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Differential sensitivity to angiotensin II in pregnant rabbits
Differential sensitivity to angiotensin II in pregnant rabbits Margaret K. McLaughlin, Ph.D., Patricia M. Quinn, M.S., and Jill S. Farnham, B.A. Burlington, Vermont Threshold concentrations for angiotensin II and barium chloride were determined in the perfused hind limb of the nonpregnant, pseudopregnant, and pregnant rabbit. The threshold concentration for these vasoconstrictors was defined as the amount of drug per milliliter of hind limb blood flow which would increase hind limb blood pressure by 4 mm Hg. There was no difference in the mean angiotensin II threshold concentration between the nonpregnant and pseudopregnant rabbits. The angiotensin II dose for the pregnant rabbits was double that of the first two groups. There was no significant difference in the mean barium chloride threshold concentration between the three groups. This difference in the pregnant angiotensin II threshold concentration with no difference in barium chloride threshold concentration indicates a change in vascular sensitivity which is dependent upon a specific angiotensin II-receptor interaction and is not inherent in the contractile response of the vessel, nor is it mimicked by pseudopregnancy. (AM. J. 0BSTET. GYNECOL. 146:633, 1983.)
Normal pregnancy in the human and a number of other species is associated with a loss in vascular response to the pressor effects of exogenously administered angiotensin II. 1- 4 Various studies have been performed in the human and sheep in an effort to determine the specific causes of this reduced vascular reactivity to angiotensin II. 2• 5· 6 The data from these studies indicate that the refractoriness to angiotensin II occurs at the level of the resistance vessels. Understanding the mechanisms involved in this angiotensin II refractoriness is important as it appears that there is a loss of refractoriness in women destined to develop pregnancy-induced hypertension. 6 The reactivity of the arterial circulation to a pressor stimulus such as angiotensin II is determined by a number of functional and structural factors in both the arterial smooth muscle and the vascular wall. These include the specific (i.e., a drug-receptor interaction) and nonspecific (i.e., the resting membrane potential) sensitivity of arterial smooth muscle to vasoactive stimuli, the inherent contractility of the arterial smooth muscle, and the viscoelasticity and geometry of the vascular wall. 7 A change in any one of these factors during pregnancy will result in an alteration of the vascular response to a particular stimulus. There is evidence in
From the Departments of Obstetrics arui GynecolOFJY and PhysiolofJY and Biophysics, The University of Vermont CoUege tf Medicine. Supported fry Vermont Heart Association Grant No. 5-26321. Presented at the Twenty-ninth Annual Meeting tf the Society for Gynecologic Investigation, Dallas, Texas, March 24-27, 1982. Received for publication June 16, 1982. Reoised]anuary 10, 1983. Accepted February 2, 1983. Reprint requests: Margaret K. McLaughlin, Ph.D., Department tf Obstetrics and GynecolofJY, Health Science Complex, C-248, University of Vermont College tf Medicine, Burlington, Vermont 05405.
different species that all of these factors can be altered by pregnancy. 4 Changes in smooth muscle contractility, vascular wall geometry, and viscoelasticity will generally affect the slope of a dose-response curve, while changes in sensitivity alter the threshold concentration of the drug, which causes a parallel shift to the left or right in the dose response curve. Therefore, if threshold doses are examined between groups, the sensitivity of the vasculature can be evaluated while the contribution of contractility, viscoelasticity, and geometry is minimized. 8 This study was designed to determine whether the vessel refractoriness to angiotension II during pregnancy is specific for angiotensin II or is the result of a nonspecific functional reduction in sensitivity to vasoactive stimuli in the peripheral vasculature. In addition to examining the pregnant state, we chose to study pseudopregnancy as well. The rabbit was chosen for these experiments as it is a reflex ovulator and easily induced to pseudopregnancy. Pseudopregnancy in the rabbit has a duration of approximately 22 days and is a function of the formation of corpora lutea following either a sterile coital stimulus or an injection of human chorionic gonadotropin (hCG). The progesterone profile in these animals is similar in form to that of the pregnant rabbit, although shorter in duration. 9 Pseudopregnancy provides a means of partially mimicking the maternal progesterone changes in pregnancy without the influence of the placental hormones. This provides an endogenous progesterone treatment and is included here to determine if this hormonal change is responsible for any of the observed results. A fixed-flow in situ hind limb perfusion technique was used for these experiments. With this technique, one vascular bed in isolated, thus circumventing the hemodynamic complication of the placental circula-
633
634
McLaughlin, Quinn, and Farnham
july 15, 1983 Am. J. Obstet. Gynecol.
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Fig. 1. Angiotensin II threshold concentrations (picograms per milliliter), barium chloride threshold concentrations (micrograms per milliliter), and the angiotensin II/barium chloride ratio in nonpregnant (open bar), pseudopregnant (hatched bar), and pregnant (solid bar) rabbits. Data are expressed as the mean ± SD.
Table I. Baseline parameters* for the treatment groups
Nonpregnant Pseudopregnant Pregnant
Limb resistance (mm Hg · min/ml)
Progesterone (nglml)
3.0 ± 0.4 2.7 ± 0.3 2.6 :t 0.4
3.8 ± 0.7 6.5 :t 0.9 8.3 :t 1.2
*Mean± SE.
tion. In a fixed-flow perfusion system the hind limb blood pressure measurement is a direct reflection of changes in the caliber of the resistance vessels and, therefore, a true reflection of vessel reactivity.
