Vol. 299, Issue 2, 729-734, November 2001

Functional Characterization of ₁-Adrenoceptor Subtypes in Human Subcutaneous Resistance Arteries

Vascular Assessment Unit, School of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, Scotland, United Kingdom (Y.P.R.J., F.J., A.M., C.H.); Western Infirmary, Glasgow, Scotland, United Kingdom (C.B., A.R.); and Autonomic Physiology Unit, Institute of Biomedical and Life Sciences, Glasgow University, Glasgow, Scotland, United Kingdom (J.C.M.)

	Abstract

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The functional characteristics of the ₁-adrenoceptor subtypes in human resistance arteries are still not clear. We recently reported that the _1A-adrenoceptor predominantly mediates contraction to norepinephrine in human skeletal muscle resistance arteries. In this study we extended these investigations to human subcutaneous resistance arteries. Arterial segments were isolated from the inguinal subcutaneous fat and mounted on a small vessel wire myograph. Potencies of agonists and antagonists were examined. N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulphonamide (A-61603) was found to be 10- and 54-fold more potent than norepinephrine and phenylephrine, respectively. Brimonidine (UK 14304) evoked significantly smaller contractile responses than norepinephrine and phenylephrine, showing the presence of a small population of ₂-adrenoceptors in these arteries, and this was confirmed by the studies with selective ₁- and ₂-adrenoceptor antagonists prazosin and (8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13a-decahydro-3-methoxyl-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]-naphthyridine (RS 79948). Prazosin, 5-methyl-urapidil, and 2-[2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane (WB 4101) shifted the potency of norepinephrine concentration dependently giving pA₂ values of 9.4, 8.9, and 10.1, respectively, showing the presence of the _1A-subtype in these arteries. Pretreatment with 1 and 10 µM chloroethylclonidine did not affect the potency of and maximum responses to norepinephrine, ruling out the presence of the _1B-subtype in these arteries. 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY 7378, 10 and 100 nM) did not affect the potency of norepinephrine but a small shift was observed by 1 µM BMY 7378, giving a pK_B value of 7.1, much less than that reported for the _1D-subtype. These results suggest the predominant involvement of _1A-adrenoceptor in the contractile responses to norepinephrine in these arteries. The physiological role of this subtype in the maintenance of peripheral arterial resistance is yet to be confirmed.

	Introduction

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Vascular postjunctional ₁-adrenoceptors play a primary role in the maintenance of peripheral vascular resistance and therefore in control of systemic arterial pressure. It is now well accepted that there are three functional ₁-adrenoceptor subtypes _1A, _1B, and _1D corresponding to the three cloned ₁-adrenoceptors, designated as _1a, _1b, and _1d (Hieble et al., 1995; Bylund et al., 1998). These three subtypes are characterized by high affinity for prazosin in functional and radioligand binding studies. ₁-Adrenoceptors with low affinity for prazosin (pA₂ < 9) have also been identified in functional studies and classified as either _1L- or _1N-based on either a low affinity or high affinity to HV 723, respectively (Flavahan and Vanhoutte, 1986; Muramatsu et al., 1990).

The relative functional importance of the subtypes in the human vascular system is not clear. Molecular biological studies provide evidence for the presence of mRNA for all the three subtypes in different conduit vessels from humans, but the functional expression was found to be limited to either one or in a few instances two of these subtypes (Hatano et al., 1994; Shibata et al., 1998; Rudner et al., 1999; Moriyama et al., 2000). Relatively little is known about the ₁-adrenoceptor subtypes present in human resistance arteries. We recently reported the predominant involvement of _1A-adrenoceptor in norepinephrine-mediated contractile responses in human skeletal muscle resistance arteries (Jarajapu et al., 2001a). In the present study we extended these investigations to the resistance arteries from the human subcutaneous vascular bed, another major vascular bed with a large contribution to peripheral vascular resistance.

