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• Research Paper Volume 12, Issue 5 pp 4163-4177

  Reduced Ca²⁺ spark activity contributes to detrusor overactivity of rats with partial bladder outlet obstruction

  Relevance score: 6.6622553

  Ji Zheng, Hao Zhou, Mengjun Yang, Siji Song, Qiang Dai, Guangju Ji, Zhansong Zhou

  Keywords: detrusor overactivity, Ca²⁺ spark, ryanodine receptor, FKBP12.6

  Published in Aging on February 29, 2020

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  We tested whether or not altered Ca²⁺ spark activity accounted for detrusor overactivity (DO) of Wistar rats after partial bladder outlet obstruction (PBOO). We constructed a DO model through PBOO and studied the Ca²⁺ spark activity of detrusor. By way of using confocal microscopy and the patch-clamp technique, Ca²⁺ sparks and spontaneous transient outward currents (STOCs) in detrusor myocytes were measured respectively. Our results indicated that Ca²⁺ spark activity and STOCs were significantly reduced in the DO detrusor myocytes compared to unafflicted control cells, and both of these had levels that were remarkably increased by applications of caffeine (10 μM), a RyR agonist, in DO myocytes. In addition, measures of detrusor contractions were also recorded by using freshly isolated detrusor strips. These results indicated that the spontaneous contraction of DO detrusor was significantly enhanced, and that the effect of caffeine (10 μM) upon detrusor contractions was reversed by applications of iberiotoxin (100 nM) which is a BK channel blocker. Western blotting (WB) analyses indicated that the levels of expression of ryanodine receptor type 2 (RyR2) and FK506 binding protein 12.6 (FKBP12.6) in bladder muscle were respectively decreased and increased in the samples from DO rats. Thus, we considered in the rat DO model wherein PBOO, the reduced Ca²⁺ spark activity in detrusor myocytes partly contributed to overactive detrusor contractions. The impaired Ca²⁺ spark activity may have resulted from decreased RyR2 expression and increased FKBP12.6 expression. Such novel findings in our research might help to provide means for better treatment outcomes for patients afflicted by bladder dysfunction.

  

  Decreased STOC frequency and amplitude in detrusor myocytes of DO rats. (A) Representative traces of STOCs in detrusor myocytes from control and DO rats. (B) Traces at a -40 mV holding potential from detrusor myocytes of control and DO rats. (C) Summarized data for measures of STOC frequency and amplitude. At all voltages, the frequencies (a) and amplitudes (b) of STOCs in DO rats were significantly lower than respective measures for rats in control groups. We used unpaired t tests for comparisons between groups. VS control *P<0.05, **P<0.01.

  
  
  

  

  Reduced properties of Ca2+ sparks in detrusor myocyte from DO model rats. (A, B) Confocal linescans of representative Ca2+ sparks in detrusor myocyte from unafflicted control (A) and DO afflicted (B) rats. (C) Summary data for Ca2+ spark related properties. Ca2+ spark frequencies decreased in detrusor myocytes of DO afflicted samples (a). Similarly, F/F0 (b), FWHM (c), rise time (d), and half-time decay (e) all were found to have been significantly reduced in DO afflicted myocytes. We used unpaired t tests for comparisons between treatment groups. VS control *P<0.05, **P<0.01.

  
  
  

  

  Unaltered SR Ca2+ load in detrusor myocytes of DO model rats. (A) Representative linescan images during 10 mM caffeine application in control (a) and DO (b) myocytes. (B) Peak amplitude of the caffeine-evoked fluorescence [Ca2+] i transient was not found to have been significantly different between DO afflicted and unafflicted control myocytes. We used unpaired t tests for comparisons between treatment groups. NS=Not significant.

  
  
  

  

  Ca2+ sparks activity was increased by caffeine and was decreased by ryanodine in both unafflicted control and DO myocyte treatment groups. (A, B) Representative Ca2+ sparks recorded in control (A) and DO (B) detrusor myocytes before and after exposure to10 μM caffeine (Ca2+ spark activator) (b) and before and after exposure to 10 μM ryanodine (Ca2+ spark inhibitor) (c). (C–G) provide summary data for Ca2+ spark frequency, F/F0, FWHM, rise time, and half-time decay of the control and DO destrusor unafflicted samples for treatments with caffeine (10 μM) and ryanodine (10 μM). To record the Ca2+ sparks, we clamped detrusor myocytes at -40 mV. We used one-way ANOVA for comparisons between groups. VS control, *P < 0.05, and **P < 0.01; VS DO, Δ P < 0.05, and ΔΔ P < 0.01.

  
  
  

  

  STOCs were increased by caffeine and decreased by ryanodine in both control and DO myocytes. (A, B) We recorded representative STOCs for control (A) and DO (B) detrusor myocytes before and after exposure to 10 μM caffeine (b) and before and after exposure to 10 μM ryanodine (c). Panels Ca and Cb provide summary data for frequencies and amplitudes of STOCs in unafflicted control and DO detrusor myocytes with regards to applications of caffeine (10 μM) and ryanodine (10 μM) (respectively). To record measures for STOCs, we clamped detrusor myocytes at -40 mV. We used one-way ANOVA for comparison between groups. VS control, *P < 0.05, and **P < 0.01; VS DO, Δ P < 0.05, and ΔΔ P < 0.01.

  
  
  

  

  Effects of Ca2+ spark regulators on detrusor contractions were reversed by the regulation of BK channels in both control and DO detrusor strips. (A, B) Representative spontaneous contractions recorded in control (A) and DO (B) bladder strips after exposure to ryanodine (Ca2+ spark antagonist), caffeine (Ca2+ spark agonist), NS1619 (BK channel agonist) and iberiotoxin (BK channel antagonist). (C) Summary data for A and B, Frequency of spontaneous contractions was significantly increased by application of ryanodine in examinations of control detrusor strips and was decreased by caffeine in examinations of DO afflicted detrusor strips. The effects of ryanodine and caffeine on detrusor contractions were found to have been reversible by way of application of NS1619 or of iberiotoxin, respectively based upon examinations of the data from control and DO detrusor strips. We used one-way ANOVA for comparisons between treatment groups. VS control, *P < 0.05, and **P < 0.01; VS DO, Δ P < 0.05, and ΔΔ P < 0.01. NS=Not significant.

  
  
  

  

  Decreased expression of ryanodine receptor-2 (RyR2) and increased expression of FKBP12.6 in detrusors of rats with DO. (A) Representative results from Western blotting analyses of RyR2, FKBP12.6, and GADPH (used as an internal control) from detrusors of unafflicted control rats and from DO afflicted rats. (B) Summarizations of levels of expression of RyR2 and FKBP12.6 proteins, given as ratios to beta actin. We used one-way ANOVA for comparisons between treatment groups. VS control, *P < 0.05, and **P < 0.01.