Biol Res 37: 641-645, 2004

ARTICLE

Regulation of capacitative and non-capacitative Ca²⁺ entry in A7r5 vascular smooth muscle cells

COLIN W. TAYLOR and ZAHID MONEER

Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, U.K.

Dirección para Correspondencia

ABSTRACT

A capacitative Ca²⁺ entry (CCE) pathway, activated by depletion of intracellular Ca²⁺ stores, is thought to mediate much of the Ca²⁺ entry evoked by receptors that stimulate phospholipase C (PLC). However, in A7r5 vascular smooth muscle cells, vasopressin, which stimulates PLC, empties intracellular Ca²⁺ stores but simultaneously inhibits their ability to activate CCE. The diacylglycerol produced with the IP₃ that empties the stores is metabolized to arachidonic and this leads to activation of nitric oxide (NO) synthase, production of NO and cyclic GMP, and consequent activation of protein kinase G. The latter inhibits CCE. In parallel, NO directly activates a non-capacitative Ca²⁺ entry (NCCE) pathway, which is entirely responsible for the Ca²⁺ entry that occurs in the presence of vasopressin. This reciprocal regulation of two Ca²⁺ entry pathways ensures that there is sequential activation of first NCCE in the presence of vasopressin, and then a transient activation of CCE when vasopressin is removed. We suggest that the two routes for Ca²⁺ entry may selectively direct Ca²⁺ to processes that mediate activation and then recovery of the cell.

Key words: Ca²⁺ entry, smooth muscle, archidonic acid, nitric oxide, cyclic GMP

INTRODUCTION

Stimulation of receptors that promote hydrolysis of phosphatidylinositol 4,5-bisphosphate often leads to complex increases in intracellular [Ca²⁺] and these Ca²⁺ signals then control a variety of cellular functions (Berridge et al., 2003). Typically, both release of Ca²⁺ from intracellular Ca²⁺ stores and an increased rate of Ca²⁺ entry across the plasma membrane contribute to these Ca²⁺ signals. The release of Ca²⁺ from intracellular stores by inositol 1,4,5-trisphosphate (IP₃) is reasonably well understood, with IP₃ and Ca²⁺ together controlling opening of the pore formed by the four subunits of the IP₃ receptor (Taylor et al., 2004). The mechanisms responsible for Ca²⁺ entry are less clear, although the idea, first proposed by Putney in 1986 (Putney, 1986), that the stores emptied by IP₃ somehow activate a Ca²⁺ entry pathway, the "capacitative Ca²⁺

CALCIUM ENTRY PATHWAYS IN A7r5 CELLS

A7r5 cells, like most other cells, express a CCE pathway that is activated by depletion of intracellular Ca²⁺ stores. In A7r5 cells, this pathway is permeable to Mn²⁺, Ca²⁺ and Ba²⁺, but not Sr²⁺; and it is selectively blocked by low concentrations of Gd³⁺ (1mM) and by 2-APB (Broad et al., 1999; Moneer & Taylor, 2002). The existence of a non-capacitative Ca²⁺ entry (NCCE) pathway is evident from experiments in which addition of AVP to A7r5 cells in which the stores have been completely emptied by treatment with thapsigargin to inhibit the Ca²⁺ pump of the ER, stimulates Sr²⁺ entry (Broad et al., 1999). Further characterization of this NCCE pathway has shown it to be permeable to Ca²⁺, Ba²⁺ and Sr²⁺, but not Mn²⁺; to be selectively blocked by SKF-96365 or LOE-908; and to be less sensitive than CCE to blockade by Gd³⁺ (Broad et al., 1999; Moneer & Taylor, 2002). The different permeation properties and susceptibilities to blockade of CCE and NCCE (summarized in Fig. 1) have proven enormously useful in dissecting the relative roles of these two Ca²⁺ entry pathways in mediating the Ca²⁺ entry evoked by AVP.

