Proc. Nat. Acad. Sca. USA
Vol. 72, No. 12, pp. 4769-4771, December 1975 Biochemistry
The effect of nerve growth factor on cyclic AMP levels in superior cervical ganglia of the rat (sympathetic nerve/cyclic nucleotide)
BRANSILAV NIKODIJEVIC*, OLGA NIKODIJEVICt, MEI-YING WONG YU, HARVEY POLLARD, AND GORDON GUROFF Section on Intermediary Metabolism, Laboratory of Biomedical Sciences, and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014
Communicated by C. B. Anfinsen, September 5, 1975 Nerve grdwth factor (NGF) produces a sevABSTRACT eral-fold increase in the cyclic AMP concentration in rat superior cervical ganglia in organ culture within 5 min. An increase can be seen with as little as 40 ng/ml of NGF. Oxidized NGF is without effect. The increase in the cAMP concentration produced by NGF is prevented by the addition of antiserum to NGF.
Nerve growth factor (NGF) is known to be involved in the growth, development, and maintenance of sympathetic and sensory ganglia (1, 2). Administration of NGF promotes outgrowth of neuronal processes both in vivo and in vitro (3, 4). Further, there is an overall stimulation of anabolic pathways (5-7). In sympathetic neurons, there is a selective increase in the activities of tyrosine hydroxylase and dopamine f3-hydroxylase, enzymes involved in the synthesis of the transmitter norepinephrine (8). Specific receptors for NGF are present in sympathetic and sensory ganglia (9-11) and at the synaptic endings of sympathetic and sensory neurons (12, 13).
However, there is no evidence concerning the intracellular events which occur between the binding of NGF to its receptors and the appearance of outgrowth or the induction of tyrouine hydroxylase. In order to obtain such information we have done experiments on the possible relationship between NGF and the concentration of the known intracellula "second messenger," cyclic 3':5'-adenosine monophosphate (cAMP). The present data show that 2.5S NGF produces a rapid and marked increase in the cAMP concentratioin in organ cultures of superior cervical ganglia from 21day-old rats. This increase occurs in response to physiological concentrations of NGF, but not to the biologically inactive derivative, oxidized NGF. The response is prevented by antiserum from sheep immunized with NGF. MATERIALS AND METHODS NGF was prepared in the 2.5S form from the salivary glands of mature male mice by the method of Bocchini and Angeletti (14) and stored frozen in small portions. The amount of NGF was. estimated by measuring the absorbance of the preparation at 280 nm (A of 1 mg/ml = 1.6) and its activity was determined using the chick embryo dorsal root ganglion ,amy (15). NGF was oxidized using N-bromosuccinimide as described by Stockel et al. (12). Antibody was produced in sheep by injection of 2.5S NGF according to a standard pro-
Rats, 21 days of age, were obtained from Zivic-Miller. The animals were killed by a blow to the head, and superior cervical ganglia were removed and decapsulated. Three pairs of ganglia were incubated in 0.5 ml of. BGJ enriched media (Fitton-Jackson Modification, Grand Island Biological Co.) for 60 min at 370 in an atmosphere of 95% 02 and 5% CO2 before the addition of NGF or of the appropriate control buffer. For cAMP assay the ganglia were rinsed in ice-cold 0.25 M sucrose for 2 min, blotted on filter paper, and homogenized in 0.6 ml of ice-cold 5% trichloroacetic acid. After 15 min the homogenate was centrifuged and the supernatant portion was washed three times with 2 volumes of ether. The aqueous phase was assayed in quadruplicate for cAMP by the competitive binding assay described by Gilman (16), using kits provided by Amersham-Searle. Each experiment included an identical set of duplicate samples with an internal standard of 0.5 pmol of cAMP. Each experiment was done at least twice, and each point represents one dish containing six ganglia. From each dish four to six samples were taken for cAMP assay. The vertical lines on the graphs represent the standard error of the cAMP determinations. RESULTS Initial experiments showed that the addition of 2.5S NGF to freshly excised superior cervical ganglia in culture produced
.20
--
An
co
-Control NGF
-
amT
.__ 0.4
0.2
tocol. Abbreviations: NGF, nerve growth factor; cAMP, cyclic 3':5'-adenoe monophosphate. * Present address: Department of Pharmacology, Medical School, University of Skopje, Yugoslavia. tPresent address: Department of Physiology, Medical School, University of Skopje, Yugoslavia.
