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Effect of the vascular endothelium on norepinephrine-induced contractions in uterine radial arteries from the nonpregnant and pregnant human uterus
Effect of the vascular endothelium on norepinephrine-induced contractions in uterine radial arteries from the nonpregnant and pregnant human uterus Simon C. Steele, Averil Y. Warren, and Ian R. Johnson, DM
Nottingham, England OBJECTIVE: Our purpose was to investigate the role of the endothelium in the human uterine arterial response to norepinephrine in the nonpregnant and pregnant states. STUDY DESIGN: Tissue was obtained from six pregnant and six nonpregnant women undergoing cesarean section or hysterectomy. Uterine radial arteries were isolated and subjected to norepinephrine dose-response curves with and without intact endothelium. RESULTS: Responses were obtained over a dose range of 10- 8 to 10- 4 norepinephrine. Initially there was no difference between vessels from pregnant and nonpregnant patients, but removal of the endothelium significantly increased the response in vessels from pregnant women. Addition of nitro-L-arginine methyl ester when the endothelium was intact did not alter the dose-response curves. CONCLUSIONS: In pregnancy human uterine radial arteries are more sensitive to norepinephrine than during the nonpregnant state. This increase is countered by an endothelium-derived relaxing factor. The factor is unlikely to be nitric oxide. (AM J OBSTET GVNECOL 1993;168:1623-8.)
Key words: Human uterine resistance vessels, sympathetic innervation, norepinephrine Vascular endothelium plays a m~or role in control of the resting tone of vascular beds and in maintenance of blood pressure. It produces a number of potent vasoconstrictor and dilator factors. One of these factors, endothelium-derived relaxing factor (EDRF), activates soluble guanylate cyclase, which results in an increase in cyclic guanosine 5'-monophosphate. This increase produces vascular relaxation by a mechanism of action that is unclear. This system is of potential importance in the control of resting arterial pressure and systemic vascular resistance in pregnancy. The pathogenesis of preeclampsia may involve a generalized vasoconstrictor disorder as a result of an abnormal endothelial cell function. I The system can be studied by removing the endothelium or by the use of N-methylarginine, which is known to block the production of nitric oxide (an EDRF) and inhibits endothelium-dependent relaxation in rat," rabbit,' and guinea pig' vessels. The purpose of this study was to investigate the role of the endothelium in the human uterine arterial re-
From the Department of Obstetrics and Gynaecology, City Hospital, Nottingham. Received for publication October 14, 1992; revised October 26, 1992; accepted October 31, 1992. Reprint requests: I.R. johnson, DM, Department of Obstetrics and Gynaecology, City Hospital, Hucknall Road, Nottingham, England NG51PB. Copyright © 1993 by Mosby-Year Book, Inc. 0002-9378/93 $1.00 + .20 6/1/43961
sponse to norepinephrine in the nonpregnant and pregnant states. Branches of the uterine arteries are the final pressure vessels controlling the supply of oxygen and nutrients to the placental bed and the fetus.
Methods Patients. Tissue was collected from six patients undergoing abdominal hysterectomy for various nonmalignant gynecologic disorders. The patients were at different stages of the menstrual cycle and ranged in age from 35 to 48 years. All were parous, and none were postmenopausal. Tissue was also collected from six pregnant patients aged 25 to 30 years who were undergoing repeat elective cesarean section at term. None of the women were hypertensive, and all of the babies weighed > 2.5 kg. Samples of myometrial tissue of approximately 2 cm' were taken from the incision in the lower segment of the uterus in the pregnant patients and from the anterior aspect of the uterus just above the isthmus in the nonpregnant patients. Immediately after collection the biopsy specimen was placed in cold physiologic salt solution (see below) and transferred to the laboratory on ice. Informed consent was obtained from each patient. This study had previously been approved by the City Hospital Nottingham Ethical Committee. Apparatus. The myograph described by Mulvany and Halpern' was used in this investigation. Each vessel was mounted as a ring preparation. The mounting wires were attached to pairs of jaws that were in turn con1623
1624 Steele, Warren, and Johnson
