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Stimulatory effects of prostaglandin E1 on rat anterior pituitary cyclic amp and luteinizing hormone release
STIM]TLATORY EFFECTS OF PROSTAGLANDIN E 1 ON RAT ANTERIOR PITUITARY CYCLIC AMP AND LUTEINIZING HORMONE RELEASE A. Ratner, M.C. Wilson, L. Srivastava and G.T. Peake Departments of Physiology and Medicine University of New Hexico School of Medicine Albuquerque, New Mexico 87131 and Department of Medicine University of Cincinnati School of Medicine Cincinnati, Ohio 45219
ABSTRACT The effect of prostaglandin E 1 (PGEI) on rat anterior pituitary cyclic AMP accumulation and luteinizing hormone (LH) release was studied both in vivo and in vitro. Addition of PGE 1 to incubation medium over a concentration range of iO -6 to 10 -4 M produced a graded increase in pituitary cyclic AFt. At the lowest concentration (10 -6 M) there was no significant increase in LH release, but proportional increments in LH release were seen with increasing concentrations of PGE I. Ten minutes after intravenous administration of 5 ~g of PGE 1 to adult male rats, pituitary cyclic AMP was substantially increased while serum LH levels were not changed. Administration of a higher dose of PGE 1 (20 ~g) produced a greater increase in pituitary cyclic AMP; and, at this dose serum LH was significantly increased. These results suggest that the PGE 1 effect on LH release is mediated by the adenyl cyclase cyclic AMP system. ACKNOWLEDGEMENTS: This work was supported by NSF Grants GB 32077 (A.R.) and USPHS/NIH Grant HD 05794 (G.T.P.). M . C . W . is a recipient of a USPH special fellowship AM 54221. The authors a c k n o w l e d g e t h e expert technical assistance of ~ . Carolyn Duszynski and Ms. Josephine A. Morris.
Accepted December 17
PROSTA~GLANDINS JANUARY 25, 1974
VOL. 5 NO. 2
165
PROSTAGLANDINS
INTRODUCTION Prostaglandin E 1 was first shown by Zor and colleagues (i, 2) to increase anterior pituitary adenyl cyclase activity and cyclic ~MP levels when added in vitro. However, the authors reported a lack of effect of PGE 1 and E 2 on LH release. Later studies have substantiated the effect of prostaglandins on pituitary cyclic AMP (3-5) and have shown that prostaglandins could increase the release of ACTH (6-8) and GH (5, 9-13) from the anterior pituitary. Evidence that prostaglandins may promote LH release in vivo has recently been reported (14, 15). However, pituitary cyclic AMP levels were not determined in these studies. Since Ratner (16) had previously observed an increase in LH release following in vitro addition of dibutyryl cyclic ~MP or aminophylline, it appears reasonable to assume that the adenyl cyclase-cyclic AMP receptor may play an important role in LH release. The purpose of the present study was to determine whether the effect of PGE I on LH release is mediated by changes in intracellular cyclic AMP concentration. MATERIALS AND METHODS Male rats of the Sprague-Dawley strain (body weight 180-220 g) from Holtzman Co. (Madison, Wisconsin) were used in all experiments. The rats were kept in temperature (24°C ± loC) and light controlled quarters (14 hr light/day) and fed rat chow and water ad libitum. PGE 1 was generously supplied by Dr. J. Weeks. Upjohn Co. For in vitro experiments, PGE 1 (0.5 mg) was dissolved in 0.3 ml ethanol and diluted with tissue culture medium 199 (Difco Laboratories). Appropriate aliquots of ethanol were added to control flasks. For i_.n_nvivq experiments, PGE 1 (i mg) was dissolved in 1 ml ethanol and diluted with sodium carbonate buffer. Control rats were injected with the ethanol-sodium carbonate diluent. IN VIVO EXPERIMENTS: The rats were lightly anesthetized with ether and given PGE 1 or the diluent via the external jugular vein over a period of 30 seconds. Ten minutes after the injection, the animal was decapitated, and the anterior pituitary quickly removed for cyclic AMP determination. Blood was collected from the decapitation site and stored overnight at 4oc° The next morning, the blood was centrifuged at 2,000 g for 30 minutes, and the serum separated and stored at -20°C until assayed for LH. IN VITRO EXPERIMENTS: The rats were killed by decapitation and the anterior pituitary removed, hemisected and randomly distributed into glass culture tubes (i0 x 65 mm) containing i ml culture medium 199. Each flask contained three hemipituitaries. Incubations were carried out a pH 7.2 in a Dubnoff metabolic shaker (60 cycles/second) under constant gassing with humidified 95% 02 - 5% CO 2 at 37C. After preincubation for one hour, the medium was replaced with the same volume of fresh medium containing the substances to be tested. Following a second hour of incubation, the hemipituitaries were assayed for cyclic AMP. The incubation medium was stored at -20oc until assayed for LH.
