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Mechanism of action of sparteine sulfate on myometrial activity in vitro
Mechanism of action of sparteine sulfate on myometrial activity in vitro S.
YAMADA,
S. B.
GUSBERG,
B.
WALKER,
H.
JAFFIN,
T.
D.
New
York,
M.D. M.D. R.N. M.D.
KERENYI, New
M.D.
York
The in vitro response of uterine muscle to sparteine sulfate was studied in 104 strips of human and 65 strips of rat uterine muscle. Sparteine sulfate influences the duration and frequency but not the maximum tension development of sfiontaneous activity in the rat myometrium. In all studies of electrically stimulated human and rat myometrium an increase of maximum tension was observed after sparteine sulfate stimulation. In the human no increase in sensitivity to sparteine sulfate was observed with advancing gestation. However, the gradual increase in sensitivity to Pitocin as pregnancy Progresses has been observed by us as well as by several other investigators. The rat uterus, unlike the human uterus, exhibits an increased sensitivity to sparteine sulfate during advancing stages of pregnancy. A greater response is demonstrable during late pregnancy and is relatively refractile during early and midgestation. The response of muscle strips obtained from placental implantation sites to sparteine sulfate were less marked than uterine muscle strips from the opposite wall of the same uterus (nonplacental sites). Furthermore, the dose response range of human uterine muscle to increasing concentrations of sparteine sulfate was narrower than with Pitocin. Larger doses of sparteine sulfate produced myometrial contracture in normal Krebs’ solution. Lower concentrations had a tendency to cause myometrial contracture on partially depolarized muscle. These studies suggest the major action of sparteine sulfate is directed at the cell membrane rather than directly on the contractile system of the uterine muscle cell.
s P A R T E I N E sulfate influences the duration and frequency but not the maximum tension development of spontaneous activity in the rat myometrium. An increase in maximum tension development in both the rat and human myometrium, when electrically stimulated, was observed after sparteine sulfate stimulation. With advancing gestation, the rat uterus,
From the De$artment of Obstetrics and Gynecology, The Mount Sinai School of Medicine and The Mount Sinai Hospital. This investigation was supported in part by Grant HD-00491-04 from the National Institutes of Health of the United States Public Health Service and by a gift from Ayerst Pharmaceutical Laboratories, New York, New York.
unlike the human uterus, exhibits an increased sensitivity to sparteine sulfate. A gradual increase in sensitivity to Pitocin as pregnancy advances was observed in the human myometrium. The response of placental site muscle strips to sparteine sulfate was less marked than nonplacental site muscle strips from the same uterus. Furthermore, the dose response range of human uterine muscle to increasing concentrations of sparteine sulfate was narrower than with Pitocin. Large doses of sparteine sulfate produced myometrial contracture in normal Krebs’ solution and lower concentrations had a tendency to cause myometrial cont.racture on partially depolarized muscle. These studies suggest that the major action of sparteine
1090
Yamada
August 15, 1968 Am. J. Obst. & Gynec.
et al.
sulfate is directed at the cell membrane rather than directly on the contractile system of the uterine muscle cell. It is notable that sparteine sulfate, a naturally occurring alkaloid, has been used as an oxytocic to induce uterine contractions or to accelerate the progress of labor. This alkaloid, purified by Stenhousel in 1851, was administered as a therapeutic agent for certain supraventricular cardiac arrhythmias and for suppression of atria1 fibrillation. In 1921, Tamba2 first described the pharmacologic effects of this drug on the myometrium. Its effectiveness on the guinea pig uterus was confirmed by Heathcote,3 in 1926, and on the pregnant cat by Kreitmiar and Sieckmann4 10 years later. In 1939 Kleine5 first reported the succesful use of sparteine sulfate in parturient women. During the same year, MahonG suggested that uterine contractions consist of neurogenie and myogenic components. The neurogenic activity is mediated through the autonomic nervous system. The sympathetic system maintains the muscle tonus, while the parasympathetic system can influence intermittent contractions of the uterus. He based these conclusions on experiments with various drugs in parturient women. In recent years it has become apparent that certain steroids (i.e., estrogens, progesterone, ovarian, and placental) have a greater influence on the regulatory mechanism of the myometrium. Several investigators have demonstrated the significant interrelationship between these hormones on membrane activity and to the contractile protein content of the cells. The concept of “progesterone block” was formulated by Csapo7* s in 1956. However, some investigators9 could not confirm the difference in membrane potential between estrogenand progesterone-dominated myometrium. Relaxin, the so-called third hormone, has also been found to have an effect on the membrane potential of the myometrium of the rabbit.lor I1 In recent years, many investigators12-15 have reported that sparteine sulfate is an effective drug for the induction or stimulation of labor. However, few studies have
been undertaken to determine the possible mode of action of sparteine sulfate on the myometrium. 16, I7 Its action on the myocardium appears to be a direct one, i.e., it is not inhibited by parasympathetic agents’s and its effect has been demonstrated on in vitro heart muscIe preparations.ls This particular work was undertaken to investigate the mode of action of sparteine sulfate on the contractile system in human and rat uterine muscle. Method
and
material
All experiments were carried out in vitro. The human muscle strips used in this study were obtained at cesarean section or hysterectomy. The pregnant, nonpregnant, and postpartum animal uteri were from the Sprague-Dawley rat.
