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Responses of isolated human uterine arteries to vasoactive drugs
Responses of isolated human uterine arteries to vasoactive drugs E. DORIS
GOUGH,
DONALD
C.
Seattle,
DYER,
M.D. PH.D.
Washington
Isolated human
uterine arteries were found to respond to several clinically important uasoactiue drugs, i.e., I-isofiroterenol, l-norepinephrine, nitroglycerin, and angiotensinamide (angiotensin). Human uterine arteries possess a&ha and beta adrenergic receptors. The contractile Potency of norepinefihrine was greater than that of angiotensin. Tachyphylaxis occurred with the use of angiotensin. The reliability of isolated human uterine arteries was quite good, which suggests that it might serve as a model for the examination of the actions of drugs on human vascular tissue.
INFORMATION CONCERNING the reaction of drugs on human uterine blood vesselsis meager. Cutchin and co-worker9 found that isolated human uterine arteries contracted to oxytocin, sparteine sulfate, ergonovine, and norepinephrine. Unfortunately, in their study, dose-responserelationship information was not provided for the agonists studied. Therefore, meaningful quantitative comparisons cannot be made for these agonists. Boeles and co-workers* have also observed contractions to ergonovine and vasopressin on isolated human uterine arteries. Because of the lack of data on the effect of vasoactive drugs on human uterine arteries, the purpose of this study was to provide such information on some clinically used drugs.
ectomy specimenswithin two hours of operation. The indications for performing the hysterectomies were for the usual gynecologic reasons.The agesof the patients ranged from 26 to 84 years, with the majority between 40 and 50 years. In this study, 50 per cent of the tissues were obtained from patients who did not receive any medications prior to surgery, 29 per cent were on estrogen hormones, 10 per cent were being treated with thyroid substitutes, and the remaining were on sedatives, diuretics, iron preparations, or a combination of these 3 drug types. We were not able to correlate sensitivity of the uterine artery to norepinephrine with premeditation. The prepared arteries used in this study were approximately 0.20 cm. wide and 12 to 14 mm. long. All strips were suspended under one-gram tension in a 10 ml. organ bath. The tissueswere bathed with a modified Krebs-Henseleit (Krebs) solution, maintained at 37’ C. and aerated with a 5 per cent CO, and 95 per cent O2 mixture. Isotonic contractions were magnified tenfold and recorded on a kymograph. The tissueswere allowed to equilibrate in the bath for at least one hour before starting the experiment. During this time, the Krebs
Methods Helically cut strips of vascular smooth muscle from human uterine arteries were prepared according to the method of Furchgott.3 Tissues were obtained from hysterFrom the Department School of Medicine, Washington. Supported
of Pharmacology, Uniuersity of
in part
by United States National of Health Grant GM 15991.
Public Wtalth Service, Institutes
625
626
Gough
and Dyer Amer.
IOO-
-
Norepinephrine
o---o
Phentolomine (10~‘g/ml1
Q....Q
Phentolomine (10-6g/mll
July 1, 1971 J. Obstet. Gynec.
80 1
Drug
concentration
(g/ml)
Fig. 2. Relaxation of norepinephrine-contracted uterine artery strips by isoproterenol. Note that at the higher concentration the relaxations to isoproterenal decreased. Vertical bars represent standard errors of 6 experiments. 10-e Agonist
10-g
I o-5 (g/ml)
10-7 10-6 concentration
10-4
Fig. 1. The effect of phentolamine on responses to norepinepherine. Phentolamine was allowed to equilibrate with the uterine artery strips for 15 min. before repeating the norepinephrine administration. Note the almost parallel and dosedependent shift of the norepinephrine dose-response curve to the right in the presence of phentolamine. The vertical bars represent standard errors of 10 experiments.
changed several times. The drug were introduced into the bath in cumulative amounts by micropipettes. The following drugs were used in this solution
was
agonists
study:
phentolamine,”
I-isoproterenol,
epinephrine, nitroglycerine, as angiotensinamide.
and
I-nor-
angiotensin
Results The smooth muscle of the human uterine arteries contracted slowly and maintained a contracted state in a response to norepi*Regitine, Jersey.
