Introduction

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Recent experimental evidence suggests a two-state receptor activation model in which G protein-coupled receptors are in equilibrium between an inactive and a spontaneously active conformation that couples to the G protein in absence of a ligand (Lefkovitz et al., 1993; Leff et al., 1997; Colquhoun, 1998). The existence of this active conformation has been revealed in artificial models as receptor mutants, systems that show an overexpression of a certain type of receptor or cloned receptors (Bond et al., 1995; Burstein et al., 1997; Gether et al., 1997; Hwa et al., 1997; Scheer et al., 1997; García Sainz and Torres-Padilla, 1999; McCune et al., 2000), but at present little is known about whether constitutively active native receptors have any physiological or pathological significance.

In previous articles (Noguera and D'Ocon, 1993; Noguera et al., 1996) we have suggested the existence of a population of constitutively active ₁-adrenoceptors in rat aorta and that some compounds traditionally used as antagonists, such as prazosin, WB 4101, and benoxathian really act as inverse agonists in this preparation. The experimental procedure that allows us to suggest the existence of this constitutive activity is a simple model in which, after depletion of intracellular calcium stores sensitive to noradrenaline, a spontaneous increase in the resting tone (IRT) of the aorta was obtained by incubation in a Ca²⁺-containing solution. This IRT does not depend on noradrenaline activation because the presence of the agonist is excluded but is selectively inhibited by the ₁-adrenoceptor antagonists cited above.

The present report deals with the analysis of this ₁-adrenoceptor constitutive activity in rat aorta, examining not only the contractile activity of this vessel but also the phosphoinositide hydrolysis as the intracellular signal linked to ₁-adrenoceptor stimulation. We also extend the study to another vessel, tail artery, to determine more about the physiological implications of the constitutive activity of ₁-adrenoceptors in the functionality of the cardiovascular system.

	Materials and Methods

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Contractile Studies. Rings of the thoracic aorta or tail artery (approximately 3-5 mm in length) of female Wistar rats (200-220 g) were denuded of endothelium by gentle rubbing and suspended in a 10-ml organ bath containing physiological solution, maintained at 37°C and gassed with 95% O₂ and 5% CO₂. An initial load of 1 g was applied to each preparation and maintained throughout a 75- to 90-min equilibration period. After this time, contractile responses to noradrenaline in Ca²⁺-containing or Ca²⁺-free solution were elicited according to the experimental procedure described in Fig. 1 (under Results). The pretension of 1 g was kept constant, but there was a loss of tension (<10-15%) when the preparations were placed in Ca²⁺-free medium. Tension was recorded isometrically by Grass FTO3 force-displacement transducers, and data were recorded on disc (MacLab). The absence of relaxant response (10%) after acetylcholine (100 µmol · l¹) addition to preparations precontracted with noradrenaline (1 µmol · l¹) indicated the absence of a functional endothelium in all the rings.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Experimental procedure designed to study the depletion of intracellular Ca2+ stores sensitive to NA in rat aorta (a) and tail artery (b) in Ca2+-free medium, and the IRT obtained in aorta by subsequent exposure to Ca2+-containing solution during the refilling of the noradrenaline-sensitive Ca2+ stores. Agonist was added in Ca2+-containing solution (Ca2+) and after W and recovery of the basal tone, the tissue was incubated for 20 min in Ca2+-free, EDTA-containing solution (Ca2+-free). After this time the agonist was applied (NA1, NA2) and washed until no contraction was induced, indicating complete depletion of internal Ca2+ stores sensitive to noradrenaline. The tissue was then incubated for 20 min in Krebs' solution and a spontaneous increase in the resting tone of aorta (not tail artery) was observed. After washing and 20 min of loading in Ca2+-free solution, a new addition of noradrenaline (NA3) was made. In the experiments designed to assess the effects of different agents on IRT, the aorta was pretreated with different concentrations of these agents 5 min before and during (20 min) the IRT was induced for second time.

