Effect of adrenergic blockade on dynamics of the pregnant primate uterus (Macaca mulatta)

Effect of adrenergic blockade on dynamics of the pregnant primate uterus (Macaca mulatta) CLTY :\f. HARBERT, JR., M.D. KENI\ETH R. SPISSO, M.D. Cllllrlottelvilll', Virginia Unanesthetized rhesus monkeys, conditioned to restraining chairs and monitored continuously by chronically implanted sensors, were used to explore the relationships between catecholamines and uterine dynamics during late gestation. Continuous intra-aortic infusion of phentolamine produced a dose-response decrease in uterine activity and blood flow. With alpha-adrenergic blockade the circadian (Fourier) variations of uterine activity were abolished and those of uterine blood flow were reversed. Placental blood flow (by the microsphere technique) was 86.1% of control values. Beta-adrenergic blockade (propranolol) accentuated the circadian variations of uterine dynamics. Vascular resistance to both the myometrium and the placentas was increased. The data indicate that the maternal placental vasculature is the primary site of uterine vascular resistance and is susceptible to adrenergic response. The data also suggest that the extrinsic resistance produced by the myometrium is of major importance in the distribution of uterine blood flow. (AM. J. OBSTET. GYNECOL. 139:767, 1981.)

WHEJ\" ANALYZED as a continuum, the spontaneous activity of both human' and nonhuman primate 2 myometrium has been shown to result in differences of uterine dynamics that modulate with periods of light and dark. During the last half of nonhuman primate pregnancy, the changes in intra-amniotic pressure, contraction frequency, aortic blood pressure, and uterine arterY blood How follow time courses that are repetitive, predictable, and conform to highly significant Fourier curves.'~ These circadian changes are incorporated in a distinct manner into the events characterizing labor and delivery." The biorhythms in uterine dynamics also engender alterations in distribution of blood How. 5 Myometrial blood How, measured during uterine diastole by the microsphere technique, is highest during periods of light and placental blood flow is highest during periods of darkness. Experiments in the

From the Department ojOb>tflrin and Gynecology. Univnsity of Virginia School of Medicine. SuptJorted in part by Research Grant HD-02798 from the !Vational h~1titutes of Health, United States Public Hralth

Sen•ice. Presf'nted bv invitation at the First Combined Annual

i\lli·eting o(The American Association of' Obstetricians and Gynecologist.\ and the Amerimn Gynecological Society, Hot Sfnings, V1rginia, September 3-6, 1980.

Rl'print requests: Cur J1. Harbert, Jr., M.D., Box 387, Unh•ersity of Virginia Hospital, Charlottesville, Virginia 22908. J002-9:l7B/Hl/0707fi7+ I!SOl..!0/0

©

Jq81 The C. V. 1\losbv Co.

nonpregnant monkey indicate that catecholamines and adrenergic receptors have a causal role in the occurrence of spontaneous variations and the establishment of circadian patterns of uterine activity. 6 The present study was undertaken to explore the effects of selective alpha- and beta-adrenergic receptor blockade on the dynamics of the pregnant nonhuman primate uterus (Macaca mulatta).

Material and methods Pregnant rhesus monkeys weighing between 6.2 and 8. 7 kg were selected from our primate colony. Duration of gestation ranged from 138 days to term ( 165 days). Dates of conception were known within 3 days. Most of the monkeys had been conditioned to restraining chairs during previous chronic experiments. Restrained animals that displayed marked agitation or depression were excluded. Animal preparation. Pressure and How sensors were implanted according to established techniques. 2-o The patterns of uterine activity were monitored through an open-end, Huid-filled catheter. Recordings of aortic blood pressure were obtained from an open-end catheter threaded into the aorta to a level approximately midway between the renal arteries and the aortic bifurcation. Venous pressure was obtained by cannulation of the femoral vein ipsilateral to the arterial pressure catheter. In selected animals, another arterial catheter

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Fig. I. Illustration of the effects of phentolamine(A) and propranolol (B) on the variations in uterine dynamics recorded over 2 consecutive days, respectively. Data bars and points represent average values for each of the 48 hours of continuous monitoring. The dark horizontal bars delineate the 24-hour periods of drug administration. The alternating 12-hour cycles of light and dark are represented by the clear and stippled background areas, respectively.

was threaded into the left ventricle for injection of radioactive microspheres to measure blood flow distribution and an ovarian vein was catheterized for measurement of uterine venous drainage. Direct measurement of uterine artery blood Row was achieved by use of a previously calibrated electromagnetic flow probe placed on the uterine artery contralateral to the pressure catheters. Periodic balancing of the flow probe was permitted by placement of an inflatable occlusive device around the hypogastric artery. The electronic signals from the pulsatile impulse of the blood flow probe were fed into a cardiotachometer for continuous measurement of maternal heart rate. Once the operations were completed, the animals were placed in restraining chairs. They were maintained in the dorsal lithotomy position for i 8 to 24 hours until fully recovered from anesthesia and then placed in an upright, sitting position. Three to five days was allowed for operative recovery before "chronic" measurements were undertaken . Throughout the operative recovery and experimental periods, the animals were maintained in a controlled environment with alternating 12-hour cycles of dark and constant-intensity light. Periodic flushing of the pressure catheters, balancing of the flow probe, determination of occlusion zero, and checks of recording system accuracy were the oniy special procedures necessary . All experimental variables were monitored continuously with the noosedated animal maintained in an upright, sitting posture. Phentolamine and propranolol, respectively, were

used to block seleclively the alpha- and beta-adrenergic receptors . The drugs were administered b~· continuous intra-aortic infusion in order to permit more accurate estimation of the amount of test agent reaching the target organ and to decrease adverse svstem ic effects. The establishmenL of alpha-adrenergic-receptor blockade was tested by administration of phenylephrine. Beta-adrenergic blockade was tested with isoproterenol. On-line electronic integration of the areas under the polygraph curves was used to measure total activity in physical units. 3 Mean pressure and flow were determined electronically with the use of a 1-second time constant.. Average values of mean pressure and Row were calculated by dividing total activity by a selected time interval in seconds. Uterine contraction frequency was expressed as the number of complete contraction-relaxation complexes rising at least I 0 mm Hg above baselin e diastolic pressure per minute. Evaluation of the data was further facilitated by use of statistical methods including the analysis of variance for regression and the analysis of variance for Fourier terms. 7 Dose-response relationships were judged on the basis of changes in mean values and of alte1·ations in configuration, amplitude. and time of maximal change or acrophase of the biorhythms. Blood flow distribution. Administration and analysis procedures adopted from the methodology employed by Rosenfeld and associates' using radioactive poly-

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Fig. 2. Presentation of the effect of alpha-adrenergic (A), beta-adrenergic (B), and both alpha- and beta-adrenergic (C) blockade on uterine activity and hemodynamics. 'The points depict the rr1ean of average values for corresponding hours measured over 5 consecutin· days for each experimental condition. PRU = Peripheral vascular units (mm Hg · ml .. 1 • min). With both alpha-adrenergic and combined adrenergic blockade. the circadian patterns of intra-amniotic pressure and contraction frequency were abolished. With beta blockade a statistically significant Fourier \·ariation was present for each measured function (intq-amniotic pressure: y = 2·!. 1 - ~~6.2 cos ct - 2!1.1 sin ct: contraction frequency: y = 0.56 + 0.18 cos ct - 1.52 sin ct; aortic blood pressure: \ = 12·1.·1 - 110.7 cos ct - 65.4 sin ct: uterine artery blood How: v = 2~t I - :~2.0 cos ct + 2fi.O sin ct; uterine vascular resistance: v = 4.25 + 16.98 cos ct + 6.~-12 sin ct).