Material and methods Animals. Studies were performed on pregnant (n = 8, 3.8 to 4.6 kg), nonpregnant (n = 6, 3.1 to 4.8 kg), and pseudopregnant (n = 7, 3.3 to 4.5 kg) New Zealand rabbits. Pregnant rabbits were studied at 28 to 31 days' gestation (term= 32 days). Pseudopregnancy was induced in nonpregnant animals by ear injection of 50 IU of hCG. 9 Studies were performed on the sixteenth or seventeenth day of pseudopregnancy. Nonpregnant rabbit studies were randomly performed between the pregnant and pseudopregnant animals. Surgical preparation. Animals were sedated with an intramuscular i~ection of 100 mg of ketamine hydrochloride (Ketaset, Bristol Lab.) and an ear vein was catheterized with the use of a local anesthetic. Anesthesia was induced with intravenous administration of 250 to 500 mg of a-chloralose (Sigma). Animals were placed on a heating pad, and tracheotomy was performed. Positive pressure ventilation with a Bird respirator was given. The left jugular vein and carotid artery were catheterized. The femoral artery
was isolated just distal to the inguinal ligament, cannulated, and perfused with blood drawn from the carotid artery using a Varistaltic roller pump (Manistat Corp.). Both the right and left hind limb saphenous arteries were catheterized. Lactated Ringer's solution (0.5 ml/min) was administered via the jugular cannula. An initial heparin dose (60 mg) at the time of the femoral artery catheterization was followed by 10 mg approximately every 30 minutes. Blood gases and pH were periodically monitored to determine the adequacy of ventilation. Blood gases were analyzed on a Radiometer blood gas analyzer. The pH and Pco 2 were maintained between 7.32 and 7.42 and between 32 and 49 mm Hg, respectively. The Po 2 was always >160 mm Hg. The systemic and the perfused hind limb blood pressures were continuously monitored via the saphenous catheters with the use of Ailtech (Eaton Corp.) pressure transducers; heart rate was derived from the pulse pressure waveform with the use of a Gould Biotach, and all measurements were recorded on a rectilinear recorder (Gould Instruments, Cleveland, Ohio). Experimental procedure. Blood flow to the hind limb was set at a rate such that hind limb blood pressure was similar to systemic blood pressure. This rate remained fixed throughout the experiment. Threshold concentration (picograms or micrograms per milliliter) was equal to the infusion rate (milliliters per minute) x drug concentration (picograms or micrograms per milliliter) + hind limb blood flow (milliliters per minute). This calculation corrects for differences in hind limb blood flow between animals. Animals were put to death with euthanasia solution, and the ovaries were checked for the presence of corpora lutea; in pregnant animals the numbers of fetuses were counted and a representative sample was weighed to help verify gestational age. In addition, blood was
Sensitivity to angiotensin II
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Fig. 3. Barium chloride (micrograms per milliliter, left ordinate, closed circles) and angiotensin II (picograms per milliliter, right ordinate, open triangles) threshold concentrations versus the plasma progesterone levels (nanograms per milliliter).
obtained just prior to sacrifice for the radioimmunoassay determination of plasma progesterone and cortisol. Statistics. Threshold doses of angiotensin II and barium chloride and the angiotensin 11/barium chloride ratio were compared with the two-way analysis of variance and Schiffs test for comparing group means. 10 Linear regression analysis was used to compare the data for progesterone versus cortisol levels, angiotensin II and barium chloride threshold doses versus progesterone levels, and angiotensin II threshold doses versus barium chloride threshold doses. Results
Mean group values for the perfused hind limb vascular resistance and plasma progesterone levels are shown in Table I. The values for the hind limb vascular resistance were calculated from the pump flow rate and the average hind limb arterial blood pressure obtained over the 5 minutes just prior to the first drug infusion (resistance== BP/Q). A comparison of threshold concentrations for angiotensin II and barium chloride for each group is shown in Fig. 1. In addition, the ratio of angiotensin I I
and barium chloride is depicted. There was no difference in the mean angiotensin II threshold concentration (picograms per milliliter, mean ± SD) between the nonpregnant (6.91 ± 0.46) and pseudopregnant rabbits (6.90 ± 1.26). The angiotensin II dose for the pregnant rabbits was nearly twice as great as that for the first two groups (13.1 ± 1.5, p < 0.01). This difference in the pregnant rabbits was maintained (p < 0.02) when the ratio of the angiotensin II dose versus the barium chloride was calculated for each animal, which is a further test for the specificity of the angiotensin II response. 10 There was no significant difference in the mean barium chloride threshold concentration (micrograms per milliliter) between the three groups: nonpregnant (2.50 ± O.l), pseudopregnant (2.60 ± 0.45), and pregnant (2.95 ± 0.2). When the angiotensin II threshold concentration for each animal was compared to the barium chloride threshold concentration, a correlation was found in the nonpregnant (r == 0.895) and pregnant (r = 0.84) rabbits. Both slopes were significantly different from zero (p < 0.01) with the pregnant slope being greater than the nonpregnant slope (Fig. 2).
636
Mclaughlin, Quinn, and Farnham
NONPREGNANT
July 15, 19~:l Am. J. Obstet. Gvnecol.
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PROGESTERONE(ng/mO Fig. 4. The correlation between plasma cortisol (nanograms per milliliter) and plasma progesterone levels (nanograms per milliliter) in each group.
