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Stimulation of cyclic AMP formation in the anterior pituitary by II-deoxyprostaglandins and their analogues
Pharmacological Research Communications, Vol. 6, No. 6, 1974
595
STIMULATION OF CYCLIC AMP FORMATION IN THE ANTERIOR PITUITARY BY I I-DEOXYPROSTAGLANDI NS AND THEIR ANALOGUES
W. Lippmann Biochemical
Pharmacology Department
Ayerst Research Laboratories Montreal, Quebec, Canada Received3 September 1974
SUMMARY.
Various l l-deoxyprostaglandins and their analogues
increased the formation of cyclic AMP by the rat anterior pituitary with their action apparently being due to a stimulation of adenyl cyclase, rather than inhibition of cyclic AMP phosphodiesterase. A structure-activity relationship was determined.
INTRODUCTION.
The importance of the activation of adenyl cyclase
and increased levels of cyclic adenosine 31:5t-monophosphate
(cyclic AMP)
in the mediation of hormonal effects in various tissues has been well documented (Sutherland and Robison,
1966).
In the anterior pituitary
the adenyl cyclase system appears to be involved in the release of gonadotrophins, adrenocorticotrophin
hormone, thyroid-stimulating
and growth hormone (Major and Kilpatrick, an involvement of prostaglandins anterior pituitary hormones.
1972).
hormone
Various findings suggest
in the mediation of the release of the
PGE I, PGA I and PGB I stimulate cyclic AMP
formation in the anterior pituitary (Zor et al., 1970). the pituitary content of LH (Labhsetwar,
1970).
antagonist (7-oxa-13-prostynoic acid)decreases
PGF2~ increases
A prostaglandin luteinizing hormone (LH)
release induced by LH-releasing factor (Amoss et al., 1970).
PGE I increases
Pharmacological Research Communications, Vol. 6, No. 6, 1974
596
the release of thyroid-stimulating hormone (TSH) and adrenocorticotrophin hormone; the induced-release by TSH-releasing hormone is decreased by the prostaglandin antagonist (Vale et al., 1971). Various prostaglandins have been shown to stimulate the formation of cyclic AMP by the anterior pituitary with large differences being observed with respect to their activities (Zor et al., 1970). Synthetic proslaglandin analogues,
i.e. ll-deoxyprostaglandins
and the!r analogues, have been shown to exhibit activities similar to natural prostaglandins, e.g. (Lippmann, 1969, 1970, 1971).
inhibition of gastric acid secretion In the present studies the ability of
various of the synthetic prostaglandin analogues (Fig.) to cause increased formation of cyclic AMP by the rat anterior pituitary in vitro has been determined.
METHODS. Cyclic AMP formation:
The determination of the incorporation of 3H-
adenine into 3H-cyclic AMP was carried out essentially as previously described (Zor et al., 1970).
Male Sprague-Dawley rats (200 gm) were
decapitated and the anterior pituitaries removed.
After the first
incubation the anterior pituitaries were washed with 0.9% NaCI solution, blotted and added to 2 ml fresh Krebs-Ringer bicarbonate buffer containing I mg per ml of glucose, IO-2M theophylline ethanolamine; prostaglandins, in a vehicle consisting of 0. I ml ethanol, 0. I ml Na2CO 3 (1.8 mg/ml) and 0.8 ml water, or vehicle were added in a volume of 40-140 ~I. incubation was carried out for 30 min at 37°C.
The
The tissues were then
blotted and added to 0.5 ml O. IN HCI and 0.I ml of a mixture of ATP, ADP, AMP and cyclic AMP (2 mg/ml each).
Pharmacological Research Communications, Vol. 6, No. 6, 1974
597
Effects of Prostaglandin Analogues on Cyclic AMP Formation Compound o
Concentration" (Mg/ml)
~H-Cyclic A M P F o r m e d 1~, o f PGE I }
I
100
0 |
25
20
99
$ 1
43 12
2O 20
1;'
20
64
PGEI OH
H
OH
R3
R~ RI
R2
- ¢ =¢ -
AY.23.578 I
II
-¢ =¢-
III
- ¢ =c --
IV
-,=-c-
/
R3
OH
H
~OH
'A'
H/ H
VI
-c-e-
M/'~""OH
-c-c-c-¢-c --¢--¢-¢-
n
II
OH
-c-.=-
VII
II
CH3/J~OH
V
VIII IX X
0
'B'
'S'
"~OH
¢H3' / ' ~
.
