Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium

Abstract

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal transduction.

Fig. 1.

Expression of transient receptor potential (TRP) channels in mouse bladder and urothelium. A: expression of message for the indicated TRP channels was detected by RT-PCR from total RNA isolated from mouse bladder. Specific TRP channel primers are given in Table 1. RT-PCR products were resolved on 2% agarose gels and visualized by staining with ethidium bromide. Numbers above or beneath the DNA bands are expected product sizes (in bp). B: RT-PCR analysis of TRP channel expression in RNA isolated from the urothelium. C: RT-PCR were performed in the absence of reverse transcriptase (left lane) or in the presence of a housekeeping gene primer (right lane) to ensure the quality of RNA and PCR. D: for TRP channels not detected in whole bladder, RT-PCR analysis was performed using RNA from positive control tissues including brain, kidney, and heart.

Fig. 2.

Western blot analysis of select TRP channels in mouse urothelium. Lysates of mouse or rat urothelium (25 μg of protein) were resolved by SDS-PAGE, and Western blots were probed with antibodies specific for the indicated TRP channel. The major protein species are indicated on the left by arrowheads, and the migration of molecular size markers is indicated to the right of each panel.

Fig. 3.

Localization of TRP channels in mouse and rat urothelium. Cryosections of mouse (A–D, F, and G) or rat (E and H–J) bladder tissue were labeled with the indicated TRP channel antibodies (green) and either rhodamine phalloidin (D and G), or antibodies to claudin 4 (E and H–J) or aquaporin-3 (AQP3; A, B, C, and F; all displayed in red). Nuclei were labeled with ToPro-3 (blue). Far right: merged images. In some panels, a dashed white line indicates the apical surface of the umbrella cells. In B, TRPC4-positive fibrils below the basal cell layer are indicated by arrows. TRPV1-labeled filaments, likely afferent nerve processes, are indicated by asterisks in C. Scale bars = 10 μm.

Fig. 3.

Localization of TRP channels in mouse and rat urothelium. Cryosections of mouse (A–D, F, and G) or rat (E and H–J) bladder tissue were labeled with the indicated TRP channel antibodies (green) and either rhodamine phalloidin (D and G), or antibodies to claudin 4 (E and H–J) or aquaporin-3 (AQP3; A, B, C, and F; all displayed in red). Nuclei were labeled with ToPro-3 (blue). Far right: merged images. In some panels, a dashed white line indicates the apical surface of the umbrella cells. In B, TRPC4-positive fibrils below the basal cell layer are indicated by arrows. TRPV1-labeled filaments, likely afferent nerve processes, are indicated by asterisks in C. Scale bars = 10 μm.

Fig. 4.

Colocalization of TRPC4 and TRPV1 with neuron-specific markers, PGP9.5 and calcitonin gene-related peptide (CGRP) in bladder tissue. Bladder sections were labeled with the either TRPC4 (A) or TRPV1 (B) antibodies (green) and PGP9.5 or CGRP antibodies (red). Nuclei were labeled with ToPro-3 (blue). Far right: merged images. The colocalization of TRPC4/TRPV1 with PGP9.5/CGRP are indicated by arrows. Scale bars = 10 μm.