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Photoaffinity labeling of the benzodiazepine receptor in bovine cerebral cortex
Life Sciences, Vol . 25, pp . 1613-1620 Printed in the U.S .A .
Pergamon Preae
PHOTOAFFINITY LABELING OF THE BENZODIAZEPINE RECEPTOR IN BOVINE CEREBRAL CORTER Richard W. Johnson and Henry I . Yamamura Departments of Biochemistry, Pharmacology and Psychiatry College of Medicine, university of Arizona Health Sciences Center Tucson, AZ 85724 (Received in final form September 24, 1979)
SUMMARY Cloaazepam, nitrazepam and flunitrazepam were found to engage in an irreversible interaction with benzodiazepine binding sites in bovine cerebral cortex homogenates upon irradiation with ultraviolet light . Photoaffinity labeling with [ H]flunitrazepam could be substantially (approx . 85%) inhibited by a number of different benzodiazepines, including clonazepam, lorazepam, Roy-3027, and non-radioactive fluaitrazepam . Spiroperidol, atropine, naltrexone, propranolol and GABA had no effect on irreversible [3A]flunitrazepam binding, indicating that this binding is to the benzodiazepine receptor as defined in previous studies . INTRODUCTION Specific, high affinity binding of benzodiazepinea has been shown to occur in brain tissue of humane and other animals (1-6) . We have undertaken to characterize the sites to which this binding occurs in order to elucidate the physiologic relevance of this phenomenon . Toward this end, it was desirable to obtain a radioactive affinity label, which would be expected to facilitate isolation and characterization of the benzodiazepine receptor . Recently, certain benzodiazepines have been reported to be capable of binding irreversibly to mammalian brain tüsue (7,8) . We therefore investigated the effects of ultraviolet irradiation oa the dissociation of [ 3H]flunitrazepam and certain nonradioactive benzodiazepines . hETHODS AND MATERIALS Bovine brains were obtained within 1 hr . of death, dissected, and stored at -20°C until used in our experiments . [3H]Flunitrazepam (specific activity 87 .5 Ci/mM) was obtained from New England Nuclear Corp . (Boston, Masa) . Clonazepam, nitrazepam, lorazepam, diazepam, Roy-3027, and non-radioactive flunitrazepam were provided by Hoffman LaRoche, Inc . Photoaffinity labeling of benzodiazepine binding sites was performed as follows : bovine tortes was homogenized using a Polytron homogenizer in 100 volumes (w/v) of ice-cold NaRPOy buffer (50 mM, pH 7 .4) and centrifuged at 49,500 x g for 10 minutes, after which the supernatant was decanted and the pellet was rehomogenized in the original whims of buffer ; 10-20 milliliters of the re0024-3205/79/181613-08$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1614
Benzodiazepine Receptors
Vol . 25, No . 18, 1979
salting homogenate was incubated with the indicated concentrations of ligand for 15 min at 0 deg~eea and then photolyzed at 0 degrees for the indicated lengths of time from above in small beakers at a distance of approximately 6 centimeters using a Mineralight model C-81 short wave ultraviolet light . Quantification of irreversible [ 3 H]flunitrazepam binding was performed as follows : after photolgsis, the tissue was centrifuged at 49,500 x g for 10 min . and rehomogenized in the original volume of buffer ; 500 microliter aliquots of the resulting homogenate were then incubated in the presence of 10 micromolar clonazepam in a total whims of 2 ml . for 60 minutes at 0 degrees, whereupon the samples were poured over GF/B filters (Whatman) and washed 3 times with 5 ml rinses of buffer . Radioactivity retained at the filters was then determined by extraction with a Triton-based scintillation cocktail and liquid scintillation spectrometry . Qualitatively similar results were obtained when the tissue, after photolysis, was subjected to repeated washing (centrifugation followed by rehomogenization) and/or exhaustive dialysis instead of displacement with clonazepam in order to remove imreacted ligand ; however both of these methods were much more time consuming than the first procedure and were accompanied by lower receptor yields . Quantification of photoaffinity labeling with non-radioactive ligands was performed by incubating 500 microliter aliquots of the photolyzed tissue, after washing 3 times as described above to remove unreacted ligand, with 1 .5 nM [3H]flimitrazepam in the presence and absence of 1 micromolar clonazepam for 60 min. at 0 degrees and then filtering as described above . Subtracting and radioactivity retained after filtering samples in which clonazepam