styrene beads with a mean diameter of 50 ± 10 p.. were used for measurements of blood How distribution. 0 Four nuclides with a specific activity of 10 mCi/gm were used: cerium-141, niobium-95, scandium-46, and strontium-85 . A nuclide was injected between 1000 and II 00 hours on 2 consecutive days during the control period before drug administration was initiated. The third and fourth nuclides were injected during the same time fraine on 2 successive days after establishment of adrenergic blockade. The order of microsphere injection \Vas designed so that the same isotope was not given to two animals in the same experimental period. The number of microspheres infused was chosen to give essentially equal count rates and varied from approximately 50 x 10~ for scandium-46 to 25 X 10 4 for strontium-85. Arterial reference samples were withdrawn from the femoral arteries starting approximately 15 seconds prior to microsphere infusion and continued for about 30 seconds after the intraventricular catheter was flushed. Venous reference samples were obtained from either a catheter in the ovarian vein or the inferior vena cava. A sarnple ,vithdrawal

rate was 1.23 ml/min with the use of a Harvard pump. Following infusion of the last suspension of microspheres, the intact uterus was remover! under operative hemostasis and prepared for counting. The weighed tissue samples were cuun ted in four channels of a Beckman Biogamma automatic gamma counter. The sets of standards for each nuclide were counted at the same time as the samples. The percentage of crossover bct\\'een nuclides ranged fron1 2.HO x 1o-:~ bctw·een

cerium-141 and niobium-!--15 to :i 1.0 between niobium95 and scandiutn-46. -rhc true counts in all satnp!es were calculated by a computer program written to separate the effects of percentage of crossover of the nuclides with the use of the solution of four equations in four unknowns. Individual tissue flows wet·e calculated by the solution of the equation advocatt'd by Makowski and associates .f' Biochemical determinations. In selected animals, measurements of aortic and ovarian vein blood and amniotic fluid concentrations of norepmephrine and epinephrine were performed. Blood and amniotic Hui
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Table I. Effect of selective alpha- and beta-adrenergic-receptor blocking agents on the diurnal (Fourier) patterns of uterine activity and hemodynamics Intra-amniotic pressure (mm Hg) Animal No.

Drug dosage (mglmin x J0- 3)

Mean of 24-hour day

Phentolamine (alpha-adrenergic blocker): M-308 0 10.8 1. 7 (2.5*) 9.2 3.4 (5.0) 7.2 6.9 (10.0) 6.0 13.8 (:!0.0) 5.2 27.7 (40.0) 5.0 Propranolol (beta-adrenergic blocket): M-504 0 20.4 17.3 (25.0) 21.8 34.6 (50.0) 22.0 69.4 (100.0) 22.7 138.8 (200.0) 22.2 22.4 277.7 (400.0)

Iamplitude Semi-

Frequency (contractions/min) Mean of 24-hour day

Iamplitude Semi-

Uterine artery blood flow (mllmin) Mean of 24-hour day

Iamplitude Semi-

Aortic blood pressure (mm Jig) Mean of 24-hour day

Iamplitude Semi-

2.9 1.9 1.9 1.2

0.49 0.46 0.41 0.41 0.40 0.39

0.12 0.10 0.12 0.05

20.4 29.0 27.2 26.2 26.6 27.2

4.0 3.8 3.6 5.6 5.9 6.3

114.0 112.0 111.5 ll0.5 95.4 94.0

11.4 10.6 11.2 12.0 14.0 14.4

6.0 6.2 5.8 5.9 7.2 6.6

0.44 0.48 0.53 0.52 0.51 0.53

0.15 0.15 0.09 0.14 0.15 0.14

36.8 36.8 35.7 34.2 34.2 34.2

7.8 8.2 8.9 8.8 9.2 8.9

125.0 124.5 124.0 125.0 125.5 124.5

22.9 22.8 25.3 23.2 21.9 22.4

*Milligrams per 24 hours. 1500, and 2300 and 2400 hours, on successive days before medication and after establishment of alpha or beta blockade. Since heparin was used to fill the catheters between sample collection periods and some degree of anticoagulation was always present, all samples were based on plasma rather than serum concentrations. Blood samples were contrifuged; the plasma was separated and stored at -900 C, and the red cells were reinfused into the animal. Procedures for plasma determinations of norepinephrine and epinephrine were modified from the radioenzymatic method of Passon and Peuler 10 with the use of a differential catecholamine assay technique with internal standards. Duplicate assays were performed on all samples. Sensitivity of the assay per 50 J.Ll sample was 5 pg for norepinephrine and 3 pg for epinephrine. Reproducibility determined by the coefhcient of correlation was 9.6% for norepinephrine and 8.0r7c for epinephrine.

Results Evaluation of the effects of selective adrenergicreceptor blockade was based on measurements obtained from 15 monkeys over a combined total of 218 days. Individual animals were monitored continuously for periods of 12 to 23 days. Fig. 1, A, charts the hourly average values of uterine function and hemodynamics measured over 2 consecutive days in late gestation. During the first 24 hours illustrated, no medications were given. Average values of intra-amniotic pressure varied from 14.0 to 22.5 mm Hg (mean 19.1 ± 2.3 mm Hg). Characteristically, lowest pressures occurred during periods of darkness and highest pressures occurred during the period of light.

Contraction frequency ranged from 0.18 to 0.84/rnin. Aortic blood pressure also varied in a diurnal pattern, ranging from 100 to 125 mm Hg with a mean of 112.6 ± 7.6 mm Hg. Uterine artery blood flow ranged between 24.5 and 31.2 ml/min with a mean of 26.9 ± 1.7 ml/min. In general, lower values were recorded during the hours when myometrial resistance to flow, as reflected by intra-amniotic pressure, was greater. At 2400 hours of the second day illustrated, continuous infusion of phentolamine was instituted. During the following 24-hour period, hourly average values of intra-amniotic pressure varied only 3 mm Hg, from 16.5 to 19.5. Contraction frequency ranged from 0.38 to 0.63 contractions per minute and showed no consistent variation in relationship to the periods of light and dark. Average values of aortic blood pressure did not show a statistical difference from the corresponding hours measured during the control period (paired Student's t test= 0.959). There was a progressive decline in hourly average values of uterine artery blood How. Upon discontinuation of phentolamine infusion, the diurnal patterns returned. The effect of continuous propranolol infusion in the same monkey is presented in Fig. 1, B. During the 24 hours of drug administration, hourly average values of intra-amniotic pressure varied in a circadian pattern between 17.5 and 26.6 mm Hg. The mean (22.4 ± 2.3 mm Hg) was significantly higher than the mean for the preceding day (18.9 ± 2.4 mm Hg, t = 6.662). Contraction frequency continued to show a diurnal pattern that did not differ significantly from control values of corresponding hours (t 1.931 ). Aortic blood pres-