166
JANUARY 25, 1974
VOL. 5 NO. 2
PROSTAGLANDINS
RADIOIi~NOASSAYS: C ~ l i c AMP: Pituitary cyclic AMP was measured by the method of Steiner et al. (17). Materials for the assay were obtained from Collaberative Research (Waltham, ~lass.). ~ne concentration of pituitary cyclic A ~ is expressed per milligram pituitary weight. LH: Serum and medium LH were measured by the method described by Niswender et al. (18). Purified ovine LH for radieiodination was kindly supplied by Dr. L.E. Reichert, Jr. Anti-ovine LH serum was kindly supplied by Dr. Niswender; rat LH standard (NI~MD-Rat-LH-RP-I) was obtained in the LH kit supplied by the Rat Pituitary Hormone Distribution Program, NIH (potency: 0.03 NIH LH-SI).
JANUARY 25, 1974
VOL. 5 NO. 2
167
PROSTAGLANDINS
RESULTS Effect_of PGE] °n LH Release a n d Pituitary Cyclic AMP Formation in vitro (Fig. 1). The addition of 10 -6 M PGE 1 to the incubation medium significantly increased the level of pituitary cyclic AMP (P < 0.01). At this dose LH release was above the control Level but the difference between experimental and control groups was not found to be statistically significant. When the concentration of PGE 1 was increased to 10 -5 M a further increase in both pituitary cyclic AMP and LH release was observed. In this case, the increase in LH was found to be significantly greater than the control value (P < 0.01). An even greater increase in pituitary cyclic AMP and LH release was seen when PGE 1 at 10 -4 M was added to the incubation medium.
t 30
[~
~3o A
LH CYCLIC AMP
25
25
2C
2o
o
I0
5 )-~ M
COgr~k -
-
to-%
Prostoglandin
Io'~M
o $
EI
Figure 1. LH release and pituitary cyclic AMP accumulation after addition of different concentrations of PGE 1 to the incubation medium. Height of bars represents the mean values and the vertical lines represent standard error of the mean. Each value represents data from 6-8 flasks.
168
J A N U A R Y 25, 1974
VOL. 5 NO. 2
PROSTAGLANDINS
Serum LH and Pituitary Cyclic AMP Accumulation after Intravenous administration of PGE I (Fig. 2 ) . Intravenous administration of 5 ~g of PGE 1 caused a significant (P < 0°01) increase in pituitary cyclic AMP ten minutes after injection, yet, there was no significant change in serum LH levels compared to the diluent injected controls. When 20 ~g of PGE 1 was administered there was an even greater increase in pituitary cyclic AMP. At this dose, serum LH levels were significantly increased (P < 0.05).
EXPERIMENT /
EXPERIMENT 2
6O
~0[ Z LH
F
C~Ct./C AMP
5O
Z /,
~o
£
5so
1 <
/"
15
~ao Io
0
e~
CONTROL
o
PGEt
20.,u~/mr
Figure 2. Serum LH levels and pituitary cyclic AMP accumulation I0 minutes after intravenous administration of two doses of PGE I. Height of bars represents the mean values and the vertical lines represent standard errors of the mean. Each value represents data from eight rats.
JANUARY 25, 1974
VOL. 5 NO. 2
169
PROSTAGLANDINS
DISCUSSION In this study, PGE 1 has been shown to be an effective stimulator of LH release in both the intact animal and when added to incubating anterior pituitary tissue. In both systems, PGE 1 also produced a marked accumulation of pituitary cyclic ~MP. In both our in viv~o and in vitro experiments the lower dose of PGE 1 effectively increased pituitary cyclic ~MP without altering LIi release. It should be pointed out that in the studies reported here and in similar studies, we are dealing with a number of different hormone producing cell types~ and, we are not directly measuring changes in LH producing cells. Prostaglandins are most probably capable of stimulating many pituitary cell types, and it very well may be that the lower concentrations of PGE 1 are stimulating the adenyl cyclase in anterior pituitary cells other than the LH producing cell. We have previously reported that PGE 1 at 10 -6 H is capable of increasing growth hormone release in vitro (5). Ti~e findings in this study support the concept that the effect of PGE 1 on LH release is mediated by the adenyl cyclase-cyclic ~ P system. However, as discussed above, it seems important to substantiate these findings by studying the actions of prostaglandin on specific LH producing cells. REFERENCES i.