was present from that retained after filtering samples in which only [3H]fluaitrazepam was present yielded the amount of specific [ 3H]flunitrazepam binding by the tissue . A decrease in this parameter after photolyzes in the presence of a particular ligand was used as an index of irreversible binding of the ligand to the receptor . Control values for [3H]flunitrazepam binding were defined as that binding to tissue treated in an identical manner in all respects except for the presence of ligand during photolysis . Saturation studies of unlabeled receptors before and after photolyzes were performed by incubating 250 microliter aliquots of tissue with various concentrations of [3H]flunitrazepam in the presence and absence of 1 micromolar clon azepam and then filtering and counting the samples as described above . As can he seen from Fig . 1, irradiation with UV light precludes removal of radioactivity from the tissue by centrifugation followed by rehomogenization and incubation in the presence of 10 micromolar clonazepam . Photoaffinity labeling could be substantially (87 .2%) inhibited by the presence of a large excess of clonazepam during photolysis . An 85% reduction of irreversible [ 3H]flunitrazepam binding could also be achieved with micromolar concentrations of lorazepam, Roy-3027, and non-radioactive flunitrazepam, however micromolar concentrations of atropine, apiroperidol, propranolol, naltrexone and GABA had no significant effect on irreversible ( 3H]flunitrazepam binding, indicating that this binding is primarily to the benzodiazepine receptor as defined in previous studies . The effects of varying concentrations of two different non-radioactive benzodiazepines on irreversible [3H]flunitrazepam binding are shown in Figure 2 . Note that a sufficiently large excess of either of these ligands will substantially inhibit photoaffinity labeling . Of particular interest, however, is the wide disparity between the ICSp values for these two compounds under these circumstances (4 .46 X 10 -1 ~ M vs . 3 .55 R 10 -B M) despite the fact that they have almost identical affinities for the benzodiazepine receptor under ordinary circumstances (9) . This enigmatic observation would be explicable if clonazepam itself was capable of engaging in an irreversible interaction with the receptor while lorazepam was not . In order to investigate this possibility, experiments were performed to quantify covalent binding of these and other benzodiazepinea
Benzodiazepine Receptors
Vol . 25, No . 18, 1979
1615
RESULTS AND DISCUSSION Figure 1 shows the effect of UV irradiation on [3H]flunitrazepam dissociation .
IOr
I "r ~N rLYNITIIAZVAr
mr aN rw Nu I~r CLOIIAi~Mr
X
n
E
_U
° z
5
0
m
a aW
N Q H Z ~
W
4 3
2
A
NO UV
4HRâ UV
Figure 1 Effect of UV li(~ht on [ 3N]flunitrazepam dissociation .
Beazodiazepine Receptors
1616
Vol . 25, No . 18, 1979
ioo
0 3
C WC C
D
~ ~ -LOY DIlPLACEH CONGfwalar)
7
Figure 2 Inhibition of irreversible [3H]flunitrazepam binding by clonazepam ( " ) and lorazepam (~ ) .
in the presence of W light . The results we obtained are presented in Fig 3, which illustrates that photolysis of brain tissue with 1 .25 nM clonazepam followed by extensive washing results in a large (86 .4X) reduction in [ 3H]flimitrazepam binding when compared to tissue photolyzed in the absence of clonazepam but treated identically in all other respects . Note that when tissue is incubated with an identical concentration of clonazepam for the same amount of time in the dark there is a negligible (5 .7X) reduction in [ 3H]flunitrazepam binding . Scatchard analysis of [ H]flunitrazepam binding to the tissue before an after photolysis showed that the reduction in binding was entirely due to a decrease in the number of available receptors,indicating a non-competitive mechanism. Thus, clonazepam appears to participate in an irreversible interaction with the benzodiazepine receptor when subjected to W irradiation .
Benzodiazepine Receptors
Vol . 25, No . 18, 1979
1617
IOOr
70
sz
ö 60 z
W 40
N C F
~ 30 W q O~~ Z _O F
~ I~ O W C
No
uv
rsHRS uv
FIGURE 3 Effect of ultraviolet irradiation on diasociatioa of clonazepam from the benzodiazepine receptor as measured by subsequent binding of [ 3A]flunitrazepam, as described in Methods and Materials . A air~~ilar experiment comparing nix different benzodiazepines is dllustrated in Figure 4, which indicates that flunitrazepam, clonazepam, and nitrazepam are all capable of binding irreversably to the benzodiazepine receptor when exposed to W light, whereas lorazepam, diazepam, and Roy-3027 are not .