nected to a force transducer (DSC 6, Eurosensor Computer Control, London) and a displacement device (a micrometer, J.P. Trading, Aarhus, Denmark), respectively. The jaws were suspended in a stainless steel chamber, for which 10 ml is sufficient to cover the vessels, and 15 ml fills the chamber. The chamber has a Perspex cover to reduce evaporation and maintain oxygen tension. Solutions can be added to the chamber through ports on the near side and in the Perspex cover. The oxygen tension and pH of the solution in the chamber were maintained by continuous aeration with 5% carbon dioxide in oxygen. The temperature of the organ bath was maintained at 37° C by water circulated from a thermostatically controlled water bath. The micrometer was used to determine the extension of the vessels, and the force transducer measured the pressure generated and enabled the wall tension to be calculated. Dissection and mounting of vessels. Intramyometrial radial arteries of internal diameter 400 to 700 j.l.m were dissected free with the aid of a stereo microscope and microsurgical instruments. The vessel segments were cut in half and then threaded on to a fine steel wire (diameter 50 j.l.m), which had been attached at one end to a sledge on the myograph. The free end of wire was then attached to the same sledge, and a second steel wire of the same size was threaded down the vessel lumen and attached to the other arm on the myograph. Mter the vessels were mounted, their length was measured with a calibrated micrometer eyepiece on a Zeiss microscope. They were then allowed to equilibrate at 37° C for 30 minutes. Solutions and drugs. The physiologic salt solution consisted of sodium chloride 119 mmol!L, sodium bicarbonate 14.9 mmol!L, potassium chloride 4.7 mmol!L, potassium dihydrogen orthophosphate 1.18 mmol!L, magnesium sulfate 1.17 mmol!L, calcium chloride 2.5 mmol!L, and glucose 5.5 mmol!L. High potassium solution (final concentration 125 mmol!L) was as for physiologic salt solution but with an equimolar substitution of potassium chloride for sodium chloride. Stock solutions of norepinephrine bitartrate were prepared in physiologic salt solution containing 0.1 gm/L ethylenediaminetetraacetic acid. The nitro-L-arginine methyl ester was made up in distilled water. Both sets of solutions were made fresh immediately before use. All of the substances were obtained from Sigma UK, Poole, U.K. Normalization procedure. Mter vessels had equilibrated they were stretched in steps of 0.5 mN/mm until the force generated by the vessel wall just exceeded 100 mm Hg (13.3 kPa). Vessels were held at each length for 1 minute, and the force generated at the internal circumference was measured immediately before the next step was taken. The points obtained could be fitted to an exponential curve. By means of the Laplace
May 1993 Am J Obstet Gynecol
equation the internal circumference at which the effective transmural pressure would be 100 mm Hg was determined. This point was defined as L lOo ' The experiments described here were performed at 0.9 L 1oo , because pilot studies showed that this point provided maximum active tension in vessels obtained from both pregnant and nonpregnant subjects with only a small amount of passive wall tension. Protocol. Before normalization one of the vessels in the organ bath was randomly selected and its endothelium removed. This was achieved by rubbing the lumen of the vessel with a human hair for 2 minutes. Preliminary experiments with acetylcholine-induced relaxation in precontracted vessels had confirmed that this procedure removed the endothelium. Mter each experiment all of the vessels were fixed and examined histologically and the absence of endothelium confirmed. Mter normalization all vessels were stimulated with the potassium-rich solution at 15-minute intervals until reproducible responses were obtained. Formal experiments were not performed if the effective active pressure at 0.9 LIOO was < 80 mm Hg. Cumulative doseresponse curves were carried out on matched pairs of rubbed and intact ring preparations with norepinephrine to induce tension; the effect of 10 mol!L of nitroL-arginine methyl ester on these dose-response curves was determined. During the course of each experiment contractions to high potassium solution were carried out to confirm the viability of the preparation. Data analysis. All data are expressed as the percent of the maximum contraction to the high potassium solution. To compare responses before and after addition of nitro-L-arginine methyl ester a paired t test was used. Results are expressed as mean ± SE. SE bars are shown on the figures.
