectasia.

Cystometric evaluation of bladder function in non-anesthetized mice with and without bladder outlet obstruction.

PURPOSE: To develop a model for cystometric study of bladder function in the awake mouse, and to characterize urodynamically and immunohistochemically the non-obstructed and infravesically obstructed mouse bladder. MATERIALS AND METHODS: Non-obstructed Balb/CJ mice, and mice with bladder outlet obstruction after surgical, partial ligation of the urethra underwent continuous cystometry as previously described for rats. Bladders were also investigated by immunohistochemistry. RESULTS: During the period of cystometry, reproducible micturition patterns were obtained. Marked differences in the urodynamic parameters between non-obstructed and obstructed mice were revealed. In mice subjected to urethral obstruction, micturition pressure (p <0.05), threshold pressure (p <0.05), bladder capacity (p <0.001), micturition volume (p <0.001), and residual volume (p <0.05) increased significantly. There was no difference in basal pressure or compliance between non-obstructed and obstructed mice. Non-voiding bladder activity was consistently recorded in obstructed mice; both frequency and amplitude increased significantly (p <0.01). Compared with non-obstructed bladders, obstructed bladders showed hypertrophy of the bladder wall and various degrees of "patchy denervation" of the detrusor. When tested in non-obstructed mice capsaicin, prostaglandin E2 (intravesical administration) and apomorphine (subcutaneous administration) induced bladder overactivity. CONCLUSIONS: Continuous cystometry can be reproducibly performed in awake, freely moving non-obstructed mice and mice with bladder outflow obstruction. The changes induced by infravesical obstruction in mice were similar to those previously found in rats. This model may be useful for investigations of genetically modified mice.

Functional characteristics of urinary tract smooth muscles in mice lacking cGMP protein kinase type I.

Nitric oxide (NO)-mediated smooth muscle relaxation is mediated by cGMP through activation of cGMP-dependent protein kinase I (cGKI). We studied the importance of cGKI for lower urinary tract function in mice lacking the gene for cGKI (cGKI-/-) and in litter-matched wild-type mice (cGKI+/+) in vitro and in vivo. cGKI deficiency did not result in any changes in bladder gross morphology or weight. Urethral strips from cGKI-/- mice showed an impaired relaxant response to nerve-derived NO. The cGMP analog 8-bromo-cGMP (8-BrcGMP) and the NO-donor SIN-1 relaxed the wild-type urethra (50-60%) but had only marginal effects in the cGKI-deficient urethra. Bladder strips from cGKI-/- mice responded normally to electrical field stimulation and to carbachol but not to 8-BrcGMP. In vivo, the cGKI-deficient mice showed bladder hyperactivity characterized by decreased intercontraction intervals and nonvoiding bladder contractions. Loss of cGKI abolishes NO-cGMP-dependent relaxations of urethral smooth muscle and results in hyperactive voiding. These data suggest that certain voiding disturbances may be associated with impaired NO-cGKI signaling.

Intravesical oxyhemoglobin initiates bladder overactivity in conscious, normal rats.

PURPOSE: To investigate whether intravesical oxyhemoglobin, a nitric oxide scavenger, changes bladder activity in normal rats. MATERIALS AND METHODS: Oxyhemoglobin was given intravesically at different concentrations to conscious, female Sprague-Dawley rats undergoing continuous cystometry. RESULTS: Intravesical oxyhemoglobin increased bladder activity in a concentration-dependent way. At a concentration of 2.5 x 10-4 M (n = 8), micturition pressure (p <0. 01), basal pressure (p <0.01), and residual volume (p <0.05) increased, and bladder capacity (p <0.001) and micturition volume (p <0.001) decreased. The effect of oxyhemoglobin was reduced or abolished by L-arginine (200 mg./kg.-1), given intra-arterially near the bladder, and was enhanced by the guanylate cyclase inhibitor, ODQ (0.5 and 1 mg./kg.-1). The K+ channel opener, ZD6169 100 ng.ml. -1, given intravesically for 1 hour prior to instillation of oxyhemoglobin, reduced or completely prevented the bladder activity induced by oxyhemoglobin. CONCLUSIONS: Intravesical oxyhemoglobin induces bladder overactivity, probably by interfering with nitric oxide (NO) generated in the urothelium or suburothelially. NO may be involved in the regulation of the threshold for afferent firing in the bladder.