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Fig. 3. Composite linear dose-response curves showing the effects of phentolamine (A) and propranolol (B). Each point represents the mean± SD for the 24-hour period . The sigmoidal lines represent the semiamplitudes of the Fourier variations. Mean values that differ significantly from the nonmedicated control measurements are marked by asterisks. The numbers enclosed within boxes are the days of monitoring upon which mean values are based. PRU = Peripheral resistance units (mm Hg · ml- ' · min) .

sure likewise did not show significant differences in hourly average variations from the control values (t = 2.022, p > 0.10). Uterine artery blood flow ranged from 27.0 to 33.0 ml/min and continued to show a diurnal variation . A composite of the hourly average values of uterine dynamics and calculated uterine vascular resistance meas ured over 5 consecutive da ys each of selective and combined adrenergic blockade in one monkey is presented in Fig. 2. The analysis of variance for Fourier terms showed that there was significant variability caused by differences between days of measurement for all tested functions under all three conditions. Under alpha blockad e (A), there was not a statistically significant Fourier variability caused by differences in ho urly average values of intra-amniotic pressure (F2 . 76 = 3.05) and contraction frequency (F 2 , 76 = 1.99) between the hours of the da y. Hourly average blood pressure values did conform to a highly significant sine curve with a mean of 114.0 mm Hg that reached a maximal variation of 8.0 mm Hg above and below the 24-hour mean (y = 114.0 + 16.1 cos ct- 82.6 sin ct. wh ere "ct" equals time in radians). The variations of hourly average values of uterine artery blood flow were statistically significant despite minimal changes which

represented only a 3.4% variation above and below the mean for the day (y = 25.9 + 0.7 cos ct- 10.5 sin ct). Uterine vascular resistance, calculated as the quotient of aortic blood pressure - intra-amniotic pressure + uterine artery blood flow, conformed to the curve y = 3.66 - 0. 70 cos ct - 1.97 sin ct. Calculation of the linear correlation coefficient by the method of least squares indicated that a statistically significant positive relationship existed between the hourly average values of aortic blood pressure and those of ute rine blood flow (r = 0.429). The correlation coefficients obtained by comparison of intra-amniotic pressure with aortic blood pressure (r = 0.372) and uterine artery blood flow (r = 0.258) were not significant (p > 0.10). Under beta blockade (Fig. 2,B), the hourly variations of all factors of uterine dynamics corresponded to highl y significant Fourier curves. The variations in uterine artery blood flow were inversely correlated with both ao rtic blood pressure (r = -0.908) and intraamniotic pressure (r = -0.838) . Both alpha blockade and beta blockade were produced by simultaneous administration of phentolamine and propranolol (Fig. 2, C). A statistically significant Fourier component of the hour-to-hour vanauon in average intra-amniotic pressure was abolished

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Table II. Values (mean ± SEM) of plasma and amniotic fluid catecholamine concentrations and of uterine dynamics obtained from three monkeys under nonmedicated and selective adrenergic blockade conditions. Values of uterine dynamics were calculated from data recorded for 10 minutes before and after sample collection Norepinephrine (nglml) Period of collection (hours)

Epinephrine (nglml)

Ovarian vein

Amniotic fluid

Aorta

Ovarian vein

A"miotic fluid

0.322 ± 0.029 0.479 ± 0.032 0.569 ± 0.040

0.045 ± 0.005 0.045 :t 0.006 0.068 ± O.Oll

0.303 ± 0.026 0.436 ± 0.047 0.292 ± 0.028

0.492 ± 0.033 0.567 ± 0.053 0.779 ± 0.035

0.053 :t 0.001 0.055 ± 0.003 0.065 0.008

Alpha-adrenergic -receptor blockade (N 5): 0.498 ± 0.056 0.523 :!: 0.062 0700-0800 0.565 :t 0.059 0.576 ± 0.061 1400-1500 0.532 ± 0.052 0.529 :t 0.046 2300-2400

0.041 ± 0.007 0.040 ± 0.006 0.056 ± 0.008

0.351 ± 0.050 0.301 ± 0.052 0.333 ± 0.062

0.426 ± 0.041 0.411 ± 0.062 0.494 ± 0.066

0.054 :t 0.003 0.056 ± 0.005 0.064 ± 0.008

Beta-adrenergic-receptor blockade (N 0.439 ± 0.021 0700-0800 0.582 :t 0.033 1400-1500 0.462 :t 0.046 2300-2400

0.051 ± 0.010 0.053 ± 0.010 0.063 ± 0.011

0.360 ± 0.043 0.488 ± 0.053 0.214 ± 0.049

0.381 ± 0.038 0.498 ± 0.048 0.202 ± 0.052

0.046 ± 0.006 0.050 ± 0.004 0.054 ± 0.006

Aorta

Nonmedicated control (N = 6): 0.506 :!: 0.022 0700-0800 0.666 :!: 0.039 1400-1500 0.565 :t 0.040 2300-2400

4):

0.343 ± 0.031 0.4 71 ± 0.044 0.376 :t 0.038

(F 2 , 16 2.98) while contraction frequency conformed to a significant two-term Fourier curve (y = 0.34 + 0.12 cos ct ~ 0.13 sin ct + 0. II cos ct ~ 0.11 sin ct). Aortic blood pressure values continued to follow a diurnal pattern that did not differ statistically from the values recorded under either alpha or beta blockade. Changes in uterine artery blood flow displayed a harmonic pattern (y 29.0 + 14.9 cos ct + 48.7 sin ct + 1.3 cos ct + 4.0 sin ct) and were linearly correlated with the hourly average variations in aortic blood pressure (r = 0. 736). The calculated values of uterine vascular resistance for corresponding hours did not conform to a Fourier curve (F 2 • 76 = 1.46) and were consistently less than the values observed during either alpha or beta blockade. Table I collates representative responses of uterine dynamics to various dosages of phentolamine and propranolol. After a control period of 2 to 3 days when no drug was given, each dosage of the adrenergic blocker was infused for 2 consecutive days. With phentolamine administration the configuration of the contractile complexes, in general, was unchanged from preinfusion patterns while contraction frequency, amplitude, and baseline pressure were decreased in relation to drug dosage. The diurnal pattern of hourly variation in intra-amniotic pressure was usually abolished at infusion rates of 0.0138 mg/min (20 mg/24 hr). Some animals continued to exhibit a diurnal variation in contraction frequency at this infusion rate. Simultaneous infusion of phenylephrine at a rate of 0.5 mg/min increased the frequency and amplitude of contractile complexes and elevated the resting baseline pressure over 10 mm Hg, indicating incomplete alpha blockade.

