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Identification of solubilised beta1 and beta2 adrenoceptors in mammalian lung
Life Sciences, Vol. 29, pp. 2527-2533 Printed in the U.S.A.
IDI~PIF!CATION A D ~ K.E.J.
Pergamon Press
OF SOITJBILIS~DBETAI AND BETA 2 INDIAN 177h~ Dickinson
and S.R.
Nahorski
Department of Pharrnac~logy and Therapeuties, Medical Sciences Building, University of Leicester, University Road, Leicester. LEI ?RH. U.K.
(Received in final form October 21, 1981)
Summry The properties of beta-adrenoceptors in solubilised and particulate preparations of rat and rabbit lung have been assessed using the specific ligand (3H)-dihydroalprenolol ((3H)-DHA). Membranes were solubilised using the detergent digitonin and the specific binding of (3H)-DHA assayed using a charcoal-centrifugation technique to separate free and bound ligand. The equilibrium dissociation constant (KD) of specific (3H)-DHA binding was very similar in particulate and soluble preparations of rat and rabbit lung. Moreover, the optical isomers of propranolol displayed virtually identical stereospecific differences in soluble and membraneous preparations. Hov~ver, the potency of various catecholamine agonists and the steepnessof the displacement curves were greater in all solubilised preparations. C~nl0uter-assisted analysis of the displacement curves generated by the highly selective betal antagonist atenolol and the beta2 antagonist ICI 118.551, revealed the co-presence of betal and beta2 adrenoceptors in solubilised rabbit lun~ preparations. Furthermore, soluble beta I adrenoceptors appear to be muchmore labile at 22°C than soluble beta2 adrenoceptors, providing support for the concept that these receptor subtypes are separate entities. The original suggestion that beta-adrenoceptors should be divided into betal and beta2 subclasses (I) has received support and has fo~ned the basis of selective therapeutic treatment in several clinical disorders. On the other hand, several observations that beta-adrenoceptor agents can display a spectrumof affinities between different tissues has led to some speculation on whether there could be a family of iso-receptors or that there is only one beta-adrenoceptor and that the apparent beta I or beta 2 selectivity relates to the ability of agents to gain access to the receptor site (see (2) for review). More recently, the use of direct receptor labelling techniques in tissue membrane preparation has providedevidence that there are only two beta-adrenoceptors (3,4,5) and that these closely correspond to the original betal and beta2 classification of Lands et al (I). Moreover, both subtypes appear to co-exist in many tissues and therefore these direct approaches could provide some explanation for the different degrees of selectivity that agents can display between tissues. However, it could still be argued that the apparent co-presence of beta-adrenoceptor subtypes in membrane preparations could result frcrntherebeing only one
0024-3205/81/242527-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2528
Soluble Beta-adrenoceptors
Vol. 29, No. 24, 1981
receptor and that apparent betal or beta2 characteristics depend upon the constraint that the membrane exerts upon the various accessory binding sites of that single receptor. For this reason we have attempted to solubilise betaadrenoceptors from mammalian lung in order to see if betal and beta2 subclasses can be identified in preparations freed of the constraints of the intact membrane. We provide evidence here, that the characteristics of betal and beta 2 adrenoceptors are n~ntained in solution and in view of the demonstration of the differential stability of the subclasses when solubilised, we suggest that they probably exist as quite separate entities. Materials
and Methods
Lungs were removed from male Wistar rats or New Zealand white rabbits and dissected free of major bronchi. Tissues were hanogenised in i0 vols of 50 mM Tris-HCl, pH 7.5, using a Polytron hcmogeniser. The hcmogenate was passed through a single layer of cheesecloth and centrifuged at 500 g for I0 min at 4oC to remove fibrous tissue. The supernatant was centrifuged at 50,000 g for 20 min and the resulting pellet washed three times with 50 ,~ Tris-HCl, pH 7.8. Suspension of this pellet in buffer formed the particulate preparation in which beta-adrenoceptors were assayed using (3H)-dihydroalprenolol ((3H)-DHA), as previously described (3). Solubilisation of lung tissue was performed by digitonin extraction of particulate preparations for 30 rain at 4°C in a median containing 0.5% digitonin (Sigma); 50 n%M Tris-HCl, pH 7.8; I00 r~M NaCI using a digitonin:protein ratio of 3:1. Following centrifugation at 50,000 g for 1 h the supernatant was used as the solubilised receptor preparation. Solubilised beta-adrenoceptors were assayed in an incubation volL~ne of 500 ~i containing (3H)-DHA, 50 n%M Tris-HCl, pH 7.8, and cor~peting drugs. Bound and free (3H)-DHA were separated by addition of 0.25 ml I. 8% Norit GSX charcoal, 0.4% BSA in 50 n~M Tris-HCl, pH 7.8, followed by centrifugation at 2,000 g for i0 rain. Aliquots of supernatant containing bound ligand were counted by liquid scintillation spectrcmetry. Specific binding was defined as the binding displaced by 200 ~M (-)-isoprenaline and represented >90% in particulate and >95% in solubilised preparations. (3H)-DHA (50 Ci/mmole) was obtained from New England Nuclear; (-)- and (+)propranolol, (±)-ICI 118.551 and (±)-atenolol were kindly donated by ICI Pharmaceuticals; and (±)-salbutamol by Glaxo-Allenburys. (-)-Isoprenaline, (-)-adrenaline, (-)-noradrenaline and digitonin were purchased from Sigma Chemical Co. Results