Both the angiotensin II and barium chloride threshold doses in the nonpregnant rabbits were correlated to the plasma progesterone levels, but this relationship was not present in the pseudopregnant or pregnant groups (Fig. 3). Since the progesterone levels in the nonpregnant rabbits without corpora lutea were higher than those reported in a basal state, 11 we measured plasma cortisol levels in the same samples to determine if an acute adrenal output contributed to these levels. There was a significant correlation between the nonpregnant plasma progesterone and cortisol levels (r = 0.81, p < 0.02) which was not as apparent in the pseudopregnant (r = 0.60) or pregnant (r = 0.59) rabbits (Fig. 4). Comment A reduced vascular response to angiotensin II in pregnancy has been demonstrated previously in the rat, rabbit, sheep, and human. In the previous studies the measured end point was the response of the systemic blood pressure to exogenous angiotensin II. 1- 4 • 12 The data reported in the sheep and human strongly suggested a specific vessel refractoriness to angiotensin II. 1' 5 ' 6 This study tested the hypothesis that in the rabbit the refractoriness to angiotensin II occurs in the arterial vasculature, is specific for angiotensin II, and occurs in a circulation other than that of the placenta. In addition, if these changes do occur, could this be duplicated in the pseudopregnant animal? The study involved two techniques: (1) a fixed-flow hind limb perfusion and (2) determination of a threshold concentration for angiotensin II and barium chloride. Determining the threshold concentration of a drug
gives an index of the responsiveness of the vasculature which should not be influenced by structural factors in the vessels being examined. 10 The use of two drugs which act by different mechanisms to initiate smooth muscle contraction allows one to draw conclusions about whether possible differences in threshold concentrations between groups are specific for one particular agent or are a nonspecific alteration in smooth muscle reactivity. In this study, there was a difference in threshold concentration for angiotensin II in the pregnant animals. However, there was no significant corresponding difference in the barium chloride threshold concentration. Angiotensin II initiates smooth muscle contraction via a drug-receptor interaction, 13 while barium chloride causes contraction by a combination of calcium displacement and blocking of potassium conductance in the membrane. 14 Since the drug-dependent stimulus to the smooth muscle is by separate mechanisms with angiotensin II and barium chloride and the only group difference observed was that with the angiotensin II in the pregnant animals, one can reasonably postulate that the alteration in these animals occurs at the level of the drug-receptor interaction. This supports the hypothesis that the refractoriness to angiotensin II observed in the pregnant rabbit is specific for angiotensin II. The individual correlation in the pregnant and nonpregnant animals between angiotensin II and barium chloride threshold concentrations indicates that the barium chloride threshold concentration does reflect the nonspecific sensitivity of the vessel (i.e., the less sensitive the vasculature was to a more general contractile stimulus, such as barium
Volume 146 Number fi
chloride, and the less sensitive it was to angiotensin II). However, by comparison of the ratio of angiotensin II to barium chloride, this factor is corrected for and the specific difference in sensitivity to angiotensin II in the pregnant rabbits is maintained. 10 No such relationship existed in the pseudopregnant animals. The data here indicate that the reduced sensitivity to angiotensin II in pregnancy is a function of drugmembrane interaction. There are a number of mechanisms which could alter this interaction. An increased rate of angiotensin II degradation would expose less drug to the muscle membrane. Angiotensin II has also been shown to regulate its receptors in the rat mesenteric arterial smooth muscle. 13 Increased amounts of angiotensin II lead to both short- and long-term alterations in angiotensin II receptor affinity and number. The short-term change in reduced binding capacity of angiotensin II (and, therefore, physiologic responsiveness) appears to be due to the binding of angiotensin II to a high-affinity, slowly reversible state, which is completely reversible in vitro within 30 minutes after the removal of angiotensin II. However, longer exposure to increased angiotensin II levels will lead to changes in kinetics of receptor turnover, a state which requires a much longer period of time to reverse itself. 13 Longterm increases in circulating angiotensin II levels would, therefore, result in reduced receptor number. Plasma angiotensin II levels are known to increase during pregnancy, 1 but the situation in the rabbit is unclear. Lumbers 15 reported increased plasma renin activity but not angiotensin II in the pregnant rabbit compared to the nonpregnant rabbit. These data correspond to a study in the same laboratory which demonstrated that pregnant rabbits were more sensitive to the pressor effects of angiotensin II than the nonpregnant controls. 16 This is in contrast to a similar study reported by Bersenbrugge and associates, 12 in which pregnant rabbits were less sensitive to angiotensin II than nonpregnant animals. Whether angiotensin II levels change in the pregnant rabbit requires further substantiation. The opposing results between our study and that reported by Lumbers cannot really be explained by differences in technique. The results of our study arc more consistent with what has been found in other species. 1 - 4 · 12 Another mechanism which has been suggested for the angiotensin II refractoriness of pregnancy is the modulation of angiotensin II -induced vasoconstriction by the stimulation of vasodilatory prostaglandins. The evidence for this comes primarily from studies in different species, including the human, in which the sensitivity to exogenous angiotensin II is increased following pretreatment with prostaglandin synthetase inhibitors. 2 • 4 • 6 The data presented here do not preclude this possibility. It would be of interest to examine
Sensitivity to angiotensin II
637
the hind limb vascular response to barium chloride following this type of drug treatment to determine if the change in sensitivity is a specific prostaglandin-inhibition phenomenon or a side effect of the synthetase inhibitor on the arterial smooth muscle. In addition to a prostaglandin-mediated modulation of angiotensin II responsiveness per se, a role for 5a-dihydroprogesterone (a progesterone metabolite), either through prostaglandin production or a steroid action which is independent of prostaglandin action. was described by Everett and associates.'' [n this study, threshold doses of angiotensin II and barium chloride were compared to plasma progestertone levels. It was only in the nonpregnant rabbits that a significant correlation was observed between threshold doses of angiotensin II and barium chloride and progesterone levels. The short-term experimental progesterone levels were higher in these nonpregnant rabbits than in animals in a basal stateY Since the adrenal gland has been shown to be a significant source of progesterone in the castrated rabbit, 11 we postulated that the elevated levels observed in this experiment were due to a stressinduced increase in adrenal outflow. The strong correlation between the nonpregnant plasma progesterone and cortisol levels tends to support th1s hypothesis. This short-term progesterone exposure appears to have had some effect upon vascular reactivity as indicated by the correlation between the threshold drug and progesterone levels. This relationship was not maintained in the other two groups. Since cortisol and progesterone levels were correlated in rhe nonpregnant rabbits, the drug threshold concentrations would be correlated to cortisol as well as progesterone in this group. A cortisol increase due to surgical stress would occur in the other two groups as well, but a similar correlation between cortisol and threshold concentrations was not found. Evidently, the long-term elevated steroid levels of both pseudopregnancy and pregnancy alter the acute effect of either progesterone or cortisol which was observed in the nonpregnant rabbits. Pseudopregnancy, in which progesterone levels are similar to those of pregnancy for 12 to I 7 days, 9 did not increase the threshold dose of angiotensin I I. This is in contrast to findings in the rat. in which both pseudopregnancy and short-term progesterone treatment reduced the whole-animal blood pressur<" response to a given dose of angiotensin Il. 17 There are a number of possible explanations as to why the pseudopregnant rabbits did not mimic the pregnant rabbits in their vascular response to angiotensin I I. The rabbits in this study were tested in the declining phase of the pseudopregnancy. which may account for the discrepancy between our study and that mentioned above in the pseudopregnant rat. The purpose here was to maximize the length of progesterone exposure. More vas-
638
McLaughlin, Quinn, and Farnham
july 15, !983
Am. J. Obstet. Gvnecol.
