To explore muscarinic receptor signaling in 4 bladder cancer cell lines, bladder urothelial cells (BUC) have been shown to release and respond to various putative neurotransmitters.

Reverse transcription-polymerase chain reaction was used to detect the presence of m1-m5 transcripts in the J82, RT4, T24, and 5637 lines of cancer BUC. Immunofluorescence was used to detect expression of m3 protein. Cancer and normal BUC were stimulated with carbachol (100 μM), a muscarinic agonist. Carbachol-evoked changes in intracellular calcium ([Ca²⁺]_i) levels were measured using fura-2 ratiometric microfluorimetry. Transfection of J82 cells with m3 plasmid was performed, and changes in carbachol-evoked [Ca²⁺]_i were re-examined.

None of the cancer cell lines expressed m3 transcripts, unlike normal BUC, which expressed m3. None of the 4 bladder cancer cell lines responded to carbachol. However, 47% of normal BUC responded to carbachol. The m3-transfected J82 cells expressed both m3 transcript and protein. Thirteen percent of m3-transfected J82 cells responded to carbachol.

This is the first description of altered muscarinic signaling in cancer BUC. Unlike normal BUC, bladder urothelial cancer cells neither expressed m3 transcript nor responded to carbachol, as measured by changes in [Ca²⁺]_i. We could partially reverse this defect in one of the cancer cell lines, J82, by transfecting these cells with the m3 plasmid. Although the effects of muscarinic receptor signaling on urothelial cell are unknown, this signaling pathway may play a role in urothelial cell adhesion similar to that in keratinocytes.