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β−Adrenergic receptor subtype expression in myocyte and non-myocyte cells in human female bladder

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Abstract

β3-Adrenergic receptor agonists are currently under clinical development for the treatment of overactive bladder, a condition that is prevalent in postmenopausal women. These agents purportedly relax bladder smooth muscle through a direct action at the myocyte β3-receptor. The aim of this study was to examine the expression of the individual beta-adrenergic receptors in full thickness sections from ageing human female bladder. We obtained a series of rabbit polyclonal antibodies generated against each of the three β-adrenergic receptors, and validated their receptor specificity in CHOK1 cells expressing each of the individual receptors. Immunostaining for β1, β2, and β3 were each more prominent in the urothelium than in the detrusor, with all receptors expressed in the same cell types, indicating co-expression of all three receptors throughout the urothelium in addition to the detrusor. Staining of all receptors was also observed in suburothelial myofibroblast-like cells, intramural ganglion cells, and in Schwann cells of intramural nerves. The β3-receptor in the human urothelium appears to be functional, as two different selective β3-receptor agonists, TAK677 and BRL37344, stimulate cAMP formation in UROtsa cells. Densitometry analysis indicates a persistent expression of all receptors throughout the bladder with increasing age, with the exception of the β2-receptor in the urothelium of the trigone, which appears to decrease slightly in older women. These data indicate that β3-receptor expression is maintained with age, but may function in concert with other β-receptors. Activation of the myocyte receptor may be influenced by action on non-myocyte structures including the intramural ganglion cells and myofibroblasts.

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Acknowledgements

We thank the staff of Asterand Ltd for excellent technical assistance with the expanded human IHC samples, and Dr. Michael P. Meredith (The Procter & Gamble Company) for help with statistical analysis during the preparation of this manuscript.

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Author notes

  1. F. Aura Kullmann

    Present address: Urogenix Inc., Durham, NC, USA

  2. Jan S. Rosenbaum

    Present address: CincyTech USA, Cincinnati, OH, USA

Authors and Affiliations

  1. Women’s Health New Business Development, Procter & Gamble Pharmaceuticals now Warner Chilcott Pharmaceuticals Inc., Mason, OH, USA

    Brian J. Limberg & Jan S. Rosenbaum

  2. Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA

    Karl-Erik Andersson

  3. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA

    F. Aura Kullmann & William C. de Groat

  4. LifeSpan BioSciences, Inc., Seattle, WA, USA

    Glenna Burmer

  5. The Procter & Gamble Company, 8700 Mason-Montgomery Road, Mason, OH, 45040, USA

    Brian J. Limberg

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Correspondence to Brian J. Limberg.

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Supplemental Figure 1

Validation of specific staining for the human β3-AR with the KG115 antibody. The antibody was generated against the human β3-AR -COOH terminus. The antibody was used at a concentration of 10 μg/ml. Staining (red) were observed in transiently transfected CHOK1 cells with the human β2-AR (b) and β3-AR (c) but not β1-AR (a) and empty vector (d). This suggests that the KG115 does not specifically recognize β3-AR by immunostaining. (GIF 179 kb)

High Resolution Image (TIFF 3062 kb)

Supplemental Figure 2

Specific Binding of β3-AR selective ligands in Mock CHOK1 cells. Whole cell binding was performed of CHOK1 cells transiently transfected with pcDNA3.1 (Mock), cDNA to illustrate endogenous expression of hβ3-AR in CHOK1 cells. The binding study was performed using 46 pM [125I] I-CYP tracer and Isoprotenerol (■) a non-selective β-AR ligand or the two β3-AR selective ligands BRL37344(∇) and TAK677(●). Relative affinity (Ki) values were 1.35 × 10-3 M, 9.35 × 10-5 M, and 1.31 × 10-5 M, respectively, for the experiment shown. The error bars represent SEM of three determinations. (GIF 3 kb)

High Resolution Image (TIFF 387 kb)

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Limberg, B.J., Andersson, KE., Aura Kullmann, F. et al. β−Adrenergic receptor subtype expression in myocyte and non-myocyte cells in human female bladder. Cell Tissue Res 342, 295–306 (2010). https://doi.org/10.1007/s00441-010-1053-x

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