Prostaglandin E1 also increased adenyl cyclase ... - Semantic Scholar

STIMULATION OF ANTERIOR PITUITARY ADENYL CYCLASE ACTIVITY AND ADENOSINE 3':5'-CYCLIC PHOSPHATE BY HYPOTHALAMIC EXTRACT AND PROSTAGLANDIN E1* BY URIEL ZOR, TOSHIO KANEKO, HERMAN P. G. SCHNEIDER, SAMUEL M. MCCANN, IRENE P. LOWE, GAIL BLOOM, BARBARA BORLAND, AND JAMES B. FIELD CLINICAL RESEARCH UNIT AND DEPARTMENT OF MEDICINE, UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE, PITTSBURGH, PENNSYLVANIA; AND DEPARTMENT OF PHYSIOLOGY, SOUTHWESTERN MEDICAL SCHOOL, DALLAS, TEXAS

Communicated by Marshall Nirenberg, April 21, 1969

Abstract.-Hypothalamic extract, containing the releasing factors for anterior pituitary hormones, within minutes stimulated adenyl cyclase activity and adenosine 3' :5'-cyclic phosphate (cyAMP) concentrations in rat anterior pituitary in vitro. Cerebral cortical extract was ineffective and hypothalamic extract had no effect on these parameters in posterior pituitary or thyroid. Prostaglandin E1 also increased adenyl cyclase activity and cyAMP levels in anterior pituitary tissue. Although NaF augmented adenyl cyclase activity, it did not elevate cyAMP. Epinephrine, norepinephrine, histamine, serotonin, dopamine, and vasopressin did not increase either adeny] cyclase or cyAMP. The increased adenyl cyclase and cyAMP produced by hypothalamic extract was associated with greater luteinizing hormone release from anterior pituitary in vitro. Activation of adenyl cyclase and increased levels of adenosine 3':5'-cyclic monophosphate (cyAMP) have been implicated in hormonal effects in many different tissues.1 Recent evidence also suggests that changes in the adenyl cyclase system may mediate anterior pituitary hormone release. Schofield2 reported that theophylline, an inhibitor of phosphodiesterase, stimulated growth hormone (GH) release from bovine pituitary slices.2 Wilber et al.1 confirmed this finding and noted that theophylline also increased thyroid stimulating hormone (TSH) release from rat pituitary halves.3 Furthermore, the dibutyryl derivative of cyAMP also augmented TSH and GH release in vitro. Bowers et al.4 reported that cyAMP and adenosine 5'-triphosphate stimulated TSH release from rat pituitary in vitro and that thyrotropin releasing factor (TRF) raised cyAMP levels. The present studies were done to test the effects of a hypothalamic extract oil adenyl cyclase activity and cyAMP concentration in rat anterior pituitary in vitro. Luteinizing hormone (LH) release was measured to assess the biologic potency of the extract. Materials and Methods.-Male Sprague-Dawley rats (200 gm) and Holtzman rats (200250 gm) were killed by cervical fracture. The anterior pituitary was rapidly removed, separated from the posterior pituitary, and placed in iced saline. The adenyl cyclase assay depends upon conversion of 32p_ or 14C-ATP to 32p_ or H4C-cyAMP. When adenyl cyclase activity was assayed in homogenates, 5-20 mg of tissue was homogenized at 40 by hand in 1 ml of 40 mM Tris-HCl (pH 7.8), 3.5 mM Mg++, 10 mM theophylline and 1 918