and Discussion
Previous experiments from these laboratories (6) have shown that rabbit and rat lung would provide excellent rich sources of betal and beta2 sites for the present study. The use of cc~uter-assisted curve fitting of the displacement curves of highly selective betal and beta 2 agents obtained in c~tition with (3H)-DHA binding to membrane preparations of these tissues has revealed that whereas about 80% of the total beta-adrenoceptors in rat lung can be classified as beta2, rabbit lung, in cc~lete contrast, possesses a predaninance of beta 1 adrenoceptors (6,7). Preliminary examination of the ability of a ntm%ber of different detergents to solubilise the receptors, revealed that only the plant glycoside digitonin provided acceptable yields of solubilised adrenoceptors. This agent has been previously used to successfully solubilise beta-adrenoceptors from several sources (8,9,10), though Brij 96 and Triton-X-100 have been used successfully by one laboratory (11,12). The soluble nature of these preparations was confirmed by the inability of further centrifugation at I00,000 g for 2 h, or passage through cellulose nitrate filters (pore size 0.2 ~m) to remove specific binding sites. Further electron microscopy of the solubilised extract failed to reveal any membraneous structures.
Vol. 29, No. 24, 1981
Soluble Beta-adrenoceptors
2529
The binding characteristics of solubilised preparations of rat and rabbit lung were cc~pared to the properties in particulate preparations (Table I ). The equilibri~n dissociation constant (KD) for the ligand (3H)-DHA was identical for solubilised and particulate preparations, although in both cases the affinity of (3H)-DHA was slightly lower in rabbit lung. The max~ n~nber of binding sites in solubilised rat lung was virtually identical to that in particulate preparations, since the yield of protein and (3H)-DHA sites was similar (ca 40%). In contrast, the density of sites in solubilised rabbit lung was less than in particulate preparations. This relates to a relatively poor yield of (3H)-DHA sites, whereas the yield of solubilised protein was similar to that obtained for rat lung. The affinities of the optical isc~ners of prepranolol were slightly weaker in solubilised than in particulate preparations of both rat and rabbit lung, but the large stereospecific difference between the isomers was maintained in solution (Table I). Both isomers generated displacement curves with slope factors close to unity. In contrast, the behaviour of various beta-adrenoceptor agonists differed both between the species and between solubilised and particulate preparations. The order of potency of the agonists isoprenaline > adrenaline > noradrenaline in rat and isoprenaline > adrenaline = noradrenaline in rabbit was similar in solubilised and particulate preparations and was indicative of an overall beta 2 classification in rat and betal in rabbit (I). However, all these agonists were more potent in solubilised preparations and it was noticed that most of the displacement curves were considerably steeper than those displayed in the m~nbrane preparations. This ccmplex behaviour could relate to high and low affinity agonist states of the beta-adrenoceptor that have been described in other preparations (13,14). It should be noted that higher agonist potencies have been previously observed in solubilised preparations (9,10) though not after purification by affinity chrcmatography (9). ~k~ther detailed analysis of the behaviour of agonists in particulate and solubilised lung preparations will be described in another manuscript. The critical question in the present study, however, was whether the copresence of beta I and beta 2 adrenoceptors previously observed in membranes could be demonstrated in solution. In order to examine this, we analysed the displac~nent curves generated by atenolol and ICI 118.551 which, respectively, demo~Istrate considerable beta I and beta2 adrenoceptor selectivity in intact (15,16) and membrane preparations (7). Computer-assisted iterative fitting of the curves obtained for all preparations are shown in Fig. I. In agre~nent with our previous studies (6), both rat and rabbit lung membrane preparations appear to possess both betal and beta 2 sites. Both atenolol and ICI 118.551 generated curves which on analysis revealed that whereas 80% of rat lung membrane sites possessed high affinity for the beta 2 antagonist and low affinity for the beta I selective drug, precisely the opposite situation was apparent in rabbit. In the solubilised receptor preparations, however, a different pattern emerged. In solubilised rat lung both atenolol and ICI 118.551 generated curves which closely approximated to a single class of binding site behaving according to the law of mass action, and which suggested an almost homogeneous beta 2 population. This suggested that either (a) there was only one type of betaadrenoceptor which corresponded to the beta 2 subclass and that an apparent small beta I population was conferred by the constraint of the membrane, or (b) there was a selective loss of beta I adrenoceptors upon solubilisation. The data obtained from rabbit lung with its much greater proportion of initial membrane betal sites, strongly suggests that the latter alternative is the correct one. Thus, in solubilised rabbit lung, the co-presence of betal and beta2 sites are clearly seen with both ICI 118. 551 and atenolol, though the relative proportion of betal sites is reduced frcm that seen in the m~nbranes.