cular beds may be affected by pregnancy than pseudopregnancy; hence, systemic pressure responses may change while the hind limb pressor response may not in the pseudopregnant rabbit. Progesterone treatment in the human will blunt the renal pressor response to angiotensin II but does not affect the overall pressor response. 18 While pseudopregnancy mimics the progesterone levels of pregnant rabbits for at least 12 to 17 days, 9 in all likelihood the cardiovascular changes of pregnancy are more profound than those in the pseudopregnant rabbit. Placental hormones cannot be discounted as modifying influences, and what the reninangiotensin II system does in the pseudopregnant rabbit is not known. In summary, the data presented here indicate that there is a reduced vascular sensitivity to exogenously administered angiotensin II in the perfused hind limb of the pregnant rabbit. This reduced sensitivity is specific to the angiotensin II-receptor interaction and is independent of vessel contractility, viscoelasticity, and wall geometry. This reduction in sensitivity is not duplicated by pseudopregnancy. In addition, progesterone levels do not correlate with the drug threshold concentrations except in nonpregnant rabbits. This effect in the nonpregnant rabbits most likely reflects the response to short-term exposure versus the long-term progesterone exposure in the other two groups. It would seem appropriate that future studies of the mechanisms involved in pregnancy-associated angiotensin II refractoriness be concerned with the functions involving the arterial smooth muscle angiotensin II- receptor interaction. REFERENCES l. Abdul-Karim, R., and Assali, N. S.: Pressor response to angiotensin in pregnant and nonpregnant women, AM.].
0BSTET. GYNECOL. 82:246, 1961. 2. Matsuura, S., Naden, R. P., Gant, N. F., Jr., Parker, C. R., Jr., and Rosenfeld, C. R.: Effect of volume expansion on pressor response to angiotension II in pregnant ewes. Am.]. Physiol. 240:H908, 1981. 3. Somlyo, A. P., and Somlyo, A. V.: Vascular smooth muscle, Pharmacal. Rev. 22:249, 1970.
4. Altura, B. T., and Altura, B. M.: Influence of sex hormones, oral contraceptives and pregnancy on vascular muscle and its reactivity, in Carrier, 0., Jr., and Shibata, S., editors: Factors Influencing Vascular Reactivity, Tokyo, 1977, Igaku-Shoin, Ltd., pp. 221-254. 5. Everett, R. B., Worley, R. ]., MacDonald, P. C., and Gant, N. F.: Modification of vascular responsiveness to angiotensin II in pregnant women by intravenously infused 5a-dihydroprogesterone, AM. J. OBSTET. GYNECOL. 131: 352, 1978. 6. Everett, R. B., Worley, R.J., MacDonald, P. C., Chand, S., and Gant, N. F.: Vascular reactivity to angiotensin II in human pregnancy, Sem. Perinatol. 2:3, 1978. 7. Johansson, B.: Determinants of vascular reactivity, Fed. Proc. 33:121, 1974. 8. Lais, L. T., and Brody, M. J.: Mechanism of vascular hyperresponsiveness in the spontaneously hypertensive rat, Circ. Res. 36(1):216, 1976. 9. Harrington, F. W., and Rothermel,]. D.: Daily changes in peripheral plasma progesterone concentrations in preg· nant and pseudopregnant rabbits, Life Sci. 20:1333, 1977. 10. Wonnacott, T. H., and Wonnacott, R. J.: Introductory Statistics, New York, 1977, John Wiley & Sons, Inc. 11. Overstrom, E. W., and Black, D. L.: Effect of ovariectomy/adrenalectomy and ovariectomy/hysterectomy in peripheral serum concentrations of estradiol and progesterone in the rabbit, presented at the Eighth New England Endocrinology Conference, Shrewsbury, Massachusetts, 1980. 12. Bersenbrugge, A. D., Goodfriend, T. L., Ball, D. L., and Rankin,]. H. G.: The effect of pregnancy on the angiotensin II pressor response in the rabbit, AM. J. 0BSTET. GYNECOL. 136:762, 1980. 13. Gunther, S., Gimbrone,Jr., M.A., and Alexander, R. W.: Regulation by angiotensin II of its receptors in resistance blood vessels, Nature 187:230, 1980. 14. Rayon, D. C., and Browick, M. S.: Effects of barium on the potassium conductance of squid axon, J. Gen. Physiol. 75:727, 1980. 15. Lumbers, E. R.: Renin and angiotensin II of extrarenal origin in the plasma offemale rabbits,]. Physiol. 234:94P, 1973. 16. Shaughn O'Brien, P. M., Filshie, G. M., and BroughtonPipkin, F.: The effect of prostaglandin E2 on the cardiovascular response to angiotensin II in pregnant rabbits, Prostaglandins 13:171,1977. 17. Hettiaratchi, E. S. G., and Pickford, J.: The effect of oestrogen and progesterone on the pressor action of angiotensin in the rat, .J. Physiol. 196:44 7, 1968. 18. Chesley, L. C., and Tepper, I. H.: Effects of progesterone and estrogen on the sensitivity to angiotensin II, J. Clin. Endocrinol. 27:576, 1967.