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mg/ml albumin (Buffer A) by using a Blaessig homogenizer. 50 jl of homogenate and appropriate substances to be tested (10 jul) were added to the assay tubes at 40. The reaction was initiated by adding 50 ,IA of 40 mM Tris-HCl (pH 7.8) containing 3.5 mM Mg++, 10 mM theophylline, 6 X 10-5 M ATP, 0.5-3 ,uc 14C- or 32P-ATP, 4 X 10-3 M cyAMP, 2 X 10-2 M phosphoenolpyruvate, 250 Mug/ml pyruvate kinase, and 1 mg/ml albumin (Buffer B). A tube containing all the reagents and boiled tissue was included as a control. Incubations were carried out for 1-16 min at 370 and were terminated by placing the tubes in a boiling H20 bath for 3 min. Adenyl cyclase activity of the intact whole anterior pituitary was also measured in vitro by the following procedure: A single anterior pituitary was added to 80 Mul of Buffer A. Substances to be tested (20 M&l) were added, and the reaction was initiated by the addition of 50 M4l of Buffer B. After 30-min incubation at 370, the reaction was terminated by placing the tubes in a boiling H20 bath for 3 min. In both assays, using homogenate and whole anterior pituitary, 0.1 ml of a mixture of ATP, ADP, AMP and cyAMP (2 mg/ml each) was added to each tube. To the tubes from the homogenate assay, 0.4 ml H20 was added, while 0.5 ml HCl (0.1 N) was added to the tubes from the assay using whole pituitary. These latter tubes were homogenized, placed in a boiling H20 bath for 15 min, and then neutralized with 0.5 N NaOH. 3H-cyAMP (20 Ml of 0.6,uCi/ml, 4800 epm) was added to all tubes to monitor recovery during purification (usually 50% recovery). After centrifugation 14C- or 32P-cyAMP was separated from other radioactive nucleotides by the method of Krishna et al.5 The supernatant solution was added to a Dowex 50 W-X4 column (hydrogen form) and then eluted with H20. The 3.5-7.5-ml fraction was collected and precipitated twice with 0.2 ml 5% ZnSO4 and 0.2 ml 0.3 N Ba(OH)2. After centrifugation, the supernatant (4.8 ml) was added to Triton X-100 phosphor (15 ml) and counted in a liquid scintillation spectrometer. For cyAMP measurements whole anterior pituitaries were weighed and preincubated for 2 hr in a Dubnoff metabolic shaker at 370 in 0.5 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 1 mg/ml glucose, 2 mg/ml albumin, and, in some cases, 10-2 M theophylline. 'The pituitaries were then transferred to 0.5 ml of fresh buffer of similar composition for a 3- to 60-min incubation. Hypothalamic and cerebral cortical extracts and other substances to be tested were added to the appropriate flasks. At the end of this incubation, the tissue was homogenized in 0.5 ml of 5% trichloroacetic acid and cyAMP was measured by a modification6 of the method of Breckenridge.7 3H-cyAMP (10 uAumoles, 9600 cpm) was added to the homogenate to monitor recovery. Trichloroacetic acid was removed by ether extraction and cyAMP was separated from ATP, ADP and 5-AMP by the method of Krishna et al.5 cyAMP was converted to ATP by incubation with phosphodiesterase, myokinase, and pyruvate kinase. A similar tube, but without phosphodiesterase, was included as the control. The resulting ATP was assayed using glucose-1-'4C as previously described.8 This latter method was modified to include pyruvate kinase and phosphoenolpyruvate so that the ADP formed would be recycled to generate more ATP. The sensitivity of the method is 10-12 mole. LH release into the medium during incubations was measured as previously described.9 Hypothalamic extract was prepared from ovine hypothalamic fragments as previously described.10 Cerebral cortical extract was similarly prepared. These extracts were dissolved in 0.1 N HCl (6 mg/0.1 ml) centrifuged and; the clear supernatant was neutralized to pH 7.4 using 5.6% NaHCO3. Prostaglandin E1, a gift from Dr. Tohn Pike, Upjohn Co., Kalamazoo, Michigan, was dissolved in 0.1 ml absolute alcohol (1 mg/0.1 ml), and 0.9 ml Na2CO3 (0.2 mg/ml) added. The monoamines (1 mg) were dissolved in 0.05 ml 0.1% sodium metabisulfite. Aliquots were added to Krebs-Ringer bicarbonate or TrisHCl buffer containing 0.1% sodium metabisulfite. Bovine TSH (2 units/mg) was provided by the Endocrinology Study Section, National Institutes of Health. Vasopressin (20 units/ml, pitressin) was purchased from ParkeDavis Co. Sterling-Winthrop, Inc. supplied i-epinephrine bitartrate and i-norepinephrine bitartrate monohydrate. Histamine dihydrochloride was obtained from Nutritional Biochemicals Corp., and serotonin creatinine sulfate monohydrate and 3-hydroxy-

PROC. N. A. S.


