Regional distribution and molecular interaction of caveolins in bladder smooth muscle.

UNLABELLED: What's known on the subject? and What does the study add? Caveolae are specialised regions of bladder smooth muscle (BSM) cell membranes where specific signalling pathways are regulated. Caveolin proteins are involved in caveolar biogenesis and function as signal transduction regulators. Expression of caveolin-1, -2, and -3 has been previously identified in the bladder; however, the distribution and relative expression of these proteins have not been defined. The present data show significant differences in the spatial distribution of caveolin proteins throughout the bladder wall. Region dependent variations in the co-localisation of caveolin subtypes in detrusor SM were also detected. These findings support the premise that the unique spatial pattern of caveolin proteins associated with BSM cells may enable regionally distinct functional responses to common stimuli. OBJECTIVE: • To determine the regional expression profile of caveolin isoforms (integral membrane proteins abundant in caveolae), the spatial relationships among caveolin proteins within specific smooth muscle (SM) regions and the extent of their molecular interactions in bladder SM (BSM). MATERIALS AND METHODS: • Regional differences in the expression of caveolin family members were determined by quantitative reverse transcriptase-polymerase chain reaction and Western blot of RNA and protein extracted from the base, body and dome of rat bladders. • To evaluate the distribution of caveolin-1 (Cav-1), Cav-2 and Cav-3 within the bladder, longitudinal tissue sections from the base to dome were processed for confocal microscopy and quantified for intensity of immunoreactivity (IR) and extent of co-localisation. • Interactions among Cav-1, Cav-2 and Cav-3 were determined by co-immunoprecipitation. RESULTS: • Differential expression of Cav-1 and Cav-3 was detected among bladder regions, with lowest expression in the bladder base relative to the dome. • Cav-1 was highly expressed in all regions, although an increase in IR from submucosa to serosa was detected in each region. • The distribution of Cav-2 IR generally paralleled Cav-1, but progressively decreased from submucosa to serosa in each region. • Cav-3 expression predominated in the medial region of BSM increasing progressively from base to dome, but was poorly expressed in the outer SM layer particularly in the dome. • Cav-1 co-precipitated extensively with both Cav-2 and Cav-3. Co-precipitation between Cav-3 and Cav-2 was also detected. CONCLUSIONS: • The isoform-specific spatial distribution and distinct molecular interactions among caveolins in BSM may contribute to the contractile heterogeneity of BSM cells and facilitate differential modulation of responses to local stimuli. • As BSM caveolae regulate key signalling processes involved in contraction, altered expression of caveolin proteins may generate a regional imbalance in contraction/relaxation responses, thus leading to bladder dysfunction.

Loss of bladder smooth muscle caveolae in the aging bladder.

AIMS: Caveolae are specialized regions of the cell membrane that modulate signal transduction and alterations in these structures affect bladder smooth muscle (BSM) contraction. Since bladder dysfunctions are common in the elderly, we evaluated the effect of aging on the morphology of caveolae and caveolin protein expression in BSM. METHODS: Caveolar morphology (number, size, and depth) in BSM was determined from electron microscopy images of young (10 weeks), adult (6-month old), and old (12-month old) rat urinary bladders. Changes in expression levels of caveolin proteins with age were investigated by Western blot and immunofluorescence microscopy. Caveolin-3 gene expression was determined by real-time RT-PCR in young and 19-month-old rat bladders. RESULTS: Twelve-month-old animals exhibited 50% fewer BSM caveolae compared to young (P < 0.01). The area of caveolae was significantly decreased at 6 and 12 months. Despite a decrease in the number of BSM caveolae at 12 months, the expression of caveolin-1 and cavin-1 were unaltered with age. In contrast, caveolin-2 and caveolin-3 protein expression and immunoreactivity were reduced in BSM at 6 and 12 months of age. Caveolin-3 gene expression was also downregulated at 19 months compared to young animals. CONCLUSION: Biological aging significantly decreases BSM caveolae number and morphology with associated selective alteration in caveolin protein expression. Since caveolae are protected membrane regions that regulate signal transduction, age-related alterations in caveolae and caveolin protein expression could alter BSM contractility resulting in bladder dysfunctions of the elderly.