Ciba
Pharm.
Products,
Inc.,
Summit,
New
nephrine similar to that of other arteries.3; 4 No spontaneous activity was observed in human uterine arteries; however, in a few vein preparations studied, some spontaneous activity was noted during exposure to norepinephrine.
Presence of alpha adrenergic receptors. The cumulative responses to norepinephrine were readily reproducible, i.e., when a second dose response was performed, there was little or no desensitization to norepinephrine. In order to establish the presence of alpha adrenergic receptors, the effects of a specific alpha adrenergic receptor antagonist, phentolamine, was added to the bath. Phentolamine produced a dose-dependent parallel shift of the norepinephrine dose-response curve to the right (Fig. 1) .
Pharmacologic effects of isoproterenol and nitroglycerin. Human uterine arteries exhibit little tone when fully equilibrated. Therefore, to investigate the presence of beta adrenergic receptors, tone was induced by adding nor-
v01umc 110 Number 5
Uterine
$ ‘2
Agonist
concentration
(g/ml)
Fig. 3. Dose-response curves to norepinephrine and isoproterenol on human uterine arteries. Note that isoproterenol does not relax the fully equilibrated artery strip and that at higher concentrations (10-s Gm. per miIliliter) it produced a concentration. Both the norepinephrineand isoproterenol-induced contractions were antagonized by phentolamine ( 10-r Gm. per milliliter). Vertical bars represent standard errors of 12 experiments.
epinephrine ( 1O-6 Gm. per milliliter) to the Krebs solution. This concentration of norepinephrine produced approximately 80 per cent of maximal contraction and maintained the contraction over a one-hour period without appreciable fade. Isoproterenol was then added to the bath in a cumulative manner. The relaxations of the norepinephrine-induced contraction were expressed as a per cent of the contraction to norepinephrine. Isoproterenol was only capable of reducing the norepinephrine contraction 30 per cent at a concentration of 3 x lo+ Gm. per milliliter (Fig. 2). Further increases in the isoproterenol concentration reduced the relaxation. The reduced relaxation at higher concentrations of isoproterenol suggested that alpha adrenergic receptors were stimulated. To investigate the possibility that isoproterenol contracted the uterine blood vessels
8
arterial
80
response to vasoactive drugs
627
1 ~Nitroglycerin
I 10-9
c-6
10-7
10-6
Drug concentration Fig. 4. The relaxant norepinephrine-contracted strips. Vertical bars 5 experiments.
I 10-S
1 10-4
(g /ml)
effects of nitroglycerin on human uterine artery represent standard errors of
at high concentrations, dose-response curves were performed to both isoproterenol and norepinephrine. The results of these cxperiments are presented in Fig. 3. It can be seen that isoproterenol contracted the uterine arteries and that phentolamine antagonized these contractions. Nitroglycerin, a nonspecific smooth muscle relaxant, also relaxed norepinephrine-contracted artery strips (Fig. 4).
Comparison of responsesto norepinephrine and angiotensin. Angiotensin, described in the literature as one of the most powerful vasoconstrictors,6 was added to strips of human uterine arteries, and the results were compared to norepinephrine (Fig. 5 ) . It is apparent that th i s tissue was less sensitive to angiotensin than to norepinephrine and that the potency of norepinephrine was greater than that of angiotensin. Repeated exposure of the arteries to angiotensin indicated that desensitization had occurred.
628
Gough
and
Dyer Amer.
7
IOP Drug
concentrotlon
(g/ml)
Fig. 5. The vasoconstrictor
effect of angiotensin compared to that of norepinephrine. Repeated administration of angiotensin indicated desensitization of the tissue to angiotensin had occurred. Vertical bars represent standard errors of 7 experiments.