920

tyramine HCl (dopamine) were purchased from Calbiochem. Phosphoenolpyruvate (tricyclohexyl ammonium salt) was a product of Sigma Chemical Co. 32P-ATP (700-1500 mCi/mmole) was supplied by International Chemical and Nuclear Corporation. 14CC-ATP (40 mCi/mmole) and 3H-cyAMP (1.4-2.3 Ci/mmole) were obtained from Schwarz BioResearch Corp. Triton X-100 phosphor contained per liter 5.5 gm 2,5-diphenyloxazole, 0.1 gm 1,4-bis[2-(4-methyl-5-phenyloxazolyl)I benzene, 333 ml Triton X-100 (Packard Corp.), and 667 ml toluene. Dowex 50 W-X4 resin (hydrogen form, 200-400 mesh) was purchased from Bio-Rad Laboratories. Phosphodiesterase was prepared from rat cerebral cortex.6 Other enzymes used were purchased from Boehringer Mannheim Corp. Results.-Adenyl cyclase activity of the anterior pituitary was proportional to

the amount of homogenate and the time of incubation up to four minutes (Table 1). Hypothalamic extract significantly increased adenyl cyclase activity within one minute and the effect persisted for at least 16 minutes. Stimulation was obtained using as little as 0.66 mg/ml, and greater effects were noted with larger amounts of the extract (Table 2). Although cerebral cortical extract was ineffective, NaF increased anterior pituitary adenyl cyclase. This substance stimulates adenyl cyclase in many tissues."-'3 Hypothalamic extract also increased anterior pituitary cyAi\IP concentration within three minutes of its addition (Table 3). Such stimulation was increased as the incubation was prolonged. 0.66 mg/ml hypothalamic extract significantly augmented cyAMP levels and maximal stimulation was produced with 2 mg/ml. The data in Table 4 demonstrate that anterior pituitary adenyl cyclase activity was doubled by hypothalamic extract, but there was no change in posterior pituitary or thyroid adenyl cyclase. TSH increased thyroid adenyl cyclase activity, and NaF stimulated posterior pituitary adenyl cyclase activity, indicating that the activity in these tissues could be increased. Hypothalamic extract increased cyAMP levels sixfold in anterior pituitaries but had no effect on posterior pituitaries removed from the same rats. Prostaglandin El stimulated adenyl cyclase activity and cyAMP concentration in anterior pituitary (Table 5). Although NaF stimulated adenyl cyclase activity, it did not elevate cyAM\P levels. A similar dissociation of NaF effects on adenyl cyclase and on cyAM1P was observed in thyroid."4 Anterior pituitary adenyl cyclase activity and cyAMP were not increased by vasopressin, epinephrine, histamine, norepinephrine, serotonin, or dopamine. TABLE 1. Effect of time and amount of tissue on adenyl cyclase activity in anterior pituitary

homogenate. Extract added ... ...

Cerebral cortex (6 mg/ml) Hypothalamic (6 mg/ml)

Amount of tissue (mg)

Adenyl Cyclase Activity (cpm 32P-cyAMP produced) Time of Incubation in Minutes 4

16

0.25 0.5

1 ... ...

2 ... ...

1

255

412

192 380 900

1

1381

...

4454

11,624

1

2340

...

7711

19,160

... ... ...

The results are the average of duplicate determinations. One pituitary homogenate was used for the time and tissue response curve, and a different one for the effects of cortical and hypothalamic extract.

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During

a

921


six-hour incubation, hypothalamic

ex-

tract caused a threefold greater release of LH, a two-

fold elevation of cyAMP, and a doubling of adenyl cyclase activity in anterior pituitary (Table 6). In this experiment anterior pituitary halves were incubated with hypothalamic extract and then homogenized for the adenyl cyclase assay. Hypothalamic extract was not added to the homogenate. Discussion.-The demonstration that crude hypothalamic extract stimulates anterior pituitary adenyl cyclase, cyAMP concentration, and LH release provides more direct proof that this system is involved in release of anterior pituitary hormones mediated by hypothalamic releasing factors. The results reported by Schofield2 and Wilber et al.A that theophylline increased GH and TSH release suggested that cyAMP might be involved in such processes. Theophylline mimics the effects of many hormones and of cyAMP on different tissues, and this has been used as evidence for cyAMP involvement in hormonal action.'5-'7 However, interpretation of such experiments may be complex. In brain, theophylline inhibited cyAMP elevation induced by electrical stimulation.'8 Although theophylline reproduced effects of antidiuretic hormone (ADH) and cyAMP on Na+ transport in frog skin, Cuthbert and Painter indicated that it was not necessary to implicate cyAMP to explain effects of either ADH or theophylline.'7 Although TSH probably regulates thyroid gland function via adenyl cyclase stimulation,'9 theophylline did not reproduce TSH effects on glucose-1-'4C oxidation or 32P-incorporation into phospholipid.20 The data that DBC3 and cyAMP4 increased TSH release provided better support for cyAMP involvement in this process. The hypothalamic extract stimulation of adenyl cyclase activity and cyAMP concentrations is rapid enough to account for release of anterior pituitary hormones. The sensitivity of the response is difficult to evaluate, since the actual concentration of releasing factors in crude hypothalamic extract is unknown. Crude hypothalamic extract containing all the releasing factors was used, since the anterior pituitary contains heterogeneous cells and each type presumably responds to a specific releasing factor. If a purified releasing factor were used, the response by its specific cell type might