Results The mounted vessels from both nonpregnant and pregnant subjects were quiescent, showing no signs of tone or spontaneous activity, when held in physiologic salt solution. The internal diameter of the vessels from nonpregnant and pregnant women at LIOO was 586 (SE 31) and 562 (SE 68) j.l.m, respectively. The active wall tension developed after 2 minutes in high potassium solution was 6.3 (SE 0.9) mN/mm in the vessels from nonpregnant subjects and 4.9 (SE 0.7) mN/mm in the vessels from pregnant women. Expressed as a more physiologic parameter, these values correspond to the vessels being able to contract against a pressure of 138 and III mm Hg, respectively. These results and parameters are summarized in Table 1. Removal of the endothelium made no difference to the active wall tension developed on exposure to high potassium solution in either the vessels from nonpre~ nant or pregnant women. The effects of removal of endothelium and the pres-
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Table I. Characteristics of the vessels Patients Pregnant (n No. of vessels Effective lumen diameter (f,Lm) Potassium stimulation Active wall tension (mN/mm) Active effective pressure (mm Hg)
ence of lO-4 mol/L of nitro-L-arginine methyl ester on the norepinephrine dose-response curve in vessels from nonpregnant women are shown in Figs. 1 and 2. The response to norepinephrine was taken as the maximum active wall force measured during a 2-minute stimulation period. Figs. 3 and 4 show the effect of removal of the endothelium and presence of lO -, mol/L nitro-Larginine methyl ester on the norepinephrine doseresponse curve in vessels from pregnant women. It can be seen that the removal of the endothelium has little effect on the norepinephrine dose-response curve in vessels from nonpregnant women, but there is a change in the dose-response curve in the vessels from pregnant
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women. The presence of lO-1 mol/L nitro-L-argmme methyl ester in the organ bath had no effect on either type of vessel. Comment
Obtaining tissue from the human uterus is difficult, technically and ethically. Inevitably patients having hysterectomies tend to be older than those having cesarean section. Although all of the patients in this study were premenopausal and the uteri were likely to be comparable, it is not possible to be certain that this was so. The samples were obtained from the incision in the uterus in pregnant patients. Attempts had previously
1626 Steele, Warren, and Johnson
May 1993
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been made to obtain samples from the placental bed, but this proved unsatisfactory. Samples were obtained from hysterectomy specimens from areas as close as possible to the analogous areas sampled in pregnant patients.
The vascular endothelium has been recognized as an important functional unit in the control of vascular smooth muscle tone ever since the discovery of endothelium-dependent vasodilatation by Furchgott and Zawadzki 6 in 1980. It has been suggested that endothe-
Steele. Warren. and Johnson
Volume 168. Number 5 Am J Obstet Gynecol
1627
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Fig. 4. Effect of addition of 10- 4 moVL nitro·L·arginine methyl ester (NAME) on norepinephrine (NE) dose-response curve in vessels with intact endothelium obtained from pregnant women (n = 6) is shown. Addition of nitro-L-arginine methyl ester has no effect. suggesting that nitric oxide plays little or no part in altering vascular reactivity to norepinephrine in these vessels in pregnancy.
lial cells secrete short-lived EDRFs-, which cause smooth muscle relaxation when stimulated by vascular agonists.7 One EDRF has recently been identified as nitric oxide or a closely related compound." In sheep the response of vascular smooth muscle to a variety of agents is altered during pregnancy.9 If EDRF activity were increased during pregnancy, this could explain some of the decline in vascular resistance and responsiveness to catecholamines during pregnancy. The current experiments were designed to determine whether the response of small intramyometrial arteries is altered by the absence of endothelium in the nonpregnant and pregnant states. Problems with handling the vessels and ensuring that the endothelium was intact in each set of experiments mean that the results have to be viewed with some caution. Similarly, the effects of repeated high potassium doses on the endothelium are uncertain. However, the techniques were standardized to minimize the differences between experiments with different vessels, to allow valid comparisons to be made. Our results did not reveal any difference in the norepinephrine sensitivity between the nonpregnant and pregnant patients. This is in accordance with the results of Svane et al. 10 Removal of the endothelium in the nonpregnant patients moved the dose-response curve slightly to the left but not significantly so. When the endothelial layer was removed from the vessels
obtained from pregnant women, there was an increase in the maximal response to norepinephrine. This could not be duplicated by the addition of 10- 4 mol/L nitroL-arginine methyl ester. This suggests that nitric oxide is not a mediator of the endothelial effect in small human intramyometrial arteries from both the nonpregnant and pregnant uterus in vitro. In pregnancy the endothelial cells appear to modifY the contractile responses of the underlying vascular smooth vessel through a mechanism other than the release of nitric oxide. This finding is particularly interesting, because there is evidence that endothelial cell injury is present in women with preeclampsia. II Even vessels with grossly normal morphologic conditions may actually have abnormalities of the endothelial cells. If the endothelial layer becomes damaged during pregnancy, our results suggest that these vessels might be more sensitive to norepinephrine and possibly other vasoconstrictors.
REFERENCES
I. RobertsjM, Taylor RN, Masci Tj, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. AM j OBSTET GYNECOL 1989;161:1200-4. 2. Umans jG, Metkus AP. N-methylarginine augments isometric contraction in rat aorta in vitro [Abstract]. Physiol. ogist 1989;32: 186. 3. Palmer RMj, Rees DD, Ashton DS, Moncada. L.Arginine is the physiological precursor for the formation of nitric
Erulkar et al.