1618
Benzodiazepine Receptors
Vol . 25, No . 18, 1979
100
80
z z
60
M=
40
m
w
0 ô w
o:
20
FIGURE 4 Effect of W irradiation in the presence or subsequent [ 3H]flunitrazepam binding . nitrazepam (12 nM) ; C - flunitrazepam (3 E = Roy-3027 (1 .4 nI~ ; F - diazepam (30
of 6 different benzodiazepines A - clonazepam (1 .25 nM) ; B nM) ; D m lorazepam (1 .2 nM) ; nM) .
A cursory examination of the structures of these compounds will reveal that a feature which is common to the first three compounds but not present in the second group is a vitro substituent at the 7 position, suggesting that this moiety, which is certainly the most likely of all the substituente present on these compounds to be capable of participation in this sort of photochemical reaction, may be responsible for photoaffinity labeling of thé benzodiazepine receptor . Figure 5 illustrates the time course of irreversible binding of non-radioactive flunitrazepam. An interesting phenomenon that is illustrated in this figure is that, while the majority of irreversible binding occurs within 4 hrs ., extended photolyais (24 hrs .) in the presence of a saturating concentration of ligând (3 nM) does not succeed in preventing 100X of the specific [ 3H]flunitrazepam binding initially exhibited by the tissue . We were interested in the possibility that this might indicate the existence of a small aubpopulation of receptors which are for some reason resistant to photoaffinity labeling . One possible explanation for the resistance of such a subpopulation of receptors to photoaffinity labeling might be that the affinity of these receptors for fhmitrazepam is less than that of the receptors which are susceptible to photoaffinity labeling . In order to pursue this possibility we performed saturation studies on brain tissue after photolyais for 24 hrs . in the presence of 3 nM flunitrazepam and compared the results tô those obtained using tissue which was treated in an identical manner except for exposure to W light . However no significant difference in affinity was found between the two groups of receptors, eliminating this factor as an explanation for the resistance of the remaining receptors to photoaffinity labeling . Thus, the reason for this residual [ 3H]flunitrazepam binding has yet to be established, however the
Vol. 25, No . 18, 1979
ßenzod~;azepine xeceptaxs
1619
possibility exists that a small fraction of the receptors retain their binding capacity even after entering into a covalent interaction with one ligand molecule . We expect that the discovery that certain benzodiazepines, some of which are available in a radioactive form, are capable of binding irreverelbly to the benzodiazepine receptor will expedite its isolation and characterization . We are currently proceeding toward this end though the use of standard biochemical techniques .
100
z
ô z m
60
z 0
cW
20
K 2
4
6
8
10
12
~~ 24
TI ME (hrs .)
FIGURE 5 Time course of photoaffinity labeling with 3 nM flunitrazepam. ACRNOWLEDGEMLNTS We wish to thank R .C . Speth, T.D . Reisine and J .W . Regan for their comments and suggestions pertaining to this manuscript . This research was supported by USPFIS Grants 2~-25257 aad MH-30626 . H .I . Yamamura is a recipient of a USPHS Research Scientist Development Award (P4i-00095) .
1620
Benzodiazepine Receptors
Vol . 25, No . 18, 1979
REFERENCES 1 . R .F . Squires and C . Braeatrup, Nature _266 : 732-734 (1977) . 2 . C . Braeatrup and R .F . Squires, Proc . Nat'1 Acad . Sci . (U .S .A .) 74 : 3805-3809 (1977) . 3 . C . Braeatrup, R . Albrechtaen and R .F . Squires, Nature _269 : 702-704 (1977) . 4 . H . Mohler and T . Okada, Life Sci . 20 : 2101-2110 (1977) 5 . H . Mohler and T . Okada, Science _198: 849-851 (1977) . 6 . À .B . Bowman, R .R . Case and P . DiStefano, FEBS Lettérs , _82 : 368-372 (1977) . 7 . K .C . Rice, A . Broswi, J . Tallman, S .M . Paul aad P . Skolnick, Nature _278 : 854-855 (1979) . 8 . H . Mohler, M . Battersby, J .G . Richarde, P . Polc, R . Cumin, L . Piers and R . Kettler, in First International Colloquies on Receptors : Neurotransmitters and Peptide Hormones ., p . 64 (1979) . 9 . R .C . Speth, G .J . Wastek, P .C . Johnson and H .I . Yamamura, Life Sci . 22 : 859866 (1978) .