Comment
The results of these studies have indicated the presence of alpha and beta adrenergic receptors in human uterine arteries. The responsesof isolated human uterine arteries to norepinephrine resemble that of isolated rabbit aortas in several ways, i.e., norepinephrine is a powerful agonist, and little or no desensitization occurs to repeated administration. Although angiotensin is reported to be the most potent vasopressor known, its ability to produce contractions of human uterine arteries was clearly less than that of norepinephrine. There is no way of knowing if the present results might pertain to these
July 1, 1971 J. Obstet. Gynec.
blood vesselsin vivo during pregnancy, but, if they did, these results would suggesttesting angiotensin in hypotensive crisis during delivery. If such were the case, the systemic blood pressure would be increased without a concomitant increase in uterine vascular resistance; hence, uterine blood flow would actually increase. The present findings are in contrast to the observations by Greiss and Wilkes,6 whose studies with angiotensin on pregnant ewes showed a marked increase of uterine vascular resistance comparable to that which results from norepinephrine. According to their experiments, the increased uterine resistance always exceeded the increase in blood pressure, thereby causing an absolute decreasein uterine blood flow. However, some caution should be used in extrapolating animal data to man as well as in vitro data to the in vivo situation. For example, in the closely linked umbilical circulation, angiotensin was a potent constrictor of isolated helically cut sheepumbilical arteries and veins,7but angiotensin had little action on similarly prepared isolated human umbilical vessels.8In the present
study it was clear
that isoproterenol
did not relax uterine arteries to the extent of nitroglycerin. At elevated concentrations, isoproterenol actually produced a contraction, which was mediated by stimulating alpha adrenergic receptors. These experiments compare favorably to those reported for the action of isoproterenol on the isolated human saphenous vein. Q The stimulation of alpha adrenergic receptors by high concentrations of isoproterenol has also been observed on other vascular smooth muscle. But our observations differ somewhat from the findings of Griess and Pick. lo, I1 In their study isoproterenol was infused into pregnant ewes, and the effects of this drug on uterine blood flow were observed. Isoproterenol produced little or no vasodilation in their studies and may have produced a slight vasoconstriction. They provided evidence that alpha adrenergic receptors were present in sheeputerine arteries, but few or no beta adrenergic receptors were present.
Volume Number
110 5
Uterine
The authors following people us in obtaining Dr. William B.
express their gratitude to the in Seattle hospitals for assisting the tissues used in this study: Hamlin and Mrs. Judy Rein
arterial
response
to vasoactive
drugs
629
(Swedish Hospital), Dr. Ralph C. Ellis and Dr. Clermont S. Powell (Doctors Hospital), and Dr. Hugh W. Jones (Virginia Mason Hospital).
REFERENCES
Cutchin, J. H., McGaughey, H. D., Scoggin, W. A., Harbert, G. M., and Thornton, W. N.: AMER.
J.
OBSTET.
GYNEC.
90:
143,
1964.
Boeler, J., Lankhorst, G. J., Beuningh, H., Stobbelaar, A. K., and Zuyderhoudr, F.: Proc. Int. Union Physiol. Sciences, No. 7, 1968. Furchgott, R. F.: In Bruner, H. D., editor: Methods in Medical Research, Chicago, 1960, Year Book Medical Publishers, Inc., vol. 8, pp. 177-186. Bohr, D. F., and Johansson, B.: Circulation Res. 19: 593, 1966.
5. 6. 7. 8.
9. 10. 11.
Page, I. H., and Bumpus, F. M.: Physiol. Rev. 41: 331, 1961. Greiss, F. Cl., and Wilkes, D. V.: Obstet. Gynec. 23: 925, 1964. Dyer, D. Cl.: J. Pharmacol. Exp. Ther. 175: 565, 1970. Somlyo, A. V., Woo, C. Y., and Somlyo, A. P.: Amer. J, Physiol. 208: 748, 1965. Coupar, I. M.: Brit. J. Pharmacol. 39: 465, 1970. Greiss, F. C., and Pick, J. R.: Obstet. Gynec. 23: 209, 1964. Greiss, F. C.: Obstet. Gynec. 21: 295, 1963.
