Most stimuli that trigger formation of inositol 1, 4, 5-trisphosphate (IP₃) evoke both release of Ca²⁺ from intracellular stores (mediated by IP₃ receptors) and enhanced Ca²⁺ entry across the plasma membrane. Store-operated Ca²⁺ entry, which is stimulated by an unknown consequence of emptying the intracellular Ca²⁺ stores, provides a major route for Ca²⁺ entry (Putney, 1999). In some cells, including A7r5 vascular smooth muscle cells, an additional Ca²⁺ entry pathway is also activated by receptors, but not by empty stores (Broad et al. 1999). In A7r5 cells, we previously demonstrated that the store-operated Ca²⁺ entry pathway (SOC) was permeable to Mn²⁺ (but not Sr²⁺) and irreversibly inhibited by low concentrations of Gd³⁺; the non-store-operated Ca²⁺ entry pathway (NSOC) was permeable to Sr²⁺ (but not Mn²⁺), less sensitive to Gd³⁺ and activated by arachidonic acid released by the sequential activities of phospholipase C and diacylglycerol lipase (Broad et al. 1999). Here we investigate the effects of a physiological stimulus, Arg⁸-vasopressin (AVP), on the activity of the SOC pathway.

The intracellular Ca²⁺ stores of A7r5 cells loaded with fura-2 were completely depleted of Ca²⁺ by incubation with thapsigargin (1 µM) and ionomycin (1 µM) for 10 min. Rates of fluorescence quenching (recorded after excitation at 360 nm) during 90 s pulses of MnCl₂ (50 µM) were then used to determine the activity of the SOC pathway. Emptying the intracellular Ca²⁺ stores increased the rate of fluorescence quenching from 0.06 ± 0.006 to 0.26 ± 0.012 % min^-1 (mean ± S.E.M., n = 3). Subsequent addition of AVP (100 nM) completely inhibited the store-regulated Mn²⁺ entry. The half-maximal effect (IC₅₀) occurred with an AVP concentration of 19 ± 2 nM (n = 3), indicating that inhibition of SOC, mobilization of intracellular Ca²⁺ stores (EC₅₀ = 22 ± 3 nM, n = 3), and activation of NSOC (EC₅₀ = 18 ± 2 nM, n = 3) are similarly sensitive to AVP. Replacement of extracellular Na⁺ with the impermeant cation N-methyl-D-glucamine (NMDG) should prevent the depolarization that would normally accompany activation of the NSOC, which are probably non-selective cation channels (Van Renterghem et al. 1988). Under these conditions, AVP still completely inhibited Mn²⁺ entry. RHC-80267, an inhibitor of diacylglycerol lipase, caused a concentration-dependent (IC₅₀ = 24 ± 4 µM, n = 3) and complete reversal of the effects of AVP on Mn²⁺ entry via SOC. We previously established a variant A7r5 cell line in which AVP failed to stimulate the NSOC pathway and attributed the defect to a loss of diacylglycerol lipase (Broad et al. 1999). In the variant cell line, AVP had no effect on Mn²⁺ entry via SOC.

We conclude that AVP both activates store-operated Ca²⁺ entry (via its ability to stimulate release of IP₃-sensitive Ca²⁺ stores) and inhibits it (probably via arachidonic acid released by the sequential activities of phospholipase C and diacylglycerol lipase). The extent to which physiological stimuli evoke store-regulated Ca²⁺ entry will be determined by the fine balance between their opposing influences on this pathway.This work was supported by The Wellcome Trust.

Broad, L.M., Cannon, T.R. & Taylor, C.W. (1999). J. Physiol. 517, 121-134. abstract

Putney, J.W. (1999). Proc. Natl Acad. Sci. USA 96, 14669-14671.

Van Renterghem, C., Romey, G. & Lazdunski, M. (1988). Proc. Natl Acad. Sci. USA 85, 9365-9369.