Pergamon Preae
PHOTOAFFINITY LABELING OF THE BENZODIAZEPINE RECEPTOR IN BOVINE CEREBRAL CORTER Richard W. Johnson and Henry I . Yamamura Departments of Biochemistry, Pharmacology and Psychiatry College of Medicine, university of Arizona Health Sciences Center Tucson, AZ 85724 (Received in final form September 24, 1979)
SUMMARY Cloaazepam, nitrazepam and flunitrazepam were found to engage in an irreversible interaction with benzodiazepine binding sites in bovine cerebral cortex homogenates upon irradiation with ultraviolet light . Photoaffinity labeling with [ H]flunitrazepam could be substantially (approx . 85%) inhibited by a number of different benzodiazepines, including clonazepam, lorazepam, Roy-3027, and non-radioactive fluaitrazepam . Spiroperidol, atropine, naltrexone, propranolol and GABA had no effect on irreversible [3A]flunitrazepam binding, indicating that this binding is to the benzodiazepine receptor as defined in previous studies . INTRODUCTION Specific, high affinity binding of benzodiazepinea has been shown to occur in brain tissue of humane and other animals (1-6) . We have undertaken to characterize the sites to which this binding occurs in order to elucidate the physiologic relevance of this phenomenon . Toward this end, it was desirable to obtain a radioactive affinity label, which would be expected to facilitate isolation and characterization of the benzodiazepine receptor . Recently, certain benzodiazepines have been reported to be capable of binding irreversibly to mammalian brain tüsue (7,8) . We therefore investigated the effects of ultraviolet irradiation oa the dissociation of [ 3H]flunitrazepam and certain nonradioactive benzodiazepines . hETHODS AND MATERIALS Bovine brains were obtained within 1 hr . of death, dissected, and stored at -20°C until used in our experiments . [3H]Flunitrazepam (specific activity 87 .5 Ci/mM) was obtained from New England Nuclear Corp . (Boston, Masa) . Clonazepam, nitrazepam, lorazepam, diazepam, Roy-3027, and non-radioactive flunitrazepam were provided by Hoffman LaRoche, Inc . Photoaffinity labeling of benzodiazepine binding sites was performed as follows : bovine tortes was homogenized using a Polytron homogenizer in 100 volumes (w/v) of ice-cold NaRPOy buffer (50 mM, pH 7 .4) and centrifuged at 49,500 x g for 10 minutes, after which the supernatant was decanted and the pellet was rehomogenized in the original whims of buffer ; 10-20 milliliters of the re0024-3205/79/181613-08$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1614
Benzodiazepine Receptors
Vol . 25, No . 18, 1979
salting homogenate was incubated with the indicated concentrations of ligand for 15 min at 0 deg~eea and then photolyzed at 0 degrees for the indicated lengths of time from above in small beakers at a distance of approximately 6 centimeters using a Mineralight model C-81 short wave ultraviolet light . Quantification of irreversible [ 3 H]flunitrazepam binding was performed as follows : after photolgsis, the tissue was centrifuged at 49,500 x g for 10 min . and rehomogenized in the original volume of buffer ; 500 microliter aliquots of the resulting homogenate were then incubated in the presence of 10 micromolar clonazepam in a total whims of 2 ml . for 60 minutes at 0 degrees, whereupon the samples were poured over GF/B filters (Whatman) and washed 3 times with 5 ml rinses of buffer . Radioactivity retained at the filters was then determined by extraction with a Triton-based scintillation cocktail and liquid scintillation spectrometry . Qualitatively similar results were obtained when the tissue, after photolysis, was subjected to repeated washing (centrifugation followed by rehomogenization) and/or exhaustive dialysis instead of displacement with clonazepam in order to remove imreacted ligand ; however both of these methods were much more time consuming than the first procedure and were accompanied by lower receptor yields . Quantification of photoaffinity labeling with non-radioactive ligands was performed by incubating 500 microliter aliquots of the photolyzed tissue, after washing 3 times as described above to remove unreacted ligand, with 1 .5 nM [3H]flimitrazepam in the presence and absence of 1 micromolar clonazepam for 60 min. at 0 degrees and then filtering as described above . Subtracting and radioactivity retained after filtering samples in which clonazepam was present from that retained after filtering samples in which only [3H]fluaitrazepam was present