Cytosolic Ca²⁺ and Phosphoinositide Hydrolysis Linked to Constitutively Active α_1d-Adrenoceptors in Vascular Smooth Muscle

G protein-coupled receptors may exist in a spontaneously active form in the absence of an agonist, i.e., constitutively active (De Ligt et al., 2000). This phenomenon has been most readily observed in cell lines in which receptors are overexpressed or mutated. Two different groups have found constitutively active cloned α_1d-adrenoceptors in stably transfected rat-1 fibroblasts (Garcia-Sainz and Torres-Padilla, 1999; McCune et al., 2000) and human embryonic kidney 293 cells (Chalothorn et al., 2002), which are mainly located in a perinuclear location (McCune et al., 2000; Chalothorn et al., 2002). In addition, we found a population of α_1d-adrenoceptors in intact rat arterial vessels such as the aorta, the iliac, or the proximal mesenteric artery that exhibit several features resembling those of constitutively active receptors (Noguera and D'Ocon, 1993; Noguera et al., 1996; Gisbert et al., 2000, 2002; Ziani et al., 2002) such as: 1) its activity occurs in the absence of an agonist; 2) it is inhibited by the α₁-adrenoceptor ligand prazosin and the selective α_1d-adrenoceptor ligand BMY 7378, which behave as inverse agonists; 3) the irreversible α₁-adrenoceptor antagonist chloroethylclonidine, acting as a neutral antagonist, inhibited noradrenaline-induced contractions in this tissue and did not affect the constitutive response but prevented its inhibition by BMY 7378 and prazosin; and 4) it is only observed in vessels (e.g., aorta, iliac, or proximal mesenteric arteries) where α_1d-adrenoceptors play a functional role. However, as opposed to constitutively active α_1d-adrenoceptors in transfected cells, this type of response in native tissues requires prior stimulation with an α₁-adrenoceptor agonist. Once the stimulus is removed, the α_1d-adrenoceptor-dependent response remains and can be inhibited by inverse agonists. A simple protocol, in which intracellular Ca²⁺ depletion by noradrenaline is followed by extracellular Ca²⁺ restoration, permits the differentiation between the agonist-induced and constitutive activation of the receptors (Noguera et al., 1996).

The aim of the present work was to analyze how the activity of these adrenoceptors is regulated by cytosolic Ca²⁺ and how it couples to membrane signals as phosphatidylinositol hydrolysis. Therefore, we examined the contractile response linked to the α_1d-adrenoceptor constitutive activity together with signals associated with stimulation of G proteins in the cell membrane, the inositol phosphate accumulation, and the changes in the cytosolic Ca²⁺ levels.

Materials and Methods

Tissue Preparation. Thoracic aorta and tail artery from Wistar rats (200–250 g) were dissected in a Krebs' solution (composition: 118 mM NaCl, 4.75 mM KCl, 25 mM NaHCO₃, 1.2 mM MgSO₄, 2.0 mM CaCl₂, 1.2 mM KH₂PO₄, and 11 mM glucose) and cut into rings (approximately 3–5 mm in length). Endothelium was removed by gently rubbing the intimal surface with a metal rod. The absence of a relaxant response after the addition of acetylcholine (10 μM) in preparations precontracted by noradrenaline (1 μM) indicated the absence of a functional endothelium.

