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Alpha and beta adrenergic receptors of canine lung tissue identification and characterization of alpha adrenergic receptors by two different ligands
Life Sciences, Vol. 30, pp. 1035-1044 Printed in the U.S.A.
Pergamon Press
ALPHA AND BETA ADRENERGIC RECEPTORS OF CANINE LUNG TISSUE IDENTIFICATION AND C}~RACTERIZATION OF ALPHA ADRENERGIC RECEPTORS BY TWO DIFFERENT LIGANDS Maki Hasegawa, M.D. and Robert G. Townley, M.D. Allergic Disease Center, Creighton University School of Medicine 2500 California Street Omaha, NE 68178 (Received in final form January 18, 1982) Summary Adrenergic receptors of canine peripheral lung tissues were measured by direct binding techniques u~in~ [3H]dihydroergocryptine ([3H]DHE), [3H]prazosim and [3H]dihydroalpreno]ol ([3H]DHA). All three ligands bound to canine lung tissue with saturabllity, stereospecificity and reversibility. Adrenergic agonists competed for binding'of [3H]DHE and [3H]prazosln in the order: l-epinephrlne > l-norepinephrine > d-epinephrine> d-norepinephrine > l-isoproterenol. Adrenerglc antagonists competed for binding of [3H]prazosln in the order: prazosin> phentolamine>yohimbine. Inhibition curves of [3H]DHE by prazosin or yohimbine were biphasic suggestlng two subtypes of binding sites. Maxlmum binding capacities of [3H]DHE ranged from 30.6 to 42.7 fmol/mg protein. [3H]prazosln from 18.3 to 26.9 fmol/mg protein and [3H]DHA from 135.2 to 359.4 fmol/mg protein. When both [3H]DHE and [3H]prazosin were used in the same membrane preparation, specific binding of [3H]DHE was always more than that of [3H]prazosin. Since [3H]prazosin is considered to bind to alpha I adrenergic receptors speciflcally and [3H]DHE is considered to bind alpha 2 adrenergic receptors nonselectively, the difference between the numbers of the specific binding sites of these two ligands should represent alpha 2 adrenergic receptors. Alpha 2 adrenergic receptor density ranged from 9.5 to 21.1 fmol/mg proteln. Our results suggest the exlstence of both alpha I and alpha 2 adrenergic receptors in canine peripheral lung tissue. Approximately 40% of alpha adrenergic receptors were alpha 2. The ratio of alpha/beta adrenrgic receptors ranged from 1:3.3 to 1:10.4. The ratio of alpha]/ beta adrenergic receptors ranged from 1:6.7 to 1:21.1. Alpha adrenergic receptors are dlvlded into two subclasses. (],2) Alpha I adrenergic receptors include postsynaptic alpha receptors which contract smooth muscles. Alpha 2 adrenerglc receptors include presynaptic alpha adrenerglc receptors which inhibit the release of norepinephrlne from sympathetic nerve endings, and postsynaptic alpha adrenergic receptors, for example alpha adrenergic receptors in human platelets. (3,4) Several investigators uslng radioligand binding techniques have reported detecting alpha I and alpha 2 receptors simultaneously in rat central nervous system (5), rat liver (6), rabbit uterus (7), and human adipocytes. (8) We prepared membrane fractions from canine lung tissue, and measured alpha adrenergic receptors using two different ligands, [3H]prazosin and [3H]dihydroergocryptine ([ 3H]DHE), to detect alph a receptors In the same membrane preparation. [3H]prazosin is a potent alpha I adrenergic antagonist and is considered to bind to alpha I adrenergic receptors specifically (9), [3H]DHE is a potent alpha 0024-3205/82/121035-10503,00/0 Copyright (c) 1982 Pergamon Press Ltd,
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antagonlst and is considered to bind to alpha I and alpha 2 receptors nonselectively. (7) When both [3H]prazosin and [3H]DHE are used in the same membrane preparation, the difference between the numbers of specific binding sites of these two ligands should represent alpha 2 adrenergic receptors. We used [3H]dihydroalprenolol ([3H]DHA) to measure beta adrenergic receptors in these same membrane preparations, and calculated the ratios between alpha and beta and between alpha I and beta receptors. To our knowledge, this is the first report describlng alpha adrenergic receptors in canine lung tissue. Methods and Materials Lungs were rapidly removed from dogs immediately after death by exsangulnation and stored at -60°C until assay. Lungs were used within 3 months. For membrane preparation, thawed lung tissues were dissected free of major bronchi, vessels and connective tissues by forceps, and minced to small pieces in 10 volumes of ice cold buffer (Tris-HCl 5mM, MgC] 2 1 mM, pH 7.4). These pieces were homogenized at full speed for 30 seconds using a Brinkmann Polytron. The homogenate was then centrifuged at 50 x g for 20 minutes. Fibrous tissues and other high density debris were removed by this centrifugatlon. The supernatant was recentrifuged at 45,000 x g for 15 minutes. The resulting pellets were resuspended in the same buffer at a concentration of approximately 1.5 mg protein/ml after filtration through a cheesecloth. All procedures were performed at 4oc. Protein concentration was determined by the method of Lowry. Membranes were incubated in triplicate tubes for ]5 minutes at 25oc with [3H]prazosin (specific radioactivity 33 Ci/m mol, Amersham), or for 20 minutes at 25°C with [3H]DHE (specific radioactivity 30.9 Ci/m mol, New England Nuclear). To each tube, 0.85 ml of membrane preparation, 0.05 ml of an appropriate concentration of radioligand and 0.I ml of either buffer or non-radioactive competitive ligand were added to make a total incubation volume of 1.0 ml. Incubations were terminated by rapid dilution with 6 ml of ice cold buffer and vacuum filtration through glass fiber filters (Whatman GF/B) which were washed 3 times with 6 ml of ice cold buffer. Filters were placed in 10 ml of toluene-based scintilant and radioactivities were determined in liquid scintilation counter (Tracor Analytic, Model 6892) at an efficiency of 45%. Specific binding was defined as total binding minus binding in the presence of 10 uM phentolamine. Incubations were also performed with [3H]DHA (specific radioactivity 45 Ci/m mol, New England Nuclear) in duplicate tubes at 25oc for 30 min. Total incubation volume was 0.25 ml: 0.175 ml of membrane preparation, 0.05 ml of an appropriate concentration of [3H]DHA and 0.025 ml of buffer or non-radioactive competitive ligand. Incubations were terminated by rapid dilution with 5 ml ice cold buffer and vacuum filtration through glass fiber filters (Whatman GF/B) which were washed 2 times with 5 ml of ice cold buffer. Specific binding was defined as total binding minus binding in the presence of i0 uM l-isoproterenol. l-Isoproterenol and other catecholamine solutions were made freshly before use to minimize the effect of oxidation. Radioactivity remaining on control filters, processed without membranes, ranged from 1 to 3% of total counts. Drugs were obtained from the following sources: l-isoproterenol, 1-epinephrine, l-norepinephrine, yohimbine, serotonin and dopamine - Sigma chemical Co.; phentolamine - Ciba Geigy Pharmaceutical Co.; d-epinephrine and d-norepinephrine - Sterling Winthrop; prazosin - Pfizer Pharmaceutical Co.