yielded the amount of specific [ 3H]flunitrazepam binding by the tissue . A decrease in this parameter after photolyzes in the presence of a particular ligand was used as an index of irreversible binding of the ligand to the receptor . Control values for [3H]flunitrazepam binding were defined as that binding to tissue treated in an identical manner in all respects except for the presence of ligand during photolysis . Saturation studies of unlabeled receptors before and after photolyzes were performed by incubating 250 microliter aliquots of tissue with various concentrations of [3H]flunitrazepam in the presence and absence of 1 micromolar clon azepam and then filtering and counting the samples as described above . As can he seen from Fig . 1, irradiation with UV light precludes removal of radioactivity from the tissue by centrifugation followed by rehomogenization and incubation in the presence of 10 micromolar clonazepam . Photoaffinity labeling could be substantially (87 .2%) inhibited by the presence of a large excess of clonazepam during photolysis . An 85% reduction of irreversible [ 3H]flunitrazepam binding could also be achieved with micromolar concentrations of lorazepam, Roy-3027, and non-radioactive flunitrazepam, however micromolar concentrations of atropine, apiroperidol, propranolol, naltrexone and GABA had no significant effect on irreversible ( 3H]flunitrazepam binding, indicating that this binding is primarily to the benzodiazepine receptor as defined in previous studies . The effects of varying concentrations of two different non-radioactive benzodiazepines on irreversible [3H]flunitrazepam binding are shown in Figure 2 . Note that a sufficiently large excess of either of these ligands will substantially inhibit photoaffinity labeling . Of particular interest, however, is the wide disparity between the ICSp values for these two compounds under these circumstances (4 .46 X 10 -1 ~ M vs . 3 .55 R 10 -B M) despite the fact that they have almost identical affinities for the benzodiazepine receptor under ordinary circumstances (9) . This enigmatic observation would be explicable if clonazepam itself was capable of engaging in an irreversible interaction with the receptor while lorazepam was not . In order to investigate this possibility, experiments were performed to quantify covalent binding of these and other benzodiazepinea
Benzodiazepine Receptors
Vol . 25, No . 18, 1979
1615
RESULTS AND DISCUSSION Figure 1 shows the effect of UV irradiation on [3H]flunitrazepam dissociation .
IOr
I "r ~N rLYNITIIAZVAr
mr aN rw Nu I~r CLOIIAi~Mr
X
n
E
_U
° z
5
0
m
a aW
N Q H Z ~
W
4 3
2
A
NO UV
4HRâ UV
Figure 1 Effect of UV li(~ht on [ 3N]flunitrazepam dissociation .
Beazodiazepine Receptors
1616
Vol . 25, No . 18, 1979
ioo
0 3
C WC C
D
~ ~ -LOY DIlPLACEH CONGfwalar)
7
Figure 2 Inhibition of irreversible [3H]flunitrazepam binding by clonazepam ( " ) and lorazepam (~ ) .
in the presence of W light . The results we obtained are presented in Fig 3, which illustrates that photolysis of brain tissue with 1 .25 nM clonazepam followed by extensive washing results in a large (86 .4X) reduction in [ 3H]flimitrazepam binding when compared to tissue photolyzed in the absence of clonazepam but treated identically in all other respects . Note that when tissue is incubated with an identical concentration of clonazepam for the same amount of time in the dark there is a negligible (5 .7X) reduction in [ 3H]flunitrazepam binding . Scatchard analysis of [ H]flunitrazepam binding to the tissue before an after photolysis showed that the reduction in binding was entirely due to a decrease in the number of available receptors,indicating a non-competitive mechanism. Thus, clonazepam appears to participate in an irreversible interaction with the benzodiazepine receptor when subjected to W irradiation .
Benzodiazepine Receptors
Vol . 25, No . 18, 1979
1617
IOOr
70
sz
ö 60 z
W 40
N C F
~ 30 W q O~~ Z _O F
~ I~ O W C
No
uv
rsHRS uv
FIGURE 3 Effect of ultraviolet irradiation on diasociatioa of clonazepam from the benzodiazepine receptor as measured by subsequent binding of [ 3A]flunitrazepam, as described in Methods and Materials . A air~~ilar experiment comparing nix different benzodiazepines is dllustrated in Figure 4, which indicates that flunitrazepam, clonazepam, and nitrazepam are all capable of binding irreversably to the benzodiazepine receptor when exposed to W light, whereas lorazepam, diazepam, and Roy-3027 are not .