Tension development during spontaneous contraction. Human myometrium was dissected into 5 cm. by 2 cm. strips. The entire rat uterus was used in 5 cm. lengths. The muscle was suspended vertically in a muscle chamber which contained oxygenated (95 per cent 0, and 5 per cent CO,) Krebs’ solution (NaCI 188.46; NaHCO, 24.87; KC1 4.47; CaCI, 2.54; KH,PO, 1.18; MgSO, 1.18; glucose 5.56; millimolar pH 7.4) at 370 c. Tension developed by the myometrium was recorded under isometric conditions with a strain gauge transducer (FTA-100-l), a carrier amplifier, and a Sanborn polyContiguous segments from each graph. uterus were tested simultaneously. After a control tracing was obtained, varying amounts of sparteine sulfate were added to the Krebs solution. All concentrations are expressed as amounts of drug per 100 ml. of chamber solution. Electrically stimulated tension. The system was the same as above. An electrical stimulus (60 cycle A.C.) of 4 volts per centimeter longitudinal field for human myometrium and 2 volts per centimeter longitudinal field for rat uterus was applied for a duration of 5 seconds and with the frequency of one stimulus every 60 seconds. The stimulus was applied by suspending a
Volume Number
101 8
Effect
of sparteine
Results
In normal Krebs’ solution. In all experiments involving human myometrium, maximum tension of the muscle strip was calculated to grams per square centimeter. Maximum tension in the spontaneous contractions was 175 to 500 Gm. per square centimeter at term, 80 to 120 Gm. per square centimeter in the nonpregnant uterus, and 38 to 60 Gm. per square centimeter in the climacteric uterus. As shown in Table I, no significant increase in maximum tension was noted in the spontaneous contracting uterus following the addition of sparteine sulfate to the chamber fluid. However, duration and frequency progressively increased with increasing concentrations (range, 1 to 50 pg per milliliter) of sparteine sulfate. In excess of 100 p,g per milliliter, a decrease in amplitude of spontaneous contractions was observed in almost all cases (Table I). In contrast, increasing doses of oxytocin resulted not only in increased duration and frequency but also in a gradual increase in amplitude as well (Table II). In the electrically stimulated tension stud-
No. of experiments
Condition
Nonplacental site at term
45 Tension Duration Frequency
Placental site
Table II. Effect
sulfate
6.622.1 5.622.0 0
3.724.5 7.824.8 10.524.2
8 Tension Duration Frequency
of oxytocin
l.Ot1.3 6.722.5 7.4t4.0
Nonplacental site
10 Tension Duration Freauencv
of I
10.9k5.2 5.7 t 5.6 11.3 2 5.3
1091
contraction
increase in tension,
of human duration,
uterus
and
frequency _---.-.-
2opg ( 30 Kg j 4OM I 50% / loo&! 4.424.6 3.225.1 2.32 5.3 -30.5 z?I12.1 4.5t6.0 9.6k6.413.3k6.9 23.3zk8.7 32.6-f 6.6 +81.5 5 15.0 12.2k7.1 14.427.6 15.4k9.7 17.051 0.4 +30.5 3t 10.1 1.5k1.5 8.454.2 10.1+4.0
on the spontaneous Percentage
in vitro
In calcium deficient Krebs’ solution. Hu-
on the spontaneous
1 IOPR
myometrium
man myometrium was observed in l/2, l/5, and l/10 of the normal calcium Krebs’ solution and, as shown in Fig. 4, spontaneous activity virtually disappeared under these conditions. However, partial return of activity was noted following the introduction
Percentage 5pg
on
ies, sparteine sulfate always produced an increase in myometrial tension, as seen in Fig. 1. Muscle strips obtained from nonplacental sites of the term myometrium responded to sparteine sulfate stimulation differently than did strips excised from the placental implantation site (Table I). Eight cases of the latter were studied. The tension developed by these strips was uniformly lowet than that of the nonplacental site strips. In similar experiments with rat myometrium, sparteine sulfate invariabIy increased the amplitude of the contractions. The sensitivity of uterine muscle to sparteine sulfate in various stages of pregnant! is seen in Fig. 2. The drug is more effective in the late pregnancy and least effective during the midpregnancy, as indicated by this graph. In the case of human myometrium, no change in sensitivity could be demonstrated at different stages of gcstation (Fig. 3).
segment of muscle in Krebs’ solution between two Ag-AgC1 wire ring electrodes.
Table I. Effect of sparteine
sulfate
1.722.4 11.0+5.6 11.1 to.1
contraction of increase
15.8k5.2 10.1 r4.0 15.9 * 9.4
1.7’3.8 22.6k6.5 14.6t2.8
of human
in tension,
20.6% 6.3 12.3 + 1 I.0 14.6 -I 77.7
duration,
22.7 2 5.5 16.7 2 9.8 11.1 t 7.7
l.lf 24.8-c 17.42 -
4.6 7.1 3.1 _.- -... --._-
uterus and frequency 24.3+ 6.0 18.8 ? 8.6 8.3 2 7.6
28.1 + 6.2 17.5-t 10.9 lO.Ok 7.5
1092
Yamada
et al.
