A non-capacitative pathway activated by arachidonic acid is the major Ca²⁺ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin

Abstract

1. Depletion of the Ca²⁺ stores of A7r5 cells stimulated Ca²⁺, though not Sr²⁺, entry. Vasopressin (AVP) or platelet-derived growth factor (PDGF) stimulated Sr²⁺ entry. The cells therefore express a capacitative pathway activated by empty stores and a non-capacitative pathway stimulated by receptors; only the former is permeable to Mn²⁺ and only the latter to Sr²⁺.

2. Neither empty stores nor inositol 1,4,5-trisphosphate (InsP₃) binding to its receptors are required for activation of the non-capacitative pathway, because microinjection of cells with heparin prevented PDGF-evoked Ca²⁺ mobilization but not Sr²⁺ entry.

3. Low concentrations of Gd³⁺ irreversibly blocked capacitative Ca²⁺ entry without affecting AVP-evoked Sr²⁺ entry. After inhibition of the capacitative pathway with Gd³⁺, AVP evoked a substantial increase in cytosolic [Ca²⁺], confirming that the non-capacitative pathway can evoke a significant increase in cytosolic [Ca²⁺].

4. Arachidonic acid mimicked the effect of AVP on Sr²⁺ entry without stimulating Mn²⁺ entry; the Sr²⁺ entry was inhibited by 100 μM Gd³⁺, but not by 1 μM Gd³⁺ which completely inhibited capacitative Ca²⁺ entry. The effects of arachidonic acid did not require its metabolism.

5. AVP-evoked Sr²⁺ entry was unaffected by isotetrandrine, an inhibitor of G protein-coupled phospholipase A₂. U73122, an inhibitor of phosphoinositidase C, inhibited AVP-evoked formation of inositol phosphates and Sr²⁺ entry. The effects of phorbol esters and Ro31-8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non-capacitative pathway. An inhibitor of diacylglycerol lipase, RHC-80267, inhibited AVP-evoked Sr²⁺ entry without affecting capacitative Ca²⁺ entry or release of Ca²⁺ stores.

6. Selective inhibition of capacitative Ca²⁺ entry with Gd³⁺ revealed that the non-capacitative pathway is the major route for the Ca²⁺ entry evoked by low AVP concentrations.

7. We conclude that in A7r5 cells, the Ca²⁺ entry evoked by low concentrations of AVP is mediated largely by a non-capacitative pathway directly regulated by arachidonic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase.

Stimulation of receptors linked to activation of phosphoinositidase C (PIC) causes an increase in free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) due to release of Ca²⁺ from intracellular stores (Berridge, 1993) and an influx of Ca²⁺ across the plasma membrane (Putney, 1997). The role of inositol 1,4,5-trisphosphate (InsP₃) in mediating Ca²⁺ release from intracellular stores is firmly established, but the mechanisms responsible for Ca²⁺ entry are less clear. In most cells, depletion of intracellular Ca²⁺ stores stimulates Ca²⁺ entry via what is commonly referred to as the ‘capacitative’ or ‘store-regulated’ Ca²⁺ entry pathway (Putney, 1997). Thapsigargin is often used to study this pathway because it selectively inhibits the Ca²⁺ pumps of intracellular stores, allowing the stores to be emptied independently of receptor activation. The capacitative pathway is thought to reflect the activity of a collection of plasma membrane Ca²⁺ channels with different ion selectivities and conductances. At least some of these channels are likely to be hetero- or homo-oligomeric assemblies of proteins encoded by mammalian homologues of the Drosophila trp and trpl genes (Montell, 1997). While depletion of the intracellular stores is sufficient to activate the capacitative pathway, the signal that passes between the stores and the plasma membrane Ca²⁺ channels remains to be unequivocally identified (Putney, 1997); nor is it clear whether the same signal regulates all capacitative pathways.

The capacitative pathway is not, however, the only pathway through which Ca²⁺ can enter cells in response to receptor activation (Clementi & Meldolesi, 1996). Additional pathways include a diverse collection of non-selective cation channels regulated by a range of intracellular messengers including protein kinase C (Oike et al. 1993), diacylglycerol (Helliwell & Large, 1997), Ca²⁺ and inositol 1,3,4,5-tetrakisphosphate (Lückhoff & Clapham, 1992), InsP₃ (Dong et al. 1995) and Ca²⁺ itself (Loirand et al. 1991). There are also receptor-regulated Ca²⁺ entry pathways, including some that are regulated by arachidonic acid (Van Delden et al. 1993; Wang et al. 1993; Shuttleworth, 1996; Munaron et al. 1997), for which the nature of the Ca²⁺ channel is unknown. In addition, numerous stimuli activate non-capacitative Ca²⁺ entry via signalling pathways that remain to be defined (Clementi & Meldolesi, 1996); these include vasopressin (AVP) in hepatocytes (Kass et al. 1994), compound 48/80 in mast cells (Fasolato et al. 1993), carbachol in PC12 cells (Clementi et al. 1992), and platelet-derived growth factor (Huang et al. 1991) or AVP (Van Renterghem et al. 1988; Byron & Taylor, 1995) in vascular smooth muscle cells.

In some cells, the Ca²⁺ entry evoked by maximal stimulation of receptors linked to InsP₃ formation is indistinguishable from that evoked by complete depletion of the intracellular Ca²⁺ stores by thapisgargin (Takemura et al. 1989; Demaurex et al. 1994; Madge et al. 1997). Patch-clamp recordings of mast cells also suggest that after maximal stimulation, the capacitative pathway accounts for most of the Ca²⁺ entry signal (Fasolato et al. 1993). These results imply that during maximal stimulation, the effect of hormones on Ca²⁺ entry may be mediated entirely by their ability to empty Ca²⁺ stores and thereby activate the capacitative pathway. However, both the existence of additional Ca²⁺ entry pathways and a suggestion that the capacitative pathway may be activated only after substantial depletion of the Ca²⁺ stores (Parekh et al. 1997; but see Hofer et al. 1998) highlight the importance of establishing which Ca²⁺ entry pathways mediate the effects of submaximal concentrations of hormones. The issue is important because under physiological conditions cells are unlikely to be maximally stimulated, and the Ca²⁺ spiking behaviour typically observed during stimulation of cells is evoked by low concentrations of hormone and sustained only while Ca²⁺ entry persists (Berridge, 1993). The lack of tools with which to clearly distinguish the contributions of different Ca²⁺ entry pathways has hitherto limited progress in defining their relative roles in mediating receptor-regulated increases in [Ca²⁺]_i during stimulation with physiologically appropriate concentrations of hormones (Clementi & Meldolesi, 1996).

