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Barium responsiveness of the rat aorta and femoral artery during pregnancy
Life Sciences, Vol. 30, pp. 163-169 Printed in the U.S.A.
Pergamon Pres~
BARIUM RESPONSIVENESS OF THE RAT AORTA AND FEMORAL ARTERY DURING PREGNANCY J. L. Hart Biology Department George Mason U n i v e r s i t y Fairfax, V i r g i n i a 22030 (Received in final form November 18, 1981)
Summary The barium responses of isolated a o r t i c s t r i p s and femoral a r t e r i e s from non-pregnant and pregnant rats were investigated. Barium caused concentration-related increases in tension of vessels from both pregnant and non-pregnant r a t s . The concentration-response curves of femoral a r t e r i e s from non-pregnant and 3 week pregnant rats were not d i f f e r e n t ; however c o n t r a c t i l i t y and slopes of concentration-response lines for thoracic aortas from I , 2 and 3 week pregnant rats ;were s i g n i f i c a n t l y less than those of aortas from non-pregnant r a t s . In a d d i t i o n , barium caused rhythmic contractions to develop in both femoral a r t e r i e s and aortas of 3 week pregnant rats more frequently than vessels from nonpregnant r a t s . Rhythmic contractions did not develop in aortas from 3 week pregnant rats rats in calcium-free Krebs. Since the effects of barium on the e l e c t r i c a l and mechanical a c t i v i t y of various muscles have been postulated to be s i m i l a r to and/or dependent on calcium, these r e s u l t s may indicate that changes in calcium s e n s i t i v i t y of vascular smooth muscle occur during pregnancy. Such changes may contribute to the blood flow r e d i s t r i bution and other cardiovascular adaptations of pregnancy. Pregnancy requires wide-spread and rapid changes in maternal physiology, e s p e c i a l l y in the cardiovascular system. An e s s e n t i a l l y new, low resistance high flow c i r c u i t develops (the utero-placental c i r c u i t ) , blood volume and cardiac output increase to meet the volume and flow requirements of t h i s new c i r c u i t , and blood flow is r e d i s t r i b u t e d and adjusted to accommodate the increased metabolic a c t i v i t y of various maternal organs(1). I t is of considerable i n t e r e s t to understand what factors contribute to the cardiovascular adaptations of normal pregnancy, since t h i s knowledge forms the basis for preventing or t r e a t i n g conditions which are the r e s u l t of these adjustments not occurring normally. Changes in vascular responsiveness to various vasoactive agents in many species have been reported to occur in pregnancy ( 2 , 3 , 4 , 5 ) . While i n v e s t i g a t i n g such changes in isolated blood vessels, i t was observed that bagium ions frequently caused normally quiescent vessels from l a t e pregnant rats to develop rhythmic contractions while i t did so only i n f r e q u e n t l y in vessels from non-pregnant rats. Barium i t s e l f is u n l i k e l y to play any role in normal cardiovascular dynamics; however, because of i t s dependence on and s i m i l a r i t i e s to many of the actions of calcium(6), i t is a useful tool to investigate vascular smooth 0024-3205/82/020163-07503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
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muscle a c t i v i t y . The present study was designed to investigate barium responsiveness of isolated blood vessels throughout pregnancy, Both the concentration-related increases in isometric tension and the rhythmogenic actions of barium were investigated. Methods Female Sprague Dawley rats (Flow Labs, V i r g i n i a ) , 3-5 months old, were used in a l l experiments. Estrous stage and pregnancy timing were determined by vaginal smears, with the day when sperm were present designated as day one of gestation. Non-pregnant rats were used only when in estrus. Pregnant rats were studied e i t h e r in l a t e pregnancy only (20-22 days) or at approximately one week i n t e r v a l s of the 3 week gestation period (6-7, 14-16 and 20-22 days), and are hereafter referred to as the I , 2, or 3 week pregnant groups. After s a c r i f i c e with ether, blood vessels to be studied were r a p i d l y removed and placed in Krebs solution of the following composition in mM: NaCl 118.2, KCl 4.6, NaHC03 24.8, KH2P04 1.2, CaCI2(2H20) 2.5, MgS04(7 H20) 1.2, glucose I0, EDTA-Na2 .03. S p i r a l l y cut s t r i p s of the thoracic aorta (l-2mm wide, 5-8mm long) were prepared according to the method of Furchgott(7). Femoral a r t e r y cylinders (3-4mm long) were mounted on two 5-0 stainless steel wire t r i a n g l e s and tied at the apexes with 6-0 s i l k suture. The vessels were then mounted in 37o K r e b s - f i l l e d tissue chambers which were continuously gassed with 95% 02 - 5% CO2. Optimal passive tensions were applied to the vessels: 1 gram for a o r t i c s t r i p s and 0.5 grams f o r femoral a r t e r y c y l i n d e r s . Isometric tension was recorded using e i t