Simultaneous Measurements of [Ca²⁺]_i and Tension. Aortic and tail artery rings were incubated for 4 to 6 and 2 to 3 h, respectively, at room temperature in Krebs' solution containing the fluorescent dye fura-2/acetoxymethyl ester (5 μM). The castor oil derivative Cremophor EL (final concentration in Krebs' 0.05%) (Sigma-Aldrich, St. Louis, MO) was used to solubilize and facilitate Fura-2/acetoxymethyl ester penetration. The adventitial layer distorts the fluorescence, so the ring has to be illuminated from the intimal side. Therefore, aortic rings were inverted so that the luminal face was exposed outward. Arterial vessels were then suspended under 1g of tension in a 5-ml organ bath containing Krebs' solution, maintained at 37°C and gassed with 95% O₂ and 5% CO₂. The bath was part of a fluorimeter (CAF 110; Jasco, Tokyo, Japan) that allows the estimation of changes in the fluorescence intensity of Fura-2 simultaneously with force development (Perez-Vizcaino et al., 1999). Rings were alternatively illuminated (128 Hz) with two excitation wave-lengths (340 and 380 nm) from a xenon lamp coupled with two monochromators. The emitted fluorescent light at the two excitation wavelengths (F340 and F380) was measured by a photomultiplier through a 510-nm filter and recorded by using data acquisition hardware (Mac Lab, model 8e; ADInstruments Pty Ltd., Castle Hill, Australia) and data recording software (Chart v3.2; ADInstruments Pty Ltd.). Force data were recorded simultaneously by an isometric force-displacement transducer coupled to the Mac Lab data acquisition system. The absolute values of [Ca²⁺]_i were estimated from the ratio of emitted fluorescence obtained at the two excitation wave-lengths(F340/F380) using the Grynkiewicz equation as described (Kanaide, 1999). The maximal and minimal F340 and F380 values for this equation were obtained by treatment with ionomycin (1.4 μM) and then with EGTA (8 mM), respectively. Autofluorescence, determined by quenching fura-2 fluorescence with MnCl₂ (1 mM) at the end of the experiment, was subtracted.

After equilibration for 30 to 45 min, the experimental procedure shown in Fig. 1 designed to evidence the constitutive activity of the α_1d-adrenoceptors was performed (Noguera and D'Ocon, 1993; Gisbert et al., 2000). Initially, a response to a maximal concentration of noradrenaline (1 μM in the aorta and 10 μM in the tail artery) was elicited. After drug washout, the preparations were placed in a Ca²⁺-free Krebs' solution (containing 0.1 mM EDTA) for 20 min, which led to a weak loss in tension (<10–15%) and a reduction in the [Ca²⁺]_i levels below resting values, and then they were exposed to noradrenaline for 5 min. This procedure was repeated twice, and after another 20 min in Ca²⁺-free solution, the bath medium was replaced by a normal Ca²⁺-containing Krebs' solution which induced an increase in [Ca²⁺]_i levels and a contractile response indicative of constitutive activity of α_1d-adrenoceptors. In some experiments, the Ca²⁺ channel blockers nimodipine and Ni²⁺ and the α_1d-adrenoceptor ligands prazosin and BMY 7378 were added during the last 10 min in Ca²⁺-free and during the exposure to the Ca²⁺-containing Krebs' solution. In the experiments where guanethidine was used it was present throughout the experiment.

Inositol Phosphate Determination. The determination of total inositol phosphate (IP) accumulation was adapted from Berridge et al. (1982) as has been previously described (Gisbert et al., 2000). Briefly, rat thoracic aortae or tail arteries were exposed to Krebs' solution containing 10 μCi · ml^-1 of myo-[³H]inositol (specific activity 70.0–100.0 Ci · mM^-1) for 2 h at 37°C and gassed with 95% O₂ plus a5%CO₂ mixture. Afterward, tissues were washed twice with Krebs' solution. Vessels were cut into rings (1 mm for aorta, 2 mm for tail artery) and pooled. Two pieces of tail artery or four rings of aorta were placed in individual tubes that were incubated at 37°C. Different experimental conditions were applied in each determination (carried out in triplicate), as detailed in Fig. 2. LiCl (10 mM) was added to inhibit the metabolism of inositol monophosphates. Incubation was stopped by placing the samples in a cold water bath (4°C) and adding 2 ml of a cold mixture of methanol/chloroform/HCl (40: 20:1, v/v/v). Samples were sonicated for 35 min at 2°–3°C in an ultrasonic water bath and, after the addition of 0.63 ml of chloroform and 1.26 ml of distilled water, centrifuged at 1500g for 10 min to facilitate phase separation. The aqueous layer was removed from the tubes to assay the IP formation. Each sample was neutralized and run through an AG1-X8 column, formate form, 100 to 200 mesh (Bio-Rad, Hercules, CA). The resin was washed successively with 6 ml of water and 6 ml of 60 mM ammonium formate/5 mM sodium tetraborate to eliminate free myo-[³H]inositol and glycerophosphoinositol, respectively. Total IPs were eluted with 3 ml of 1 M ammonium formate/0.1 M formic acid. The eluent fractions were collected and counted in a scintillation counter. The lipid layer remaining after removal of the aqueous phase was used for measurement of [³H]phosphatidylinositols. Accumulation of [³H]IP was routinely calculated as a percentage (dpm%) of total [³H]inositol-labeled lipids in each individual sample to correct interexperimental variations in label incorporation and sample sizes or was expressed as a percentage above the unstimulated [³H]IP accumulation (basal).

