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Characteristics of vascular smooth muscle in the maternal resistance circulation during pregnancy in the rat
Characteristics of vascular smooth muscle in the maternal resistance circulation during pregnancy in the rat Marjorie C. Meyer, MD: Joseph E. Brayden, PhD,b and Margaret K. McLaughlin, PhDc
Burlington, Vermont, and Pittsburgh, Pennsylvania OBJECTIVE: Our purpose was to determine if pregnancy results in a decrease in arterial sensitivity to receptor-independent stimuli and a change in vascular smooth muscle membrane potential. STUDY DESIGN: Mesenteric resistance arteries from late pregnant (n = 19) and age-matched virgin control (n = 20) Sprague-Dawley rats were studied in a pressurized arteriograph system or isometric myograph. RESULTS: Arteries from pregnant rats were less sensitive to membrane depolarization by K+ than were those from nonpregnant rats (mean effective concentration that produced a 50% response 49 vs 39 mmol/L, pregnant vs nonpregnant, p < 0.05). Arterial basal tone and the myogenic response to increasing pressure steps were also reduced in arteries from pregnant rats compared with nonpregnant controls. The vascular smooth muscle membrane of the arteries from the pregnant rats was hyperpolarized compared with that from the control rats (- 64 mV from pregnant rats vs - 57 mV from nonpregnant rats, p < 0.01). This was associated with a reduction in vasomotion in the arteries from the pregnant rats (10% for pregnant rats vs 45% from nonpregnant rats, p < 0.01). CONCLUSION: Pregnancy results in alterations of the vascular smooth muscle, including changes in the regulation of membrane potential and a reduced sensitivity to receptor-independent stimuli. (AM J OBSTET GYNECOL 1993;169:1510-6.)
Key words: Pregnancy, resistance arteries, vascular reactivity, rat, membrane potential, myogenic, vasomotion The mechanisms responsible for the decrease in peripheral vascular resistance in pregnancy are not known. Potential contributors to the reduced vascular tonus include increased production of vasodilatory substances, such as endothelium-dependent relaxing factor (EDRF) (nitric oxide) and prostaglandins and decreases in vascular smooth muscle sensitivity to vasoactive stimuli such as adrenergic agonists.!" Whether a decreased sensitivity to vasoconstrictors represents an inherent difference in vascular smooth muscle contractile capability, compared with a difference in the second messenger systems related to receptor activity, is not clear. In this study vascular reactivity in pregnancy was examined independently of receptor function with two ap-
From the Departments of Obstetrics and Gynecology" and Pharmacology, b University of Vermont College of Medicine, and the Department of Obstetrics and Gynecology, Magee-Women's Hospital, University of Pittsburgh.' Supported in part by United States Public Health Service grant No. HD18162. Presented in part at the Thirty-seventh Annual Meeting ofthe Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990, and at the Fortieth Annual Meeting of the Society for Gynecologic Investigation, Toronto, Ontario, Canada, March 31April 3, 1993. Reprint requests: Marjorie C. Meyer, MD, Department of Obstetrics and Gynecology, Medical Center Hospital of Vermont, Burlington, VT 05405. Copyright © 1993 by Mosby-Year Book, Inc. 0002-9378/93 $1.00 + .20 6/6/50993
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proaches. In the first approach tone was altered by elevating the extracellular potassium concentration, which depolarizes the vascular smooth muscle cells. A decrease in the sensitivity to this direct stimulation would indicate that factors independent of receptor function are involved in pregnancy adaptation. In a second approach vascular tone was changed by increasing intraluminal pressure. Vascular tone is an important component of peripheral vascular resistance. Tone represents the inherent contraction in a vessel in response to intraluminal pressure. Tone can be basal (related to a constant intraluminal pressure) or myogenic (related to an increase in intraluminal pressure). Myogenic tone is thought to be an important component of autoregulation." This study was designed to test the hypotheses that late pregnancy is associated with (l) a decrease in arterial sensitivity to cell membrane depolarizationinduced smooth muscle contraction, (2) a decrease in arterial tone, both basal and myogenic, in the resistance-sized mesenteric arteries of the rat, and (3) a change in the vascular smooth muscle membrane potential that is associated with both decreased sensitivity and tone.
Methods Animal model. Virgin Sprague-Dawley rats (225 to 250 gm) were housed and bred in the University of
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Vermont College of Medicine Animal Facility (accredited by the American Association for Accreditation of Laboratory Animal Care), Two or three cycling female rats were housed with a male for 24 hours, and the presence of sperm in vaginal smears confirmed day 1 of pregnancy (term 22 days). Pregnant rats were studied at 17 to 20 days' gestation; age-matched virgins were used as controls. The animal protocol was approved by the University of Vermont Institutional Animal Care and Use Committee. Vessel preparation. Ras were decapitated while under light anesthesia with methohexital sodium (50 mg/kg body weight). For the study of myogenic tone and potassium chloride sensitivity, a section of the mesentery 5 to 10 cm distal to the pylorus was rapidly removed and placed in ice-cold physiologic saline solution. Resistance-sized (= 200 urn relaxed diameter) mesenteric arteries were dissected from the surrounding tissue, transferred to an arteriograph, and mounted on two microcannulas. Residual blood was flushed from the lumen, and the distal cannula was occluded to prevent flow. The proximal cannula was attached to a pressure transducer and a motor-driven syringe. The signal from the pressure transducer was compared with an internal reference voltage determined by the investigator. The polarity of the output voltage from the comparator determined the direction of the motor attached to the syringe plunger. This servopressure system advanced or retracted the syringe plunger to establish a desired intraluminal pressure. After an artery was mounted on the microcannulas, the arteriograph was placed on the stage of a compound microscope. A video camera attached to the viewing tube provided an image of the artery on a television monitor. A video dimension analyzing system (Living Systems, Burlington, Vt.) processed a selected vidicon line to provide lumen diameter and wall thickness measurements. Transmural pressure and lumen diameter measurements were directly available as either digital readouts or analog signals. Both the pressure and dimensional parameters were calibrated at the beginning of each experiment. The arteriograph chamber was perfused at a rate of 40 ml/min with bathing solution maintained at a temperature, pH, and P0 2 of 37 ± OS C, 7.4 ± 0.2, and 150 mm Hg, respectively. This system is described further elsewhere." The study of mesenteric vascular smooth muscle membrane potential and vasomotion was performed with an isometric wire myograph system. Resistance size (= 200 urn) arteries were dissected from the surrounding tissue and threaded onto 16 urn wires. These wires were fastened to two stainless-steel blocks that were mounted in a specially designed myograph system. One block was attached to a Kulite strain-gauge force transducer (Kulite Semiconductor Products, Ridgefield,