RECIPROCAL REGULATION OF CCE AND NCCE

A simple experiment highlights the problem. Earlier work with fura 2-loaded A7r5 cells had suggested that NCCE might be more important than CCE in mediating the Ca²⁺ entry evoked by AVP (Broad et al., 1999). Yet the Ca²⁺ entry signal (CCE) evoked by depletion of intracellular stores using thapsigargin was 5 times greater than that evoked by maximal activation of NCCE alone using AVP in the presence of 1 mM Gd³⁺ to inhibit CCE. How can the small Ca²⁺ signals generated by NCCE be more important than the large Ca²⁺ signals evoked by CCE? The answer is that under physiological conditions, receptors stimulate both formation of IP₃ (which empties stores and so activates CCE) and diacyglycerol (DAG), and the latter provides signals that both inhibit CCE and activate NCCE. In effect, physiological stimuli apply both the accelerator (IP₃) and the brakes (signals from DAG) to the CCE pathway, whereas thapsigargin (universally used to activate CCE) provides only a heavy foot on the accelerator. The experiment that most clearly demonstrates this inhibition of CCE by AVP exploits our observation that Mn²⁺ (which quenches fura 2 fluorescence) permeates the CCE, but not the NCCE, pathway. In cells with stores emptied by thapsigargin, there is an increased rate of Mn²⁺ entry (detected as a quench of fura 2 fluorescence at its Ca²⁺-insensitive wavelength) and that is completely inhibited in a concentration-dependent fashion by AVP (half-maximal inhibitory concentration, IC₅₀ = 19 ± 2 nM). Analogous experiments measuring Ba²⁺ or Ca²⁺ entry have confirmed this inhibition of CCE by AVP (Moneer & Taylor, 2002).

The most important question, of course, is to ask which of the two Ca²⁺ entry pathways mediates the Ca²⁺ entry evoked by physiological stimuli. To address this we recorded Ca²⁺ signals from cells as we stimulated them with maximal or sub-maximal concentrations of AVP before rapidly removing the AVP (and simultaneously adding an antagonist of the AVP receptor). In parallel, the experiments were performed under control conditions, with only CCE blocked (1 mM Gd³⁺), with only NCCE blocked (30 mM LOE-908), or with all Ca²⁺ entry blocked (100 mM Gd³⁺). The results with a sub-maximal (i.e. physiological) concentration of AVP are most informative. Under these conditions, all Ca²⁺ entry occurred via NCCE when AVP was present; when AVP was removed, NCCE shut down and there was a transient phase of Ca²⁺ entry via CCE (Moneer & Taylor, 2002). There is persuasive evidence that different physiological responses respond differently to Ca²⁺ signals generated by different Ca²⁺ entry pathways: adenylyl cyclase, for example, is often found to be selectively regulated by Ca²⁺ entering cells via CCE (Cooper, 2003). We suggest therefore that the sequential activation of first NCCE and then CCE by AVP may have important physiological consequences by first directing Ca²⁺ (from NCCE) to processes that initiate the cellular response (contraction for vascular smooth muscle), and then (from CCE) to processes that actively promote cellular recovery (e.g. relaxation). In this way, both addition of AVP and its removal may actively contribute to controlling cellular activity by regulating whether Ca²⁺ enters cells via NCCE or CCE.

Figure 1. Properties of CCE and NCCE in A7r5 cells. a. The signaling sequence linking AVP to formation of IP3 and DAG and then to arachidonic acid is shown; hammerheads indicate inhibitors. Arachidonic acid (via NOS, see text) activates NCCE and inhibits CCE. b,c. Gd3+ (1 mM) selectively inhibits CCE without blocking NCCE (b), and LOE-908 (30 mM) selectively inhibits NCCE without affecting CCE (c). Solid bars denote the presence of Gd3+ (b) or LOE-908 (c)