GOUGH,
DONALD
C.
Seattle,
DYER,
M.D. PH.D.
Washington
Isolated human
uterine arteries were found to respond to several clinically important uasoactiue drugs, i.e., I-isofiroterenol, l-norepinephrine, nitroglycerin, and angiotensinamide (angiotensin). Human uterine arteries possess a&ha and beta adrenergic receptors. The contractile Potency of norepinefihrine was greater than that of angiotensin. Tachyphylaxis occurred with the use of angiotensin. The reliability of isolated human uterine arteries was quite good, which suggests that it might serve as a model for the examination of the actions of drugs on human vascular tissue.
INFORMATION CONCERNING the reaction of drugs on human uterine blood vesselsis meager. Cutchin and co-worker9 found that isolated human uterine arteries contracted to oxytocin, sparteine sulfate, ergonovine, and norepinephrine. Unfortunately, in their study, dose-responserelationship information was not provided for the agonists studied. Therefore, meaningful quantitative comparisons cannot be made for these agonists. Boeles and co-workers* have also observed contractions to ergonovine and vasopressin on isolated human uterine arteries. Because of the lack of data on the effect of vasoactive drugs on human uterine arteries, the purpose of this study was to provide such information on some clinically used drugs.
ectomy specimenswithin two hours of operation. The indications for performing the hysterectomies were for the usual gynecologic reasons.The agesof the patients ranged from 26 to 84 years, with the majority between 40 and 50 years. In this study, 50 per cent of the tissues were obtained from patients who did not receive any medications prior to surgery, 29 per cent were on estrogen hormones, 10 per cent were being treated with thyroid substitutes, and the remaining were on sedatives, diuretics, iron preparations, or a combination of these 3 drug types. We were not able to correlate sensitivity of the uterine artery to norepinephrine with premeditation. The prepared arteries used in this study were approximately 0.20 cm. wide and 12 to 14 mm. long. All strips were suspended under one-gram tension in a 10 ml. organ bath. The tissueswere bathed with a modified Krebs-Henseleit (Krebs) solution, maintained at 37’ C. and aerated with a 5 per cent CO, and 95 per cent O2 mixture. Isotonic contractions were magnified tenfold and recorded on a kymograph. The tissueswere allowed to equilibrate in the bath for at least one hour before starting the experiment. During this time, the Krebs
Methods Helically cut strips of vascular smooth muscle from human uterine arteries were prepared according to the method of Furchgott.3 Tissues were obtained from hysterFrom the Department School of Medicine, Washington. Supported
of Pharmacology, Uniuersity of
in part
by United States National of Health Grant GM 15991.
Public Wtalth Service, Institutes
625
626
Gough
and Dyer Amer.
IOO-
-
Norepinephrine
o---o
Phentolomine (10~‘g/ml1
Q....Q
Phentolomine (10-6g/mll
July 1, 1971 J. Obstet. Gynec.
80 1
Drug
concentration
(g/ml)
Fig. 2. Relaxation of norepinephrine-contracted uterine artery strips by isoproterenol. Note that at the higher concentration the relaxations to isoproterenal decreased. Vertical bars represent standard errors of 6 experiments. 10-e Agonist
10-g
I o-5 (g/ml)
10-7 10-6 concentration
10-4
Fig. 1. The effect of phentolamine on responses to norepinepherine. Phentolamine was allowed to equilibrate with the uterine artery strips for 15 min. before repeating the norepinephrine administration. Note the almost parallel and dosedependent shift of the norepinephrine dose-response curve to the right in the presence of phentolamine. The vertical bars represent standard errors of 10 experiments.
changed several times. The drug were introduced into the bath in cumulative amounts by micropipettes. The following drugs were used in this solution
was
agonists
study:
phentolamine,”
I-isoproterenol,
epinephrine, nitroglycerine, as angiotensinamide.
and
I-nor-
angiotensin
Results The smooth muscle of the human uterine arteries contracted slowly and maintained a contracted state in a response to norepi*Regitine, Jersey.