Meyer, Brayden, and Mclaughlin
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N.J.), the other connected to a displacement device. The blocks rested in a bath that was perfused continuously with a bathing solution, as described above. Arteries mounted in these two systems relax between 95% and 100% from a preconstructed baseline to 3 urnol/L methacholine. Solutions and drugs. The bathing solution was a modified Krebs-Ringer solution consisting of (millimoles per liter): sodium chloride 142, potassium chloride 4.7, magnesium sulfate 1.17, calcium chloride 1.56, potassium phosphate 1.18, sodium bicarbonate 24, and glucose 5.5. Potassium chloride solutions were made with equimolar substitutions of potassium chloride for sodium chloride. Relaxing solution was similar to the bathing solution except that calcium chloride was removed and 1 mmol/L ethylene glycol-bistjs-aminoethyl ether)N,N,N/,N/-tetraacetic acid and 0.1 mmol/L papaverine were added. Experimental protocol Potassium sensitivity. The mesenteric arteries were mounted and equilibrated at 50 mm Hg for 30 minutes before a dose-response curve to potassium chloride was generated. The diameter of each artery was monitored in response to increasing concentrations of potassium chloride (6 to 124 mmol/L). All dose-response curves were performed in the presence of 2 umol/L prazosin (Sigma) to block any effect of norepinephrine release from nerve terminals within the artery in response to potassium chloride exposure. Tone and myogenic response. A second series of experiments was performed to determine both the basal tone for each artery and its myogenic response to increasing pressure steps. The arterial segment was mounted and equilibrated at 50 mm Hg for 30 minutes. The intraluminal pressure was increased to 75 mm Hg for 15 minutes for the measurement of basal tone. This pressure was chosen to approximate the mean arterial pressure experienced by these arteries in vivo. The intraluminal pressure was then set to 25 mm Hg for 10 minutes, after which the pressure was increased in rapid (2-second) 25 mm Hg pressure steps with the arterial diameter measured at 5 minutes up to 100 mm Hg. After generation of the myogenic response curve each artery was placed in the relaxing solution, and the pressure steps were repeated. The inhibition of smooth muscle reactivity was verified by the lack of a response to 124 mmol/L potassium chloride. In a third series of experiments the basal tone and myogenic response measurements were obtained before and after the inhibition of nitric oxide synthase with 0.24 mmol/L Nconitro-L-arginine. Endothelial integrity was verified in these arteries by obtaining 90% to 100% relaxation response to 3 urnol/L methacholine. Membrane potential and vasomotion. Experiments were performed on a separate set of mesenteric arteries from
1512 Meyer, Brayden, and McLaughlin
December 1993 Am J Obstet Gynecol
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Fig. 1. Concentration-response curves to potassium for mesenteric arteries fro m pr egn ant (n = 5, closed circles) and non pregn ant (n = 5, open circles) ra ts. Change in diamet er is exp resse d as percentage of maxi mu m pot assium res po nse . Dat a re present mean ± SE.
pregn ant an d nonpregnant ra ts, to measure the membran e potential in individ ual vascular smooth muscle cells and to de scribe th e d ifferences in vasomo tor behavior be tween groups. The m embran e potential and vasomo tor ac tivity were m easu red in ar teries stretched to an identical point on th e ir passive len gth tension s curv es . 10 Membranes p ot ential was re co rded with intrace llular microelectrodes filled with 0.5 mol/L potassium chloride with tip resi stances of 80 to 120 Mil and tip pot entials < 5 mV. The arterial medi a was approached fro m the adventitial side of th e vesse l with the site of impalement at the midpoint of the arterial segment. Five to seven cells were impaled in each artery. Voltage was recorded with an ele ctrom et er (W P1) and a dat a acquisition system (Axon topc, Axon In struments). The m embran e p otential re cor di ngs were acce p ted onl y if th e membran e potential measu rem ent rem ained stable for 3 minutes and no cha nge occurred in the micr oel ectrode tip re sistance before and after each impalement. The vasomotor ac tivity was m easured in a set of ex periments in whic h a norepinephrine do se-response curve was performed on eac h artery. The vasomotion was qu antitated by calculating th e p ercent oscilla tion in force arou nd the me an force ge nera te d at the norepinephrine median effec tive concentration for ea ch ar tery . Data analysis. The p ercent tone of the ind ivid ual ar teries was calcula te d at eac h p res sure ste p by means of th e follo wing equation: % Tone = (Dr - Dpss)/D r' 100
where Dr is the relaxed internal diameter and D pss is th e internal diameter in physiolo gic saline solution. T he constrictor response to the potassium chloride
depolarization was calculated by mean s of the absolute reduc tion from control diameter at each concentration of potassium chloride, wh ere the con trol diameter was that measured just befor e the add ition of potassium chlo ride . This difference in diameter at ea ch concen tr at ion was then expressed as a p ercent of th e maximal co nstric tion to 124 mrnol/L pot assium chloride. The med ian effective concentrat ion was derived from each individual dose-response curve . T he median effective conc en tration to potassium chloride and the basal tone at 75 mm Hg was compared between the two groups with an unpaired Student t test. The myogenic response of th e ar teries was compared between the two groups by mean s of analysis of vari ance with repeated measures. T he effect of Nco-nitro-t-arginine on the myogenic resp on se and the membrane p otential an d degree of vaso motio n was compared with an unpaired Student t test. Differences betw een mean s were cons id ered significant at p < 0.05. Results
There were no significant differences in the relaxed arterial di ameters betw een th e pregnant an d nonpregnant rats (197 ± 9 urn (n = 14) vers us 199 ± 7 urn (n = 16, mean ± SEM ). Potassium sensitivity. The dose-re sponse curve to potassium chlorid e is depicted in Fig. 1. The doseres po nse curve of th e ar te ries from th e pregnant ani m als was shi fted to th e right of the arteries from the nonpregnant animals, with the median effective conce n tra tion from pregnant animals (n = 5, 49 ± 5.4 mmol/L) being significantly greater than that from nonpregnant rats (n = 5, 39 ± 2.6 mmol/L, m ean ± SE, p < 0.05).