'A"
I~
"
OH
n
~ H ~ " "~ OH H" ~' OH
O,H OjH
I
5
15
20 5
41 23
20 20
69 18
20 20 20
4 1 2
O II
'-.~-' ~
~ C O O H
()H
H
104
I
PGE2 OH
0 3
77
01
26
R2 R2
R3
"
XI
XII
¢
¢
- l =• -
H/
H
0
20 5 I
7
O
20
40
OH "8"
OH 'A'
II
69 28
5
~3
1
7
Added as vMume of 40 pl.
The columns employed were Dowex 50W X-8 (200-400 mesh, H + form, 3.5 x 0.4 cm) (Krishna et al.,
1968)0
The supernatant fluids from the
precipitation procedures were placed on the columns followed by 2 ml water; 3 ml water were then added and collected.
The supernatant fluid
from the subsequent precipitation steps was added to 15 ml Bray scintillation medium (Bray,
1960).
Quadruplicate determinations were per-
formed and each analogue was tested at least twice.
Pharmacological Research Communications, Vol. 6, No. 6, 1974
598 Malerials:
The synthetic prostaglandin analogues studied (Fig.) were
prepared by Drs. J.F. Bagli, T. Bogri and H.A. Abraham of Ayerst Laboratories.
The designation e refers to the same stereochemistry as it
occurs at tile C-15 of the natural prostaglandin,
i.e. (nat), and the A
refers to the opposite stereochemistry,
i.e. (epi).
purchased from Ono Pharmaceuticals Co.
The other materials utilized were:
[8-3H] adenine,
and PGE 2 were
17 Curies/mmole, Schwarz/Mann; theophylline monoethanol-
amine, K & K Laboratories,
RESULTS.
PGE
Dowex 50W-X8, Bio-Rad Laboratories.
PGE i stimulated cycl ic AMP formation
in a dose-dependent manner as shown in the Fig.
in the anterior pituitary
Based upon PGE I at
I Pg/ml stimulation of cyclic AMP formation was about one-half with PGE at 0.3 pg/ml.
I
In furfiler studies under similar conditions the Michael is
constant (Kin) (Lineweaver and Burk, 1934) was determined to be 0.8 x IO-6M. Max.imum activity observed with the synthetic prostaglandin analogues was obtained with AY-23,578 (rac-ll-deoxy PGEI)[I], comparable activities to PGE I being observed at 20 and 5 ~g/ml', respectively.
The stereochemistry
am C-15 was of importance since the enantio~orph of I, i.e. rac-15-ePill-deoxy PGE I [11], was ineffective at 20 pg/ml.
That the hydrogen
on C-15 was of significance, was shown by the findings that replacement by a methyl group yielded a compound (III) exhibiting only slight activity at 20 ~g/ml. Reduction of the double bond at C-13, activity
i.e. IV and V, decreased the
in relation to I, although both of the analogues showed high
activity at 20 pg/ml
in contrast to II.
Thus, greater specificity was
obtained when the C-13 bond was unsaturated.
As with the analogues
containing the double bond at C-13, the presence of the hydrogen atom at C-15 was of importance since VI, which contains
IV and V, was highly
active whereas the analogue containing a methyl group at C-15 [VII]
Pharmaco~g~al Research Commun~ations, Vol. & No. 6, 1974 exhibited only s l i g h t
activity.
Also,
in place of the secondary alcohol
599
i n t r o d u c t i o n of a carbonyl group
at C-15 yielded an i n a c t i v e d e r i v a t i v e
[viii]. The PGF type analogues containing a saturated bond at C-13, i.e. IX and X, did not exhibit activity at 20 pg/ml in contrast to the PGE analogues, i.e. IV and V, indicating the importance of the nature of the substituent on C-9. PGE 2 was a potent stimulator and exhibited activity in a similar range to that of PGE I.
The PGE 2 type synthetic analogues corresponding
to I and II, i.e. XI and XII, both exhibited high activities as did the PGE I type analogues.