Art.
Fig. 1. Normal conditions as control. A, Human myometrium at fourteenth myometrium at sixteenth week. C, Human myometrium at term. D, Human term with oxytocin for comparison.
I 0
I /
Fig. 2. Effect imum tension myometrium.
I 2 of sparteine of electrically
I
I
3
4
sulfate on stimulated
pEND the
maxhuman
EFFECT OF 3Ouqkc SPARTEINE SUiATEONTHERAT MYOMETRIUM
O-SPONTANEOUS
CONTRACTION
l ~ELECTRICALLYSTIYULATEO TENSION
Days
of Prtgnoncy
Fig. 3. Estrus, pregnant, and postpartum with 30 pg per milliliter of sparteine Sparteine sulfate has least effect in the as is seen here.
rat uteri sulfate. midstage,
of 100 pg sparteine sulfate. The appearance of sparteine sulfate-induced contracture was noted in l/10 calcium deficient solution. The response to 100 PU per cubic centimeter oxytocin was even more marked on the same muscle strip and a regular contraction pattern developed.
August 1.5. t968 J. Obst. & Gynrc
week. B, Human myometrium at
Fig. 5 demonstrates the effect of increasing doses of sparteine on rat uterine muscle at different stages of gestation in l/10 Ca++ ion concentrations. Smaller doses of sparteine sulfate appear to be effective on the estrogen dominated uterus (Fig. 5, A). When rat myometrium is progesterone dominated, at the fifteenth day of pregnancy (Fig. 5, B), the same dose of sparteine sulfate is much less effective. As pregnancy approaches term, on the twentieth day (Fig. 5, C), the response is somewhat increased but does not equal that of the unblocked, estrogen-dominated myometrium. The percentage increase in uterine tension in normal, l/2, l/5, and l/10 Ca++ Krebs’ solution, after the addition of sparteine sulfate, on the electrically stimulated rat myometrium is seen in Fig. 6. The response is inversely proportiona to the degree of depolarization. In K+ excess and deficiency solutions. A comparison of effects of sparteine sulfate on the human myometrium in normal Krebs’ and in 5 x K solution is made in Fig. 7. Depolarization of myometrial cells was noted and increases in uterine active tension were observed in the 5 x K experiments. Maximum tension of the electrically stimuIated myometrium (4 voIts per square centimeter) at varying sparteine sulfate concentrations were then plotted. In 5 x K solution, the maximum myometrial tension
Fig. 4. Human myometrium in calcium-deficient solutions. A, Spontaneous contraction in normal Krebs’ solution with sparteine sulfate. B, In l/2 Ca” Krebs’ solution after 100 pg per solution after 100 pg per milliliter sparteine milliliter sparteine sulfate. C, In l/5 Cat+ Krebs’ sulfate. D, In l/10 Ca++ Krebs’ solution after 100 pg per milliliter sparteine sulfate. E, In l/10 Cat+ Krebs’ solution after 100 aU oxytocin.
Fig. 5. Rat gen-dominated ometrium.
myometrium estrus
in calcium-deficient Krebs’ solution with myometrium; B, fifteenth day myometrium;
! B 2 oz p.
’
1
1
,
IOug 2Oug 30ug 40ug 5Oug 60ug Dose of Sparteina Sulfate ug./ ml.
Ca*FREE
s ?
70ug
I”
Fig. 6. Increasing Ca++ deficiency interferes with the stimulatory effect of sparteine sulfate on the electrically stimulated rat myometrium.
L
5o
L
A, Estroday my-
:.,FoL~K+s~uT,~
0
--I-x-x-x4 I
1;:
sparteine sulfate. C, twentieth
0 IN NORYAL KREES SOLUTION t
I
Control 2oug Dose of Sporteine
I
*
40ug Sutfote
1
.
6Ouq ug/ml.
Fig. 7. Comparison of dose response sparteine sulfate on electrically stimulated myometrium in normal Krebs’ solution 5 x K.
effect of human and in
1094
Yamada
August 15, 1968 Am. J. Obst. & Gyncc.
et al.
EFFECTSOFSPAATEINE SULFATEON RAT MYOMETRIUM INVARIOUS K+CONTENT SOLUTIONS A l8fh Day of Pregmcy B. tzlfrs Posfpmfum .-IN NORYALKREBSSOLUTION o-lN2lK+SolUTlON q INSrK+ SOLUTIOK 8 a- IN KRINGERsolurloN ‘Zc IIN K~EFI~IENTSOLUTION !z!50-
I
Dose of Sparteine
Sulfate
Fig. 8. Percentage increase development of electrically depolarized rat myometrium.
2
uglml
4
8
I6
32
64
in Sdution
in maximum stimulated,
tension partially
was greater at all levels of drug concentration. The rat myometrium in K” excess solution showed greater percentage increase in maximum tension in late pregnancy than it did postpartum (Fig. 8) .