Chemicals. Acetylcholine, (-)-noradrenaline, prazosin, lithium chloride, NiCl₂, Cremophor EL, nimodipine, and guanethidine were purchased from Sigma-Aldrich, BMY 7378 from Sigma/RBI (Natick, MA), myo-[³H]inositol from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK), and Fura-2/acetoxymethyl ester (1 mM solution in dimethyl sulfoxide) from Calbiochem (La Jolla, CA). Other reagents were of analytical grade. All compounds were dissolved in distilled water.

Statistical Analysis. The results are presented as the mean ± S.E.M. for n determinations obtained from different animals. Where analysis of variance showed significant differences (P < 0.05), the results were further analyzed using the Student-Newman-Keuls test (GraphPad Software, Inc., San Diego, CA). Differences between phasic and tonic responses were analyzed by means of a paired Student's t test.

Results

[Ca²⁺]_i Signal and Contractility Linked to the Constitutively Active Population of α_1d-Adrenoceptors. Representative traces of the changes in contractile force and [Ca²⁺]_i obtained using the protocol designed to evidence the constitutive activity of the α_1d-adrenoceptors in Fura-2-loaded arteries are shown in Fig. 1. The contractile responses were similar to those previously obtained in tissues not loaded with fura-2 and mounted in conventional organ baths (Noguera et al., 1996). The basal [Ca²⁺]_i values were 120 ± 19 and 129 ± 15 nM, in the aorta and tail artery, respectively (n = 4). Noradrenaline (NA1 in Figs. 1, 3, and 4) evoked a rapid and transient (“phasic response”) increase in [Ca²⁺]_i (224 ± 33 and 251 ± 17 nM, respectively), followed by a decrease to a sustained level (“tonic response”) (193 ± 45 and 187 ± 14 nM, respectively, after 5 min). Changes in intracellular calcium levels were accompanied by a slower contractile response (7.86 ± 1.25 and 9.87 ± 1.38 mN, respectively). The responses measured at 5 min were used as a control (100%) of the responses obtained thereafter in each preparation. After washing and recovery of basal contractile tone the bathing medium was changed to a Ca²⁺-free solution, and a decrease in the basal tone (-10 ± 5%, n = 8, and -5 ± 4%, n = 5, in the aorta and tail artery, respectively, P > 0.05) and in the [Ca²⁺]_i (-81 ± 12 and -87 ± 26% in the aorta and tail artery, respectively, P < 0.05 for both) was observed. After 20 min under these conditions, the addition of noradrenaline (NA2 in Figs. 1, 3, and 4) induced a transient increase in [Ca²⁺]_i, which represents an index of the content of agonist-sensitive intracellular Ca²⁺ stores, and a phasic contraction. Washing with Ca²⁺-free medium induced a further decrease in [Ca²⁺]_i. Upon a second application of the agonist in Ca²⁺-free solution (NA3 in Figs. 1, 3, and 4), only weak increases in contractile force and [Ca²⁺]_i were evoked, which indicated an almost complete depletion of internal Ca²⁺ stores sensitive to noradrenaline. In the aorta, after repeated washing with Ca²⁺-free solution in the absence of noradrenaline for 20 min, restoring the Ca²⁺-containing solution induced a transient increase (phasic) followed by a decrease to a sustained level (tonic) in [Ca²⁺]_i, so that the [Ca²⁺]_i returned to the initial resting levels. This was associated with a contractile response (60.2 ± 11.2% of the control response to noradrenaline, n = 8) (Figs. 1 and 3). In contrast, in the tail artery, upon restoring the Ca²⁺-containing solution, [Ca²⁺]_i also returned to the initial resting values (Figs. 1 and 4), but the response was not clearly biphasic as in the aorta and only a slight increase in force was observed (8.8 ± 2.1% of the control response to noradrenaline, n = 5).