Volume 169, Number 6 Am J Obstet Gynecol
Meyer, Brayden, and Mclaughlin
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Tone. The basal tone produced in arteries at 75 mm Hg was significantly less in the arteries from the pregnant animals (pregnant 5% ± 1% vs nonpregnant 22% ± 3%, P < 0.05) (Fig. 2). As shown in Fig. 3, the myogenic response was significantly blunted in the arteries from the pregnant rats. There was a significant increase in tone with increased pressure in the arteries from nonpregnant animals (p < 0.05) that was lacking in the arteries from pregnant animals (p > 0.05). Arteries were incubated in the presence of Nw-nitro-Larginine to test whether an inhibitor of nitric oxide would increase myogenic tone. There was no change in myogenic tone in either group with nitric oxide synthase inhibition. The tone in the nonpregnant rats at 100 mm Hg was 20% ± 6% in physiologic saline solu-
tion versus 14% ± 9% in Nw-nitro-L-arginine (n = 4, not significant). In the late pregnant rats tone was 4% ± 9% in physiologic saline solution versus 3% ± 2% in Nw-nitro-L-arginine (n = 5, not significant). Membrane potential. Two to six smooth muscle cells were impaled in each artery from each rat. The means for the individual arteries are listed in Table 1. The SD for the resting membrane potential between the smooth muscle cells from any individual artery was < 1 mY. The smooth muscle cells from the pregnant rat arteries were hyperpolarized 7 mV compared with those from the nonpregnant rats (p < 0.001). Vasomotion. There was a striking difference in the degree of vasomotion exhibited in the norepinephrinecontracted arteries from the two groups. Fig. 4 is a
1514 Meyer, Brayden, and McLaughlin
December 1993 Am J Obstet Gynecol
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and pregnant (bottom panel) rat. Arrows, Increasing norepinephrine concentrations.
Table I. Resting membrane potential in mesenteric resistance arteries Animal No.
I 2 3 4
Mean ± SD
Nonpregnant (mV)
-56 (6) -56 (2) -58 (3) -58 (5) - 57 ± -1
Animal No.
I 2 3 4
Pregnant (mV)
-64 -59 -66 -66 -64 ±
(4) (4) (3) (5)
-3*
The number in parentheses is the number of measurements in each artery. *Pregnant versus nonpregnant, p < 0.01. tracing that depicts the vasomotion in an artery from a pregnant rat and one from a nonpregnant animal. On average, at the median effective concentration for norepinephrine in each artery (= 3 X 10- 6 mol/L), the arteries from the pregnant rats oscillated 10% ± 6% around their mean tension, whereas those from the nonpregnant rats oscillated 45% ± 8% (mean ± SD, P < 0.01). There was no significant difference in the oscillatory rate between the two groups. Comment
The major conclusion reached from these studies is that pregnancy modifies the vascular smooth muscle of the mesenteric vasculature independently of receptormediated processes. This is evidenced in the arteries from the late gestation rats by (1) the reduced basal tone and myogenic response, (2) the blunted response
to K+ depolarization, and (3) the decrease in spontaneous oscillatory behavior. The difference in the membrane potential in the vascular smooth muscle between the pregnant and nonpregnant rats provides one potential explanation for these observed differences in the behavior of the resistance-sized mesenteric arteries. The basal tone of these arteries is determined by the steady-state force generation of the vascular wall. There is a steep relationship between the internal calcium [Ca ++]; and steady-state force generation in small arteries. II The intracellular calcium concentration is tightly regulated in both the resting and activated state. The activity of the voltage-dependent calcium channel is a major determinant of the [Ca ++]; during activation of the vascular smooth muscle cell. The open state probability of this channel is influenced markedly by the membrane potential. The open-state probability of these channels increases 2.7-fold for every 7 to 9 mV depolarization of the vascular smooth muscle. II, 12 Thus the 7 mV increase in the membrane potential in the vascular smooth muscle of the pregnant rat arteries would be expected to reduce the open-state probability of these channels, thereby affecting intracellular calcium and vessel tone. In addition, the myogenic response is associated with cell membrane depolarization and calcium entry through calcium channels in a number of arteries." A 7 mV hyperpolarization would increase the magnitude of depolarization required and hence the magnitude of pressure stimulus required to reach the membrane potential range over which the myogenic response occurs.
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The difference in vasomotion during pregnancy is potentially explained by the same mechanism (i.e., the hyperpolarization-reducing calcium influx). Vascular smooth muscle cells have an inherent ability to regulate their own intracellular ([Ca ++t) by spontaneously discharging calcium from the sarcoplasmic reticulum. This discharge of calcium has been shown to be closely associated with an influx of extracellular calcium through specific membrane Ca + + channels. This oscillatory behavior in [Ca + + t provides a biochemical explanation for the vasomotion that is observed in vivo and in vitro. 13 That such vasomotor activity is blunted in vitro during pregnancy (our observation) is consistent with a membrane hyperpolarization reducing the driving force for extracellular Ca + + movement into the cell. The difference in membrane potential will aiso modifY the response to either the non-receptor-mediated (K+) or receptor-mediated (norepinephrine) vasoactive agents because an influx of extracellular calcium is a component of the contractile response to both agonists. Therefore the change in membrane potential could explain both the observed decrease of basal tone during pregnancy (at least in the rat) and the reduction in systemic vascular reactivity. Thus during pregnancy there appears to be an alteration in the regulation of the vascular smooth muscle membrane, causing a hyperpolarization that will reduce calcium entry into the cell to a given stimulus. Whether calcium entry is actually different in these vascular smooth muscle cells during pregnancy remains to be examined. Previous studies in the pig have shown that catechol estrogens can alter calcium entry in the uterine artery through voltage-dependent calcium channels. 14 Although the main mesenteric artery appeared steroid insensitive in those particular studies, there are likely to be both vessel size and species differences with regard to steroidogenic effects on vascular smooth muscle function. It is not known if the catechol-estrogen modification of voltage-dependent calcium channels was caused by changes in vascular smooth muscle membrane potential. While the change in membrane potential provides a likely explanation for the gestational effects on vessel behavior, there are numerous other possibilities. An alternative modulator of the myogenic response is the endothelium. Numerous studies examining the role of the endothelium in the myogenic behavior of arteries have suggested that the myogenic response is endothelium dependent, whereas others have shown that the myogenic response is endothelium independent.P''" There is a wide discrepancy between methods, arteries, and species studied in these reports. Further, there are a variety of endothelial-derived factors that modulate vascular smooth muscle function. Because plasma and urinary levels of cyclic guanosine 5/-monophosphate