Comment These in vitro experiments have shown that an adequate amount of sparteine sulfate has a direct effect on myometrial activity. It stimulates tension development, frequency, and duration in the spontaneousIy contracting rat uterus. In human myometrium, under similar circumstances, increases in frequency and duration of contractions are noted but the effect on maximum tension development is negligible. Experiments on electrically stimulated uterine muscle showed that sparteine sulfate increased tension in both human and rat uteri. From these findings, it is quite apparent the effect of sparteine sulfate on the
Stenhouse, Tamba, Kioto 4: 3. Heathcote, Therap. 4. Kreitmiar, Jahresb.
The authors wish to extend thanks to Dr. John Sproul and Ayerst Laboratories for their interest and support and to Trent Pharmaceuticals, Inc., for the Tocosamine (sparteine sulfate, Trent) they so generously supplied for this study.
5.
REFERENCES
1. 2.
mechanical activity of the myometrium is of a stimulatory nature. Rosenblum and SteinZO reported that iu cases of human uterine muscle “depolarized” by potassium Ringer’s solution sparteine sulfate acts primarily on the cell membrane rather than directly on the contractile elements. Our experiments also suggest that the sparteine sulfate effect is mediated by altering the membrane potential. Once tetanus is brought about by K+ excess depolarization, no further sparteine sulfate effect is possible, Similarly, Ca++-induced depolarization produces a narrowing of the range where sparteine sulfate exhibits a stimulatory effect. Tetanic contractions developed rapidly and at comparatively lower doses. To minimize variables inherent when testing different pharmacologic agents, contiguous strips of the same myometrium were used GmultaneousIy, one for sparteine sulfate and the other for oxytocin. The majority of investigators agree that the most pronounced effect of oxytocin is mediated through the changes in membrane potentials.21‘2s However, some reports point out the possibility of additional components as well. Berger and MarshalIZ reported the action of oxytocin on the contractile protein of the rat myometrium with depolarized muscle, and Yamada30 has reported that oxytocin appears to have some slight effect on the contractile system of the myometrium using glycerol-treated muscle.
J.: Liebig’s Ann. 78: 20, 1851. G.: Acta scholae med. univ. imp. in 85, 1921. R. St. A.: J. Pharmacol. & Exper. 27: 431, 1936. H., and Sieckmann, W.: Merck’s 50: 111, 1936.
6. 7. 8. 9.
Kleine, H. 0.: Klin. Wchnschr. 18: 360: 1939. Mahon, R.: Gynec. et obst. 39: 177, 1939. Csapo, A.: Am. J. Anat. 98: 273, 1956a. Csapo, A.: Recent Progr. Hormone Res. 12: 405, 1956b. Kao, C. Y., and Nishiyama, A.: Am. J. Physiol. 207: 793, 1964.
V&me
IiN
Number
8
10. 11.
12.
13. 14. 15.
16. 17.
1x. 19.
Effect
Yamada, S.: Texas Rep. Biol. & Med. 22: 209, 1964. Anderson, N., and Zarrow, M. X.: Presented at FASEB Meeting, Chicago, Illinois, April, 1964. Plentl, A. A., Friedman, E. A., and Gray, M. F.: AM. J. OBST. & GYNEC. 82: 1331, 1961. Bedrosian, L., and Gamble, J. J.: Obst. & Gynec. 21: 400, 1963. Boysen, H.: Obst. & Gynec. 21: 403, 1963. Cromer, D. W., Reeves, B. D., and Danforth, D. N.: AM. J. OBST. & GYNEC. 89: 268, 1964. Stander, R. W.: Obst. & Gynec. 26: 876, 1965. Landesman, R., Wilson, K. H., La Russa, R., and Silverman, F.: Obst. & Gynec. 23: 2, 1964. Lu, G.: Arch. Internat. Pharmacodyn. 76: 367, 1948. Soula, L. C., and Delas, J.: J, de mtd. de Paris xliv: 901, 1925.
of sparteine
20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
sulfate
on
myometrium
in vitro
1095
Rosenblum, I., and Stein, A. A.: J. Pharmacol. & Exper. Therap. 144: 138, 1964. Woodbury, J. W., and McIntyre, D. M.: Am. J. Physiol. 177: 355, 1954. Woodbury; J. W., and McIntyre, D. M.: Am. T. Phvsiol. 187: 338. 1956. Jung,’ H.: Arch. GynPk. 190: 194, 1957. Landa, J. F., West, T. C., and Thiersrh, J. B.: Am. J. Physiol. 196: 905, 1959. Kuriyama, H., and Csapd, A.: Biol. Bull. Woods Hole 177: 417. 1959. Csapo, A.: Ann. New York Acad. Sr 75: 790, 1959. Coutinho. E., and Csapo, A.: J. Gen. Physiol. 43: 13, 1959. Kuriyama, H., and Csapo, A.: Endocrinology 68: 1010, 1961. Berger, E., and Marshall, J. M : Am. J. Physiol. 201: 931, 1961. Yamada. S.: Jikei-Ishi (Jikei M. J.? 74: 2711, 1959.