IP Accumulation Linked to the Constitutively Active Population of α_1d-Adrenoceptors. The experiments of IP accumulation were performed following a protocol previously described by us (Gisbert et al., 2000), which attempts to reproduce the conditions of the contractility studies (Fig. 2).

Basal IP accumulation (sample 1 in Fig. 2) in rat aorta (9.55 ± 0.62 dpm%, n = 14) was significantly higher (P < 0.001) than that obtained in rat tail artery (6.96 ± 0.44 dpm%, n = 14). To test whether this increase in activity was due to the constitutive activity of α_1d-adrenoceptors in rat aorta, we analyzed the effects of two α₁ ligands on this basal activity. Prazosin (1 μM) and BMY 7378 (10 μM), which exhibit selective affinity for α₁- and α_1d-adrenoceptors, respectively, did not inhibit the basal accumulation of IP in rat aorta (prazosin: 98.0 ± 7.7% with respect to control, n = 5; BMY 7378: 102.7 ± 7.7% with respect to control, n = 4).

Noradrenaline increased IP accumulation in Ca²⁺-containing solution in the aorta (19.9 ± 1.6 dpm%, n = 16) and tail artery (79.8 ± 1.7 dpm%, n = 5). Despite this, the increase in [Ca²⁺]_i was progressively reduced upon successive applications of noradrenaline in Ca²⁺-free medium, and the IP accumulation in Ca²⁺-free medium was similar to that in Ca²⁺-containing medium (NA1, NA2, and NA3 in Figs. 3 and 4). These results confirm that the progressive reduction in [Ca²⁺]_i responses to successive applications of noradrenaline in Ca²⁺-free medium was due to Ca²⁺ store depletion but not to changes in IP accumulation. After the depletion of the intracellular Ca²⁺ stores sensitive to noradrenaline, restoration of extracellular Ca²⁺ (sample 6, S) induced a significant increase in the IP accumulation in the aorta (Fig. 3C) but not in the tail artery (Fig. 4C).

Effects of α₁-Adrenoceptor Ligands and Guanethidine. The effects of prazosin (1 μM) and BMY 7378 (0.1 μM) were tested to analyze whether the responses observed upon restoration of extracellular Ca²⁺ were due to the constitutive activity of α₁-adrenoceptors. In the rat aorta, prazosin and BMY 7378 added 10 min before and during the exposure to Ca²⁺-containing solution had no effect on basal [Ca²⁺]_i or resting tone but produced strong (>80%) inhibitory effects on the contraction induced by restoring extracellular Ca²⁺ (Fig. 5). In the presence of prazosin or BMY 7378, the initial component of the [Ca²⁺]_i signal (phasic in Fig. 5) was similar to the sustained increase in [Ca²⁺]_i (tonic in Fig. 5) induced by restoring extracellular Ca²⁺. Both of them were similar to the sustained increase in [Ca²⁺]_i observed in the control aortae. The time courses of the [Ca²⁺]_i signals and contractile responses in control and in BMY 7378-treated arteries are compared in Fig. 7.

To rule out the possibility that the release of noradrenaline from nerve terminals could play a role on the [Ca²⁺]_i signals and contractile responses upon restoring extracellular Ca²⁺, the aortae were treated with guanethidine (5 μM) throughout the experiment in the presence of this drug, the transient and sustained changes in [Ca²⁺]_i and the contractile responses induced by restoring extracellular Ca²⁺ were similar to those in parallel control experiments (85 ± 10, 86 ± 6, and 102 ± 7%, respectively, n = 3).

In the tail artery, prazosin (1 μM) had no effect on either the increase in the [Ca²⁺]_i levels or the contractile response induced by restoration of extracellular Ca²⁺ (n = 3).

