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Gene expression of muscarinic, tachykinin, and purinergic receptors in porcine bladder: comparison with cultured cells.

Bahadory F, Moore KH, Liu L, Burcher E - Front Pharmacol (2013)

Bottom Line: The gene expression of all receptors decreased in culture compared with the fresh tissue, although the reduction in cultured urothelial cells appeared less significant compared to suburothelial and detrusor cells.Cultured myofibroblasts and detrusor cells did not contract in response to the agonists acetylcholine, neurokinin A, and β,γ-MeATP, up to concentrations of 0.1 and 1 mM.The significant reduction of M3, NK2, and P2X1 receptors under culture conditions may be associated with the unresponsiveness of cultured suburothelial and detrusor cells to their respective agonists.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medical Sciences, University of New South Wales Sydney, NSW, Australia.

ABSTRACT
Urothelial cells, myofibroblasts, and smooth muscle cells are important cell types contributing to bladder function. Multiple receptors including muscarinic (M3/M5), tachykinin (NK1/NK2), and purinergic (P2X1/P2Y6) receptors are involved in bladder motor and sensory actions. Using female pig bladder, our aim was to differentiate between various cell types in bladder by genetic markers. We compared the molecular expression pattern between the fresh tissue layers and their cultured cell counterparts. We also examined responses to agonists for these receptors in cultured cells. Urothelial, suburothelial (myofibroblasts), and smooth muscle cells isolated from pig bladder were cultured (10-14 days) and identified by marker antibodies. Gene (mRNA) expression level was demonstrated by real-time PCR. The receptor expression pattern was very similar between suburothelium and detrusor, and higher than urothelium. The gene expression of all receptors decreased in culture compared with the fresh tissue, although the reduction in cultured urothelial cells appeared less significant compared to suburothelial and detrusor cells. Cultured myofibroblasts and detrusor cells did not contract in response to the agonists acetylcholine, neurokinin A, and β,γ-MeATP, up to concentrations of 0.1 and 1 mM. The significant reduction of M3, NK2, and P2X1 receptors under culture conditions may be associated with the unresponsiveness of cultured suburothelial and detrusor cells to their respective agonists. These results suggest that under culture conditions, bladder cells lose the receptors that are involved in contraction, as this function is no longer required. The study provides further evidence that cultured cells do not necessarily mimic the actions exerted by intact tissues.

No MeSH data available.


Related in: MedlinePlus

(A) Histological staining (A) and fluorescent immunostaining (B–D) of intact segments of porcine bladder. (A) Masson's staining showing urothelium (u), smooth muscle bundles (sm), and blood vessels (bv). (B) Immunocytochemical characterization with AE1/AE3 (cytokeratin marker) shows staining of urothelium only. (C) Immunostaining with vimentin shows labeling of large spindle-shaped cells (arrowheads) and a cell layer (*) under the urothelium. (D) Immunostaining with α-SMA (smooth muscle marker) shows labeling of smooth muscle bundles, the outer perimeter of blood vessels as well as part of a cell layer (*, suburothelium) under the urothelium. All cells were double stained with DAPI (cell nuclei, blue). All panels are shown at the same magnification. Bar = 50 μm.
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Figure 1: (A) Histological staining (A) and fluorescent immunostaining (B–D) of intact segments of porcine bladder. (A) Masson's staining showing urothelium (u), smooth muscle bundles (sm), and blood vessels (bv). (B) Immunocytochemical characterization with AE1/AE3 (cytokeratin marker) shows staining of urothelium only. (C) Immunostaining with vimentin shows labeling of large spindle-shaped cells (arrowheads) and a cell layer (*) under the urothelium. (D) Immunostaining with α-SMA (smooth muscle marker) shows labeling of smooth muscle bundles, the outer perimeter of blood vessels as well as part of a cell layer (*, suburothelium) under the urothelium. All cells were double stained with DAPI (cell nuclei, blue). All panels are shown at the same magnification. Bar = 50 μm.

Mentions: Adjacent sections of whole porcine bladder were stained histologically with Masson's (Figure 1A) to reveal histological features, and immunostained with marker antibodies (Figures 1B–D). The urothelium was stained by the cytokeratin marker AE1/AE3 (Figure 1B) but not by vimentin (Figure 1C) or by α-SMA (Figure 1D). A cell layer 40–50 μm thick under the urothelium was stained moderately by both vimentin and α-SMA. Blood vessels in the lamina propria were stained strongly by α-SMA and weakly by vimentin. Detrusor muscle bundles were stained strongly by α-SMA and not at all by vimentin. However, large spindle shaped cells throughout the lamina propria were stained only by vimentin.