The A7r5 clonal cell line was orginally derived from rat thoracic aorta and the cells retain many characteristics of vascular smooth muscle. AVP, acting via V_1a receptors, stimulates several signalling cascades including PIC, phospholipases A₂ and D (Thibonnier et al. 1991), and various Ca²⁺ transport processes, including InsP₃-mediated release of intracellular Ca²⁺ stores and consequent activation of capacitative Ca²⁺ entry, stimulation of Ca²⁺ extrusion, and activation of a non-capacitative divalent cation entry pathway (Byron & Taylor, 1995). The non-capacitative pathway almost certainly reflects the activity of the Ca²⁺-permeable non-selective cation channels identified by patch-clamp analyses (Van Renterghem et al. 1988; Iwasawa et al. 1997). These channels are stimulated by AVP through a pathway that is insensitive to both pertussis toxin and L-type Ca²⁺ channel antagonists, but they are not activated by thapsigargin or intracellular application of InsP₃ or Ca²⁺ (Van Renterghem et al. 1988; Krautwurst et al. 1994; Nakajima et al. 1996). The inability to selectively inhibit the capacitative and non-capacitative pathways, and the stimulation of Ca²⁺ extrusion by AVP had prevented a direct assessment of either the Ca²⁺ permeability of the non-capacitative pathway or its contribution to the increase in [Ca²⁺]_i evoked by hormones. We now demonstrate that the non-capacitative pathway is permeable to Ca²⁺, that it is activated by arachidonic acid released from the diacylglycerol formed by PIC, that its stimulation does not require depletion of intracellular Ca²⁺ stores, and that it is at least as important as capacitative Ca²⁺ entry in mediating the increases in [Ca²⁺]_i evoked by physiological concentrations of AVP.

METHODS

Materials

U73122 (1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)-hexyl]-1H-pyrrole-2,5-dione), U73343 (1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione), isotet-randrine and arachidonic acid were from Calbiochem. RHC-80267 and Ro31-8220 were from the Alexis Corporation Ltd (Nottingham, UK). Arg⁸-vasopressin (AVP), aspirin, phorbol 12,13-dibutyrate, indomethacin, heparin, stearic acid and nordihydroguaiaretic acid (NDGA) were from Sigma. Fura-2 AM and Cascade-Blue-labelled dextran were from Molecular Probes (Leiden, Netherlands). Other materials were from the suppliers reported previously (Byron & Taylor, 1993, 1995). The intracellular targets of the inhibitors are shown in Figs 6A and 10.

Figure 6. Arachidonic acid mediates the effects of AVP on the non-capacitative pathway.

A, targets of the inhibitors used are shown by hammerheads (⊊); pm, plasma membrane. B, inhibition of DAG lipase by RHC-80267 (50 μM, 15 min) selectively inhibited AVP (100 nM)-evoked Sr²⁺ entry without significantly affecting either AVP-evoked release of Ca²⁺ from intracellular stores or the capacitative Ca²⁺ entry evoked by stores that had been emptied by thapsigargin (Tg) (means ±s.e.m., n = 3). C and D, effects of AVP (100 nM) and arachidonic acid (AA, 50 μM) on Sr²⁺ entry to single A7r5 cells that had been pretreated with ionomycin and thapsigargin are shown for normal cells (C) and for the cell line in which AVP fails to stimulate Sr²⁺ entry (D). E and F, the results show that both AVP and arachidonic acid stimulate Sr²⁺ entry which is unaffected by 1 μM Gd³⁺, but fully inhibited by 100 μM Gd³⁺. G, in sub-confluent cells, continuous perfusion with arachidonic acid (50 μM) stimulated a sustained Ca²⁺ entry in almost all cells (thick line; average response from 100 cells), but even 5 μM arachidonic acid (thin line, average response from 5 cells) stimulated a more modest Ca²⁺ entry in 5 ± 1 % (n = 3 coverslips). The traces are typical of those from 3 independent experiments. H, arachidonic acid (50 μM) did not stimulate Mn²⁺ influx, indicating that it affected neither membrane integrity nor the capacitative pathway. Traces C-F and H are the average responses of at least 20 individual cells from a single experiment and are typical of 3 independent experiments.

Figure 10. Capacitative and non-capacitative Ca²⁺ entry pathways.

The targets of the inhibitors (hammerheads, ⊊) and the proposed means (arrows) whereby receptors stimulate the two Ca²⁺ entry pathways are shown.

Cell culture and spectrofluorimetry

A7r5 cells were cultured, plated onto glass coverslips, and loaded with fura-2 as previously described (Byron & Taylor, 1995). Briefly, A7r5 cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum, 3.8 mM L-glutamine and 0.9 % non-essential amino acids; they were passaged every 7 days when they were confluent. Cells were subcultured in the same medium onto round (22 mm diameter, for single cell imaging) or rectangular (9 mm × 22 mm, for cell population measurements) glass coverslips and used for experiments after 1-5 days. Confluent cultures of cells were loaded with fura-2 in Hepes-buffered saline (HBS (mM): 135 NaCl, 5.9 KCl, 1.2 MgCl₂, 1.5 CaCl₂, 11.6 Hepes, 11.5 glucose; pH 7.3; 22°C) supplemented with fura-2 AM (2 μM), Pluronic F-127 (0.02 %), and bovine serum albumin (1 mg ml⁻¹) for 2 h at 22°C, and then incubated for a further hour in the absence of fura-2 before being used for experiments. Previous experiments (Byron & Taylor, 1995) had established that with this loading protocol > 95 % of the fura-2 was cytosolic.

For measurements of [Ca²⁺]_i in cell populations, coverslips were mounted vertically in an optical cuvette in a Hitachi F4500 spectrofluorimeter. Light of appropriate excitation wavelength (340-380 nm), was provided by a monochromator and emitted fluorescence (510 nm) was collected at 0.5 s intervals. The cuvette was continuously perfused with HBS at 22°C. At the perfusion rates used (10-15 ml min⁻¹), the medium exchanged with a half-time of ∼11 s. Autofluorescence was determined at the end of each experiment by addition of ionomycin (1 μM) and MnCl₂(1 mM) in Ca²⁺-free HBS. Fluorescence ratios (340 nm/380 nm; shown as F₃₄₀/F₃₈₀ in figures) were calculated after subtraction of autofluorescence and calibrated to free [Ca²⁺] using a look-up table created from Ca²⁺ standards (Molecular Probes).