Therefore,
in addition to the PGE
series,
natural and synthetic PGE 2 analogues also exhibit the stimulatory activity. The analogues which caused an increased formation of cyclic AMP were examined essentially as described previously (Zor et al., 1969; Zor et al., 1970) for their effects on cyclic AMP phosphodiesterase activity in the anterior pituitary.
Theophylline, at the concentration
employed in the cyclic AMP formation studies, prevented the decrease. I, IV, V, VI, XI and XII, at the highest level examined for their ability to increase cyclic AMP formation, did not cause appreciable changes in cyclic AMP degradation. with PGE I and PGE 2.
Inhibition was also not observed
Thus in contrast to theophylline, the prostaglandin
analogues did not appear to be inhibitors of the phosphodiesterase.
DISCUSSION.
The increase in cyclic AMP formation observed with the
natural pros~aglandins and the ll-deoxyprostaglandins and their analogues appears to be due to stimulation of adenyl cyclase, rather than inhibition of cyclic AMP phosphodiesterase, as none of the pros~aglandins examined blocked the degradation of cyclic AMP.
In addition, theophylline, which
inhibited the cyclic AMP phosphodiesterase, was present in the determinations of the effects of the prostaglandins on cyclic AMP formation.
Pharmaco/ogica/ Research Communications, Vo/. 6, IVo. 6, 1974
600
In the present studies differences have been observed with regard to the abil ily of synthetic prostaglandin analogues to cause stimulation of cyclic AMP formation.
Differences have also been reported for the
natural prostaglandins as in the anterior pituitary PGE I was found to be the most potent examined with PGA I and PGB I exhibiting much less activil-y; PGFI~ was ineffective (Zor __et__al', 1970).
In this regard,
it is of interest that, with respect 4o the synthetic prostaglandin analogues
in the present studies, the PGE analogues,
were highly active whereas the PGF analogues, exhibit activity.
In another- tissue,
i.e. IV and V,
i.e. IX and X, did not
i.e. dog thyroid, differences
have also been observed as PGE I was effective whereas PGFI~ and PGB I were not (Zor et al., 1969).
In addition,
in human term placental
homogenate preparations the relative potencies were PGE I > PGE 2 > PGA I > PGB 2, PGF2~, PGA2; PGFI~ was not appreciably active in relation to PGE I (Satoh and Ryan,
1972).
In view of the present findings and those showing that the ll-deoxyprostaglandins exhibit activities similar to natural prostaglandins (Lippmann,
1969, 1970, 1971), the effects of the ll-deoxyprostaglandins
and related synthetic prostaglandin analogues on the release of anterior pituitary hormones are of interest.
ACKNOWLEDGEMENT.
The author acknowledges the technical assistance
of Miss Gall Carew-Gibson.
Pharmaco/ogica/ Research Commun/cations, Vo/. 6, No. 6, 1974
601
REFERENCES Amoss, M., Blackwell, R., Vale, W., Burgus, R. and Guillemin, R. (1971) intern. Cong. Phys. Sci. 9, 17, abst. 37. Bray, G.A. (1960) Anal. Biochem. I, 279. Krishna, G., Weiss, B. and Brodie, R.B. (1968) J. Pharmacol. Exp. Therap. 163, 379. Labhsetwar, A.P. (1970) J. Reprod. Fertil. 23, 155. Lineweaver, H. and Burk, D. (1934) J. Amer. Chemo Soc. 56, 658. Lippmann, W. (1969) J. Pharm. Pharmac. 21, 335. Lippmann, W. (1970) J. Pharm. Pharmac. 22L, 65. Lippmann, W.. (1971) Ann. N.Y. Acad. Sci. 180, 332. Major, P.W. and Kilpatrick, R. (1972) J. Endocrinol. 5_22, 593. Satoh, K. and Ryan, K.J. (1972) J. Clin. Invest. 51, 456. Sutherland, E.W. and Robison, G.A. (1966) Pharmacol. Rev. 18, 145. Vale, W., Rivier, C. and Guillemin, R. (1971) Fed. Proc. 30, 363, abst. 960. Zor, U., Kaneko, T., Lowe, I.P., Bloom, G. and Field, J.B. (1969) J. Biol. Chem. 244 , 5189. Zor, U., Kaneko, T°, Schneider, H.P.G., McCann, S.M. and Field, J.B. (1970) J. Biol. Chem. 245, 2883.