27 (± 3) 245 (± 25) 1430 (± 180) 660 (± 130)
(-)-Isoprenaline
(-)-Adrenaline
(-)-Noradrenaline
(±)-Salbutamol
360 (± 52)
470 (± 60)
32 (± 5)
8 (± 1.2)
91 (± 2)
0.86 (± 0.02)
1400 (± 205)
385 (± 41)
603 (± 80)
24 (± 2)
85 (± 5)
0.97 (± 0.I)
1.04 (± 0.03)
390 (± 19)
0.59 (± 0.05)
Particulate
Rabbit
in Rat and Rabbit Lung
Soluble
1300
33
58
2.6
250
2.2
(± 120)
(± 6)
(± I0)
(± 0.4)
(± 20)
(± 0.I)
1.12 (± 0.04)
190 (-+ 5)
0.67 (± 0.06)
Lung
Apparent equilibrium dissociation constants, binding site maxima and Hill coefficients were calculated from Scatchard and Hill plots of saturation data. IC50 values of ccrr~0eting drug were determined graphically by Hill analyses and inhibition constants calculated using the equation Ki = IC50/I + S/KD, where S is the concentration of (3H)-DHA used; KD is the dissociation constant for (3H)-DHA. All data represent the mean ± ~qE of 3-8 experiments conducted in duplicate (displac~nent experiments) or triplicate (saturation experiments).
55 (± 2)
(+)-Propranolol
l - -
K. (nM)
I.I0 (± 0.03)
1.05 (± 0.04)
0.32 (± 0.01)
Soluble
440 (± 50)
Rat Lung
410 (± 20)
0.35 (± 0.01)
0.69 (± 0.05)
constants
prot)
I
and Soluble Beta-Adrenoceptors
Particulate
of Particulate
(-)-Propranolol
Inhibition
Kill slope (n H)
Bma x (fmoles/mg
K D (nM)
(3H)-DHA
A~ent/DetezTnination
Characteristics
TABLE
0o
Z O
O
0 o ,.~
I
cr
Vol. 29, No. 24, 1981
lOO
Soluble Beta-adrenoceptors
2531
RABBITLUNG
o// . o//
80 60
/
/ o/°
oi .°/
./
/
i18,551. / ~ / ~ / " / ' / . / ~ / ' / . / . / / " / "
IC'
40
S°
20 =,
. o _.......-~,~~ ' ~ ' ~
/
./
j
./ ~,/
- LOG
B
ioo
A'~TACON I ST
M
RATLUNG
ICl i18,551
60
///
////~:/"
///
/
40
/ /
f:/
20
i0
9
/~/° / 8
7 - LOG
6
//" 5
ATENOLOL
q
3
ANTAGONIST M
FIG. 1 Inhibition of specific (3H)-DHA binding to soluble (filled symbols) and particulate (open symbols) preparations of rabbit and rat lung by the beta2-selective antagonist ICI 118. 551 and the beta lselective antagonist atenolol. (3H)-DHA (1-1.5 nM) and competing drug were incubated with each preparation and specific binding of (3H)-DHA was determined as described in Table I. The curves shown are the mean of three separate experiments performed in duplicate and the S~ on all the points was <10%. The mean curves were analysed using computer-assisted iterative curve fitting (7), arriving at the best fit parameters by minimisation of the sum of squares. One or two non-interacting site models were tested for each curve. IC50 values and proportions of sites generated in this way were: Rabbit Lung (particulate) atenolol - betal site 800 nM, 79%, beta 2 site 41,000 raM, 21%; (soluble) betal site 2,700 nM, 58%, beta 2 site 63,000 nM, 4270; ICI 118.551 (particulate) beta 1 site 210 nM, 81%, beta2 site 6 riM, 19%; (soluble) beta I site 840 nM, 60%, beta2 site 13 nM, 40%. Rat Lung (particulate) atenolol betal site 210 nM, 20%, beta2 site 31,000 nM, 80%; (soluble) beta 2 site 61,000 raM, 100%; ICI 118.551 (particulate) beta I site 530 nM, 17~, beta 2 site 8.5 nM, 83%; (soluble) beta I site 2,200 nM, 6%, beta2 site 12 D_M, 94%.
2532
Soluble Beta-adrenoceptors
Vol. 29, No. 24, 1981
Identical proportions of betal and beta2 adrenoceptor sites were obtaine~d with both of these agents and the different inhibition constants for each subclass were quite independent of the source or proportion of receptor subtype. Moreover, use of other beta I (betaxolol) or beta2 (procaterol) selective agents provided virtually identical proportions of sites (not shown).