Ciba
Pharm.
Products,
Inc.,
Summit,
New
nephrine similar to that of other arteries.3; 4 No spontaneous activity was observed in human uterine arteries; however, in a few vein preparations studied, some spontaneous activity was noted during exposure to norepinephrine.
Presence of alpha adrenergic receptors. The cumulative responses to norepinephrine were readily reproducible, i.e., when a second dose response was performed, there was little or no desensitization to norepinephrine. In order to establish the presence of alpha adrenergic receptors, the effects of a specific alpha adrenergic receptor antagonist, phentolamine, was added to the bath. Phentolamine produced a dose-dependent parallel shift of the norepinephrine dose-response curve to the right (Fig. 1) .
Pharmacologic effects of isoproterenol and nitroglycerin. Human uterine arteries exhibit little tone when fully equilibrated. Therefore, to investigate the presence of beta adrenergic receptors, tone was induced by adding nor-
v01umc 110 Number 5
Uterine
$ ‘2
Agonist
concentration
(g/ml)
Fig. 3. Dose-response curves to norepinephrine and isoproterenol on human uterine arteries. Note that isoproterenol does not relax the fully equilibrated artery strip and that at higher concentrations (10-s Gm. per miIliliter) it produced a concentration. Both the norepinephrineand isoproterenol-induced contractions were antagonized by phentolamine ( 10-r Gm. per milliliter). Vertical bars represent standard errors of 12 experiments.
epinephrine ( 1O-6 Gm. per milliliter) to the Krebs solution. This concentration of norepinephrine produced approximately 80 per cent of maximal contraction and maintained the contraction over a one-hour period without appreciable fade. Isoproterenol was then added to the bath in a cumulative manner. The relaxations of the norepinephrine-induced contraction were expressed as a per cent of the contraction to norepinephrine. Isoproterenol was only capable of reducing the norepinephrine contraction 30 per cent at a concentration of 3 x lo+ Gm. per milliliter (Fig. 2). Further increases in the isoproterenol concentration reduced the relaxation. The reduced relaxation at higher concentrations of isoproterenol suggested that alpha adrenergic receptors were stimulated. To investigate the possibility that isoproterenol contracted the uterine blood vessels
8
arterial
80
response to vasoactive drugs
627
1 ~Nitroglycerin
I 10-9
c-6
10-7
10-6
Drug concentration Fig. 4. The relaxant norepinephrine-contracted strips. Vertical bars 5 experiments.
I 10-S
1 10-4
(g /ml)
effects of nitroglycerin on human uterine artery represent standard errors of
at high concentrations, dose-response curves were performed to both isoproterenol and norepinephrine. The results of these cxperiments are presented in Fig. 3. It can be seen that isoproterenol contracted the uterine arteries and that phentolamine antagonized these contractions. Nitroglycerin, a nonspecific smooth muscle relaxant, also relaxed norepinephrine-contracted artery strips (Fig. 4).
Comparison of responsesto norepinephrine and angiotensin. Angiotensin, described in the literature as one of the most powerful vasoconstrictors,6 was added to strips of human uterine arteries, and the results were compared to norepinephrine (Fig. 5 ) . It is apparent that th i s tissue was less sensitive to angiotensin than to norepinephrine and that the potency of norepinephrine was greater than that of angiotensin. Repeated exposure of the arteries to angiotensin indicated that desensitization had occurred.
628
Gough
and
Dyer Amer.
7
IOP Drug
concentrotlon
(g/ml)
Fig. 5. The vasoconstrictor
effect of angiotensin compared to that of norepinephrine. Repeated administration of angiotensin indicated desensitization of the tissue to angiotensin had occurred. Vertical bars represent standard errors of 7 experiments.