Gene expression of muscarinic, tachykinin, and purinergic receptors in porcine bladder: comparison with cultured cells.

Bahadory F, Moore KH, Liu L, Burcher E - Front Pharmacol (2013)

(A) Histological staining (A) and fluorescent immunostaining (B–D) of intact segments of porcine bladder. (A) Masson's staining showing urothelium (u), smooth muscle bundles (sm), and blood vessels (bv). (B) Immunocytochemical characterization with AE1/AE3 (cytokeratin marker) shows staining of urothelium only. (C) Immunostaining with vimentin shows labeling of large spindle-shaped cells (arrowheads) and a cell layer (*) under the urothelium. (D) Immunostaining with α-SMA (smooth muscle marker) shows labeling of smooth muscle bundles, the outer perimeter of blood vessels as well as part of a cell layer (*, suburothelium) under the urothelium. All cells were double stained with DAPI (cell nuclei, blue). All panels are shown at the same magnification. Bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842897&req=5

Figure 1: (A) Histological staining (A) and fluorescent immunostaining (B–D) of intact segments of porcine bladder. (A) Masson's staining showing urothelium (u), smooth muscle bundles (sm), and blood vessels (bv). (B) Immunocytochemical characterization with AE1/AE3 (cytokeratin marker) shows staining of urothelium only. (C) Immunostaining with vimentin shows labeling of large spindle-shaped cells (arrowheads) and a cell layer (*) under the urothelium. (D) Immunostaining with α-SMA (smooth muscle marker) shows labeling of smooth muscle bundles, the outer perimeter of blood vessels as well as part of a cell layer (*, suburothelium) under the urothelium. All cells were double stained with DAPI (cell nuclei, blue). All panels are shown at the same magnification. Bar = 50 μm.
Mentions: Adjacent sections of whole porcine bladder were stained histologically with Masson's (Figure 1A) to reveal histological features, and immunostained with marker antibodies (Figures 1B–D). The urothelium was stained by the cytokeratin marker AE1/AE3 (Figure 1B) but not by vimentin (Figure 1C) or by α-SMA (Figure 1D). A cell layer 40–50 μm thick under the urothelium was stained moderately by both vimentin and α-SMA. Blood vessels in the lamina propria were stained strongly by α-SMA and weakly by vimentin. Detrusor muscle bundles were stained strongly by α-SMA and not at all by vimentin. However, large spindle shaped cells throughout the lamina propria were stained only by vimentin.

Bottom Line: The gene expression of all receptors decreased in culture compared with the fresh tissue, although the reduction in cultured urothelial cells appeared less significant compared to suburothelial and detrusor cells.Cultured myofibroblasts and detrusor cells did not contract in response to the agonists acetylcholine, neurokinin A, and β,γ-MeATP, up to concentrations of 0.1 and 1 mM.The significant reduction of M3, NK2, and P2X1 receptors under culture conditions may be associated with the unresponsiveness of cultured suburothelial and detrusor cells to their respective agonists.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medical Sciences, University of New South Wales Sydney, NSW, Australia.

ABSTRACT
Urothelial cells, myofibroblasts, and smooth muscle cells are important cell types contributing to bladder function. Multiple receptors including muscarinic (M3/M5), tachykinin (NK1/NK2), and purinergic (P2X1/P2Y6) receptors are involved in bladder motor and sensory actions. Using female pig bladder, our aim was to differentiate between various cell types in bladder by genetic markers. We compared the molecular expression pattern between the fresh tissue layers and their cultured cell counterparts. We also examined responses to agonists for these receptors in cultured cells. Urothelial, suburothelial (myofibroblasts), and smooth muscle cells isolated from pig bladder were cultured (10-14 days) and identified by marker antibodies. Gene (mRNA) expression level was demonstrated by real-time PCR. The receptor expression pattern was very similar between suburothelium and detrusor, and higher than urothelium. The gene expression of all receptors decreased in culture compared with the fresh tissue, although the reduction in cultured urothelial cells appeared less significant compared to suburothelial and detrusor cells. Cultured myofibroblasts and detrusor cells did not contract in response to the agonists acetylcholine, neurokinin A, and β,γ-MeATP, up to concentrations of 0.1 and 1 mM. The significant reduction of M3, NK2, and P2X1 receptors under culture conditions may be associated with the unresponsiveness of cultured suburothelial and detrusor cells to their respective agonists. These results suggest that under culture conditions, bladder cells lose the receptors that are involved in contraction, as this function is no longer required. The study provides further evidence that cultured cells do not necessarily mimic the actions exerted by intact tissues.

No MeSH data available.


Related in: MedlinePlus