h e r Harvard (no. 363) or Narco Biosystem (F-60) force transducers. After 90-120 min e q u i l i b r a t i o n , cumulative concentration-response curves to barium chloride (BaCI2-2H20, Sigma) were carried out. The BaCl2 stock solutions were prepared in pR 4, S04-free Krebs to prevent p r e c i p i t a t i o n . I t was added in small volumes, without osmotic compensation, to the tissue chambers to achieve the f i n a l bath concentrations which are expressed in molar (M) concentrations in figures. In experiments on the possible role of external calcium in barium-induced rhythmic contractions, 2 a o r t i c s t r i p s from each of f i v e 3 week pregnant rats were used. One s t r i p was equilibrated for 2 hours in calcium-free Krebs with repeated washings at 10-15 min i n t e r v a l s , and then exposed three times to IO-7M epinephrine in order to deplete loosely bound i n t r a c e l l u l a r calcium. After t h i s prbcedure, cumulative barium concentration-response curves were done as described above. The second s t r i p was equilibrated for 3 hours in normal calcium-containing Krebs before barium exposure. At the end of a l l experiments, vessel dimensions were measured and a f t e r 20-24 hours of dessication, dry weight was determined. Isometric tension was then expressed as mg of tension/mg dry weight. For determining differences between proportions, the Fisher exact test was used (8). Maximum tension development ( c o n t r a c t i l i t y ) was compared between groups by the t - t e s t or, i f more than 2 groups were being analyzed, by analysis of variance. I f analysis of v~riance indicated s i g n i f i c a n t differences, Dunnett's t e s t for comparison of a control (non-pregnant) mean to each other group mean (1,2,3 week pregnant) was used(g). Regression analysis of concentration-response curves and comparisons of slopes were done by the methods of T a l l a r i d a and Murray(lO). All means are expressed plus and minus standard errors (SE). Results Bariumcaused rhythmic contractions to develop in 85% (18 of 22) a o r t i c s t r i p s from 3 week pregnant rats but only 15% (3 of 20) of aortas from nonpregnant r a t s . This was a highly s i g n i f i c a n t difference (p<.O01). The
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PG
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FIG. 1 Record o f isometric tension of a o r t i c s t r i p s from 3 d i f f e r e n t 3 week pregnant (PG) and I non-pregnant (N-PG) r a t . Barium was added at the A and B arrows. A l l vessels from pregnant r a t s show rhythmic c o n t r a c t i o n s . frequencies and patterns of rhythmic contractions varied considerably with frequencies normally between 4 and 12/min. However, as can be seen in Fig. 1 (upper t r a c e ) , some preparations also developed concurrent f a s t 4-12/min) and slow (.15-.25/min) rhythms. The most common pattern seen when doing complete concentration-response curves was t h a t i l l u s t r a t e d in the upper trace o f Fig. 2, where rhythmic contractions developed at intermediate concentrat i o n s , but stopped at higher concentrations as t o t a l tension continued to i n crease, and then reappeared during the wash out. Rhythmic contractions during barium exposure did not develop in any of 6 a o r t i c s t r i p s from one week pregnant r a t s and in only 1 o f 6 from two week pregnant r a t s . These rates of occurrence were not s i g n i f i c a n t l y d i f f e r e n t (p>.05) from those o f aortas from non-pregnant r a t s . In order to determine i f barium r h y t h m i c i t y occurred in vessels other than the e l a s t i c a o r t a , the more muscular femoral a r t e r y was also studied. Rhythmic contractions developed in a l l 5 femoral a r t e r i e s from 3 week pregnant r a t s during barium exposure, while occurring in only 2 o f 5 femorals from non-pregnant r a t s . Because of the small numbers used, t h i s was not a s i g n i f i c a n t d i f f e r e n c e at the .05 l e v e l , but was at the .I0 l e v e l . The f r e quencies of femoral a r t e r y rhythmic contractions ranged between 3 and 8/min and u s u a l l y occurred throughout the barium exposure with higher concentrations causing slower rhythmic contractions of a l a r g e r magnitude (Fig. 3).
166
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FIG. 2 Records o f isometric tension o f 2 a o r t i c s t r i p s from the same 3 week pregnant r a t during cumulative a d d i t i o n s o f barium to give f i n a l bath c o n c e n t r a t i o n s f o r both i n d i c a t e d above the upper arrow. Upper t r a c i n g is of a o r t i c s t r i p in normal calcium; lower t r a c i n g is o f s t r i p in c a l c i u m - f r e e Krebs.
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FIG. 3 Records o f c o n t r a c t i l e responses of femoral a r t e r i e s from 2 d i f f e r e n t 3 week pregnant r a t s during barium exposure. Records are not continuous; p o r t i o n s have been deleted in preparing f i g u r e .