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TABLE 3. Time and dose responses of hypothalamic extract stimulation of 3'5'-cyclic AMP levels in anterior pituitary slices. -3'5'-Cyclic AMP (momoles/gm)Extract added

Diluent Diluent + albumin Cerebral cortex Hypothalamic

mg/ml ... 6 6 6

3

Times of Incubation in Minutes 10 60

... ... 17.4 40

15.6 13.0 13.2 92.9 116 37.6 23.6

...

5 158

2 ... ... ... 0.66 ... 0.22 ... ... 13.6 ... 0.074 ... The results are the averages of duplicate determinations. The slices were preincubated for 2 hr in 0.5 ml of KRB buffer containing glucose (1 mg/ml), albumin (2mg/ml) and theophylline (10-2 M). The time of incubation refers to the length of time the slices were incubated with the appropriate It

extract.

be masked by the failure of the remaining cells to respond. Since hypothalamic extract caused a much greater increase in cyAMP levels than in adenyl cyclase, the former would be a better parameter to measure when more purified hypothalamic releasing factors are tested. The hypothalamic extract stimulation of anterior pituitary adenyl cyclase and cyAMP demonstrates some specificity, since cerebral cortex extract did not increase these parameters. Furthermore, the hypothalamic extract did not modify cyAMP concentration in posterior pituitary or adenyl cyclase activity in this tissue or in thyroid. In addition to hypothalamic extract, prostaglandin El also increased anterior pituitary adenyl cyclase activity and cyAMP levels (Table 5). Stimulation of cyAMP by prostaglandin El has now been observed in a variety of tissues.'4' 21 However, it is not known whether or nor prostaglandin El releases anterior pituitary hormones. The failure of NaF, which augmented anterior pituitary adenyl cyclase activity, to increase cyAMP concentration is difficult to explain. Although Wilber et al.3 reported that 4.5 X 10-4 M epinephrine increased both GH and TSH release from anterior pituitary halves, similar concentrations of this substance did not augment cyAMP levels (Table 5). This suggests that TABLE 4. Failure of hypothalamic extract to stimulate adenyl cyclase activity and 3'5'cyclic AMP levels in posterior pituitary and thyroid tissue. Adenyl Cyclase Activity

(4C-cyAMP cpm produced) Cerebral Tissue

Hypo-

thalamic cortex NaF, 10-2 M extract extract TSH 742 + 15 1413 4- 43 ... ... 764 ±h 7 738 + 69 1992 + 80 ... 2614 ± 67 737 + 15 703 -i 9 ...

3'5'-Cyclic AMP

(momoles/gm)

Cerebral cortex extract

Hypothalamic extract

21.3 120 18.5 22.6 ... ... Thyroid The results are the averages of duplicate or triplicate determinations. The concentration of cerebral cortex and hypothalamic extract was 6 mg/ml. Anterior and posterior pituitary tissue was obtained from the same group of rats. When adenyl cyclase activity was measured, 0.5-1 mg of anterior pituitary and posterior pituitary homogenate was incubated for 4 and 8 min, respectively. Adenyl cyclase activity in thyroid was measured using homogenate equivalent to 10 mg of beef thyroid incubated for 8 min. The concentration of TSH was 2 units/ml. When cyAMP was measured, whole anterior or posterior pituitary was preincubated for 2 hr in 0.5 ml KRB buffer containing glucose (1 mg/ml), albumin (2 mg/ml), and theophylline (10-2 M). The glands were then incubated with the appropriate extract for 10 min.

Anterior pituitary Posterior pituitary


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TABLE 5. Effect of various substances on adenyl cyclase activity and 3'5'-cyclic AMP levels in whole anterior pituitary. Adenyl cyclase activity (cpm 14C-cyAMP

Expt.

(Cerebral cortex extract Hypothalamic extract Prostaglandin E1

Amount 6 mg/ml 6 mg/ml 40 ,ug/ml

NaF

10-2M

Substance

no.

