cADP-ribose (cADPR) has been shown to regulate calcium release from intracellular stores in a variety of mammalian cells. However, little is known regarding possible actions of the phosphate analogue, cADPR-P. The aim of this study was to investigate any possible effects of cADPR-P on guinea-pig ventricular myocytes.

Male guinea-pigs were killed by cervical dislocation following stunning, and myocytes isolated enzymatically from the ventricles of the hearts. Cells were superfused with physiological solution containing 2.5 mM calcium (36 °C) and patch clamped (using either conventional whole-cell or amphotericin-permeabilized configurations). Action potentials were stimulated at 1 Hz. Cell shortening was measured from a video image of the cell using an edge-detection system. Sarcoplasmic reticulum (SR) loading was assessed under voltage-clamp conditions, by measuring the integral of the inward current generated in response to rapid application of 20 mM caffeine. Data are quoted as means ± S.E.M. and statistical significance assessed using Student’s paired t test.

cADPR-P caused a concentration-dependent (100 nM to 100 mM in the pipette) increase in cell contraction when applied using conventional whole-cell patch (control measurements made within 30 s of membrane rupture when little cADPR-P is expected to have entered the cytoplasm). 10 mM cADPR-P increased contraction by 32 ± 8 % after 3 min (n = 6, P < 0.05). In a second series of experiments, control measurements were made in the permeabilized patch after which the membrane was ruptured to allow cADPR-P entry to the cytoplasm. At low concentrations (10 nM to 1 mM), there was a significant decrease in cell contraction. 100 nM cADPR-P decreased cell contraction by 36 ± 7 % after 3 min (n = 6, P < 0.05). At higher concentrations (10 and 100 mM) there was a significant increase in cell contraction. 10 mM cADPR-P increased cell contraction by 26 ± 5 % after 3 min (n = 10, P < 0.05). In six of these cells in which 10 mM cADPR-P increased contraction by 28 ± 5 % (P < 0.05), there was no significant change in SR loading (6 ± 7 %, n = 6, P > 0.05). In contrast, there were no significant changes in cell contraction over this time period if cADPR-P was excluded from the patch pipette in either series of experiments.

These data are consistent with a biphasic action of cADPR-P on contraction of guinea-pig ventricular myocytes, with low concentrations reducing contraction magnitude, and higher concentrations enhancing it. The enhancement of contraction was not associated with an increase in loading of the SR with calcium. Whether cADPR-P exerts its effects through the same mechanism as cADPR or independently remains to be determined.

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

All procedures accord with current UK legislation.