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Results Density and affinity of binding sites. The binding of [3H3prazosin to canine lung membrane was saturable. Specific binding reached a plateau between 3 to 4 nM of [3H3prazosin, and was generally 50 to 70% of the total binding at concentration up to 0.5 ruM. Scatchard analysis showed a high affinity binding sites with mean equilibrium dissociation constant (Kd) of 0.48 ± 0.3 nM (mean ± i S.E.M.) and mean maximal binding capacity (Bmax) of 22.1 ± 1.05 fmol/mg protein (mean ± i S.E.M.) (Table I). Hill plots indicated the absences of cooperativity. Fig. i shows a saturation curve and Scatchard plot of the data from Dog I. TABLE I Binding Capacities and Affinities of Adrenergic Receptors of Canine Lung
[3H]prazosin
Bmax
Kd
[3H3DHE
Bmax
[3H]DHA
Kd
Bmax
Kd
Dog I
21.6±1.0
0.36±0.03
Dog II
26.9±1.9
0.47±0.08
33.5±0.5
1.24±0.28
278.0±48.0
0.88±0.31
Dog III
18.9±1.1
0.43±0.04
36.3±4.8
1.19±0.29
180.4±18.4
0.98±0.08
Dog IV
24.9±0.5
0.48±0.12
42.7±3.9
0.98±0.13
359.4±10.9
0.92±0.15
Dog V
20.3
0.60
30.3
1.31
156.2
0.38
Dog VI
21.5±1.2
0.57±0.16
42.3±4.3
1.74±0.13
191.2±31.0
0.77±0.09
Dog VII
18.3±2.6
0.46±0.06
30.6±5.4
1.41±0.26
135.2±4.2
0.32±0.12
Dog VIII
20.5±3.2
0.61±0.11
34.1±0.2
1.51±0.08
280.4±66.4
0.78±0.33
Dog IX
20.9±0.3
0.47±0.07
31.6±2.3
1.29±0.23
247.6±52.8
0.78±0.04
Each value is the mean of 2-4 separate experiments (±i S.E.M.). In most experiments, specific binding of all three ligands were measured simultaneously in the same lung membrane preparation. Bmax is maximum binding in fmol/mg protein; Kd is the equilibrium dissociation constant in nM. The binding of [3H]DHE to canine lung membrane was also saturable. Specific binding reached a plateau between 4 to 5 nM of [3HSDHE, and was generally 40 to 60% of the total binding at concentration up to 1.0 nM. Scatachard analysis showed a high affinity binding sites with mean Kd of 1.33±0.09 nM and mean Bmax of 36.4±1.8 fmol/mg protein (Table I). Fig. 2 shows a saturation curve and a Scatchard plot of the data from Dog II. The specific binding of [3HSDHE was always more than that of [3H3prazosin when assayed in the same membrane preparation. Repeated measurement of receptor densities on the same animal lung over the time showed no significant difference. Kinetics of bindin 8. Specific binding of [3H]prazosin reached a plateau in I0 minutes at 25°C, remained at a steady state for I0 minutes and was reversible on the addition of 10 uM phentolamine with a t½ dissociation of 2.8 minutes (Fig.3)~ The pseudo-first order rate constant (Kob) was calculated as 0.33 min. -I and the second first order rate constant (K2) as 0.26 min. -I. The association constant (KI) was calculated as 0.16 x 109 M -I min. -I. using the formula [(Kob - K2)/(S)] where (S) is the concentration of [3B~prazosin used in the assay (0.43-nM)° The kinetic dissociation constant (Kd) from the ratio K2/K1 was calculated as 1.6 nM.
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Adrenergic Receptors
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Vol. 30, No. 12, 1982
3.20
5 ~ .
u
(£)
/ ~
B
o
u
n
e
Kd • 0"40 nM
,o,
d
(fmol/mg protein)
I
I
i
I
I
2
3
4
pH- prazosin]
nM
FIG. 1 Speclfic [3H]prazosin binding with increasing concentrations of [3H]prazosin to lung membranes of Dog I. Inset: Scatchard analysis with a s]ope -i/Kd determined by linear regression analysis. Bmax is calculated from the intercept of the plot with the abscissa as 23.2 fmol/mg protein. A
.5 30 2
Q,
m
°E2 0
~
el,-
c "o
c
2
Kd • 0"96 nM r - 0"93
/
.0, m°
Bound
u o
I0I 20I 30 , ( frnol/mg protein )
I
J
I
;
I
I
2
3
4
5
[ ' H - DHE]
n.
FIG. 2 Specific [3H]DHE binding with increasing concentrations of [3H]DHE to the lung membranes of Dog II. Inset: Scatchard analysis with a slope -i/Kd. Bmax is the intercept of the plot with abscissa.
Vol. 30, No.
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Adrenergic Receptors
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I00
"10
c o
50
,,..- TI/-Z = 1"8 rain min
c w o N O Ib
a, !
