title: Elevated polyamines in urothelial cells from OAB subjects mediate oxotremorine-evoked rapid intracellular calcium rise and delayed acetylcholine release.
abstract: Increased polyamine signaling in bladder urothelial cells (BUC) may play a role in the pathophysiology of overactive bladder (OAB). We quantitated intracellular polyamine levels in cultured BUC from OAB and asymptomatic (NB) subjects. We assessed whether polyamines modulated rapid intracellular calcium ([Ca(2+)]i) changes and delayed acetylcholine (ACh) release evoked by oxotremorine (OXO, a muscarinic agonist). BUC were cultured from cystoscopic biopsies. High-performance liquid chromatography (HPLC) quantitated intracellular putrescine, spermidine, and spermine levels. Five-millimeter difluoromethylornithine (DFMO), and one-millimeter methylglyoxalbisguanylhydrazone (MGBG) treatments were used to deplete intracellular polyamines. Ten micrometers of OXO were used to increase [Ca(2+)]i levels (measured by fura 2 microfluorimetry) and trigger extracellular ACh release (measured by ELISA). Polyamine levels were elevated in OAB compared with NB BUC (0.5 ± 0.15 vs. 0.16 ± 0.03 nmol/mg for putrescine, 2.4 ± 0.21 vs. 1.01 ± 0.13 nmol/mg for spermidine, and 1.90 ± 0.27 vs. 0.86 ± 0.26 nmol/mg for spermine; P < 0.05 for all comparisons). OXO evoked greater [Ca(2+)]i rise in OAB (205.10 ± 18.82% increase over baseline) compared with in NB BUC (119.54 ± 13.01%; P < 0.05). After polyamine depletion, OXO evoked [Ca(2+)]i rise decreased in OAB and NB BUC to 43.40 ± 6.45 and 38.82 ± 3.5%, respectively. OXO tended to increase ACh release by OAB vs. NB BUC (9.02 ± 0.1 vs. 7.04 ± 0.09 μM, respectively; P < 0.05). Polyamine depletion reduced ACh release by both OAB and NB BUC. In conclusion, polyamine levels were elevated twofold in OAB BUC. OXO evoked greater increase in [Ca(2+)]i and ACh release in OAB BUC, although these two events may be unrelated. Depletion of polyamines caused OAB BUC to behave similarly to NB BUC.
authors.affiliation: Dept. of Urology, Yale School of Medicine, 789 Howard Ave., FMP 300, P.O. Box 208058, New Haven, CT 06520-8058. toby.chai@yale.edu.
authors.forename: Mingkai
authors.initials: M
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.forename: Noboru
authors.initials: N
authors.lastname: Tomiya
authors.forename: Yuchao
authors.initials: Y
authors.lastname: Hsu
authors.forename: Toby C
authors.initials: TC
authors.lastname: Chai
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D010095:Oxotremorine / Q000494:pharmacology; D011073:Polyamines / Q000378:metabolism*; D011700:Putrescine / Q000378:metabolism; D015398:Signal Transduction; D013095:Spermidine / Q000378:metabolism; D013096:Spermine / Q000378:metabolism; D053201:Urinary Bladder, Overactive / Q000378:metabolism*; D019459:Urothelium / Q000378:metabolism*
chemical_list: D011073:Polyamines; D013096:Spermine; D010095:Oxotremorine; D000109:Acetylcholine; D002118:Calcium; D013095:Spermidine; D011700:Putrescine
keywords: intracellular calcium; muscarinic signaling; overactive bladder; polyamine; urothelial cells