Normal pregnancy in the human and a number of other species is associated with a loss in vascular response to the pressor effects of exogenously administered angiotensin II. 1- 4 Various studies have been performed in the human and sheep in an effort to determine the specific causes of this reduced vascular reactivity to angiotensin II. 2• 5· 6 The data from these studies indicate that the refractoriness to angiotensin II occurs at the level of the resistance vessels. Understanding the mechanisms involved in this angiotensin II refractoriness is important as it appears that there is a loss of refractoriness in women destined to develop pregnancy-induced hypertension. 6 The reactivity of the arterial circulation to a pressor stimulus such as angiotensin II is determined by a number of functional and structural factors in both the arterial smooth muscle and the vascular wall. These include the specific (i.e., a drug-receptor interaction) and nonspecific (i.e., the resting membrane potential) sensitivity of arterial smooth muscle to vasoactive stimuli, the inherent contractility of the arterial smooth muscle, and the viscoelasticity and geometry of the vascular wall. 7 A change in any one of these factors during pregnancy will result in an alteration of the vascular response to a particular stimulus. There is evidence in
From the Departments of Obstetrics arui GynecolOFJY and PhysiolofJY and Biophysics, The University of Vermont CoUege tf Medicine. Supported fry Vermont Heart Association Grant No. 5-26321. Presented at the Twenty-ninth Annual Meeting tf the Society for Gynecologic Investigation, Dallas, Texas, March 24-27, 1982. Received for publication June 16, 1982. Reoised]anuary 10, 1983. Accepted February 2, 1983. Reprint requests: Margaret K. McLaughlin, Ph.D., Department tf Obstetrics and GynecolofJY, Health Science Complex, C-248, University of Vermont College tf Medicine, Burlington, Vermont 05405.
different species that all of these factors can be altered by pregnancy. 4 Changes in smooth muscle contractility, vascular wall geometry, and viscoelasticity will generally affect the slope of a dose-response curve, while changes in sensitivity alter the threshold concentration of the drug, which causes a parallel shift to the left or right in the dose response curve. Therefore, if threshold doses are examined between groups, the sensitivity of the vasculature can be evaluated while the contribution of contractility, viscoelasticity, and geometry is minimized. 8 This study was designed to determine whether the vessel refractoriness to angiotension II during pregnancy is specific for angiotensin II or is the result of a nonspecific functional reduction in sensitivity to vasoactive stimuli in the peripheral vasculature. In addition to examining the pregnant state, we chose to study pseudopregnancy as well. The rabbit was chosen for these experiments as it is a reflex ovulator and easily induced to pseudopregnancy. Pseudopregnancy in the rabbit has a duration of approximately 22 days and is a function of the formation of corpora lutea following either a sterile coital stimulus or an injection of human chorionic gonadotropin (hCG). The progesterone profile in these animals is similar in form to that of the pregnant rabbit, although shorter in duration. 9 Pseudopregnancy provides a means of partially mimicking the maternal progesterone changes in pregnancy without the influence of the placental hormones. This provides an endogenous progesterone treatment and is included here to determine if this hormonal change is responsible for any of the observed results. A fixed-flow in situ hind limb perfusion technique was used for these experiments. With this technique, one vascular bed in isolated, thus circumventing the hemodynamic complication of the placental circula-
633
634
McLaughlin, Quinn, and Farnham
july 15, 1983 Am. J. Obstet. Gynecol.
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Fig. 1. Angiotensin II threshold concentrations (picograms per milliliter), barium chloride threshold concentrations (micrograms per milliliter), and the angiotensin II/barium chloride ratio in nonpregnant (open bar), pseudopregnant (hatched bar), and pregnant (solid bar) rabbits. Data are expressed as the mean ± SD.
Table I. Baseline parameters* for the treatment groups
Nonpregnant Pseudopregnant Pregnant
Limb resistance (mm Hg · min/ml)
Progesterone (nglml)
3.0 ± 0.4 2.7 ± 0.3 2.6 :t 0.4
3.8 ± 0.7 6.5 :t 0.9 8.3 :t 1.2
*Mean± SE.
tion. In a fixed-flow perfusion system the hind limb blood pressure measurement is a direct reflection of changes in the caliber of the resistance vessels and, therefore, a true reflection of vessel reactivity.