Zor, U., T. Kaneko, II.P.G. Schneider, S.M. McCann, I.P. Lowe, G. Bloom, B. Borland, and J.B. Field. Stimulation of anterior pituitary adenyl cyclase activity and adenosine 3',5'-cyclic phosphate by hypothalamic extract and prostaglandin El, Proc. Nat. Acad. Sci. 63:918, 1969.
2.
Zor, U., T. Kaneko, Further studies of 3',5'-monophosphate glandins, J. Biol.
3.
Cooper, R.H., M. McPherson, and J.G. Schofield. ~ e effect of prostaglandins on ox pituitary content of adenosine 3',5'-cyclic monophosphate and the release of growth hormone, Biochem. J. 127:143, 1972.
4.
Makino, T. Study on the intracellular mechanism of LH release in the anterior pituitary. Am. J. Obstet. Gynecol. 115:606, 1973.
5.
Ratner, A., M.C. Wilson, and G.T. Peake. Antagonism of prostaglandin promoted pituitary cyclic AMP accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid, Prostaglandins. 3:413, 1973.
6.
De Wied, D., A. Witter, D.N.G. Versteeg, and A.H. Mulder. Release of ACTH by substances of central nervous system origin, Endocrinology, 85:561, 1969.
170
H.P.G. Schneider, S.M. McCann, and J.B. Field. stimulation of anterior pituitary cyclic adenosine formation by hypothalamic extract and prostaChem. 245:2883, 1970.
JANUARY 25, 1974
VOL. 5 NO. 2
PROSTAGLANDINS
7.
Peng, T., K.M. Six and P.L° Munson. Effects of prostaglandin E 1 on the hypothalamo-hypop~yseal-adrenocortical axis in rats, Endocrinology, 86:202, 1970.
8.
Hedge~ G.A. ACTH secretion due to nypothalamo-pituitary effects of adenosine-3',5'-monophosphate and related substances, Endocrinology, 89:500, 1971.
9.
MacLeod, R.M. and J.E. Lehmeyer. Release of pituitary growth hormone by prostaglandins and dibutyryl adenosine cyclic 3',5'-monophosphate in the absence of protein synthesis. Proc. Nat. Acad. Sci. 67:1172, 1970.
i0.
Hertelendy, F. Studies on growth hormone secretion II. Stimulation by prostaglandins in vitro, Acta Endocrinologica, 68:355, 1971.
ii.
Ito, H., G. Momose, T. Katayama, H. Takagishi, L. Ito, H. Nakajima and Y. Takei. Effect of prostaglandin on the secretion of human growth hormone. J. Clin. Endocrinol. Metab. 32:857, 1971.
12.
Hertelendy, F., H. Todd, K. Ehrhart and R. Blute. Studies on growth hormone secretion; IV. In vivo effects of prostaglandin El, Prostaglandins, 2:79, 1972.
13.
Kato, Y., J. Dupre and J.C. Beck. Plasma growth hormone in the anesthetized rat: Effects of dibutyryl cyclic ~ P , prostaglandin El, adrenergic agents~ vasopressin, ehlorpromazine, amphetamine and L-Dopa, Endocrinology, 93:135, 1973.
14.
Spies, H.C. and R.L. Norman. Luteinizing hormone release and ovulation induced by the intraventricular infusion of prostaglandin E 1 into pentobarbital-blocked rats, Prostaglandins, 4:131, 1973.
15.
Tsafriri, A., Y. Koch and H.R. Lindner. Ovulation rate and serum LH levels in rats treated with indomethacin or prostaglandin E2, Prostaglandins, 3:461, 1973.
16.
Ratner, A. Stimulation of luteinizing hormone release in vitro by dibutyryl-cyclic-AMP and theophylline, Life Sciences, 9:1221, 1970.
17.
Steiner, A.L., D.W. Kipnis, P. Utinger, and C.W. Parker. Radioimmunoassay for the measurement of adenosine 3',5'-cyclic phosphate, Proc. Nat. Acad. Sci. 64:367, 1969.
18.