Measurements of [Ca²⁺]_i in single A7r5 cells were performed in a small perfusion chamber on the stage of a Nikon Diaphot inverted epifluorescence microscope. Light of appropriate excitation wavelength (340, 358 or 380 nm) was provided by a high pressure xenon arc lamp mounted behind a rapidly rotating filter wheel fitted with narrow band-pass filters. Emitted fluorescence was collected by an intensified CCD camera (Photonic Science, Milham, UK) after passage through a 480 nm long-pass barrier filter (Byron & Taylor, 1995). Images were collected at 1 s intervals and analysed using IonVision III software (Improvision, Coventry, UK). Most figures show results from cell populations because single cell analyses confirmed that ∼95 % of the cells gave similar responses to hormonal stimuli.

Rates of Mn²⁺ entry were measured by recording the quench of fura-2 fluorescence with excitation at 358 nm. Sr²⁺ entry was usually measured in Ca²⁺-free HBS containing EGTA (1 mM) to minimize the effects of contaminating Ca²⁺, and supplemented with SrCl₂ (2.5 mM, free [Sr²⁺]∼1.5 mM). In order to avoid chelation of Mn²⁺ or Gd³⁺, which bind EGTA with high affinity, EGTA was omitted from all media containing them. In parallel experiments, there was no detectable Ca²⁺ entry in nominally Ca²⁺-free HBS, irrespective of the presence of EGTA. Nimodipine (100 nM) or verapamil (10 μM) were included in all media to inhibit the L-type Ca²⁺ channels that generate spontaneous Ca²⁺ spikes in A7r5 cells (Byron & Taylor, 1993).

Cells were loaded with heparin by microinjection (Femtotip, Eppendorf, Hamburg, Germany) of medium containing (mM): 27 K₂HPO₄, 8 NaHPO₄, 26 KH₂PO₄; pH 7.3, and 2.5 mM Cascade-Blue-labelled dextran (to identify microinjected cells) and 1 or 10 mg ml⁻¹ heparin. The cells were then loaded with fura-2 AM and the [Ca²⁺]_i of single cells recorded as described above.

Formation of [³H]inositol phosphates

A7r5 cells were grown to 50-80 % confluence in 24-well multiplates and then cultured for 72 h in inositol-free Dulbecco's modified Eagle's medium supplemented with 1 % fetal calf serum and 2 μCi ml⁻¹ of [2-³H]inositol. The cells were washed twice with HBS containing 50 mM LiCl and incubated in the same medium for a further 10 min (22°C) before replacement of the medium with Ca²⁺-free, Li⁺-containing HBS supplemented with appropriate additions. Incubations were terminated by addition of 0.5 ml of ice-cold trichloroacetic acid (1 M), and left on ice for 30 min. The samples were neutralized using a mixture of 1,1,2-trichlorotrifluoroethane and tri-n-octylamine (1:1), loaded onto anion exchange columns and the [³H]inositol phosphates eluted.

RESULTS

Capacitative and non-capacitative Ca²⁺ entry pathways

Addition of thapsigargin (1 μM) to A7r5 cells in Ca²⁺-free medium caused a transient increase in [Ca²⁺]_i as their intracellular Ca²⁺ stores completely emptied. Restoration of extracellular Ca²⁺ then caused a rapid and substantial increase in [Ca²⁺]_i reflecting entry of Ca²⁺ through the capacitative pathway (Fig. 1A). Addition of AVP (100 nM) to cells in which [Ca²⁺]_i had been increased by capacitative Ca²⁺ entry caused [Ca²⁺]_i to rapidly decrease (Fig. 1A). The decrease in [Ca²⁺]_i did not result from opening of non-selective cation channels causing depolarization and a decrease in the electrochemical gradient for Ca²⁺ entry because while AVP stimulated opening of these cation channels in the parental cell line, it did not stimulate them in a subclone of the cells (Fig. 1D and see below) which nevertheless responded to AVP with a decrease in [Ca²⁺]_i (Fig. 1C). The decrease in [Ca²⁺]_i resulted from stimulation of Ca²⁺ extrusion because the decline of [Ca²⁺]_i after rapid removal of extracellular Ca²⁺ was faster after addition of AVP (half-times = 22 and 17 s before and after AVP addition, respectively; L. M. Broad, T. R. Cannon & C. W. Taylor, in preparation). The effect of AVP on [Ca²⁺]_i therefore reflects the balance between its ability to regulate Ca²⁺ fluxes into the cytosol and Ca²⁺ removal from it. In the present study, we focus solely on the regulation of Ca²⁺ entry across the plasma membrane by AVP, and by performing all experiments in the presence of nimodipine or verapamil, we exclude the contributions of L-type Ca²⁺ channels.

Figure 1. Capacitative and non-capacitative pathways in A7r5 cells.

A and B, cells were treated with thapsigargin (1 μM, Tg) or thapsigargin and ionomycin (1 μM, I/Tg) in Ca²⁺-free HBS to empty their intracellular stores before addition of either Sr²⁺ or Ca²⁺ (1.5 mM) and then AVP (100 nM). A, thapsigargin evoked a transient increase in [Ca²⁺]_i as the intracellular Ca²⁺ stores released their entire Ca²⁺ content. The empty stores failed to stimulate Sr²⁺ entry, but stimulated a substantial entry of Ca²⁺ through the capacitative pathway. Addition of AVP then caused a decrease in [Ca²⁺]_i due to stimulation of Ca²⁺ extrusion. B, while pretreatment with ionomycin and thapsigargin to empty the Ca²⁺ stores failed to stimulate Sr²⁺ entry, addition of AVP (100 nM) stimulated Sr²⁺ entry. The same pretreatment was used in all subsequent experiments to empty intracellular Ca²⁺ stores. C and D, experiments identical to those describd for A and B were performed on the subclone of cells in which the effects of AVP on Sr²⁺ entry were selectively lost. During many subsequent passages, the cells retained this phenotype and never recovered an ability to respond to AVP with Sr²⁺ entry. Traces are each representative of at least 10 recordings. In each of the traces recording Sr²⁺ entry (A-D and Fig. 3), the fluorescence ratios have not been calibrated to [Sr²⁺]_i because although the increase in fluorescence ratio is entirely due to Sr²⁺ entry (see Fig. 2), the initial fluorescence results from Ca²⁺ bound to fura-2. It should, however, be noted that fura-2 has a much lower affinity for Sr²⁺ (K_d= 7.6 μM) than for Ca²⁺ (K_d= 227 nM) (Byron & Taylor, 1995).