1

produced) 167 i 30 720 i 30 393 4± 26 1462 -t 257 110 i 3 167 i 30 720 + 30 123 i 15 130 i 38

3'5' Cyclic AMP

(mpmoles/gm) 15.5 64.7 169 13.3 21.3 20.8 95.7 27.1 26.6 24.0 11.6 68.1 12.6 6.1 9.1

2 U/ml 6 mg/ml 6 mg/ml Hypothalamic extract 6 X 10-4 M 2 iEpinephrine 6 X 10-4M iLNorepinephrine 5 X 10-4M ... ISerotonin 6 mg/ml [Cerebral cortex extract 252 + 12 6 mg/mil Hypothalamic extract 825 ± 9 3 NaF 10-2M 1531 4- 129 1.6 X 10-4 ... Histamine 1.6 X 10-4 ... (Dopamine For the assay of adenyl cyclase whole anterior pituitary (5-6 mg) was incubated in 0.15 ml 0.04 M Tris-HCl buffer containing 3.5 mM Mg++, 10 mM theophylline, 1 mg/ml albumin, 6 X 10-5 M ATP, 0.5 ,uc 14C-ATP, 4 X 10-3M cyAMP, 2 X 10-2 M phosphoenolpyruvate, and 250 ,ug/ml pyruvate kinase. The results are the mean + sEM of triplicate determinations. For cyAMP determinations, the whole anterior pituitary was preincubated for 2 hr in 0.5 ml KRB buffer and then transferred to 0.5 ml buffer containing the appropriate extract or substance for a lO-min incubation. The results are the average of duplicate determinations.

Vasopressin

Cerebral cortex extract

epinephrine-induced release of these hormones may be mediated via some other mechanism. The experiments of Kakiuchi and RalJ22' 23 indicate that catecholamines may effect cyAMP concentrations differently in different tissues. Although norepinephrine stimulated cyAMP in rabbit cerebral cortex and cerebellum, the effects tended to decrease in the latter tissue after six minutes of incubation but persisted in the former one. Catecholamines stimulated adenyl cyclase activity in pineal gland and this effect could be inhibited by $-blocking agents and serotonin.24 Although histamine depletes FSH in rat pituitary in vivo and has been identified in hypothalamic extracts,25 it did not increase cyAMP in anterior pituitary. Histamine elevated cyAMP concentration in cerebral cortex and cerebellum.22 23 In the pineal it did not stimulate adenyl cyclase TABLE 6. Effect of hypothalamic extract on adenyl cyclase activity, 3'5'-cyclic AMP concentration, and LH release in anterior pituitary slices.

Treatment

Diluent

Hypothalamic extract

Adenyl cyclase activity (cpm 32P-cyAMP produced) 1409 +33 2794 + 39

3'5'-Cyclic AMP

LH Release

(m;&moles/gm)

(Ag/mg tissue)

2.5

0.18(0.14-0.23)*

6.1

0.59(0.43-0.90)

The slices were preincubated for 30 min in 2 ml of TC 199 and then transferred to a similar medium containing either diluent or hypothalamic extract (4.5 mg/ml) for a 360-min incubation. At the end of the incubation the tissue was removed and homogenized for assay of adenyl cyclase activity and cyAMP concentration. The adenyl cyclase results are the mean 4 sEM of triplicate determinations. Homogenate equivalent to 1 mg of anterior pituitary tissue was used and the incubation period was 4 min. The cyAMP levels are the average of duplicate determinations. LH release was measured using the incubation medium after removal of the pituitary halves. * 95% confidence limits.

924


PROC. N. A. S.