Comment
The results of these studies have indicated the presence of alpha and beta adrenergic receptors in human uterine arteries. The responsesof isolated human uterine arteries to norepinephrine resemble that of isolated rabbit aortas in several ways, i.e., norepinephrine is a powerful agonist, and little or no desensitization occurs to repeated administration. Although angiotensin is reported to be the most potent vasopressor known, its ability to produce contractions of human uterine arteries was clearly less than that of norepinephrine. There is no way of knowing if the present results might pertain to these
July 1, 1971 J. Obstet. Gynec.
blood vesselsin vivo during pregnancy, but, if they did, these results would suggesttesting angiotensin in hypotensive crisis during delivery. If such were the case, the systemic blood pressure would be increased without a concomitant increase in uterine vascular resistance; hence, uterine blood flow would actually increase. The present findings are in contrast to the observations by Greiss and Wilkes,6 whose studies with angiotensin on pregnant ewes showed a marked increase of uterine vascular resistance comparable to that which results from norepinephrine. According to their experiments, the increased uterine resistance always exceeded the increase in blood pressure, thereby causing an absolute decreasein uterine blood flow. However, some caution should be used in extrapolating animal data to man as well as in vitro data to the in vivo situation. For example, in the closely linked umbilical circulation, angiotensin was a potent constrictor of isolated helically cut sheepumbilical arteries and veins,7but angiotensin had little action on similarly prepared isolated human umbilical vessels.8In the present
study it was clear
that isoproterenol
did not relax uterine arteries to the extent of nitroglycerin. At elevated concentrations, isoproterenol actually produced a contraction, which was mediated by stimulating alpha adrenergic receptors. These experiments compare favorably to those reported for the action of isoproterenol on the isolated human saphenous vein. Q The stimulation of alpha adrenergic receptors by high concentrations of isoproterenol has also been observed on other vascular smooth muscle. But our observations differ somewhat from the findings of Griess and Pick. lo, I1 In their study isoproterenol was infused into pregnant ewes, and the effects of this drug on uterine blood flow were observed. Isoproterenol produced little or no vasodilation in their studies and may have produced a slight vasoconstriction. They provided evidence that alpha adrenergic receptors were present in sheeputerine arteries, but few or no beta adrenergic receptors were present.
Volume Number
110 5
Uterine
The authors following people us in obtaining Dr. William B.
express their gratitude to the in Seattle hospitals for assisting the tissues used in this study: Hamlin and Mrs. Judy Rein
arterial
response
to vasoactive
drugs
629
(Swedish Hospital), Dr. Ralph C. Ellis and Dr. Clermont S. Powell (Doctors Hospital), and Dr. Hugh W. Jones (Virginia Mason Hospital).
REFERENCES
Cutchin, J. H., McGaughey, H. D., Scoggin, W. A., Harbert, G. M., and Thornton, W. N.: AMER.
J.
OBSTET.
GYNEC.
90:
143,
1964.
Boeler, J., Lankhorst, G. J., Beuningh, H., Stobbelaar, A. K., and Zuyderhoudr, F.: Proc. Int. Union Physiol. Sciences, No. 7, 1968. Furchgott, R. F.: In Bruner, H. D., editor: Methods in Medical Research, Chicago, 1960, Year Book Medical Publishers, Inc., vol. 8, pp. 177-186. Bohr, D. F., and Johansson, B.: Circulation Res. 19: 593, 1966.
5. 6. 7. 8.
9. 10. 11.
Page, I. H., and Bumpus, F. M.: Physiol. Rev. 41: 331, 1961. Greiss, F. Cl., and Wilkes, D. V.: Obstet. Gynec. 23: 925, 1964. Dyer, D. Cl.: J. Pharmacol. Exp. Ther. 175: 565, 1970. Somlyo, A. V., Woo, C. Y., and Somlyo, A. P.: Amer. J, Physiol. 208: 748, 1965. Coupar, I. M.: Brit. J. Pharmacol. 39: 465, 1970. Greiss, F. C., and Pick, J. R.: Obstet. Gynec. 23: 209, 1964. Greiss, F. C.: Obstet. Gynec. 21: 295, 1963.