title: Beta-adrenergic receptor activation in immortalized human urothelial cells stimulates inflammatory responses by PKA-independent mechanisms.
abstract: BACKGROUND
  Interstitial cystitis (IC) is a debilitating disease characterized by chronic inflammation of the urinary bladder, yet specific cellular mechanisms of inflammation in IC are largely unknown. Multiple lines of evidence suggest that beta-adrenergic receptor (AR) signaling is increased in the inflamed urothelium, however the precise effects of these urothelial cell signals have not been studied. In order to better elucidate the AR signaling mechanisms of inflammation associated with IC, we have examined the effects of beta-AR stimulation in an immortalized human urothelial cell line (UROtsa). For these studies, UROtsa cells were treated with effective concentrations of the selective beta-AR agonist isoproterenol, in the absence or presence of selective inhibitors of protein kinase A (PKA). Cell lysates were analyzed by radioimmunoassay for generation of cAMP or by Western blotting for induction of protein products associated with inflammatory responses.
  
  
  RESULTS
  Radioligand binding demonstrated the presence of beta-ARs on human urothelial UROtsa cell membranes. Stimulating UROtsa cells with isoproterenol led to concentration-dependent increases of cAMP production that could be inhibited by pretreatment with a blocking concentration of the selective beta-AR antagonist propranolol. In addition, isoproterenol activation of these same cells led to significant increases in the amount of phosphorylated extracellular signal-regulated kinase (pERK), inducible nitric oxide synthase (iNOS) and the induced form of cyclooxygenase (COX-2) when compared to control. Moreover, preincubation of UROtsa cells with the selective PKA inhibitors H-89 or Rp-cAMPs did not diminish this isoproterenol mediated phosphorylation of ERK or production of iNOS and COX-2.
  
  
  CONCLUSIONS
  Functional beta-ARs expressed on human urothelial UROtsa cell membranes increase the generation of cAMP and production of protein products associated with inflammation when activated by the selective beta-AR agonist isoproterenol. However, the increased production of iNOS and COX-2 by isoproterenol is not blocked when UROtsa cells are preincubated with inhibitors of PKA. Therefore, UROtsa cell beta-AR activation significantly increases the amount of iNOS and COX-2 produced by a PKA-independent mechanism. Consequently, this immortalized human urothelial cell line can be useful in characterizing potential AR signaling mechanisms associated with chronic inflammatory diseases of the bladder.
authors.affiliation: Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota, School of Medicine & Health Sciences, Grand Forks, ND 58202-9037, USA. eharmon@medicine.nodak.edu
authors.forename: Erin B
authors.initials: EB
authors.lastname: Harmon
authors.forename: Jill M
authors.initials: JM
authors.lastname: Porter
authors.forename: James E
authors.initials: JE
authors.lastname: Porter