Abstract
After depletion of intracellular calcium stores sensitive to noradrenaline, a spontaneous increase in the resting tone (IRT) when incubated in Ca2+-containing solution was observed in isolated rat aorta, but not in tail artery. This IRT does not depend on agonist activation of α1-adrenoceptors but it is inhibited by prazosin. A close relationship was found between the inhibitory potencies of prazosin (pIC50 = 9.833), BMY 7378 (pIC50 = 8.924), and 5-methylurapidil (pIC50 = 7.883) against IRT and their affinities for cloned α1D-adrenoceptors. Chloroethylclonidine (100 μmol · l−1) did not inhibit the IRT. After depletion of internal calcium stores by noradrenaline in absence of the agonist, loading in Ca2+-containing solution also brings about an increase in the inositol phosphate (IP) levels in rat aorta (not seen in tail artery) that is inhibited by prazosin (1 μmol · l−1), BMY 7378 (10 μmol · l−1), and 5-methylurapidil (10 μmol · l−1), thus confirming the results obtained in contractile studies. Chloroethylclonidine (100 μmol · l−1) did not inhibit this IP accumulation. The fact that the IRT and the IP accumulation related to it can be selectively inhibited by different α1-adrenoceptor antagonists suggests the existence of a population of α1D-adrenoceptors that show constitutive activity in rat aorta, not in tail artery.
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; Garcı́a 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 α1-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 α1-adrenoceptor antagonists cited above.
The present report deals with the analysis of this α1-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 α1-adrenoceptor stimulation. We also extend the study to another vessel, tail artery, to determine more about the physiological implications of the constitutive activity of α1-adrenoceptors in the functionality of the cardiovascular system.
Materials and Methods
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 · l−1) addition to preparations precontracted with noradrenaline (1 μmol · l−1) indicated the absence of a functional endothelium in all the rings.
The composition of the physiological Ca2+-containing solution was as follows: 118 mM NaCl, 4.75 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11 mM glucose. Ca2+-free solution had the same composition except that CaCl2 was omitted and EDTA (0.1 mM) was added.
Contractions in Ca2+-containing solution were expressed in milligrams of developed tension and, when elicited in Ca2+-free medium, as a percentage of the noradrenaline-induced contractions obtained in Ca2+-containing solution. Increases in resting tone were also expressed as a percentage of the noradrenaline-induced contraction in Ca2+-containing solution. The concentration (−log [M]) needed to produce 50% relaxation or inhibition (pIC50) was obtained from a nonlinear regression plot (GraphPad Software; San Diego, CA). It was impossible to calculate the S.E. of the mean of the pIC50values for antagonists relative to the inhibition of the IRT. The other results are presented as the mean ± S.E. for ndeterminations obtained from different animals.
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 · l−1 of myo-[3H]inositol (specific activity 70.0 Ci · mmol−1) 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 · l−1) in Ca2+-containing solution and stimulated with noradrenaline (1 μmol · l−1 or 10 μmol · l−1 in aorta or tail samples, respectively) for 30 min. LiCl (10 mmol · l−1) 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 2500gfor 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 · l−1ammonium formate-5 mmol · l−1 sodium tetraborate to eliminate free myo-[3H]inositol and glycerophosphoinositol, respectively. Total IPs were eluted with 3 ml of 1 mol · l−1 ammonium formate-0.1 mol · l−1 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
Contractile Studies in Rat Aorta or Tail Artery.
Table1 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 · l−1 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).
We then changed to a Ca2+-free solution and after 20 min in this medium, the addition of noradrenaline also induced a contraction (NA1, Table 1; Fig. 1) that was used as an index for the content of agonist-sensitive intracellular stores. No contraction was evoked upon a second application of the agonist (NA2, Fig. 1) in the same solution, which indicates complete depletion of internal Ca2+ stores sensitive to noradrenaline. The tissue was then incubated for 20 min in Ca2+-containing solution to refill the intracellular Ca2+ stores, and a spontaneous increase in the resting tone (IRT = 53.3 ± 4.9% of noradrenaline control) was observed in rat aorta but not in tail artery, whereas only a slight increase in the baseline was obtained (8.8 ± 2.1% of the control response to noradrenaline).
The IRT observed in aorta is not sustained, and takes 1057 ± 54 s to reach the baseline (n = 15). It decreases as slowly as the control response to noradrenaline in Ca2+-containing solution disappears after washing (Fig. 2). Returning the tissues to a Ca2+-free solution reduced the tension to baseline, and further application of noradrenaline (NA3) 20 min later reproduced the contractile response elicited first in Ca2+-free solution, which indicates a complete refilling of internal stores.
