Posterior urethral valves (PUV) result in congenital bladder outflow obstruction exclusively affecting boys (Woolf & Thiruchelvam, 2001). As a consequence of this prenatal obstruction, these boys often have persistent postnatal bladder dysfunction in later life, with resultant end-stage renal failure and delayed achievement of urinary continence. To understand the pathophysiology of PUV, this study examined the role of intracellular Ca²⁺ ([Ca²⁺]_i) regulation in altered contractility using a fetal lamb model with induced in utero bladder outflow obstruction.

Partial bladder outflow obstruction was induced in male fetal sheep by placement of an omega-shaped urethral ring and urachal ligation midway through gestation, at 75 days (full-term 150 days) under 2Ð3 % halothane anaesthesia in a NO/O₂ mixture. Animals were killed 30 days after surgery (105 days) with I.V. sodium pentobarbitone. Procedures accorded to UK legislation. Sham-operated control fetuses underwent urethral and urachal exposure only. Detrusor smooth muscle was obtained from the mid-region of bladders after removal of mucosa and serosa. Isometric twitch tension was measured from small muscle strips (less than 1 mm in diameter) and [Ca²⁺]_i measured in single dissociated myocytes loaded with the fluorescent indicator fura-2 (Wu & Fry, 1998). Contraction and [Ca²⁺]_i rise were elicited by carbachol, an analogue of the functional neurotransmitter acetylcholine. Data were expressed as means ± S.E.M. and Student’s unpaired t test used to test the statistical significance between data sets.

In multicellular preparations, the force of contraction in response to carbachol (10 mM) was significantly reduced in obstructed fetal bladders (obstructed: 2.4 ± 0.5 mN mg^-1, n = 4; sham-operated control: 4.7 ± 0.2 mN mg^-1, n = 3; P < 0.05). In isolated detrusor cells, 10 mM carbachol elicited a [Ca²⁺]_i transient from a resting value of 118 ± 8 nM with an net increase of 629 ± 86 nM (n = 16), whilst in cells from obstructed bladders, the net rise was only 414 ± 59 nM from a resting value of 144 ± 2 nM (n = 20, P < 0.05). Further experiments determined the concentration dependence of the carbachol-induced [Ca²⁺]_i rise over a range from 0.03 to 100 mM. The minimal effective concentration was around 0.1 mM and the maximal effect around 10 mM. An EC₅₀ of 0.8 ± 0.2 mM (n = 6) was obtained from the doseÐresponse relationship for the control group, whilst the relationship was shifted to the right in the obstruction group (EC₅₀ 2.4 ± 0.5 mM, n = 6; P < 0.05).

These results demonstrate that bladder outflow obstruction during fetal development results in a reduced muscarinic receptor efficacy and sensitivity in detrusor myocytes. The impaired Ca²⁺ regulation coupled to the receptor activation in these cells may in part account for the reduced bladder contractility found after in utero bladder obstruction.

We thank the Royal College of Surgeons (England) for financial assistance.

All procedures accord with current UK legislation.