The organisation of InsP₃ receptors (IP₃R) and ryanodine receptors (RyR) on intracellular Ca²⁺ stores was examined in single, voltage-clamped myocytes from the portal veins of guinea-pigs.

Guinea-pigs of ~500 g were humanely killed by stunning then immediate exsanguination. Increases in cytosolic Ca²⁺ ([Ca²⁺]_c) were produced by flash photolysis of caged IP₃ (25 µM) or by pressure ejection of caffeine (10 mM). Results are shown as means ± S.E.M. and statistical significance was determined by Student’s paired t test, where P < 0.05 was considered significant.

In ryanodine (50 µM) the amplitudes of IP₃-evoked Ca²⁺ transients were unaffected (ΔF/F₀ = 0.8 ± 0.1 units in control and ΔF/F₀ = 0.9 ± 0.2 units in ryanodine, n = 4) indicating that IP₃-evoked Ca²⁺ release does not evoke Ca²⁺-induced Ca²⁺ release from RyR. Depletion of Ca²⁺ stores, which possess RyR, by caffeine which opens RyR, in the presence of ryanodine (50 µM) abolished Ca²⁺ transients evoked by IP₃, suggesting that IP₃R and RyR were located on the same intracellular Ca²⁺ store. IP₃-evoked Ca²⁺ transients were abolished in a Ca²⁺-free extracellular bathing solution, but caffeine-evoked Ca²⁺ transients persisted at constant amplitude (ΔF/F₀ = 1.3 ± 0.4 units in control and ΔF/F₀ = 1.3 ± 0.3 units after abolition of IP₃ responses in Ca²⁺-free solution, n = 3, P > 0.05). Thus, in addition to a store containing RyR and IP₃R, a separate store with RyR alone existed. Despite an extended time period (> 15 min) in Ca²⁺-free extracellular solution, caffeine-evoked Ca²⁺ transients remained (ΔF/F₀ = 1.1 ± 0.3 units, n = 3) suggesting that the separate RyR-only store was able to refill itself from the cytosolic Ca²⁺ pool. Both IP₃– and caffeine-evoked Ca²⁺ transients were abolished by the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (500 nM, n = 5, P < 0.05).

These results suggest that the guinea-pig portal vein contains two types of Ca²⁺ store; one has only RyR and can be refilled from a cytosolic Ca²⁺ source; the other has both RyR and IP₃R and is dependent for refilling on extracellular Ca²⁺.

This work was funded by the British Heart Foundation and The Wellcome Trust.