The potencies of the agonists norepinephrine (nonselective), phenylephrine (₁-selective), brimonidine (UK 14304, ₂-selective) (Cambridge, 1981), and A-61603 (_1A-selective) (Knepper et al., 1995) were determined. The affinities of the reversible competitive antagonists prazosin (₁-selective) (Cambridge et al., 1977), 5-methyl-urapidil (_1A-selective) (Gross et al., 1988), WB 4101 (selective for _1A- and _1D-subtypes) (Morrow and Creese, 1986; Kenny et al., 1995), and BMY 7378 (_1D-selective) (Goetz et al., 1995) were examined. Inactivation by the _1B-adrenoceptor alkylating agent chloroethylclonidine (Han et al., 1987) was also evaluated.

	Materials and Methods

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Preparation of Human Subcutaneous Resistance Arteries. Biopsies of subcutaneous fat from inguinal areas from subjects who were undergoing inguinal hernia operations were collected in physiological saline solution (PSS; see below for composition) and transported to the laboratory under ice-cold conditions. Resistance arteries (normalized diameter 300 ± 8 microns; n = 85/31; no. of arterial segments/no. of subjects) were dissected out under a microscope (Zeiss Welwyn, Garden City, UK) within an hour. All the subjects were male and were aged from 45 to 70 years. None of the patients had any underlying disease such as diabetes or hypertension. The study was approved by the appropriate ethical committee and all the subjects gave informed consent.

Small Vessel Wire Myography. Arterial segments of 2-mm length were mounted in a four-channel small vessel wire myograph (Danish Myotech, Aarhus, Denmark) for isometric tension measurements. The vessel segments were incubated in PSS of the following composition 119 mM NaCl, 4.5 mM KCl, 25 mM NaHCO₃, 1 mM KH₂PO₄, 1 mM MgSO₄(7H₂O), 11 mM (+)-glucose, and 2.5 mM CaCl₂, at 37°C and gassed with carbogen. One hour after mounting, the resting tension  internal circumference relation was determined for each vessel segment (Mulvany and Halpern, 1977). The resting tension was then set to a normalized internal circumference of L_0.9 where L_0.9 = 0.9L₁₀₀ and L₁₀₀ is the internal circumference that the vessel would have under a transmural pressure of 100 mm Hg (13.3 kPa). The software program Myodaq-Myodata (Danish Myoteq, Aarhus, Denmark) was used for data acquisition. Subsequently, vessel viability was checked by exposure to high-potassium solution (123 mM) twice and then to 10 µM norepinephrine in the presence of high-potassium solution. Arterial segments were considered viable if they produced an effective pressure of more than 100 mm Hg (13.3 kPa) when stimulated with 123 mM KCl. Effective pressure was calculated from the Laplace equation as follows:
which corrects for differences in length and diameter of arterial segments (Mulvany and Halpern, 1977). All the vessels were found to be viable according to this criterion. The presence of functional endothelium was checked with 1 µM carbachol after precontracting with 1 µM norepinephrine. All the vessels in the study produced >60% relaxation.

After an equilibration period of 1 h, two to four concentration-response curves (CRCs) were obtained in each arterial segment. No significant changes in maximum responses (percentage of CRC1 maximum: CRC2, 103 ± 6; CRC3, 101 ± 11; and CRC4, 99 ± 12, n = 5) or pEC₅₀ values (CRC1, 6.6 ± 0.1; CRC2, 7.0 ± 0.1; CRC3, 6.7 ± 0.2; and CRC4, 6.7 ± 0.2, n = 5) of norepinephrine were observed, showing that repeated CRCs were reproducible and no corrections for time-dependent changes were required. The first CRC was taken as control and the subsequent CRCs were obtained after incubating the arterial segments for 30 min with antagonists at different concentrations. In the experiments with chloroethylclonidine the arterial segments were exposed to chloroethylclonidine (1 or 10 µM) for 30 min and then washed for 60 min (three times every 15 min) (Hancock, 1996). Propranolol (1 µM), cocaine (3 µM), and corticosterone (3 µM) were added to the PSS when CRCs to norepinephrine were obtained (to block -adrenoceptors, and neuronal and non-neuronal uptake of norepinephrine, respectively). EDTA (0.023 mM) and ascorbic acid (0.3 mM) were included in the PSS to prevent oxidation of norepinephrine. RS 79948 (0.1 µM), a selective ₂-adrenoceptor antagonist (Brown et al., 1993; Uhlen et al., 1998) was present in PSS during the experimental protocols for ₁-adrenoceptor subtype characterization.