Niswender, G.D., A.R. Midgley, Jr., S.E. Monroe and L.E. Reichert, Jr. Radioimmunoassay rat luteinizing hormone with anti-ovine LH serum and ovine LH -131 I, Proc. Soc. Exptl. Biol. Med. 128:807, 1968.
JANUARY 25, 1974
VOL. 5 NO. 2
171
ABSTRACT The effect of prostaglandin E 1 (PGEI) on rat anterior pituitary cyclic AMP accumulation and luteinizing hormone (LH) release was studied both in vivo and in vitro. Addition of PGE 1 to incubation medium over a concentration range of iO -6 to 10 -4 M produced a graded increase in pituitary cyclic AFt. At the lowest concentration (10 -6 M) there was no significant increase in LH release, but proportional increments in LH release were seen with increasing concentrations of PGE I. Ten minutes after intravenous administration of 5 ~g of PGE 1 to adult male rats, pituitary cyclic AMP was substantially increased while serum LH levels were not changed. Administration of a higher dose of PGE 1 (20 ~g) produced a greater increase in pituitary cyclic AMP; and, at this dose serum LH was significantly increased. These results suggest that the PGE 1 effect on LH release is mediated by the adenyl cyclase cyclic AMP system. ACKNOWLEDGEMENTS: This work was supported by NSF Grants GB 32077 (A.R.) and USPHS/NIH Grant HD 05794 (G.T.P.). M . C . W . is a recipient of a USPH special fellowship AM 54221. The authors a c k n o w l e d g e t h e expert technical assistance of ~ . Carolyn Duszynski and Ms. Josephine A. Morris.
Accepted December 17
PROSTA~GLANDINS JANUARY 25, 1974
VOL. 5 NO. 2
165
PROSTAGLANDINS
INTRODUCTION Prostaglandin E 1 was first shown by Zor and colleagues (i, 2) to increase anterior pituitary adenyl cyclase activity and cyclic ~MP levels when added in vitro. However, the authors reported a lack of effect of PGE 1 and E 2 on LH release. Later studies have substantiated the effect of prostaglandins on pituitary cyclic AMP (3-5) and have shown that prostaglandins could increase the release of ACTH (6-8) and GH (5, 9-13) from the anterior pituitary. Evidence that prostaglandins may promote LH release in vivo has recently been reported (14, 15). However, pituitary cyclic AMP levels were not determined in these studies. Since Ratner (16) had previously observed an increase in LH release following in vitro addition of dibutyryl cyclic ~MP or aminophylline, it appears reasonable to assume that the adenyl cyclase-cyclic AMP receptor may play an important role in LH release. The purpose of the present study was to determine whether the effect of PGE I on LH release is mediated by changes in intracellular cyclic AMP concentration. MATERIALS AND METHODS Male rats of the Sprague-Dawley strain (body weight 180-220 g) from Holtzman Co. (Madison, Wisconsin) were used in all experiments. The rats were kept in temperature (24°C ± loC) and light controlled quarters (14 hr light/day) and fed rat chow and water ad libitum. PGE 1 was generously supplied by Dr. J. Weeks. Upjohn Co. For in vitro experiments, PGE 1 (0.5 mg) was dissolved in 0.3 ml ethanol and diluted with tissue culture medium 199 (Difco Laboratories). Appropriate aliquots of ethanol were added to control flasks. For i_.n_nvivq experiments, PGE 1 (i mg) was dissolved in 1 ml ethanol and diluted with sodium carbonate buffer. Control rats were injected with the ethanol-sodium carbonate diluent. IN VIVO EXPERIMENTS: The rats were lightly anesthetized with ether and given PGE 1 or the diluent via the external jugular vein over a period of 30 seconds. Ten minutes after the injection, the animal was decapitated, and the anterior pituitary quickly removed for cyclic AMP determination. Blood was collected from the decapitation site and stored overnight at 4oc° The next morning, the blood was centrifuged at 2,000 g for 30 minutes, and the serum separated and stored at -20°C until assayed for LH. IN VITRO EXPERIMENTS: The rats were killed by decapitation and the anterior pituitary removed, hemisected and randomly distributed into glass culture tubes (i0 x 65 mm) containing i ml culture medium 199. Each flask contained three hemipituitaries. Incubations were carried out a pH 7.2 in a Dubnoff metabolic shaker (60 cycles/second) under constant gassing with humidified 95% 02 - 5% CO 2 at 37C. After preincubation for one hour, the medium was replaced with the same volume of fresh medium containing the substances to be tested. Following a second hour of incubation, the hemipituitaries were assayed for cyclic AMP. The incubation medium was stored at -20oc until assayed for LH.