Molecular reactions and ultrastructural damage in the chronically ischemic bladder.

PURPOSE: Clinical and basic research data suggest that pelvic ischemia may contribute to bladder overactivity. We characterized the molecular and ultrastructural reactions of the chronically ischemic bladder. MATERIALS AND METHOD: A model of pelvic ischemia was developed by creating iliohypogastric/pudendal arterial atherosclerosis in rabbits. At 12 weeks conscious urinary frequency was examined, bladder blood flow was recorded and cystometrograms were done using general anesthesia. Bladder tissue was processed for molecular and ultrastructural analysis using quantitative real-time polymerase chain reaction, Western blot and transmission electron microscopy. RESULTS: Conscious urinary frequency and the frequency of spontaneous bladder contractions significantly increased in animals with pelvic ischemia. Bladder ischemia up-regulated the gene and protein expression of hypoxia inducible factor-1α, transforming growth factor-ß and nerve growth factor B. Vascular endothelial growth factor gene expression also increased but protein levels were unchanged. Transmission electron microscopy of ischemic bladder samples showed swollen mitochondria with degraded granules, thickened epithelium, deformed muscle fascicles, collagen deposition and impaired microvasculature with thickened intima and disrupted endothelial cell junctions. Degenerating axonal and Schwann cell profiles, and myelin sheath splitting around axons and Schwann cells were evident in ischemic bladders. CONCLUSIONS: Interrupting pelvic blood flow resulted in an ischemic overactive bladder and significant increase in conscious urinary frequency. Molecular responses involving hypoxia inducible factor, transforming growth factor-ß, vascular endothelial growth factor and nerve growth factor were associated with mitochondrial injury, fibrosis, microvasculature damage and neurodegeneration. Ischemia may have a key role in bladder overactivity and lower urinary tract symptoms.

Oxidative stress and neurodegeneration in penile ischaemia.

OBJECTIVE: To seek markers of oxidative stress and examine neural structural integrity in chronic penile ischaemia using a rabbit model of arteriogenic erectile dysfunction (ED), as the role of ischaemia in penile neuropathy and the oxidative mechanism of neurodegeneration in ED remains unknown. MATERIALS AND METHODS: A rabbit model of atherosclerosis-induced ED was developed by partial balloon de-endothelialization of the iliac arteries. After 10 weeks, intracavernosal blood flow and erectile function in the arteriogenic ED group were compared with age-matched controls. Erectile tissues were processed for analysis of oxidative stress markers and nerve fibre density using enzyme immunoassay and immunohistochemical staining, respectively. Oxidative stress-sensitive genes were determined with quantitative real-time polymerase chain reaction. Tissue ultrastructure was examined by transmission electron microscopy. RESULTS: Significant erectile tissue ischaemia, erectile dysfunction, increased levels of oxidative products, and marked nitrotyrosine immunoreactivity was evident in the ED group. Oxidative stress-sensitive genes encoding hypoxia inducible factor-1alpha (HIF-1alpha), superoxide dismutase (SOD), aldose reductase (AR) and nerve growth factor (NGF) were up-regulated in the ischaemic erectile tissue. These changes were associated with collapsed axonal and Schwann cell profiles, neurodegeneration, mitochondrial structural damage, increased caveolae, loss of endothelium, and sporadic vacuolization. CONCLUSIONS: Neuropathy appears to follow the vascular insult in arteriogenic ED. Neural injury in penile ischaemia involves a neurovascular phenomenon mediated by oxidative free radicals. Mitochondrial structural damage and increased HIF-1alpha gene expression may be early signals of oxidative stress and neurodegeneration in ED. Up-regulation of SOD, AR and NGF may be a coordinated defensive reaction to oxidative radicals that seems to fail to prevent neural injury in the ischaemic penis. Our study introduces the concept of oxidative neurodegeneration in the pathophysiology of arteriogenic ED. Therapeutic strategies to protect penile nerves from free radical incursion may enhance the efficacy of surgical and pharmacological interventions in arteriogenic ED.

