Regulation of Ca²⁺-sensitive adenylyl cyclases (AC) by Ca²⁺ requires capacitative Ca²⁺ entry (CCE) (Mons et al. 1998), but whether Ca²⁺-sensitive phosphodiesterases (PDEs) are similarly discriminating has not been addressed. Activation of muscarinic receptors inhibits isoprenaline (ISO)-evoked cAMP accumulation in 1321N1 human astrocytoma cells (Gross and Clark 1977; Meeker and Harden 1982). The effect is mediated by Ca²⁺-CaM-dependent phosphodiesterase (PDE1), which is activated by an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) (Tanner et al. ,. 1986). Here, we attempt to determine whether PDE1 exhibits selectivity for either Ca²⁺ released from intracellular stores or Ca²⁺ entering the cell.

In fura-2-loaded 1321N1 cells, carbachol (CCh, 1 mM) evoked a rapid and transient increase in [Ca²⁺]_i (t = 24 ± 2 s, n = 5) followed by a slower, sustained phase of Ca²⁺ entry (t_1/2 = 93 ± 29, n = 5). The peak increase in [Ca²⁺]_i by the two phases were 1143 ± 152 nM (n = 5) and 712 ± 109 nM (n = 5), respectively. In analogous experiments, cells were stimulated with ISO (10 µM, 5 min) in the presence and absence of CCh. The inhibition of cAMP response by CCh was largely dependent on extracellular Ca²⁺ (15 ± 8 % (n = 17) of the total inhibition was dependent on Ca²⁺ from intracellular stores and 86 ± 8 % (n = 17) on Ca²⁺ entry). Both effects were reversed by the PDE inhibitors IBMX and 8-methoxymethyl-3-isobutyl-1-methylxanthine (PDE1) but not rolipram (PDE4). We conclude that PDE1 activation requires a sustained elevation in intracellular free Ca²⁺ concentration which is only achieved though by an influx of Ca²⁺ into the cell. Gd³⁺ (10 µM, 5 min) and 2-APB (100 µM, 5 min) inhibited CCE in thapsigargin (1 µM, 15 min) pre-treated cells by 89 ± 2 % (n = 16) and 86 ± 5 % (n = 10), respectively. In parallel experiments, CCE inhibited ISO-evoked cAMP accumulation; the effect was reversed by IBMX and CCE blockers. In ionomycin pre-treated cells (10 µM, 15 min), the presence of 30 µM Ca²⁺ in the extracellular medium produced a similar rise in [Ca²⁺]_i as 3 mM Ca²⁺ in thapsigargin pre-treated cells (576 ± 89 nM, n = 9 and 527 ± 86 nM, n = 10, respectively). However, only the latter effect was blocked by CCE inhibitors. Ionomycin-mediated Ca²⁺ entry also inhibited ISO-evoked cAMP accumulation and the effect also was reversed by IBMX. Thus PDE1 requires sustained Ca²⁺ entry for activation, but unlike ACs, it does not discriminate between the different routes of Ca²⁺ entry.

This work was supported by The Wellcome Trust.