Increasing the dosage of phentolamine to 0.0277 mg/min (40 mg/24 hr) abolished the diurnal patterns of contraction frequency in all animals. Beta blockade was usually accomplished at a propranolol infusion rate of 0.0694 mg/min ( 100 rng/24 hr). One monkey required the infusion of propranolol at a rate of 0.1388 mg/min (200 mg/24 hr) before a response to isoproterenol was abolished. With complete beta blockade, baseline pressure and amplitude of contractions increased. The 24-hour mean values of uterine artery blood flow decreased and the semiamplitudes of the diurnal components increased with increasing dosage. Aortic blood pressure values remained unchanged or increased slightly from preinfusion levels. The dose-response relationships of uterine dynamics to the infusion of phentolamine or propranolol are displayed graphically in Fig. 3, A and B, respectively. Even before alpha blockade was established at a phentolamine infusion rate of 0.0138 mg/min (20 mg/24 hr) or more, decreases noted in the 24-hour mean values for intra-amniotic pressure (41.2%), contraction frequency (9.4%), and aortic blood pressure (7.2%) were statistically significant compared to preinfusion mean values. A statistical increase ( 10.3%) in uterine vascular resistance occurred at a drug infusion rate of 0.0069 mg/min (10 mg/24 hr). With alpha blockade, uterine artery blood flow was 15.3% below the preinfusion mean flow rate. The semiamplitude of the Fourier variations for neither aortic blood pressure nor uterine vascular resistance varied statistically from control days at any drug dosage. The Fourier pattern of uterine artery blood flow persisted but became positively corre-

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Uterine dynamics Intra-amniotic

pressure (mm Hg)

Uterine artery blood flow (ml/min)

Aortic blood

pressure (mm Hg)

19.1 ± 0.9

28.3 ± 0.2

23.1 ± 0.6

24.5 ± 0.7

17.4±0.4

25.8 ± 0.4

ll7.3 ± 1.4 123.0± 1.0 II 1.6 ± 2 5

17.:~ ± 1.2 17.0 ± 1.4 17.4 ± 1.4

28.1 ± 1.2 27.1±0.4 25.6 ± 0.8

ll8.0 ± 2.5 126.4 ± 1.2 115.2 ± 1.9

22.2 ± .05 27.2 ± 1.3 21.9 ± 0.6

27.6 ± 1.3 23.5 ± 1.9 29.3 ± 2.0

115.0:!: 3.7 128.5 ± 3.9 117.0 ± 1.1

0.607) with the aortic blood pressure varilated (• ations. Although beta blockade was not produced until the propranolol infusion rate reached at least 0.0694 mg/min (100 mg/24 hours), ;.~ significant increase (5.7%) in mean contraction frequency was noted at an infusion rate of 0.0173 mg/min (25 mg/24 hr). Statistically significant changes in 24-hour mean values of intra-amniotic pressure (6.2% increase) and uterine artery blood flow (5. 7% decrease) were recorded at a propranolol infusion rate of 0.0346 mg/min (50 mg/24 hr). At a dosage rate of 0.1388 mg/min (200 mg/24 hr), there was a statistical increase in aortic blood pressure (4.:3%) and a decrease in maternal heart rate from !69.3 ± 37.5 to 134.6 ± 34.0 beats/min. A significant increase ( 10.3%) in merine vascular resistance was noted prior to beta blockade. However, perfusion pressure (aortic blood pressure - intra-amniotic pressure) did not differ statistically from nonmedicated control values ar. any infusion rate for either propranolol or phentolamine. Measurements of plasma and amniotic fluid concentrations of norepinephrine and epinephrine were made in three monkeys during non medicated control periods and after alpha or beta blockade (Table II). Under non medicated conditions, intravascular concentrations of both nom-epinephrine and epinephrine were significantly higher in samples collected between 1400 and 1500 hours than in those collected between 0700 and 0800 hours. In general, the ovarian vein concentrations of norepinephrine were significantiy iower and rhose of epinephrine were higher than the aortic blood concentrations. Intra-amniotic pressure \vas positively corre-

lated with the concentration of norepinephrine in the aorta (r == 0. 708) and negatively correlated with the concentration of epinephrine in rhe ovarian vein (r == -0.551 ). None of the vascular neurohormone concentrations ;.vas statistically correlated \Vith either uterine artery blood How or aortic blood pressure. Under conditions of alpha blockade, there were no statistically significant differences of neurohormone concentrations between the three collection periods or between aortic and ovarian vein samples. However, the ovarian vein samplings of epinephrine remained significantly correlated with intra-amniotic fluid pressure changes (r = 0. 753). With blockade of beta receptors, concentrations of norepinephrine were significantly less than samplings obtained from the same animal during nonmedicated control periods (t = 5.414) but again displayed statistically higher values between 1400 and 1500 hours. The norepinephrine concentration in aortic blood was correlated posiriveiy with imra-amnioric pressure changes (r = 0. 90 I) and inversely with uterine artery blood flow (r = -0.636). Aortic and ovarian vein concentrations of epinephrine were neither statistically different nor correlated with any measurements of uterine dynamics. Amniotic fluid concentrations of norepinephrine and epinephrine did not vary significantly between either the various collection periods or the three sampling conditions and were not significantly correlated with any of the measurements of uterine dynamics. The distribution of blood flows to placental and nonplacental uterine tissues was measured in four animals under alpha blockade and in three monkeys after establishment of beta blockade (Table III). The eight measurements made during alpha blockade were compared to measurements made during the control penoa. !Vlean values sraristicaiiy less during alpha blockade were: total uterine blood flow (11.3%), placental blood flow (13.8%), cervical blood flow (47.7%), intra-amniotic pressure (34.3%), and aortic blood pressure (3.7%). Despite a statistical difference in total uterine blood flow, the percentage distribution to the placental and endometrial tissues did not differ significantly between alpha blockade and control measurements. Proportionate blood flow to the myometrium was increased 19.7%. Vascular resistance to the uterus and placentas was calculated by the formula: aortic blood pressure intra-amniotic pressure + blood flow: that to other uterine tissues was determined by the formula: aortic blood pressure+ blood How. On the average, total uterine vascular resistance was increased 13.9% and piacentai vascular resistance was increased 15.5%. Calculation of vascular resistance by multiplying proportionate individual tissue fi
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Table III. Values (mean ± SEM) of uterine hemodynamics calculated from the microsphere data measured during relaxation on nonmedicated control days and on days with adrenergic blockade Actual flow (mllmin) Variable

I

Control

Percent flow

Drug

Control

I

Vascular resistance (PRU)

Drug

Control

T

Drug

Alpha-adrenergic-receptor blockade (N = 8)

Uterine blood flow Cervix Myometrium Endometrium Placentas Intra-amniotic pressure (mm Hg) Aortic blood pressure (mm Hg)

92.6 4.4 13.6 2.4 72.2 12.8

± ± ± ± ± ±

0.8 0.9 0.7 0.3 l.1 0.4

119.0 ± 2.3

81.2* 2.3* 14.5 2.2 62.2* 8.4*

± 2.0 ± 0.4 ± l.5 ± 0.3 ± 0.9 ± 0.9

1.1

2.9* 17 .6* 2.8 76.7

± ± ± ±

0.6 l.3 0.4 0.9

1.15 38.07 8.90 55.40 1.48

± ± ± ± ±

0.04 9.04 0.52 6.69 0.06

0.4 0.6 0.3 0.8

4.8* 13.2* 2.8 79.2

± ± ± ±

0.3 0.6 0.2 0.9

l.04 29.83 8.60 44.84 l.32

± ± ± ± ±

0.06 3.78 0.51 4.90 0.06

4.8 14.7 2.6 77.9

± ± ± ±

l.O 0.6 0.3

4.4 14.6 2.9 78.1

± ± ± ±

1.31*±0.05 60.67 ± 8.69 8.58 ± 0.98 ~)6.52

± 3.5._j-

1.71 ± 0.05

114.6* ± 2.0

Beta-adrenergic-receptor blockade (N = 6):