Sensitivity of the Intracellular Signals to Ca²⁺Channel Blockers in the Rat Aorta. To analyze the role of extracellular Ca²⁺ entry on intracellular signals ([Ca²⁺]_i and IP accumulation), another set of experiments were performed in the presence of Ni²⁺ (1 mM) or the specific L-type voltage-dependent Ca²⁺ channel blocker nimodipine (0.1 nM). After depletion of internal Ca²⁺ stores by noradrenaline, the Ca²⁺ channel blockers were added 10 min before and during the exposure to Ca²⁺-containing solution. In the experiments addressed to determine the IP accumulation, the Ca²⁺ channel blockers were added in sample 7 (Fig. 2). Neither nimodipine nor Ni²⁺ modified resting [Ca²⁺]_i or basal tone. Figure 6 shows that nimodipine inhibited the contractile response (Fig. 6A), but not the increase in the IP levels (Fig. 6C) upon restoration of extracellular Ca²⁺. In addition, nifedipine (0.1 μM) was also without effect on the IP accumulation in the aorta (41.2 ± 7.7%, n = 5 versus 55.5 ± 6.1% above basal values, n = 13, P > 0.05). In the presence of nimodipine, the initial increase in [Ca²⁺]_i (phasic in Fig. 6B) was similar to the sustained response (tonic in Fig. 6B), and both of them were similar to the sustained component of the control (Fig. 6B). This can be more clearly observed in Fig. 7, which also shows that the time course of the changes in [Ca²⁺]_i and tone in the presence of nimodipine was very similar to that observed in the presence of BMY 7378. Addition of Ni²⁺ produced a very strong inhibitory effect (>90%) on all the signals associated with the restoration of extracellular Ca²⁺, i.e., the increases in contractile tone, the phasic and tonic increases in [Ca²⁺]_i, and the IP levels (Fig. 6). The IP accumulation in Ni²⁺-treated arteries was very similar to that observed when Ca²⁺ was not included in the medium (sample 8 in Fig. 2; Fig. 6C).

Discussion

We have previously shown that in vessels where the α_1d-adrenoceptors play a functional role, following an adrenergic stimulus, a population of α_1d-adrenoceptors temporarily remains in a constitutively active state when the stimulus disappears (Noguera et al., 1996; Gisbert et al., 2000). There is an experimental procedure that allows us to easily differentiate this constitutive activity from the response induced by an adrenergic stimulus. The assumption that the contractile response observed after removal of the agonist is due to the constitutive activity of α_1d-adrenoceptors is based on previous evidence as described above (see Introduction). In addition, the present results show that guanethidine, a noradrenergic neuron-blocking drug, had no effect on these responses, ruling out a possible role for noradrenaline release from nerve terminals. However, despite noradrenaline not being present during the constitutive response, it is absolutely necessary as a previous α₁-adrenoceptor stimulus. In fact, a similar response was obtained by depletion of internal Ca²⁺ stores by other α₁-adrenoceptor agonists such as methoxamine and phenylephrine, whereas clonidine, serotonin, caffeine, ryanodine, thapsigargin, and cyclopiazonic acid, which deplete Ca²⁺ stores in an α₁-adrenoceptor-independent manner, did not elicit any contractile response when extracellular calcium was restored (Noguera and D'Ocon, 1993; Noguera et al., 1996, 1997, 1998).

α₁-Adrenoceptors signal through both pertussis toxin-sensitive G proteins and G_q/11 proteins located in the cell membrane (Garcia-Sainz et al., 1999; Piascik and Perez, 2001). They mobilize intracellular Ca²⁺ as a consequence of the IP accumulation and activate Ca²⁺ influx via voltage-dependent and independent Ca²⁺ channels (Minneman, 1988; Zhong and Minneman, 1999; Inoue et al., 2001). The α_1d-adrenoceptor subtype is located mainly intracellularly in a perinuclear orientation (McCune et al., 2000; Chalothorn et al., 2002). Then, the receptors must migrate to the cell membrane to interact with G_q/11 proteins located in it; therefore, the mechanisms involved in this migration would control the contractile response related to the constitutively active α_1D-adrenoceptors. This hypothesis prompted us to analyze the increase in the contractile tone and the intracellular signaling ([Ca²⁺]_i and IP accumulation) associated with the constitutive activity of the α_1d subtype.