Effects of intravesical administration of the K+ channel opener, ZD6169, in conscious rats with and without bladder outflow obstruction.

PURPOSE: To investigate the urodynamic effects of the new K(ATP) channel opener, ZD6169, given intravesically, in rats with and without bladder outflow obstruction. MATERIALS AND METHODS: Female, conscious Sprague-Dawley rats, normal or with bladder hypertrophy and overactivity secondary to bladder outflow obstruction, were given ZD6169 intravesically (10 or 100 ng./ml.), and intra-arterially (1 mg./kg.). Continuous cystometry was performed. RESULTS: In normal and obstructed rats, intravesical ZD6169 had similar, dose-dependent effects on bladder function. In obstructed rats, ZD6169 (100 ng./ml.) significantly decreased micturition pressure (17%), and increased bladder capacity (32%), micturition volume (18%), residual volume (145%), and inter-contraction interval (71%). There was a marked decrease in both frequency (40%) and amplitude (43%) of the spontaneous bladder activity. When given intra-arterially in obstructed rats ZD6169 increased bladder capacity (19%) and residual volume (47%) and decreased amplitude (51%), but not frequency, of the spontaneous bladder activity. CONCLUSIONS: In both normal and obstructed rats, intravesical ZD6169, at the investigated doses, significantly affected bladder function. In obstructed rats, the drug markedly reduced bladder overactivity. If the results have clinical validity, intravesical ZD6169 may offer an alternative way of treating bladder overactivity in selected patients.

Atm inactivation results in aberrant telomere clustering during meiotic prophase.

A-T (ataxia telangiectasia) individuals frequently display gonadal atrophy, and Atm-/- mice show spermatogenic failure due to arrest at prophase of meiosis I. Chromosomal movements take place during meiotic prophase, with telomeres congregating on the nuclear envelope to transiently form a cluster during the leptotene/zygotene transition (bouquet arrangement). Since the ATM protein has been implicated in telomere metabolism of somatic cells, we have set out to investigate the effects of Atm inactivation on meiotic telomere behavior. Fluorescent in situ hybridization and synaptonemal complex (SC) immunostaining of structurally preserved spermatocytes I revealed that telomere clustering occurs aberrantly in Atm-/- mice. Numerous spermatocytes of Atm-/- mice displayed locally accumulated telomeres with stretches of SC near the clustered chromosome ends. This contrasted with spermatogenesis of normal mice, where only a few leptotene/zygotene spermatocytes I with clustered telomeres were detected. Pachytene nuclei, which were much more abundant in normal mice, displayed telomeres scattered over the nuclear periphery. It appears that the timing and occurrence of chromosome polarization is altered in Atm-/- mice. When we examined telomere-nuclear matrix interactions in spermatocytes I, a significant difference was observed in the ratio of soluble versus matrix-associated telomeric DNA sequences between meiocytes of Atm-/- and control mice. We propose that the severe disruption of spermatogenesis during early prophase I in the absence of functional Atm may be partly due to altered interactions of telomeres with the nuclear matrix and distorted meiotic telomere clustering.

A common molecular basis for rearrangement disorders on chromosome 22q11.