Meyer, Brayden, and Mclaughlin
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(the secondary messenger for EDRF) are elevated during gestation in the rat," we hypothesized that increased EDRF may be responsible for the reduced myogenic response of the mesenteric arteries from the pregnant rats. Because the methods for endothelial removal in larger arteries are known to affect the experimental outcome; 1 we chose to examine nitric oxide alone in these very small arteries by means of pharmacologic inhibition of nitric oxide synthase. The acute inhibition of nitric oxide synthesis did not increase either the basal or myogenic tone in the arteries from the late pregnant rats. This observed lack of an increased basal EDRF influence during pregnancy is consistent with our finding that basal EDRF production does not appear to be responsible for the blunted adrenergic responsiveness during pregnancy in the mesenteric arteries of rats. 1 However, our study design does not preclude the possibility that other endothelialdependent agents (i.e., prostaglandins or endothelialderived hyperpolarizing factor) may mediate the reduced tone and myogenic response during pregnancy. In addition, EDRF could have as yet unidentified, longer-term effects on the vascular smooth muscle that may not be reversed with acute inhibition of nitrate synthase. For example, nitric oxide has recently been shown to react with membrane-bound thiol groups on the N-methyl-n-aspartate subtype of the glutamate receptor, resulting in a persistent blockage of N-methylo-aspartate responses." It also may be that EDRF modulates in vivo myogenic responses in a manner that is not apparent when it is studied in vitro. Flow-induced shear stress in vivo can cause the production and release of EDRF from the endothelium-a situation that may not be apparent under these in vitro no-flow conditions. We have observed that the stimulated release of EDRF, as opposed to basal production, is increased during pregnancy. The interaction between flow and myogenic behavior during pregnancy requires further study. Thus the data from this and previous studies continue to support the idea that pregnancy results in an alteration of the vascular smooth muscle function that contributes to the hemodynamic profile of pregnancy. It would appear that one alteration involves changes in the regulation of membrane potential. This in turn may modify calcium movement into the vascular smooth muscle. That the observed change in membrane potential is actually influencing the calcium movement in these cells under different activation states requires further direct study. In addition, the question remains as to the cause for the membrane hyperpolarization. The easiest means to achieve this change would be through an alteration in the potassium conductance, which is regulated by a variety of channels. An attractive possibility is that pregnancy induces a change in the
1516 Meyer, Brayden, and McLaughlin
activity of the adenosine 5'-triphosphate-dependent potassium channel, recently identified in the vascular smooth muscle. The adenosine 5'-triphosphate-dependent K + channel, through its dependence on adenosine 5'-triphosphate for its regulation, could provide a link between metabolism and the regulation of blood flOW. 2 3 Such a possibility provides an interesting future direction for this work. REFERENCES 1. Davidge ST, McLaughlin MK. Endogenous modulation of the blunted adrenergic response in resistance-sized mesenteric arteries from the pregnant rat. AM j OBSTET GYNECOL 1992;167:1691-8. 2. Harrison GL, Moore LG. Blunted vasoreactivity in pregnant guinea pigs is not restored by meclofenamate. AM j OBSTET GYNECOL 1989;160:258-64. 3. jansakul C, Boura ALA, King RG. Effect of endothelial cell removal on constrictor and dilator responses of aortae of pregnant rats. J Auton Pharmacol 1989;9:93-101. 4. Weiner C, Liu KZ, Thompson L, Herrig j, Chestnut D. Effect of pregnancy on endothelium and smooth muscle: their role in reduced adrenergic sensitivity. Am j Physiol 1991;261:HI275-83. 5. Crandall ME, Keve TM, McLaughlin MK. Characterization of norepinephrine sensitivity in the maternal splanchnic circulation during pregnancy. AM j OBSTET GYNECOL 1990; 162: 1296-301. 6. St-Louis j, Sicotte B. Prostaglandin- or endothelium-mediated vasodilation is not involved in the blunted responses to vasoconstrictors in pregnant rats. AM j OBSTET GYNECOL 1992;166:684-92. 7. Pallor MS. Mechanism of decreased responsiveness to angiotensin II, NE and vasopressin in pregnant rats. Am j Physiol 1984;247:HlOO-8. 8. johnsson PC. The myogenic response. In: Handbook of physiology. The cardiovascular system. Vascular smooth muscle. Bethesda, Maryland: American Physiological Society, 1980:409-42. 9. Halpern W, Osol G, Coy GS. Mechanical behavior of pressurized in vitro prearteriolar vessels determined with a video system. Ann Biomed Engineering 1984;12:463-79. 10. McLaughlin MK, Keve TM. Pregnancy-induced changes
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11.
12. 13. 14.
15.
16. 17. 18. 19.
20. 21. 22. 23.
in resistance blood vessels. AM j OBSTET GYNECOL 1986; 155:1296-9. Nelson MT, Patlak jB, Worley jF, Standen NB. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am j Physiol 1990;259:C318. Brayden jE, Nelson MT. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science 1992;256:532-5. Weissberg PL, Little Pj, Bobik A. Spontaneous oscillations in cytoplasmic calcium concentrations in vascular smooth muscle. Am J Physiol 1989;256:C951-7. Stice SL, Ford SP, Rosazza jP, Van Orden DE. Role of 4-hydroxylated estradiol in reducing Ca 2 + uptake of uterine arterial smooth muscle cells through potential sensitive channels. Bioi Reprod 1987;36:361-8. Harder DR. Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact endothelium. Circ Res 1987;60:102-7. Katusic ZS, Shepherd jT, Vanhoutte PM. Endotheliumdependent contraction to stretch in canine basilar arteries. Am J Physiol 1987;252:H671-3. Hwa jj, Bevan jA. Stretch-dependent (myogenic) tone in rabbit ear resistance arteries. Am j Physiol 1986;250:H8795. Kuo L, Chilian WM, Davis MJ. The coronary arteriolar myogenic response is independent of the endothelium. Circ Res 1990;66:860-6. Nakayama K, Tanaka T, Fujishima K. Potentiation of stretch-induced myogenic tone of dog cerebral artery by hemolysate and the inhibitory action of calcium antagonists. Eur J Pharmacol 1990;169:33-42. Conrad KP, Vernier KA. Plasma level, urinary excretion, and metabolic production of cGMP during gestation in rats. Am J Physiol 1989;257:R847-53. MacPherson RD, McLeod L], Rasiah RL. Myogenic response of isolated pressurized rabbit ear artery is independent of endothelium. Am j Physiol 1991;260:H779-84. Lei SZ, Pan ZH, Aggarwal SK, et al. Effect of nitric oxide production in the redox modulatory site of the NMDA receptor-channel complex. Neuron 1992;8: 1087-99. Standen NB, Quayle jM, Davies NW, Brayden jE, Huang Y, Nelson MT. Hyperpolarizing vasodilators activate ATPsensitive K + channels in arterial smooth muscle. Science 1989;245: 177-80.
