Recent results challenge the long-standing idea that capacitative Ca²⁺ entry (CCE) is the major receptor-regulated pathway for Ca²⁺ entry into cells (Broad et al. 1999; Mignen et al. 2001). In A7r5 vascular smooth muscle cells, vasopressin, a potent vasoconstricting hormone, stimulates formation of IP₃ and diacylglycerol (DAG); both messengers ultimately regulate Ca²⁺ entry. IP₃, by emptying Ca²⁺ stores, generates the signal that activates CCE, while the arachidonic acid released from DAG both stimulates non-capacitative Ca²⁺ entry (NCCE) and simultaneously inhibits CCE (Moneer & Taylor, 2002). This reciprocal regulation of the two pathways ensures that all Ca²⁺ entry is via NCCE when vasopressin is present, while CCE is transiently activated only after removal of vasopressin (Moneer & Taylor, 2002). Here we demonstrate that endogenous production of nitric oxide (NO) downstream of arachidonic acid mediates this reciprocal regulation of the two Ca²⁺ entry pathways.

Western blot analysis showed that type III NO synthase (NOS III) was expressed in A7r5 cell membranes. In A7r5 cells loaded with fura-2 to allow measurement of cytoplasmic Ca²⁺, we used selective blockers of CCE or NCCE (Moneer & Taylor, 2002) to examine the effects of vasopressin on each pathway. L-NAME, a competitive inhibitor of NOS, reversed the inhibitory effect of vasopressin (100 nM) on CCE in a concentration-dependent manner (IC₅₀ = 39 ± 5 mM; mean ± S.E.M., n = 3). With the CCE pathway blocked, vasopressin activated NCCE (Δ[Ca²⁺]_i = 83 ± 9 nM, n = 4); that activation was also blocked (Δ[Ca²⁺]_i = 5 ± 3 nM, n = 4) by L-NAME (700 mM). In both cases the effects of L-NAME were reversed by addition of L-arginine (2.1 mM), the endogenous NOS substrate. The effects of vasopressin on the two Ca²⁺ entry pathways were mimicked by the slow-releasing NO donor, NOC-18 and by NO solution (1 mM). We conclude that reciprocal regulation of CCE and NCCE by vasopressin is mediated by NOS, activated directly or indirectly by arachidonic acid.

The membrane-permeant analogue of cyclic GMP, 8-bromo-cyclic GMP, completely inhibited CCE (the increase in cytosolic [Ca²⁺] fell from 567 ± 63 to 12 ± 5 nM, n = 4), but it did not activate NCCE ([Ca²⁺]_i rise = 7 ± 4 nM, n = 3). Furthermore, selective inhibitors of soluble guanylyl cyclase (ODQ, IC₅₀ = 44 ± 8 nM, n = 3) and cyclic GMP-dependent protein kinase (PKG) (KT5823, IC₅₀ = 86 ± 10 nM, n = 3) potently inhibited the ability of vasopressin to inhibit CCE, without affecting activation of NCCE. Thus the activation of soluble guanylyl cyclase by NO and subsequent activation of PKG are essential only for inhibition of CCE.

In addition to its role as endothelium-derived relaxing factor, we conclude that NO also fulfils an essential role in co-ordinating two receptor-regulated Ca²⁺ entry pathways.

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