4. 5. 6. 7.
oxide in endothelium dependent relaxation. Biochem Biophys Res Commun 1988;153:1251-6. Sakuma I, Stuehr DJ, Gross SS, Nathan C, Levi R. Identification of arginine as a precursor of endothelium derived relaxing factor. Proc Nat! Acad Sci USA 1988;85:8664-7. Mulvany MJ, Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res 1977 ;41: 19-26. Furchgott RE, Zawadzki ]V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288:373-6. Furchgott RF. Role of endothelium in responses of vascular smooth muscle. Circ Res 1983;53:557-73.
May 1993 Am J Obstet Gynecol
8. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide accounts for the biological activity of endothelium derived relaxing factor. Nature 1987;327:524-6. 9. Griendling KK, Fuller ED, Cox RH. Pregnancy induced changes in sheep uterine and carotid arteries. Am J Physiol 1985;17:H658-65. 10. Svane D, Sk~aa K, Andersson K-E, Forman A. Vascular responses in term pregnant and nonpregnant human uterus. Placenta 1991;12:47-54. 11. Roberts JM. Pregnancy-related hypertension. In: Creasy RK, Resnick R, eds. Maternal-fetal medicine: principles and practice. Philadelphia: WB Saunders, 1989.
Expression of different potassium channels in cells isolated from human myometrium and leiomyomas Solomon D. Erulkar, PhD: Jack Ludmir, MD,b Boris Ger, MD: and Rosanne D. Nori, MAa Philadelphia, Pennsylvania OBJECTIVE: Electrophysiologic characteristics of human myometrial and leiomyomatous cells isolated in culture were investigated. Both types of cell were shown to be smooth muscle cells by immunofluorescence. STUDY DESIGN: Voltage-activated potassium currents were recorded by whole-cell patch-clamp techniques and analyzed for differences in expression, voltage-dependence, kinetics, and inactivation. RESULTS: Depolarizing-voltage steps from - 90 mV to + 30 mV elicited two types of noninactivating outward currents that differed in their kinetics in 83% (n = 36) of normal cells in culture for 3 to 5 days; 6% responded with fast (3.5 milliseconds) outward inactivating currents; 11 % in culture for 1 day responded only with long-lasting inactivating currents (33.2 ± 7.2 milliseconds). Cells isolated from leiomyomas responded preferentially (65%. n = 31) with fast (3.3 ± 0.1 milliseconds) outward inactivating currents; 35% responded with noninactivating outward currents similar to those from normal cells. CONCLUSION: Different potassium channel currents. noninactivating and inactivating. are predominantly expressed in cells isolated from human myometrium and leiomyomas. respectively. (AM J OBSTET GVNECOL 1993; 168: 1628-39.)
Key words: Potassium channels, human, leiomyomas, myometrium Uterine leiomyomas (fibroids) occur in approximately 30% to 40% of women of reproductive age. I These benign tumors can cause infertility, pregnancy complications, and abnormal uterine bleeding and are
From the Departments of Pharmacology" and Obstetrics and Gynecology, b University of Pennsylvania Medical School. Supported by National Institutes of Health grant No. NSI2211, University of Pennsylvania Research Foundation grant No. 3-7513, and Biomedical Research Support grant No. RR 07083. Received for publication july 31,1992; revised October 16, 1992; accepted November 16, 1992. Reprint requests: S.D. Erulkar, PhD, DPhil, Department of Pharmacology, University of Pennsylvania Medical School, Philadelphia, PA 19104. Copyright © 1993 by Mosby-Year Book, Inc. 0002-9378/93 $1.00 + .20 6/1/44282
1628
responsible for many hysterectomies. 2 Unfortunately, relatively little is known about the cause of these common, benign uterine muscle tumors. Fibroids appear to be clonally derived.' Cytogenic aberrations in leiomyoma cells, frequently involving chromosome 12, have been reported, suggesting that spontaneous chromosome rearrangement may be responsible for initiation and proliferation of leiomyoma, in some cases but no consistent pattern has emerged to suggest involvement of a specific gene locus in the abnormal cell proliferation,,· 7 Attempts to characterize myometrial and leiomyoma cells in culture have shown differences in binding and growth responses to epidermal growth factor and, to a lesser degree, platelet-derived growth factor. B , 9 These
Nottingham, England OBJECTIVE: Our purpose was to investigate the role of the endothelium in the human uterine arterial response to norepinephrine in the nonpregnant and pregnant states. STUDY DESIGN: Tissue was obtained from six pregnant and six nonpregnant women undergoing cesarean section or hysterectomy. Uterine radial arteries were isolated and subjected to norepinephrine dose-response curves with and without intact endothelium. RESULTS: Responses were obtained over a dose range of 10- 8 to 10- 4 norepinephrine. Initially there was no difference between vessels from pregnant and nonpregnant patients, but removal of the endothelium significantly increased the response in vessels from pregnant women. Addition of nitro-L-arginine methyl ester when the endothelium was intact did not alter the dose-response curves. CONCLUSIONS: In pregnancy human uterine radial arteries are more sensitive to norepinephrine than during the nonpregnant state. This increase is countered by an endothelium-derived relaxing factor. The factor is unlikely to be nitric oxide. (AM J OBSTET GVNECOL 1993;168:1623-8.)