Oxidative modification of mitochondrial integrity and nerve fiber density in the ischemic overactive bladder.

PURPOSE: To our knowledge the mechanism of neurodegeneration in the overactive bladder remains unknown. We examined mitochondrial integrity and searched for markers of oxidative neural injury in the ischemic overactive bladder. MATERIALS AND METHODS: A rabbit model of overactive bladder was developed by inducing moderate pelvic ischemia. After 16 weeks cystometrograms and blood flow recordings from overactive bladders were compared with those in age matched controls. Bladder tissues were processed to assess oxidative products, oxidative stress sensitive genes and nerve fiber density using enzyme immunoassay, quantitative real-time polymerase chain reaction and immunohistochemical staining, respectively. Tissue ultrastructure was examined by transmission electron microscopy. RESULTS: Ischemia increased spontaneous bladder contractions and led to cyclic ischemia-reperfusion. Tissue levels of oxidative and nitrosative products, and oxidative stress sensitive genes encoding superoxide dismutase and aldose reductase were up-regulated in the overactive bladder. Transmission electron microscopy of overactive bladder tissues showed mitochondria with distinctive morphological features, characterized by swollen membranes, decreased granules, a total loss of granules and sporadic membrane damage. These changes were associated with sporadic loss of epithelial mucosal membrane, twisted smooth muscle cells, diffused vacuolization and marked neurodegeneration. CONCLUSIONS: Our findings suggest free radical mediated ultrastructural damage and neurodegeneration in the overactive bladder. Overactivity associated mitochondrial stress may have a central role in epithelial damage, smooth muscle cell injury and neurodegeneration. Superoxide dismutase and aldose reductase up-regulation in the overactive bladder imply intrinsic defensive reaction against free radicals that apparently fails to prevent oxidative damage and neurodegeneration. Therapeutic strategies targeting basic mitochondrial processes such as energy metabolism or free radical generation may help better manage wall degeneration and neuropathy in the overactive bladder.

Effects of ischemia on tachykinin-containing nerves and neurokinin receptors in the rabbit bladder.

OBJECTIVES: Our previous studies showed marked changes in efferent nerve structure and reactivity in the ischemic bladder. The goal of this study was to examine the effects of bladder ischemia on tachykinin (TK) containing sensory nerves and neurokinin receptors (NKR) in a rabbit model. METHODS: We recorded bladder blood flow and spontaneous contractions in treated animals at week 8 after the induction of iliac arteries atherosclerosis and in age-matched controls. Bladder tissues were processed for studies of isometric smooth muscle tension in the organ bath, NK2R gene expression using quantitative real-time polymerase chain reaction (PCR), immunohistochemical staining of TK containing nerves and epithelial TK expression, and transmission electron microscopy. RESULTS: Atherosclerosis-induced ischemia significantly increased the frequency of spontaneous bladder contractions in vivo. Electrical field stimulation (EFS)-induced smooth muscle contractions were significantly greater in the ischemic tissues. Inhibition of NK1R diminished contractions to low-frequency EFS in control tissues while having no significant effect on the ischemic tissues. In contrast, NK2R inhibition significantly decreased contractions to both low- and high-frequency EFS in the ischemic tissues. Inhibition of NK3R had no significant effect on EFS-induced contractions. Real-time PCR showed a significant increase in NK2R gene expression in the ischemic bladder. The number of TK immunopositive nerves and epithelial TK immunoreactivity were significantly greater in the ischemic bladder. These alterations were associated with marked ultrastructural reactions to bladder ischemia. CONCLUSIONS: Alterations of NK2R reactivity and gene expression, increased number of TK immunopositive nerves, and greater epithelial TK immunoreactivity may imply activated bladder afferents to signal ischemic insult.