-r-
I
I
I
I
I
I0
20
:50
4.0
50
|
60
Time (minutes) FIG. 3 Timecourse of specific binding of [3H]prazosin to the lung membranes of Dog I at 25oC at a concentration of 0.43 riM. Reversibility of binding was measured after the addition of I0 uM phentolamine. Specific bindlng of [3H]DHE reached plateau in 15 min at 25°C, remained at a steady state for 40 minutes and was reversible on the addition of 10 uM phentolamine with t½ dissociation of 26 mlnutes (Fig. 4). The Kob was 0.13 min. -I and the K 2 was 0.023 min. -I. The Kd was calculated as 0.24 riM. Speciflcity of bindin$. The binding was sterospecific (Table II). The order of potency of catecholamine agonists in competing for [3H]prazosin binding sites showed 1-epinephrine >l-norepinephrine >d-epinephrine> d-norepinephr~ne. The blnding of [3H]DHE also showed stereospecificity in same order as that of [3H]prazosin (Table II). Both of these orders of potency are consistent with the classical pattern of alpha receptors. The potencies of alpha adrenerglc antagonists in competing for [3H]prazosin binding sltes showed prazosin >phentolamine >yohimbine (Table II). Inhibitlon curve of [3H]DHE binding by prazosln or yohimbine was blphasic suggesting that these antagonists interact with two distinct receptor populations (Fig. 5). Indirect Hill plots gave a slope factor of 0.19 for the curve by prazosin and 0.44 for the curve by yohimbine. In contrast, indirect Hill plots gave a slope factor of 0.92 for inhibition curve of [ 3 H ~ r a z o s i n binding by prazosin, 0.97 by phentolamine and 1.02 by yohimbine. Comparison of alpha and beta adrener$ic receptors. Binding of [3H]DHA was also saturable. Scatchard analysis showed high affinity binding sites with mean Kd of 0.83±0.06 nM and mean Bmax of 242.4±28.8 fmol/mg proteln. Fig. 6 shows saturation curve and a Scatchard plot of data from Dog IX.
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Adrenergic Receptors in Canine Lung
Vol. 30, No. 12, 1982
O
o
I00
q~ c s 0 i,J
-26rain
50
0 i
I
i
I0
20
I
30
Time
I
40
I
50
I
60
(minutes)
FIG. 4 Timecourse of specific binding of [3H]DHE to the lung membranes of Dog VIII at 25°C at concentration of 1.17 nM. Reversibility of binding was measured after the addition of 10 uM phentolamine. TABLE II Ki* Values for Agonists and Antagonists Determined by Inhibition Study [3H]prazosin
[3H]DHE
1-epinephrine
l°8x10 -7
1.8x10 -7
d-epinephrine
1.9x10 -6
3.6xi0 -6
l-norepinephrine
5.1x10 -7
2.7xi0 -7
d-norepinephrine
1.4x10 -5
1.0xl0 -5
l-isoproterenol
1.7x10 -5
2.8xi0 -5
Serontonin
l.lxl0 -3
Dopamine
5.4xi0 -4
Prazosin
3.0x10-Ii
Phentolamine
8.2xi0 -8
Yohimbine
2.2xi0-6
*Ki = ED50/(I+S/Kd); ED50: Molar concentration of agent causing 50% inhibition; S: Molar concentration of radioligand; Kd: Dissociation constant of radioligand. Each value is the mean of 2-4 separate experiments.
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Adrenergic Receptors in Canine Lung
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I00
°°I "U c ,q 0
50
50-
u.I -to I -r
m
I0
9
8
7
6
5
4
-IoQ [prozo,i@
I0
9 -log
8
7
6
5
4
[yohimbine]
FIG. 5 Inhibition curves of [3H]DHE binding to canine lung membrane by prazosin and yohimbine. The curves represent a typical experiment. Biphasic curves suggest two subtypes of [3H]DHE binding sites. The curves were eye-fitted. Ratios between alpha and beta adrenergic receptors calculated for each sample ranged from 1:3.3 to 1:10.3 (mean 1:5.8±1.0). The ratios between alpha 1 and alpha adrenergic receptors ranged from i:i.i to 1:2.6 (mean i:i.7±0.i). The ratio between alpha I and beta adrenergic receptors ranged from 1:6.7 to 1:21.1 (mean 1:10.2±0.9). Discussion Specific bindings of both [3H]prazosin and [3H]DHE to canine lung membrane were saturable, stereospecific and reversible. These findings satisfy the essential characteristics expected for alpha adrenergic receptors. Bmax of [3H]prazosin in an individual dog ranged from 18.3 to 26.9 fmol/mg protein, and Bmax of [3H]DHE ranged from 30.3 to 42.7 fmol/mg protein (Table I). When both ligands were used in the same membrane preparation, specific binding of [3H]DHE was always more than that of [3H]prazosin (p< 0.001). [3H]prazosin is considered to bind to alpha I adrenergic receptors specifically. (9,10) As shown in Table II, in the competition study, prazosin was much more potent than yohimbine in displacing the [3H]prazosin binding to canine lung membrane in our experimental system. Phentolamine was between prazosin and yohimbine. Inhibition curves by these three compounds were monophasic. These findings indicate that [3H]prazosin bound to alpha I adrenergic receptors of canine lung membrane specifically. (11,12) [3H]DHE is considered to bind to alpha adrenergic receptors nonselectively. (7) In our experimental system, [3H]DHE displacement curves by prazosin and yohimbine were biphasic, indicating two subtypes of binding sites. (7) Slope factors from indirect Hill plots for these curves were much less than one and different from slope factors for inhihibitior curve of [3H]prazos~n by prazosin, phentolamine and yohimbine which showed a slope
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Adrener~ic Receptors
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12, 1982
.E 300 e 41-
0
A
K E 1"5
-6 2 0 0 E
0 " 7 3 nM
/
c
/
'0 I=
..o.. ~
_ _
"~
emox-300.3
I00 IJ
Bound
o ¢,n
I
~.