Burlington, Vermont, and Pittsburgh, Pennsylvania OBJECTIVE: Our purpose was to determine if pregnancy results in a decrease in arterial sensitivity to receptor-independent stimuli and a change in vascular smooth muscle membrane potential. STUDY DESIGN: Mesenteric resistance arteries from late pregnant (n = 19) and age-matched virgin control (n = 20) Sprague-Dawley rats were studied in a pressurized arteriograph system or isometric myograph. RESULTS: Arteries from pregnant rats were less sensitive to membrane depolarization by K+ than were those from nonpregnant rats (mean effective concentration that produced a 50% response 49 vs 39 mmol/L, pregnant vs nonpregnant, p < 0.05). Arterial basal tone and the myogenic response to increasing pressure steps were also reduced in arteries from pregnant rats compared with nonpregnant controls. The vascular smooth muscle membrane of the arteries from the pregnant rats was hyperpolarized compared with that from the control rats (- 64 mV from pregnant rats vs - 57 mV from nonpregnant rats, p < 0.01). This was associated with a reduction in vasomotion in the arteries from the pregnant rats (10% for pregnant rats vs 45% from nonpregnant rats, p < 0.01). CONCLUSION: Pregnancy results in alterations of the vascular smooth muscle, including changes in the regulation of membrane potential and a reduced sensitivity to receptor-independent stimuli. (AM J OBSTET GYNECOL 1993;169:1510-6.)
Key words: Pregnancy, resistance arteries, vascular reactivity, rat, membrane potential, myogenic, vasomotion The mechanisms responsible for the decrease in peripheral vascular resistance in pregnancy are not known. Potential contributors to the reduced vascular tonus include increased production of vasodilatory substances, such as endothelium-dependent relaxing factor (EDRF) (nitric oxide) and prostaglandins and decreases in vascular smooth muscle sensitivity to vasoactive stimuli such as adrenergic agonists.!" Whether a decreased sensitivity to vasoconstrictors represents an inherent difference in vascular smooth muscle contractile capability, compared with a difference in the second messenger systems related to receptor activity, is not clear. In this study vascular reactivity in pregnancy was examined independently of receptor function with two ap-
From the Departments of Obstetrics and Gynecology" and Pharmacology, b University of Vermont College of Medicine, and the Department of Obstetrics and Gynecology, Magee-Women's Hospital, University of Pittsburgh.' Supported in part by United States Public Health Service grant No. HD18162. Presented in part at the Thirty-seventh Annual Meeting ofthe Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990, and at the Fortieth Annual Meeting of the Society for Gynecologic Investigation, Toronto, Ontario, Canada, March 31April 3, 1993. Reprint requests: Marjorie C. Meyer, MD, Department of Obstetrics and Gynecology, Medical Center Hospital of Vermont, Burlington, VT 05405. Copyright © 1993 by Mosby-Year Book, Inc. 0002-9378/93 $1.00 + .20 6/6/50993
1510
proaches. In the first approach tone was altered by elevating the extracellular potassium concentration, which depolarizes the vascular smooth muscle cells. A decrease in the sensitivity to this direct stimulation would indicate that factors independent of receptor function are involved in pregnancy adaptation. In a second approach vascular tone was changed by increasing intraluminal pressure. Vascular tone is an important component of peripheral vascular resistance. Tone represents the inherent contraction in a vessel in response to intraluminal pressure. Tone can be basal (related to a constant intraluminal pressure) or myogenic (related to an increase in intraluminal pressure). Myogenic tone is thought to be an important component of autoregulation." This study was designed to test the hypotheses that late pregnancy is associated with (l) a decrease in arterial sensitivity to cell membrane depolarizationinduced smooth muscle contraction, (2) a decrease in arterial tone, both basal and myogenic, in the resistance-sized mesenteric arteries of the rat, and (3) a change in the vascular smooth muscle membrane potential that is associated with both decreased sensitivity and tone.
Methods Animal model. Virgin Sprague-Dawley rats (225 to 250 gm) were housed and bred in the University of
Volume 169, Number 6 Am J Obstet Gynecol
Vermont College of Medicine Animal Facility (accredited by the American Association for Accreditation of Laboratory Animal Care), Two or three cycling female rats were housed with a male for 24 hours, and the presence of sperm in vaginal smears confirmed day 1 of pregnancy (term 22 days). Pregnant rats were studied at 17 to 20 days' gestation; age-matched virgins were used as controls. The animal protocol was approved by the University of Vermont Institutional Animal Care and Use Committee. Vessel preparation. Ras were decapitated while under light anesthesia with methohexital sodium (50 mg/kg body weight). For the study of myogenic tone and potassium chloride sensitivity, a section of the mesentery 5 to 10 cm distal to the pylorus was rapidly removed and placed in ice-cold physiologic saline solution. Resistance-sized (= 200 urn relaxed diameter) mesenteric arteries were dissected from the surrounding tissue, transferred to an arteriograph, and mounted on two microcannulas. Residual blood was flushed from the lumen, and the distal cannula was occluded to prevent flow. The proximal cannula was attached to a pressure transducer and a motor-driven syringe. The signal from the pressure transducer was compared with an internal reference voltage determined by the investigator. The polarity of the output voltage from the comparator determined the direction of the motor attached to the syringe plunger. This servopressure system advanced or retracted the syringe plunger to establish a desired intraluminal pressure. After an artery was mounted on the microcannulas, the arteriograph was placed on the stage of a compound microscope. A video camera attached to the viewing tube provided an image of the artery on a television monitor. A video dimension analyzing system (Living Systems, Burlington, Vt.) processed a selected vidicon line to provide lumen diameter and wall thickness measurements. Transmural pressure and lumen diameter measurements were directly available as either digital readouts or analog signals. Both the pressure and dimensional parameters were calibrated at the beginning of each experiment. The arteriograph chamber was perfused at a rate of 40 ml/min with bathing solution maintained at a temperature, pH, and P0 2 of 37 ± OS C, 7.4 ± 0.2, and 150 mm Hg, respectively. This system is described further elsewhere." The study of mesenteric vascular smooth muscle membrane potential and vasomotion was performed with an isometric wire myograph system. Resistance size (= 200 urn) arteries were dissected from the surrounding tissue and threaded onto 16 urn wires. These wires were fastened to two stainless-steel blocks that were mounted in a specially designed myograph system. One block was attached to a Kulite strain-gauge force transducer (Kulite Semiconductor Products, Ridgefield,