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Vol. 30, No. 2, 1982
Vascular Ba 2+ Responses During Pregnancy
NON'PREGNANT
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FIG. 4 Barium c o n c e n t r a t i o n - r e s p o n s e curves (+ SE) o f a o r t i c s t r i p s from 14 non-pregnant ( e s t r u s ) , 6 one ~ e k , 6 two week, and 24 three week pregnant r a t s . Concentration response curves o f a o r t i c s t r i p s from non-pregnant and 1,2, and 3 week pregnant r a t s are graphed in Fig. 4. A l l s t r i p s contracted in a c o n c e n t r a t i o n - r e l a t e d manner. However, maximum tension development (contractility) was less at a l l stages of pregnancy, s i g n i f i c a n t l y so at 1 and 2 weeks ( p < . 0 5 ) , and the slopes o f the ECpn-EC80 l i n e s , as determined by r e gression a n a l y s i s ( I 0 ) , were s i g n i f i c a n ~ T y less at a l l stages o f pregnancy than t h a t o f aortas from non-pregnant r a t s , w h i l e EC-50's were not s i g n i f i cantly different. However, the slope, e l e v a t i o n , and maximum tension of the barium c o n c e n t r a t i o n - r e s p o n s e curves f o r femoral a r t e r i e s from 3 week pregnant r a t s were not s i g n i f i c a n t l y d i f f e r e n t from those o f the femorals from nonpregnant r a t s (p>.05). In order to determine i f e x t e r n a l calcium was involved in the b a r i u m - i n duced rhythmic c o n t r a c t i o n s , two a o r t i c s t r i p s from each of f i v e 3 week pregnant r a t s were studied - one i n calcium " f r e e " and the o t h e r in normal calcium (2.5mM) Krebs. The records from one o f these experiments is in Fig. 2. A l l f i v e o f the aortas in the c a l c i u m - c o n t a i n i n g Krebs developed rhythmic cont r a c t i o n s , w h i l e none o f these in c a l c i u m - f r e e Krebs d i d . Mean maximum tension f o r the aortas in the c a l c i u m - f r e e Krebs was 780 + 40 mg/ mg dry w e i g h t , which was o n l y 39% o f the mean maximum tensions f o r t h e aortas in normal c a l cium. Discussion The e f f e c t s o f pregnancy on two aspects o f v a s c u l a r smooth muscle responsivesness to barium were i n v e s t i g a t e d : i n d u c t i o n o f rhythmic c o n t r a c t i o n s and sustained i s o m e t r i c c o n t r a c t i l e responses. Both aortas and femoral a r t e r i e s
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from 3 week pregnant rats developed rhythmic contractions during barium exposure more frequently than did those from non-pregnant estrus r a t s . Rhythmic contractions did not, however, develop more frequently in aortas from 1 and 2 week pregnant r a t s . Therefore, this increased s u s c e p t i b i l i t y to barium-induced r h y t h m i c i t y appears to be c h a r a c t e r i s t i c of l a t e , but not earl i e r , stages of pregnancy. Patterns of r h y t h m i c i t y varied in aortas, with concurrent fast and slow contractions occurring in some preparations. Although the basis for this was not investigated, i t has been reported ( I I ) that the patterns of rhythmic mechanical a c t i v i t y of isolated r a t a o r t i c s t r i p s depend on the number, f r e quency, and r e g u l a r i t y of active pacemaker sites w i t h i n the muscle, which can be related to the size of the s t r i p s . S t r i p size did vary some in the current study, and may have contributed to the d i f f e r e n t patterns of barium-induced contractions observed. Tension development of the aorta in reponse to barium was also modified by pregnancy, with maximum tension ( c o n t r a c t i l i t y ) and concentration-response curve slopes depressed at a l l stages of pregnancy studied indicating a reduced maximum responsiveness of the aorta to barium during pregnancy. Femoral a r t e r i e s , however, which were studied in l a t e pregnancy only, did not show any differences in tension development when compared to femorals from non-pregnant r a t s . This suggests that there are regional differences in changes in barium s e n s i t i v i t y during pregnancy. Barium has been postulated to act on vascular smooth muscle by at l e a s t two mechanisms. F i r s t , barium, a d i v a l e n t cation s i m i l a r to calcium, acts i n t r a c e l l u r l a r l y d i r e c t l y on the c o n t r a c t i l e mechanism, although with a lower a f f i n i t y than calcium, mimicking the a c t i v a t i n g action of calcium(6). Second, barium has been shown to influence the membrane e l e c t r i c a l properties of smooth muscle, thus i n d i r e c t l y influencing contraction where, along with other e f f e c t s , i t lowers potassium conductance, increases calcium conductance, and induces rhythmic contractions in some normally quiescent vessels (12,13, 14). Although the basis for the rhythmogenic effects of barium on vascular smooth muscle has not been c l e a r l y established, i t has been reported that the pacemaker-like action potentials that develop in r a b b i t and k i t t e n v e n t r i cular p a p i l l a r y muscles when exposed to