Minutes of Incubation
FIG. 1. Temporal effect of NGF on cAMP level in rat superior cervical ganglia (60 min preincubation). 2.5S NGF was added at a concentration of 3 Ag/ml. A comparable amount of the appropriate buffer was added in the control experiments. Mean I SEM.
4769
4770
Biochemistry: Nikodijevic et al.
Proc. Nat. Acad. Sci. USA 72 (1975) 2.0 F
1.75
1.50 1.5
0
,c
-e
1.25
c
E a.
1.00
1.0 _ C1
0~
0.75
0.50
0.5 _ 0.04
0.4
4.0
40.0
Concentration of NGF (j.I g/ml)
FIG. 2. Effect of NGF concentration on cAMP level. Rat superior cervical ganglia were preincubated for 60 min at 37°. Then 2.55 NGF was added and the ganglia were incubated for an additional 5 min. Control value for cAMP was 0.68 I 0.031. Mean I SEM.
an increase in cAMP. The cAMP concentration, however, was also somewhat elevated for a few minutes by the mere act of removal of the ganglia from the animal and their introduction into the culture medium. A preliminary incubation of 60 min was instituted in order to stabilize the cAMP concentration in the ganglia before the addition of NGF. During this incubation the cAMP concentrations in the ganglia decreased markedly from about 2 pmol per ganglion to 0.8 pmol per ganglion, and NGF produced a 400% increase within 5 min (Fig. 1). Introduction of comparable amounts of the appropriate buffer without NGF gave no change in the cAMP content. A concentration-dependent sigmoid response to NGF could be shown (Fig. 2) with a threshold at concentrations as low as 40 ng/ml. Oxidized NGF at concentrations as high as 10 MAg/Ml had no effect on cAMP concentrations (Fig. 3), nor did antiserum from sheep immunized with 2.5S NGF. However, when 2.5S NGF was preincubated for 15 min at room temperature with the sheep antiserum, its action on cAMP was inhibited by about 90% (Fig. 3).
I
Control
NGF
Oxid. NGF
Antiser. Antiser. + NGF
FIG. 3. cAMP levels in ganglia in the presence of oxidized NGF and NGF + NGF-antiserum. Superior cervical ganglia were preincubated for 60 min at 370. Then 4 ,g/ml 2.5S NGF and/or inhibitors were added and the ganglia were incubated for an additional 5 min. Oxidized NGF concentration was 10 Aig/ml. Antiserum, 5 mg, was preincubated with 2 ,ug of 2.5S NGF at room temperature for 15 min and the mixture was added to the ganglia for an additional 5 min of incubation. NGF without antiserum was kept at room temperature for 15 min for the control. Mean 4 SEM.
