Human bladder biopsy samples were obtained at open surgery, placed in Ca-free HEPES buffer and used immediately for experiments. Local ethical committee approval and patient consent were obtained for each specimen. For immunoconfocal microscopy of cryosections, full-thickness specimens (3–5 mm2) were placed in a mould containing Cryo-M-Bed (Bright Instrument Co. Ltd, UK) and frozen directly in liquid nitrogen. For electrophysiological measurements and Northern blotting, mucosa was dissected from detrusor in Ca-free HEPES buffer. Samples of both portions were frozen for Northern blots and detrusor strips cut for impedance measurements. Samples were classified into two categories: (i) stable, i.e. from patients with bladder cancer and no symptomatic evidence of detrusor instability, taken distant from the malignant sites; (ii) patients with urodynamically confirmed idiopathic detrusor instability. Three samples were also obtained from patients with hyper-reflexia and their data are reported separately.
Impedance was measured at 37°C in Tyrode's solution (mmol/L): NaCl, 118; KCl, 4.0; NaHCO3, 24; NaH2PO4, 0.4; MgCl2, 1.0; CaCl2, 1.8; glucose, 6.0; Na pyruvate, 5.0; pH 7.4 with 5% CO2/95% O2. Tissues were transported in a Ca-free HEPES-buffered solution with NaHCO3 replaced by 10 mmol/L HEPES and 14 mmol/L NaCl, pH 7.1 with NaOH.
For impedance measurements, a three-compartment oil-gap chamber was used, previously validated with guinea-pig detrusor muscle and myocardium [10,17]. A detrusor strip (diameter ≈ 1 mm, length 4–7 mm) was pulled through rubber partitions separating the compartments, with at least 1 mm in the outer chambers containing Tyrode solution; the middle chamber was filled with mineral oil. Constant amplitude alternating current (frequency, f 20 Hz−300 kHz; ω = 2 πf) flowed between platinum-black electrodes in the outer chambers and thus was constrained to flow via the preparation intracellular space, with a proportion through an extracellular shunt. Resistance, r, and capacitance, c, of the system were recorded with a balanced Wien bridge (Wayne-Kerr 6425, UK) assuming a parallel rc configuration . Two sets of recordings were made 10 min apart; values were always within 5% and the average used. Platinum-black electrode resistance, re, and capacitance, ce were measured separately in a large volume of Tyrode solution. The resistivity of Tyrode solution (RT, 49 ± 1 Ω.cm; 37°C, n = 3) was measured in a conductivity cell (length 1.0 cm, cross-sectional area 0.070 cm2).
Electrode polarization resistance, rp, and capacitance, cp, were calculated from re and ce[10,19]:
Preparation impedance, zs, was initially calculated as resistive, rs, and reactive, xs, components , using r and c values and electrode polarization properties:
Extracellular shunt correction, rec, to yield detrusor impedance zd, was obtained from the relation:
Detrusor impedance, zd, was finally separated into resistive, rd (= zs.cosφ) and reactive, xd (=zs.sinφ) components, where φ= tan−1(xs/rs). Values of zd, rd and xd (Ω.cm−1) were converted to specific values Z, Rd and Xd (Ω.cm) on multiplication by muscle cross-sectional area.
Measurement of extracellular shunt resistance. An electrical model of the preparation assumed an extracellular shunt, rec, in parallel with the detrusor impedance. Two needle platinum-black electrodes were placed in the preparation within the oil-gap at different distances apart; rec (Ω.cm−1) was determined from the slope of the resistance vs distance plot. The equivalent cross-sectional area of strip contributing to rec, assuming it was filled with Tyrode soultion, is RT/rec (Ω.cm/(cm−1) = cm2) and is expressed as a proportion of total preparation cross-sectional area.
To detect connexin43 by immunoconfocal microscopy a commercially available mouse monoclonal antibody of proven specificity was used [12,20] (Chemicon International Ltd, UK; dilution 1 : 1000). Connexin40 and connexin45 were detected with polyclonal antibodies . For connexin40, rabbit polyclonal antibody S15C(R83) was used at 1 : 250; for connexin45, guinea-pig polyclonal antibodies Q14E(GP42) or Q14E(GP42B) were used at 1 : 100 and 1 : 500, respectively. All polyclonal anticonnexin antibodies raised in the National Heart & Lung Institute laboratories were confirmed be isotype and gap junction-specific by immunofluorescence and Western blotting of connexin transfectants, and by electron-microscopic immunogold labelling . Q14E(GP42B) is a newly purified batch of the original antiserum Q14E(GP42) raised in the same guinea-pig , which was characterized by Western blot and immunofluorescence against the original Q14E(GP42) for comparison.
The fluorescent secondary detection systems were: donkey antimouse-Cy3 (1 : 250; Chemicon) or Texas Red (1 : 500; Jackson ImmunoResearch Laboratory, Inc, USA) for connexin43; antirabbit TRITC (1 : 25, DAKO Ltd, UK) for connexin40; and donkey antiguinea-pig-Cy3 (1 : 250; Chemicon) for connexin45. The use of different fluorochromes ensured clear visualization against the natural autofluorescence of the tissue. Both primary and secondary antibodies were diluted in PBS containing 1% BSA and 5% human serum.
Sections (12 µm thick) of directly frozen (not chemically fixed) specimens were prepared using a cryomicrotome, mounted on poly l-lysine-coated slides and stored at − 80°C until use. The unfixed sections were immersed in methanol at −20°C for 10 min, rinsed three times in PBS for 10 min and blocked in PBS-buffered 1% BSA for 1 h before primary antibody incubation for 2 h at room temperature. The sections were then washed three times in PBS and incubated with the matching fluorochrome-conjugated secondary antibody for 1 h at room temperature. Negative immunolabelling controls omitted the primary antibody; positive controls used sections of rat heart in which the distribution of the three connexin isotypes is established .
Immunolabelled sections were examined by confocal laser scanning microscopy, using a system equipped with argon, krypton and helium-neon lasers. To distinguish autofluorescence (e.g. that from elastic laminae) from specific immunolabel signal, the fluorescein isothiocyanate channel detector was used. Series of images were sequentially recorded for each channel to avoid signal crossover.
For thin-section electron microscopy, samples were fixed in 2.5% glutaraldehyde, followed by 2% osmium tetroxide in cacodylate buffer. After dehydration through an ethanol series, the samples were embedded in epoxy resin (Araldite CY212, Agar Scientific Ltd, UK). Thin sections were stained with uranyl acetate and lead citrate, and examined in an electron microscope.
For Northern blot analysis, total cellular RNA was purified from frozen, pulverized tissues using a modified guanidinium isothiocyanate/acid phenol extraction [22,23]. Equal amounts (5 µg/lane) of each sample were run in formaldehyde-agarose gels and capillary-transferred onto nylon membrane (Hybond N, Amersham Int., UK). High stringency hybridization was used (65 °C, 5 × saline-citrate) with random primed probes generated from gel-purified inserts (connexin43, connexin40, connexin45) radiolabelled using 32P . Northern blots were quantified by densitometric scanning of autoradiograms, using 10 samples in each group; multiple exposures were obtained to ensure linearity of the film response.