The chromosome 22q11 region is susceptible to rearrangements that are associated with congenital anomaly disorders and malignant tumors. Three congenital anomaly disorders, cat-eye syndrome, der() syndrome and velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) are associated with tetrasomy, trisomy or monosomy, respectively, for part of chromosome 22q11. VCFS/DGS is the most common syndrome associated with 22q11 rearrangements. In order to determine whether there are particular regions on 22q11 that are prone to rearrangements, the deletion end-points in a large number of VCFS/DGS patients were defined by haplotype analysis. Most VCFS/DGS patients have a similar 3 Mb deletion, some have a nested distal deletion breakpoint resulting in a 1.5 Mb deletion and a few rare patients have unique deletions or translocations. The high prevalence of the disorder in the population and the fact that most cases occur sporadically suggest that sequences at or near the breakpoints confer susceptibility to chromosome rearrangements. To investigate this hypothesis, we developed hamster-human somatic hybrid cell lines from VCFS/DGS patients with all three classes of deletions and we now show that the breakpoints occur within similar low copy repeats, termed LCR22s. To support this idea further, we identified a family that carries an interstitial duplication of the same 3 Mb region that is deleted in VCFS/DGS patients. We present models to explain how the LCR22s can mediate different homologous recombination events, thereby generating a number of rearrangements that are associated with congenital anomaly disorders. We identified five additional copies of the LCR22 on 22q11 that may mediate other rearrangements leading to disease.

Spinal and peripheral mechanisms contributing to hyperactive voiding in spontaneously hypertensive rats.

The influence of noradrenergic mechanisms involved in micturition in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was investigated using continuous cystometry in in vivo and in vitro studies on isolated bladder and urethral tissues. Compared with WKY rats, SHR had a significantly lower bladder capacity (SHR: 0.7 +/- 0. 05 ml; WKY rats: 1.3 +/- 0.06 ml; P < 0.001), micturition volume (SHR: 0.4 +/- 0.04 ml, WKY rats: 1.2 +/- 0.05 ml; P < 0.001), and an increased amplitude of nonvoiding (unstable) bladder contractions. The effects of intrathecal and intra-arterial doxazosin on cystometric parameters were more pronounced in SHR than in WKY rats. There was a marked reduction in nonvoiding contractions after intrathecal (but not intra-arterial) doxazosin in SHR. Norepinephrine (0.1 microM-1 mM) failed to evoke contractions in bladder strips from WKY rats, in contrast to a weak contractile response in SHR. The response to electrical field stimulation was significantly less in bladder strips from SHR than from WKY rats. In WKY rats, norepinephrine produced concentration-dependent inhibition (87 +/- 5%, n = 6) of nerve-evoked bladder contractions. Almost no inhibition (11 +/- 8%, n = 6) was found in SHR. Alterations in bladder function of SHR appear to be associated with changes in the noradrenergic control of the micturition reflex, in addition to an increased smooth muscle and decreased neuronal responsiveness to norepinephrine. The marked reduction in nonvoiding contractions after intrathecal doxazosin suggests that the bladder hyperactivity in SHR has at least part of its origin in supraspinal and/or spinal structures.

Effects of polyamine synthesis inhibition on polyamines, growth and mechanical properties in hypertrophic rat urinary bladder.

The polyamines putrescine, spermidine and spermine, are ubiquitous intracellular metabolites associated with growth and protein synthesis. In this study effects of polyamine synthesis inhibition on bladder growth, polyamine levels and mechanical properties were investigated in rat urinary bladder subjected to partial outflow obstruction that causes bladder hypertrophy. The S-adenosyl methionine decarboxylase inhibitor CGP-48664 (5 and 20 mg kg-1) was administered alone or in combination with the ornithine decarboxylase inhibitor DFMO (500 mg kg-1), starting one day before creation of partial outflow obstruction and then daily for 7 days. The bladder muscle level of putrescine was increased 38 times and that of spermine reduced by 4 times while spermidine was unchanged after treatment with CGP-48664 (20 mg kg-1). The increase in putrescine was abolished in animals receiving CGP-48664 in combination with DFMO. Treatment with polyamine synthesis inhibitors could not prevent or reduce the hypertrophy of the bladder as judged by bladder wet weight and protein contents. The effects on polyamine quantities were not associated with changes in Ca(2+)-force relationship or in agonist and electrically stimulated force. In summary, treatment of rats with polyamine synthesis inhibitors resulted in changes in polyamine levels in the growing urinary bladder but did not affect growth or mechanical properties.