Key words: Human uterine resistance vessels, sympathetic innervation, norepinephrine Vascular endothelium plays a m~or role in control of the resting tone of vascular beds and in maintenance of blood pressure. It produces a number of potent vasoconstrictor and dilator factors. One of these factors, endothelium-derived relaxing factor (EDRF), activates soluble guanylate cyclase, which results in an increase in cyclic guanosine 5'-monophosphate. This increase produces vascular relaxation by a mechanism of action that is unclear. This system is of potential importance in the control of resting arterial pressure and systemic vascular resistance in pregnancy. The pathogenesis of preeclampsia may involve a generalized vasoconstrictor disorder as a result of an abnormal endothelial cell function. I The system can be studied by removing the endothelium or by the use of N-methylarginine, which is known to block the production of nitric oxide (an EDRF) and inhibits endothelium-dependent relaxation in rat," rabbit,' and guinea pig' vessels. The purpose of this study was to investigate the role of the endothelium in the human uterine arterial re-
From the Department of Obstetrics and Gynaecology, City Hospital, Nottingham. Received for publication October 14, 1992; revised October 26, 1992; accepted October 31, 1992. Reprint requests: I.R. johnson, DM, Department of Obstetrics and Gynaecology, City Hospital, Hucknall Road, Nottingham, England NG51PB. Copyright © 1993 by Mosby-Year Book, Inc. 0002-9378/93 $1.00 + .20 6/1/43961
sponse to norepinephrine in the nonpregnant and pregnant states. Branches of the uterine arteries are the final pressure vessels controlling the supply of oxygen and nutrients to the placental bed and the fetus.
Methods Patients. Tissue was collected from six patients undergoing abdominal hysterectomy for various nonmalignant gynecologic disorders. The patients were at different stages of the menstrual cycle and ranged in age from 35 to 48 years. All were parous, and none were postmenopausal. Tissue was also collected from six pregnant patients aged 25 to 30 years who were undergoing repeat elective cesarean section at term. None of the women were hypertensive, and all of the babies weighed > 2.5 kg. Samples of myometrial tissue of approximately 2 cm' were taken from the incision in the lower segment of the uterus in the pregnant patients and from the anterior aspect of the uterus just above the isthmus in the nonpregnant patients. Immediately after collection the biopsy specimen was placed in cold physiologic salt solution (see below) and transferred to the laboratory on ice. Informed consent was obtained from each patient. This study had previously been approved by the City Hospital Nottingham Ethical Committee. Apparatus. The myograph described by Mulvany and Halpern' was used in this investigation. Each vessel was mounted as a ring preparation. The mounting wires were attached to pairs of jaws that were in turn con1623
1624 Steele, Warren, and Johnson
nected to a force transducer (DSC 6, Eurosensor Computer Control, London) and a displacement device (a micrometer, J.P. Trading, Aarhus, Denmark), respectively. The jaws were suspended in a stainless steel chamber, for which 10 ml is sufficient to cover the vessels, and 15 ml fills the chamber. The chamber has a Perspex cover to reduce evaporation and maintain oxygen tension. Solutions can be added to the chamber through ports on the near side and in the Perspex cover. The oxygen tension and pH of the solution in the chamber were maintained by continuous aeration with 5% carbon dioxide in oxygen. The temperature of the organ bath was maintained at 37° C by water circulated from a thermostatically controlled water bath. The micrometer was used to determine the extension of the vessels, and the force transducer measured the pressure generated and enabled the wall tension to be calculated. Dissection and mounting of vessels. Intramyometrial radial arteries of internal diameter 400 to 700 j.l.m were dissected free with the aid of a stereo microscope and microsurgical instruments. The vessel segments were cut in half and then threaded on to a fine steel wire (diameter 50 j.l.m), which had been attached at one end to a sledge on the myograph. The free end of wire was then attached to the same sledge, and a second steel wire of the same size was threaded down the vessel lumen and attached to the other arm on the myograph. Mter the vessels were mounted, their length was