595
STIMULATION OF CYCLIC AMP FORMATION IN THE ANTERIOR PITUITARY BY I I-DEOXYPROSTAGLANDI NS AND THEIR ANALOGUES
W. Lippmann Biochemical
Pharmacology Department
Ayerst Research Laboratories Montreal, Quebec, Canada Received3 September 1974
SUMMARY.
Various l l-deoxyprostaglandins and their analogues
increased the formation of cyclic AMP by the rat anterior pituitary with their action apparently being due to a stimulation of adenyl cyclase, rather than inhibition of cyclic AMP phosphodiesterase. A structure-activity relationship was determined.
INTRODUCTION.
The importance of the activation of adenyl cyclase
and increased levels of cyclic adenosine 31:5t-monophosphate
(cyclic AMP)
in the mediation of hormonal effects in various tissues has been well documented (Sutherland and Robison,
1966).
In the anterior pituitary
the adenyl cyclase system appears to be involved in the release of gonadotrophins, adrenocorticotrophin
hormone, thyroid-stimulating
and growth hormone (Major and Kilpatrick, an involvement of prostaglandins anterior pituitary hormones.
1972).
hormone
Various findings suggest
in the mediation of the release of the
PGE I, PGA I and PGB I stimulate cyclic AMP
formation in the anterior pituitary (Zor et al., 1970). the pituitary content of LH (Labhsetwar,
1970).
antagonist (7-oxa-13-prostynoic acid)decreases
PGF2~ increases
A prostaglandin luteinizing hormone (LH)
release induced by LH-releasing factor (Amoss et al., 1970).
PGE I increases
Pharmacological Research Communications, Vol. 6, No. 6, 1974
596
the release of thyroid-stimulating hormone (TSH) and adrenocorticotrophin hormone; the induced-release by TSH-releasing hormone is decreased by the prostaglandin antagonist (Vale et al., 1971). Various prostaglandins have been shown to stimulate the formation of cyclic AMP by the anterior pituitary with large differences being observed with respect to their activities (Zor et al., 1970). Synthetic proslaglandin analogues,
i.e. ll-deoxyprostaglandins
and the!r analogues, have been shown to exhibit activities similar to natural prostaglandins, e.g. (Lippmann, 1969, 1970, 1971).
inhibition of gastric acid secretion In the present studies the ability of
various of the synthetic prostaglandin analogues (Fig.) to cause increased formation of cyclic AMP by the rat anterior pituitary in vitro has been determined.
METHODS. Cyclic AMP formation:
The determination of the incorporation of 3H-
adenine into 3H-cyclic AMP was carried out essentially as previously described (Zor et al., 1970).
Male Sprague-Dawley rats (200 gm) were
decapitated and the anterior pituitaries removed.
After the first
incubation the anterior pituitaries were washed with 0.9% NaCI solution, blotted and added to 2 ml fresh Krebs-Ringer bicarbonate buffer containing I mg per ml of glucose, IO-2M theophylline ethanolamine; prostaglandins, in a vehicle consisting of 0. I ml ethanol, 0. I ml Na2CO 3 (1.8 mg/ml) and 0.8 ml water, or vehicle were added in a volume of 40-140 ~I. incubation was carried out for 30 min at 37°C.
The
The tissues were then
blotted and added to 0.5 ml O. IN HCI and 0.I ml of a mixture of ATP, ADP, AMP and cyclic AMP (2 mg/ml each).
Pharmacological Research Communications, Vol. 6, No. 6, 1974
597
Effects of Prostaglandin Analogues on Cyclic AMP Formation Compound o
Concentration" (Mg/ml)
~H-Cyclic A M P F o r m e d 1~, o f PGE I }
I
100
0 |
25
20
99
$ 1
43 12
2O 20
1;'
20
64
PGEI OH
H
OH
R3
R~ RI
R2
- ¢ =¢ -
AY.23.578 I
II
-¢ =¢-
III
- ¢ =c --
IV
-,=-c-
/
R3
OH
H
~OH
'A'
H/ H
VI
-c-e-
M/'~""OH
-c-c-c-¢-c --¢--¢-¢-
n
II
OH
-c-.=-
VII
II
CH3/J~OH
V
VIII IX X
0
'B'
'S'
"~OH
¢H3' / ' ~
.