Concentration-response curves of relaxation to prazosin (0.001 nmol · l−1–1 μmol · l−1), BMY 7378 (0.001 nmol · l−1–1 μmol · l−1), 5-methylurapidil (0.001 nmol · l−1–10 μmol · l−1), or chloroethylclonidine (0.001 μmol · l−1–100 μmol · l−1) were obtained by addition of cumulative concentrations of the compounds to tissues in which sustained contractions had been induced by maximal concentrations of noradrenaline (1 μmol · l−1 in rat aorta and 10 μmol · l−1 in tail artery). Relaxations were expressed as a percentage of the maximum increment of tension obtained by agonist addition and the pIC50 of relaxation obtained for each antagonist on aorta or tail artery are summarized in Table 2. Concentration-response curves of inhibition to the same compounds were obtained by addition of concentrations of antagonist 15 min before and during the loading period in Ca2+-containing solution that permits the refilling of internal Ca2+ stores previously depleted by noradrenaline (Fig. 1). The magnitude of the IRT observed in rat aorta during this period in presence of each concentration of antagonist (Fig. 3) was expressed as a percentage of the reference IRT obtained in absence of any agent and the pIC50 calculated was also summarized in Table2. In this case, chloretylclonidine at the higher concentration assayed (100 μmol · l−1), which completely relaxed noradrenaline-induced contraction of rat aorta, had no effect on IRT. Moreover, when rings of rat aorta were exposed to chloroethylclonidine (100 μmol · l−1) for 30 min and then washed for 20 min to remove the antagonist an IRT was observed of similar magnitude (n = 5) with respect to the reference IRT obtained in absence of any agent. When we compare the pIC50 obtained for each antagonist on IRT or noradrenaline-induced contractile response in aorta and tail artery with the pKi obtained in competition experiments on cloned α1-adrenoceptors ( Kenny et al., 1995; Schwinn et al., 1995), we can observe a close relationship between the results obtained on IRT in aorta and cloned α1D-adrenoceptors, or between tail artery and cloned α1A-adrenoceptors (Table 2). The low affinity of BMY 7378 excludes participation of α1D-adrenoceptors in the functional response of the tail artery to noradrenaline.
IP Determination.
To find out whether the IRT observed in functional studies is really due to an activated state of α1D-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 · l−1 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 · l−1 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).
A new experimental procedure designed by us attempts to reproduce the conditions developed in contractile studies. After incubation for 2 h in physiological solution containing myo-[3H]inositol, tissues were placed in individual tubes with physiological solution free of CaCl2 for 20 min. We prepared 10 different samples, each of which was subjected to the different experimental conditions that are summarized in Fig.5.
The samples numbered 1 and 2 in Fig. 5 were used as controls to determine the influence on the IP accumulation of the incubation time in Ca2+-free medium (90 min total, sample 1) or successive washings (W) during this incubation time (sample 2). The results obtained showed that the level of IP was slightly decreased by these parameters (Fig. 6).
The samples numbered 3 to 5 (Fig. 5) were used as controls of the IP formation induced by noradrenaline after two successive additions and washings of noradrenaline in Ca2+-free medium. For this purpose, the samples were incubated for 5 min in presence of noradrenaline (NA1) and washed for 10 min before a new incubation period (5 min), also in presence of noradrenaline (NA2). This was followed by a second washing (10 min). Finally, LiCl was added to samples 3 and 4 but not to 5. An antagonist (A), prazosin at 1 μmol · l−1, was also added to sample 4. Ten minutes later, noradrenaline was added again to the three samples for 30 min. The incubation was then stopped and the IP formation determined. The results obtained were similar to the control response to noradrenaline in Ca2+-containing medium (Fig.6). Prazosin also inhibits the IP formation elicited by noradrenaline in these conditions and, as has been previously shown in Ca2+-containing medium, IP accumulation cannot be detected in the sample that does not include LiCl.
Samples 6 to 7 represent an attempt to reproduce the experimental procedure used in contraction studies in which IRT was observed. Sample 6 was incubated in presence of noradrenaline two times (5 min each with 10-min washing) before LiCl was added and incubation prolonged 10 min more. Finally, CaCl2 was added during the last 30 min as in the contraction studies. The results obtained are summarized in Fig. 6 and show that after depletion of the intracellular Ca2+ stores by addition of noradrenaline in a Ca2+-free medium, when CaCl2 was included in the incubating solution, a significant increase in the IP accumulation was detected (sample 6 versus 2) in aorta, but not in tail artery, that reproduced the IRT observed in the contraction studies. This accumulation was inhibited by addition of 1 μmol · l−1 prazosin (sample 7 versus 6), as also occurs in the contraction studies.