I [~'H- DH A]
I O0 200 300 (fmoI/mg protein) I
2
nM
FIG. 6 Specific [3H]DHA binding with Increaslng concentrations of [3H]DHA to the lung membranes of Dog IX. Inset: Scatchard analysis with a slope -I/Kd. Bm,~x is the intercept of the plet with the abscissa. factor close to one as mentioned in results. [3H]DHE have been reported to bind to serotonin and dopamine receptors in addition to alpha adrenergic receptors ~n rat brain. (13) However, lower concentrations of [3H]DHE ( <5 nM) should speciflcally label the alpha receptors. It is not likely that [3H]DHE Interacted with serotonln or dopamine receptors in our experimental system, because specific binding of [3H]DHE satlsfied the characteristics expected for alpha adrenerglc receptors, and because serotonin and dopamlne were much less potent than 1-epinephrine and l-norepinephrlne in dlsplaclng [3H]DHE from the membrane binding sites. Thus, the difference between numbers of [3H]DHE binding sites and [SH]prazosin binding sites should represent alpha 2 adrenergic receptors. From the experimental data, numbers of alpha 2 adrenergic receptors of canlne lung tissue ranged from 4.3 to 31.1 fmol/mg protein. Appro-~imately 40% of alpha adrenergic receptors in canlne lung membranes were alpha 2 receptors. Our data suggests the existence of alpha 2 adrenergic receptors in canine lung tissue. The signiflcance of this observation is not clear now. The exlstence of non-presynaptic alpha 2 receptors has been reported in several mammalian tissues: brain (5), platelets (3,4), uterus (7), and adipocytes. (8) Alpha 2 adrenergic receptors have been reported to inhibit adenylate cyclase activity in platelets (4), adiDocytes (8,15) and thyroid tissue. (16) Several authors have reported physiologic alpha adrenergic respon~ive~ess in pulmonary tissue in mammalian trachea (17), human and canine tracheal and bronchial smooth muscle (18), and in rat peripheral lung strip. (19)
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These reports, however, did not distinguish between alpha I and alpha 2 adrenergic responsiveness. More recently, alpha I and alpha 2 adrenergic responses have been reported in canine trachea. (20) We determined the ratios alpha/beta 1:5.8 and alphal/beta 1:10.2. These values compared with the ratio alphal/beta 1:16 reported by Barnes, et al., in guinea pig lung membranes. (I0) They also reported reduced beta and increased alpha adrenergic receptor density in the lung tissue of guinea pig with experimentally induced asthma. (21) Szentivanyi observed adrenergic receptors shift from beta to alpha in lung tissues and lymphocytes from patients with asthma. (22) We observed the same tendency in lymphocytes from asthmatics compared to normal controls. (23) Szentivanyi proposed the hypothesis of imbalance of the autonomic nervous system with a beta adrenergic defect to explain the pathophysiology of asthma. (24) If this is the case, it is important to examine the numbers and ratios of alpha and beta adrenergic receptors in humans and in animals under various conditions. For example, changes of adrenergic receptor densities and affinities could be examined in asthmatics with or without drugs, immunological treatments and attacks. The membrane preparations used in these experiments were homogenates of whole lung, which included several different tissues which may have adrenergic receptors such as bronchioles, alveoli, blood vessels and parenchyma. It is difficult to determine the exact distribution of adrenergic receptors in lung tissues by direct binding techniques. We did not determine whether both alpha and beta adrenergic receptors are present on the same°cells. We determined the density and radioligand binding characteristics of alpha, alpha I, and beta adrenergic receptors and calculated their ratios in canine lung tissue. References I. 2. 3. 4. 5. 6. 7. 8.
9. i0. ii. 12. 13. 14. 15. 16. 17. 18.
S.Z. LANGER, Biochem. Pharmacol. 23 1893-1800 (1974). S. BERTHELSEN and W.A. PETTINGER, Life Sci. 21 595-606 (1977). K.D. NEWMAN, L.T. WILLIAMS, N.H. BISHOPRIC and R.J. LEFKOWITZ, J. Clin. Invest. 61 395-402 (1978). R.W. ALEXANDER, B. COPPER, and R.I. HANDIN, J. Clin. Invest. 61 11361144 (1978). A. HAGGI, D.C. U'PRICHARD and S.J. ENNA, Science 207 645-647 (1980). B.B. HOFFMAN, D.F. DUKES and R.J. LEFKOWITZ, Life Sci. 28 265-272 (1981). B.B. HOFFMAN, A. DE LEAN, C.L. WOOD, D.D. SCHOCKEN and R.J. LEFKOWITZ, Life Sci. 24 1793-1746 (1979). T.W. BURNES, P.E. LANGLEY, B.E. TERRY, D.B. BYLUND, B.B. HOFFMAN, M.D. THARP, R.J. LEFKOWITZ, J.A. GARCIA-SAINZ and J.N. FAIN, J° Clin. Invest. 67 467-475 (1981). P. GREENGRASS and R. BREMER, Eur. J. Pharmacol. 55 323-326 (1979). P. BARNES, J. KARLINER, C. HAMILTON and C. DOLLERY, Life Sci. 25 12071214 (1979). C.L. WOOD, C.D. ARNETT, W.R. CLARK, B.S. TSAI and R.J. LEFKOWITZ, Biochem. Pharmacol. 28 1277-1282 (1979). B.B. HOFFMAN and R.J. LEFKOWITZ, N. Eng. J. Med. 302 1390-1396 (1980). J.N. DAVIS, W. STRITTMATTER, E. HOYLER and R.J. LEFKOWITZ, Brain Res. 132 327-336 (1977). D.A. GREENBERG and S.H. SNYDER, Life Sci. 20 927-931 (1977). J.A. GARCIA-SAINZ, B.B. HOFFMAN, SHIH-YING LI, J. LEFKOWITZ and J.N. FAIN, Life Sci. 27 953-961 (1980). K. YAMASHITA, S. YAMASHITA and Y. ARIYOSHI, Life Sci. 27 1127-1130 (1980). J.H. FLEISCH, H.M. MALING and B. BRODIE, Am. J. Physiol. 218 596-599 (1970). M.P. KNUSSL and J.B. RICHARDSON, J. Appl. Physiol. 45 307-311 (1978).
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N. Chand, Res. Com. Chem. Path. Pharmacol. 25 215-226 (1979). K. LEE, A. BEWTRA and R.G. TOWNLEY, FASEB 235 (1981) (abst). P.J. BARNES, C.T. DOLLERY and J. MAC DERMOT, Nature 285 569-571 (1980). A. SZENTIVANYI, O. HEIM and P.S. SCHULTZE, Ann. N.Y. Acd. Sci. 332 295298 (1979). 23. Y. SANO, K. KROKOS, J.B. CHENG, A. BEWTRA and R.G. TOWNLEY, Clin. Res. 29 172A (1981) (abst). 24. A. SZENTIVANYI, J. Allergy 42 203-232 (1968).
