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Inhibition of uterine contraction by synthetic parathyroid hormone fragment
Life Sciences, Vol. 28, pp. 1317-1321 Printed in the U.S.A.
Pergamon Press
INHIBITION OF UTERINE CONTRACTION BY SYNTHETIC PARATHYROID HORMONE FRAGMENT Peter K.T. Pang I, Ronald L. Shew 2, and Wilbur H. Sawyer 3 iDepartment of Pharmacology and Therapeutics, and2Department of Anatomy, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, and3Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, N.Y. (Received in final form January 13, 1981) Summary The inhibitory effect of synthetic bovine parathyroid hormone fragment [bPTH-(I-34)] on rat uterine contraction was studied in vitro. Oxytocin, prostaglandin F2~ and acetylcholine produced log dose-related contraction. The addition of bPTH-(I-34) shifted the dose-response curves of the three agonists to the right. Two doses of bPTH-(I-34) were tested. The higher dose (400 ng/ml) caused a greater inhibition of the agonists than did the lower dose (40 ng/ ml). bPTH-(I-34) also inhibited the uterine contraction elicited by electrical stimulation of the tissue. We suggest that bPTH-(I-34) has a non-specific depressing effect on the contractile mechanism of the uterine tissue. The influence of parathyroid hormone (PTH) on mineral metabolism has been well studied and firmly established. Charbon (1,2) reported that PTH had a vasodilatory action and lowered the blood pressure in dogs. Our recent investigations have extended these observations to other species and shown that synthetic bovine PTH containing the amino terminal 1-34 amino acids [bPTH-(I-34)] had a direct vasodilatory action on specific vascular beds (3,4,5,6). It appears that PTH relaxes vascular smooth muscle. Since many vasoactive peptides, such as bradykinin, are also capable of acting on non-vascular smooth muscle, we attempted to determine whether bPTH-(I-34) can affect another smooth muscle, i.e., that of the uterus. Methods Adult female Sherman rats weighing between 180-220 grams were injected i.p. with diethylstilbestrol (50 ~g) 14-16 hours before being killed by cervical dislocation. The uterine horns were dissected out immediately and suspended in a tissue chamber in Van Dyke-Hastings solution containing 0.5 m M M g C I 2, 113.8 mM NaCI, 6.0 mMKCI, 5.0 mM CaCI2, 29.8 mM NaHC03, .72 m M N a 2 H P O 4, .22 m M N a H 2 P O 4, 2.8 mM glucose, equilibrated with 5% carbon dioxide and 95% oxygen (pH 7.4) in . a constant temperature bath (32°C) (7). A resting tension of I gram was applied to the suspended uterine horns and contractions were measured isometrically with a Grass force-dlsplacement transducer (model FT 03) and recorded with a Grass polygraph (model 7D).
0024-3205/81/121317-05502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
1318
Parathyroid Hormone and Uterine Activity
Vol. 28, No.
12, 1981
All solutions were injected directly into the tissue bath. The following agonists were used to cause contraction of uterine smooth muscle: (i) oxytocin (Syntocinon, Sandoz, 0.I mU to 5.0 mU), (2) prostaglandin F2e (0.i ug to 2.0 ug), and (3) acetylcholine bromide (200 ng to 2,000 ng). Electrical stimulation through the surrounding medium, 2 volts, 60 Hz, repeated every minute for a duration of 2.5 seconds was also used to produce contraction in the uterine horn. Four series of experiments were performed. In the first experiment, we investigated the effect of bPTH-(I-34) (Peninsula Laboratories) on oxytocinstimulated uterine contraction. Oxytocin was added to the bath and the contractile force was recorded. After thorough rinsing, the same dose of oxytocin was tested. If similar responses were observed, the tissue was again rinsed thoroughly and bPTH-(I-34) was added to the bath to give a concentration of 40 or 400 ng/ml of bath medium. One minute later, the same dose of oxytocin was tested again. Using this procedure, we could determine the lo~-dose response curves for oxytocin in the presence or absence of bPTH-(i-34). Solvent and the same molar concentration of calcitonin were tested. They showed no inhibitory action (Shew, Yee and Pang, unpublished data). In the second and third series of experiments, we used the same method to determine the effects of 40 and 400 ng of bPTH-(I-34) per ml bath medium on uterine contractions in response to acetylcholine or prostaglandin F2u. In