barium are eliminated when calcium is removed from the bathing solution(15). This report is consistent with the result~ of the current study in which rhythmic contractions of aortas from 3 week pregnant rats f a i l e d to develop in calcium-free Krebs. Thus in this t i s sue, barium r h y t h m i c i t y also depends on the presence of external calcium. The fact that c o n t r a c t i l i t y was reduced, but not abolished in calciumfree Krebs, adds support to the proposed dual mechanism of action of barium. The tension induced in calcium-free Krebs (which was only 39% of that with calcium present) could be due to the d i r e c t action of barium on the contracti l e mechanism, while that in calcium containing Krebs is the sum of the d i r e c t action plus the i n d i r e c t effects on membrane e l e c t r i c a l a c t i v i t y . However, since the procedure used does not assure the removal of calcium from a l l of i t s c e l l u l a r stores, and tissue calcium measurements were not done, these results do not c l e a r l y establish t h i s . However, the fact that the increased s u s c e p t i b i l i t y of the aorta to barium r h y t h m i c i t y occurred only in l a t e pregnancy while the reduction in tension development was greater at 1 and 2 weeks of pregnancy, suggests that these two effects of barium are being modified independently by pregnancy. Since barium i t s e l f almost c e r t a i n l y plays no role in normal or pregnancy c i r c u l a t o r y dynamics, these results are of i n t e r e s t because there is evidence that barium produces e f f e c t s which are in part dependent on and s i m i l a r to
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those of calcium on the e l e c t r i c a l and mechanical properties of various types of muscles (15,16). Previous studies have indicated that vascular responsiveness to various vasoactive agents is modified in pregnancy. Thus decreased vasoconstrictor responses to angiotensin I I (2), norepinephrine (3), histamine (4), and prostaglandins (5) during pregnancy have been reported. I t is postulated that t~ese changes in vascular responsiveness contribute to the maintenance of the high, uninterrupted uteroplacental blood flow needed to support normal fetal growth, as well as to the r e d i s t r i b u t i o n of maternal blood flow and modified f l u i d dynamics of normal pregnancies. The results of the current study provide additional evidence of altered vascular function during pregnancy. Although the basis for the modified barium responsiveness has not been i d e n t i f i e d , other reports have suggested that the sex hormones involved in the maintenance of pregnancy play a role in modulating membrane e l e c t r i c a l a c t i v i t i e s of excitable tissues (17). Batra recently reported that estrogen, which is elevated in most species during pregnancy, decreases the movement of calcium into vascular smooth muscle (18). Since barium has been postulated to act in part by increasing plasma membrane calcium conductance, this e f f e c t of estrogen could help to explain the reduced barium-induced tension of blood vessels during pregnancy assuming that this e f f e c t o f estrogen is one that is maintained during in v i t r o studies. Such a change in calcium dynamics could also be involved in the pregnancyassociated decreases in vascular responsiveness to endogenous vasoactive agents whose actions depend on transmembrane calcium movements ( 2 , 3 , 4 , 5 ) . Acknowledgements This research was supported in part by a g r a n t - i n - a i d from the American Heart Association, Northern Virginia Chapter of the Virginia A f f i l i a t e . Stat i s t i c a l analyses were done on a Hewlett-Packard 9825-A Computer (NSF Grant DEB 7905844). References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18.
B NUWAYHID, Am. J. Obstet. Gynec. 135:590-596 (1979). S.M. LIEB, T. CABALUM, M ZUGAIB, R. ERKOLLA, K. TABSH, E. USHIODA, B.S. NUWAYHID, C.R. BRINKMAN, and N.S. ASSALI. Am. J. Physiol. 238: 209-213 (1980). J. DOGTEROMand W. DEJONG, Eur. J. Pharmacol. 25:267-269 (1974). J. L. HART, Fed. Proc. 39:390 (1980). L. MOOREand J.T. REEVES-~Am. J. Physiol. 239:H297-301 (1980). D.F. BOHR, Fed. Proc. 33:127-132 (1974). R.F. FURCHGOTTand S. BHADRAKOM, J. Pharmacol. Exp. Ther. 142: 39-58. (1953). J.H. ZAR, B i o s t a t i s t i c a l Analysis, p. 291, Prentice Hall, New Jersey (1974). I b i d . , p. 151. R.J. TALLARDIA and'R.B. MURRAY, Manual of Pharmacological Calculations, p. I I , Springer-Verlag, New York I - - ~ ) _ _ G. BIAMINO and P. KRUKENBERG, Am. J. Physiol. 217:376-382 (1969). D.R. HARDERand N. SPERELAKIS, Pflugers Archiv. 378:111-119 (1978). B. UVELIUS, S.R. SIGURDSSON, and B. JOHANSSON. Blood Vessels I I : 245259 (1974). K. HERMSMEYER,Am. J. Physiol. 230:1031-1036 (1976). Y. SAEKI, T. SHIBATA, and K. SHIOZAWA, Am. J. Physiol. 240:216-221 (1981) D.C. EATON and M.S. BRODWICK, J. Gen. Physiol. 75:727-750 (1980). D.R. HARDERand P.B. COULSON, J. Cell. P h y s i o l . 7 0 0 : 3 7 5 - 3 8 2 (1979). S. BATRA, Trends in Pharmacol. Sci. Oct.: 388-391 (1980).