Inositol Phosphate (IP) Determination. The determination of total inositol phosphate accumulation was adapted from Berridge et al. (1982). Briefly, rat thoracic aortae or tail arteries (four or five animals were sacrificed) were exposed to Ca²⁺-containing solution containing 1 µmol · l¹ of myo-[³H]inositol (specific activity 70.0 Ci · mmol¹) for 2 h at 37°C and gassed with 95% O₂ plus a 5% CO₂ mixture. After this incubation, the tissue was washed twice with physiological solution. The 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 (performed in triplicate), as are detailed under Results. In control experiments, tissues were incubated for 10 min with saline or prazosin (1 µmol · l¹) in Ca²⁺-containing solution and stimulated with noradrenaline (1 µmol · l¹ or 10 µmol · l¹ in aorta or tail samples, respectively) for 30 min. LiCl (10 mmol · l¹) was added 30 s before treatment 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). The samples were sonicated for 35 min at 2-3°C and, after addition of 0.63 ml of chloroform and 1.26 ml of distilled water, were centrifuged at 2500g 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 mmol · l¹ ammonium formate-5 mmol · l¹ sodium tetraborate to eliminate free myo-[³H]inositol and glycerophosphoinositol, respectively. Total IPs were eluted with 3 ml of 1 mol · l¹ ammonium formate-0.1 mol · l¹ formic acid. The eluent fractions were collected and counted in a scintillation counter. We normalized IP radioactivities in terms of free myo-[³H]inositol in void volume fractions in each experiment to correct for differences in the amount of tissue and myo-[³H]inositol labeling in different rings. The IP accumulation was expressed as percentage of basal release in each case and where ANOVA showed significant differences (P < .05), the results were further analyzed using the Student-Newman-Keuls test.

Chemicals. The following drugs were obtained from Sigma (St. Louis, MO): acetylcholine, ()-noradrenaline, prazosin, chloroethylclonidine, and lithium chloride, or Research Biochemicals International (Natick MA): BMY 7378 and 5-methylurapidil. myo-[³H]Inositol was from Amersham (Buckinghamshire, England). Other reagents were of analytical grade. All compounds were dissolved in distilled water.

	Results

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Contractile Studies in Rat Aorta or Tail Artery. Table 1 summarizes the results and Fig. 1 shows the experimental procedure designed to study the depletion of intracellular Ca²⁺ stores sensitive to noradrenaline and the IRT obtained by subsequent exposure to Ca²⁺-containing physiological solution during the refilling of these stores. Noradrenaline at 1 or 10 µmol · l¹ evoked a sustained contraction in rat aorta or tail artery, respectively, that was used as a control of the maximal response obtained with this agonist in each preparation. After careful washing, the return to the baseline was slower in aorta than in tail artery. Aorta takes 1191 ± 65 s (n = 15) to recovery the basal tone, whereas tail artery only takes 283 ± 16 s (n = 15) (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Time course of the decay in the maximal contractile response to noradrenaline after removal of the agonist in thoracic aorta and tail artery. The resting tone was measured 1, 3, 5, 8, 10, 15, and 20 min after noradrenaline removal. The decay of the IRT observed in aorta after depletion of intracellular calcium stores sensitive to noradrenaline and posterior loading in Ca2+-free medium was also measured at the times given above.

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View this table: [in this window] [in a new window]   TABLE 2 Comparison of pIC50 values of the agents tested with their published pKi values for 1-adrenoceptor subtypes Values are expressed as mean ± S.E.M. pIC50 values of the agents tested on the IRT of rat aorta and on noradrenaline-induced contraction in aorta and tail artery. pKi values for cloned and expressed 1-adrenoceptor subtypes.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Modification induced by different concentrations of BMY 7378 in the IRT obtained in aorta after the experimental procedure described in Fig. 1. a, representative tracings showing the IRT observed in absence (IRT 1) or presence (IRT 2) of different concentrations of BMY 7378. NA1 and NA2, 1 µmol · l1. b, concentration-response curve of inhibition by BMY 7378 of the IRT observed in rat aorta.