Meyer, Brayden, and Mclaughlin
1511
N.J.), the other connected to a displacement device. The blocks rested in a bath that was perfused continuously with a bathing solution, as described above. Arteries mounted in these two systems relax between 95% and 100% from a preconstructed baseline to 3 urnol/L methacholine. Solutions and drugs. The bathing solution was a modified Krebs-Ringer solution consisting of (millimoles per liter): sodium chloride 142, potassium chloride 4.7, magnesium sulfate 1.17, calcium chloride 1.56, potassium phosphate 1.18, sodium bicarbonate 24, and glucose 5.5. Potassium chloride solutions were made with equimolar substitutions of potassium chloride for sodium chloride. Relaxing solution was similar to the bathing solution except that calcium chloride was removed and 1 mmol/L ethylene glycol-bistjs-aminoethyl ether)N,N,N/,N/-tetraacetic acid and 0.1 mmol/L papaverine were added. Experimental protocol Potassium sensitivity. The mesenteric arteries were mounted and equilibrated at 50 mm Hg for 30 minutes before a dose-response curve to potassium chloride was generated. The diameter of each artery was monitored in response to increasing concentrations of potassium chloride (6 to 124 mmol/L). All dose-response curves were performed in the presence of 2 umol/L prazosin (Sigma) to block any effect of norepinephrine release from nerve terminals within the artery in response to potassium chloride exposure. Tone and myogenic response. A second series of experiments was performed to determine both the basal tone for each artery and its myogenic response to increasing pressure steps. The arterial segment was mounted and equilibrated at 50 mm Hg for 30 minutes. The intraluminal pressure was increased to 75 mm Hg for 15 minutes for the measurement of basal tone. This pressure was chosen to approximate the mean arterial pressure experienced by these arteries in vivo. The intraluminal pressure was then set to 25 mm Hg for 10 minutes, after which the pressure was increased in rapid (2-second) 25 mm Hg pressure steps with the arterial diameter measured at 5 minutes up to 100 mm Hg. After generation of the myogenic response curve each artery was placed in the relaxing solution, and the pressure steps were repeated. The inhibition of smooth muscle reactivity was verified by the lack of a response to 124 mmol/L potassium chloride. In a third series of experiments the basal tone and myogenic response measurements were obtained before and after the inhibition of nitric oxide synthase with 0.24 mmol/L Nconitro-L-arginine. Endothelial integrity was verified in these arteries by obtaining 90% to 100% relaxation response to 3 urnol/L methacholine. Membrane potential and vasomotion. Experiments were performed on a separate set of mesenteric arteries from
1512 Meyer, Brayden, and McLaughlin
December 1993 Am J Obstet Gynecol
100
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Fig. 1. Concentration-response curves to potassium for mesenteric arteries fro m pr egn ant (n = 5, closed circles) and non pregn ant (n = 5, open circles) ra ts. Change in diamet er is exp resse d as percentage of maxi mu m pot assium res po nse . Dat a re present mean ± SE.
pregn ant an d nonpregnant ra ts, to measure the membran e potential in individ ual vascular smooth muscle cells and to de scribe th e d ifferences in vasomo tor behavior be tween groups. The m embran e potential and vasomo tor ac tivity were m easu red in ar teries stretched to an identical point on th e ir passive len gth tension s curv es . 10 Membranes p ot ential was re co rded with intrace llular microelectrodes filled with 0.5 mol/L potassium chloride with tip resi stances of 80 to 120 Mil and tip pot entials < 5 mV. The arterial medi a was approached fro m the adventitial side of th e vesse l with the site of impalement at the midpoint of the arterial segment. Five to seven cells were impaled in each artery. Voltage was recorded with an ele ctrom et er (W P1) and a dat a acquisition system (Axon topc, Axon In struments). The m embran e p otential re cor di ngs were acce p ted onl y if th e membran e potential measu rem ent rem ained stable for 3 minutes and no cha nge occurred in the micr oel ectrode tip re sistance before and after each impalement. The vasomotor ac tivity was m easured in a set of ex periments in whic h a norepinephrine do se-response curve was performed on eac h artery. The vasomotion was qu antitated by calculating th e p ercent oscilla tion in force arou nd the me an force ge nera te d at the norepinephrine median effec tive concentration for ea ch ar tery . Data analysis. The p ercent tone of the ind ivid ual ar teries was calcula te d at eac h p res sure ste p by means of th e follo wing equation: % Tone = (Dr - Dpss)/D r' 100
where Dr is the relaxed internal diameter and D pss is th e internal diameter in physiolo gic saline solution. T he constrictor response to the potassium chloride
depolarization was calculated by mean s of the absolute reduc tion from control diameter at each concentration of potassium chloride, wh ere the con trol diameter was that measured just befor e the add ition of potassium chlo ride . This difference in diameter at ea ch concen tr at ion was then expressed as a p ercent of th e maximal co nstric tion to 124 mrnol/L pot assium chloride. The med ian effective concentrat ion was derived from each individual dose-response curve . T he median effective conc en tration to potassium chloride and the basal tone at 75 mm Hg was compared between the two groups with an unpaired Student t test. The myogenic response of th e ar teries was compared between the two groups by mean s of analysis of vari ance with repeated measures. T he effect of Nco-nitro-t-arginine on the myogenic resp on se and the membrane p otential an d degree of vaso motio n was compared with an unpaired Student t test. Differences betw een mean s were cons id ered significant at p < 0.05. Results
There were no significant differences in the relaxed arterial di ameters betw een th e pregnant an d nonpregnant rats (197 ± 9 urn (n = 14) vers us 199 ± 7 urn (n = 16, mean ± SEM ). Potassium sensitivity. The dose-re sponse curve to potassium chlorid e is depicted in Fig. 1. The doseres po nse curve of th e ar te ries from th e pregnant ani m als was shi fted to th e right of the arteries from the nonpregnant animals, with the median effective conce n tra tion from pregnant animals (n = 5, 49 ± 5.4 mmol/L) being significantly greater than that from nonpregnant rats (n = 5, 39 ± 2.6 mmol/L, m ean ± SE, p < 0.05).
Volume 169, Number 6 Am J Obstet Gynecol
Meyer, Brayden, and Mclaughlin
1513
25 20
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Fig. 3. Degree of tone present in response to increasing transmural pressure in arteries from nonpregnant (n = 5, open bars) and pregnant (n = 5, hatched bars) rats. Data represent mean ± SE.