Oxytocin-induced stimulation and inhibition of bladder activity in normal, conscious rats--influence of nitric oxide synthase inhibition.

The role of the oxytocin-containing projections to the autonomic nuclei of the spinal cord for lower urinary tract function has not been clarified. The hypothesis was tested that oxytocin acts as a mediator of bladder contraction at the spinal cord level. In conscious female rats undergoing continuous cystometry, intrathecal oxytocin (30 ng approximately 30 pmoles) significantly increased micturition pressure (P<0.001), and decreased bladder capacity (P<0.01) and micturition volume (P<0.01). Residual volume increased (P<0.05), and so did the amplitude and frequency of non-voiding contractions (P<0.01). Immediately after administration of oxytocin, the animals showed frequent stretching movements and yawning, and they licked their tails. The effects of oxytocin were dose-dependent; high concentrations (100 ng) were ineffective. Intra-arterial injection of oxytocin (30 ng) near the bladder had no effect. In isolated detrusor strips, oxytocin caused a concentration-dependent contraction; the concentration response curve was concentration-dependently shifted to the right by the oxytocin antagonist, 1-deamino, 2-D-Tyr(OEt), 4-Thr, 8-Orn-OT. Intrathecal injection of the antagonist (500 ng), had per se no effect on micturition. However, when the antagonist was given intrathecally 4-5 min prior to intrathecal oxytocin (30 ng), the effects of oxytocin were reduced or completely prevented. When given after intrathecal administration of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester, intrathecal oxytocin (30 ng) abolished micturition within 5-7 min; all animals developed overflow incontinence, and paralysis of the hindlimbs. These results suggests that in the rat, oxytocin, released from descending pathways, may act as a modulator of the micturition reflex at the spinal level, and that it may interact with nitric oxide. The physiological implications of the findings remain to be established.

Testosterone-induced prostatic growth in the rat causes bladder overactivity unrelated to detrusor hypertrophy.

BACKGROUND: Testosterone treatment of rats produces prostatic hypertrophy and detrusor overactivity. Whether or not the detrusor overactivity can be related to an increase in the responsiveness of lower urinary tract smooth muscles is not known. METHODS: Male Sprague-Dawley rats were given daily injections of testosterone propionate for 2 weeks. Effects on cystometric parameters and on the responsiveness of isolated detrusor, urethral, and prostate smooth muscle preparations to drugs and electrical field stimulation were investigated. RESULTS: Testosterone treatment increased prostatic weight twofold (controls, 768 mg; testosterone-treated, 1,478 mg), but not bladder weight (103 mg vs. 116 mg). Micturition pressure (77%), bladder capacity (75%), residual volume (56%), and micturition volume (83%) increased significantly in treated animals, and bladder overactivity developed. No effect of intraarterial doxazosin on these changes was observed. The differences in urodynamic parameters between control and testosterone-treated rats could not be correlated with changes in bladder, urethral, or prostate excitatory innervation, as revealed by responses to electrical field stimulation, or by smooth muscle responses to different contractant drugs. CONCLUSIONS: Some of the urodynamic effects seen after testosterone treatment seem to be caused by the mechanical obstruction of the enlarged prostate. Since there were no changes in smooth muscle responsiveness, it is suggested that the bladder overactivity observed can partly be related to testosterone-induced changes of the micturition reflex at the lower urinary tract, spinal, and/or supraspinal levels.

Effects of the K+ channel opener, ZD6169, on volume and PGE2-stimulated bladder activity in conscious rats.