YAMADA,
S. B.
GUSBERG,
B.
WALKER,
H.
JAFFIN,
T.
D.
New
York,
M.D. M.D. R.N. M.D.
KERENYI, New
M.D.
York
The in vitro response of uterine muscle to sparteine sulfate was studied in 104 strips of human and 65 strips of rat uterine muscle. Sparteine sulfate influences the duration and frequency but not the maximum tension development of sfiontaneous activity in the rat myometrium. In all studies of electrically stimulated human and rat myometrium an increase of maximum tension was observed after sparteine sulfate stimulation. In the human no increase in sensitivity to sparteine sulfate was observed with advancing gestation. However, the gradual increase in sensitivity to Pitocin as pregnancy Progresses has been observed by us as well as by several other investigators. The rat uterus, unlike the human uterus, exhibits an increased sensitivity to sparteine sulfate during advancing stages of pregnancy. A greater response is demonstrable during late pregnancy and is relatively refractile during early and midgestation. The response of muscle strips obtained from placental implantation sites to sparteine sulfate were less marked than uterine muscle strips from the opposite wall of the same uterus (nonplacental sites). Furthermore, the dose response range of human uterine muscle to increasing concentrations of sparteine sulfate was narrower than with Pitocin. Larger doses of sparteine sulfate produced myometrial contracture in normal Krebs’ solution. Lower concentrations had a tendency to cause myometrial contracture on partially depolarized muscle. These studies suggest the major action of sparteine sulfate is directed at the cell membrane rather than directly on the contractile system of the uterine muscle cell.
s P A R T E I N E sulfate influences the duration and frequency but not the maximum tension development of spontaneous activity in the rat myometrium. An increase in maximum tension development in both the rat and human myometrium, when electrically stimulated, was observed after sparteine sulfate stimulation. With advancing gestation, the rat uterus,
From the De$artment of Obstetrics and Gynecology, The Mount Sinai School of Medicine and The Mount Sinai Hospital. This investigation was supported in part by Grant HD-00491-04 from the National Institutes of Health of the United States Public Health Service and by a gift from Ayerst Pharmaceutical Laboratories, New York, New York.
unlike the human uterus, exhibits an increased sensitivity to sparteine sulfate. A gradual increase in sensitivity to Pitocin as pregnancy advances was observed in the human myometrium. The response of placental site muscle strips to sparteine sulfate was less marked than nonplacental site muscle strips from the same uterus. Furthermore, the dose response range of human uterine muscle to increasing concentrations of sparteine sulfate was narrower than with Pitocin. Large doses of sparteine sulfate produced myometrial contracture in normal Krebs’ solution and lower concentrations had a tendency to cause myometrial cont.racture on partially depolarized muscle. These studies suggest that the major action of sparteine
1090
Yamada
August 15, 1968 Am. J. Obst. & Gynec.
et al.
sulfate is directed at the cell membrane rather than directly on the contractile system of the uterine muscle cell. It is notable that sparteine sulfate, a naturally occurring alkaloid, has been used as an oxytocic to induce uterine contractions or to accelerate the progress of labor. This alkaloid, purified by Stenhousel in 1851, was administered as a therapeutic agent for certain supraventricular cardiac arrhythmias and for suppression of atria1 fibrillation. In 1921, Tamba2 first described the pharmacologic effects of this drug on the myometrium. Its effectiveness on the guinea pig uterus was confirmed by Heathcote,3 in 1926, and on the pregnant cat by Kreitmiar and Sieckmann4 10 years later. In 1939 Kleine5 first reported the succesful use of sparteine sulfate in parturient women. During the same year, MahonG suggested that uterine contractions consist of neurogenie and myogenic components. The neurogenic activity is mediated through the autonomic nervous system. The sympathetic system maintains the muscle tonus, while the parasympathetic system can influence intermittent contractions of the uterus. He based these conclusions on experiments with various drugs in parturient women. In recent years it has become apparent that certain steroids (i.e., estrogens, progesterone, ovarian, and placental) have a greater influence on the regulatory mechanism of the myometrium. Several investigators have demonstrated the significant interrelationship between these hormones on membrane activity and to the contractile protein content of the cells. The concept of “progesterone block” was formulated by Csapo7* s in 1956. However, some investigators9 could not confirm the difference in membrane potential between estrogenand progesterone-dominated myometrium. Relaxin, the so-called third hormone, has also been found to have an effect on the membrane potential of the myometrium of the rabbit.lor I1 In recent years, many investigators12-15 have reported that sparteine sulfate is an effective drug for the induction or stimulation of labor. However, few studies have
been undertaken to determine the possible mode of action of sparteine sulfate on the myometrium. 16, I7 Its action on the myocardium appears to be a direct one, i.e., it is not inhibited by parasympathetic agents’s and its effect has been demonstrated on in vitro heart muscIe preparations.ls This particular work was undertaken to investigate the mode of action of sparteine sulfate on the contractile system in human and rat uterine muscle. Method
and
material
All experiments were carried out in vitro. The human muscle strips used in this study were obtained at cesarean section or hysterectomy. The pregnant, nonpregnant, and postpartum animal uteri were from the Sprague-Dawley rat.