Tone. The basal tone produced in arteries at 75 mm Hg was significantly less in the arteries from the pregnant animals (pregnant 5% ± 1% vs nonpregnant 22% ± 3%, P < 0.05) (Fig. 2). As shown in Fig. 3, the myogenic response was significantly blunted in the arteries from the pregnant rats. There was a significant increase in tone with increased pressure in the arteries from nonpregnant animals (p < 0.05) that was lacking in the arteries from pregnant animals (p > 0.05). Arteries were incubated in the presence of Nw-nitro-Larginine to test whether an inhibitor of nitric oxide would increase myogenic tone. There was no change in myogenic tone in either group with nitric oxide synthase inhibition. The tone in the nonpregnant rats at 100 mm Hg was 20% ± 6% in physiologic saline solu-
tion versus 14% ± 9% in Nw-nitro-L-arginine (n = 4, not significant). In the late pregnant rats tone was 4% ± 9% in physiologic saline solution versus 3% ± 2% in Nw-nitro-L-arginine (n = 5, not significant). Membrane potential. Two to six smooth muscle cells were impaled in each artery from each rat. The means for the individual arteries are listed in Table 1. The SD for the resting membrane potential between the smooth muscle cells from any individual artery was < 1 mY. The smooth muscle cells from the pregnant rat arteries were hyperpolarized 7 mV compared with those from the nonpregnant rats (p < 0.001). Vasomotion. There was a striking difference in the degree of vasomotion exhibited in the norepinephrinecontracted arteries from the two groups. Fig. 4 is a
1514 Meyer, Brayden, and McLaughlin
December 1993 Am J Obstet Gynecol
400 iii
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E
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iii
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Fig. 4. Tracing of typical pattern of vasomotion in mesenteric artery from nonpregnant (top panel)
and pregnant (bottom panel) rat. Arrows, Increasing norepinephrine concentrations.
Table I. Resting membrane potential in mesenteric resistance arteries Animal No.
I 2 3 4
Mean ± SD
Nonpregnant (mV)
-56 (6) -56 (2) -58 (3) -58 (5) - 57 ± -1
Animal No.
I 2 3 4
Pregnant (mV)
-64 -59 -66 -66 -64 ±
(4) (4) (3) (5)
-3*
The number in parentheses is the number of measurements in each artery. *Pregnant versus nonpregnant, p < 0.01. tracing that depicts the vasomotion in an artery from a pregnant rat and one from a nonpregnant animal. On average, at the median effective concentration for norepinephrine in each artery (= 3 X 10- 6 mol/L), the arteries from the pregnant rats oscillated 10% ± 6% around their mean tension, whereas those from the nonpregnant rats oscillated 45% ± 8% (mean ± SD, P < 0.01). There was no significant difference in the oscillatory rate between the two groups. Comment
The major conclusion reached from these studies is that pregnancy modifies the vascular smooth muscle of the mesenteric vasculature independently of receptormediated processes. This is evidenced in the arteries from the late gestation rats by (1) the reduced basal tone and myogenic response, (2) the blunted response
to K+ depolarization, and (3) the decrease in spontaneous oscillatory behavior. The difference in the membrane potential in the vascular smooth muscle between the pregnant and nonpregnant rats provides one potential explanation for these observed differences in the behavior of the resistance-sized mesenteric arteries. The basal tone of these arteries is determined by the steady-state force generation of the vascular wall. There is a steep relationship between the internal calcium [Ca ++]; and steady-state force generation in small arteries. II The intracellular calcium concentration is tightly regulated in both the resting and activated state. The activity of the voltage-dependent calcium channel is a major determinant of the [Ca ++]; during activation of the vascular smooth muscle cell. The open state probability of this channel is influenced markedly by the membrane potential. The open-state probability of these channels increases 2.7-fold for every 7 to 9 mV depolarization of the vascular smooth muscle. II, 12 Thus the 7 mV increase in the membrane potential in the vascular smooth muscle of the pregnant rat arteries would be expected to reduce the open-state probability of these channels, thereby affecting intracellular calcium and vessel tone. In addition, the myogenic response is associated with cell membrane depolarization and calcium entry through calcium channels in a number of arteries." A 7 mV hyperpolarization would increase the magnitude of depolarization required and hence the magnitude of pressure stimulus required to reach the membrane potential range over which the myogenic response occurs.
Volume 169, Number 6 Am J Obstet Gynecol
The difference in vasomotion during pregnancy is potentially explained by the same mechanism (i.e., the hyperpolarization-reducing calcium influx). Vascular smooth muscle cells have an inherent ability to regulate their own intracellular ([Ca ++t) by spontaneously discharging calcium from the sarcoplasmic reticulum. This discharge of calcium has been shown to be closely associated with an influx of extracellular calcium through specific membrane Ca + + channels. This oscillatory behavior in [Ca + + t provides a biochemical explanation for the vasomotion that is observed in vivo and in vitro. 13 That such vasomotor activity is blunted in vitro during pregnancy (our observation) is consistent with a membrane hyperpolarization reducing the driving force for extracellular Ca + + movement into the cell. The difference in membrane potential will aiso modifY the response to either the non-receptor-mediated (K+) or receptor-mediated (norepinephrine) vasoactive agents because an influx of extracellular calcium is a component of the contractile response to both agonists. Therefore the change in membrane potential could explain both the observed decrease of basal tone during pregnancy (at least in the rat) and the reduction in systemic vascular reactivity. Thus during pregnancy there appears to be an alteration in the regulation of the vascular smooth muscle membrane, causing a hyperpolarization that will reduce calcium entry into the cell to a given stimulus. Whether calcium entry is actually different in these vascular smooth muscle cells during pregnancy remains to be examined. Previous studies in the pig have shown that catechol estrogens can alter calcium entry in the uterine artery through voltage-dependent calcium channels. 14 Although the main mesenteric artery appeared steroid insensitive in those particular studies, there are likely to be both vessel size and species differences with regard to steroidogenic effects on vascular smooth muscle function. It is not known if the catechol-estrogen modification of voltage-dependent calcium channels was caused by changes in vascular smooth muscle membrane potential. While the change in membrane potential provides a likely explanation for the gestational effects on vessel behavior, there are numerous other possibilities. An alternative modulator of the myogenic response is the endothelium. Numerous studies examining the role of the endothelium in the myogenic behavior of arteries have suggested that the myogenic response is endothelium dependent, whereas others have shown that the myogenic response is endothelium independent.P''" There is a wide discrepancy between methods, arteries, and species studied in these reports. Further, there are a variety of endothelial-derived factors that modulate vascular smooth muscle function. Because plasma and urinary levels of cyclic guanosine 5/-monophosphate