I00 7=
80
6g
g ,-
20
6
5
FIG.
q
~
2
Inhibition of specific (3H)-DHA binding to a soluble preparation of rabbit lung by the betal-selective antagonist atenolol. Soluble preparations were assayed immediately following solubilisation (m) or after 1½ h (D), 5 h (A) or 5½ h (O) incubation at 22oc. Chn~es were analysed as described in Fig. I and generated the following parameters: No incubation - betal site 1,900 nM, 53~, beta2 site 46,000 nM, 477o; 1½ h - betal site 1,500 nM, 36%, beta 2 site 62,000 nM 64%; 5 h - betal site 2,300 nM, 15%, beta2 site 64,000 r~M, 85%; 5½ h - beta2 site 56,000 nM, 100%. The possibility that beta I sites are much more labile than beta2 adrenoceptors in rabbit lung at 22°C was clearly confirmed by the data shown in Fig. 2. In these studies solubilised rabbit lung preparations were analysed with atenolol either inm~diately following solubilisation or after the total number of (3H)DHA sites were reduced following storage for various periods at 22°C. Computerassisted fitting of the generated curves clearly d~onstrates a preferential loss of betal sites with time in these 'aged' preparations. Similar results have been demonstrated using the beta2-selective antagonist ICI 118.551 to analyse the proportions of beta I and beta2 adrenoceptors. Furthermore, prolonged incubations of solubilised rabbit lung preparations (>5 h) yield an adrenoceptor population for which adrenaline has an affinity almost 20 times greater than noradrenaline, and which is suggestive of a beta 2 adrenoceptor. It seems certain that the lo~gr recovery of total beta-adrenoceptors from rabbit lung (Table I) reflects a selective loss of the high initial proportion of betal sites in this preparation. In conclusion therefore, the present studies have den~nstrated for the first time that beta I and beta2 adrenoceptors can be identified together in solubilised preparations of lung. The co-presence of the two beta-adrenoceptor subtypes in preparations free of the constraints of the ~ a n e together with the observation that, at least in rabbit lung, betal sites are much more labile than beta 2 receptors, suggests the sites may be separate entities. Support for
Vol. 29, No. 24, 1981
Soluble Beta-adrenoceptors
2533
this concept has come from Venter and colleagues (12) who have shown that autoantibodies present in ser~n of patients with allergic rhinitis or asthma inhibit ligand binding to dog or calf lung beta~adrenoceptors (mostly beta2) but not heart (mostly beta I) receptors. However, definitive evidence on this point requires the physical separation of the subtypes frcm solubilised preparations. Preliminary studies from other laboratories (17) suggested a difference in molecular weight of beta-adrenoceptors from canine heart and liver, though surprisingly pharmacological differences in the preparations were not apparent. Since most manrnalian tissues probably contain both receptor subtypes (18) it is difficult to interpret the significance of these results. At present we are atten~0ting to physically separate beta I and beta 2 adrenoceptors from solubilised preparations of rabbit lung. Acknowledgements We would like to thank the Science Research Jenny Bell for manuscript preparation.
Council
for financial
support
and
References i. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18.
A.M. LA~S, A. ARNOLD, J.P. McAULIFF, F.P. LADUENA and T.G. E~)WN, Nature 214 597-598 (1967). M.J. DALY and G.P. LEVY, Trends in Autonomic Pharmacology, Vol. I, ed. S. Kalsner, p. 347-385, Urban & Schwarzenberg, Baltimore-Munich (1979). D.B. BARNEtt, E. RUGG and S.R. NAI~RSKI, Nature 273 166-168 (1978). S.R. NARDRSKI, Eur. J. Pharmacol. 51 199-209 (1978). K.P. MINN--, A. HEDRERG and P.B. MOLINOFF, J. Pharmacol. Exp. Ther. 211 502-508 (1979). E. F~JGG, D.B. BARNErr and S.R. NAHORSKI, Mol. Pharmacol. 14 996-1005 (1978) K.E.J. DICKINSON, A. RICHARDSON and S.R. NA}K)RSKI, Mol. Pha~nacol. 19 194204 (1981). M.G. CARC~ and R.J. LEFKOWITZ, J. Biol. Chem. 251 2374-2384 (1976). G. VAUQUELIN, P. GEYNET, J. HAN(~ and D. S T Y , Proc. Natl. Acad. Sci 74 3710-3714 (1977). J. KLEIN~IN and H. GLOSSMAN, Naunyn-Schmiedeberg's Arch. Pharmacol. 305 191 (1978). W.L. STRAUSS, G. GHAI, C.M. FRASER and J.C. VENTER, Arch. Biochem. Biophys. 196 566-573 (1979). J.C. VENTER, C.M. FRASER and L.C. HARRISON, Science 207 1361-1362 (1980). R.S. KENT, A. DE LEAN and R.J. LEFKOWITZ, Mol. Pharmacol. 17 14-23 (1980). A. DE LEAN, J.M. STADEL and R.J. LEFKOWITZ, J. Biol. Chem. 255 7108-7117 (1980). A.M. BARRETT, J. CARTER, J.D. FITZ~, R. HULL and D. LE COUNT, Br. J. Pharmacol. 48 340P (1973). A. BILSKI, S. DORRIF~S, J.D. F r l ~ , R. JESSUP, H. ~IJCKER and J. WADE, Br. J. Pharmacol. 69 292P-293P (1980). C.M. FRASh~, G. GHAI, A.J. CAVE and J.C. VENl~, Fed. Proc. 37 684 (1978). S.R. NAHORSKI, Trends Pharm. Sci. 2 95-98 (1981).