In agreement with previous observations (Byron & Taylor, 1995), addition of Sr²⁺ to the extracellular medium of cells with empty intracellular Ca²⁺ stores had no effect on fura-2 fluorescence, but subsequent addition of AVP (100 nM) caused a substantial increase in the fura-2 fluorescence ratio (Fig. 1B). Because the excitation wavelength at which fura-2 fluorescence is unaffected by binding of divalent cations is different for Sr²⁺ (363 nm) and Ca²⁺ (358 nm) (Byron & Taylor, 1993), these isoemissive wavelengths can be used to resolve whether the changes in fura-2 fluorescence are wholly attributable to an increase in [Sr²⁺]_i or to changes in [Ca²⁺]_i triggered by Sr²⁺ entry. The results show the increase in [Ca²⁺]_i caused by addition of thapsigargin and ionomycin had no effect on the fura-2 fluorescence recorded at the isoemissive wavelength for Ca²⁺ (358 nm), but caused the expected large decrease in the fluorescence recorded at the longer isoemissive wavelength for Sr²⁺ (363 nm) (Fig. 2). During AVP-evoked Sr²⁺ entry, the fluorescence recorded at the isoemissive wavelength for Sr²⁺ was unchanged while that recorded at the isoemissive wavelength for Ca²⁺ increased. The results thereby establish that the increase in fura-2 fluorescence evoked by AVP in the presence of extracellular Sr²⁺ is entirely due to an increase in [Sr²⁺]_i (Fig. 2). These results confirm the existence of two distinct divalent cation entry pathways in A7r5 cells: a capacitative pathway activated by empty Ca²⁺ stores, and a second pathway stimulated by receptor activation. We refer to the latter as a non-capacitative pathway because, in contrast to the capacitative pathway (Putney, 1997) (Fig. 1A), empty Ca²⁺ stores do not provide a sufficient stimulus for its activation (Fig. 1B). Only the capacitative pathway is permeable to Mn²⁺ (Byron & Taylor, 1995) and only the non-capacitative pathway is detectably permeable to Sr²⁺. In subsequent experiments, the intracellular Ca²⁺ stores were emptied using the protocol shown in Fig. 1B.

Figure 2. The fura-2 fluorescence changes evoked by AVP reflect an increase in [Sr²⁺]_i.

In an experiment similar to that shown in Fig. 1B, fura-2 fluorescence was simultaneously recorded at four excitation wavelengths: 340 and 380 nm to provide the fluorescence ratio (panel i; F_340/380) used in all experiments, and at the isoemissive wavelengths for Ca²⁺ (358 nm) and Sr²⁺ (363 nm) (panel ii). The results show that the fluorescence changes that follow stimulation of cells with AVP in the presence of extracellular Sr²⁺ result wholly from an increase in [Sr²⁺]_i.

In the continued presence of a maximal concentration of AVP, both the enhancement of Ca²⁺ extrusion (Fig. 1A) and Sr²⁺ entry (Fig. 1B) were relatively transient, although the non-capacitative pathway remained active to a lesser degree for as long as AVP was present (Fig. 9A). We have not extensively investigated the mechanisms underlying the relatively transient nature of the increase in [Sr²⁺]_i evoked by AVP: because Sr²⁺ is effectively handled by Ca²⁺ pumps, it may result from a combination of partial desensitization of the AVP-regulated Sr²⁺ entry pathway and activation of the plasma membrane Ca²⁺ pump by AVP (Byron & Taylor, 1995; L. M. Broad, T. R. Cannon & C. W. Taylor, in preparation). Addition of platelet-derived growth factor (PDGF, 10 nM), another agonist that stimulates InsP₃ formation, after the response to a maximal concentration of AVP had waned evoked further Sr²⁺ entry (Fig. 3A), suggesting that the declining response to AVP is partially attributable to desensitization of the V_1a receptor. The desensitization is probably mediated, at least in part, by protein kinase C (see below) (Pfeilschifter et al. 1989; Plevin et al. 1992). The Sr²⁺ entry evoked by a low concentration of AVP (1 nM) was more sustained than that evoked by a maximal concentration (Fig. 3B), suggesting that responses to physiological stimuli are likely to be more sustained, and presumably thereby more dependent on Ca²⁺ entry, than the responses evoked by the supramaximal stimulation commonly used experimentally.

Figure 9. Low concentrations of AVP preferentially activate non-capacitative Ca²⁺ entry.

A, each trace shows the average change in [Ca²⁺]_i recorded from 4 coverslips of cells stimulated with 1 μM (i), 1 nM (ii), or 100 pM (iii) AVP in the presence (red and black) or absence (blue) of extracellular Ca²⁺. Blue traces are the responses that result solely from release of intracellular Ca²⁺ stores. Red traces show responses from cells that were not treated with Gd³⁺ and therefore represent the normal responses to AVP. Cells shown by the black traces were pretreated with 1 μM Gd³⁺ for 600 s and therefore represent responses without capacitative Ca²⁺ entry. B, the Ca²⁺ signal resulting from Ca²⁺ mobilization has been subtracted to reveal the total Ca²⁺ entry component (red) and the Ca²⁺ entry that persists after Gd³⁺ treatment (black); the latter therefore represents non-capacitative Ca²⁺ entry. C, the fraction of the Ca²⁺ entry mediated by the non-capacitative (Gd³⁺-resistant; black traces) and capacitative (total Ca²⁺ entry less that remaining after Gd³⁺ treatment; red traces) pathways was determined as described in the text. D, the relative contributions of the two pathways 600 s after AVP addition are summarized and show that at low concentrations of AVP, the non-capacitative pathway is the major route for Ca²⁺ entry (means ±s.e.m., n = 3). E, in the subclone of cells lacking AVP-evoked Sr²⁺ entry, all Ca²⁺ entry evoked by 1 nM AVP is blocked by 1 μM Gd³⁺, indicating that it occurs solely via the capacitative pathway (lines are coded as in A). F, the relative magnitudes (% maximal response) of the Ca²⁺ mobilization and capacitative Ca²⁺ entry evoked by three concentrations of AVP suggest that store depletion is tightly coupled to stimulation of the capacitative pathway.

Figure 3. The effect of a low concentration of AVP on Sr²⁺ entry is more sustained than that of a maximal concentration.

A, the Sr²⁺ entry evoked by AVP (100 nM) is transient, but subsequent addition of PDGF (10 nM) evokes further Sr²⁺ entry. B, the Sr²⁺ entry evoked by a submaximal concentration of AVP (1 nM, i) is more sustained than that evoked by a maximal concentration (100 nM, ii).

We established a subclone of A7r5 cells in which the capacitative pathway and both the Ca²⁺ mobilization and Ca²⁺ efflux evoked by AVP were indistinguishable from the parent cells, but in which AVP failed to evoke Sr²⁺ entry (Fig. 1C and D). Besides establishing the independence of the two divalent cation entry pathways and confirming that proteins downstream of the V_1a receptor are required to mediate Sr²⁺ entry, this cell line has also proven useful in analyses of the mechanisms underlying activation of the non-capacitative pathway.