Ca²⁺Signal. In rat aorta, after depletion of noradrenaline-sensitive Ca²⁺ stores and restoration of extracellular Ca²⁺ in the absence of the agonist, the increase in [Ca²⁺]_i represents a complex phenomenon in which transient and sustained components could be differentiated. To pharmacologically analyze the changes in [Ca²⁺]_i, we tested the effects of two Ca²⁺ channel blockers, nimodipine and Ni²⁺, an α₁-adrenoceptor ligand, prazosin, and a selective α_1d-adrenoceptor ligand BMY 7378.

In the presence of nimodipine, the transient component of [Ca²⁺]_i increase was not evident and the contractile response was strongly inhibited. Therefore, Ca²⁺ entry through L-type voltage-operated channels is responsible for the transient component of [Ca²⁺]_i increase, and it is involved in the contractile process. The transient and sustained components of Ca²⁺ entry as well as the associated contractile response were blocked by Ni²⁺, a nonspecific Ca²⁺ channel blocker. This cation is commonly used to block store-operated Ca²⁺ channels (Jung et al., 2000; Kukkonen and Akerman, 2001), nonselective cationic channels which activate upon Ca²⁺-store depletion. Despite store-operated Ca²⁺ channels being the most reasonable targets for Ni²⁺ in our conditions, the present experiments do not exclude other Ca²⁺ entry pathways such as the store depletion-independent Ca²⁺ entry pathway recently associated with α₁-adrenoceptors (Inoue et al., 2001).

In presence of prazosin or BMY 7378, the transient component has not been observed, but the sustained increase in [Ca²⁺]_i was not affected, which confirmed that both components of calcium entry could be pharmacologically distinguished. The α₁-adrenoceptor ligand-sensitive increase in [Ca²⁺]_i was remarkably similar to the nimodipine-sensitive one, suggesting that the constitutive activity of α_1d-adrenoceptors is required for the activation of L-type channels, permitting calcium entry, which contributes to contraction. The sustained increase in [Ca²⁺]_i restores cytosolic calcium levels but is not involved in the contractile response.

In tail artery, where a functional role of α_1d-adrenoceptors can be excluded (Lachnit et al., 1997; Gisbert et al., 2000), only a sustained increase in [Ca²⁺]_i was observed upon restoration of extracellular Ca²⁺. This response was insensitive to α₁-adrenoceptor ligands and was not accompanied by contraction. The lack of functional α_1d-adrenoceptors in the tail artery is likely to be responsible for the absence of the transient [Ca²⁺]_i increase and the contractile response observed in this tissue.

IP Signal. Previous (Gisbert et al., 2000) and present results indicate that, in the absence of the agonist, restoration of extracellular Ca²⁺ after depletion of noradrenaline-sensitive intracellular stores increased IP accumulation in rat aorta but not in tail artery. This IP accumulation was inhibited by prazosin and BMY 7378 (Gisbert et al., 2000), which confirmed the dependence of the signal on α_1d-adrenoceptor activity. Interestingly, as present results show, the IP accumulation observed in absence of the agonist is only obtained when extracellular calcium entry restores cytosolic calcium levels. Moreover, Ni²⁺, which almost suppressed the increase in [Ca²⁺]_i, also abolished the IP accumulation upon restoration of extracellular Ca²⁺. The fact that nimodipine, which inhibits Ca²⁺ entry through L-channels, strongly inhibited the associated contractile response, but neither affected the restoration of cytosolic Ca²⁺ levels nor abolished the IP accumulation, confirms that Ca²⁺ entry through L-type channels is the consequence of the constitutive activity of α_1d-adrenoceptors and is essential for contraction but not for IP accumulation due to constitutively active α_1d-adrenoceptors. Therefore, and this is a crucial point of this study, these results suggest that the constitutively active α_1d-adrenoceptors, located intracellularly, require a physiological level of cytosolic Ca²⁺ to promote IP accumulation.