DISCUSSION The present data show that NGF in physiological amounts produces an in vitro increase in the cAMP concentration in sympathetic ganglia. This observation raises the further question of the relationship, if any, of this rise in cAMP level to the other actions of NGF, particularly those concerning the induction of tyrosine hydroxylase. In this system, i.e., superior cervical ganglion from 21-day-old rats in BGJ medium, tyrosine hydroxylase activity is significantly higher for several days in the presence of NGF than in its absence (17,*). It may be possible to correlate the rise in cAMP with the effects on tyrosine hydroxylase. It must be pointed out, however, that the general question of the involvement of cyclic nucleotides in the induction of tyrosine hydroxylase in other systems is controversial (18, 19). The lack of an effect of oxidized NGF indicates that the rise in cAMP produced by NGF is related to its physiological action. Oxidation of NGF in this manner destroys the accessible tryptophan residues. The oxidized NGF is inactive in promoting neurite outgrowth in sensory ganglia and does not react with NGF-antiserum (20). Further, it does not
maintain tyrosine hydroxylase activity in cultured sympathetic gangliat. The present data are somewhat analogous to those obtained with insulin in other systems. Under certain conditions insulin can be shown to influence cyclic nucleotide levels (21), although with insulin cAMP levels decrease. The reported changes in response to insulin are quite rapid, as are those shown here in response to NGF. However, the significance of these insulin-induced changes in terms of the overall cellular response to insulin remains to be elucidated. The comparison between insulin and NGF becomes more meaningful in view of the relationship recently shown in the primary and secondary structures of the two proteins (22, 23). cAMP and its dibutyryl derivative have been shown to produce a significant enhancement of outgrowth in chick embryo dorsal root ganglia (24) and fetal rat or mouse sensory ganglia (25). But, although the authors of these studies suggested that NGF might act through cAMP as a "second messenger," it has not been possible, so far, to implicate cAMP convincingly in the action of NGF. In fact, investigations specifically designed to detect changes in cAMP in response to the administration of NGF have provided convincing negative results (26, 27). Previous studies, however, have involved sensory ganglia, and no previous experiments relating NGF to cAMP in sympathetic ganglia have appeared. The present data show, in contrast, an effect of NGF on cAMP concentration. This effect is by far the most rapid shown for NGF, and is exhibited by NGF at concentrations comparable to those exerting its other, well-documented effects. A large number of other questions remain such as the effect of the 7S form of NGF, the changes in other nucleotides, and the possible effects of NGF on adenylate cyclase or phosphodiesterase. But primarily it is of interest to understand how, and indeed whether, these 5-min changes relate to the much later alterations in either the enzymatic composition or the morphology of the neurons.
$ B. Nikodijevic, V. Rowe, M. W. Yu, and G. Guroff, in preparation.
1. Levi-Montalcini, R. (1966) Harvey Lect. 60, 217-259. 2. Levi-Montalcini, R. & Angeletti, P. U. (1968) Physiol. Rev. 48, 534-569.
Biochemistry: Nikodijevic et al. 3. Angeletti, P. U. & Levi-Montalcini, R. (1963) Dev. Biol. 7, 653-659. 4. Johnson, D. G., Silberstein, S. D., Hanbauer, I. & Kopin, I. J. (1972) J. Neurochem. 19,2025-2029. 5. Angeletti, P. U., Gandini-Attardi, D., Toschi, G. & Levi-Montalcini, R. (1965) Biochim. Biophys. Acta 95, 111-120. 6. Liuzzi, A., Angeletti, P. U. & Levi-Montalcini, R. (1965) J. Neurochem. 12, 705-708. 7. Luizzi, A., Porchiari, F. & Angeletti, P. U. (1968) Brain Res. 7,452-454. 8. Thoenen, H., Angeletti, P. U., Levi-Montalcini, R. & Kettler, R. (1971) Proc. Nat. Acad. Sci. USA 68, 1598-1602. 9. Banerjee, S. P., Snyder, S. H., Cuatrecasas, P. & Greene, L. A. (1973) Proc. Nat. Acad. Sci. USA 70, 2519-2523. 10. Frazier, W. A., Boyd, L. F. & Bradshaw, R. A. (1974) J. Biol. Chem. 249,5513-5519. 11. Herrup, K. & Shooter, E. M. (1973) Proc. Nat. Acad. Sci. USA
70,3884-3888.
Proc. Nat. Acad. Sci. USA 72 (1975) 15. 16. 17. 18.
19. 20. 21. 22. 23. 24. 25.