What are the signals that emanate from the V_1A-vasopressin receptor that allow it to reciprocally regulate NCCE and CCE? U73122 (an inhibitor of phospholipase C, IC₅₀ = 1 mM) and RHC 80267 (an inhibitor of DAG lipase, IC₅₀ = 24 mM) blocked the ability of AVP to both inhibit CCE and activate NCCE, and arachidonic acid (or its stable analogue, ETYA) mimicked the effects of AVP on the two pathways. We therefore concluded that arachidonic acid, released by DAG lipase from the DAG produced with IP₃, provides the signal that leads to reciprocal regulation of the two Ca²⁺ entry pathways. We had assumed, in keeping with results from others showing direct regulation of NCCE by arachidonic acid (Mignen & Shuttleworth, 2000), that both pathways would be directly regulated by arachidonic acid, but the regulation proved to be more complex. Selective inhibitors of nitric oxide synthase (NOS, L-NAME), soluble guanylyl cyclase (ODQ) and cyclic GMP-dependent protein kinase (PKG, KT5823) as well as mimics of the endogenous signals (NO, NO donors, 8-bromo-cyclic GMP) provide persuasive evidence that the effects of arachidonic acid are mediated by activation of NOS (Moneer et al., 2003). This conclusion is further supported by our observation that NOS III (also known as endothelial- or e-NOS) is expressed in A7r5 cells and that biochemical assays show activation of NOS by AVP. We have not yet established how arachidonic acid promotes activation of NOS-III. Our current scheme (Fig. 2) suggests that arachidonic acid activates NOS-III and that the NO produced then directly stimulates NCCE, possibly via S-nitrosylation of a critical thiol group on the channel or associated protein. NO also stimulates soluble guanylyl cyclase, producing cGMP which then activates PKG; each of these steps is required for inhibition of CCE (though not for activation of CCE). It is likely therefore that phosphorylation of either the CCE channel or an associated protein mediates the inhibition by AVP. This pattern of regulation has important implications. Because inhibition of CCE is mediated by signals generated further down the signaling cascade than activation of NCCE, the former benefits from greater amplification than the latter. This is important in ensuring that CCE will always be completely inhibited before NCCE can be activated. Our analysis of the signaling sequence thus provides a rational explanation for our observation that in the presence of AVP all Ca²⁺ entry occurs via the NCCE pathway, with CCE coming transiently into play only after removal of AVP.

Figure 2. The role of NOS in mediating reciprocal regulation of CCE and NCCE by AVP.

We conclude that in A7r5 cells, AVP reciprocally regulates two Ca²⁺ entry pathways. By stimulating phospholipase C, AVP produces two intracellular messengers, IP₃ and DAG, both of which are important in regulating Ca²⁺ signals. IP₃ binds to its intracellular receptor and stimulates release of Ca²⁺ from intracellular stores, thereby generating both the initial Ca²⁺ signal and the signal (empty stores) that activates CCE. Arachidonic acid, released from the DAG by DAG lipase, activates NOS-III and that leads to activation of NCCE (by NO itself) and to inhibition of CCE (via PKG), the latter benefiting from a more amplifying signal cascade, which ensures complete inhibition of CCE whenever NCCE is activated. The result of this reciprocal regulation is that when AVP is present all Ca²⁺ entry occurs via NCCE and this is followed by a transient activation of CCE only after removal of AVP. This strictly sequential activation of the two Ca²⁺ entry pathways may serve selectively to direct Ca²⁺ (from NCCE) first to the proteins that mediate activation and then (from CCE) to proteins that mediate recovery. Clearly it would be inappropriate to ask whether NCCE or CCE is the more important route for Ca²⁺ entry: both are likely to be important in mediating different phases of the response to AVP.

ACKNOWLEDGEMENTS

Supported by grants from the Wellcome Trust, British Heart Foundation and BBSRC.

REFERENCES

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Corresponding author: Colin W Taylor, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, U.K.. Phone/Fax: 01223 334058, E-mail: cwt1000@cam.ac.uk

Received: June 29, 2004. Accepted: March 12, 2004.