IDI~PIF!CATION A D ~ K.E.J.
Pergamon Press
OF SOITJBILIS~DBETAI AND BETA 2 INDIAN 177h~ Dickinson
and S.R.
Nahorski
Department of Pharrnac~logy and Therapeuties, Medical Sciences Building, University of Leicester, University Road, Leicester. LEI ?RH. U.K.
(Received in final form October 21, 1981)
Summry The properties of beta-adrenoceptors in solubilised and particulate preparations of rat and rabbit lung have been assessed using the specific ligand (3H)-dihydroalprenolol ((3H)-DHA). Membranes were solubilised using the detergent digitonin and the specific binding of (3H)-DHA assayed using a charcoal-centrifugation technique to separate free and bound ligand. The equilibrium dissociation constant (KD) of specific (3H)-DHA binding was very similar in particulate and soluble preparations of rat and rabbit lung. Moreover, the optical isomers of propranolol displayed virtually identical stereospecific differences in soluble and membraneous preparations. Hov~ver, the potency of various catecholamine agonists and the steepnessof the displacement curves were greater in all solubilised preparations. C~nl0uter-assisted analysis of the displacement curves generated by the highly selective betal antagonist atenolol and the beta2 antagonist ICI 118.551, revealed the co-presence of betal and beta2 adrenoceptors in solubilised rabbit lun~ preparations. Furthermore, soluble beta I adrenoceptors appear to be muchmore labile at 22°C than soluble beta2 adrenoceptors, providing support for the concept that these receptor subtypes are separate entities. The original suggestion that beta-adrenoceptors should be divided into betal and beta2 subclasses (I) has received support and has fo~ned the basis of selective therapeutic treatment in several clinical disorders. On the other hand, several observations that beta-adrenoceptor agents can display a spectrumof affinities between different tissues has led to some speculation on whether there could be a family of iso-receptors or that there is only one beta-adrenoceptor and that the apparent beta I or beta 2 selectivity relates to the ability of agents to gain access to the receptor site (see (2) for review). More recently, the use of direct receptor labelling techniques in tissue membrane preparation has providedevidence that there are only two beta-adrenoceptors (3,4,5) and that these closely correspond to the original betal and beta2 classification of Lands et al (I). Moreover, both subtypes appear to co-exist in many tissues and therefore these direct approaches could provide some explanation for the different degrees of selectivity that agents can display between tissues. However, it could still be argued that the apparent co-presence of beta-adrenoceptor subtypes in membrane preparations could result frcrntherebeing only one
0024-3205/81/242527-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2528
Soluble Beta-adrenoceptors
Vol. 29, No. 24, 1981
receptor and that apparent betal or beta2 characteristics depend upon the constraint that the membrane exerts upon the various accessory binding sites of that single receptor. For this reason we have attempted to solubilise betaadrenoceptors from mammalian lung in order to see if betal and beta2 subclasses can be identified in preparations freed of the constraints of the intact membrane. We provide evidence here, that the characteristics of betal and beta 2 adrenoceptors are n~ntained in solution and in view of the demonstration of the differential stability of the subclasses when solubilised, we suggest that they probably exist as quite separate entities. Materials
and Methods
Lungs were removed from male Wistar rats or New Zealand white rabbits and dissected free of major bronchi. Tissues were hanogenised in i0 vols of 50 mM Tris-HCl, pH 7.5, using a Polytron hcmogeniser. The hcmogenate was passed through a single layer of cheesecloth and centrifuged at 500 g for I0 min at 4oC to remove fibrous tissue. The supernatant was centrifuged at 50,000 g for 20 min and the resulting pellet washed three times with 50 ,~ Tris-HCl, pH 7.8. Suspension of this pellet in buffer formed the particulate preparation in which beta-adrenoceptors were assayed using (3H)-dihydroalprenolol ((3H)-DHA), as previously described (3). Solubilisation of lung tissue was performed by digitonin extraction of particulate preparations for 30 rain at 4°C in a median containing 0.5% digitonin (Sigma); 50 n%M Tris-HCl, pH 7.8; I00 r~M NaCI using a digitonin:protein ratio of 3:1. Following centrifugation at 50,000 g for 1 h the supernatant was used as the solubilised receptor preparation. Solubilised beta-adrenoceptors were assayed in an incubation volL~ne of 500 ~i containing (3H)-DHA, 50 n%M Tris-HCl, pH 7.8, and cor~peting drugs. Bound and free (3H)-DHA were separated by addition of 0.25 ml I. 8% Norit GSX charcoal, 0.4% BSA in 50 n~M Tris-HCl, pH 7.8, followed by centrifugation at 2,000 g for i0 rain. Aliquots of supernatant containing bound ligand were counted by liquid scintillation spectrcmetry. Specific binding was defined as the binding displaced by 200 ~M (-)-isoprenaline and represented >90% in particulate and >95% in solubilised preparations. (3H)-DHA (50 Ci/mmole) was obtained from New England Nuclear; (-)- and (+)propranolol, (±)-ICI 118.551 and (±)-atenolol were kindly donated by ICI Pharmaceuticals; and (±)-salbutamol by Glaxo-Allenburys. (-)-Isoprenaline, (-)-adrenaline, (-)-noradrenaline and digitonin were purchased from Sigma Chemical Co. Results
and Discussion
Previous experiments from these laboratories (6) have shown that rabbit and rat lung would provide excellent rich sources of betal and beta2 sites for the present study. The use of cc~uter-assisted curve fitting of the displacement curves of highly selective betal and beta 2 agents obtained in c~tition with (3H)-DHA binding to membrane preparations of these tissues has revealed that whereas about 80% of the total beta-adrenoceptors in rat lung can be classified as beta2, rabbit lung, in cc~lete contrast, possesses a predaninance of beta 1 adrenoceptors (6,7). Preliminary examination of the ability of a ntm%ber of different detergents to solubilise the receptors, revealed that only the plant glycoside digitonin provided acceptable yields of solubilised adrenoceptors. This agent has been previously used to successfully solubilise beta-adrenoceptors from several sources (8,9,10), though Brij 96 and Triton-X-100 have been used successfully by one laboratory (11,12). The soluble nature of these preparations was confirmed by the inability of further centrifugation at I00,000 g for 2 h, or passage through cellulose nitrate filters (pore size 0.2 ~m) to remove specific binding sites. Further electron microscopy of the solubilised extract failed to reveal any membraneous structures.