Uterine blood flow Cervix Myometrium Endometrium Placentas Intraamniotic pressure (mm Hg) Aortic blood pressure (mm Hg)

94.5 4.2 13.8 2.7 73.8 18.9

± ± ± ± ± ±

2.1 0.4 0.8 0.3 l.3 2.1

116.2 ± 3.0

*p < 0.05 (paired Student's t test); PRU

=

84.7* 4.1 ll.2* 2.4 67.0* 22.0*

± ± ± ± ± ±

2.7 0.4 0.6 0.2 2.2 l.9

1.16* 30.43 10.91* 50.95 l.47*

± ± ± ± ±

0.06 3.16 0.65 2.95 0.08

120.2 ± 2.1 mm Hg · ml- 1

·

min.

uterine artery blood flow and evaluation of the data based on flow per gram of tissue did not alter the statistical relationships. Beta blockade resulted in a reduction of blood flow to the myometrium (18.8%) and placentas (8.5%). The percentage of uterine blood How distributed to the cervix w·as significantly increased (9.0%) 'vhile that going to the myometrium was decreased (9.5%). Total uterine vascular resistance was increased 11.5%. Resistance of blood flow to the myometrium was increased 26.9% and that to the placentas was increased 11.4% without a statistically significant change in resistance to flow for either the cervix or the endometrium. The hourly average values of uterine dynamics measured over 48 hours in pregnancies terminating in spontaneous labor and delivery in two monkeys receiving continuous drug infusions are illustrated in Fig. 4. With alpha blockade (Aj, the hourly average values of intra-amniotic pressure ranged from 9.4 to 42.0 mm Hg. Except for the period of active labor, the values were consistently below the 2.5% confidence level calculated from recordings made in 15 nonmedicated monkcys. 4 In general, the hourly average values of contraction frequency were below the mean for corresponding hours recorded in non medicated animals but within the 95% confidence interval. The analysis of variance for regression indicated that aortic blood pressure values did not differ significantly from those of the nonmedicated monkeys. The majority of the hourly average values of uterine artery blood How were

below the 2.5% confidence level for nonmedicated animals and did not show a rhythmic variation. During the final hour oflabor, the average How rate decreased to 52.9% of the mean value for the 48-hour period (24.6 ± 4.1 ml/min). Under beta blockade (Fig. 4, B), intra-amniotic pressure values were above the 97.5% confidence level in many instances but continued to follow an overall pattern similar to that observed in nonmedicated animals. The hourly average values of contraction frequency and aortic blood pressure did not vary appreciably from values in the nonmedicated monkeys. In general, uterine artery blood flow values were at or below the 2.5% confidence level with a progressive decrease over the last 20 to 24 hours of pregnancy, reaching 66.5% of the mean value for the 48-hour period (24.8 ± 3.2 ml/min). Uterine vascular resistance was significantly lower in the animals with beta biockade than in those receiving phentolamine (t = 7. 164). The linear correlations calculated by comparison of intra~amniotic pressure and aortic blood pressure with uterine artery blood How were still highly significant and inversely related under both alpha and beta blockade. Two other monkeys were observed throughout spontaneous labor and delivery while receiving adrenergic blocking agents. One animal was infused with phentolamine at a rate of 0.0277 mg/min (40 mg/24 hr) and the other received propranolol at 0.2777 mg/min (400 mg/24 hr). Both animals displayed patterns in uterine dynamics comparable for the respec-

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T

775

(HOU

Fig. 4. Graphic presentation of the vanauons in hourly average values of uterine actlVIty and hemodynamics measured over 48 hours in two monkeys during spontaneous labor and delivery. The shaded background areas represent the 95% confidence intervals determined for individudal mean values of each function measured in 15 nonmedicated monkeys. PRU = Peripheral resistance units (mm Hg · ml - ' · min) calculated by the formula: aortic blood pressure - intra-amniotic pressure+ uterine artery blood flow. Monkey-401 (A) delivered under conditions of alpha-adrenergic blockade. Monkey-706 (B) delivered under conditions of beta-adrenergic blockade.

tive drugs to those depicted in Fig. 4, suggesting that neither norepinephrine nor epinephrine is the primary factor in the initiation and progression of labor. Comment

The present data indicate involvement of both adrenergic neuroreceptors and catecholamines in the occurrence of hour-to-hour variations and circadian (Fourier) patterns of spontaneous activity of the nonhuman primate uterus during late pregnancy. Administration of an alpha-adrenergic antagonist, phentolamine, decreased contraction frequency, amplitude, and baseline pressure with a resultant reduction in average intra-amniotic pressure values. These responses are similar to those reported for the human uterus" and consistent with inhibition of the stimulatory effect of norepinephrine on the myometrium. Administration of the beta blocker propranolol increased contraction frequency, amplitude, and baseline pressure; the changes were comparable to other values observed in rhe pregnant human .'' Removal of the inhibitory effect of epinephrine would permit an increased myometrial response to norepinephrine. Whether these alterations are direct results of the infused agents or reflect selec-

tive blockade and the influence of circulating catecholamines is not specifically delineated. Support of the influence of circulating catecholamines is evidenced by the observation that aortic concentrations of norepinephrine are higher during periods of increased uterine activity (Table II). The observation in both nonmedicated monkeys and those under beta blockade that ovarian vein concentrations of norepinephrine are statistically lower than those in arterial blood is consistent with the uptake of norepinephrine by tissues and receptors within the uterus . The higher concentrations of epinephrine in the ovarian vein than in arterial blood, together with the significant negative linear correlations between ovarian vein epinephrine levels and intra-amniotic pressure , suggest either manufacture of this neurohormone or its release from within the uterus in association with changes of myometrial activity. With the administration of either phentolamine or propranolol, the alterations in uterine dynamics became statistically significantly different at dosages below those which produced complete neuroreceptor blockade. Although the changes in uterine dynamics are dose dependent (Table I, Fig. 3). the infusion rates

776

Harbert and Spisso

.-\ptil I, Am.