PURPOSE: To investigate the effects of the new K(ATP) channel opener, ZD6169, shown to have an in vivo selectivity for the bladder, on bladder activity in rats. MATERIALS AND METHODS: ZD6169 was given intra-arterially (i.a., 0.1 and 1 mg./kg.) or orally (3 mg./kg.) to conscious Sprague-Dawley rats undergoing continuous cystometry. Investigations were also performed before and after stimulation of bladder activity by intravesical prostaglandin (PG) E2. RESULTS: Intra-arterial ZD6169 increased residual volume, but caused no changes in other cystometric parameters. In rats receiving oral ZD6169, cystometric parameters were compared (every hour up to five hours) to those recorded in rats receiving oral vehicle. No differences were found, except in threshold pressure, which was significantly increased. Intravesical PGE2 20 microM increased micturition and basal pressures, and decreased bladder capacity and micturition volume. ZD6169 1 mg./kg., given i.a., reduced or completely prevented the activity induced by intravesical PGE2. Three hours after orally administered ZD6169 (3 mg./kg.), intravesical PGE2 20 microM had no effect. Three hours after oral administration of vehicle, the effects of PGE2 were attenuated, but still statistically significant. CONCLUSIONS: ZD6169, given i.a. or orally, increased threshold pressure, but had otherwise little effect on volume-induced micturition. However, the drug markedly reduced or prevented PGE2-induced bladder activity when given i.a.; it was also effective when given orally. If ZD6169 has inhibiting effects on bladder contraction in man without any cardiovascular actions, the drug may represent a novel, promising way of treating bladder overactivity.

Stimulation of bladder activity by volume, L-dopa and capsaicin in normal conscious rats--effects of spinal alpha 1-adrenoceptor blockade.

To study possible differences in alpha 1-adrenoceptor involvement in the spinal mechanisms mediating bladder activity induced by volume (bladder filling), central (L-dopa), and peripheral (capsaicin) stimulation, we investigated if these types of bladder activity were modified by intrathecal (i.t.) or intra-arterial (i.a.) administration of the alpha 1-adrenoceptor antagonist, indoramin. Indoramin is selective for the alpha 1A-adrenoceptor subtype, whereas most clinically used alpha 1-adrenoceptor antagonists, including doxazosin, have no subtype selectivity. The drug effects were studied by continuous cystometry in normal, conscious rats and rats with bladder activity evoked by intraperitoneal L-dopa (50 mg/kg after carbidopa pretreatment), or by intravesical capsaicin (30 microM). I.t. indoramin (50 nmol) significantly decreased micturition pressure, and increased bladder capacity and micturition volume. Dribbling incontinence due to urinary retention was observed in one of ten rats. L-dopa-stimulated bladder overactivity was significantly attenuated by i.t. or i.a. indoramin (50 nmol). Similar effects of i.t. and i.a. doxazosin (50 nmol) have been reported previously. Intravesical capsaicin (30 microM) caused bladder activity, which was attenuated by i.t. indoramin (50 nmol), but not by i.t. doxazosin (50 nmol). I.a. indoramin did not reduce capsaicin-induced bladder activity; doxazosin was moderately effective. The results suggest that the bulbospinal micturition reflex evoked by bladder filling and L-dopa involves a descending pathway where transmission is partly mediated by spinal alpha 1-adrenoceptors. Bladder overactivity evoked by intravesical capsaicin, which elicits a vesicospinal-vesical reflex, was not affected by i.t. doxazosin in a dose that attenuates activity mediated through the bulbo-spinal pathway. This suggests less involvement of spinal alpha 1-adrenoceptors in the vesico-spinal-vesical than in the bulbo-spinal voiding reflex.

Effect of penclomedine (NSC-338720) on telomere fusions, chromatin blebbing, and cell viability with and without telomerase activity and abrogated p53 function.