Pergamon Pres~
BARIUM RESPONSIVENESS OF THE RAT AORTA AND FEMORAL ARTERY DURING PREGNANCY J. L. Hart Biology Department George Mason U n i v e r s i t y Fairfax, V i r g i n i a 22030 (Received in final form November 18, 1981)
Summary The barium responses of isolated a o r t i c s t r i p s and femoral a r t e r i e s from non-pregnant and pregnant rats were investigated. Barium caused concentration-related increases in tension of vessels from both pregnant and non-pregnant r a t s . The concentration-response curves of femoral a r t e r i e s from non-pregnant and 3 week pregnant rats were not d i f f e r e n t ; however c o n t r a c t i l i t y and slopes of concentration-response lines for thoracic aortas from I , 2 and 3 week pregnant rats ;were s i g n i f i c a n t l y less than those of aortas from non-pregnant r a t s . In a d d i t i o n , barium caused rhythmic contractions to develop in both femoral a r t e r i e s and aortas of 3 week pregnant rats more frequently than vessels from nonpregnant r a t s . Rhythmic contractions did not develop in aortas from 3 week pregnant rats rats in calcium-free Krebs. Since the effects of barium on the e l e c t r i c a l and mechanical a c t i v i t y of various muscles have been postulated to be s i m i l a r to and/or dependent on calcium, these r e s u l t s may indicate that changes in calcium s e n s i t i v i t y of vascular smooth muscle occur during pregnancy. Such changes may contribute to the blood flow r e d i s t r i bution and other cardiovascular adaptations of pregnancy. Pregnancy requires wide-spread and rapid changes in maternal physiology, e s p e c i a l l y in the cardiovascular system. An e s s e n t i a l l y new, low resistance high flow c i r c u i t develops (the utero-placental c i r c u i t ) , blood volume and cardiac output increase to meet the volume and flow requirements of t h i s new c i r c u i t , and blood flow is r e d i s t r i b u t e d and adjusted to accommodate the increased metabolic a c t i v i t y of various maternal organs(1). I t is of considerable i n t e r e s t to understand what factors contribute to the cardiovascular adaptations of normal pregnancy, since t h i s knowledge forms the basis for preventing or t r e a t i n g conditions which are the r e s u l t of these adjustments not occurring normally. Changes in vascular responsiveness to various vasoactive agents in many species have been reported to occur in pregnancy ( 2 , 3 , 4 , 5 ) . While i n v e s t i g a t i n g such changes in isolated blood vessels, i t was observed that bagium ions frequently caused normally quiescent vessels from l a t e pregnant rats to develop rhythmic contractions while i t did so only i n f r e q u e n t l y in vessels from non-pregnant rats. Barium i t s e l f is u n l i k e l y to play any role in normal cardiovascular dynamics; however, because of i t s dependence on and s i m i l a r i t i e s to many of the actions of calcium(6), i t is a useful tool to investigate vascular smooth 0024-3205/82/020163-07503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
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Vascular Ba 2+ Responses During Pregnancy
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muscle a c t i v i t y . The present study was designed to investigate barium responsiveness of isolated blood vessels throughout pregnancy, Both the concentration-related increases in isometric tension and the rhythmogenic actions of barium were investigated. Methods Female Sprague Dawley rats (Flow Labs, V i r g i n i a ) , 3-5 months old, were used in a l l experiments. Estrous stage and pregnancy timing were determined by vaginal smears, with the day when sperm were present designated as day one of gestation. Non-pregnant rats were used only when in estrus. Pregnant rats were studied e i t h e r in l a t e pregnancy only (20-22 days) or at approximately one week i n t e r v a l s of the 3 week gestation period (6-7, 14-16 and 20-22 days), and are hereafter referred to as the I , 2, or 3 week pregnant groups. After s a c r i f i c e with ether, blood vessels to be studied were r a p i d l y removed and placed in Krebs solution of the following composition in mM: NaCl 118.2, KCl 4.6, NaHC03 24.8, KH2P04 1.2, CaCI2(2H20) 2.5, MgS04(7 H20) 1.2, glucose I0, EDTA-Na2 .03. S p i r a l l y cut s t r i p s of the thoracic aorta (l-2mm wide, 5-8mm long) were prepared according to the method of Furchgott(7). Femoral a r t e r y cylinders (3-4mm long) were mounted on two 5-0 stainless steel wire t r i a n g l e s and tied at the apexes with 6-0 s i l k suture. The vessels were then mounted in 37o K r e b s - f i l l e d tissue chambers which were continuously gassed with 95% 02 - 5% CO2. Optimal passive tensions were applied to the vessels: 1 gram for a o r t i c s t r i p s and 0.5 grams f o r femoral a r t e r y c y l i n d e r s . Isometric tension was recorded using e i t h e r Harvard (no. 363) or Narco Biosystem (F-60) force transducers. After 90-120 min e q u i l i b r a t i o n , cumulative concentration-response curves to barium chloride (BaCI2-2H20, Sigma) were carried out. The BaCl2 stock solutions were prepared in pR 4, S04-free Krebs to prevent p r e c i p i t a t i o n . I t was added in small volumes, without osmotic compensation, to the tissue chambers to achieve the f i n a l bath concentrations which are expressed in molar (M) concentrations in figures. In experiments on the possible role of external calcium in barium-induced rhythmic contractions, 2 a o r t i c s t r i p s from each of f i v e 3 week pregnant rats were used. One s t r i p was equilibrated for 2 hours in calcium-free Krebs with repeated washings at 10-15 min i n t e r v a l s , and then exposed three times to IO-7M epinephrine in order to deplete loosely bound i n t r a c e l l u l a r calcium. After t h i s prbcedure, cumulative barium concentration-response curves were done as described above. The second s t r i p was equilibrated for 3 hours in normal calcium-containing Krebs before barium exposure. At the end of a l l experiments, vessel dimensions were measured and a f t e r 20-24 hours of dessication, dry weight was determined. Isometric tension was then expressed as mg of tension/mg dry weight. For determining differences between proportions, the Fisher exact test was used (8). Maximum tension development ( c o n t r a c t i l i t y ) was compared between groups by the t - t e s t or, i f more than 2 groups were being analyzed, by analysis of variance. I f analysis of v~riance indicated s i g n i f i c a n t differences, Dunnett's t e s t for comparison of a control (non-pregnant) mean to each other group mean (1,2,3 week pregnant) was used(g). Regression analysis of concentration-response curves and comparisons of slopes were done by the methods of T a l l a r i d a and Murray(lO). All means are expressed plus and minus standard errors (SE). Results Bariumcaused rhythmic contractions to develop in 85% (18 of 22) a o r t i c s t r i p s from 3 week pregnant rats but only 15% (3 of 20) of aortas from nonpregnant r a t s . This was a highly s i g n i f i c a n t difference (p<.O01). The
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Vascular Ba 2+ Responses During Pregnancy
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PG
A
B
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FIG. 1 Record o f isometric tension of a o r t i c s t r i p s from 3 d i f f e r e n t 3 week pregnant (PG) and I non-pregnant (N-PG) r a t . Barium was added at the A and B arrows. A l l vessels from pregnant r a t s show rhythmic c o n t r a c t i o n s . frequencies and patterns of rhythmic contractions varied considerably with frequencies normally between 4 and 12/min. However, as can be seen in Fig. 1 (upper t r a c e ) , some preparations also developed concurrent f a s t 4-12/min) and slow (.15-.25/min) rhythms. The most common pattern seen when doing complete concentration-response curves was t h a t i l l u s t r a t e d in the upper trace o f Fig. 2, where rhythmic contractions developed at intermediate concentrat i o n s , but stopped at higher concentrations as t o t a l tension continued to i n crease, and then reappeared during the wash out. Rhythmic contractions during barium exposure did not develop in any of 6 a o r t i c s t r i p s from one week pregnant r a t s and in only 1 o f 6 from two week pregnant r a t s . These rates of occurrence were not s i g n i f i c a n t l y d i f f e r e n t (p>.05) from those o f aortas from non-pregnant r a t s . In order to determine i f barium r h y t h m i c i t y occurred in vessels other than the e l a s t i c a o r t a , the more muscular femoral a r t e r y was also studied. Rhythmic contractions developed in a l l 5 femoral a r t e r i e s from 3 week pregnant r a t s during barium exposure, while occurring in only 2 o f 5 femorals from non-pregnant r a t s . Because of the small numbers used, t h i s was not a s i g n i f i c a n t d i f f e r e n c e at the .05 l e v e l , but was at the .I0 l e v e l . The f r e quencies of femoral a r t e r y rhythmic contractions ranged between 3 and 8/min and u s u a l l y occurred throughout the barium exposure with higher concentrations causing slower rhythmic contractions of a l a r g e r magnitude (Fig. 3).