Pergamon Press
ALPHA AND BETA ADRENERGIC RECEPTORS OF CANINE LUNG TISSUE IDENTIFICATION AND C}~RACTERIZATION OF ALPHA ADRENERGIC RECEPTORS BY TWO DIFFERENT LIGANDS Maki Hasegawa, M.D. and Robert G. Townley, M.D. Allergic Disease Center, Creighton University School of Medicine 2500 California Street Omaha, NE 68178 (Received in final form January 18, 1982) Summary Adrenergic receptors of canine peripheral lung tissues were measured by direct binding techniques u~in~ [3H]dihydroergocryptine ([3H]DHE), [3H]prazosim and [3H]dihydroalpreno]ol ([3H]DHA). All three ligands bound to canine lung tissue with saturabllity, stereospecificity and reversibility. Adrenergic agonists competed for binding'of [3H]DHE and [3H]prazosln in the order: l-epinephrlne > l-norepinephrine > d-epinephrine> d-norepinephrine > l-isoproterenol. Adrenerglc antagonists competed for binding of [3H]prazosln in the order: prazosin> phentolamine>yohimbine. Inhibition curves of [3H]DHE by prazosin or yohimbine were biphasic suggestlng two subtypes of binding sites. Maxlmum binding capacities of [3H]DHE ranged from 30.6 to 42.7 fmol/mg protein. [3H]prazosln from 18.3 to 26.9 fmol/mg protein and [3H]DHA from 135.2 to 359.4 fmol/mg protein. When both [3H]DHE and [3H]prazosin were used in the same membrane preparation, specific binding of [3H]DHE was always more than that of [3H]prazosin. Since [3H]prazosin is considered to bind to alpha I adrenergic receptors speciflcally and [3H]DHE is considered to bind alpha 2 adrenergic receptors nonselectively, the difference between the numbers of the specific binding sites of these two ligands should represent alpha 2 adrenergic receptors. Alpha 2 adrenergic receptor density ranged from 9.5 to 21.1 fmol/mg proteln. Our results suggest the exlstence of both alpha I and alpha 2 adrenergic receptors in canine peripheral lung tissue. Approximately 40% of alpha adrenergic receptors were alpha 2. The ratio of alpha/beta adrenrgic receptors ranged from 1:3.3 to 1:10.4. The ratio of alpha]/ beta adrenergic receptors ranged from 1:6.7 to 1:21.1. Alpha adrenergic receptors are dlvlded into two subclasses. (],2) Alpha I adrenergic receptors include postsynaptic alpha receptors which contract smooth muscles. Alpha 2 adrenerglc receptors include presynaptic alpha adrenerglc receptors which inhibit the release of norepinephrlne from sympathetic nerve endings, and postsynaptic alpha adrenergic receptors, for example alpha adrenergic receptors in human platelets. (3,4) Several investigators uslng radioligand binding techniques have reported detecting alpha I and alpha 2 receptors simultaneously in rat central nervous system (5), rat liver (6), rabbit uterus (7), and human adipocytes. (8) We prepared membrane fractions from canine lung tissue, and measured alpha adrenergic receptors using two different ligands, [3H]prazosin and [3H]dihydroergocryptine ([ 3H]DHE), to detect alph a receptors In the same membrane preparation. [3H]prazosin is a potent alpha I adrenergic antagonist and is considered to bind to alpha I adrenergic receptors specifically (9), [3H]DHE is a potent alpha 0024-3205/82/121035-10503,00/0 Copyright (c) 1982 Pergamon Press Ltd,
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antagonlst and is considered to bind to alpha I and alpha 2 receptors nonselectively. (7) When both [3H]prazosin and [3H]DHE are used in the same membrane preparation, the difference between the numbers of specific binding sites of these two ligands should represent alpha 2 adrenergic receptors. We used [3H]dihydroalprenolol ([3H]DHA) to measure beta adrenergic receptors in these same membrane preparations, and calculated the ratios between alpha and beta and between alpha I and beta receptors. To our knowledge, this is the first report describlng alpha adrenergic receptors in canine lung tissue. Methods and Materials Lungs were rapidly removed from dogs immediately after death by exsangulnation and stored at -60°C until assay. Lungs were used within 3 months. For membrane preparation, thawed lung tissues were dissected free of major bronchi, vessels and connective tissues by forceps, and minced to small pieces in 10 volumes of ice cold buffer (Tris-HCl 5mM, MgC] 2 1 mM, pH 7.4). These pieces were homogenized at full speed for 30 seconds using a Brinkmann Polytron. The homogenate was then centrifuged at 50 x g for 20 minutes. Fibrous tissues and other high density debris were removed by this centrifugatlon. The supernatant was recentrifuged at 45,000 x g for 15 minutes. The resulting pellets were resuspended in the same buffer at a concentration of approximately 1.5 mg protein/ml after filtration through a cheesecloth. All procedures were performed at 4oc. Protein concentration was determined by the method of Lowry. Membranes were incubated in triplicate tubes for ]5 minutes at 25oc with [3H]prazosin (specific radioactivity 33 Ci/m mol, Amersham), or for 20 minutes at 25°C with [3H]DHE (specific radioactivity 30.9 Ci/m mol, New England Nuclear). To each tube, 0.85 ml of membrane preparation, 0.05 ml of an appropriate concentration of radioligand and 0.I ml of either buffer or non-radioactive competitive ligand were added to make a total incubation volume of 1.0 ml. Incubations were terminated by rapid dilution with 6 ml of ice cold buffer and vacuum filtration through glass fiber filters (Whatman GF/B) which were washed 3 times with 6 ml of ice cold buffer. Filters were placed in 10 ml of toluene-based scintilant and radioactivities were determined in liquid scintilation counter (Tracor Analytic, Model 6892) at an efficiency of 45%. Specific binding was defined as total binding minus binding in the presence of 10 uM phentolamine. Incubations were also performed with [3H]DHA (specific radioactivity 45 Ci/m mol, New England Nuclear) in duplicate tubes at 25oc for 30 min. Total incubation volume was 0.25 ml: 0.175 ml of membrane preparation, 0.05 ml of an appropriate concentration of [3H]DHA and 0.025 ml of buffer or non-radioactive competitive ligand. Incubations were terminated by rapid dilution with 5 ml ice cold buffer and vacuum filtration through glass fiber filters (Whatman GF/B) which were washed 2 times with 5 ml of ice cold buffer. Specific binding was defined as total binding minus binding in the presence of i0 uM l-isoproterenol. l-Isoproterenol and other catecholamine solutions were made freshly before use to minimize the effect of oxidation. Radioactivity remaining on control filters, processed without membranes, ranged from 1 to 3% of total counts. Drugs were obtained from the following sources: l-isoproterenol, 1-epinephrine, l-norepinephrine, yohimbine, serotonin and dopamine - Sigma chemical Co.; phentolamine - Ciba Geigy Pharmaceutical Co.; d-epinephrine and d-norepinephrine - Sterling Winthrop; prazosin - Pfizer Pharmaceutical Co.