Telomeres, or chromosome ends, are essential in maintaining chromosomal integrity. Telomeres consist of a short hexameric sequence, 3'-TTAGGG-5', repeated in tandem arrays added to chromosomes by the ribonucleoprotein enzyme telomerase. In this study, we assessed whether penclomedine, a novel synthetic pyridine compound presently being evaluated in clinical trials for its anticancer activity, influences telomere fusions (chromosome end-to-end associations) and telomerase activity in cells in culture. We found that penclomedine reduced the mitotic index, induced chromosome end associations in all phases of the cell cycle, and rapidly induced chromatin blebbing in a concentration-dependent manner in both cervical carcinoma (HeLa) cells and in normal human fibroblasts (AG1522). However, the effectiveness of the drug was much more pronounced in HeLa cells. In addition, there was a drug-mediated, concentration-dependent decline in telomerase activity noted in the HeLa cells that correlated with a decrease in mitotic index and an increase in telomere fusions. Interestingly, when the mitotic index, chromatin blebbing, and telomere fusions were compared between the telomerase positive (HeLa) and negative (AG1522) cell types, penclomedine affected chromatin stability to a greater extent in those cells with detectable telomerase activity. In addition, telomerase positive colorectal carcinoma cells with abrogated p53 (RC-10.3 cells) were more sensitive to penclomedine than were telomerase positive cells with wild-type p53 (RKO cells). These studies suggest that penclomedine may have a therapeutic advantage in killing tumor cells that are positive for telomerase activity and defective in p53 function.

Capsaicin-induced bladder overactivity and nociceptive behaviour in conscious rats: involvement of spinal nitric oxide.

To investigate the role of nitric oxide (NO) in spinal regulation of lower urinary tract function and bladder nociceptive behaviour, cystometry was performed in conscious rats. The effects of intra-arterial and intrathecal administration of the NO synthase (NOS)-inhibitor, L-NG-nitroarginine methyl ester (L-NAME), were studied on volume- and capsaicin-induced micturitions. The incidence of nociceptive behaviour after intravesical capsaicin was investigated in the absence and presence of L-NAME. Intrathecal L-NAME (0.5 mg) had no effect on the normal, volume-induced micturition. Intravesical capsaicin (30 microM) increased the micturition pressure (p < 0.01), the basal pressure (p < 0.01) and decreased the bladder capacity (p < 0.01) and the micturition volume (p < 0.01). Administration of L-NAME intrathecally (0.5 mg) or intra-arterially (25 mg/kg) had no effects on the capsaicin-induced bladder activity. During capsaicin-infusion, the rats showed signs of distress such as licking and head-turning directed toward the abdomen. This nociceptive behaviour was shown during 31 +/- 3% (n = 6) of the observation period. The capsaicin-induced nociceptive behaviour was markedly reduced by intrathecal and to a less extent by intra-arterial, administration of L-NAME. The percentage time spent licking and head-turning was reduced to 11 +/- 2%, n = 6 (p < 0.001) and 18 +/- 3%, n = 6 (p < 0.05) in rats treated with intrathecal and intra-arterial L-NAME, respectively. The results suggest that NO is not involved in the spinal regulation of the volume- or capsaicin-induced micturition. In contrast, the nociceptive behaviour evoked by intravesical capsaicin seems to involve spinal NO.

Angiotensin II and bladder obstruction in the rat: influence on hypertrophic growth and contractility.

