The contribution of intracellular Ca2+ release to contraction in human bladder smooth muscle.
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1. The importance of Ca2+ release from the sarcoplasmic reticulum (SR) in excitation contraction (EC) coupling in human detrusor muscle remains controversial. In this paper the contribution of Ca2+ release to agonist induced contraction is assessed. 2. Dose response curves to carbachol (0.01 - 10 microM) were constructed before and after exposure to 200 nM Thapsigargin (Tg). Tg pre-treatment reduced the force of contraction at all agonist concentrations however, the reduction was dose dependent. At 0.1 microM the contractions were reduced to 14.5 +/- 7% (mean +/- s.e.mean) of controls (n = 8) while at 10 microM the contractions were only reduced to 92 +/- 3% of controls (n = 10). 3. The role of external Ca2+ was examined by measuring the magnitude of contraction to low and high doses of agonist in the presence and absence of external Ca2+. With (0.1-0.3 microM) carbachol the contractions in nominally Ca2+ free media were 4+/-4% of controls (n = 7) whilst with (1 - 10 microM) carbachol the contractions were 36 +/- 8% of controls (n=7) suggesting that at low agonist concentrations the release of Ca2+ has a requirement for external Ca2+. 4. Pre-treatment of muscle strips with the Ca2+ channel blocking agent diltiazem reduced the contractile responses to carbachol. Contractions induced by 0.1 microM were reduced to 29+/-11% (P<0.05) of controls while those activated by 10 microM were reduced to 86+/-6% (P= 0.1) of controls (n = 4) suggesting the Ca2+ influx needed to activate internal store release at low agonist stimulation is through L-type Ca2+ channels. 5. These observations confirm the importance of thapsigargin sensitive intracellular Ca2+ store release in the activation of contraction of detrusor smooth muscle and suggest the overall contribution of this store depends upon the magnitude of the agonist stimulation. (+info)
Developmental expression of the four plasma membrane calcium ATPase (Pmca) genes in the mouse.
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The plasma membrane calcium ATPases are critical components in the regulation of cellular calcium homeostasis and signaling. In mammals, there are 4 Pmca genes, and information on the cellular and tissue distribution of their expression during development will provide insight into the regulation and possible function of each Pmca isoform. Using specific probes and in situ hybridization, we found that the four Pmca genes are expressed in spatially overlapping but distinct patterns in the mouse embryo. The dynamic temporal patterns of expression indicate that the individual isoforms are subject to both positive and negative regulation. The differential and restricted expression of Pmca genes supports the notion that they play unique functional roles in mammalian development. (+info)
Basic fibroblast growth factor increases long-term survival of spinal motor neurons and improves respiratory function after experimental spinal cord injury.
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Acute focal injection of basic fibroblast growth factor (FGF2) protects ventral horn (VH) neurons from death after experimental contusive spinal cord injury (SCI) at T8. Because these neurons innervate respiratory muscles, we hypothesized that respiratory deficits resulting from SCI would be attenuated by FGF2 treatment. To test this hypothesis we used a head-out plethysmograph system to evaluate respiratory parameters in conscious rats before and at 24 hr and 7, 28, and 35 d after SCI. Two groups of rats (n = 8 per group) received either FGF2 (3 microg) beginning 5 min after injury or vehicle (VEH) solution alone. We found significantly increased respiratory rate and decreased tidal volume at 24 hr and 7 d after SCI in the VEH-treated group. Ventilatory response to breathing 5 or 7% CO(2) was also significantly reduced. Recovery took place over time. Respiration remained normal in the FGF2-treated group. At 35 d after injury, histological analyses were used to compare long-term neuron survival. FGF2 treatment doubled the survival of VH neurons adjacent to the injury site. Because the number of surviving VH neurons rostral to the injury epicenter was significantly correlated to the ventilatory response to CO(2), it is likely that the absence of respiratory deficits in FGF2-treated rats was caused by its neuroprotective effect. Our results demonstrate that FGF2 treatment prevents the respiratory deficits produced by thoracic SCI. Because FGF2 also reduced the loss of preganglionic sympathetic motoneurons after injury, this neurotrophic factor may have broad therapeutic potential for SCI. (+info)
AP-1 mediates stretch-induced expression of HB-EGF in bladder smooth muscle cells.
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Mechanical induction of growth factor synthesis may mediate adaptive responses of smooth muscle cells (SMC) to increases in physical load. We previously demonstrated that cyclic mechanical stretch induces expression of the SMC, fibroblast, and epithelial cell mitogen heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bladder SMC, an observation that suggests that this growth factor may be involved in compensatory bladder hypertrophy. In the present study we provide evidence that the activator protein-1 (AP-1) transcription factor plays a critical role in this mechanoinduction process. Rat bladder SMC were transiently transfected with a series of 5' deletion mutants of a promoter-reporter construct containing 1. 7 kb of the mouse HB-EGF promoter that was previously shown to be stretch responsive. The stretch-mediated increase in promoter activity was completely ablated with deletion of nucleotide positions -1301 to -881. Binding of AP-1, as evaluated by electrophoretic mobility shift assay, to a synthetic oligonucleotide containing an AP-1 binding site increased in response to stretch, and binding was inhibited by excess unlabeled DNA corresponding to nucleotides -993 to -973 from the HB-EGF promoter, a region that contains a previously recognized composite AP-1/Ets site. Stretch-induced promoter activity was significantly inhibited by site-directed mutagenesis of the AP-1 or Ets components of this site. Consistent with the promoter and gel-shift studies, curcumin, an inhibitor of AP-1 activation, suppressed the HB-EGF mRNA induction after stretch. Stretch also specifically increased mRNA levels for matrix metalloproteinase (MMP)-1, the promoter of which contains a functional AP-1 element, but not for MMP-2, the promoter of which does not contain an AP-1 element. The stretch response of the MMP-1 gene was also completely inhibited by curcumin. Collectively, these findings indicate that AP-1-mediated transcription plays an important role in the regulation of gene expression in bladder muscle in response to mechanical forces. (+info)
Role of CYP1A2 in the toxicity of long-term phenacetin feeding in mice.