An important issue which arises from the present results is whether the α_1d-adrenoceptors are “truly” constitutively active in native tissues if they need calcium to evidence their activity. We can suppose that calcium acts directly on the receptor, changing its conformation from an inactive to an active state. However, our results do not sustain this hypothesis because neither prazosin nor BMY 7378 affected the basal accumulation of IP in the aorta, indicating that in presence of physiological levels of cytosolic calcium, we cannot observe constitutive activity in vessels not previously stimulated by an α₁-adrenoceptor agonist. This evidence suggests that calcium plays a role in facilitating the coupling of constitutively active α_1d-adrenoceptors to G proteins but does not act directly on changing the conformation of the receptor. It is well known that α_1d-adrenoceptors are intracellularly located in a perinuclear orientation (McCune et al., 2000; Chalothorn et al., 2002), but G_q/11 proteins, which mediate IP accumulation, are in the cell membrane then, if α_1d-adrenoceptors are in an active conformation but intracellularly located, they need to migrate to the cell membrane to couple to G_q/11 proteins and induce IP accumulation. We propose that calcium allows this migration, and when cytosolic calcium levels are very low, the constitutively active receptors cannot migrate; therefore, membrane signals such as IP accumulation cannot be observed.

Another essential question is the exact role that the previous adrenergic stimulus played in the population of α_1d-adrenoceptors. We can exclude that residual noradrenaline could be activating the receptors giving a “persistent activation” instead of a “constitutive activity” for the following evidences. First, it is clear from previous works (Noguera et al., 1996, 1997; Gisbert et al., 2000) that noradrenaline-induced contractile responses and constitutively active α_1d-adrenoceptor-induced responses are pharmacologically distinguishable. The first is chloroethylclonidine-sensitive and nimodipine-insensitive, whereas the second is chloroethylclonidine-insensitive and nimodipine-sensitive. Second, noradrenaline-induced IP accumulation was not dependent on [Ca²⁺]_i because noradrenaline induced similar increases in IP accumulation in Ca²⁺-containing (NA1 in Fig. 3) and Ca²⁺-free medium (NA2 and NA3 in Fig. 3) despite [Ca²⁺]_i levels being much lower in the latter conditions. However, as has been previously discussed, IP accumulation related to constitutively active α_1d-adrenoceptors depends on calcium then, if NA persistently activates α_1d-adrenoceptors, Ca²⁺ would not be needed to observe the IP accumulation.

If noradrenaline is not present, two different possibilities must be considered. One of them is the possibility that intracellularly located α_1d-adrenoceptors were activated by adrenergic agonists and remained temporarily in an active conformation when the agonist disappeared. In this case, the constitutive activity of the α_1d-adrenoceptors is temporal, not an intrinsic property of the receptor, and depends directly on a previous stimulus. The other possibility is the consideration that the α_1d-adrenoceptors would always be in an active conformation but intracellularly located, and the adrenergic stimulus was needed as a signal for its recruitment to the external membrane permitting the coupling to G proteins located in it. This agonist-induced recruitment of cytosolic adrenoceptors has been previously described with regard to the α_1A subtype (Holtbäck et al., 1999). Our present results do not allow us to differentiate between whether the constitutive activity of α_1d-adrenoceptors is a temporal or an essential property of these receptors. Previous studies (Garcia-Sainz and Torres-Padilla, 1999; McCune et al., 2000) using cloned α_1d-adrenoceptors expressed in different cell lines have shown constitutive activity for this subtype, independent of previous adrenergic stimulus, and then, we can suggest that this is an essential property of the receptor also in native tissues.

In conclusion, our experiments demonstrated that, after an adrenergic stimulus, a population of α_1d-adrenoceptors remains in an active conformation. In Ca²⁺-free medium, the low level of cytosolic Ca²⁺ reached following the depletion of noradrenaline-sensitive intracellular Ca²⁺ stores does not permit the display of the membrane signals associated with the constitutive activity of α_1d-adrenoceptors. When extracellular Ca²⁺ is restored, Ca²⁺ enters into aortic smooth muscle cells by means of two processes with different time courses and pharmacology. An Ni²⁺-sensitive increase in [Ca²⁺]_i is required to evidence the constitutive activity of α_1d-adrenoceptors in native tissues as indicated by increased IP accumulation. An additional [Ca²⁺]_i increase through L-type channels is triggered by constitutively active α_1d-adrenoceptors and is responsible of the contractile response.

Under physiological conditions, the cytosolic Ca²⁺ levels are high enough to permit the coupling of the constitutively active α_1d-adrenoceptors to the membrane signals, and its participation in the contractile response. These processes allow that the contraction, triggered by an adrenergic stimulus, would be temporarily sustained even when the stimulus was removed (Ziani et al., 2002).