Because AVP also stimulates Ca²⁺ extrusion (Byron & Taylor, 1995) (Fig. 1A and C), it had been difficult to determine either the Ca²⁺ permeability of the non-capacitative pathway or its contribution to Ca²⁺ signalling under physiological conditions. To address these problems, we assessed the abilities of known Ca²⁺ channel blockers to discriminate between the capacitative and non-capacitative pathways: Gd³⁺ proved the most useful. Pretreatment of A7r5 cells with Gd³⁺ (1 μM) completely abolished capacitative Ca²⁺ entry (Fig. 4A) without affecting the Sr²⁺ entry evoked by AVP (100 nM) (Fig. 4B). At higher concentrations, Gd³⁺ (100 μM) also inhibited the Sr²⁺ entry pathway. Figure 4C shows that capacitative Ca²⁺ entry is about 150-fold more sensitive to blockade by Gd³⁺ (IC₅₀= 34 ± 5 nM) than is non-capacitative Sr²⁺ entry (IC₅₀= 5.3 ± 0.7 μM). The difference parallels differences between the trp and trpl channels of Drosophila photoreceptors; the former, a Ca²⁺-selective channel, is ∼100-fold more sensitive to inhibition by Gd³⁺ than the latter, which is a non-selective cation channel (Sinkins et al. 1996). Furthermore, whereas blockade of the non-capacitative pathway in A7r5 cells by Gd³⁺ was reversible (Fig. 4E), blockade of the capacitative pathway was essentially irreversible. Pretreatment of naive cells with Gd³⁺ (3 μM, 400 s) had no effect on the basal rate of Mn²⁺ entry, but completely blocked capacitative Mn²⁺ (not shown) or Ca²⁺ entry (Fig. 4D) when the stores were subsequently emptied in the absence of Gd³⁺. Even 20 min after removal of Gd³⁺, there was no detectable reversal of the block of the capacitative pathway. Since the capacitative pathway is permeable to Mn²⁺ (Byron & Taylor, 1995) and irreversibly inhibited by Gd³⁺ (Fig. 4D), the lack of effect of Gd³⁺ on the basal rate of Mn²⁺ entry indicates first that the capacitative pathway is wholly inactive in unstimulated A7r5 cells, and second that Gd³⁺ is capable of irreversibly inhibiting closed capacitative Ca²⁺ entry channels. In contrast, inhibition of the non-capacitative pathway by higher concentrations of Gd³⁺ (100 μM) was more readily reversible (Fig. 4E).

Figure 4. Selective block of the capacitative pathway by Gd³⁺.

A and B, methods similar to those of Fig. 1 were used to examine the effects of Gd³⁺ (1 μM) on capacitative Ca²⁺ entry (A) and AVP-evoked Sr²⁺ entry (B); only the capacitative pathway was blocked by 1 μM Gd³⁺. C, the concentration-dependent effects of Gd³⁺ on capacitative Ca²⁺ entry (○) and AVP-induced Sr²⁺ entry (•) (means ±s.e.m., n = 3). D and E, Gd³⁺ (3 μM) irreversibly inhibited capacitative Ca²⁺ entry (D), but the inhibition of AVP-evoked Sr²⁺ entry by 100 μM Gd³⁺ was reversible (E). F, despite the counteracting effect of the stimulation of Ca²⁺ extrusion by AVP (i, no Gd³⁺), AVP stimulated an increase in [Ca²⁺]_i when added to cells in which the capacitative pathway had been fully inhibited by Gd³⁺ (ii, 1 μM). Each trace is typical of at least 3 experiments.

Our ability to irreversibly inhibit the capacitative pathway by pretreatment with 1-3 μM Gd³⁺ without affecting the non-capacitative pathway (Fig. 4C) allowed us to directly assess the Ca²⁺ permeability of the latter. The results demonstrate that despite a counteracting ability of AVP to stimulate Ca²⁺ extrusion (Fig. 1A and C), addition of AVP to cells in which the capacitative pathway had been fully inhibited evoked a substantial increase in [Ca²⁺]_i that was entirely dependent on Ca²⁺ entry (Fig. 4F). As expected, in the subclone of cells in which AVP failed to stimulate Sr²⁺ entry, AVP also failed to evoke Ca²⁺ entry (not shown). We conclude, in keeping with the suggestion that the non-capacitative pathway is a non-selective cation channel (Van Renterghem et al. 1988), that the pathway is permeable to Ca²⁺ and furthermore capable of evoking a significant increase in [Ca²⁺]_i.

The non-capacitative pathway is stimulated by arachidonic acid produced after activation of phosphoinositidase C

Receptor-mediated stimulation of phospholipase A₂ (PLA₂) has previously been suggested to regulate Ca²⁺ entry (Wang et al. 1993; Shuttleworth, 1996; Munaron et al. 1997), but in A7r5 cells isotetrandrine (10 μM), a selective inhibitor of G protein-coupled phospholipase A₂ (Akiba et al. 1992), had no effect on the Sr²⁺ entry evoked by AVP (100 nM). The peak increase in [Sr²⁺]_i in the presence of isotetrandrine was 105 ± 12 % (n = 3) of that in its absence. Other agonists that stimulate PIC, either via receptors linked to G proteins, namely bombesin (100 nM) and 5-hydroxytryptamine (50 μM), or via receptors with tyrosine kinase activity, namely PDGF (5 nM, Fig. 1D), mimicked the effects of AVP on both Ca²⁺ mobilization and Sr²⁺ influx. These results are consistent with the shared ability of these receptors to stimulate PIC underlying their activation of the non-capacitative pathway. We attempted to test this suggestion directly using U73122, an inhibitor of PIC (Bleasdale et al. 1990).

In A7r5 cells perfused with Ca²⁺-free HBS, U73122 (5 μM) caused an increase in [Ca²⁺]_i that was not mimicked by the analogue, U73343, which does not inhibit PIC (Taylor & Broad, 1998). Others have encountered similar problems with U73122 and attributed them to inhibition of the endoplasmic reticulum Ca²⁺ pump (De Moel et al. 1995). To circumvent the direct effect of U73122 on Ca²⁺ stores, we examined the effect of U73122 on the accumulation of inositol phosphates evoked by AVP and compared that with its effect on Sr²⁺ entry. A maximally effective concentration of U73122 (30 μM) fully inhibited the effect of AVP (50 nM) on the formation of inositol phosphates and Sr²⁺ entry, and half-maximally inhibited each at concentrations of 0.9 ± 0.1 and 1.1 ± 0.2 μM, respectively (n = 3), in line with previous reports of the sensitivity of PIC to U73122 (Macrez-Lepetre et al. 1996) (Fig. 5A and B). In view of the direct effects of U73122 on Ca²⁺ stores and of previous reports that both it and U73343 may inhibit capacitative Ca²⁺ entry (Taylor & Broad, 1998), we were concerned that the inhibition of Sr²⁺ entry by U73122 might result from a direct inhibition of the non-capacitative pathway. We believe this to be unlikely because when the non-capacitative pathway was directly activated by addition of arachidonic acid (see below), U73122 (10 μM) had no effect on the resulting Sr²⁺ entry (Fig. 5C). We conclude that activation of PIC is necessary for receptors to stimulate the non-capacitative pathway and since InsP₃ has already been shown not to activate the non-selective cation current of A7r5 cells (Van Renterghem et al. 1988; Nakajima et al. 1996), we considered the possibility that activation of protein kinase C by diacylglycerol might be responsible.