needed to produce blockade are considerably higher than those required in the nonpregnant monkey. 6 Catecholamine sensitivity has been shown to be related to progesterone versus estrogen dominance 12 and plasma concentrations of progesterone and estrogen are known to increase during pregnancy in the rhesus monkey. 1' 3 In addition to their effects on myometrial activity, adrenergic receptors and catecholamines appear to have distinct effects on uterine vasculature and blood flow distribution. While proportionate decreases in 24-hour mean values of aortic blood (9%) and intraamniotic (54%) pressures were considerably different, the actual pressure differentials between nonmedicated and alpha-blockade conditions were, in general, less disparate (Tables I and III). As a result, perfusion pressure (aortic blood pressure - intra-amniotic pressure) remained unchanged. The reduction in blood pressure may reflect vasodilatation in systemic vessels below the level of the infusion catheter or return of the drug to the general circulation with resultant systemic vasodilatation. However, significant changes in maternal heart rate were not detected. An alternative possibility is that the reduction in myometrial pressure engendered a decrease in extrinsic vascular resistance that was reflected in the systemic blood pressure. This is supported by the observation that under both alpha-adrenergic (Figs. 2, A, and 3, A) and combined adrenergic (Fig. 2, C) blockade, uterine artery blood flow became positively correlated with the Fourier pattern of aortic blood pressure. In each circumstance, the head of pressure in the arterial system appeared to be a principal factor in the circadian patterns of blood flow to the uterus. According to the formula for total uterine vascular resistance (Ri [intrinsic resistance] + Re [extrinsic resistance] = aortic blood pressure - intra-amniotic pressure 7 uterine blood flow), the increase in total resistance with decreased intra-amniotic pressure noted with alpha blockade would suggest the occurrence of an even greater increase in intrinsic resistance. If, however, intra-amniotic pressure is considered to be equivalent to extrinsic resistance, the presence of a constant perfusion pressure would result in a reduction of blood flow in association with a decrease of both aortic blood and intra-amniotic pressures. The magnitude of the increase in total uterine vascular resistance could be tempered by an actual decrease in intrinsic resistance secondary to blocking the vasoconstrictive action of alpha-adrenergic neurohormones. Under conditions of beta blockade, the increased pressures (Fig. 3, B) may reflect unopposed direct alpha-adrenergic responses. Another factor which could influence the rise in aortic blood pressure is the increase in extrinsic resis-

J

!~HI

Ohsln. (;mcml.

tance engendered by the elevation in intra-amniotic pressure. The absolute values of the increase ill the two pressures \vere similar. Additiunal suppori {an be derived from the parallel increases noted in intraamniotic pressure and total uterine vascular resistance. At a propranolol infusion rate of 0.0694 mg/min. the mean intra-amniotic pressure was II 0.6'7< and uterine vascular resistance was ll ().4w to the myometrium and the placentas. Since the proportionate decrease in myometrial blood flow ( 18.Wlc) approximated the increase observed in the intra-amniotic pressure values ( 16.4'/c ), the 26.8% increase in resistance suggests that the myometrial vasculature is capable of responding to the vasoconstrictor effects of norepinephrine. The increased resistance and decreased flm,· to the placentas in response to both alpha and beta blockade indicate that these supplying vessels are susceptible to factors in addition to extrinsic rnyornetrial pressure and from the major site of resistance to total uterine blood flow. Since blood How to the placentas must pass through the myometrium and the endometrium, either the site of resistance is not near the mvoendometrial junction 14 or, as in other species, the vessels underlying the placentas react differently from those in other portions of the uterus. 13 · 16 The variations in response may be related to differences in steroid hormones and adrenergic receptor concentrations at placental and nonplacental sites and to involvement of other vasoactive substances such as prostaglandins and prostacyclin. \Vhen one considers that both the human and the

Effect of adrenergic blockade on uterine dynamics

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nonhuman primate uterus contract throughout pregnancy, the role of extrinsic myometrial resistance and constant perfusion pressure in the presence of adrenergic blockade may have profound homeostatic significance. This also may be of physiologic importance in comparative studies between species. Extrapo-

777

lation to pregnancy in higher primates raises certain clinical implications. Among these are the use of adrenergic agents in pregnancies complicated by hypertension, in treatment of premature labor, and in conjunction with obstetric anesthesia.

REFERENCES !. Harbert, G. M.: Diurnal patterns in uterine dynamics, In

Comline, K. S., Cross, K. W., Dawes, G. S., and Nathanielsz, P. W.: Foetal and Neonatal Physiology, Proceedings, Sir Joseph Barcroft Centenary Symposium, Cambridge, July 24-27, 1972; Cambridge, 1973, University Press, p. 4/:1. """

2. Harbert, G. M., Cornell, G. W., and Thornton, W. N.: Diurnal variation of spontaneous uterine activity in nonpregnant primates (Macaca mulatta), Science 170:82, 1970. 3. Harbert, G. M.: Biorhythms of the pregnant uterus (Macaca mulatta), AM. J. 0BSTET. GYNECOL. 129:401, 1977. 4. Harbert, G. M., and Spisso, K. R.: Biorhythms of the primate uterus (Macaca mulatta) during labor and delivery, AM. J. 0BSTET. GYNECOL. 138:686, 1980. 5. Harbert, G. M., Croft, B. Y., and Spisso, K. R.: Effects of biorhythms on blood flow distribution in the pregnant uterus (Macaca mu{atta), AM. j. 0BSTET. GYNECOL. 135: 828, 1979. 6. Harbert, G. M., and Zuspan, F. P.: Relationship between catecholamines and the periodicity of spontaneous uterine activity in a nonpre!!nant primate (Macaca mulatta), AM. J. 0BSTET. GYNECOL 129:51, 1977.' . 7. Bliss, G. I.: Statistics in Biology, New York, 1970, vol. 2, McGraw-Hill Book Company, Inc. 8. Rosenfeld, C. R., Killam, A. P., Battaglia, F. C., Makowski, E. L., and Meschia, G.: Effect of estradiol-17-beta on the magnitude and distribution of uterine blood flow in nonpregnant, oophorectomized ewes, Pediatr. Res. 7:139, 1973.

Discussion DR. NICHOLAS S.

Los Angeles, California. Dr. Harbert and his associates are to be coni1Tatulated for their attempt to devise a chronically instrumented experimental model for the study of some physiologic aspects of the pregnant uterus in primates. Frankly, I had been under the impression that the monkey could not be restrained sufficiently to obtain stable baseline cardiovascular functions for a prolonged time, but after reading this article, I think I have to change my mind. . ASSALI,

If I understand it correctly, the main objective of these extremely complex studies is to investigate the contributions of the adrenergic limb of the autonomic nervous system to the dynamics of the pregnant uterus during the latter part of gestation. Specifically, the work is centered around the role of the alpha- and beta-adrenergic-receptor systems in the generation of the diurnal variations of "circadian rhythm" in myometrial activity as well as in the uteroplacental circulation.

9. Makowski, E. L., Meschia, G., Droegemueller, W., and Battaglia, F. C.: Measurement of umbilical arterial blood flow to the sheep placenta and fetus in utero, Circ. Res. 23:623, 1968. . 10. Passon, P. G., and Peuler, J. D.: A simplified radiometric assay for plasma norepinephrine and epinephrine, Anal. Biochem. 51:618, 1973. II. Wansbrough, H., Nakanishi, H., and Wood, C.: The effect of adrenergic receptor blocking drugs on the human uterus,J. Obstet. Gynaecol. Br. Commonw. 75:189, 1968. 12. Miller, M.D., and Marshall, J. M.: Uterine response to nerve stimulation; relation to hormonal status and catecholamines, Am.]. Physiol. 209:859, 1965. 13. Challis, J. R. G., Davies, I. J., Benirschke, K., Hendricks, A. G., and Ryan, K. J.: The concentrations of progesterone, estrone and estradiol-17{3 in the peripheral plasma of the rhesus monkey during the final third of gestation and after the induction of abortion with PGF2 , Endocrinology 95:547, 1974. 14. Moll, W., Wallenburg, H. C. S., Kastendieck, E., and VasJar, M.: The flow re~istance of the spiral artery and the related intervillous space in the rhesus monkey placenta, Pflue!!ers Arch 377:255. 1978. 15. Rose;feld, C. R., Barton, M.D., and Meschia, G.: Effects of epinephrine on distribution of blood flow in the pregnant ewe, AM.J. 0BSTET. GYNECOL. 124:156, 1976. 16. Anderson, S. G., Still,]. G., and Greiss, F. C.: Differential reactivity of the gravid uterine vasculatures: effects of norepinephrine, i~:~.M:. j. 0BSTET. GYNECOL. 129:292, 1977.