Vol. 30, No. 12, 1982
Adrenergic Receptors in Canine Lung
1037
Results Density and affinity of binding sites. The binding of [3H3prazosin to canine lung membrane was saturable. Specific binding reached a plateau between 3 to 4 nM of [3H3prazosin, and was generally 50 to 70% of the total binding at concentration up to 0.5 ruM. Scatchard analysis showed a high affinity binding sites with mean equilibrium dissociation constant (Kd) of 0.48 ± 0.3 nM (mean ± i S.E.M.) and mean maximal binding capacity (Bmax) of 22.1 ± 1.05 fmol/mg protein (mean ± i S.E.M.) (Table I). Hill plots indicated the absences of cooperativity. Fig. i shows a saturation curve and Scatchard plot of the data from Dog I. TABLE I Binding Capacities and Affinities of Adrenergic Receptors of Canine Lung
[3H]prazosin
Bmax
Kd
[3H3DHE
Bmax
[3H]DHA
Kd
Bmax
Kd
Dog I
21.6±1.0
0.36±0.03
Dog II
26.9±1.9
0.47±0.08
33.5±0.5
1.24±0.28
278.0±48.0
0.88±0.31
Dog III
18.9±1.1
0.43±0.04
36.3±4.8
1.19±0.29
180.4±18.4
0.98±0.08
Dog IV
24.9±0.5
0.48±0.12
42.7±3.9
0.98±0.13
359.4±10.9
0.92±0.15
Dog V
20.3
0.60
30.3
1.31
156.2
0.38
Dog VI
21.5±1.2
0.57±0.16
42.3±4.3
1.74±0.13
191.2±31.0
0.77±0.09
Dog VII
18.3±2.6
0.46±0.06
30.6±5.4
1.41±0.26
135.2±4.2
0.32±0.12
Dog VIII
20.5±3.2
0.61±0.11
34.1±0.2
1.51±0.08
280.4±66.4
0.78±0.33
Dog IX
20.9±0.3
0.47±0.07
31.6±2.3
1.29±0.23
247.6±52.8
0.78±0.04
Each value is the mean of 2-4 separate experiments (±i S.E.M.). In most experiments, specific binding of all three ligands were measured simultaneously in the same lung membrane preparation. Bmax is maximum binding in fmol/mg protein; Kd is the equilibrium dissociation constant in nM. The binding of [3H]DHE to canine lung membrane was also saturable. Specific binding reached a plateau between 4 to 5 nM of [3HSDHE, and was generally 40 to 60% of the total binding at concentration up to 1.0 nM. Scatachard analysis showed a high affinity binding sites with mean Kd of 1.33±0.09 nM and mean Bmax of 36.4±1.8 fmol/mg protein (Table I). Fig. 2 shows a saturation curve and a Scatchard plot of the data from Dog II. The specific binding of [3HSDHE was always more than that of [3H3prazosin when assayed in the same membrane preparation. Repeated measurement of receptor densities on the same animal lung over the time showed no significant difference. Kinetics of bindin 8. Specific binding of [3H]prazosin reached a plateau in I0 minutes at 25°C, remained at a steady state for I0 minutes and was reversible on the addition of 10 uM phentolamine with a t½ dissociation of 2.8 minutes (Fig.3)~ The pseudo-first order rate constant (Kob) was calculated as 0.33 min. -I and the second first order rate constant (K2) as 0.26 min. -I. The association constant (KI) was calculated as 0.16 x 109 M -I min. -I. using the formula [(Kob - K2)/(S)] where (S) is the concentration of [3B~prazosin used in the assay (0.43-nM)° The kinetic dissociation constant (Kd) from the ratio K2/K1 was calculated as 1.6 nM.
1038
Adrenergic Receptors
in Canlne Lung
Vol. 30, No. 12, 1982
3.20
5 ~ .
u
(£)
/ ~
B
o
u
n
e
Kd • 0"40 nM
,o,
d
(fmol/mg protein)
I
I
i
I
I
2
3
4
pH- prazosin]
nM
FIG. 1 Speclfic [3H]prazosin binding with increasing concentrations of [3H]prazosin to lung membranes of Dog I. Inset: Scatchard analysis with a s]ope -i/Kd determined by linear regression analysis. Bmax is calculated from the intercept of the plot with the abscissa as 23.2 fmol/mg protein. A
.5 30 2
Q,
m
°E2 0
~
el,-
c "o
c
2
Kd • 0"96 nM r - 0"93
/
.0, m°
Bound
u o
I0I 20I 30 , ( frnol/mg protein )
I
J
I
;
I
I
2
3
4
5
[ ' H - DHE]
n.
FIG. 2 Specific [3H]DHE binding with increasing concentrations of [3H]DHE to the lung membranes of Dog II. Inset: Scatchard analysis with a slope -i/Kd. Bmax is the intercept of the plot with abscissa.
Vol. 30, No.
12, 1982
Adrenergic Receptors
in Canine Lung
1039
I00
"10
c o
50
,,..- TI/-Z = 1"8 rain min
c w o N O Ib
a, !