12. Stockel, K., Paravicini, U. & Thoenen, H. (1974) Brain Res.
76,413-421. 13. Stoeckel, K., Schwab, M. & Thoenen, H. (1975) Brain Res. 89, 1-14. 14. Bocchini, V. & Angeletti, P. U. (1969) Proc. Nat. Acad. Sci. USA 64, 787-794.
26. 27.
4771
Fenton, E. L. (1970) Exp. Cell Res. 59, 383-392. Gilman, A. G. (1970) Proc. Nat. Acad. Sci. USA 67,305-312. Mackay, A. V. P. (1974) Br. J. Pharmacol. 51, 509-520. Guidotti, A., Kurosawa, A., Chuang, D. M. & Costa, E. (1975) Proc. Nat. Acad. Sci. USA 72, 1152-1156. Otten, U., Mueller, R. A., Oesch, F. & Thoenen, H., (1974) Proc. Nat. Acad. Sci. USA 71, 2217-2221. Angeletti, R. H. (1970) Biochim. Biophys. Acta 214,478-482. Hollenberg, M. D. & Cuatrecasas, P. (1975) Fed. Proc. 34, 1556-1564. Frazier, W. A., Angeletti, R. H. & Bradshaw, R. A. (1972) Science 176,482-488. Frazier, W. A., Hogue-Angeletti, R. H., Sherman, R. & Bradshaw, R. A. (1973) Biochemistry 12,3281-3293. Roisen, F. J., Murphy, R. A., Pichichero, M. E. & Braden, W. G. (1972) Science 175, 73-74. Haas, D. C., Hier, D. B., Arnason, B. G. W. & Young, M. (1972) Proc. Soc. Exp. Biol. Med. 140, 45-47. Hier, D. B., Arnason, B. G. W. & Young, M. (1973) Science 182,79-81. Frazier, W. A., Ohlendorf, C. E., Boyd, L. F., Oloe, L., Johnson, E. M., Ferrendelli, J. A. & Bradshaw, R. A. (1973) Proc. Nat. Acad. Sci. USA 70,2448-2452.
Vol. 72, No. 12, pp. 4769-4771, December 1975 Biochemistry
The effect of nerve growth factor on cyclic AMP levels in superior cervical ganglia of the rat (sympathetic nerve/cyclic nucleotide)
BRANSILAV NIKODIJEVIC*, OLGA NIKODIJEVICt, MEI-YING WONG YU, HARVEY POLLARD, AND GORDON GUROFF Section on Intermediary Metabolism, Laboratory of Biomedical Sciences, and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014
Communicated by C. B. Anfinsen, September 5, 1975 Nerve grdwth factor (NGF) produces a sevABSTRACT eral-fold increase in the cyclic AMP concentration in rat superior cervical ganglia in organ culture within 5 min. An increase can be seen with as little as 40 ng/ml of NGF. Oxidized NGF is without effect. The increase in the cAMP concentration produced by NGF is prevented by the addition of antiserum to NGF.
Nerve growth factor (NGF) is known to be involved in the growth, development, and maintenance of sympathetic and sensory ganglia (1, 2). Administration of NGF promotes outgrowth of neuronal processes both in vivo and in vitro (3, 4). Further, there is an overall stimulation of anabolic pathways (5-7). In sympathetic neurons, there is a selective increase in the activities of tyrosine hydroxylase and dopamine f3-hydroxylase, enzymes involved in the synthesis of the transmitter norepinephrine (8). Specific receptors for NGF are present in sympathetic and sensory ganglia (9-11) and at the synaptic endings of sympathetic and sensory neurons (12, 13).