Results are expressed as mean ± S.E.M where n is the number of subjects. Agonist potency is expressed as pEC₅₀ (the negative logarithm of the concentration required to produce 50% of the maximum response). pEC₅₀ values and maximum responses were calculated using the software program GraphPad Prism (GraphPad Software, San Diego, CA), which fits CRCs to the four-parameter logistic equation given below:
where X is the logarithm of the molar concentration of the agonist, Y is the response and P is the Hill slope. Antagonist affinities are expressed either as pK_B or pA₂ values. pK_B was used when one concentration of the antagonist was used to obtain the affinity and calculated using the following equation (Schild, 1949):
where K_B is the dissociation constant, [B] is the molar concentration of the antagonist, and r is the ratio of EC₅₀ of the agonist in the presence of the antagonist to that in the absence. pA₂ values were obtained when three different concentrations of the antagonist were used. These values were obtained from the x-intercept of the plot of log(r  1) and log[B] (Arunlakshana and Schild, 1959) after linear regression, using GraphPad prism.

Drugs. ()-Norepinephrine (arterenol) bitartrate, brimonidine (UK 14304), propranolol hydrochloride, corticosterone acetate, WB 4101, and prazosin HCl were obtained from Sigma Chemical (Poole, Dorset, UK); cocaine HCl was obtained from Thornton and Ross Ltd. (Huddersfield, UK); RS 79948 and A-61603 were obtained from Tocris (Avonmouth, Bristol, UK); 5-methyl-urapidil, chloroethylclonidine 2HCl, and BMY 7378 were obtained from Sigma/RBI (Natick, MA). The stock solution of 5-methyl-urapidil was prepared in 5% dimethyl sulfoxide and that of corticosterone acetate was prepared in 25% absolute ethanol. Stock solutions of all the other drugs were prepared in distilled water. PSS containing 123 mM KCl was prepared by replacing NaCl with an equimolar quantity of KCl.

Statistics. pEC₅₀ values and maximum responses were compared by using paired t test or two-way analysis of variance followed by the Newman-Keuls range test for multiple comparisons. Confidence limits (CLs) were obtained from GraphPad prism.

	Results

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Contractile Responses to Different Adrenoceptor Agonists in Human Subcutaneous Resistance Arteries. Norepinephrine, phenylephrine, brimonidine, and A-61603 produced concentration-dependent contractile responses in the human subcutaneous resistance arteries (Fig. 1). Maximum responses, expressed as percentage of KCl (123 mM) response, produced by norepinephrine (96 ± 6), phenylephrine (87 ± 7), and A-61603 (98 ± 8) were not significantly different but significantly higher than that of brimonidine (33 ± 4; P < 0.01). Higher pEC₅₀ values were observed with A-61603 (7.8 ± 0.1) compared with norepinephrine (6.8 ± 0.1; P < 0.01) and phenylephrine (6.1 ± 0.1; P < 0.01). A-61603 was found to be 10 and 54 times more potent than norepinephrine and phenylephrine, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Contractile responses to the agonists A-61603 (n = 7), norepinephrine (n = 10), phenylephrine (n = 10), and brimonidine (n = 10) in human subcutaneous resistance arteries.