'A"
I~
"
OH
n
~ H ~ " "~ OH H" ~' OH
O,H OjH
I
5
15
20 5
41 23
20 20
69 18
20 20 20
4 1 2
O II
'-.~-' ~
~ C O O H
()H
H
104
I
PGE2 OH
0 3
77
01
26
R2 R2
R3
"
XI
XII
¢
¢
- l =• -
H/
H
0
20 5 I
7
O
20
40
OH "8"
OH 'A'
II
69 28
5
~3
1
7
Added as vMume of 40 pl.
The columns employed were Dowex 50W X-8 (200-400 mesh, H + form, 3.5 x 0.4 cm) (Krishna et al.,
1968)0
The supernatant fluids from the
precipitation procedures were placed on the columns followed by 2 ml water; 3 ml water were then added and collected.
The supernatant fluid
from the subsequent precipitation steps was added to 15 ml Bray scintillation medium (Bray,
1960).
Quadruplicate determinations were per-
formed and each analogue was tested at least twice.
Pharmacological Research Communications, Vol. 6, No. 6, 1974
598 Malerials:
The synthetic prostaglandin analogues studied (Fig.) were
prepared by Drs. J.F. Bagli, T. Bogri and H.A. Abraham of Ayerst Laboratories.
The designation e refers to the same stereochemistry as it
occurs at tile C-15 of the natural prostaglandin,
i.e. (nat), and the A
refers to the opposite stereochemistry,
i.e. (epi).
purchased from Ono Pharmaceuticals Co.
The other materials utilized were:
[8-3H] adenine,
and PGE 2 were
17 Curies/mmole, Schwarz/Mann; theophylline monoethanol-
amine, K & K Laboratories,
RESULTS.
PGE
Dowex 50W-X8, Bio-Rad Laboratories.
PGE i stimulated cycl ic AMP formation
in a dose-dependent manner as shown in the Fig.
in the anterior pituitary
Based upon PGE I at
I Pg/ml stimulation of cyclic AMP formation was about one-half with PGE at 0.3 pg/ml.
I
In furfiler studies under similar conditions the Michael is
constant (Kin) (Lineweaver and Burk, 1934) was determined to be 0.8 x IO-6M. Max.imum activity observed with the synthetic prostaglandin analogues was obtained with AY-23,578 (rac-ll-deoxy PGEI)[I], comparable activities to PGE I being observed at 20 and 5 ~g/ml', respectively.
The stereochemistry
am C-15 was of importance since the enantio~orph of I, i.e. rac-15-ePill-deoxy PGE I [11], was ineffective at 20 pg/ml.
That the hydrogen
on C-15 was of significance, was shown by the findings that replacement by a methyl group yielded a compound (III) exhibiting only slight activity at 20 ~g/ml. Reduction of the double bond at C-13, activity
i.e. IV and V, decreased the
in relation to I, although both of the analogues showed high
activity at 20 pg/ml
in contrast to II.
Thus, greater specificity was
obtained when the C-13 bond was unsaturated.
As with the analogues
containing the double bond at C-13, the presence of the hydrogen atom at C-15 was of importance since VI, which contains
IV and V, was highly
active whereas the analogue containing a methyl group at C-15 [VII]
Pharmaco~g~al Research Commun~ations, Vol. & No. 6, 1974 exhibited only s l i g h t
activity.
Also,
in place of the secondary alcohol
599
i n t r o d u c t i o n of a carbonyl group
at C-15 yielded an i n a c t i v e d e r i v a t i v e
[viii]. The PGF type analogues containing a saturated bond at C-13, i.e. IX and X, did not exhibit activity at 20 pg/ml in contrast to the PGE analogues, i.e. IV and V, indicating the importance of the nature of the substituent on C-9. PGE 2 was a potent stimulator and exhibited activity in a similar range to that of PGE I.
The PGE 2 type synthetic analogues corresponding
to I and II, i.e. XI and XII, both exhibited high activities as did the PGE I type analogues.