Material and methods Animals. Studies were performed on pregnant (n = 8, 3.8 to 4.6 kg), nonpregnant (n = 6, 3.1 to 4.8 kg), and pseudopregnant (n = 7, 3.3 to 4.5 kg) New Zealand rabbits. Pregnant rabbits were studied at 28 to 31 days' gestation (term= 32 days). Pseudopregnancy was induced in nonpregnant animals by ear injection of 50 IU of hCG. 9 Studies were performed on the sixteenth or seventeenth day of pseudopregnancy. Nonpregnant rabbit studies were randomly performed between the pregnant and pseudopregnant animals. Surgical preparation. Animals were sedated with an intramuscular i~ection of 100 mg of ketamine hydrochloride (Ketaset, Bristol Lab.) and an ear vein was catheterized with the use of a local anesthetic. Anesthesia was induced with intravenous administration of 250 to 500 mg of a-chloralose (Sigma). Animals were placed on a heating pad, and tracheotomy was performed. Positive pressure ventilation with a Bird respirator was given. The left jugular vein and carotid artery were catheterized. The femoral artery
was isolated just distal to the inguinal ligament, cannulated, and perfused with blood drawn from the carotid artery using a Varistaltic roller pump (Manistat Corp.). Both the right and left hind limb saphenous arteries were catheterized. Lactated Ringer's solution (0.5 ml/min) was administered via the jugular cannula. An initial heparin dose (60 mg) at the time of the femoral artery catheterization was followed by 10 mg approximately every 30 minutes. Blood gases and pH were periodically monitored to determine the adequacy of ventilation. Blood gases were analyzed on a Radiometer blood gas analyzer. The pH and Pco 2 were maintained between 7.32 and 7.42 and between 32 and 49 mm Hg, respectively. The Po 2 was always >160 mm Hg. The systemic and the perfused hind limb blood pressures were continuously monitored via the saphenous catheters with the use of Ailtech (Eaton Corp.) pressure transducers; heart rate was derived from the pulse pressure waveform with the use of a Gould Biotach, and all measurements were recorded on a rectilinear recorder (Gould Instruments, Cleveland, Ohio). Experimental procedure. Blood flow to the hind limb was set at a rate such that hind limb blood pressure was similar to systemic blood pressure. This rate remained fixed throughout the experiment. Threshold concentration (picograms or micrograms per milliliter) was equal to the infusion rate (milliliters per minute) x drug concentration (picograms or micrograms per milliliter) + hind limb blood flow (milliliters per minute). This calculation corrects for differences in hind limb blood flow between animals. Animals were put to death with euthanasia solution, and the ovaries were checked for the presence of corpora lutea; in pregnant animals the numbers of fetuses were counted and a representative sample was weighed to help verify gestational age. In addition, blood was
Sensitivity to angiotensin II
Volume !46 Number 6
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4
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PLASMA PROGESTERONE
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Fig. 3. Barium chloride (micrograms per milliliter, left ordinate, closed circles) and angiotensin II (picograms per milliliter, right ordinate, open triangles) threshold concentrations versus the plasma progesterone levels (nanograms per milliliter).
obtained just prior to sacrifice for the radioimmunoassay determination of plasma progesterone and cortisol. Statistics. Threshold doses of angiotensin II and barium chloride and the angiotensin 11/barium chloride ratio were compared with the two-way analysis of variance and Schiffs test for comparing group means. 10 Linear regression analysis was used to compare the data for progesterone versus cortisol levels, angiotensin II and barium chloride threshold doses versus progesterone levels, and angiotensin II threshold doses versus barium chloride threshold doses. Results
Mean group values for the perfused hind limb vascular resistance and plasma progesterone levels are shown in Table I. The values for the hind limb vascular resistance were calculated from the pump flow rate and the average hind limb arterial blood pressure obtained over the 5 minutes just prior to the first drug infusion (resistance== BP/Q). A comparison of threshold concentrations for angiotensin II and barium chloride for each group is shown in Fig. 1. In addition, the ratio of angiotensin I I
and barium chloride is depicted. There was no difference in the mean angiotensin II threshold concentration (picograms per milliliter, mean ± SD) between the nonpregnant (6.91 ± 0.46) and pseudopregnant rabbits (6.90 ± 1.26). The angiotensin II dose for the pregnant rabbits was nearly twice as great as that for the first two groups (13.1 ± 1.5, p < 0.01). This difference in the pregnant rabbits was maintained (p < 0.02) when the ratio of the angiotensin II dose versus the barium chloride was calculated for each animal, which is a further test for the specificity of the angiotensin II response. 10 There was no significant difference in the mean barium chloride threshold concentration (micrograms per milliliter) between the three groups: nonpregnant (2.50 ± O.l), pseudopregnant (2.60 ± 0.45), and pregnant (2.95 ± 0.2). When the angiotensin II threshold concentration for each animal was compared to the barium chloride threshold concentration, a correlation was found in the nonpregnant (r == 0.895) and pregnant (r = 0.84) rabbits. Both slopes were significantly different from zero (p < 0.01) with the pregnant slope being greater than the nonpregnant slope (Fig. 2).
636
Mclaughlin, Quinn, and Farnham
NONPREGNANT
July 15, 19~:l Am. J. Obstet. Gvnecol.
PSEUDOPREGNANT
PREGNANT
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•
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• 6
8
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PROGESTERONE(ng/mO Fig. 4. The correlation between plasma cortisol (nanograms per milliliter) and plasma progesterone levels (nanograms per milliliter) in each group.