Figure 5. Phosphoinositidase C is required for AVP to stimulate the non-capacitative pathway.

A and B, concentration-dependent effects of U73122 (•) and U73343 (○) on AVP (50 nM)-evoked formation of [³H]inositol phosphates (A) and Sr²⁺ entry (B). Results are means ±s.e.m. of 3 independent experiments. C, U73122 (10 μM) abolished the Sr²⁺ entry evoked by AVP (100 nM) in thapsigargin-treated cells without affecting that evoked by arachidonic acid (AA, 50 μM). Results are typical of those from 3 similar experiments.

Activation of protein kinase C with the phorbol ester phorbol 12,13-dibutyrate (PDBu, 1 μM, 10 min) failed to stimulate Sr²⁺ entry in A7r5 cells with empty Ca²⁺ stores (not shown), although a similar treatment of naive cells (1 μM PDBu, 15 min) substantially inhibited (by 68 ± 3 %, n = 3) the ability of AVP (100 nM) to stimulate release of intracellular Ca²⁺ stores and completely abolished (-2 ± 2 %) AVP-stimulated Sr²⁺ entry to cells with empty stores. The inactive phorbol ester, 4-α-phorbol 12,13 didecanoate (1 μM, 15 min), had no effect. These results suggest that rather than mimicking the effects of AVP on the non-capacitative pathway, protein kinase C exerts a negative feedback on the V_1a receptor. Previous analyses of primary cultures of rat aorta and A10 vascular smooth muscle cells have also implicated protein kinase C in such negative feedback (Pfeilschifter et al. 1989; Plevin et al. 1992). This suggestion gains further support from our observation that Ro31-8220 (15 μM, 10 min), an inhibitor of protein kinase C (Plevin et al. 1992), massively potentiated (to 347 ± 39 % of control, n = 3) the Sr²⁺ influx evoked by AVP in A7r5 cells.

The evidence so far suggests that an intracellular signal downstream of PIC is likely to mediate the effects of receptors on non-capacitative Ca²⁺ entry, but the signal is neither protein kinase C nor InsP₃ (Van Renterghem et al. 1988; Nakajima et al. 1996). Arachidonic acid (or perhaps other unsaturated fatty acids) released from diacylglycerol (DAG) by diacylglycerol lipase, the major route for DAG metabolism in vascular smooth muscle cells (Lee & Severson, 1994), is a potential candidate.

An inhibitor of DAG lipase, RHC-80267 (50 μM) (Sutherland & Amin, 1982) (Fig. 6A), did not itself stimulate Sr²⁺ entry, but it inhibited that evoked by AVP by 73 ± 7 % (n = 3). Further increasing the concentration of RHC-80267 to 100 μM caused no further inhibition. RHC-80267 (50 μM, 15 min) had no effect on basal [Ca²⁺]_i or capacitative Ca²⁺ entry (106 ± 20 % of control, n = 3) and only minimally inhibited (90 ± 9 % of control, n = 3) AVP-stimulated release of intracellular Ca²⁺ stores (Fig. 6B), indicating, in line with previous reports, that PIC was not inhibited. The inhibition of AVP-evoked Sr²⁺ entry occurred at concentrations of RHC-80267 (50-100 μM) similar to those used to block DAG lipase in other cells in which its effects on PIC and PLA₂ were minimal (Sutherland & Amin, 1982). The incomplete inhibition of AVP-evoked Sr²⁺ entry by RHC-80267 may result from incomplete inhibition of DAG lipase, the mechanism of which is unknown (Sutherland & Amin, 1982), or suggest that DAG lipase may not be the only route through which AVP regulates the non-capacitative pathway. Arachidonic acid can be released by at least three mechanisms: the sequential action of PIC and DAG lipase; the action of PLA₂ on phospholipids; and the action of a lipase on phosphatidic acid. The latter pathways may perhaps play minor roles in mediating AVP-stimulated Sr²⁺ entry.

In fourteen experiments, addition of arachidonic acid (10-50 μM) to A7r5 cells with empty Ca²⁺ stores stimulated Sr²⁺ entry (Fig. 6C). In each of three experiments, the response to arachidonic acid was completely inhibited by Gd³⁺ at a concentration (100 μM) which fully inhibited AVP-evoked Sr²⁺ entry (Fig. 6E), but it was unaffected by a lower concentration of Gd³⁺ (1 μM) that fully inhibited capacitative Ca²⁺ entry (Fig. 6F). The effects of AVP and arachidonic acid on Sr²⁺ entry were not additive: the response to the latter was 124 ± 19 % of that evoked by AVP (n = 4). Furthermore, U73122 (10 μM), which is proposed to inhibit AVP-evoked Sr²⁺ entry by preventing formation of arachidonic acid, did not inhibit the Sr²⁺ entry evoked by arachidonic acid (Fig. 5C). Previous studies suggested that arachidonic acid was most effective when applied to cells at low density (Alonso-Torre & Garciá-Sancho, 1997): low concentrations of arachidonic acid (< 10 μM) stimulated Ca²⁺ entry in freshly dispersed cells (Wang et al. 1993) or membrane patches (Munaron et al. 1997), whereas higher concentrations (> 10 μM) were required for confluent cells (Van Delden et al. 1993). Consistent with these observations, when arachidonic acid (5 μM) was continuously applied to sub-confluent cultures of A7r5 cells in the presence of 1 μM Gd³⁺ to block the capacitative pathway, it stimulated Ca²⁺ entry, though in only 5 ± 1 % of cells (Fig. 6G). These findings support the idea that the effect of AVP on the non-capacitative pathway may be mediated by arachidonic acid.

