We have studied the long-term effects of the membrane-soluble cAMP analogue (8-CPT-cAMP) on the properties of voltage-gated Ca²⁺ channels expressed in cultured rat chromaffin cells from humanely killed rats. Whole-cell Ca²⁺ currents were measured in bath solutions containing 10 mM Ca²⁺ plus 300 nM TTX to block voltage-gated Na⁺ channels. In most experiments, cells were held at -80 mV holding potential (V_h). In these conditions, the inward Ca²⁺ currents measured in cells of 3Ð8 days in culture had similar voltage-dependent characteristics. The currents started activating at about -30 mV, reached maximal amplitude at +15 mV and reversed around +75 mV. The I/V characteristics showed a single negative peak at +15 mV and maximal voltage dependence at -12 mV. Ca²⁺ currents were fully blocked by 100 mM Cd²⁺ and were not affected by replacement of Na⁺ with TRIS⁺ or by adding 50 mM Ni²⁺. This suggests a dominance of high-threshold Ca²⁺ currents and little or no contribution of both low-threshold (T-type) and Na⁺ TTX-insensitive currents in control conditions. Nifedipine (3 mM) blocked 62 ± 1.8 % (mean ± S.E.M., n = 33 cells) of the total current at 0 mV (V_h = -40 mV), suggesting a large contribution of L-channels to the high-threshold Ca²⁺ currents (Hern‡ndez-Guijo et al. 1999).

Addition of 8-CPT-cAMP (200 mM for 1Ð4 days to cells in culture) caused the appearance of a new inward current component, which started activating at ~-50 mV and was quickly inactivating. The time course of inactivation varied from cell to cell. It was either fast (t_f = 3 ± 0.6 ms at -20 mV, n = 5), slow (t_s = 21 ± 1.1 ms, n = 5) or a mixture of the two. The amplitude of this component increased with the day of culture and with the duration of 8-CPT-cAMP exposure. In some cells, after 4 days exposure, the contribution was comparable or even larger than the high-threshold Ca²⁺ currents available in normal conditions. Pharmacological dissection revealed the existence of two distinct components: (1) a fast inactivating TTX-insensitive Na⁺ current, which disappeared when external Na⁺ was replaced with TRIS⁺ and remained unaltered in the presence of 50 mM Ni²⁺ or when external Ca²⁺ was lowered to 0.5 mM and, (2) a T-type low-threshold Ca²⁺ current which persisted when TRIS⁺ replaced external Na⁺. This component was blocked by 50 mM Ni²⁺ and preserved with 30 mM Cd²⁺. A conditioning pulse of 80 ms to -30 mV largely inactivated both transient currents. 8-CPT-cAMP had no action on the amplitude and voltage dependence of high-threshold Ca²⁺ currents available in control conditions. L-type channels, which contributed to 62 % of the currents in control conditions, were found not to be significantly different after 2 days of 8-CPT-cAMP incubation (61 ± 2.3 %, P > 0.05, paired t test, n = 30). It is concluded that 8-CPT-cAMP selectively stimulates the functional expression of ion channels (Ca²⁺ and Na⁺), which lower the threshold of firing rate in rat chromaffin cells.

The work was supported by the Italian MIUR (2001.55324) and the CNR (01.00443.ST97).

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