measured with a calibrated micrometer eyepiece on a Zeiss microscope. They were then allowed to equilibrate at 37° C for 30 minutes. Solutions and drugs. The physiologic salt solution consisted of sodium chloride 119 mmol!L, sodium bicarbonate 14.9 mmol!L, potassium chloride 4.7 mmol!L, potassium dihydrogen orthophosphate 1.18 mmol!L, magnesium sulfate 1.17 mmol!L, calcium chloride 2.5 mmol!L, and glucose 5.5 mmol!L. High potassium solution (final concentration 125 mmol!L) was as for physiologic salt solution but with an equimolar substitution of potassium chloride for sodium chloride. Stock solutions of norepinephrine bitartrate were prepared in physiologic salt solution containing 0.1 gm/L ethylenediaminetetraacetic acid. The nitro-L-arginine methyl ester was made up in distilled water. Both sets of solutions were made fresh immediately before use. All of the substances were obtained from Sigma UK, Poole, U.K. Normalization procedure. Mter vessels had equilibrated they were stretched in steps of 0.5 mN/mm until the force generated by the vessel wall just exceeded 100 mm Hg (13.3 kPa). Vessels were held at each length for 1 minute, and the force generated at the internal circumference was measured immediately before the next step was taken. The points obtained could be fitted to an exponential curve. By means of the Laplace
May 1993 Am J Obstet Gynecol
equation the internal circumference at which the effective transmural pressure would be 100 mm Hg was determined. This point was defined as L lOo ' The experiments described here were performed at 0.9 L 1oo , because pilot studies showed that this point provided maximum active tension in vessels obtained from both pregnant and nonpregnant subjects with only a small amount of passive wall tension. Protocol. Before normalization one of the vessels in the organ bath was randomly selected and its endothelium removed. This was achieved by rubbing the lumen of the vessel with a human hair for 2 minutes. Preliminary experiments with acetylcholine-induced relaxation in precontracted vessels had confirmed that this procedure removed the endothelium. Mter each experiment all of the vessels were fixed and examined histologically and the absence of endothelium confirmed. Mter normalization all vessels were stimulated with the potassium-rich solution at 15-minute intervals until reproducible responses were obtained. Formal experiments were not performed if the effective active pressure at 0.9 LIOO was < 80 mm Hg. Cumulative doseresponse curves were carried out on matched pairs of rubbed and intact ring preparations with norepinephrine to induce tension; the effect of 10 mol!L of nitroL-arginine methyl ester on these dose-response curves was determined. During the course of each experiment contractions to high potassium solution were carried out to confirm the viability of the preparation. Data analysis. All data are expressed as the percent of the maximum contraction to the high potassium solution. To compare responses before and after addition of nitro-L-arginine methyl ester a paired t test was used. Results are expressed as mean ± SE. SE bars are shown on the figures.
Results The mounted vessels from both nonpregnant and pregnant subjects were quiescent, showing no signs of tone or spontaneous activity, when held in physiologic salt solution. The internal diameter of the vessels from nonpregnant and pregnant women at LIOO was 586 (SE 31) and 562 (SE 68) j.l.m, respectively. The active wall tension developed after 2 minutes in high potassium solution was 6.3 (SE 0.9) mN/mm in the vessels from nonpregnant subjects and 4.9 (SE 0.7) mN/mm in the vessels from pregnant women. Expressed as a more physiologic parameter, these values correspond to the vessels being able to contract against a pressure of 138 and III mm Hg, respectively. These results and parameters are summarized in Table 1. Removal of the endothelium made no difference to the active wall tension developed on exposure to high potassium solution in either the vessels from nonpre~ nant or pregnant women. The effects of removal of endothelium and the pres-
Steele, Warren, and Johnson
Volume 168. Number 5 Am J Obstet Gynecol
1625
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Log [NE] Fig. 1. Effect of removal of vascular endothelium on norepinephrine (NE) dose-response curve in vessels obtained from nonpregnant women (n = 6) is shown. When endothelium is removed, dose-response curve is moved to left but not significantly.
