AMP-activated protein kinase (AMPK) has been implicated in contractility changes in bladders with partial bladder outlet obstruction (PBOO), but the role of AMPK in the contractile response of normal bladder remains unclear. We investigated the phosphorylation of AMPKα and expression of the involved upstream AMPK kinases (AMPKKs) in a model of bladders with PBOO and sought to determine whether the pharmacological inhibition of these two factors affected detrusor contractility in normal bladders, using female Sprague–Dawley rats. Cystometry and Western blot analysis were performed in rats that were subjected to PBOO induction or a sham operation. Cystometry was performed in normal rats that received selective inhibitors of AMPKα and Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKKβ) (compound C and STO-609, respectively) at doses determined in the experiments. In the PBOO bladders, bladder weight and micturition pressure (MP) were higher and AMPKα phosphorylation (T172) and CaMKKβ expression was significantly reduced. Compound C and STO-609 increased MP. The increased contractile response in bladders with PBOO-induced hypertrophy was related to decreased CaMKKβ/AMPK signaling activity, and the pharmacological inhibition of this pathway in normal bladders increased detrusor contractility, implying a role of CaMKKβ/AMPK signaling in the bladder in the regulation of detrusor contractility.

The involuntary dual control systems of the autonomic nervous system (ANS) in the bladder of awake spontaneously hypertensive rats (SHRs) were investigated through simultaneous registrations of intravesical and intraabdominal pressures to observe detrusor overactivity (DO) objectively as a core symptom of an overactive bladder. SHRs (n = 6) showed the features of overactive bladder syndrome during urodynamic study, especially DO during the filling phase. After injection of the nonselective sympathetic blocking agent labetalol, DO disappeared in 3 of 6 SHRs (50%). DO frequency decreased from 0.98 ± 0.22 min⁻¹ to 0.28 ± 0.19 min⁻¹ (p < 0.01), and DO pressure decreased from 3.82 ± 0.57 cm H₂O to 1.90 ± 0.86 cm H₂O (p < 0.05). This suggests that the DO originating from the overactive parasympathetic nervous system is attenuated by the nonselective blocking of the sympathetic nervous system. The detailed mechanism behind this result is still not known, but parasympathetic overactivity seems to require overactive sympathetic nervous system activity in a kind of balance between these two systems. These findings are consistent with recent clinical findings suggesting that patients with idiopathic overactive bladder may have ANS dysfunction, particularly a sympathetic dysfunction. The search for newer and better drugs than the current anticholinergic drugs as the mainstay for overactive bladder will be fueled by our research on these sympathetic mechanisms. Further studies of this principle are required.

Purpose: Adenosine monophosphate-activated protein kinase (AMPK) is thought to inhibit cell proliferation or promote cell death, but the details remain unclear. In this study, we propose that AMPK inhibits the expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) by relying on the hypoxia-inducible factor 1 alpha (HIF-1α)-induced caveolin-1 (Cav-1) expression pathway in noninvasive human bladder tumor (RT4) cells.Methods: In cells exposed to a hypoxic environment (0.5% oxygen), the levels of expression and phospho-activity of the relevant signaling enzymes were examined via Western blots and reverse transcription-polymerase chain reaction. Cell proliferation was assessed using a Cell Counting Kit-8 assay.Results: The level of expression of Cav-1 was very low or undetectable in RT4 cells. Hypoxia was associated with significantly decreased cell growth, along with marked induction of HIF-1α and Cav-1 expression; additionally, it suppressed the expression of the antiapoptotic marker Bcl-2 while leaving AMPK activity unchanged. Under hypoxic conditions, HIF-1α acts as a transcription factor for Cav-1 mRNA gene expression. The cell growth and Bcl-2 expression suppressed under hypoxia were reversed along with decreases in the induced HIF-1α and Cav-1 levels by AMPK activation with metformin (1mM) or phenformin (0.1mM). In addition, pretreatment with AMPK small interfering RNA not only increased the hypoxia-induced expression of HIF-1α and Cav-1, but also reversed the suppression of Bcl-2 expression. These results suggest that HIF-1α and Cav-1 expression in hypoxic environments is regulated by basal AMPK activity; therefore, the inhibition of Bcl-2 expression cannot be expected when AMPK activity is suppressed, even if Cav-1 expression is elevated.Conclusions: For the first time, we find that AMPK activation can regulate HIF-1α induction as well as HIF-1α-induced Cav1 expression, and the hypoxia-induced inhibitory effect on the antiapoptotic pathway in RT4 cells is due to Cav-1-dependent AMPK activity.

Vascular endothelial growth factor (VEGF), produced by most tumors, not only plays an important role in tumor angiogenesis but also can inhibit the maturation of dendritic cells (DCs) from hematopoietic progenitors. In this study, we investigated the relationship between the clinical characteristics of patients with acute myeloid leukemia (AML) and the generation of DCs from the leukemic cells, and found that cellular VEGF level in leukemic cells affected the generation of leukemic-DCs in vitro. Leukemic-DCs were successfully generated from 16 of 21 AML patients, and generated leukemic DCs exhibited high level of HLA-DR and CD54 expression, and intermediate level of CD80, CD86, CD83, and CD1a expression. Leukemic-DCs also were able to stimulate allogeneic T cell proliferation and cytotoxic activities against autologous leukemic cells. The failure of generation of leukemic-DCs from leukemic cells in some AML patients was related with its cellular VEGF levels. The leukemic cells that failed to generate leukemic-DCs had significantly higher cellular VEGF levels than that of generated leukimic-DCs (p = 0.005), and neutralizing of VEGF with anti-VEGF Ab inhibited the cellular VEGF level at the transcription level as determined by RT-PCR analysis, and at the protein level determined by West blotting and ELISA. Inhibition of cellular VEGF levels induced the generation of leukemic-DCs determined by increased levels of surface markers, namely, CD40, CD80, CD86, and HLA-DR. Moreover, inhibition of cellular VEGF levels also increased IL-12 production and allosimulatory capacity of leukemic-DCs, suggesting functional differentiation of leukemic-DCs. Interestingly, cellular VEGF levels also are inversely related to the achievement of complete remission for induction therapy (p = 0.02). Taken together, these results suggest that cellular VEGF levels during the differentiation of AML cells to leukemic-DCs are a predictive marker of the positive response of the AML patients to the remission-inducing therapy, and down-regulation of cellular VEGF levels can be one target of cancer immunotherapy by generating of sufficient number of DCs in AML patients.