Conversion of arachidonic acid to active metabolites is unlikely to be required for stimulation of Sr²⁺ entry because in each of at least three experiments, inhibitors of the most important enzymes, cyclo-oxygenase (indomethacin, 100 μM; or aspirin, 500 μM) and lipoxygenase (NDGA, 50 μM, which also inhibits cytochrome P450 cyclo-oxygenase), failed to inhibit the Sr²⁺ entry evoked by either AVP (100 nM) (Fig. 7A and B) or arachidonic acid (50 μM) (not shown). Indeed, while NDGA had no effect on Ca²⁺ mobilization, it potentiated the effect of AVP on Sr²⁺ entry by prolonging the response for as long as NDGA remained: 500 s after AVP addition, the fluorescence signal reflecting Sr²⁺ entry remained only 0.04 ± 0.02 units (n = 3) above baseline in control cells, but was elevated by 0.42 ± 0.06 units (n = 3) in cells treated with NDGA (Fig. 7A). We conclude that arachidonic acid is likely to directly stimulate the non-capacitative pathway.

Figure 7. Arachidonic acid metabolism is not required for it to stimulate the non-capacitative pathway.

A, in the presence of NDGA (50 μM, thick trace), the Sr²⁺ entry evoked by AVP (100 nM) was substantially enhanced relative to control (thin trace). The histograms (means ±s.e.m., n = 3) show the fluorescence signal recorded 500 s after additon of AVP in control cells and cells treated with NDGA. B, aspirin (500 μM, thick trace) had no effect on AVP-evoked Sr²⁺ entry (control, thin trace). Traces are typical records from 3 independent measurements of cell populations.

Although AVP failed to stimulate Sr²⁺ entry in the unresponsive subclone of A7r5 cells (Fig. 1D), the cells did respond to arachidonic acid (Fig. 6D), suggesting that they express the non-capacitative Ca²⁺ entry pathway, but lack the link between AVP-stimulated PIC and the channel. We speculate that the unresponsive cells lack DAG lipase.

In view of the relatively high concentrations of arachidonic acid used in this (> 5 μM) and other studies (typically ∼10 μM or more) (Van Delden et al. 1993; Wang, et al. 1993; Shuttleworth, 1996) to overcome the acknowledged problems of applying a liphophilic messenger to cells in physiological saline, we were concerned to eliminate any possible artefacts by demonstrating a selective activation of the non-capacitative pathway. First, even the highest concentration of arachidonic acid used (50 μM) did not stimulate Mn²⁺ entry, confirming both the integrity of the plasma membrane and the lack of effect of arachidonic acid on the capacitative pathway (Fig. 6H). Second, lower concentrations of arachidonic acid evoked Ca²⁺ entry, but in only a fraction (5 ± 1 %) of the cells (Fig. 6G). Third, a concentration of Gd³⁺ (1 μM) that fully inhibited capacitative Ca²⁺ entry did not inhibit arachidonic acid-evoked Sr²⁺ entry (Fig. 6F). Fourth, the effects of maximally effective concentrations of AVP and arachidonic acid on Sr²⁺ entry were similar (Fig. 6C) and non-additive (not shown). Fifth, a concentration of Gd³⁺ that blocked AVP-evoked Sr²⁺ entry also fully inhibited the response to arachidonic acid (Fig. 6E). Sixth, AVP-evoked Sr²⁺ entry was potentiated when arachidonic acid metabolism was inhibited (Fig. 7A); this is an important result because it confirms that arachidonic acid lies on the pathway linking the V_1a receptor to the non-capacitative pathway. Finally, in three experiments the effects of arachidonic acid were not mimicked by stearic acid (100 μM) (not shown).

We conclude that arachidonic acid directly mediates the effects of AVP on the non-capacitative pathway in A7r5 cells, consistent with several previous reports in which metabolism of arachidonic acid was not required for it to stimulate Ca²⁺ entry (Van Delden et al. 1993; Wang et al. 1993; Shuttleworth, 1996; Munaron et al. 1997). Our conclusion is also consistent with the proven ability of AVP to stimulate arachidonic acid release in A7r5 cells (Thibonnier et al. 1991; Cane et al. 1997) and with arachidonic acid stimulating Ca²⁺ entry in DDT₁MF-2 cells (Van Delden et al. 1993) and epithelial cells (Shuttleworth, 1996) in the absence of store emptying. In neither of those studies, however, is it clear whether the Ca²⁺ enters via the same or a different pathway to that activated by empty stores. Our results provide the first unequivocal demonstration that arachidonic acid stimulates a non-capacitative Ca²⁺ entry pathway. Furthermore, the link between receptors and non-capacitative Ca²⁺ entry in A7r5 cells is unusual in that the arachidonic acid derives largely from the sequential activities of PIC and DAG lipase (Figs 5 and 6). The same route from PIC to arachidonate links activation of the fibroblast growth factor receptor to Ca²⁺ entry in neurones (Doherty & Walsh, 1996), but in all previous studies of arachidonic acid-regulated Ca²⁺ entry into non-neuronal cells, PLA₂ has been proposed to be responsible for the arachidonic acid formation (Wang et al. 1993; Shuttleworth, 1996; Munaron et al. 1997).

Because AVP and arachidonic acid also stimulate release of intracellular Ca²⁺ stores in both A7r5 and other cells (Van Delden et al. 1993), the experiments so far have not demonstrated whether arachidonic acid is a sufficient stimulus for activation of the non-capacitative pathway in the absence of store depletion. We were unable to resolve the issue with Xestospongin, which was recently shown to block type 1 InsP₃ receptors (Gafni et al. 1997), because it also caused emptying of the intracellular Ca²⁺ stores. Instead, cells were microinjected with heparin, which is a competitive antagonist of InsP₃ receptors (Taylor & Broad, 1998). The microinjected cells were then stimulated with PDGF, an agonist that stimulates InsP₃ formation without requiring activation of a G protein, rather than with AVP in order to avoid problems arising from the ability of heparin to uncouple receptors from their G proteins (Taylor & Broad, 1998). In three experiments, heparin (10 mg ml⁻¹ in the injection pipette) completely abolished PDGF-evoked Ca²⁺ mobilization without affecting the Sr²⁺ entry evoked by PDGF (Fig. 8). Subsequent addition of ionomycin and thapsigargin confirmed that PDGF had stimulated Sr²⁺ entry into cells with replete Ca²⁺ stores. PDGF evoked the normal Ca²⁺ mobilization and Sr²⁺ entry in cells injected with a lower concentration of heparin (1 mg ml⁻¹), confirming that the results were not an artefact of the microinjection procedures. We conclude that depletion of intracellular Ca²⁺ stores is not required for PDGF to stimulate the non-capacitative Ca²⁺ entry pathway.

Figure 8. Depletion of intracellular Ca²⁺ stores is not required for activation of the non-capacitative pathway.