166
JANUARY 25, 1974
VOL. 5 NO. 2
PROSTAGLANDINS
RADIOIi~NOASSAYS: C ~ l i c AMP: Pituitary cyclic AMP was measured by the method of Steiner et al. (17). Materials for the assay were obtained from Collaberative Research (Waltham, ~lass.). ~ne concentration of pituitary cyclic A ~ is expressed per milligram pituitary weight. LH: Serum and medium LH were measured by the method described by Niswender et al. (18). Purified ovine LH for radieiodination was kindly supplied by Dr. L.E. Reichert, Jr. Anti-ovine LH serum was kindly supplied by Dr. Niswender; rat LH standard (NI~MD-Rat-LH-RP-I) was obtained in the LH kit supplied by the Rat Pituitary Hormone Distribution Program, NIH (potency: 0.03 NIH LH-SI).
JANUARY 25, 1974
VOL. 5 NO. 2
167
PROSTAGLANDINS
RESULTS Effect_of PGE] °n LH Release a n d Pituitary Cyclic AMP Formation in vitro (Fig. 1). The addition of 10 -6 M PGE 1 to the incubation medium significantly increased the level of pituitary cyclic AMP (P < 0.01). At this dose LH release was above the control Level but the difference between experimental and control groups was not found to be statistically significant. When the concentration of PGE 1 was increased to 10 -5 M a further increase in both pituitary cyclic AMP and LH release was observed. In this case, the increase in LH was found to be significantly greater than the control value (P < 0.01). An even greater increase in pituitary cyclic AMP and LH release was seen when PGE 1 at 10 -4 M was added to the incubation medium.
t 30
[~
~3o A
LH CYCLIC AMP
25
25
2C
2o
o
I0
5 )-~ M
COgr~k -
-
to-%
Prostoglandin
Io'~M
o $
EI
Figure 1. LH release and pituitary cyclic AMP accumulation after addition of different concentrations of PGE 1 to the incubation medium. Height of bars represents the mean values and the vertical lines represent standard error of the mean. Each value represents data from 6-8 flasks.
168
J A N U A R Y 25, 1974
VOL. 5 NO. 2
PROSTAGLANDINS
Serum LH and Pituitary Cyclic AMP Accumulation after Intravenous administration of PGE I (Fig. 2 ) . Intravenous administration of 5 ~g of PGE 1 caused a significant (P < 0°01) increase in pituitary cyclic AMP ten minutes after injection, yet, there was no significant change in serum LH levels compared to the diluent injected controls. When 20 ~g of PGE 1 was administered there was an even greater increase in pituitary cyclic AMP. At this dose, serum LH levels were significantly increased (P < 0.05).
EXPERIMENT /
EXPERIMENT 2
6O
~0[ Z LH
F
C~Ct./C AMP
5O
Z /,
~o
£
5so
1 <
/"
15
~ao Io
0
e~
CONTROL
o
PGEt
20.,u~/mr
Figure 2. Serum LH levels and pituitary cyclic AMP accumulation I0 minutes after intravenous administration of two doses of PGE I. Height of bars represents the mean values and the vertical lines represent standard errors of the mean. Each value represents data from eight rats.
JANUARY 25, 1974
VOL. 5 NO. 2
169
PROSTAGLANDINS
DISCUSSION In this study, PGE 1 has been shown to be an effective stimulator of LH release in both the intact animal and when added to incubating anterior pituitary tissue. In both systems, PGE 1 also produced a marked accumulation of pituitary cyclic ~MP. In both our in viv~o and in vitro experiments the lower dose of PGE 1 effectively increased pituitary cyclic ~MP without altering LIi release. It should be pointed out that in the studies reported here and in similar studies, we are dealing with a number of different hormone producing cell types~ and, we are not directly measuring changes in LH producing cells. Prostaglandins are most probably capable of stimulating many pituitary cell types, and it very well may be that the lower concentrations of PGE 1 are stimulating the adenyl cyclase in anterior pituitary cells other than the LH producing cell. We have previously reported that PGE 1 at 10 -6 H is capable of increasing growth hormone release in vitro (5). Ti~e findings in this study support the concept that the effect of PGE 1 on LH release is mediated by the adenyl cyclase-cyclic ~ P system. However, as discussed above, it seems important to substantiate these findings by studying the actions of prostaglandin on specific LH producing cells. REFERENCES i.