Tension development during spontaneous contraction. Human myometrium was dissected into 5 cm. by 2 cm. strips. The entire rat uterus was used in 5 cm. lengths. The muscle was suspended vertically in a muscle chamber which contained oxygenated (95 per cent 0, and 5 per cent CO,) Krebs’ solution (NaCI 188.46; NaHCO, 24.87; KC1 4.47; CaCI, 2.54; KH,PO, 1.18; MgSO, 1.18; glucose 5.56; millimolar pH 7.4) at 370 c. Tension developed by the myometrium was recorded under isometric conditions with a strain gauge transducer (FTA-100-l), a carrier amplifier, and a Sanborn polyContiguous segments from each graph. uterus were tested simultaneously. After a control tracing was obtained, varying amounts of sparteine sulfate were added to the Krebs solution. All concentrations are expressed as amounts of drug per 100 ml. of chamber solution. Electrically stimulated tension. The system was the same as above. An electrical stimulus (60 cycle A.C.) of 4 volts per centimeter longitudinal field for human myometrium and 2 volts per centimeter longitudinal field for rat uterus was applied for a duration of 5 seconds and with the frequency of one stimulus every 60 seconds. The stimulus was applied by suspending a
Volume Number
101 8
Effect
of sparteine
Results
In normal Krebs’ solution. In all experiments involving human myometrium, maximum tension of the muscle strip was calculated to grams per square centimeter. Maximum tension in the spontaneous contractions was 175 to 500 Gm. per square centimeter at term, 80 to 120 Gm. per square centimeter in the nonpregnant uterus, and 38 to 60 Gm. per square centimeter in the climacteric uterus. As shown in Table I, no significant increase in maximum tension was noted in the spontaneous contracting uterus following the addition of sparteine sulfate to the chamber fluid. However, duration and frequency progressively increased with increasing concentrations (range, 1 to 50 pg per milliliter) of sparteine sulfate. In excess of 100 p,g per milliliter, a decrease in amplitude of spontaneous contractions was observed in almost all cases (Table I). In contrast, increasing doses of oxytocin resulted not only in increased duration and frequency but also in a gradual increase in amplitude as well (Table II). In the electrically stimulated tension stud-
No. of experiments
Condition
Nonplacental site at term
45 Tension Duration Frequency
Placental site
Table II. Effect
sulfate
6.622.1 5.622.0 0
3.724.5 7.824.8 10.524.2
8 Tension Duration Frequency
of oxytocin
l.Ot1.3 6.722.5 7.4t4.0
Nonplacental site
10 Tension Duration Freauencv
of I
10.9k5.2 5.7 t 5.6 11.3 2 5.3
1091
contraction
increase in tension,
of human duration,
uterus
and
frequency _---.-.-
2opg ( 30 Kg j 4OM I 50% / loo&! 4.424.6 3.225.1 2.32 5.3 -30.5 z?I12.1 4.5t6.0 9.6k6.413.3k6.9 23.3zk8.7 32.6-f 6.6 +81.5 5 15.0 12.2k7.1 14.427.6 15.4k9.7 17.051 0.4 +30.5 3t 10.1 1.5k1.5 8.454.2 10.1+4.0
on the spontaneous Percentage
in vitro
In calcium deficient Krebs’ solution. Hu-
on the spontaneous
1 IOPR
myometrium
man myometrium was observed in l/2, l/5, and l/10 of the normal calcium Krebs’ solution and, as shown in Fig. 4, spontaneous activity virtually disappeared under these conditions. However, partial return of activity was noted following the introduction
Percentage 5pg
on
ies, sparteine sulfate always produced an increase in myometrial tension, as seen in Fig. 1. Muscle strips obtained from nonplacental sites of the term myometrium responded to sparteine sulfate stimulation differently than did strips excised from the placental implantation site (Table I). Eight cases of the latter were studied. The tension developed by these strips was uniformly lowet than that of the nonplacental site strips. In similar experiments with rat myometrium, sparteine sulfate invariabIy increased the amplitude of the contractions. The sensitivity of uterine muscle to sparteine sulfate in various stages of pregnant! is seen in Fig. 2. The drug is more effective in the late pregnancy and least effective during the midpregnancy, as indicated by this graph. In the case of human myometrium, no change in sensitivity could be demonstrated at different stages of gcstation (Fig. 3).
segment of muscle in Krebs’ solution between two Ag-AgC1 wire ring electrodes.
Table I. Effect of sparteine
sulfate
1.722.4 11.0+5.6 11.1 to.1
contraction of increase
15.8k5.2 10.1 r4.0 15.9 * 9.4
1.7’3.8 22.6k6.5 14.6t2.8
of human
in tension,
20.6% 6.3 12.3 + 1 I.0 14.6 -I 77.7
duration,
22.7 2 5.5 16.7 2 9.8 11.1 t 7.7
l.lf 24.8-c 17.42 -
4.6 7.1 3.1 _.- -... --._-
uterus and frequency 24.3+ 6.0 18.8 ? 8.6 8.3 2 7.6
28.1 + 6.2 17.5-t 10.9 lO.Ok 7.5
1092
Yamada
et al.