Therefore,
in addition to the PGE
series,
natural and synthetic PGE 2 analogues also exhibit the stimulatory activity. The analogues which caused an increased formation of cyclic AMP were examined essentially as described previously (Zor et al., 1969; Zor et al., 1970) for their effects on cyclic AMP phosphodiesterase activity in the anterior pituitary.
Theophylline, at the concentration
employed in the cyclic AMP formation studies, prevented the decrease. I, IV, V, VI, XI and XII, at the highest level examined for their ability to increase cyclic AMP formation, did not cause appreciable changes in cyclic AMP degradation. with PGE I and PGE 2.
Inhibition was also not observed
Thus in contrast to theophylline, the prostaglandin
analogues did not appear to be inhibitors of the phosphodiesterase.
DISCUSSION.
The increase in cyclic AMP formation observed with the
natural pros~aglandins and the ll-deoxyprostaglandins and their analogues appears to be due to stimulation of adenyl cyclase, rather than inhibition of cyclic AMP phosphodiesterase, as none of the pros~aglandins examined blocked the degradation of cyclic AMP.
In addition, theophylline, which
inhibited the cyclic AMP phosphodiesterase, was present in the determinations of the effects of the prostaglandins on cyclic AMP formation.
Pharmaco/ogica/ Research Communications, Vo/. 6, IVo. 6, 1974
600
In the present studies differences have been observed with regard to the abil ily of synthetic prostaglandin analogues to cause stimulation of cyclic AMP formation.
Differences have also been reported for the
natural prostaglandins as in the anterior pituitary PGE I was found to be the most potent examined with PGA I and PGB I exhibiting much less activil-y; PGFI~ was ineffective (Zor __et__al', 1970).
In this regard,
it is of interest that, with respect 4o the synthetic prostaglandin analogues
in the present studies, the PGE analogues,
were highly active whereas the PGF analogues, exhibit activity.
In another- tissue,
i.e. IV and V,
i.e. IX and X, did not
i.e. dog thyroid, differences
have also been observed as PGE I was effective whereas PGFI~ and PGB I were not (Zor et al., 1969).
In addition,
in human term placental
homogenate preparations the relative potencies were PGE I > PGE 2 > PGA I > PGB 2, PGF2~, PGA2; PGFI~ was not appreciably active in relation to PGE I (Satoh and Ryan,
1972).
In view of the present findings and those showing that the ll-deoxyprostaglandins exhibit activities similar to natural prostaglandins (Lippmann,
1969, 1970, 1971), the effects of the ll-deoxyprostaglandins
and related synthetic prostaglandin analogues on the release of anterior pituitary hormones are of interest.
ACKNOWLEDGEMENT.
The author acknowledges the technical assistance
of Miss Gall Carew-Gibson.
Pharmaco/ogica/ Research Commun/cations, Vo/. 6, No. 6, 1974
601
REFERENCES Amoss, M., Blackwell, R., Vale, W., Burgus, R. and Guillemin, R. (1971) intern. Cong. Phys. Sci. 9, 17, abst. 37. Bray, G.A. (1960) Anal. Biochem. I, 279. Krishna, G., Weiss, B. and Brodie, R.B. (1968) J. Pharmacol. Exp. Therap. 163, 379. Labhsetwar, A.P. (1970) J. Reprod. Fertil. 23, 155. Lineweaver, H. and Burk, D. (1934) J. Amer. Chemo Soc. 56, 658. Lippmann, W. (1969) J. Pharm. Pharmac. 21, 335. Lippmann, W. (1970) J. Pharm. Pharmac. 22L, 65. Lippmann, W.. (1971) Ann. N.Y. Acad. Sci. 180, 332. Major, P.W. and Kilpatrick, R. (1972) J. Endocrinol. 5_22, 593. Satoh, K. and Ryan, K.J. (1972) J. Clin. Invest. 51, 456. Sutherland, E.W. and Robison, G.A. (1966) Pharmacol. Rev. 18, 145. Vale, W., Rivier, C. and Guillemin, R. (1971) Fed. Proc. 30, 363, abst. 960. Zor, U., Kaneko, T., Lowe, I.P., Bloom, G. and Field, J.B. (1969) J. Biol. Chem. 244 , 5189. Zor, U., Kaneko, T°, Schneider, H.P.G., McCann, S.M. and Field, J.B. (1970) J. Biol. Chem. 245, 2883.