activity.24 Although these and other amines have been implicated in the release of anterior pituitary hormones,26 27 they, serotonin, and dopamine did not increase cyAMP concentration in anterior pituitary. These studies do not completely exclude a role for such substances in anterior pituitary hormone release as their function could be a permissive one. Vasopressin increases ACTH28 and GH29 release in vivo and TSH release in vitro,1° but it did not modify anterior pituitary adenyl cyclase activity or cyAMP levels (Table 5). The increased release of LH from anterior pituitary halves incubated with hypothalamic extract demonstrates that such extracts have biologic activity. However, only the use of purified releasing factors will indicate whether the substance responsible for hormone release also causes adenyl cyclase activation and cyAMP elevation. Despite the fact that hypothalamic extract increases adenyl cyclase activity and cyAMP in anterior pituitary in a matter of minutes, augmented LH release could not be detected in incubation of less than 60 minutes9 while the in vivo response to LH releasing factor was evident within ten minutes.3' The more complex details as to how increased cyAMP mediates hormone release remain to be elucidated. Nonetheless, the present results provide strong support for the hypothesis that hypothalamic releasing factors act through a mechanism involving increased adenyl cyclase activity and elevation of cyAMP concentrations. Dr. Gopal Krishna kindly provided helpful information concerning the assay of adenyl cyclase. Abbreviations: DBC, N6-2'-O-dibutyryl cyclic adenosine 3',5' monophosphate; FSH, follicle stimulating hormone; KRB, Krebs-Ringer bicarbonate buffer containing theophylline (10-2 M). * Supported by grants from the National Institutes of Health (AM-06865 and AM-10073) and the Ford Foundation. ' Sutherland, E. W., and G. A. Robison, Pharmacol. Rev., 18, 145 (1966). 2 Schofield, G. J., Nature, 215, 1382 (1967). 8 Wilber, F. J., G. T. Peake, and R. D. Utiger, Clin. Res., 41, 99 (1968). 4 Bowers, Y. C., G. A. Robison, K. L. Lee, F. Verster, and A. V. Schally, in Program of the American Thyroid Association Meeting, Washington, D.C., 1968, p. 55. 6 Krishna, G., B. Weiss, and B. B. Brodie, J. Pharmacol. Exptl. Therap., 163, 379 (1968). 6 Kaneko, T., and J. B. Field, submitted to Biochemistry. 7Breckenridge, B. M., these PROCEEDINGS, 52, 1580 (1964). 8 Ohta, M., R. J. Jarrett, and J. B. Field, J. Lab. Clin. Med., 67, 1013 (1966). 9 Crighton, D. B., S. Watanabe, A. P. S. Dhariwal, and S. M. McCann, Proc. Soc. Exptl. Biol. Med., 128, 537 (1968). 10Dhariwal, A. P. S., J. Antunes-Rodrigues, and S. M. McCann, Proc. Soc. Exptl. Biol. Med., 118, 999 (1965). "Sutherland, E. W., T. W. Rall, and T. Menon, J. Biol. Chem., 237, 1220 (1962). 12 Taunton, D. O., J. Roth, and I. Pastan, Biochem. Biophys. Res. Commun., 29, 1 (1967). 13Pastan, I., V. Macchia, and R. Katzen, Endocrinology, 83, 157 (1968). 14Kaneko, T., U. Zor, and J. B. Field, Science, 163, 1062 (1969). 15 Turtle, J. R., and D. M. Kipnis, Biochem. Biophys. Res. Commun., 28, 797 (1967). 16 Brodie, B. B., J. I. Davies, S. Hynie, G. Krishna, and B. Weiss, Pharmacol. Rev., 18, 273 (1966). 17 Cuthbert, A. W., and E. Painter, J. Physiol., 199, 593 (1968). 18 Rall, T. W., and A. Sattin, in Proc. 24th Intern. Congr. Physiol., p. 167 (1968). 19 Pastan, I., and R. Katzen, Biochem. Biophys. Res. Commun., 29, 792 (1967). 20 Zor, U., G. Bloom, I. P. Lowe, and J. B. Field, Endocrinology, 84, 1082 (1969).

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Butcher, R. W., and C. E. Baird, J. Biol. Chem., 243, 1713 (1968). 22 Kakiuchi, S., and T. W. Rall, Mol. Pharmacol., 4, 367 (1968). 23 Ibid., p. 379. 24Weiss, B., and E. Costa, J. Pharmacol. Exptl. Therap., 161, 310 (1968). 2 White, W. F., A. I. Cohen, R. H. Rippel, J. C. Story, and A. V. Schally, Endocrinology, 82, 742 (1968). 26 Lippmann, W., R. Leonardi, J. Ball, and J. A. Coppola, J. Pharmacol. Exptl. Therap., 156, 258 (1967). 27 Meyerson, B. J., and C. H. Sawyer, Endocrinology, 83, 170 (1968). 28 Yates, F. E., and J. Urquhart, Physiol. Rev., 42, 359 (1962). 29 Meyer, V., and E. Knobil, Endocrinology, 79, 1016 (1966). 30 LaBella, F. S., Can. J. Physiol. Pharmacol., 42, 75 (1964). 31McCann, S. M., and V. D. Ramirez, Recent Progr. Hormone Res., 20, 131 (1964). 21