166
Vascular
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Vol. 30, No. 2, 1982
Vascular Ba 2+ Responses During Pregnancy
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FIG. 4 Barium c o n c e n t r a t i o n - r e s p o n s e curves (+ SE) o f a o r t i c s t r i p s from 14 non-pregnant ( e s t r u s ) , 6 one ~ e k , 6 two week, and 24 three week pregnant r a t s . Concentration response curves o f a o r t i c s t r i p s from non-pregnant and 1,2, and 3 week pregnant r a t s are graphed in Fig. 4. A l l s t r i p s contracted in a c o n c e n t r a t i o n - r e l a t e d manner. However, maximum tension development (contractility) was less at a l l stages of pregnancy, s i g n i f i c a n t l y so at 1 and 2 weeks ( p < . 0 5 ) , and the slopes o f the ECpn-EC80 l i n e s , as determined by r e gression a n a l y s i s ( I 0 ) , were s i g n i f i c a n ~ T y less at a l l stages o f pregnancy than t h a t o f aortas from non-pregnant r a t s , w h i l e EC-50's were not s i g n i f i cantly different. However, the slope, e l e v a t i o n , and maximum tension of the barium c o n c e n t r a t i o n - r e s p o n s e curves f o r femoral a r t e r i e s from 3 week pregnant r a t s were not s i g n i f i c a n t l y d i f f e r e n t from those o f the femorals from nonpregnant r a t s (p>.05). In order to determine i f e x t e r n a l calcium was involved in the b a r i u m - i n duced rhythmic c o n t r a c t i o n s , two a o r t i c s t r i p s from each of f i v e 3 week pregnant r a t s were studied - one i n calcium " f r e e " and the o t h e r in normal calcium (2.5mM) Krebs. The records from one o f these experiments is in Fig. 2. A l l f i v e o f the aortas in the c a l c i u m - c o n t a i n i n g Krebs developed rhythmic cont r a c t i o n s , w h i l e none o f these in c a l c i u m - f r e e Krebs d i d . Mean maximum tension f o r the aortas in the c a l c i u m - f r e e Krebs was 780 + 40 mg/ mg dry w e i g h t , which was o n l y 39% o f the mean maximum tensions f o r t h e aortas in normal c a l cium. Discussion The e f f e c t s o f pregnancy on two aspects o f v a s c u l a r smooth muscle responsivesness to barium were i n v e s t i g a t e d : i n d u c t i o n o f rhythmic c o n t r a c t i o n s and sustained i s o m e t r i c c o n t r a c t i l e responses. Both aortas and femoral a r t e r i e s
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from 3 week pregnant rats developed rhythmic contractions during barium exposure more frequently than did those from non-pregnant estrus r a t s . Rhythmic contractions did not, however, develop more frequently in aortas from 1 and 2 week pregnant r a t s . Therefore, this increased s u s c e p t i b i l i t y to barium-induced r h y t h m i c i t y appears to be c h a r a c t e r i s t i c of l a t e , but not earl i e r , stages of pregnancy. Patterns of r h y t h m i c i t y varied in aortas, with concurrent fast and slow contractions occurring in some preparations. Although the basis for this was not investigated, i t has been reported ( I I ) that the patterns of rhythmic mechanical a c t i v i t y of isolated r a t a o r t i c s t r i p s depend on the number, f r e quency, and r e g u l a r i t y of active pacemaker sites w i t h i n the muscle, which can be related to the size of the s t r i p s . S t r i p size did vary some in the current study, and may have contributed to the d i f f e r e n t patterns of barium-induced contractions observed. Tension development of the aorta in reponse to barium was also modified by pregnancy, with maximum tension ( c o n t r a c t i l i t y ) and concentration-response curve slopes depressed at a l l stages of pregnancy studied indicating a reduced maximum responsiveness of the aorta to barium during pregnancy. Femoral a r t e r i e s , however, which were studied in l a t e pregnancy only, did not show any differences in tension development when compared to femorals from non-pregnant r a t s . This suggests that there are regional differences in changes in barium s e n s i t i v i t y during pregnancy. Barium has been postulated to act on vascular smooth muscle by at l e a s t two mechanisms. F i r s t , barium, a d i v a l e n t cation s i m i l a r to calcium, acts i n t r a c e l l u r l a r l y d i r e c t l y on the c o n t r a c t i l e mechanism, although with a lower a f f i n i t y than calcium, mimicking the a c t i v a t i n g action of calcium(6). Second, barium has been shown to influence the membrane e l e c t r i c a l properties of smooth muscle, thus i n d i r e c t l y influencing contraction where, along with other e f f e c t s , i t lowers potassium conductance, increases calcium conductance, and induces rhythmic contractions in some normally quiescent vessels (12,13, 14). Although the basis for the rhythmogenic effects of barium on vascular smooth muscle has not been c l e a r l y established, i t has been reported that the pacemaker-like action potentials that develop in r a b b i t and k i t t e n v e n t r i cular p a p i l l a r y muscles when exposed to barium are eliminated when calcium is removed