Table I. Characteristics of the vessels Patients Pregnant (n No. of vessels Effective lumen diameter (f,Lm) Potassium stimulation Active wall tension (mN/mm) Active effective pressure (mm Hg)
ence of lO-4 mol/L of nitro-L-arginine methyl ester on the norepinephrine dose-response curve in vessels from nonpregnant women are shown in Figs. 1 and 2. The response to norepinephrine was taken as the maximum active wall force measured during a 2-minute stimulation period. Figs. 3 and 4 show the effect of removal of the endothelium and presence of lO -, mol/L nitro-Larginine methyl ester on the norepinephrine doseresponse curve in vessels from pregnant women. It can be seen that the removal of the endothelium has little effect on the norepinephrine dose-response curve in vessels from nonpregnant women, but there is a change in the dose-response curve in the vessels from pregnant
= 6)
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women. The presence of lO-1 mol/L nitro-L-argmme methyl ester in the organ bath had no effect on either type of vessel. Comment
Obtaining tissue from the human uterus is difficult, technically and ethically. Inevitably patients having hysterectomies tend to be older than those having cesarean section. Although all of the patients in this study were premenopausal and the uteri were likely to be comparable, it is not possible to be certain that this was so. The samples were obtained from the incision in the uterus in pregnant patients. Attempts had previously
1626 Steele, Warren, and Johnson
May 1993
Am J Obstet Gynccol
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Log [NE] Fig. 3. Effect of removing endothelium on norepinephrine (NE) dose-response CUIVe in vessels obtained from pregnant women (n = 6) is shown. Sensitiviry to norepinephrine is significantly increased.
been made to obtain samples from the placental bed, but this proved unsatisfactory. Samples were obtained from hysterectomy specimens from areas as close as possible to the analogous areas sampled in pregnant patients.
The vascular endothelium has been recognized as an important functional unit in the control of vascular smooth muscle tone ever since the discovery of endothelium-dependent vasodilatation by Furchgott and Zawadzki 6 in 1980. It has been suggested that endothe-
Steele. Warren. and Johnson
Volume 168. Number 5 Am J Obstet Gynecol
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Fig. 4. Effect of addition of 10- 4 moVL nitro·L·arginine methyl ester (NAME) on norepinephrine (NE) dose-response curve in vessels with intact endothelium obtained from pregnant women (n = 6) is shown. Addition of nitro-L-arginine methyl ester has no effect. suggesting that nitric oxide plays little or no part in altering vascular reactivity to norepinephrine in these vessels in pregnancy.
lial cells secrete short-lived EDRFs-, which cause smooth muscle relaxation when stimulated by vascular agonists.7 One EDRF has recently been identified as nitric oxide or a closely related compound." In sheep the response of vascular smooth muscle to a variety of agents is altered during pregnancy.9 If EDRF activity were increased during pregnancy, this could explain some of the decline in vascular resistance and responsiveness to catecholamines during pregnancy. The current experiments were designed to determine whether the response of small intramyometrial arteries is altered by the absence of endothelium in the nonpregnant and pregnant states. Problems with handling the vessels and ensuring that the endothelium was intact in each set of experiments mean that the results have to be viewed with some caution. Similarly, the effects of repeated high potassium doses on the endothelium are uncertain. However, the techniques were standardized to minimize the differences between experiments with different vessels, to allow valid comparisons to be made. Our results did not reveal any difference in the norepinephrine sensitivity between the nonpregnant and pregnant patients. This is in accordance with the results of Svane et al. 10 Removal of the endothelium in the nonpregnant patients moved the dose-response curve slightly to the left but not significantly so. When the endothelial layer was removed from the vessels
obtained from pregnant women, there was an increase in the maximal response to norepinephrine. This could not be duplicated by the addition of 10- 4 mol/L nitroL-arginine methyl ester. This suggests that nitric oxide is not a mediator of the endothelial effect in small human intramyometrial arteries from both the nonpregnant and pregnant uterus in vitro. In pregnancy the endothelial cells appear to modifY the contractile responses of the underlying vascular smooth vessel through a mechanism other than the release of nitric oxide. This finding is particularly interesting, because there is evidence that endothelial cell injury is present in women with preeclampsia. II Even vessels with grossly normal morphologic conditions may actually have abnormalities of the endothelial cells. If the endothelial layer becomes damaged during pregnancy, our results suggest that these vessels might be more sensitive to norepinephrine and possibly other vasoconstrictors.