the fourth series of experiments, uterine horns were stimulated electrically, bPTH-(I-34) was then added to the bath and the change in contractile force was recorded. After thorough rinsing, the contractile force returned to the pre-treatment level and a second dose of bPTH-(I-34) was tested. Each uterine horn was tested with several doses of bPTH-(I-34) and the % change in contractile force was calculated and plotted separately for each piece of tissue. Results Figure I shows the log dose-response to oxytocin in the presence of 40 or 400 ng bPTH-(I-34) per ml, or in the absence of this peptide. Each point represents the mean of data from at least six uterine horns. The vertical bars designate standard errors. The data clearly demonstrate the antagonistic action of bPTH-(I-34). Increasing the concentrations of bPTH-(I-34) shifts the log dose-response curve more to the right. This is also evident in uterine activity stimulated by acetylcholine or prostaglandin F2~ (Figures 2 and 3). In Figure 4, the antagonistic action of bPTH-(I-34) on electrically stimulated uterine contraction is apparent. In all five uterine horns studied, increasing the doses of bPTH-(I-34) resulted in increased inhibition. Discussion The relaxing influence of bPTH-(I-34) on vascular smooth muscle is now well established (1,2,3,4,5,6). It has been shown that bPTH-(I-34) has a direct vascular action (4) which is unrelated to the hypercalcemic action of the hormone (8,9,10). Investigations with analogs and fragments of bPTH suggested that the amino acids 24-28 may be involved in the hypotensive action. The present study is the first to report the action of bPTH-(I-34) on another smooth muscle tissue, the uterus. One interesting feature of this investigation is the ability of bPTH-(I-34) to antagonize the contraction produced by three chemicals and by electrical stimulation. This indicates thenonspeciflc antagonistic action of the peptide. It would also suggest that bPTH-(I-34) has a very basic mechanism of action of the contractile apparatus of the tissue. It will be very interesting to determine whether PTH can influence in vivo uterine contraction.
Vol. 28, No. 12, 1981
Parathyroid Hormone and Uterine Activity
1319
3Z u~
T
o
ZuJ k-
CONTROL~"~/~
2 ~
~'//~0
ng PTH/ml
q¢ rf (3
2= m uJ
1--
(3 Z.¢ 3: (J
0I
DOSE
I
OF
I
I
I
I
ACETYLCHOLINE [IJM/m,] Figure i
Inhibition of in vitro rat uterine contraction produced by oxytocin. The preparation of PTH used is the synthetic bPTH-(I-34). Data are expressed as mean ± SEM.
z
~3w
(3 Z
w
(Z3 1 ! O 0-
0.02
o12 DOSEOFOXYT~IN
lmU/ml l
Figure 2 Inhibition of in vitro rat uterine contraction produced by acetylcholine, m e preparation of P ~ used is the syntheti c bP~-(l-34). Data are ~presse~ as mean ± S ~ .
1320
Parathyroid Hormone and Uterine Activity
Vol. 28, No. 12, 1981
1
T E~ / ' , ! /
~•
/
/
52z T ~ i
/ ./4o.g p1".~ ~i/ ,"400 .g PTM m ,
O-
002
o11
o2
o4
DOSE OF PGF2o' [l~g ml!
Figure 3 Inhibition of in vitro rat uterine contraction produced by PGF2a. The preparation of PTH used is the synthetic bPTH-(I-34). Data are expressed as mean ± SEM.
ELECTRICAL STIMULATION (2 V)
-20-
~.r "-400 -~ - 6 0 , -80-
-I00 13
27
DOSE O~ ,-34 ~ o ~
l,~/m,I
Figure 4 Inhibition of the in vitro rat uterine contraction produced by electrical stimulation. The preparation of P T H u s e d is the synthetic bPTH-(I-34). Acknowledgements This investigation was supported by NSF Grant # PCM 78-17695 to PKTP and NSF Grant # PCM 76-18689 and NIAMD Grant # AM 01940 to WHS. References 1. 2. 3.
G.A. CHARBON, Eur. J. Pharmacol. ~ 275-278 (1968). G.A. CHARBON and P.F. HULSTAERT, Endocrinology 95 621-626 (1974). P.K.T. PANG, M. YANG, C. OGURO, J.G. PHILLIPS and J.A. YEE, Gett. Comp.
Vol. 28, No. 12, 1981
Parathyroid Hormone and Uterine Activity
1321
Endocrinol. 4-1 135-138 (1980). P.K.T. PANG, T.E. TENNER, Jr., J.A. YEE, M. YANG and H.F. JANSSEN, Proc. Nat. Acad. Sci. USA 77 675-678 (1980). 5. P.K.T. PANG, H.F. JANSSEN and J.A. YEE, Pharmacology, in press. 6. M.F. CRASS, III and P.K.T. PANG, Science 207 1087-1089 (1980). 7. R.A. MUNSICK, Endocrinology 66 451-457 (1960). 8. P.K.T. PANG, M. YANG and A.D. KENNY, Calc. Tiss. Int. 3-1 74 (1980). 9. A.D. KENNY and P.K.T. PANG, Calc. Tiss. Int. 31 73 (1980). i0. P.K.T. PANG, M. YANG, M.C. KHOSLA and F.M. BUMPUS, Proc. VII Int. Conf. on Calcium Regulating Hormones, Estes Park, Colorado, Sept. 5-9 (1980).