Vol. 29, No. 24, 1981
Soluble Beta-adrenoceptors
2529
The binding characteristics of solubilised preparations of rat and rabbit lung were cc~pared to the properties in particulate preparations (Table I ). The equilibri~n dissociation constant (KD) for the ligand (3H)-DHA was identical for solubilised and particulate preparations, although in both cases the affinity of (3H)-DHA was slightly lower in rabbit lung. The max~ n~nber of binding sites in solubilised rat lung was virtually identical to that in particulate preparations, since the yield of protein and (3H)-DHA sites was similar (ca 40%). In contrast, the density of sites in solubilised rabbit lung was less than in particulate preparations. This relates to a relatively poor yield of (3H)-DHA sites, whereas the yield of solubilised protein was similar to that obtained for rat lung. The affinities of the optical isc~ners of prepranolol were slightly weaker in solubilised than in particulate preparations of both rat and rabbit lung, but the large stereospecific difference between the isomers was maintained in solution (Table I). Both isomers generated displacement curves with slope factors close to unity. In contrast, the behaviour of various beta-adrenoceptor agonists differed both between the species and between solubilised and particulate preparations. The order of potency of the agonists isoprenaline > adrenaline > noradrenaline in rat and isoprenaline > adrenaline = noradrenaline in rabbit was similar in solubilised and particulate preparations and was indicative of an overall beta 2 classification in rat and betal in rabbit (I). However, all these agonists were more potent in solubilised preparations and it was noticed that most of the displacement curves were considerably steeper than those displayed in the m~nbrane preparations. This ccmplex behaviour could relate to high and low affinity agonist states of the beta-adrenoceptor that have been described in other preparations (13,14). It should be noted that higher agonist potencies have been previously observed in solubilised preparations (9,10) though not after purification by affinity chrcmatography (9). ~k~ther detailed analysis of the behaviour of agonists in particulate and solubilised lung preparations will be described in another manuscript. The critical question in the present study, however, was whether the copresence of beta I and beta 2 adrenoceptors previously observed in membranes could be demonstrated in solution. In order to examine this, we analysed the displac~nent curves generated by atenolol and ICI 118.551 which, respectively, demo~Istrate considerable beta I and beta2 adrenoceptor selectivity in intact (15,16) and membrane preparations (7). Computer-assisted iterative fitting of the curves obtained for all preparations are shown in Fig. I. In agre~nent with our previous studies (6), both rat and rabbit lung membrane preparations appear to possess both betal and beta 2 sites. Both atenolol and ICI 118.551 generated curves which on analysis revealed that whereas 80% of rat lung membrane sites possessed high affinity for the beta 2 antagonist and low affinity for the beta I selective drug, precisely the opposite situation was apparent in rabbit. In the solubilised receptor preparations, however, a different pattern emerged. In solubilised rat lung both atenolol and ICI 118.551 generated curves which closely approximated to a single class of binding site behaving according to the law of mass action, and which suggested an almost homogeneous beta 2 population. This suggested that either (a) there was only one type of betaadrenoceptor which corresponded to the beta 2 subclass and that an apparent small beta I population was conferred by the constraint of the membrane, or (b) there was a selective loss of beta I adrenoceptors upon solubilisation. The data obtained from rabbit lung with its much greater proportion of initial membrane betal sites, strongly suggests that the latter alternative is the correct one. Thus, in solubilised rabbit lung, the co-presence of betal and beta2 sites are clearly seen with both ICI 118. 551 and atenolol, though the relative proportion of betal sites is reduced frcm that seen in the m~nbranes.