-r-
I
I
I
I
I
I0
20
:50
4.0
50
|
60
Time (minutes) FIG. 3 Timecourse of specific binding of [3H]prazosin to the lung membranes of Dog I at 25oC at a concentration of 0.43 riM. Reversibility of binding was measured after the addition of I0 uM phentolamine. Specific bindlng of [3H]DHE reached plateau in 15 min at 25°C, remained at a steady state for 40 minutes and was reversible on the addition of 10 uM phentolamine with t½ dissociation of 26 mlnutes (Fig. 4). The Kob was 0.13 min. -I and the K 2 was 0.023 min. -I. The Kd was calculated as 0.24 riM. Speciflcity of bindin$. The binding was sterospecific (Table II). The order of potency of catecholamine agonists in competing for [3H]prazosin binding sites showed 1-epinephrine >l-norepinephrine >d-epinephrine> d-norepinephr~ne. The blnding of [3H]DHE also showed stereospecificity in same order as that of [3H]prazosin (Table II). Both of these orders of potency are consistent with the classical pattern of alpha receptors. The potencies of alpha adrenerglc antagonists in competing for [3H]prazosin binding sltes showed prazosin >phentolamine >yohimbine (Table II). Inhibitlon curve of [3H]DHE binding by prazosln or yohimbine was blphasic suggesting that these antagonists interact with two distinct receptor populations (Fig. 5). Indirect Hill plots gave a slope factor of 0.19 for the curve by prazosin and 0.44 for the curve by yohimbine. In contrast, indirect Hill plots gave a slope factor of 0.92 for inhibition curve of [ 3 H ~ r a z o s i n binding by prazosin, 0.97 by phentolamine and 1.02 by yohimbine. Comparison of alpha and beta adrener$ic receptors. Binding of [3H]DHA was also saturable. Scatchard analysis showed high affinity binding sites with mean Kd of 0.83±0.06 nM and mean Bmax of 242.4±28.8 fmol/mg proteln. Fig. 6 shows saturation curve and a Scatchard plot of data from Dog IX.
1040
Adrenergic Receptors in Canine Lung
Vol. 30, No. 12, 1982
O
o
I00
q~ c s 0 i,J
-26rain
50
0 i
I
i
I0
20
I
30
Time
I
40
I
50
I
60
(minutes)
FIG. 4 Timecourse of specific binding of [3H]DHE to the lung membranes of Dog VIII at 25°C at concentration of 1.17 nM. Reversibility of binding was measured after the addition of 10 uM phentolamine. TABLE II Ki* Values for Agonists and Antagonists Determined by Inhibition Study [3H]prazosin
[3H]DHE
1-epinephrine
l°8x10 -7
1.8x10 -7
d-epinephrine
1.9x10 -6
3.6xi0 -6
l-norepinephrine
5.1x10 -7
2.7xi0 -7
d-norepinephrine
1.4x10 -5
1.0xl0 -5
l-isoproterenol
1.7x10 -5
2.8xi0 -5
Serontonin
l.lxl0 -3
Dopamine
5.4xi0 -4
Prazosin
3.0x10-Ii
Phentolamine
8.2xi0 -8
Yohimbine
2.2xi0-6
*Ki = ED50/(I+S/Kd); ED50: Molar concentration of agent causing 50% inhibition; S: Molar concentration of radioligand; Kd: Dissociation constant of radioligand. Each value is the mean of 2-4 separate experiments.
Vol. 30, No. 12, 1982
Adrenergic Receptors in Canine Lung
1041
I00
°°I "U c ,q 0
50
50-
u.I -to I -r
m
I0
9
8
7
6
5
4
-IoQ [prozo,i@
I0
9 -log
8
7
6
5
4
[yohimbine]
FIG. 5 Inhibition curves of [3H]DHE binding to canine lung membrane by prazosin and yohimbine. The curves represent a typical experiment. Biphasic curves suggest two subtypes of [3H]DHE binding sites. The curves were eye-fitted. Ratios between alpha and beta adrenergic receptors calculated for each sample ranged from 1:3.3 to 1:10.3 (mean 1:5.8±1.0). The ratios between alpha 1 and alpha adrenergic receptors ranged from i:i.i to 1:2.6 (mean i:i.7±0.i). The ratio between alpha I and beta adrenergic receptors ranged from 1:6.7 to 1:21.1 (mean 1:10.2±0.9). Discussion Specific bindings of both [3H]prazosin and [3H]DHE to canine lung membrane were saturable, stereospecific and reversible. These findings satisfy the essential characteristics expected for alpha adrenergic receptors. Bmax of [3H]prazosin in an individual dog ranged from 18.3 to 26.9 fmol/mg protein, and Bmax of [3H]DHE ranged from 30.3 to 42.7 fmol/mg protein (Table I). When both ligands were used in the same membrane preparation, specific binding of [3H]DHE was always more than that of [3H]prazosin (p< 0.001). [3H]prazosin is considered to bind to alpha I adrenergic receptors specifically. (9,10) As shown in Table II, in the competition study, prazosin was much more potent than yohimbine in displacing the [3H]prazosin binding to canine lung membrane in our experimental system. Phentolamine was between prazosin and yohimbine. Inhibition curves by these three compounds were monophasic. These findings indicate that [3H]prazosin bound to alpha I adrenergic receptors of canine lung membrane specifically. (11,12) [3H]DHE is considered to bind to alpha adrenergic receptors nonselectively. (7) In our experimental system, [3H]DHE displacement curves by prazosin and yohimbine were biphasic, indicating two subtypes of binding sites. (7) Slope factors from indirect Hill plots for these curves were much less than one and different from slope factors for inhihibitior curve of [3H]prazos~n by prazosin, phentolamine and yohimbine which showed a slope
1042
Adrener~ic Receptors
in Canine Lun~
Vol.
30, No.
12, 1982
.E 300 e 41-
0
A
K E 1"5
-6 2 0 0 E
0 " 7 3 nM
/
c
/
'0 I=
..o.. ~
_ _
"~
emox-300.3
I00 IJ
Bound
o ¢,n
I
~.
