Materials and Methods

Reverse Transcription–PCR. Poly (A) RNA was isolated from tissue or cultured cells by homogenization in Trizol (Life Technologies, Grand Island, NY) followed by selection on Oligotex columns (Qiagen, Chatsworth, CA). Twenty nanograms of polyA RNA were reverse transcribed with an oligo (dT) primer in 50 mM TrisCl75 mM KCl3 mM MgCl2 0.01 M DTT20 units of RNase inhibitor0. 5 mM dNTP by using 200 units of Superscript II (Life Technologies), then treated with 1 unit of RNase H. PCR amplification (94 C for 2 min; 35 cycles of 94 C for 30 s, 60 C for 30 s, 72 C for 1 min; 72 C for 2 min) was conducted in 20 mM TrisCl50 mM KCl1. 5 mM MgCl2 0.2 mM dNTP0. 4 μM primer pairs, using 1.25 units of platinum Taq (Life Technologies) and the following primers: VR-1, 5-caaggctgtcttcatcatcc-3 and 5-attcccacacacctcccagttc-3; VRL-1, 5-gatgaagagggcaatgc-3, 5-ccattcagtctccatgaagc-3. Amplification products were visualized on a 1% agarose gel with ethidium bromide and sequenced to confirm identity.

Cell Culture. Preparation and characterization of dorsal root ganglia and urothelial cultures has been described in previous reports (1–3). Briefly, bladders excised from anesthetized Sprague–Dawley rats, VR1 null mice (4), or wild-type littermates were cut open and gently stretched (urothelial side down). Muscle was dissected away, and the urothelium was incubated overnight in MEM (Cellgro), penicillinstreptomycinfungizone and 2.5 mg ml 1 dispase (GIBCO). The urothelium was gently scraped from underlying tissue, treated with 0.25% trypsin, and resuspended in keratinocyte medium (GIBCO). The single cell suspension (0.1 ml, 50,000–150,000 cells per ml) was plated on the surface of collagen-coated dishes. All cells in these cultures were cytokeratin positive (cytokeratins 17 and 20, Dako) and, therefore, were presumably of epithelial origin. All procedures involving rats and mice were conducted in accordance with Institutional Animal Care and Use Committee policies.