4.
Pergamon Press
INHIBITION OF UTERINE CONTRACTION BY SYNTHETIC PARATHYROID HORMONE FRAGMENT Peter K.T. Pang I, Ronald L. Shew 2, and Wilbur H. Sawyer 3 iDepartment of Pharmacology and Therapeutics, and2Department of Anatomy, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, and3Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, N.Y. (Received in final form January 13, 1981) Summary The inhibitory effect of synthetic bovine parathyroid hormone fragment [bPTH-(I-34)] on rat uterine contraction was studied in vitro. Oxytocin, prostaglandin F2~ and acetylcholine produced log dose-related contraction. The addition of bPTH-(I-34) shifted the dose-response curves of the three agonists to the right. Two doses of bPTH-(I-34) were tested. The higher dose (400 ng/ml) caused a greater inhibition of the agonists than did the lower dose (40 ng/ ml). bPTH-(I-34) also inhibited the uterine contraction elicited by electrical stimulation of the tissue. We suggest that bPTH-(I-34) has a non-specific depressing effect on the contractile mechanism of the uterine tissue. The influence of parathyroid hormone (PTH) on mineral metabolism has been well studied and firmly established. Charbon (1,2) reported that PTH had a vasodilatory action and lowered the blood pressure in dogs. Our recent investigations have extended these observations to other species and shown that synthetic bovine PTH containing the amino terminal 1-34 amino acids [bPTH-(I-34)] had a direct vasodilatory action on specific vascular beds (3,4,5,6). It appears that PTH relaxes vascular smooth muscle. Since many vasoactive peptides, such as bradykinin, are also capable of acting on non-vascular smooth muscle, we attempted to determine whether bPTH-(I-34) can affect another smooth muscle, i.e., that of the uterus. Methods Adult female Sherman rats weighing between 180-220 grams were injected i.p. with diethylstilbestrol (50 ~g) 14-16 hours before being killed by cervical dislocation. The uterine horns were dissected out immediately and suspended in a tissue chamber in Van Dyke-Hastings solution containing 0.5 m M M g C I 2, 113.8 mM NaCI, 6.0 mMKCI, 5.0 mM CaCI2, 29.8 mM NaHC03, .72 m M N a 2 H P O 4, .22 m M N a H 2 P O 4, 2.8 mM glucose, equilibrated with 5% carbon dioxide and 95% oxygen (pH 7.4) in . a constant temperature bath (32°C) (7). A resting tension of I gram was applied to the suspended uterine horns and contractions were measured isometrically with a Grass force-dlsplacement transducer (model FT 03) and recorded with a Grass polygraph (model 7D).
0024-3205/81/121317-05502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
1318
Parathyroid Hormone and Uterine Activity
Vol. 28, No.
12, 1981
All solutions were injected directly into the tissue bath. The following agonists were used to cause contraction of uterine smooth muscle: (i) oxytocin (Syntocinon, Sandoz, 0.I mU to 5.0 mU), (2) prostaglandin F2e (0.i ug to 2.0 ug), and (3) acetylcholine bromide (200 ng to 2,000 ng). Electrical stimulation through the surrounding medium, 2 volts, 60 Hz, repeated every minute for a duration of 2.5 seconds was also used to produce contraction in the uterine horn. Four series of experiments were performed. In the first experiment, we investigated the effect of bPTH-(I-34) (Peninsula Laboratories) on oxytocinstimulated uterine contraction. Oxytocin was added to the bath and the contractile force was recorded. After thorough rinsing, the same dose of oxytocin was tested. If similar responses were observed, the tissue was again rinsed thoroughly and bPTH-(I-34) was added to the bath to give a concentration of 40 or 400 ng/ml of bath medium. One minute later, the same dose of oxytocin was tested again. Using this procedure, we could determine the lo~-dose response curves for oxytocin in the presence or absence of bPTH-(i-34). Solvent and the same molar concentration of calcitonin were tested. They showed no inhibitory action (Shew, Yee and Pang, unpublished data). In the second and third series of experiments, we used the same method to determine the effects of 40 and 400 ng of bPTH-(I-34) per ml bath medium on uterine contractions in response to acetylcholine or prostaglandin F2u. In the fourth series of experiments, uterine horns were stimulated electrically, bPTH-(I-34) was then added to the bath and the change in contractile force was recorded. After thorough rinsing, the contractile force returned to the pre-treatment level and a second dose of bPTH-(I-34) was tested. Each uterine horn was tested with several doses of bPTH-(I-34) and the % change in contractile force was calculated and plotted separately for each piece of tissue. Results Figure I shows the log dose-response to oxytocin in the presence of 40 or 400 ng bPTH-(I-34) per ml, or in the absence of this peptide. Each point represents the mean of data from at least six uterine horns. The vertical bars designate standard errors. The data clearly demonstrate the antagonistic action of bPTH-(I-34). Increasing the concentrations of bPTH-(I-34) shifts the log dose-response curve more to the right. This is also evident in uterine activity stimulated by acetylcholine or prostaglandin F2~ (Figures 2 and 3). In Figure 4, the antagonistic action of bPTH-(I-34) on electrically stimulated uterine contraction is apparent. In all five uterine horns studied, increasing the doses of bPTH-(I-34) resulted in increased inhibition. Discussion The relaxing influence of bPTH-(I-34) on vascular smooth muscle is now well established (1,2,3,4,5,6). It has been shown that bPTH-(I-34) has a direct vascular action (4) which is unrelated to the hypercalcemic action of the hormone (8,9,10). Investigations with analogs and fragments of bPTH suggested that the amino acids 24-28 may be involved in the hypotensive action. The present study is the first to report the action of bPTH-(I-34) on another smooth muscle tissue, the uterus. One interesting feature of this investigation is the ability of bPTH-(I-34) to antagonize the contraction produced by three chemicals and by electrical stimulation. This indicates thenonspeciflc antagonistic action of the peptide. It would also suggest that bPTH-(I-34) has a very basic mechanism of action of the contractile apparatus of the tissue. It will be very interesting to determine whether PTH can influence in vivo uterine contraction.