IP Determination. To find out whether the IRT observed in functional studies is really due to an activated state of _1D-adrenoceptors we tested the second messenger production, or IP formation, linked to activation of these receptors (Graham et al., 1996). In aorta and tail artery, noradrenaline concentration-dependently increased IP accumulation and the maximal response in both tissues was obtained with 10 µmol · l¹ noradrenaline (291.8 ± 23.2% related to basal release, n = 11 in aorta and 1318.2 ± 6.3%, n = 3 in tail artery). Prazosin at 1 µmol · l¹ inhibited the maximal accumulation of IP induced by noradrenaline in both tissues. When similar experiments were performed in absence of CaCl₂ in the incubating medium, the results obtained were identical (Fig. 4). When LiCl was not present during the incubation time in presence of noradrenaline, the accumulation of IP due to this agonist was not detectable (Fig. 4).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   IP accumulation in Ca2+-containing () and Ca2+-free medium (), obtained in thoracic aorta (a) and tail artery (b) after incubation with NA (10 µmol · l1), NA (10 µmol · l1) + prazosin (10 µmol · l1) (NA + P), or NA (10 µmol · l1) in absence of LiCl in the incubating medium [NA Li ()]. Results were expressed as a percentage with regard to the basal level in Ca2+-containing medium. Basal = 3573 ± 393 dpm · mg of protein1 in aorta (n = 5) and 5200 ± 295 dpm · mg of protein1 in tail artery (n = 6). ***P < .001, **P < .01 versus basal level in Ca2+-containing medium, Student-Newman-Keuls test.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   Description of the experimental procedure designed to determine IP accumulation under different experimental conditions in Ca2+-free medium: samples 1 to 9. NA1, first addition of noradrenaline (10 µmol · l1) in Ca2+- and Li+-free solution (5 min). W (10 min). NA2, second addition of noradrenaline (10 µmol · l1) in Ca2+- and Li+-free solution (5 min). A represents the addition of a concentration of antagonist in Ca2+-free, Li+-containing solution (10 min). NA3, addition of noradrenaline (10 µmol · l1) in Ca2+- and Li+-containing solution (30 min). All samples were incubated in Ca2+- and Li+-free solution (), and in samples 3 to 7 noradrenaline was added twice (NA1 and NA2) followed by Ws to promote depletion of calcium from internal stores. The exclusion of LiCl from the incubating medium permits IP formation due to noradrenaline activation but prevents it from accumulating. LiCl (10 mmol · l1) was then added to all samples (light gray bar) except sample 5, and A was added to samples 4, 7, and 9 for 10 min. Finally, 1.8 mM CaCl2 was added to samples 6 to 9 (), and noradrenaline (NA3) was included in samples 3 to 5 for 30 min.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Determination of the inositol phosphate accumulation obtained in thoracic aorta (a) and tail artery (b) incubated in Ca2+-free medium. Numbers 1 to 9 indicate different samples that receive different treatments according to the experimental procedure described in Fig. 4. Sample 3 represents noradrenaline-induced accumulation of IP. Sample 6 represents IP accumulation observed in absence of the agonist after depletion of internal calcium stores sensitive to noradrenaline and posterior loading in Ca2+-containing solution. This accumulation correlates with the IRT observed in contractile studies in thoracic aorta but not in tail artery (Fig. 1). In samples 4, 7, and 9 the antagonist tested was prazosin (10 µmol · l1). Values are expressed as percentages of the basal level obtained in Ca2+-containing solution. The discontinuous line represents this value. ***P < .001 versus basal level in Ca2+-containing medium, Student-Newman-Keuls test.

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View larger version (18K): [in this window] [in a new window]   Fig. 7.   Modification of the IP accumulation elicited by NA (10 µmol · l1) (a), and related to the IRT (b) observed in contraction studies after treatment for 40 min with prazosin (1 µmol · l1) (P), BMY 7378 (10 µmol · l1) (BMY), chloroethylclonidine (100 µmol · l1) (CEC), and 5-methylurapidil (10 µmol · l1) (5-M). Results are represented as a percentage of activation with regard to stimulation of NA or IRT. NA-induced accumulation of IP = 11619 ± 1272 dpm · mg of protein1. IRT-induced accumulation of IP = 7020 ± 312 dpm · mg of protein1. ***P < .001, *P < .05 versus noradrenaline-induced accumulation (a) or IRT (b), Student-Newman-Keuls test.

	Discussion

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The present results show that in rat aorta, noradrenaline, through activation of ₁-adrenoceptors, induces an IP accumulation that releases Ca²⁺ from internal stores (Berridge, 1992; Graham et al., 1996), and that these stores are depleted by successive additions of this agonist in a Ca²⁺-free medium. When emptied, the stores can be rapidly replenished by Ca²⁺ influx during the incubation in Ca²⁺-containing solution in the absence of the agonist (Putney, 1990; Noguera and D'Ocon, 1993; Noguera et al., 1996, 1997, 1998), and this process manifests itself not only by the recovery of the response to noradrenaline in Ca²⁺ free medium but also by the increase in the resting tone observed (IRT in Fig. 1).