Zor, U., T. Kaneko, II.P.G. Schneider, S.M. McCann, I.P. Lowe, G. Bloom, B. Borland, and J.B. Field. Stimulation of anterior pituitary adenyl cyclase activity and adenosine 3',5'-cyclic phosphate by hypothalamic extract and prostaglandin El, Proc. Nat. Acad. Sci. 63:918, 1969.
2.
Zor, U., T. Kaneko, Further studies of 3',5'-monophosphate glandins, J. Biol.
3.
Cooper, R.H., M. McPherson, and J.G. Schofield. ~ e effect of prostaglandins on ox pituitary content of adenosine 3',5'-cyclic monophosphate and the release of growth hormone, Biochem. J. 127:143, 1972.
4.
Makino, T. Study on the intracellular mechanism of LH release in the anterior pituitary. Am. J. Obstet. Gynecol. 115:606, 1973.
5.
Ratner, A., M.C. Wilson, and G.T. Peake. Antagonism of prostaglandin promoted pituitary cyclic AMP accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid, Prostaglandins. 3:413, 1973.
6.
De Wied, D., A. Witter, D.N.G. Versteeg, and A.H. Mulder. Release of ACTH by substances of central nervous system origin, Endocrinology, 85:561, 1969.
170
H.P.G. Schneider, S.M. McCann, and J.B. Field. stimulation of anterior pituitary cyclic adenosine formation by hypothalamic extract and prostaChem. 245:2883, 1970.
JANUARY 25, 1974
VOL. 5 NO. 2
PROSTAGLANDINS
7.
Peng, T., K.M. Six and P.L° Munson. Effects of prostaglandin E 1 on the hypothalamo-hypop~yseal-adrenocortical axis in rats, Endocrinology, 86:202, 1970.
8.
Hedge~ G.A. ACTH secretion due to nypothalamo-pituitary effects of adenosine-3',5'-monophosphate and related substances, Endocrinology, 89:500, 1971.
9.
MacLeod, R.M. and J.E. Lehmeyer. Release of pituitary growth hormone by prostaglandins and dibutyryl adenosine cyclic 3',5'-monophosphate in the absence of protein synthesis. Proc. Nat. Acad. Sci. 67:1172, 1970.
i0.
Hertelendy, F. Studies on growth hormone secretion II. Stimulation by prostaglandins in vitro, Acta Endocrinologica, 68:355, 1971.
ii.
Ito, H., G. Momose, T. Katayama, H. Takagishi, L. Ito, H. Nakajima and Y. Takei. Effect of prostaglandin on the secretion of human growth hormone. J. Clin. Endocrinol. Metab. 32:857, 1971.
12.
Hertelendy, F., H. Todd, K. Ehrhart and R. Blute. Studies on growth hormone secretion; IV. In vivo effects of prostaglandin El, Prostaglandins, 2:79, 1972.
13.
Kato, Y., J. Dupre and J.C. Beck. Plasma growth hormone in the anesthetized rat: Effects of dibutyryl cyclic ~ P , prostaglandin El, adrenergic agents~ vasopressin, ehlorpromazine, amphetamine and L-Dopa, Endocrinology, 93:135, 1973.
14.
Spies, H.C. and R.L. Norman. Luteinizing hormone release and ovulation induced by the intraventricular infusion of prostaglandin E 1 into pentobarbital-blocked rats, Prostaglandins, 4:131, 1973.
15.
Tsafriri, A., Y. Koch and H.R. Lindner. Ovulation rate and serum LH levels in rats treated with indomethacin or prostaglandin E2, Prostaglandins, 3:461, 1973.
16.
Ratner, A. Stimulation of luteinizing hormone release in vitro by dibutyryl-cyclic-AMP and theophylline, Life Sciences, 9:1221, 1970.
17.
Steiner, A.L., D.W. Kipnis, P. Utinger, and C.W. Parker. Radioimmunoassay for the measurement of adenosine 3',5'-cyclic phosphate, Proc. Nat. Acad. Sci. 64:367, 1969.
18.
Niswender, G.D., A.R. Midgley, Jr., S.E. Monroe and L.E. Reichert, Jr. Radioimmunoassay rat luteinizing hormone with anti-ovine LH serum and ovine LH -131 I, Proc. Soc. Exptl. Biol. Med. 128:807, 1968.
JANUARY 25, 1974
VOL. 5 NO. 2
171