Both the angiotensin II and barium chloride threshold doses in the nonpregnant rabbits were correlated to the plasma progesterone levels, but this relationship was not present in the pseudopregnant or pregnant groups (Fig. 3). Since the progesterone levels in the nonpregnant rabbits without corpora lutea were higher than those reported in a basal state, 11 we measured plasma cortisol levels in the same samples to determine if an acute adrenal output contributed to these levels. There was a significant correlation between the nonpregnant plasma progesterone and cortisol levels (r = 0.81, p < 0.02) which was not as apparent in the pseudopregnant (r = 0.60) or pregnant (r = 0.59) rabbits (Fig. 4). Comment A reduced vascular response to angiotensin II in pregnancy has been demonstrated previously in the rat, rabbit, sheep, and human. In the previous studies the measured end point was the response of the systemic blood pressure to exogenous angiotensin II. 1- 4 • 12 The data reported in the sheep and human strongly suggested a specific vessel refractoriness to angiotensin II. 1' 5 ' 6 This study tested the hypothesis that in the rabbit the refractoriness to angiotensin II occurs in the arterial vasculature, is specific for angiotensin II, and occurs in a circulation other than that of the placenta. In addition, if these changes do occur, could this be duplicated in the pseudopregnant animal? The study involved two techniques: (1) a fixed-flow hind limb perfusion and (2) determination of a threshold concentration for angiotensin II and barium chloride. Determining the threshold concentration of a drug
gives an index of the responsiveness of the vasculature which should not be influenced by structural factors in the vessels being examined. 10 The use of two drugs which act by different mechanisms to initiate smooth muscle contraction allows one to draw conclusions about whether possible differences in threshold concentrations between groups are specific for one particular agent or are a nonspecific alteration in smooth muscle reactivity. In this study, there was a difference in threshold concentration for angiotensin II in the pregnant animals. However, there was no significant corresponding difference in the barium chloride threshold concentration. Angiotensin II initiates smooth muscle contraction via a drug-receptor interaction, 13 while barium chloride causes contraction by a combination of calcium displacement and blocking of potassium conductance in the membrane. 14 Since the drug-dependent stimulus to the smooth muscle is by separate mechanisms with angiotensin II and barium chloride and the only group difference observed was that with the angiotensin II in the pregnant animals, one can reasonably postulate that the alteration in these animals occurs at the level of the drug-receptor interaction. This supports the hypothesis that the refractoriness to angiotensin II observed in the pregnant rabbit is specific for angiotensin II. The individual correlation in the pregnant and nonpregnant animals between angiotensin II and barium chloride threshold concentrations indicates that the barium chloride threshold concentration does reflect the nonspecific sensitivity of the vessel (i.e., the less sensitive the vasculature was to a more general contractile stimulus, such as barium
Volume 146 Number fi
chloride, and the less sensitive it was to angiotensin II). However, by comparison of the ratio of angiotensin II to barium chloride, this factor is corrected for and the specific difference in sensitivity to angiotensin II in the pregnant rabbits is maintained. 10 No such relationship existed in the pseudopregnant animals. The data here indicate that the reduced sensitivity to angiotensin II in pregnancy is a function of drugmembrane interaction. There are a number of mechanisms which could alter this interaction. An increased rate of angiotensin II degradation would expose less drug to the muscle membrane. Angiotensin II has also been shown to regulate its receptors in the rat mesenteric arterial smooth muscle. 13 Increased amounts of angiotensin II lead to both short- and long-term alterations in angiotensin II receptor affinity and number. The short-term change in reduced binding capacity of angiotensin II (and, therefore, physiologic responsiveness) appears to be due to the binding of angiotensin II to a high-affinity, slowly reversible state, which is completely reversible in vitro within 30 minutes after the removal of angiotensin II. However, longer exposure to increased angiotensin II levels will lead to changes in kinetics of receptor turnover, a state which requires a much longer period of time to reverse itself. 13 Longterm increases in circulating angiotensin II levels would, therefore, result in reduced receptor number. Plasma angiotensin II levels are known to increase during pregnancy, 1 but the situation in the rabbit is unclear. Lumbers 15 reported increased plasma renin activity but not angiotensin II in the pregnant rabbit compared to the nonpregnant rabbit. These data correspond to a study in the same laboratory which demonstrated that pregnant rabbits were more sensitive to the pressor effects of angiotensin II than the nonpregnant controls. 16 This is in contrast to a similar study reported by Bersenbrugge and associates, 12 in which pregnant rabbits were less sensitive to angiotensin II than nonpregnant animals. Whether angiotensin II levels change in the pregnant rabbit requires further substantiation. The opposing results between our study and that reported by Lumbers cannot really be explained by differences in technique. The results of our study arc more consistent with what has been found in other species. 1 - 4 · 12 Another mechanism which has been suggested for the angiotensin II refractoriness of pregnancy is the modulation of angiotensin II -induced vasoconstriction by the stimulation of vasodilatory prostaglandins. The evidence for this comes primarily from studies in different species, including the human, in which the sensitivity to exogenous angiotensin II is increased following pretreatment with prostaglandin synthetase inhibitors. 2 • 4 • 6 The data presented here do not preclude this possibility. It would be of interest to examine
Sensitivity to angiotensin II
637
the hind limb vascular response to barium chloride following this type of drug treatment to determine if the change in sensitivity is a specific prostaglandin-inhibition phenomenon or a side effect of the synthetase inhibitor on the arterial smooth muscle. In addition to a prostaglandin-mediated modulation of angiotensin II responsiveness per se, a role for 5a-dihydroprogesterone (a progesterone metabolite), either through prostaglandin production or a steroid action which is independent of prostaglandin action. was described by Everett and associates.'' [n this study, threshold doses of angiotensin II and barium chloride were compared to plasma progestertone levels. It was only in the nonpregnant rabbits that a significant correlation was observed between threshold doses of angiotensin II and barium chloride and progesterone levels. The short-term experimental progesterone levels were higher in these nonpregnant rabbits than in animals in a basal stateY Since the adrenal gland has been shown to be a significant source of progesterone in the castrated rabbit, 11 we postulated that the elevated levels observed in this experiment were due to a stressinduced increase in adrenal outflow. The strong correlation between the nonpregnant plasma progesterone and cortisol levels tends to support th1s hypothesis. This short-term progesterone exposure appears to have had some effect upon vascular reactivity as indicated by the correlation between the threshold drug and progesterone levels. This relationship was not maintained in the other two groups. Since cortisol and progesterone levels were correlated in rhe nonpregnant rabbits, the drug threshold concentrations would be correlated to cortisol as well as progesterone in this group. A cortisol increase due to surgical stress would occur in the other two groups as well, but a similar correlation between cortisol and threshold concentrations was not found. Evidently, the long-term elevated steroid levels of both pseudopregnancy and pregnancy alter the acute effect of either progesterone or cortisol which was observed in the nonpregnant rabbits. Pseudopregnancy, in which progesterone levels are similar to those of pregnancy for 12 to I 7 days, 9 did not increase the threshold dose of angiotensin I I. This is in contrast to findings in the rat. in which both pseudopregnancy and short-term progesterone treatment reduced the whole-animal blood pressur<" response to a given dose of angiotensin Il. 17 There are a number of possible explanations as to why the pseudopregnant rabbits did not mimic the pregnant rabbits in their vascular response to angiotensin I I. The rabbits in this study were tested in the declining phase of the pseudopregnancy. which may account for the discrepancy between our study and that mentioned above in the pseudopregnant rat. The purpose here was to maximize the length of progesterone exposure. More vas-
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Am. J. Obstet. Gvnecol.