Art.
Fig. 1. Normal conditions as control. A, Human myometrium at fourteenth myometrium at sixteenth week. C, Human myometrium at term. D, Human term with oxytocin for comparison.
I 0
I /
Fig. 2. Effect imum tension myometrium.
I 2 of sparteine of electrically
I
I
3
4
sulfate on stimulated
pEND the
maxhuman
EFFECT OF 3Ouqkc SPARTEINE SUiATEONTHERAT MYOMETRIUM
O-SPONTANEOUS
CONTRACTION
l ~ELECTRICALLYSTIYULATEO TENSION
Days
of Prtgnoncy
Fig. 3. Estrus, pregnant, and postpartum with 30 pg per milliliter of sparteine Sparteine sulfate has least effect in the as is seen here.
rat uteri sulfate. midstage,
of 100 pg sparteine sulfate. The appearance of sparteine sulfate-induced contracture was noted in l/10 calcium deficient solution. The response to 100 PU per cubic centimeter oxytocin was even more marked on the same muscle strip and a regular contraction pattern developed.
August 1.5. t968 J. Obst. & Gynrc
week. B, Human myometrium at
Fig. 5 demonstrates the effect of increasing doses of sparteine on rat uterine muscle at different stages of gestation in l/10 Ca++ ion concentrations. Smaller doses of sparteine sulfate appear to be effective on the estrogen dominated uterus (Fig. 5, A). When rat myometrium is progesterone dominated, at the fifteenth day of pregnancy (Fig. 5, B), the same dose of sparteine sulfate is much less effective. As pregnancy approaches term, on the twentieth day (Fig. 5, C), the response is somewhat increased but does not equal that of the unblocked, estrogen-dominated myometrium. The percentage increase in uterine tension in normal, l/2, l/5, and l/10 Ca++ Krebs’ solution, after the addition of sparteine sulfate, on the electrically stimulated rat myometrium is seen in Fig. 6. The response is inversely proportiona to the degree of depolarization. In K+ excess and deficiency solutions. A comparison of effects of sparteine sulfate on the human myometrium in normal Krebs’ and in 5 x K solution is made in Fig. 7. Depolarization of myometrial cells was noted and increases in uterine active tension were observed in the 5 x K experiments. Maximum tension of the electrically stimuIated myometrium (4 voIts per square centimeter) at varying sparteine sulfate concentrations were then plotted. In 5 x K solution, the maximum myometrial tension
Fig. 4. Human myometrium in calcium-deficient solutions. A, Spontaneous contraction in normal Krebs’ solution with sparteine sulfate. B, In l/2 Ca” Krebs’ solution after 100 pg per solution after 100 pg per milliliter sparteine milliliter sparteine sulfate. C, In l/5 Cat+ Krebs’ sulfate. D, In l/10 Ca++ Krebs’ solution after 100 pg per milliliter sparteine sulfate. E, In l/10 Cat+ Krebs’ solution after 100 aU oxytocin.
Fig. 5. Rat gen-dominated ometrium.
myometrium estrus
in calcium-deficient Krebs’ solution with myometrium; B, fifteenth day myometrium;
! B 2 oz p.
’
1
1
,
IOug 2Oug 30ug 40ug 5Oug 60ug Dose of Sparteina Sulfate ug./ ml.
Ca*FREE
s ?
70ug
I”
Fig. 6. Increasing Ca++ deficiency interferes with the stimulatory effect of sparteine sulfate on the electrically stimulated rat myometrium.
L
5o
L
A, Estroday my-
:.,FoL~K+s~uT,~
0
--I-x-x-x4 I
1;:
sparteine sulfate. C, twentieth
0 IN NORYAL KREES SOLUTION t
I
Control 2oug Dose of Sporteine
I
*
40ug Sutfote
1
.
6Ouq ug/ml.
Fig. 7. Comparison of dose response sparteine sulfate on electrically stimulated myometrium in normal Krebs’ solution 5 x K.
effect of human and in
1094
Yamada
August 15, 1968 Am. J. Obst. & Gyncc.
et al.
EFFECTSOFSPAATEINE SULFATEON RAT MYOMETRIUM INVARIOUS K+CONTENT SOLUTIONS A l8fh Day of Pregmcy B. tzlfrs Posfpmfum .-IN NORYALKREBSSOLUTION o-lN2lK+SolUTlON q INSrK+ SOLUTIOK 8 a- IN KRINGERsolurloN ‘Zc IIN K~EFI~IENTSOLUTION !z!50-
I
Dose of Sparteine
Sulfate
Fig. 8. Percentage increase development of electrically depolarized rat myometrium.
2
uglml
4
8
I6
32
64
in Sdution
in maximum stimulated,
tension partially
was greater at all levels of drug concentration. The rat myometrium in K” excess solution showed greater percentage increase in maximum tension in late pregnancy than it did postpartum (Fig. 8) .