Effect of Selective ₁- and ₂-Adrenoceptor Antagonists on Contractile Responses to Norepinephrine, Phenylephrine, and Brimonidine in Human Subcutaneous Resistance Arteries. The selective ₂-adrenoceptor antagonist RS 79948 (0.1 µM) decreased the sensitivity of norepinephrine by 11-fold (Fig. 2a). Prazosin (0.1 µM) produced a nonparallel rightward shift of the CRC to norepinephrine, resulting in a biphasic CRC (Fig. 2a). Further addition of RS 79948 (0.1 µM) shifted the lower part of the CRC, making it parallel to that of the control (Fig. 2a). In the presence of 0.1 µM RS 79948, prazosin (0.1 µM) produced a further parallel shift of the CRC to norepinephrine by 611-fold, giving a pK_B value of 9.7. The sensitivity of phenylephrine was not affected by RS 79948 (0.1 µM) but was shifted by prazosin by 193-fold, giving a pK_B value of 9.3 (Fig. 2b).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Antagonism of contractile responses to norepinephrine (n = 6) (a), phenylephrine (n = 3) (b), and brimonidine (n = 4-8) (c) by RS 79948, prazosin, and their combination in human subcutaneous resistance arteries.

CRCs to brimonidine were obtained only in the concentration range of 1 nM to 30 µM. Exact EC₅₀ values could not be obtained in the presence of antagonists because the CRCs did not achieve the maximum responses in the concentration range of the agonist used. Prazosin (0.1 µM) had little effect on the lower part of the CRC to brimonidine but reduced responses to higher concentrations of brimonidine (Fig. 2c). RS 79948 (0.1 µM) produced an approximately 200-fold shift in the sensitivity of brimonidine. Further addition of prazosin (0.1 µM) reduced responses to only the highest concentrations of brimonidine used (30 µM) (percentage of control maximum responses: RS 79948 (0.1 µM), 66 ± 13 and RS 79948 (0.1 µM) + prazosin (0.1 µM), 32 ± 7; P < 0.05).

Affinities of Nonselective and Subtype-Selective ₁-Adrenoceptor Antagonists in Human Subcutaneous Resistance Arteries. Prazosin produced concentration-dependent parallel rightward shifts in the sensitivity of norepinephrine (Fig. 3a) without significantly affecting the maximum responses. The Schild regression analysis (Fig. 3b) gave a pA₂ value of 9.4 with a slope of 1.1 (95% CL, 0.8-1.4).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   a, antagonism of norepinephrine-mediated contractile responses by prazosin in human subcutaneous resistance arteries (n = 10). b, Schild plot for the antagonism of norepinephrine-mediated contractile responses by prazosin (n = 30).

5-Methyl-urapidil antagonized norepinephrine-mediated contractile responses concentration dependently (Fig. 4a) without affecting the maximum responses. Schild regression analysis (Fig. 4b) gave a pA₂ value of 8.9 with a slope of 1.0 (95% CL, 0.7-1.2). Incubation of arterial segments with 5-methyl-urapidil for 30 min did not increase the basal tension ruling out a possible agonist effect on 5-hydroxytryptamine_1A receptors (Schoeffter and Hoyer, 1988).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   a, antagonism of norepinephrine-mediated contractile responses by 5-methyl-urapidil in human subcutaneous resistance arteries (n = 10). b, Schild plot for the antagonism of norepinephrine-mediated contractile responses by 5-methyl-urapidil (n = 30).

WB 4101 produced concentration-dependent rightward shifts in the sensitivity to norepinephrine (Fig. 5a) without affecting the maximum responses. Schild regression analysis (Fig. 5b) gave a pA₂ value of 10.1 with a slope of 0.8 (95% CL, 0.5-1.2).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   a, antagonism by WB 4101 of norepinephrine-mediated contractile responses in human subcutaneous resistance arteries (n = 6). b, Schild plot for the antagonism by WB 4101 by norepinephrine-mediated contractile responses (n = 18).