Because of time limitation, I shall limit my discussion to some aspects of the experimental design, of methodology and data interpretation. The main background of these studies was the observation made by the authors in the nonpregnant monkey that alpha-adrenergic blockade reduced the magnitude and frequency of uterine contractions \vhereas beta blockade produced the opposite. Here again, I must confess that I did not know that the nonpregnant uterus of the monkey produces, in the resting state, rhythmic contractions of s~ch intensity as to allm:: their accurate and reproducible recording by presently available techniques. At any rate, these observations led the authors to conclude that adrenergic receptors and their respective transmitters, norepinephrine, play a causal role in the circadian patterns of uterine activity. When we talk about the influences of the autonomic nervous system on a contractile organ such as the uterus, we should remember that autonomic receptors, whether alpha or beta adrenergic, may be present not

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only around the myometrial ceiis but also in the waiis of the vascular bed supplying that organ. These receptors are also present in a larger or smaller degree in different parts of the regional and central cardiovascular system. Therefore, when one tries to manipulate, by pharmacologic means, the autonomic receptors of the uterus, the net results of this manipulation may depend not only on its effects on the myometrium itself and its blood supply but also on the algebraic sum of all regional and central hemodynamic changes. I am sure the authors had in mind all these complex factors, which are extremely difficult to separate, when they designed the experimental protocol. In addition to intra-amniotic pressure, which reflects roughly the behavior of the contracting uterine element, they monitored the perfusing pressure and uterine blood How. With these three parameters. they proceeded to estimate the vascular resistances in resting control periods of light and darkness and again during alphaand beta-adrenergic blockades. In order to minimize the "adverse systemic effects which could cause rapid deterioration of the experimental preparation" and to "permit more accurate estimation of the amount of test agents reaching the target organ", the authors decided to administer the drugs by continuous intra-aortic infusion. In my opinion, this reasoning is extremely naive for two reasons: First, any amount of drug deposited into the aorta, even as a bolus injection, will reach the systemic circulation within 20 seconds. When the agent is given by continuous infusion for days, it is illogical to consider the resulting action as being caused by intraarterial and not systemic administration. Second, as far as I know, there is no conclusive evidence to show that adrenergic-blocking agents when instilled into the aorta have enough time to diffuse through the intima and reach the receptor regions in a sufficient dose to exert a blocking action. Therefore, for all practical and scientific purposes, the results presented in this paper should be considered as being caused by systemic administration of phentolamine and propranolol. Another problem in the experimental design and methodology is concerned with what the authors call a dose-response relationship. In pharmacologic research, dose-response curves to any vasoactive agent are usually constructed by: ( 1) selecting the most specific action of the agent and observing the changes in the magnitude of that action produced hy an accurately determined dose and (2) having absolute certainty that the effects of the first dose have vanished before testing another dose. The experimental design of this work fulfills neither of these requirements. The response to the agent is taken as the conglomerate changes in several interrelated parameters over a long period of time and the drug was given continuously in such a way as to produce cumulative effects. Probably because of the confused understanding of the meaning of dose-response relationship, the graphically presented data in Fig. 2 are supposed to illustrate

April I. 191<1 Am. j, Obstct. Cvneml.

the proportionality between the dose and the changes in the measured parameters. However, careful analysis of the figures shows that such a proportionality does not exist. For instance, doubling the rate of infusion of phentolamine (from 10 to 20 mg/24 hours) did not change significantly the frequency of uterine contractions, the uterine blood How, or the intra-amniotic pressure; during the infusion nf twice that dose (front 20 to 40 mg/24 hours), the curves of these parameters were totally flat, indicating no increased responses. The same thing is seen with propranolol. This is what, in fact, one expects to see with receptor blockers; once the blockade is complete, no more effect is seen with Illcreasing doses. Now I would like to comment briefly on some of the measuring techniques and the results obtained. The authors monitored the uterine blood How with an electromagnetic flowmeter that required frequent blood vessel occlusions to correct for baseline shift. judging by the magnitude of blood How depicted in Fig. I and listed in the table, the blood vessel must have been relatively small (between 1 and 2 mm). With this low flow and this type of A. ow meter, the inherent cnor in the electromagnetic method may exceed 20%, depending on the conditions surrounding the transducer, yet the authors give significance to changes between consecutive measurements which range hom 2% to 5%. In my experience, no instrument currently available can measure How with such a degree of accuracy, and no amount of statistical manipulation, no matter how sophisticated it may be, can improve the significance of the measurements when the error IS inherent in the method itself. Perhaps these factors account for the misinterpretation of the uterine How data obtained with phentolamine and illustrated in Fig. 1. The authors state that the figures on blood flow show cyclic variations between dark and light. However, if one analyzes the data carefully, it is easy to sec: (I) that the hlood How was decreasing progressively throughout the entire period of the experiment, whether it was day or night, and (2) that the slope of the descending curve during the control period was not different from that observed during phentolamine infusion. All of these factors indicate clearly the absence of stability of the experimental preparation with respect to the flow-measuring technique. Another part of the data which bothers me somewhat pertains to the values of intra-amniotic pressure recorded in the resting state and without any drug. Some of the pressure values exceed 20 mm Hg and this must certainly be disturbing for the fetal circulation for the following reason: If we assume that the mean aortic pressure of the unstressed fetal monkey is close to 50 mm Hg (similar to that of the fetal lamb) and that the mean umbilical vein pressure is about 15 mm Hg, this leaves a pressure gradient between the umbilical artery and vein of about 35 mm Hg. If we deduct from this gradient an intra-amniotic pressure of :!0 to 23 mm

Effect of adrenergic blockade on uterine dynamics

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sponses must be similar within the mammalian species. Our attention must be focused on the resistance factors pertinent to each vascular bed under consider;nion. During a myometrial contraction, extrinsic ,·ascular resistance (Re) increases. In the myometrial vasculature, this is counterbalanced by a reduction in intrinsic resistance (R;) secondary to metabolic activity so that the net resistive change is nil and blood Hovv to the myometrium changes little. Similar autoregulatory responses in R; have not been demonstrated in the placental vasculature so that the net change would be a positive one and placental blood flow would decrease. During myometrial relaxation from normal levels, reverse changes might be expected but these have not been evaluated experimentally. Adrenergic antagonists may have both a direct and an indirect effect upon vascular smooth muscle (Ri). We have observed a direct dose-related increase in R; during adrenergic blockade with phenoxybenzamine but no significant effects during beta-adrenergic blockade. Indirect effects of adrenergic blockade on a given vascular bed 'vill be dependent upon normal chronic levels of alpha stimulation. For example, the myometrial vasculature has relatively high levels of alphaadrenergic tonus. Therefore, alpha-adrenergic blockade would be expected to cause a significant decrease in Ri. Aiternately, the placental vasculature is widely dilated with little resting alpha-adrenergic tonus. Therefore, little change in R, would be expected during alpha-adrenergic blockade. Finally, it has been shown that the myometrial vasculature is much more sensitive to alpha-adrenergic stimulation than the placental vessels. Therefore, the effects of alpha agonists or antagonists lNould be expected to be magnified in the more sensitive bed. Predicted responses from these considerations to adrenergic blockade in the rhesus macaque are shown in Table I and correlated with those observed by Dr. Harbert and associates. The similarity is obvious and confirms, I believe, the validity of comparative physiology, at least in this particular area. Dr. I Iarbert probably said it best when he sent me his manuscript many months ago. In the accompanying letter he said, "'It looks as though the monkey and the sheep are not so different after all." I have but one question. It is my understanding that epinephrine is produced primarily by chromaffin tissues of the adrenal medulla and para-aortic tissues ·with