1618
Benzodiazepine Receptors
Vol . 25, No . 18, 1979
100
80
z z
60
M=
40
m
w
0 ô w
o:
20
FIGURE 4 Effect of W irradiation in the presence or subsequent [ 3H]flunitrazepam binding . nitrazepam (12 nM) ; C - flunitrazepam (3 E = Roy-3027 (1 .4 nI~ ; F - diazepam (30
of 6 different benzodiazepines A - clonazepam (1 .25 nM) ; B nM) ; D m lorazepam (1 .2 nM) ; nM) .
A cursory examination of the structures of these compounds will reveal that a feature which is common to the first three compounds but not present in the second group is a vitro substituent at the 7 position, suggesting that this moiety, which is certainly the most likely of all the substituente present on these compounds to be capable of participation in this sort of photochemical reaction, may be responsible for photoaffinity labeling of thé benzodiazepine receptor . Figure 5 illustrates the time course of irreversible binding of non-radioactive flunitrazepam. An interesting phenomenon that is illustrated in this figure is that, while the majority of irreversible binding occurs within 4 hrs ., extended photolyais (24 hrs .) in the presence of a saturating concentration of ligând (3 nM) does not succeed in preventing 100X of the specific [ 3H]flunitrazepam binding initially exhibited by the tissue . We were interested in the possibility that this might indicate the existence of a small aubpopulation of receptors which are for some reason resistant to photoaffinity labeling . One possible explanation for the resistance of such a subpopulation of receptors to photoaffinity labeling might be that the affinity of these receptors for fhmitrazepam is less than that of the receptors which are susceptible to photoaffinity labeling . In order to pursue this possibility we performed saturation studies on brain tissue after photolyais for 24 hrs . in the presence of 3 nM flunitrazepam and compared the results tô those obtained using tissue which was treated in an identical manner except for exposure to W light . However no significant difference in affinity was found between the two groups of receptors, eliminating this factor as an explanation for the resistance of the remaining receptors to photoaffinity labeling . Thus, the reason for this residual [ 3H]flunitrazepam binding has yet to be established, however the
Vol. 25, No . 18, 1979
ßenzod~;azepine xeceptaxs
1619
possibility exists that a small fraction of the receptors retain their binding capacity even after entering into a covalent interaction with one ligand molecule . We expect that the discovery that certain benzodiazepines, some of which are available in a radioactive form, are capable of binding irreverelbly to the benzodiazepine receptor will expedite its isolation and characterization . We are currently proceeding toward this end though the use of standard biochemical techniques .
100
z
ô z m
60
z 0
cW
20
K 2
4
6
8
10
12
~~ 24
TI ME (hrs .)
FIGURE 5 Time course of photoaffinity labeling with 3 nM flunitrazepam. ACRNOWLEDGEMLNTS We wish to thank R .C . Speth, T.D . Reisine and J .W . Regan for their comments and suggestions pertaining to this manuscript . This research was supported by USPFIS Grants 2~-25257 aad MH-30626 . H .I . Yamamura is a recipient of a USPHS Research Scientist Development Award (P4i-00095) .
1620
Benzodiazepine Receptors
Vol . 25, No . 18, 1979
REFERENCES 1 . R .F . Squires and C . Braeatrup, Nature _266 : 732-734 (1977) . 2 . C . Braeatrup and R .F . Squires, Proc . Nat'1 Acad . Sci . (U .S .A .) 74 : 3805-3809 (1977) . 3 . C . Braeatrup, R . Albrechtaen and R .F . Squires, Nature _269 : 702-704 (1977) . 4 . H . Mohler and T . Okada, Life Sci . 20 : 2101-2110 (1977) 5 . H . Mohler and T . Okada, Science _198: 849-851 (1977) . 6 . À .B . Bowman, R .R . Case and P . DiStefano, FEBS Lettérs , _82 : 368-372 (1977) . 7 . K .C . Rice, A . Broswi, J . Tallman, S .M . Paul aad P . Skolnick, Nature _278 : 854-855 (1979) . 8 . H . Mohler, M . Battersby, J .G . Richarde, P . Polc, R . Cumin, L . Piers and R . Kettler, in First International Colloquies on Receptors : Neurotransmitters and Peptide Hormones ., p . 64 (1979) . 9 . R .C . Speth, G .J . Wastek, P .C . Johnson and H .I . Yamamura, Life Sci . 22 : 859866 (1978) .