That this IRT is closely related to ₁-adrenoceptors and not just to the emptying of intracellular Ca²⁺ pools is demonstrated by the fact that depletion of internal Ca²⁺ stores by methoxamine and phenylephrine also elicits an IRT, whereas clonidine, 5-hydroxytryptamine, caffeine, ryanodine, thapsigargine, and cyclopiazonic acid, which also depleted internal Ca²⁺ stores, did not elicit any IRT (Noguera and D'Ocon, 1993; Noguera et al., 1996, 1998).

If we assume that endogenous agonists are not present, the fact that this IRT was selectively inhibited by prazosin suggests the existence of a population of ₁-adrenoceptors in a constitutively active state, as we previously proposed (Noguera et al., 1996, 1998). The questions that arise from these results are as follows: Is this a general model that can be shown in different vascular smooth muscles? Which subtype of ₁-adrenoceptor is involved? Is the inositol phosphate accumulation implicated in this process? Can we say that we are dealing with constitutive ₁-adrenoceptor activity? and What is the role of this process in the functionality of a vessel?

To answer the first question, we have analyzed this model in another vascular tissue, rat tail artery. The experimental procedure was the same as the one used in aorta, but the results were not similar. After depletion of internal Ca²⁺ stores sensitive to noradrenaline, no increase in the resting tone was observed (Fig. 1), which means that the IRT found in aorta is specifically related to the ₁-adrenoceptors present in this tissue.

Rat tail artery contracts in response to noradrenaline via activation of at least two adrenoceptor subtypes, one of which displays the pharmacology of the _1A-adrenoceptor (Villalobos-Molina and Ibarra, 1996; Lachnit et al., 1997; Mita and Walsh, 1997). Our results confirm participation of _1A-adrenoceptors in the contractile response to noradrenaline in this vessel but also show that _1D-adrenoceptor is not involved in the response. In any case, the _1A-adrenoceptor or the undefined one has not shown constitutive activity in our experimental conditions.

The subtype(s) of ₁-adrenoceptors present in the rat thoracic aorta has been the subject of extensive research (Hieble et al., 1995). Finally, a selective _1D-antagonist BMY 7378 was described, and it was demonstrated that in the rat aorta, the functional activity of this antagonist correlates well with binding affinities for cloned _1D-adrenoceptors (Kenny et al., 1995; Saussy et al., 1996; Hussain and Marshall, 1997). This suggests that the _1D-adrenoceptor plays a functional role in this tissue without excluding participation of the other subtypes. Therefore, the subtype of ₁-adrenoceptor that shows constitutive activity in our experimental model could be the _1D-adrenoceptor.

To confirm this hypothesis, we assayed the activity of three antagonists acting selectively on ₁-adrenoceptor subtypes: BMY 7378, which, as has been mentioned before, acts on the _1D-subtype; 5-methylurapydil, which acts on the _1A-subtype; and chloroethylclonidine, an irreversible antagonist of the _1B- and _1D-subtypes (Hieble et al., 1995; Schwinn et al., 1995).

The results obtained with the three compounds assayed confirm that the population of ₁-adrenoceptors that intervenes in the functional response of rat aorta to noradrenaline belongs, at least in part, to the _1D-subtype. Moreover, the present results show that the IRT that we attribute to the constitutive activity of ₁-adrenoceptors is selectively blocked by BMY 7378, and this compound's potency as an inhibitor of the IRT correlates well with its affinity estimated at the cloned _1D-subtype. This confirms the hypothesis that this subtype of ₁-adrenoceptor can show constitutive activity in our model and makes it clear that BMY 7378 acts as an inverse agonist. 5-Methylurapydil also acts as an inverse agonist, with an inhibitory potency on IRT that correlates well with its affinity estimated at the cloned _1D-subtype and that is lower than the potency shown at the _1A-adrenoceptor subtype. Chloroethylclonidine lacks activity on IRT but inhibits in a concentration-dependent manner the noradrenaline-induced contraction, thus suggesting that it does not act on _1D-adrenoceptors as an inverse agonist but as a neutral antagonist.