However, there is no evidence concerning the intracellular events which occur between the binding of NGF to its receptors and the appearance of outgrowth or the induction of tyrouine hydroxylase. In order to obtain such information we have done experiments on the possible relationship between NGF and the concentration of the known intracellula "second messenger," cyclic 3':5'-adenosine monophosphate (cAMP). The present data show that 2.5S NGF produces a rapid and marked increase in the cAMP concentratioin in organ cultures of superior cervical ganglia from 21day-old rats. This increase occurs in response to physiological concentrations of NGF, but not to the biologically inactive derivative, oxidized NGF. The response is prevented by antiserum from sheep immunized with NGF. MATERIALS AND METHODS NGF was prepared in the 2.5S form from the salivary glands of mature male mice by the method of Bocchini and Angeletti (14) and stored frozen in small portions. The amount of NGF was. estimated by measuring the absorbance of the preparation at 280 nm (A of 1 mg/ml = 1.6) and its activity was determined using the chick embryo dorsal root ganglion ,amy (15). NGF was oxidized using N-bromosuccinimide as described by Stockel et al. (12). Antibody was produced in sheep by injection of 2.5S NGF according to a standard pro-
Rats, 21 days of age, were obtained from Zivic-Miller. The animals were killed by a blow to the head, and superior cervical ganglia were removed and decapsulated. Three pairs of ganglia were incubated in 0.5 ml of. BGJ enriched media (Fitton-Jackson Modification, Grand Island Biological Co.) for 60 min at 370 in an atmosphere of 95% 02 and 5% CO2 before the addition of NGF or of the appropriate control buffer. For cAMP assay the ganglia were rinsed in ice-cold 0.25 M sucrose for 2 min, blotted on filter paper, and homogenized in 0.6 ml of ice-cold 5% trichloroacetic acid. After 15 min the homogenate was centrifuged and the supernatant portion was washed three times with 2 volumes of ether. The aqueous phase was assayed in quadruplicate for cAMP by the competitive binding assay described by Gilman (16), using kits provided by Amersham-Searle. Each experiment included an identical set of duplicate samples with an internal standard of 0.5 pmol of cAMP. Each experiment was done at least twice, and each point represents one dish containing six ganglia. From each dish four to six samples were taken for cAMP assay. The vertical lines on the graphs represent the standard error of the cAMP determinations. RESULTS Initial experiments showed that the addition of 2.5S NGF to freshly excised superior cervical ganglia in culture produced
.20
--
An
co
-Control NGF
-
amT
.__ 0.4
0.2
tocol. Abbreviations: NGF, nerve growth factor; cAMP, cyclic 3':5'-adenoe monophosphate. * Present address: Department of Pharmacology, Medical School, University of Skopje, Yugoslavia. tPresent address: Department of Physiology, Medical School, University of Skopje, Yugoslavia.
Minutes of Incubation
FIG. 1. Temporal effect of NGF on cAMP level in rat superior cervical ganglia (60 min preincubation). 2.5S NGF was added at a concentration of 3 Ag/ml. A comparable amount of the appropriate buffer was added in the control experiments. Mean I SEM.
4769
4770
Biochemistry: Nikodijevic et al.
Proc. Nat. Acad. Sci. USA 72 (1975) 2.0 F
1.75
1.50 1.5
0
,c
-e
1.25
c
E a.
1.00
1.0 _ C1
0~
0.75
0.50
0.5 _ 0.04
0.4
4.0
40.0
Concentration of NGF (j.I g/ml)
FIG. 2. Effect of NGF concentration on cAMP level. Rat superior cervical ganglia were preincubated for 60 min at 37°. Then 2.55 NGF was added and the ganglia were incubated for an additional 5 min. Control value for cAMP was 0.68 I 0.031. Mean I SEM.
an increase in cAMP. The cAMP concentration, however, was also somewhat elevated for a few minutes by the mere act of removal of the ganglia from the animal and their introduction into the culture medium. A preliminary incubation of 60 min was instituted in order to stabilize the cAMP concentration in the ganglia before the addition of NGF. During this incubation the cAMP concentrations in the ganglia decreased markedly from about 2 pmol per ganglion to 0.8 pmol per ganglion, and NGF produced a 400% increase within 5 min (Fig. 1). Introduction of comparable amounts of the appropriate buffer without NGF gave no change in the cAMP content. A concentration-dependent sigmoid response to NGF could be shown (Fig. 2) with a threshold at concentrations as low as 40 ng/ml. Oxidized NGF at concentrations as high as 10 MAg/Ml had no effect on cAMP concentrations (Fig. 3), nor did antiserum from sheep immunized with 2.5S NGF. However, when 2.5S NGF was preincubated for 15 min at room temperature with the sheep antiserum, its action on cAMP was inhibited by about 90% (Fig. 3).