REFERENCES
I. RobertsjM, Taylor RN, Masci Tj, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. AM j OBSTET GYNECOL 1989;161:1200-4. 2. Umans jG, Metkus AP. N-methylarginine augments isometric contraction in rat aorta in vitro [Abstract]. Physiol. ogist 1989;32: 186. 3. Palmer RMj, Rees DD, Ashton DS, Moncada. L.Arginine is the physiological precursor for the formation of nitric
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oxide in endothelium dependent relaxation. Biochem Biophys Res Commun 1988;153:1251-6. Sakuma I, Stuehr DJ, Gross SS, Nathan C, Levi R. Identification of arginine as a precursor of endothelium derived relaxing factor. Proc Nat! Acad Sci USA 1988;85:8664-7. Mulvany MJ, Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res 1977 ;41: 19-26. Furchgott RE, Zawadzki ]V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288:373-6. Furchgott RF. Role of endothelium in responses of vascular smooth muscle. Circ Res 1983;53:557-73.
May 1993 Am J Obstet Gynecol
8. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide accounts for the biological activity of endothelium derived relaxing factor. Nature 1987;327:524-6. 9. Griendling KK, Fuller ED, Cox RH. Pregnancy induced changes in sheep uterine and carotid arteries. Am J Physiol 1985;17:H658-65. 10. Svane D, Sk~aa K, Andersson K-E, Forman A. Vascular responses in term pregnant and nonpregnant human uterus. Placenta 1991;12:47-54. 11. Roberts JM. Pregnancy-related hypertension. In: Creasy RK, Resnick R, eds. Maternal-fetal medicine: principles and practice. Philadelphia: WB Saunders, 1989.
Expression of different potassium channels in cells isolated from human myometrium and leiomyomas Solomon D. Erulkar, PhD: Jack Ludmir, MD,b Boris Ger, MD: and Rosanne D. Nori, MAa Philadelphia, Pennsylvania OBJECTIVE: Electrophysiologic characteristics of human myometrial and leiomyomatous cells isolated in culture were investigated. Both types of cell were shown to be smooth muscle cells by immunofluorescence. STUDY DESIGN: Voltage-activated potassium currents were recorded by whole-cell patch-clamp techniques and analyzed for differences in expression, voltage-dependence, kinetics, and inactivation. RESULTS: Depolarizing-voltage steps from - 90 mV to + 30 mV elicited two types of noninactivating outward currents that differed in their kinetics in 83% (n = 36) of normal cells in culture for 3 to 5 days; 6% responded with fast (3.5 milliseconds) outward inactivating currents; 11 % in culture for 1 day responded only with long-lasting inactivating currents (33.2 ± 7.2 milliseconds). Cells isolated from leiomyomas responded preferentially (65%. n = 31) with fast (3.3 ± 0.1 milliseconds) outward inactivating currents; 35% responded with noninactivating outward currents similar to those from normal cells. CONCLUSION: Different potassium channel currents. noninactivating and inactivating. are predominantly expressed in cells isolated from human myometrium and leiomyomas. respectively. (AM J OBSTET GVNECOL 1993; 168: 1628-39.)
Key words: Potassium channels, human, leiomyomas, myometrium Uterine leiomyomas (fibroids) occur in approximately 30% to 40% of women of reproductive age. I These benign tumors can cause infertility, pregnancy complications, and abnormal uterine bleeding and are
From the Departments of Pharmacology" and Obstetrics and Gynecology, b University of Pennsylvania Medical School. Supported by National Institutes of Health grant No. NSI2211, University of Pennsylvania Research Foundation grant No. 3-7513, and Biomedical Research Support grant No. RR 07083. Received for publication july 31,1992; revised October 16, 1992; accepted November 16, 1992. Reprint requests: S.D. Erulkar, PhD, DPhil, Department of Pharmacology, University of Pennsylvania Medical School, Philadelphia, PA 19104. Copyright © 1993 by Mosby-Year Book, Inc. 0002-9378/93 $1.00 + .20 6/1/44282
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responsible for many hysterectomies. 2 Unfortunately, relatively little is known about the cause of these common, benign uterine muscle tumors. Fibroids appear to be clonally derived.' Cytogenic aberrations in leiomyoma cells, frequently involving chromosome 12, have been reported, suggesting that spontaneous chromosome rearrangement may be responsible for initiation and proliferation of leiomyoma, in some cases but no consistent pattern has emerged to suggest involvement of a specific gene locus in the abnormal cell proliferation,,· 7 Attempts to characterize myometrial and leiomyoma cells in culture have shown differences in binding and growth responses to epidermal growth factor and, to a lesser degree, platelet-derived growth factor. B , 9 These