27 (± 3) 245 (± 25) 1430 (± 180) 660 (± 130)
(-)-Isoprenaline
(-)-Adrenaline
(-)-Noradrenaline
(±)-Salbutamol
360 (± 52)
470 (± 60)
32 (± 5)
8 (± 1.2)
91 (± 2)
0.86 (± 0.02)
1400 (± 205)
385 (± 41)
603 (± 80)
24 (± 2)
85 (± 5)
0.97 (± 0.I)
1.04 (± 0.03)
390 (± 19)
0.59 (± 0.05)
Particulate
Rabbit
in Rat and Rabbit Lung
Soluble
1300
33
58
2.6
250
2.2
(± 120)
(± 6)
(± I0)
(± 0.4)
(± 20)
(± 0.I)
1.12 (± 0.04)
190 (-+ 5)
0.67 (± 0.06)
Lung
Apparent equilibrium dissociation constants, binding site maxima and Hill coefficients were calculated from Scatchard and Hill plots of saturation data. IC50 values of ccrr~0eting drug were determined graphically by Hill analyses and inhibition constants calculated using the equation Ki = IC50/I + S/KD, where S is the concentration of (3H)-DHA used; KD is the dissociation constant for (3H)-DHA. All data represent the mean ± ~qE of 3-8 experiments conducted in duplicate (displac~nent experiments) or triplicate (saturation experiments).
55 (± 2)
(+)-Propranolol
l - -
K. (nM)
I.I0 (± 0.03)
1.05 (± 0.04)
0.32 (± 0.01)
Soluble
440 (± 50)
Rat Lung
410 (± 20)
0.35 (± 0.01)
0.69 (± 0.05)
constants
prot)
I
and Soluble Beta-Adrenoceptors
Particulate
of Particulate
(-)-Propranolol
Inhibition
Kill slope (n H)
Bma x (fmoles/mg
K D (nM)
(3H)-DHA
A~ent/DetezTnination
Characteristics
TABLE
0o
Z O
O
0 o ,.~
I
cr
Vol. 29, No. 24, 1981
lOO
Soluble Beta-adrenoceptors
2531
RABBITLUNG
o// . o//
80 60
/
/ o/°
oi .°/
./
/
i18,551. / ~ / ~ / " / ' / . / ~ / ' / . / . / / " / "
IC'
40
S°
20 =,
. o _.......-~,~~ ' ~ ' ~
/
./
j
./ ~,/
- LOG
B
ioo
A'~TACON I ST
M
RATLUNG
ICl i18,551
60
///
////~:/"
///
/
40
/ /
f:/
20
i0
9
/~/° / 8
7 - LOG
6
//" 5
ATENOLOL
q
3
ANTAGONIST M
FIG. 1 Inhibition of specific (3H)-DHA binding to soluble (filled symbols) and particulate (open symbols) preparations of rabbit and rat lung by the beta2-selective antagonist ICI 118. 551 and the beta lselective antagonist atenolol. (3H)-DHA (1-1.5 nM) and competing drug were incubated with each preparation and specific binding of (3H)-DHA was determined as described in Table I. The curves shown are the mean of three separate experiments performed in duplicate and the S~ on all the points was <10%. The mean curves were analysed using computer-assisted iterative curve fitting (7), arriving at the best fit parameters by minimisation of the sum of squares. One or two non-interacting site models were tested for each curve. IC50 values and proportions of sites generated in this way were: Rabbit Lung (particulate) atenolol - betal site 800 nM, 79%, beta 2 site 41,000 raM, 21%; (soluble) betal site 2,700 nM, 58%, beta 2 site 63,000 nM, 4270; ICI 118.551 (particulate) beta 1 site 210 nM, 81%, beta2 site 6 riM, 19%; (soluble) beta I site 840 nM, 60%, beta2 site 13 nM, 40%. Rat Lung (particulate) atenolol betal site 210 nM, 20%, beta2 site 31,000 nM, 80%; (soluble) beta 2 site 61,000 raM, 100%; ICI 118.551 (particulate) beta I site 530 nM, 17~, beta 2 site 8.5 nM, 83%; (soluble) beta I site 2,200 nM, 6%, beta2 site 12 D_M, 94%.
2532
Soluble Beta-adrenoceptors
Vol. 29, No. 24, 1981
Identical proportions of betal and beta2 adrenoceptor sites were obtaine~d with both of these agents and the different inhibition constants for each subclass were quite independent of the source or proportion of receptor subtype. Moreover, use of other beta I (betaxolol) or beta2 (procaterol) selective agents provided virtually identical proportions of sites (not shown).