I
Control
NGF
Oxid. NGF
Antiser. Antiser. + NGF
FIG. 3. cAMP levels in ganglia in the presence of oxidized NGF and NGF + NGF-antiserum. Superior cervical ganglia were preincubated for 60 min at 370. Then 4 ,g/ml 2.5S NGF and/or inhibitors were added and the ganglia were incubated for an additional 5 min. Oxidized NGF concentration was 10 Aig/ml. Antiserum, 5 mg, was preincubated with 2 ,ug of 2.5S NGF at room temperature for 15 min and the mixture was added to the ganglia for an additional 5 min of incubation. NGF without antiserum was kept at room temperature for 15 min for the control. Mean 4 SEM.
DISCUSSION The present data show that NGF in physiological amounts produces an in vitro increase in the cAMP concentration in sympathetic ganglia. This observation raises the further question of the relationship, if any, of this rise in cAMP level to the other actions of NGF, particularly those concerning the induction of tyrosine hydroxylase. In this system, i.e., superior cervical ganglion from 21-day-old rats in BGJ medium, tyrosine hydroxylase activity is significantly higher for several days in the presence of NGF than in its absence (17,*). It may be possible to correlate the rise in cAMP with the effects on tyrosine hydroxylase. It must be pointed out, however, that the general question of the involvement of cyclic nucleotides in the induction of tyrosine hydroxylase in other systems is controversial (18, 19). The lack of an effect of oxidized NGF indicates that the rise in cAMP produced by NGF is related to its physiological action. Oxidation of NGF in this manner destroys the accessible tryptophan residues. The oxidized NGF is inactive in promoting neurite outgrowth in sensory ganglia and does not react with NGF-antiserum (20). Further, it does not
maintain tyrosine hydroxylase activity in cultured sympathetic gangliat. The present data are somewhat analogous to those obtained with insulin in other systems. Under certain conditions insulin can be shown to influence cyclic nucleotide levels (21), although with insulin cAMP levels decrease. The reported changes in response to insulin are quite rapid, as are those shown here in response to NGF. However, the significance of these insulin-induced changes in terms of the overall cellular response to insulin remains to be elucidated. The comparison between insulin and NGF becomes more meaningful in view of the relationship recently shown in the primary and secondary structures of the two proteins (22, 23). cAMP and its dibutyryl derivative have been shown to produce a significant enhancement of outgrowth in chick embryo dorsal root ganglia (24) and fetal rat or mouse sensory ganglia (25). But, although the authors of these studies suggested that NGF might act through cAMP as a "second messenger," it has not been possible, so far, to implicate cAMP convincingly in the action of NGF. In fact, investigations specifically designed to detect changes in cAMP in response to the administration of NGF have provided convincing negative results (26, 27). Previous studies, however, have involved sensory ganglia, and no previous experiments relating NGF to cAMP in sympathetic ganglia have appeared. The present data show, in contrast, an effect of NGF on cAMP concentration. This effect is by far the most rapid shown for NGF, and is exhibited by NGF at concentrations comparable to those exerting its other, well-documented effects. A large number of other questions remain such as the effect of the 7S form of NGF, the changes in other nucleotides, and the possible effects of NGF on adenylate cyclase or phosphodiesterase. But primarily it is of interest to understand how, and indeed whether, these 5-min changes relate to the much later alterations in either the enzymatic composition or the morphology of the neurons.