cular beds may be affected by pregnancy than pseudopregnancy; hence, systemic pressure responses may change while the hind limb pressor response may not in the pseudopregnant rabbit. Progesterone treatment in the human will blunt the renal pressor response to angiotensin II but does not affect the overall pressor response. 18 While pseudopregnancy mimics the progesterone levels of pregnant rabbits for at least 12 to 17 days, 9 in all likelihood the cardiovascular changes of pregnancy are more profound than those in the pseudopregnant rabbit. Placental hormones cannot be discounted as modifying influences, and what the reninangiotensin II system does in the pseudopregnant rabbit is not known. In summary, the data presented here indicate that there is a reduced vascular sensitivity to exogenously administered angiotensin II in the perfused hind limb of the pregnant rabbit. This reduced sensitivity is specific to the angiotensin II-receptor interaction and is independent of vessel contractility, viscoelasticity, and wall geometry. This reduction in sensitivity is not duplicated by pseudopregnancy. In addition, progesterone levels do not correlate with the drug threshold concentrations except in nonpregnant rabbits. This effect in the nonpregnant rabbits most likely reflects the response to short-term exposure versus the long-term progesterone exposure in the other two groups. It would seem appropriate that future studies of the mechanisms involved in pregnancy-associated angiotensin II refractoriness be concerned with the functions involving the arterial smooth muscle angiotensin II- receptor interaction. REFERENCES l. Abdul-Karim, R., and Assali, N. S.: Pressor response to angiotensin in pregnant and nonpregnant women, AM.].
0BSTET. GYNECOL. 82:246, 1961. 2. Matsuura, S., Naden, R. P., Gant, N. F., Jr., Parker, C. R., Jr., and Rosenfeld, C. R.: Effect of volume expansion on pressor response to angiotension II in pregnant ewes. Am.]. Physiol. 240:H908, 1981. 3. Somlyo, A. P., and Somlyo, A. V.: Vascular smooth muscle, Pharmacal. Rev. 22:249, 1970.
4. Altura, B. T., and Altura, B. M.: Influence of sex hormones, oral contraceptives and pregnancy on vascular muscle and its reactivity, in Carrier, 0., Jr., and Shibata, S., editors: Factors Influencing Vascular Reactivity, Tokyo, 1977, Igaku-Shoin, Ltd., pp. 221-254. 5. Everett, R. B., Worley, R. ]., MacDonald, P. C., and Gant, N. F.: Modification of vascular responsiveness to angiotensin II in pregnant women by intravenously infused 5a-dihydroprogesterone, AM. J. OBSTET. GYNECOL. 131: 352, 1978. 6. Everett, R. B., Worley, R.J., MacDonald, P. C., Chand, S., and Gant, N. F.: Vascular reactivity to angiotensin II in human pregnancy, Sem. Perinatol. 2:3, 1978. 7. Johansson, B.: Determinants of vascular reactivity, Fed. Proc. 33:121, 1974. 8. Lais, L. T., and Brody, M. J.: Mechanism of vascular hyperresponsiveness in the spontaneously hypertensive rat, Circ. Res. 36(1):216, 1976. 9. Harrington, F. W., and Rothermel,]. D.: Daily changes in peripheral plasma progesterone concentrations in preg· nant and pseudopregnant rabbits, Life Sci. 20:1333, 1977. 10. Wonnacott, T. H., and Wonnacott, R. J.: Introductory Statistics, New York, 1977, John Wiley & Sons, Inc. 11. Overstrom, E. W., and Black, D. L.: Effect of ovariectomy/adrenalectomy and ovariectomy/hysterectomy in peripheral serum concentrations of estradiol and progesterone in the rabbit, presented at the Eighth New England Endocrinology Conference, Shrewsbury, Massachusetts, 1980. 12. Bersenbrugge, A. D., Goodfriend, T. L., Ball, D. L., and Rankin,]. H. G.: The effect of pregnancy on the angiotensin II pressor response in the rabbit, AM. J. 0BSTET. GYNECOL. 136:762, 1980. 13. Gunther, S., Gimbrone,Jr., M.A., and Alexander, R. W.: Regulation by angiotensin II of its receptors in resistance blood vessels, Nature 187:230, 1980. 14. Rayon, D. C., and Browick, M. S.: Effects of barium on the potassium conductance of squid axon, J. Gen. Physiol. 75:727, 1980. 15. Lumbers, E. R.: Renin and angiotensin II of extrarenal origin in the plasma offemale rabbits,]. Physiol. 234:94P, 1973. 16. Shaughn O'Brien, P. M., Filshie, G. M., and BroughtonPipkin, F.: The effect of prostaglandin E2 on the cardiovascular response to angiotensin II in pregnant rabbits, Prostaglandins 13:171,1977. 17. Hettiaratchi, E. S. G., and Pickford, J.: The effect of oestrogen and progesterone on the pressor action of angiotensin in the rat, .J. Physiol. 196:44 7, 1968. 18. Chesley, L. C., and Tepper, I. H.: Effects of progesterone and estrogen on the sensitivity to angiotensin II, J. Clin. Endocrinol. 27:576, 1967.