Cells were microinjected with heparin (10 mg ml⁻¹ in injection pipette; thin trace) and then stimulated with PDGF (2.5 nM) in Ca²⁺-free HBS. Intracellular heparin abolished the Ca²⁺ mobilization evoked by PDGF (2.5 nM) without affecting Sr²⁺ entry. Subsequent addition of ionomycin and thapsigargin (I/Tg) confirmed that whereas PDGF had fully emptied the intracellular Ca²⁺ stores of the control cells (thick trace), the stores of the cells injected with heparin (thin trace) had retained their Ca²⁺. The traces show results from individual cells typical of 3 similar experiments.

The non-capacitative pathway is the major Ca²⁺ entry pathway stimulated by low concentrations of AVP

In a final series of experiments, we used Gd³⁺ (1 μM) to establish the relative contributions of capacitative (Gd³⁺-sensitive) and non-capacitative (Gd³⁺-insensitive) Ca²⁺ entry to the Ca²⁺ signals evoked by different concentrations of AVP. The Ca²⁺ mobilization evoked by AVP (0.1 nM or 1 μM) was unaffected by Gd³⁺: the peak [Ca²⁺]_i was 1283 ± 199 nM with 1 μM AVP and 71 ± 5 nM with 0.1 nM AVP, and in the presence of Gd³⁺ the corresponding responses were 1254 ± 104 nM and 73 ± 5 nM (n = 3).

Naive A7r5 cells were stimulated with AVP (0.1-1000 nM) in the absence of extracellular Ca²⁺ to establish the magnitude of InsP₃-evoked Ca²⁺ release (blue lines in Fig. 9A). In parallel experiments cells were stimulated with the same concentrations of AVP in the presence of extracellular Ca²⁺ either with (black lines in Fig. 9A) or without (red lines) pretreatment with Gd³⁺ (600 s, 1 μM). The red traces therefore represent the normal responses to AVP. In each experiment, as expected, a subsequent addition of thapsigargin evoked capacitative Ca²⁺ entry only in cells that had not been exposed to Gd³⁺ (not shown). By subtracting the responses to AVP in the absence of extracellular Ca²⁺ from those in its presence, the effects of AVP on Ca²⁺ entry were determined (Fig. 9B). Then by subtracting the Ca²⁺ entry recorded in the presence of Gd³⁺ (black lines in Fig. 9B) from that in its absence (red lines), the relative contributions of the non-capacitative and capacitative pathways were established. The results are shown in Fig. 9C in which red lines denote the percentage of the total Ca²⁺ entry occurring through the capacitative pathway and black lines denote that occurring via the non-capacitative pathway. In the subclone of cells lacking AVP-evoked Sr²⁺ entry, pretreatment with Gd³⁺ (1 μM) completely abolished the Ca²⁺ influx evoked by AVP (1 nM): the response was indistinguishable from that in the absence of extracellular Ca²⁺(Fig. 9E).

The results of this analysis (Fig. 9) demonstrate that even after maximal stimulation with AVP, ∼25 % of the Ca²⁺ entry occurs via the non-capacitative pathway. At lower concentrations of AVP, similar to those likely to occur physiologically, the non-capacitative pathway provides the major route for Ca²⁺ entry accounting for up to 90 % of the Ca²⁺ entry evoked by 100 pM AVP (Fig. 9D). The relatively greater contribution from non-capacitative Ca²⁺ entry at lower concentrations of AVP is consistent with Sr²⁺ entry (EC₅₀= 15 ± 5 nM, n = 3) being slightly, though significantly (P < 0.05), more sensitive to AVP than Ca²⁺ release from intracellular stores (EC₅₀= 32 ± 5 nM, n = 3). While the Ca²⁺ content of the stores regulates the capacitative pathway, its activation has been proposed to occur only after substantial depletion of the stores (Parekh et al. 1997), in which case the disparity between the AVP sensitivities of the two Ca²⁺ entry pathways would be larger than suggested by our comparison of Ca²⁺ release and Sr²⁺ entry. Although we have not extensively characterized the relationship between store depletion and capacitative Ca²⁺ entry, the effects of these three AVP concentrations on Ca²⁺ mobilization and capacitative Ca²⁺ entry suggest a close relationship: even modest Ca²⁺ mobilization evokes capacitative Ca²⁺ entry (Fig. 9F), consistent with a recent study in which the luminal free [Ca²⁺] of the stores and stimulation of the capacitative pathway were tightly correlated (Hofer et al. 1998).

Conclusions

By exploiting the different permeabilities of the non-capacitative and capacitative pathways of A7r5 cells, we have established that arachidonic acid (and perhaps other unsaturated fatty acids) released by the sequential actions of receptor-activated PIC and DAG lipase directly stimulates the non-capacitative pathway, that depletion of intracellular Ca²⁺ stores is not required for activation of the pathway, that it is Ca²⁺ permeable, and that low concentrations of Gd³⁺ selectively block the capacitative pathway (Fig. 10). The selective effect of Gd³⁺ allowed us to address the relative contributions of capacitative and non-capacitative Ca²⁺ entry to the Ca²⁺ signals evoked by a physiological stimulus and establish that non-capacitative Ca²⁺ entry is most important (Fig. 9). Our results challenge the prevailing view that the capacitative pathway provides the major route for receptor-regulated Ca²⁺ entry - a view deriving largely from studies in which hormones failed to increase [Ca²⁺]_i beyond that observed when the capacitative pathway was maximally activated by thapsigargin (Takemura et al. 1989; Demaurex et al. 1994; Madge et al. 1997). They are also consistent with observations from endothelial cells in which Ca²⁺ entry is essential to sustain Ca²⁺ spiking, but the episodic discharge of intracellular stores is not accompanied by bursts of capacitative Mn²⁺ entry (Jacob, 1990), and with suggestions that empty stores are not responsible for stimulating the Ca²⁺ entry that sustains Ca²⁺ oscillations in avian exocrine glands (Shuttleworth, 1996). The major role of non-selective cation channels in vascular smooth muscle cells has previously been supposed to be to cause depolarization and so activation of voltage-gated Ca²⁺ channels. We have not attempted to compare the relative effects on [Ca²⁺]_i of L-type Ca²⁺ channels (which for practical reasons were always inhibited in our experiments) with the pathways directly regulate by AVP. Our results do, however, indicate that even when voltage-gated Ca²⁺ channels are inactive, non-selective cation channels are likely to provide a major route for receptor-regulated Ca²⁺ entry in vascular smooth muscle and so allow them to play an important role in regulating [Ca²⁺]_i even when the membrane is hyperpolarized. We conclude that Ca²⁺ entry through non-capacitative pathways is likely to be a more important element of the Ca²⁺ entry evoked by physiological stimuli than hitherto supposed and that it may thereby play a major role in providing the Ca²⁺ required to sustain the Ca²⁺ spiking behaviour evoked by such stimuli (Berridge, 1993).