Meyer, Brayden, and Mclaughlin
1515
(the secondary messenger for EDRF) are elevated during gestation in the rat," we hypothesized that increased EDRF may be responsible for the reduced myogenic response of the mesenteric arteries from the pregnant rats. Because the methods for endothelial removal in larger arteries are known to affect the experimental outcome; 1 we chose to examine nitric oxide alone in these very small arteries by means of pharmacologic inhibition of nitric oxide synthase. The acute inhibition of nitric oxide synthesis did not increase either the basal or myogenic tone in the arteries from the late pregnant rats. This observed lack of an increased basal EDRF influence during pregnancy is consistent with our finding that basal EDRF production does not appear to be responsible for the blunted adrenergic responsiveness during pregnancy in the mesenteric arteries of rats. 1 However, our study design does not preclude the possibility that other endothelialdependent agents (i.e., prostaglandins or endothelialderived hyperpolarizing factor) may mediate the reduced tone and myogenic response during pregnancy. In addition, EDRF could have as yet unidentified, longer-term effects on the vascular smooth muscle that may not be reversed with acute inhibition of nitrate synthase. For example, nitric oxide has recently been shown to react with membrane-bound thiol groups on the N-methyl-n-aspartate subtype of the glutamate receptor, resulting in a persistent blockage of N-methylo-aspartate responses." It also may be that EDRF modulates in vivo myogenic responses in a manner that is not apparent when it is studied in vitro. Flow-induced shear stress in vivo can cause the production and release of EDRF from the endothelium-a situation that may not be apparent under these in vitro no-flow conditions. We have observed that the stimulated release of EDRF, as opposed to basal production, is increased during pregnancy. The interaction between flow and myogenic behavior during pregnancy requires further study. Thus the data from this and previous studies continue to support the idea that pregnancy results in an alteration of the vascular smooth muscle function that contributes to the hemodynamic profile of pregnancy. It would appear that one alteration involves changes in the regulation of membrane potential. This in turn may modify calcium movement into the vascular smooth muscle. That the observed change in membrane potential is actually influencing the calcium movement in these cells under different activation states requires further direct study. In addition, the question remains as to the cause for the membrane hyperpolarization. The easiest means to achieve this change would be through an alteration in the potassium conductance, which is regulated by a variety of channels. An attractive possibility is that pregnancy induces a change in the
1516 Meyer, Brayden, and McLaughlin
activity of the adenosine 5'-triphosphate-dependent potassium channel, recently identified in the vascular smooth muscle. The adenosine 5'-triphosphate-dependent K + channel, through its dependence on adenosine 5'-triphosphate for its regulation, could provide a link between metabolism and the regulation of blood flOW. 2 3 Such a possibility provides an interesting future direction for this work. REFERENCES 1. Davidge ST, McLaughlin MK. Endogenous modulation of the blunted adrenergic response in resistance-sized mesenteric arteries from the pregnant rat. AM j OBSTET GYNECOL 1992;167:1691-8. 2. Harrison GL, Moore LG. Blunted vasoreactivity in pregnant guinea pigs is not restored by meclofenamate. AM j OBSTET GYNECOL 1989;160:258-64. 3. jansakul C, Boura ALA, King RG. Effect of endothelial cell removal on constrictor and dilator responses of aortae of pregnant rats. J Auton Pharmacol 1989;9:93-101. 4. Weiner C, Liu KZ, Thompson L, Herrig j, Chestnut D. Effect of pregnancy on endothelium and smooth muscle: their role in reduced adrenergic sensitivity. Am j Physiol 1991;261:HI275-83. 5. Crandall ME, Keve TM, McLaughlin MK. Characterization of norepinephrine sensitivity in the maternal splanchnic circulation during pregnancy. AM j OBSTET GYNECOL 1990; 162: 1296-301. 6. St-Louis j, Sicotte B. Prostaglandin- or endothelium-mediated vasodilation is not involved in the blunted responses to vasoconstrictors in pregnant rats. AM j OBSTET GYNECOL 1992;166:684-92. 7. Pallor MS. Mechanism of decreased responsiveness to angiotensin II, NE and vasopressin in pregnant rats. Am j Physiol 1984;247:HlOO-8. 8. johnsson PC. The myogenic response. In: Handbook of physiology. The cardiovascular system. Vascular smooth muscle. Bethesda, Maryland: American Physiological Society, 1980:409-42. 9. Halpern W, Osol G, Coy GS. Mechanical behavior of pressurized in vitro prearteriolar vessels determined with a video system. Ann Biomed Engineering 1984;12:463-79. 10. McLaughlin MK, Keve TM. Pregnancy-induced changes
December 1993 Am J Obstet Gynecol
11.
12. 13. 14.
15.
16. 17. 18. 19.
20. 21. 22. 23.
in resistance blood vessels. AM j OBSTET GYNECOL 1986; 155:1296-9. Nelson MT, Patlak jB, Worley jF, Standen NB. Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone. Am j Physiol 1990;259:C318. Brayden jE, Nelson MT. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science 1992;256:532-5. Weissberg PL, Little Pj, Bobik A. Spontaneous oscillations in cytoplasmic calcium concentrations in vascular smooth muscle. Am J Physiol 1989;256:C951-7. Stice SL, Ford SP, Rosazza jP, Van Orden DE. Role of 4-hydroxylated estradiol in reducing Ca 2 + uptake of uterine arterial smooth muscle cells through potential sensitive channels. Bioi Reprod 1987;36:361-8. Harder DR. Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact endothelium. Circ Res 1987;60:102-7. Katusic ZS, Shepherd jT, Vanhoutte PM. Endotheliumdependent contraction to stretch in canine basilar arteries. Am J Physiol 1987;252:H671-3. Hwa jj, Bevan jA. Stretch-dependent (myogenic) tone in rabbit ear resistance arteries. Am j Physiol 1986;250:H8795. Kuo L, Chilian WM, Davis MJ. The coronary arteriolar myogenic response is independent of the endothelium. Circ Res 1990;66:860-6. Nakayama K, Tanaka T, Fujishima K. Potentiation of stretch-induced myogenic tone of dog cerebral artery by hemolysate and the inhibitory action of calcium antagonists. Eur J Pharmacol 1990;169:33-42. Conrad KP, Vernier KA. Plasma level, urinary excretion, and metabolic production of cGMP during gestation in rats. Am J Physiol 1989;257:R847-53. MacPherson RD, McLeod L], Rasiah RL. Myogenic response of isolated pressurized rabbit ear artery is independent of endothelium. Am j Physiol 1991;260:H779-84. Lei SZ, Pan ZH, Aggarwal SK, et al. Effect of nitric oxide production in the redox modulatory site of the NMDA receptor-channel complex. Neuron 1992;8: 1087-99. Standen NB, Quayle jM, Davies NW, Brayden jE, Huang Y, Nelson MT. Hyperpolarizing vasodilators activate ATPsensitive K + channels in arterial smooth muscle. Science 1989;245: 177-80.