Comment These in vitro experiments have shown that an adequate amount of sparteine sulfate has a direct effect on myometrial activity. It stimulates tension development, frequency, and duration in the spontaneousIy contracting rat uterus. In human myometrium, under similar circumstances, increases in frequency and duration of contractions are noted but the effect on maximum tension development is negligible. Experiments on electrically stimulated uterine muscle showed that sparteine sulfate increased tension in both human and rat uteri. From these findings, it is quite apparent the effect of sparteine sulfate on the
Stenhouse, Tamba, Kioto 4: 3. Heathcote, Therap. 4. Kreitmiar, Jahresb.
The authors wish to extend thanks to Dr. John Sproul and Ayerst Laboratories for their interest and support and to Trent Pharmaceuticals, Inc., for the Tocosamine (sparteine sulfate, Trent) they so generously supplied for this study.
5.
REFERENCES
1. 2.
mechanical activity of the myometrium is of a stimulatory nature. Rosenblum and SteinZO reported that iu cases of human uterine muscle “depolarized” by potassium Ringer’s solution sparteine sulfate acts primarily on the cell membrane rather than directly on the contractile elements. Our experiments also suggest that the sparteine sulfate effect is mediated by altering the membrane potential. Once tetanus is brought about by K+ excess depolarization, no further sparteine sulfate effect is possible, Similarly, Ca++-induced depolarization produces a narrowing of the range where sparteine sulfate exhibits a stimulatory effect. Tetanic contractions developed rapidly and at comparatively lower doses. To minimize variables inherent when testing different pharmacologic agents, contiguous strips of the same myometrium were used GmultaneousIy, one for sparteine sulfate and the other for oxytocin. The majority of investigators agree that the most pronounced effect of oxytocin is mediated through the changes in membrane potentials.21‘2s However, some reports point out the possibility of additional components as well. Berger and MarshalIZ reported the action of oxytocin on the contractile protein of the rat myometrium with depolarized muscle, and Yamada30 has reported that oxytocin appears to have some slight effect on the contractile system of the myometrium using glycerol-treated muscle.
J.: Liebig’s Ann. 78: 20, 1851. G.: Acta scholae med. univ. imp. in 85, 1921. R. St. A.: J. Pharmacol. & Exper. 27: 431, 1936. H., and Sieckmann, W.: Merck’s 50: 111, 1936.
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Kleine, H. 0.: Klin. Wchnschr. 18: 360: 1939. Mahon, R.: Gynec. et obst. 39: 177, 1939. Csapo, A.: Am. J. Anat. 98: 273, 1956a. Csapo, A.: Recent Progr. Hormone Res. 12: 405, 1956b. Kao, C. Y., and Nishiyama, A.: Am. J. Physiol. 207: 793, 1964.
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1x. 19.
Effect
Yamada, S.: Texas Rep. Biol. & Med. 22: 209, 1964. Anderson, N., and Zarrow, M. X.: Presented at FASEB Meeting, Chicago, Illinois, April, 1964. Plentl, A. A., Friedman, E. A., and Gray, M. F.: AM. J. OBST. & GYNEC. 82: 1331, 1961. Bedrosian, L., and Gamble, J. J.: Obst. & Gynec. 21: 400, 1963. Boysen, H.: Obst. & Gynec. 21: 403, 1963. Cromer, D. W., Reeves, B. D., and Danforth, D. N.: AM. J. OBST. & GYNEC. 89: 268, 1964. Stander, R. W.: Obst. & Gynec. 26: 876, 1965. Landesman, R., Wilson, K. H., La Russa, R., and Silverman, F.: Obst. & Gynec. 23: 2, 1964. Lu, G.: Arch. Internat. Pharmacodyn. 76: 367, 1948. Soula, L. C., and Delas, J.: J, de mtd. de Paris xliv: 901, 1925.
of sparteine
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sulfate
on
myometrium
in vitro
1095
Rosenblum, I., and Stein, A. A.: J. Pharmacol. & Exper. Therap. 144: 138, 1964. Woodbury, J. W., and McIntyre, D. M.: Am. J. Physiol. 177: 355, 1954. Woodbury; J. W., and McIntyre, D. M.: Am. T. Phvsiol. 187: 338. 1956. Jung,’ H.: Arch. GynPk. 190: 194, 1957. Landa, J. F., West, T. C., and Thiersrh, J. B.: Am. J. Physiol. 196: 905, 1959. Kuriyama, H., and Csapd, A.: Biol. Bull. Woods Hole 177: 417. 1959. Csapo, A.: Ann. New York Acad. Sr 75: 790, 1959. Coutinho. E., and Csapo, A.: J. Gen. Physiol. 43: 13, 1959. Kuriyama, H., and Csapo, A.: Endocrinology 68: 1010, 1961. Berger, E., and Marshall, J. M : Am. J. Physiol. 201: 931, 1961. Yamada. S.: Jikei-Ishi (Jikei M. J.? 74: 2711, 1959.