I00 7=
80
6g
g ,-
20
6
5
FIG.
q
~
2
Inhibition of specific (3H)-DHA binding to a soluble preparation of rabbit lung by the betal-selective antagonist atenolol. Soluble preparations were assayed immediately following solubilisation (m) or after 1½ h (D), 5 h (A) or 5½ h (O) incubation at 22oc. Chn~es were analysed as described in Fig. I and generated the following parameters: No incubation - betal site 1,900 nM, 53~, beta2 site 46,000 nM, 477o; 1½ h - betal site 1,500 nM, 36%, beta 2 site 62,000 nM 64%; 5 h - betal site 2,300 nM, 15%, beta2 site 64,000 r~M, 85%; 5½ h - beta2 site 56,000 nM, 100%. The possibility that beta I sites are much more labile than beta2 adrenoceptors in rabbit lung at 22°C was clearly confirmed by the data shown in Fig. 2. In these studies solubilised rabbit lung preparations were analysed with atenolol either inm~diately following solubilisation or after the total number of (3H)DHA sites were reduced following storage for various periods at 22°C. Computerassisted fitting of the generated curves clearly d~onstrates a preferential loss of betal sites with time in these 'aged' preparations. Similar results have been demonstrated using the beta2-selective antagonist ICI 118.551 to analyse the proportions of beta I and beta2 adrenoceptors. Furthermore, prolonged incubations of solubilised rabbit lung preparations (>5 h) yield an adrenoceptor population for which adrenaline has an affinity almost 20 times greater than noradrenaline, and which is suggestive of a beta 2 adrenoceptor. It seems certain that the lo~gr recovery of total beta-adrenoceptors from rabbit lung (Table I) reflects a selective loss of the high initial proportion of betal sites in this preparation. In conclusion therefore, the present studies have den~nstrated for the first time that beta I and beta2 adrenoceptors can be identified together in solubilised preparations of lung. The co-presence of the two beta-adrenoceptor subtypes in preparations free of the constraints of the ~ a n e together with the observation that, at least in rabbit lung, betal sites are much more labile than beta 2 receptors, suggests the sites may be separate entities. Support for
Vol. 29, No. 24, 1981
Soluble Beta-adrenoceptors
2533
this concept has come from Venter and colleagues (12) who have shown that autoantibodies present in ser~n of patients with allergic rhinitis or asthma inhibit ligand binding to dog or calf lung beta~adrenoceptors (mostly beta2) but not heart (mostly beta I) receptors. However, definitive evidence on this point requires the physical separation of the subtypes frcm solubilised preparations. Preliminary studies from other laboratories (17) suggested a difference in molecular weight of beta-adrenoceptors from canine heart and liver, though surprisingly pharmacological differences in the preparations were not apparent. Since most manrnalian tissues probably contain both receptor subtypes (18) it is difficult to interpret the significance of these results. At present we are atten~0ting to physically separate beta I and beta 2 adrenoceptors from solubilised preparations of rabbit lung. Acknowledgements We would like to thank the Science Research Jenny Bell for manuscript preparation.
Council
for financial
support
and
References i. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18.
A.M. LA~S, A. ARNOLD, J.P. McAULIFF, F.P. LADUENA and T.G. E~)WN, Nature 214 597-598 (1967). M.J. DALY and G.P. LEVY, Trends in Autonomic Pharmacology, Vol. I, ed. S. Kalsner, p. 347-385, Urban & Schwarzenberg, Baltimore-Munich (1979). D.B. BARNEtt, E. RUGG and S.R. NAI~RSKI, Nature 273 166-168 (1978). S.R. NARDRSKI, Eur. J. Pharmacol. 51 199-209 (1978). K.P. MINN--, A. HEDRERG and P.B. MOLINOFF, J. Pharmacol. Exp. Ther. 211 502-508 (1979). E. F~JGG, D.B. BARNErr and S.R. NAHORSKI, Mol. Pharmacol. 14 996-1005 (1978) K.E.J. DICKINSON, A. RICHARDSON and S.R. NA}K)RSKI, Mol. Pha~nacol. 19 194204 (1981). M.G. CARC~ and R.J. LEFKOWITZ, J. Biol. Chem. 251 2374-2384 (1976). G. VAUQUELIN, P. GEYNET, J. HAN(~ and D. S T Y , Proc. Natl. Acad. Sci 74 3710-3714 (1977). J. KLEIN~IN and H. GLOSSMAN, Naunyn-Schmiedeberg's Arch. Pharmacol. 305 191 (1978). W.L. STRAUSS, G. GHAI, C.M. FRASER and J.C. VENTER, Arch. Biochem. Biophys. 196 566-573 (1979). J.C. VENTER, C.M. FRASER and L.C. HARRISON, Science 207 1361-1362 (1980). R.S. KENT, A. DE LEAN and R.J. LEFKOWITZ, Mol. Pharmacol. 17 14-23 (1980). A. DE LEAN, J.M. STADEL and R.J. LEFKOWITZ, J. Biol. Chem. 255 7108-7117 (1980). A.M. BARRETT, J. CARTER, J.D. FITZ~, R. HULL and D. LE COUNT, Br. J. Pharmacol. 48 340P (1973). A. BILSKI, S. DORRIF~S, J.D. F r l ~ , R. JESSUP, H. ~IJCKER and J. WADE, Br. J. Pharmacol. 69 292P-293P (1980). C.M. FRASh~, G. GHAI, A.J. CAVE and J.C. VENl~, Fed. Proc. 37 684 (1978). S.R. NAHORSKI, Trends Pharm. Sci. 2 95-98 (1981).