from the bathing solution(15). This report is consistent with the result~ of the current study in which rhythmic contractions of aortas from 3 week pregnant rats f a i l e d to develop in calcium-free Krebs. Thus in this t i s sue, barium r h y t h m i c i t y also depends on the presence of external calcium. The fact that c o n t r a c t i l i t y was reduced, but not abolished in calciumfree Krebs, adds support to the proposed dual mechanism of action of barium. The tension induced in calcium-free Krebs (which was only 39% of that with calcium present) could be due to the d i r e c t action of barium on the contracti l e mechanism, while that in calcium containing Krebs is the sum of the d i r e c t action plus the i n d i r e c t effects on membrane e l e c t r i c a l a c t i v i t y . However, since the procedure used does not assure the removal of calcium from a l l of i t s c e l l u l a r stores, and tissue calcium measurements were not done, these results do not c l e a r l y establish t h i s . However, the fact that the increased s u s c e p t i b i l i t y of the aorta to barium r h y t h m i c i t y occurred only in l a t e pregnancy while the reduction in tension development was greater at 1 and 2 weeks of pregnancy, suggests that these two effects of barium are being modified independently by pregnancy. Since barium i t s e l f almost c e r t a i n l y plays no role in normal or pregnancy c i r c u l a t o r y dynamics, these results are of i n t e r e s t because there is evidence that barium produces e f f e c t s which are in part dependent on and s i m i l a r to
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Vascular Ba 2+ Responses During Pregnancy
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those of calcium on the e l e c t r i c a l and mechanical properties of various types of muscles (15,16). Previous studies have indicated that vascular responsiveness to various vasoactive agents is modified in pregnancy. Thus decreased vasoconstrictor responses to angiotensin I I (2), norepinephrine (3), histamine (4), and prostaglandins (5) during pregnancy have been reported. I t is postulated that t~ese changes in vascular responsiveness contribute to the maintenance of the high, uninterrupted uteroplacental blood flow needed to support normal fetal growth, as well as to the r e d i s t r i b u t i o n of maternal blood flow and modified f l u i d dynamics of normal pregnancies. The results of the current study provide additional evidence of altered vascular function during pregnancy. Although the basis for the modified barium responsiveness has not been i d e n t i f i e d , other reports have suggested that the sex hormones involved in the maintenance of pregnancy play a role in modulating membrane e l e c t r i c a l a c t i v i t i e s of excitable tissues (17). Batra recently reported that estrogen, which is elevated in most species during pregnancy, decreases the movement of calcium into vascular smooth muscle (18). Since barium has been postulated to act in part by increasing plasma membrane calcium conductance, this e f f e c t of estrogen could help to explain the reduced barium-induced tension of blood vessels during pregnancy assuming that this e f f e c t o f estrogen is one that is maintained during in v i t r o studies. Such a change in calcium dynamics could also be involved in the pregnancyassociated decreases in vascular responsiveness to endogenous vasoactive agents whose actions depend on transmembrane calcium movements ( 2 , 3 , 4 , 5 ) . Acknowledgements This research was supported in part by a g r a n t - i n - a i d from the American Heart Association, Northern Virginia Chapter of the Virginia A f f i l i a t e . Stat i s t i c a l analyses were done on a Hewlett-Packard 9825-A Computer (NSF Grant DEB 7905844). References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18.
B NUWAYHID, Am. J. Obstet. Gynec. 135:590-596 (1979). S.M. LIEB, T. CABALUM, M ZUGAIB, R. ERKOLLA, K. TABSH, E. USHIODA, B.S. NUWAYHID, C.R. BRINKMAN, and N.S. ASSALI. Am. J. Physiol. 238: 209-213 (1980). J. DOGTEROMand W. DEJONG, Eur. J. Pharmacol. 25:267-269 (1974). J. L. HART, Fed. Proc. 39:390 (1980). L. MOOREand J.T. REEVES-~Am. J. Physiol. 239:H297-301 (1980). D.F. BOHR, Fed. Proc. 33:127-132 (1974). R.F. FURCHGOTTand S. BHADRAKOM, J. Pharmacol. Exp. Ther. 142: 39-58. (1953). J.H. ZAR, B i o s t a t i s t i c a l Analysis, p. 291, Prentice Hall, New Jersey (1974). I b i d . , p. 151. R.J. TALLARDIA and'R.B. MURRAY, Manual of Pharmacological Calculations, p. I I , Springer-Verlag, New York I - - ~ ) _ _ G. BIAMINO and P. KRUKENBERG, Am. J. Physiol. 217:376-382 (1969). D.R. HARDERand N. SPERELAKIS, Pflugers Archiv. 378:111-119 (1978). B. UVELIUS, S.R. SIGURDSSON, and B. JOHANSSON. Blood Vessels I I : 245259 (1974). K. HERMSMEYER,Am. J. Physiol. 230:1031-1036 (1976). Y. SAEKI, T. SHIBATA, and K. SHIOZAWA, Am. J. Physiol. 240:216-221 (1981) D.C. EATON and M.S. BRODWICK, J. Gen. Physiol. 75:727-750 (1980). D.R. HARDERand P.B. COULSON, J. Cell. P h y s i o l . 7 0 0 : 3 7 5 - 3 8 2 (1979). S. BATRA, Trends in Pharmacol. Sci. Oct.: 388-391 (1980).