Hg. we would have a net perfusing pressure of 10 to 15 mm Hg. I do not know how can a fetus survive and gro\'\T \vith such a !o\v pressure. I am not challenging the hypothesis made by the authors that the autonomic nervous system plays a significant role in the homeostatic tuning of our physiologic functions during our daily life. I am almost certain that the background for their hypothesis is sound and the concepts are legitimate, and I am indeed very sorry if I appeared to be too critical in analyzing this paper. My intention in raising so many questions regarding experimental design. methodology, and data interpretation is to alert investigators in this difficult and complex biological field to some pitfalls of which a person can become avvare only through years of experience and through many trials and errors. I sincerely hope that Dr. Harbert and his co-workers will accept my comments with magnanimity and in the same scientific spirit in which they were made. DR. FRANK C. GREISS, JR., Winston-Salem, North Carolina. Elucidation of homeostatic control mechanisms requires an experimental preparation \vhich maximizes information to be derived and simultaneously minimizes the experimental nature of the preparation. To this end, the chronic animal model has been developed. In this and previous experiments, Dr. Harbert and associates have demonstrated the abiiity to use a sophisticated array of minimally invasive and/or chronic experimental techniques in a capricious subhuman primate to obtain remarkably subtle data which by their very consistency suggest that we are looking at the real thing. Last year, Dr. Harbert described circadian rhythms in myometrial activity and hemodynamic alterations associated with then1 during the last tritnester of pregnancy in the rhesus macaque. Today, he has shown us that, as in the nonpregnant monkey, these rhythms are dependent upon catecholamines and adrenergic receptors within the various uterine tissues. In his written discussion, he has commented upon the implications these results may have upon the use of adrenergic agonists and antagonists in clinical practice. While such speculations are of obvious importance, I have been unable to come up with what I think may be practical clinical applications. Therefore, I have chosen to comment upon the results as they relate to previous observations in other animal species since it has been my hypothesis over the years that, except for differences in placentation, basic uterine hemodynamic rP-

Table I. Predicted responses from reported data Ri (metabolic)

Ri (direct)

Ri (indirect)

0

ti tt

H

1/0(?)

0

0 0

0

il i il t

Alpha blockade:

Placenta Myometrium Beta blockade:

Placenta Myometrium

1 1

i i

t(?) 1

0

i

Total R (predicted/observed)

il t

780

Harbert and Spisso

.\pril l.

i•J~

t

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some small producuon m the central nervous system. In addition, placental monamine oxidase and catechol-0-methyltransferasc degrade epinephrine and norepinephrine so that no significant plan'IIta! transfn occurs. Where then do ) ou postulate the source of the epinephrine necessan to produce the consistently higher ovarian vein levels over aortic levels that you observed? DR. ROBERT JAFFE, San Francisco. l :alifornia. There is a large nun1ber of biologic rhythn1s in pregnancy. \Ve

have found diurnal variation in corticosteroids in the long-term catheterized rhesus monkev letus. Others have found diurnal \ariation in dehydroepiandrosterone sulfate in the fetal rhesus monkev circulation. \Vurtman has presented data suggesting that corticosteroids from the ktal adr-enal cortex increase the enzynH::\ involved in the conversion uf ill;repinephrine to epinephrine in the fetal adrenal medulla. \Vhilt> I do not know the origin of the catecholamincs in the maternal circulation, it is possible that there is also a biologic rhythm in catccholamines in the fetal nt-culation. DR. CALVIN HOBEL, Torrance, California. I think this is a very exciting P'-lpt~r. It h;_ts bruught up Illctll~

interesting questions. The data on increased epinephrine levels in ovarian blood are very exciting. [ would like to address nn comments to the possibilitv that norepinephrine is converted into epinephrine in uterine tissues (a new concept). l~ecently \VC identihed signitlcarn an1ounts of phenylethanolamine-;\;-methvltransferase in uterine tissues. l'he presence of this en>yme in both the myometrium and the endometrium could cause peripheral conversion of norepinephrine to epinephrine. and the elevated epinephrine levds in ovarian \ein blood reponed by Dr. Harbert and associates. DR. HARBERT (Closing). I di~agree with some of Dr. Assali's comments on the experimental protocol. for instance, his belief that imra-aortic drug administration is the equivalent of systemic administration. Certainly, "·ith the large doses ol propranolol. \H' did see a signiticarll decrease in maternal heart rate, from 160 to approximately 12{0 beats/min. indicating that there was a svstemic efleci. With the administration of phentolamine ''e did not see
basi( doses that produced either alpha- or betaadrenergic blockade. \ly apparent confusion, from Dr. Assali's aspc( t. ot the definition and the use of a dose-response tune cannot be satisfactorily or adequately worked out between the two of us at this opportunity. Likewise, Dr. Assali and I han: discussed, on <>ther occasions, the problems that are involved with the electromagnetic How probe in an artery of 2 to :! 1h mm in diameter. the app1oximate site of the uterine anen· in the pregnant monkey. It is nn feeling that while there is a \·ariation in the reliabilitv oft hese Hm, probes that may be as much as 207c. this is a random variation bv definition. Conse uol b~ exaci in quaniity, the comparative proportion and direction of the changes are real obsenat ions. Dr. Assali. through Dr. Brinkman, also commented on the high resting intra-amniotic pres~urC's in the nonmedicated animals that were reported in one particular table of the paper. He questioned what effect ihi,; may have on perfusion pressure in the fetus. There is t>vidence that the increase in the amniotic pressures causes ;; parallel increase' in botll umbilical artery and umbilical vein pressures so that perfusion pressure associated with these changes does not van on the fetal side of the circulation. In reply to Dr. Greiss's comments, I think Dr. Jaffe and Dr. l:lobel have givt"n the besl anslvers to his 1n~jor

question: V\'hat is the source of the high concentrations of epinephrine in the ovarian vessels n~rsus the arterial twPt'n - - - - - - - - rhP - - - - nvan ----·--- thP ---uterine fundus and should not pick up blood drainage from the' ovary itself. There is always the possibi!itv of a methodologic artifact. Howe\el', these tests ar·e relati\dy specific, and the consistencies were such that I am ih;t \ ...·illing.

at this point. io (ll"ccpt n1ethodulug1c t:rrof

as tlw cause. Dr. <_;reiss said he could not sec.> a ··practie