Although confirmed in sea urchin eggs, the ability of cADPR to release Ca²⁺ from mammalian smooth muscle SR is disputed. Even where cADPR is acknowledged to release Ca²⁺ from the SR, its precise mechanism of action is unresolved. In particular, the contribution of the FK506 binding protein 12.6 (FKBP12.6) in mediating the cADPR response is unclear. In the present study the ability of cADPR to modulate Ca²⁺ release from the SR was examined in a smooth muscle that lacked FKBP12.6 but contained functional RyR and IP₃R, which may modulate the physiological response of the tissue to excitatory agonists (McCarron et al. 2002). Male guinea-pigs were humanely killed by cervical dislocation followed by exsanguination according to the requirements of the Animal (Scientific Procedures) Act, 1986. Data presented are means ± S.E.M. A Student’s paired t test was used to test for significant difference (P < 0.05). The absence of FKBP12.6 in colonic smooth muscle and the protein’s presence in brain (both guinea-pig) were confirmed by Western blots. In single guinea-pig colonic myocytes voltage-clamped in the whole-cell configuration and in which the cytosolic Ca²⁺ ([Ca²⁺]_c) was measured using fluo-3, photolysed IP₃ (25 mM) and caffeine (10 mM; by hydrostatic application) each significantly (P < 0.01) increased [Ca²⁺]_c (ΔF/F₀ = 1.3 ± 0.2 and 1.3 ± 0.1 units, respectively, n = 12). However, photolysed cADPR (50 or 500 mM) increased neither bulk average [Ca²⁺]_c (ΔF/F₀ = 0.01 ± 0.01 units, n = 11) nor local subsarcolemma Ca²⁺ (the frequency and amplitude of spontaneous transient outward currents were unaltered). Nor did cADPR increase Ca²⁺ transients evoked by depolarisation (-70 to +10 mV, n = 6), even though low concentrations of caffeine (~500 mM) were effective in this respect (depolarisation-evoked response increased to 174 ± 40 %, n = 10, P < 0.01, control value was 100 %). These results suggest that cADPR did not enable Ca²⁺-induced Ca²⁺ release. Furthermore, cADPR did not increase the Ca²⁺ signal evoked by caffeine in concentrations that produced submaximal responses. In contrast to its failure to alter [Ca²⁺]_c in smooth muscle, flash photolysis of caged cADPR increased [Ca²⁺]_c significantly (P < 0.05) in sea urchin eggs (ΔF/F₀ = 3.1 ± 0.9 units, n = 10). Together these results suggest that in mammalian smooth muscle lacking FKBP12.6 cADPR does not regulate Ca²⁺ release.

We would like to thank Professor M. Whitaker and Dr C. Leckie, Department of Physiological Sciences, Medical School, University of Newcastle upon Tyne, UK for their advice and assistance on the preparation of sea urchin eggs and Dr T. Seidler, Klinikum Göttingen, Göttingen, Germany for the kind gift of anti-FKBP12.6 antibody. This work was supported by the British Heart Foundation and The Wellcome Trust.

All procedures accord with current UK legislation.