Neither the sensitivity nor the maximum contractile responses to norepinephrine was affected by 1 and 10 µM chloroethylclonidine (Fig. 6). Incubation of arterial segments with chloroethylclonidine had no effect on baseline tension, ruling out any agonist action at ₁-adrenoceptors (Docherty and O'Rourke, 1997; Ibarra et al., 2000). The potency of norepinephrine was not affected by 0.1 and 1 nM BMY 7378 but decreased 11-fold by 1 µM BMY 7378, giving a pK_B of 7.1 ± 0.3 (Fig. 7).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of pretreatment with chloroethylclonidine on norepinephrine-mediated contractile responses in human subcutaneous resistance arteries (n = 7).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Antagonism of norepinephrine-mediated contractile responses by BMY 7378 in human subcutaneous resistance arteries (n = 7).

	Discussion

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The present study shows that the contractile responses to norepinephrine in human subcutaneous resistance arteries are predominantly mediated by the _1A-adrenoceptor. This study also shows a small contribution of ₂-adrenoceptors to the norepinephrine-mediated contractile responses in these arteries.

Postjunctional ₁- and ₂-Adrenoceptors in Human Subcutaneous Resistance Arteries. Results with the agonists show the presence of both ₁- and ₂-adrenoceptors in these arteries. The contribution of ₁-adrenoceptors to norepinephrine-mediated responses is greater than that of ₂-adrenoceptors because the contractile responses evoked by the ₂-selective full agonist brimonidine (Cambridge, 1981; Thaina et al., 1999) were significantly smaller than that of norepinephrine and phenylephrine. Studies with ₁- and ₂-adrenoceptor-selective antagonists also show evidence for a minor involvement of ₂-adrenoceptors in norepinephrine-mediated responses. RS 79948 produced an 11-fold decrease in the potency of norepinephrine. CRCs to norepinephrine in the presence of 0.1 µM prazosin were not parallel to that of control but were made parallel by the addition of 0.1 µM RS 79948, showing the involvement of an ₂-adrenoceptor population that is activated by norepinephrine in the lower concentration range.

As observed in the earlier studies on human skeletal muscle resistance arteries (Jarajapu et al., 2001b), RS 79948 was found to be selective for ₂-adrenoceptors because it did not affect the potency of phenylephrine, an ₁-selective agonist, but shifted the potency of brimonidine by approximately 200-fold. Prazosin shifted the upper part of the CRC to brimonidine. These observations indicate activation of ₁-adrenoceptors by brimonidine in the higher concentration range and this is in agreement with our findings in human skeletal muscle resistance arteries (Jarajapu et al., 2001b).

Nielson et al. (1990, 1991) reported predominant ₂-adrenoceptor-mediated contractile responses in human resistance arteries, which is in contrast to the present study. Our studies in arteries from different human vascular beds show that the contribution of postjunctional ₂-adrenoceptors to norepinephrine-mediated responses varies with the vascular bed. For example, the ₂-selective agonist brimonidine was found to be equipotent and equi-efficacious to norepinephrine in gluteal subcutaneous arteries but evoked significantly smaller contractile responses, compared with norepinephrine and phenylephrine, in inguinal subcutaneous and skeletal muscle resistance arteries (Jarajapu et al., 2001c).

Postjunctional ₁-Adrenoceptor Subtypes in Human Subcutaneous Resistance Arteries. A-61603, a selective _1A-adrenoceptor agonist (Knepper et al., 1995), produced contractile responses in these arteries with 10- and 54-fold greater potency than norepinephrine and phenylephrine, respectively. The higher potency of A-61603 may suggest the presence of a predominant population of _1A-adrenoceptors in these arteries.

Prazosin produced concentration-dependent rightward shifts in the sensitivity of norepinephrine without affecting the maximum responses. The Schild slope of 1.1 indicates the competitive nature of the antagonism. The pA₂ value (>9) indicates that the receptors are of the type at which prazosin shows higher affinity, ruling out the presence of _1L- and _1N-adrenoceptors (Flavahan and Vanhoutte, 1986; Muramatsu et al., 1990).