$ B. Nikodijevic, V. Rowe, M. W. Yu, and G. Guroff, in preparation.
1. Levi-Montalcini, R. (1966) Harvey Lect. 60, 217-259. 2. Levi-Montalcini, R. & Angeletti, P. U. (1968) Physiol. Rev. 48, 534-569.
Biochemistry: Nikodijevic et al. 3. Angeletti, P. U. & Levi-Montalcini, R. (1963) Dev. Biol. 7, 653-659. 4. Johnson, D. G., Silberstein, S. D., Hanbauer, I. & Kopin, I. J. (1972) J. Neurochem. 19,2025-2029. 5. Angeletti, P. U., Gandini-Attardi, D., Toschi, G. & Levi-Montalcini, R. (1965) Biochim. Biophys. Acta 95, 111-120. 6. Liuzzi, A., Angeletti, P. U. & Levi-Montalcini, R. (1965) J. Neurochem. 12, 705-708. 7. Luizzi, A., Porchiari, F. & Angeletti, P. U. (1968) Brain Res. 7,452-454. 8. Thoenen, H., Angeletti, P. U., Levi-Montalcini, R. & Kettler, R. (1971) Proc. Nat. Acad. Sci. USA 68, 1598-1602. 9. Banerjee, S. P., Snyder, S. H., Cuatrecasas, P. & Greene, L. A. (1973) Proc. Nat. Acad. Sci. USA 70, 2519-2523. 10. Frazier, W. A., Boyd, L. F. & Bradshaw, R. A. (1974) J. Biol. Chem. 249,5513-5519. 11. Herrup, K. & Shooter, E. M. (1973) Proc. Nat. Acad. Sci. USA
70,3884-3888.
Proc. Nat. Acad. Sci. USA 72 (1975) 15. 16. 17. 18.
19. 20. 21. 22. 23. 24. 25.
12. Stockel, K., Paravicini, U. & Thoenen, H. (1974) Brain Res.
76,413-421. 13. Stoeckel, K., Schwab, M. & Thoenen, H. (1975) Brain Res. 89, 1-14. 14. Bocchini, V. & Angeletti, P. U. (1969) Proc. Nat. Acad. Sci. USA 64, 787-794.
26. 27.
4771
Fenton, E. L. (1970) Exp. Cell Res. 59, 383-392. Gilman, A. G. (1970) Proc. Nat. Acad. Sci. USA 67,305-312. Mackay, A. V. P. (1974) Br. J. Pharmacol. 51, 509-520. Guidotti, A., Kurosawa, A., Chuang, D. M. & Costa, E. (1975) Proc. Nat. Acad. Sci. USA 72, 1152-1156. Otten, U., Mueller, R. A., Oesch, F. & Thoenen, H., (1974) Proc. Nat. Acad. Sci. USA 71, 2217-2221. Angeletti, R. H. (1970) Biochim. Biophys. Acta 214,478-482. Hollenberg, M. D. & Cuatrecasas, P. (1975) Fed. Proc. 34, 1556-1564. Frazier, W. A., Angeletti, R. H. & Bradshaw, R. A. (1972) Science 176,482-488. Frazier, W. A., Hogue-Angeletti, R. H., Sherman, R. & Bradshaw, R. A. (1973) Biochemistry 12,3281-3293. Roisen, F. J., Murphy, R. A., Pichichero, M. E. & Braden, W. G. (1972) Science 175, 73-74. Haas, D. C., Hier, D. B., Arnason, B. G. W. & Young, M. (1972) Proc. Soc. Exp. Biol. Med. 140, 45-47. Hier, D. B., Arnason, B. G. W. & Young, M. (1973) Science 182,79-81. Frazier, W. A., Ohlendorf, C. E., Boyd, L. F., Oloe, L., Johnson, E. M., Ferrendelli, J. A. & Bradshaw, R. A. (1973) Proc. Nat. Acad. Sci. USA 70,2448-2452.