Vol. 28, No. 12, 1981
Parathyroid Hormone and Uterine Activity
1319
3Z u~
T
o
ZuJ k-
CONTROL~"~/~
2 ~
~'//~0
ng PTH/ml
q¢ rf (3
2= m uJ
1--
(3 Z.¢ 3: (J
0I
DOSE
I
OF
I
I
I
I
ACETYLCHOLINE [IJM/m,] Figure i
Inhibition of in vitro rat uterine contraction produced by oxytocin. The preparation of PTH used is the synthetic bPTH-(I-34). Data are expressed as mean ± SEM.
z
~3w
(3 Z
w
(Z3 1 ! O 0-
0.02
o12 DOSEOFOXYT~IN
lmU/ml l
Figure 2 Inhibition of in vitro rat uterine contraction produced by acetylcholine, m e preparation of P ~ used is the syntheti c bP~-(l-34). Data are ~presse~ as mean ± S ~ .
1320
Parathyroid Hormone and Uterine Activity
Vol. 28, No. 12, 1981
1
T E~ / ' , ! /
~•
/
/
52z T ~ i
/ ./4o.g p1".~ ~i/ ,"400 .g PTM m ,
O-
002
o11
o2
o4
DOSE OF PGF2o' [l~g ml!
Figure 3 Inhibition of in vitro rat uterine contraction produced by PGF2a. The preparation of PTH used is the synthetic bPTH-(I-34). Data are expressed as mean ± SEM.
ELECTRICAL STIMULATION (2 V)
-20-
~.r "-400 -~ - 6 0 , -80-
-I00 13
27
DOSE O~ ,-34 ~ o ~
l,~/m,I
Figure 4 Inhibition of the in vitro rat uterine contraction produced by electrical stimulation. The preparation of P T H u s e d is the synthetic bPTH-(I-34). Acknowledgements This investigation was supported by NSF Grant # PCM 78-17695 to PKTP and NSF Grant # PCM 76-18689 and NIAMD Grant # AM 01940 to WHS. References 1. 2. 3.
G.A. CHARBON, Eur. J. Pharmacol. ~ 275-278 (1968). G.A. CHARBON and P.F. HULSTAERT, Endocrinology 95 621-626 (1974). P.K.T. PANG, M. YANG, C. OGURO, J.G. PHILLIPS and J.A. YEE, Gett. Comp.
Vol. 28, No. 12, 1981
Parathyroid Hormone and Uterine Activity
1321
Endocrinol. 4-1 135-138 (1980). P.K.T. PANG, T.E. TENNER, Jr., J.A. YEE, M. YANG and H.F. JANSSEN, Proc. Nat. Acad. Sci. USA 77 675-678 (1980). 5. P.K.T. PANG, H.F. JANSSEN and J.A. YEE, Pharmacology, in press. 6. M.F. CRASS, III and P.K.T. PANG, Science 207 1087-1089 (1980). 7. R.A. MUNSICK, Endocrinology 66 451-457 (1960). 8. P.K.T. PANG, M. YANG and A.D. KENNY, Calc. Tiss. Int. 3-1 74 (1980). 9. A.D. KENNY and P.K.T. PANG, Calc. Tiss. Int. 31 73 (1980). i0. P.K.T. PANG, M. YANG, M.C. KHOSLA and F.M. BUMPUS, Proc. VII Int. Conf. on Calcium Regulating Hormones, Estes Park, Colorado, Sept. 5-9 (1980).
4.