5-Methyl-urapidil and WB 4101 also produced rightward shifts in the sensitivity of norepinephrine without affecting the maximum responses. Schild slopes of 1.0 and 0.8 observed with 5-methyl-urapidil and WB 4101, respectively, confirm the competitive nature of the antagonism. The pA₂ values of 8.9 and 10.1 for 5-methyl-urapidil and WB 4101, respectively, observed in this study are in agreement with the reported affinity values for the mammalian _1a-adrenoceptor subtype expressed in rat fibroblasts (Ford et al., 1996).

Because WB 4101 shows similar affinity to human _1a- and _1d-subtypes (Kenny et al., 1995; Ford et al., 1996) the presence of the functional _1D-subtype was also studied by using BMY 7378, a selective _1D-antagonist (Goetz et al., 1995). Sensitivity to norepinephrine was not affected by 10 and 100 nM BMY 7378, showing the lack of contribution of the _1D-subtype to norepinephrine-mediated responses. The shift produced by 1 µM BMY 7378 gave a pK_B value of 7.1, which is much less than the reported affinity for the human _1d-subtype (9.4) but similar to that obtained for human _1b- (7.2) and _1a-subtypes (6.6) expressed in rat fibroblasts (Goetz et al., 1995).

Chloroethylclonidine was first identified as a reliable tool to subclassify ₁-adrenoceptor subtypes with preference for the _1B-subtype (Han et al., 1987). However, in the present study neither the sensitivity nor the maximum response to norepinephrine was affected by 1 and 10 µM chloroethylclonidine, ruling out the involvement of the _1B-subtype in the norepinephrine-mediated responses in these arteries. The lack of sensitivity of ₁-adrenoceptors to chloroethylclonidine in human subcutaneous arteries is in contrast to that observed in skeletal muscle resistance arteries in which the maximum responses to norepinephrine as well as A-61603 were decreased by chloroethylclonidine to a similar extent (Jarajapu et al., 2001b).

These results with the agonists and antagonists clearly indicate that the contractile responses to norepinephrine in human subcutaneous resistance arteries are predominantly mediated by the _1A-adrenoceptor subtype. This, together with our previous studies in human skeletal muscle resistance arteries (Jarajapu et al., 2001a), implies the predominant functional expression of _1A-adrenoceptors in human resistance vasculature. In apparent contrast to this, selective _1A-adrenoceptor antagonists are considered to be a potential treatment for benign prostatic hypertrophy, lacking in blood pressure-lowering side effects (Akiyama et al., 1999; Williams et al., 1999). In clinical studies, lack of cardiovascular side effects of tamsulosin was attributed to its selectivity to the _1A-adrenoceptor subtype (Djavan and Marberger, 1999; Harada and Fujimura, 2000). This selectivity is doubtful, however, with other studies showing that tamsulosin has either equal affinity to all the three subtypes (Buckner et al., 1996; Harada et al., 2000; Piao et al., 2000) or about 10-fold selectivity over the _1B-subtype but not the _1D-subtype (Ford et al., 1996; Williams et al., 1999). The importance of the _1A-adrenoceptor subtype in the maintenance of peripheral vascular resistance and the regulation of systemic arterial pressure therefore requires further study. The functional ₁-adrenoceptor subtypes in resistance arteries from other important vascular beds, e.g., mesenteric, renal, hepatic, remain to be characterized.

	Footnotes

Accepted for publication August 14, 2001.

Received for publication June 7, 2001.

Y.P.R.J. is supported by School of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, Scotland, UK.

Address correspondence to: Dr. Chris Hillier, School of Biological and Biomedical Sciences, Glasgow Caledonian University, 70 Cowcaddens Rd., Glasgow G4 0BA, Scotland, UK. E-mail: c.Hillier{at}gcal.ac.uk

	Abbreviations

UK 14304, 5-bromo-N-[2-imidazolin-2-yl]-6-quinoxalinamine; A-61603, N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulphonamide; WB 4101, 2-[2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane; BMY 7378, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione; PSS, physiological saline solution; CRC, concentration-response curve; RS 79948, (8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]-naphthyridine; CL, confidence limit.

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