To clarify the role of the depletion of intracellular Ca2+ stores and/or Ca2+entry in this process, samples 8 and 9 included CaCl2 in the last 30 min but without previous depletion of intracellular Ca2+ stores sensitive to noradrenaline. The results obtained indicate that on changing the tissues from an incubating medium free of Ca2+ to a Ca2+-containing one, a slight increase in the basal formation of IP is observed, but this increase is not inhibited by prazosin (Fig. 6). The magnitude of the increase when Ca2+ was added correlates well with the slight decrease previously observed in IP formation when Ca2+ was eliminated (Fig. 6, samples 1 and 8 versus control 100%).
To analyze the activity of the selective α1-adrenoceptor antagonists on this IP accumulation observed in absence of agonist, similar experiments were performed to test BMY 7378 (10 μmol · l−1), 5-methylurapidil (10 μmol · l−1), and chloroethylclonidine (100 μmol · l−1) on this accumulation as well as the activity of these compounds on noradrenaline-induced IP formation in Ca2+-free medium. The results obtained indicate that BMY 7378 and 5-methylurapidil inhibit both noradrenaline-induced IP accumulation (Fig. 7a) and the increase in the IP levels observed in absence of the agonist (Fig. 7b). Chloroethylclonidine, which inhibits the noradrenaline-induced IP signal, did not modify the IP accumulation observed in absence of agonist (Fig. 7).
Discussion
The present results show that in rat aorta, noradrenaline, through activation of α1-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 α1-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 α1-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 α1-adrenoceptor is involved? Is the inositol phosphate accumulation implicated in this process? Can we say that we are dealing with constitutive α1-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 α1-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 α1A-adrenoceptor (Villalobos-Molina and Ibarra, 1996; Lachnit et al., 1997; Mita and Walsh, 1997). Our results confirm participation of α1A-adrenoceptors in the contractile response to noradrenaline in this vessel but also show that α1D-adrenoceptor is not involved in the response. In any case, the α1A-adrenoceptor or the undefined one has not shown constitutive activity in our experimental conditions.
The subtype(s) of α1-adrenoceptors present in the rat thoracic aorta has been the subject of extensive research (Hieble et al., 1995). Finally, a selective α1D-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 α1D-adrenoceptors (Kenny et al., 1995; Saussy et al., 1996; Hussain and Marshall, 1997). This suggests that the α1D-adrenoceptor plays a functional role in this tissue without excluding participation of the other subtypes. Therefore, the subtype of α1-adrenoceptor that shows constitutive activity in our experimental model could be the α1D-adrenoceptor.
To confirm this hypothesis, we assayed the activity of three antagonists acting selectively on α1-adrenoceptor subtypes: BMY 7378, which, as has been mentioned before, acts on the α1D-subtype; 5-methylurapydil, which acts on the α1A-subtype; and chloroethylclonidine, an irreversible antagonist of the α1B- and α1D-subtypes (Hieble et al., 1995; Schwinn et al., 1995).
The results obtained with the three compounds assayed confirm that the population of α1-adrenoceptors that intervenes in the functional response of rat aorta to noradrenaline belongs, at least in part, to the α1D-subtype. Moreover, the present results show that the IRT that we attribute to the constitutive activity of α1-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 α1D-subtype. This confirms the hypothesis that this subtype of α1-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 α1D-subtype and that is lower than the potency shown at the α1A-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 α1D-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 α1-adrenoceptor activation that can be interpreted as the first functional evidence of the constitutive activity of native α1D-adrenoceptors in vascular tissues. Recently, in rat-1 fibroblasts stably expressing α1D-adrenoceptors spontaneous ligand-independent activity has been shown (Garcı́a-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 α1-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 α1D-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 α1D-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 α1D-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 α1D-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 α1D-adrenoceptors do not seem to play a functional role. Therefore, the presence of a population of α1D-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 α1D-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 α1-adrenoceptor subtypes are present in different vessels and indicate that they are involved in the different tissue functions.
Footnotes
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Send reprint requests to: Pilar D'Ocon, Departamento de Farmacologı́a, Facultad de Farmacia, Universitat de València, Avda Vicent Andres estelles s/n 46100 Burjassot, València, Spain. E-mail: m.pilar.docon{at}uv.es
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↵1 This work was supported by a research grant from the Spanish Comisión Interministerial de Ciencia y Tecnologı́a (SAF98-0123).
- Abbreviations:
- IRT
- increase in the resting tone
- IP
- inositol phosphate
- NA
- noradrenaline
- W
- washing
- A
- antagonist
- Received April 4, 2000.
- Accepted August 1, 2000.
- The American Society for Pharmacology and Experimental Therapeutics