In response to the second question, the present results demonstrate the existence of a mechanical response of rat aorta after depletion of internal Ca²⁺ stores sensitive to ₁-adrenoceptor activation that can be interpreted as the first functional evidence of the constitutive activity of native _1D-adrenoceptors in vascular tissues. Recently, in rat-1 fibroblasts stably expressing _1D-adrenoceptors spontaneous ligand-independent activity has been shown (García-Sainz and Torres-Padilla, 1999; McCune et al., 2000), confirming our previous and present results in native receptors (Noguera et al., 1996, 1998).

The next question to analyze is the intervention of IP accumulation in this process as an intracellular signal of receptor activation. Experiments were performed to mimic the procedure used in the organ bath experiments, and the results obtained give clear evidence of the existence of IP accumulation after depletion of intracellular calcium stores sensitive to noradrenaline, in absence of the agonist and when calcium was added again to the incubation medium (Fig. 5, sample 6). This IP accumulation can be inhibited by prazosin, BMY 7378, and 5-methylurapidil, but chloroethylclonidine does not inhibit it. This is consistent with the observations in organ bath experiments and shows the dependence of the signal on ₁-adrenoceptor activation. Moreover, if internal calcium stores sensitive to noradrenaline were not previously depleted, addition of calcium to the incubation medium only slightly increased the level of IP, and this slight increase, which corresponds in magnitude to the decrease observed when calcium was removed from the medium, is not inhibited by prazosin. The fact that BMY 7378 and 5-methylurapidil, which inhibit IRT in contractile studies, also inhibit this IP response, and that chloroethylclonidine does not inhibit IRT or IP formation indicates a close relationship between the two signals.

In conclusion, the IRT observed in contractile experiments and the IP accumulation related to it can be inhibited by prazosin, BMY 7378, and 5-methylurapidil in conditions in which the presence of exogenous noradrenaline can be ruled out. This observation strongly suggests the existence of a population of _1D-adrenoceptors with constitutive activity. Prazosin, BMY 7378, and 5-methylurapidil behave as "inverse agonists", as has been previously proposed for prazosin (Noguera et al., 1996) and 5-methylurapidil (Lee et al., 1997). Chloroethylclonidine, which inhibits the noradrenaline-induced functional response in both systems (contraction and IP formation), did not affect the IRT or the correlated IP accumulation. This compound did not show inverse agonist activity but these results then demonstrate that endogenous noradrenaline is also not involved in this process. Excluding the participation of an agonist, the model could be considered representative of the functional behavior of a population of constitutively active _1D-adrenoceptors.

Moreover, the data provided in the present study suggest a possible answer to our final question about the physiological role of this constitutive activity of _1D-adrenoceptors. Figure 1 and the results summarized in Fig. 2 show that if we compare the noradrenaline-induced contractile responses in aorta and tail artery in Ca²⁺-containing solution, we can observe that after removing the agonist, contraction disappears in aorta as slowly as IRT decreases, but that in tail artery the decay of the response to noradrenaline is faster. These results can be interpreted as be due to differences in histology or lipid content between the two vessels but, from our observation about IRT we could also extrapolate that in physiological conditions, after noradrenaline activity and removal, a population of _1D-adrenoceptors could remain temporally in a constitutively active state and could be responsible for the slow disappearance of the contractile response to the agonist. This mechanism is not observed in tail artery, where _1D-adrenoceptors do not seem to play a functional role. Therefore, the presence of a population of _1D-adrenoceptors in a vessel can signify that the contractile responses of this tissue can be sustained even when the agonist is removed, and this would in turn modulate the contractile activity in this vessel, thus preventing abrupt changes in caliber when the agonist disappears. In contrast, an imbalance in this modulating mechanism could give rise to pathologies as hypertension or age-related vascular diseases, in which a possible role of _1D-adrenoceptors in their pathogenesis and/or maintenance has been postulated (Villalobos-Molina and Ibarra, 1996; Villalobos-Molina et al., 1999; Ibarra et al., 1997, 1998; Xu et al., 1998). We are currently investigating this exciting hypothesis.

Further studies are needed to determine the importance of this phenomenon in the contractile response to agonists in physiological or pathological situations and in different vascular beds, but our observations could explain why different ₁-adrenoceptor subtypes are present in different vessels and indicate that they are involved in the different tissue functions.