I [~'H- DH A]
I O0 200 300 (fmoI/mg protein) I
2
nM
FIG. 6 Specific [3H]DHA binding with Increaslng concentrations of [3H]DHA to the lung membranes of Dog IX. Inset: Scatchard analysis with a slope -I/Kd. Bm,~x is the intercept of the plet with the abscissa. factor close to one as mentioned in results. [3H]DHE have been reported to bind to serotonin and dopamine receptors in addition to alpha adrenergic receptors ~n rat brain. (13) However, lower concentrations of [3H]DHE ( <5 nM) should speciflcally label the alpha receptors. It is not likely that [3H]DHE Interacted with serotonln or dopamine receptors in our experimental system, because specific binding of [3H]DHE satlsfied the characteristics expected for alpha adrenerglc receptors, and because serotonin and dopamlne were much less potent than 1-epinephrine and l-norepinephrlne in dlsplaclng [3H]DHE from the membrane binding sites. Thus, the difference between numbers of [3H]DHE binding sites and [SH]prazosin binding sites should represent alpha 2 adrenergic receptors. From the experimental data, numbers of alpha 2 adrenergic receptors of canlne lung tissue ranged from 4.3 to 31.1 fmol/mg protein. Appro-~imately 40% of alpha adrenergic receptors in canlne lung membranes were alpha 2 receptors. Our data suggests the existence of alpha 2 adrenergic receptors in canine lung tissue. The signiflcance of this observation is not clear now. The exlstence of non-presynaptic alpha 2 receptors has been reported in several mammalian tissues: brain (5), platelets (3,4), uterus (7), and adipocytes. (8) Alpha 2 adrenergic receptors have been reported to inhibit adenylate cyclase activity in platelets (4), adiDocytes (8,15) and thyroid tissue. (16) Several authors have reported physiologic alpha adrenergic respon~ive~ess in pulmonary tissue in mammalian trachea (17), human and canine tracheal and bronchial smooth muscle (18), and in rat peripheral lung strip. (19)
Vol. 30, No.
12, 1982
Adrenergic Receptors
in Canine Lung
1043
These reports, however, did not distinguish between alpha I and alpha 2 adrenergic responsiveness. More recently, alpha I and alpha 2 adrenergic responses have been reported in canine trachea. (20) We determined the ratios alpha/beta 1:5.8 and alphal/beta 1:10.2. These values compared with the ratio alphal/beta 1:16 reported by Barnes, et al., in guinea pig lung membranes. (I0) They also reported reduced beta and increased alpha adrenergic receptor density in the lung tissue of guinea pig with experimentally induced asthma. (21) Szentivanyi observed adrenergic receptors shift from beta to alpha in lung tissues and lymphocytes from patients with asthma. (22) We observed the same tendency in lymphocytes from asthmatics compared to normal controls. (23) Szentivanyi proposed the hypothesis of imbalance of the autonomic nervous system with a beta adrenergic defect to explain the pathophysiology of asthma. (24) If this is the case, it is important to examine the numbers and ratios of alpha and beta adrenergic receptors in humans and in animals under various conditions. For example, changes of adrenergic receptor densities and affinities could be examined in asthmatics with or without drugs, immunological treatments and attacks. The membrane preparations used in these experiments were homogenates of whole lung, which included several different tissues which may have adrenergic receptors such as bronchioles, alveoli, blood vessels and parenchyma. It is difficult to determine the exact distribution of adrenergic receptors in lung tissues by direct binding techniques. We did not determine whether both alpha and beta adrenergic receptors are present on the same°cells. We determined the density and radioligand binding characteristics of alpha, alpha I, and beta adrenergic receptors and calculated their ratios in canine lung tissue. References I. 2. 3. 4. 5. 6. 7. 8.
9. i0. ii. 12. 13. 14. 15. 16. 17. 18.
S.Z. LANGER, Biochem. Pharmacol. 23 1893-1800 (1974). S. BERTHELSEN and W.A. PETTINGER, Life Sci. 21 595-606 (1977). K.D. NEWMAN, L.T. WILLIAMS, N.H. BISHOPRIC and R.J. LEFKOWITZ, J. Clin. Invest. 61 395-402 (1978). R.W. ALEXANDER, B. COPPER, and R.I. HANDIN, J. Clin. Invest. 61 11361144 (1978). A. HAGGI, D.C. U'PRICHARD and S.J. ENNA, Science 207 645-647 (1980). B.B. HOFFMAN, D.F. DUKES and R.J. LEFKOWITZ, Life Sci. 28 265-272 (1981). B.B. HOFFMAN, A. DE LEAN, C.L. WOOD, D.D. SCHOCKEN and R.J. LEFKOWITZ, Life Sci. 24 1793-1746 (1979). T.W. BURNES, P.E. LANGLEY, B.E. TERRY, D.B. BYLUND, B.B. HOFFMAN, M.D. THARP, R.J. LEFKOWITZ, J.A. GARCIA-SAINZ and J.N. FAIN, J° Clin. Invest. 67 467-475 (1981). P. GREENGRASS and R. BREMER, Eur. J. Pharmacol. 55 323-326 (1979). P. BARNES, J. KARLINER, C. HAMILTON and C. DOLLERY, Life Sci. 25 12071214 (1979). C.L. WOOD, C.D. ARNETT, W.R. CLARK, B.S. TSAI and R.J. LEFKOWITZ, Biochem. Pharmacol. 28 1277-1282 (1979). B.B. HOFFMAN and R.J. LEFKOWITZ, N. Eng. J. Med. 302 1390-1396 (1980). J.N. DAVIS, W. STRITTMATTER, E. HOYLER and R.J. LEFKOWITZ, Brain Res. 132 327-336 (1977). D.A. GREENBERG and S.H. SNYDER, Life Sci. 20 927-931 (1977). J.A. GARCIA-SAINZ, B.B. HOFFMAN, SHIH-YING LI, J. LEFKOWITZ and J.N. FAIN, Life Sci. 27 953-961 (1980). K. YAMASHITA, S. YAMASHITA and Y. ARIYOSHI, Life Sci. 27 1127-1130 (1980). J.H. FLEISCH, H.M. MALING and B. BRODIE, Am. J. Physiol. 218 596-599 (1970). M.P. KNUSSL and J.B. RICHARDSON, J. Appl. Physiol. 45 307-311 (1978).
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Adrenergic Receptors in Canine Lung
Vol. 30, No. 12, 1982
N. Chand, Res. Com. Chem. Path. Pharmacol. 25 215-226 (1979). K. LEE, A. BEWTRA and R.G. TOWNLEY, FASEB 235 (1981) (abst). P.J. BARNES, C.T. DOLLERY and J. MAC DERMOT, Nature 285 569-571 (1980). A. SZENTIVANYI, O. HEIM and P.S. SCHULTZE, Ann. N.Y. Acd. Sci. 332 295298 (1979). 23. Y. SANO, K. KROKOS, J.B. CHENG, A. BEWTRA and R.G. TOWNLEY, Clin. Res. 29 172A (1981) (abst). 24. A. SZENTIVANYI, J. Allergy 42 203-232 (1968).