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The mechanisms underlying phenacetin-induced toxicity and carcinogenicity are not clear. In particular, it is not known whether these effects are mediated by metabolic activation of the drug. CYP1A2 is known to metabolize phenacetin in vitro. To determine the role of this enzyme in vivo, the toxicity and carcinogenicity of phenacetin was examined in Cyp1a2-null mice (that lack CYP1A2). Six- to 8-week-old wild type (+/+) or null (-/-) mice were fed either a control diet, or one containing 1.25% phenacetin, ad libitum for up to 67 weeks. Representative groups of mice were examined for phenacetin-induced toxicity and carcinogenicity after 36, 48, 58, or 67 weeks of feeding. Consistent with the known role of CYP1A2 in phenacetin metabolism, plasma levels of phenacetin were higher and acetaminophen levels lower in the (-/-) mice fed phenacetin compared to phenacetin-fed (+/+) controls. Weight gain was significantly depressed in both groups of phenacetin-fed mice after 4 weeks of feeding, and continued to be lower for the remainder of the experiment, compared to controls. Hepatomegaly and splenomegaly were more severe in (-/-) mice but present in both genotypes fed phenacetin at all time points assessed. Histological analysis of liver, kidney, spleen, and urogenital tract also revealed a differential response in the (-/-) mice fed phenacetin compared to (+/+) mice fed the same diet. Further, mortality was the most severe in the (-/-) mice fed phenacetin than in all other groups. Despite significant toxicity in (-/-) mice fed phenacetin, only one renal carcinoma was found among them. Results from this work demonstrate that, in the absence of CYP1A2, phenacetin is more toxic than in controls. This provides evidence that metabolism of phenacetin by CYP1A2 alters toxicity in vivo, and suggests that alternate CYP1A2-independent metabolic pathways contribute to its toxicity. (+info)
Neuronal nitric oxide synthase in the neural pathways of the urinary bladder.
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Nitric oxide (NO) is a unique biological messenger molecule. It serves, in part, as a neurotransmitter in the central and peripheral nervous systems. Neurons containing NO have been identified histochemically by the presence of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) reactivity or immunohistochemically by the antibody for neuronal NO synthase (n-NOS). Previous histochemical or pharmacological studies have raised the possibility that NO may play an important role in the neural pathways of the lower urinary tract. There is also considerable evidence to suggest that n-NOS is plastic and could be upregulated following certain lesions in the lower urinary tract. The present review summarises the distribution of n-NOS containing neurons innervating the urinary bladder and the changes of the enzyme expression in some experimentally induced pathological conditions. (+info)
Specific p53 gene mutations in urinary bladder epithelium after the Chernobyl accident.
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After the Chernobyl accident, the incidence of urinary bladder cancers in the Ukraine population increased gradually from 26.2 to 36.1 per 100,000 between 1986 and 1996. Urinary bladder epithelium biopsied from 45 male patients with benign prostatic hyperplasia living in radiocontaminated areas of Ukraine demonstrated frequent severe urothelial dysplasia, carcinoma in situ, and a single invasive transitional cell carcinoma, combined with irradiation cystitis in 42 cases (93%). No neoplastic changes (carcinoma in situ or transitional cell carcinoma) were found in 10 patients from clean areas (areas without radiocontamination). DNA was extracted from the altered urothelium of selected paraffin-embedded specimens that showed obviously abnormal histology (3 cases) or intense p53 immunoreactivity (15 cases), and mutational analysis of exons 5-8 of the p53 gene was performed by PCR-single-strand conformational polymorphism analysis followed by DNA sequencing. Nine of 17 patients (53%) had one or more mutations in the altered urothelium. Urine sediment samples were also collected from the patients at 4-27 months after biopsy and analyzed by PCR-single-strand conformational polymorphism analysis or yeast functional assay, and identical or additional p53 mutations were found in four of five cases. Interestingly, a relative hot spot at codon 245 was found in five of nine (56%) cases with mutations, and 11 of the 13 mutations determined (73%) were G:C to A:T transitions at CpG dinucleotides, reported to be relatively infrequent (approximately 18%) in human urinary bladder cancers. Therefore, the frequent and specific p53 mutations found in these male patients may alert us to a future elevated occurrence of urinary bladder cancers in the radiocontaminated areas. (+info)
Internal iliac artery embolisation for intractable bladder haemorrhage in the peri-operative phase.
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Intractable haemorrhage from the bladder wall during transurethral resection of bladder tumour is uncommon but potentially catastrophic. Internal iliac artery embolisation is a minimally invasive technique, which is now widely practised to stop bleeding from branches of these arteries is situations including pelvic malignancy, obstetric and gynaecological emergencies and trauma. We report its successful use peri-operatively, in an unfit, elderly patient with uncontrolled bleeding. (+info)