The mechanisms and mediators of hypertrophic growth secondary to infravesical urinary outflow obstruction are unknown. The renin-angiotensin system has been implicated in vascular and cardiac hypertrophy, but the involvement of angiotensin II (ANG II) as a trophic factor in the lower urinary tract has not been investigated. In this study, the ANG II subtype AT1 receptor antagonist losartan (DuP 753) was administered perorally (15 mg.kg-1.day-1) for 28 days to rats subjected to partial urethral obstruction or sham surgery. Partial urethral obstruction caused a 3.5-fold increase in bladder weight and a 3-fold increase in bladder protein content compared with sham rats. However, no difference was observed in bladder weight or bladder protein content between losartan-treated rats and rats receiving no drug. Cystometric evaluation of bladder function revealed significant increases in micturition volume, bladder capacity, bladder compliance, and spontaneous contractile activity in rats subjected to partial urethral obstruction compared with sham rats. However, bladder function in rats treated with losartan was not different from bladder function in rats receiving no drug. In vitro studies of isolated bladder tissue showed a weak contractile response to ANG II (1 microM) that amounted to 4.4 +/- 1.0% of the response to K+ (124 mM). The ANG II-induced contraction was abolished by losartan (10 microM) and indomethacin (10 microM). The contractile response to ANG II (1 microM), K+ (124 mM), and transmural nerve stimulation (2 Hz) was reduced in bladder strips from obstructed rats. In conclusion, no evidence was found for involvement of ANG II in development of bladder hypertrophy. The effect of ANG II on bladder smooth muscle tone was minor but was mediated by stimulation of the AT1 subtype receptor.

Chromosome end-to-end associations and telomerase activity during cancer progression in human cells after treatment with alpha-particles simulating radon progeny.

Chromosome end-to-end associations seen at metaphase involve telomeres and are commonly observed in cells derived from individuals with ataxia telangiectasia and most types of human tumors. The associations may arise because of short telomeres and/or alterations of chromatin structure. There is a growing consensus that telomere length is stabilized by the activity of telomerase in immortal cells; however, it is not clear why some immortal cells display chromosome end-to-end associations. In the present study we evaluated chromosome end-to-end associations, telomere length and telomerase activity with the tumorigenic status of human bronchial epithelial cells immortalized with human papillomavirus. Oncogenic transformation was initiated using radon simulated alpha-particles and cells evaluated as primary, secondary and metastatic transformants. The fewest chromosome end associations and lowest telomerase activity were observed in the parental immortalized cells. However, increased levels of telomerase activity were detected in alpha-particle survivors while robust telomerase activity was seen in the tumorigenic cell lines. The tumorigenic cells that were telomerase positive and had the highest frequency of cells with chromosome end-to-end associations were also metastatic. No correlation was found between telomere length and the different stages of carcinogenicity.

Perfadex is superior to Euro-Collins solution regarding 24-hour preservation of vascular function.

BACKGROUND: The aim of this study was to compare Perfadex with Euro-Collins solution regarding 24-hour preservation of endothelium-dependent relaxation and vascular smooth muscle function. METHODS: The infrarenal aorta of 72 isogenic rats was studied in organ baths as fresh controls, after 24 hours of cold (4 degrees C) storage, and after 24-hour storage followed by transplantation and examination after 7 or 30 days. The thromboxane A2 analogue U-46619 was used to test contractility. Acetylcholine chloride was used to elicit endothelium-dependent relaxation and papaverine hydrochloride, to elicit endothelium-independent relaxation. RESULTS: With both solutions, all grafts were patent after 7 and 30 days. Vessels preserved in Euro-Collins solution for 24 hours lost 95% (p < 0.001) of their contractility compared with fresh controls; 7 days after transplantation, they had regained 40% of initial contractility, and after 30 days, there was no significant decrease in contractility. Vessels preserved in Perfadex manifested no significant decrease in contractility at any time. Endothelium-dependent relaxation could not be evaluated in vessels stored for 24 hours in Euro-Collins solution because they had lost almost all contractility; 7 days after transplantation, endothelium-dependent relaxation was reduced by 65% (p < 0.001), but at 30 days after transplantation, there was no significant decrease in endothelium-dependent relaxation. Vessels preserved in Perfadex for 24 hours lost 17% (p < 0.05) of endothelium-dependent relaxation, but 7 and 30 days after transplantation, there was no significant decrease in endothelium-dependent relaxation. CONCLUSIONS: Perfadex, but not Euro-Collins solution, has the capacity to preserve vascular function after 24 hours of storage followed by in vivo reperfusion.