Berchuck et al.
nism of activation of latent recombinant transforming growth factor ~ 1 by plasmin. J Cell Bioi 1990; 110: 1361-7. 24. Sha X, Brunner AM, Purchio AF, Gentry LE. Transforming growth factor ~ 1: importance of glycosylation and acidic proteases for processing and secretion. Mol Endocrinol 1989;3: 1090-8. 25. Sipes NJ, Lyons RM, Moses HL. Isolation and characterization of Kirsten murine sarcoma virus-transformed mouse keratinocytes resistant to transforming growth factor~. Mol Carcino 1990;3:12-9.
February 1992 Am J Obstet Gynecol
26. Valverius EM, Walker-Jones D, Bates SE, et at. Production of and responsiveness to transforming growth factor-~ in normal and oncogene-transformed human mammary epithelial cells. Cancer Res 1989;49:6269-74. 27. Hubbs AF, Hahn FF, Thomassen DG. Increased resistance to transforming growth factor ~ accompanied neoplastic progression of rat tracheal epithelial cells. Carcinogenesis 1989;10;1599-605.
Prostaglandin- or endothelium-mediated vasodilation is not involved in the blunted responses of blood vessels to vasoconstrictors in pregnant rats Jean St-Louis, PhD, and Benoit Sicotte, BSc Montreal, Quebec, Canada Pregnancy is associated with decreases of blood pressure and vascular sensitivity to vasopressor agents. We have hypothesized that the increased liberation of endogenous vasodilator(s) by the vascular endothelium or other structures could mediate these blunted responses. Thoracic aorta rings of nonpregnant, 21 days pregnant, and first day post partum rats respond similarly to acetylcholine, an endothelium-dependent vasorelaxant. In contrast, the potency of the response to sodium nitroprusside, an endothelium-independent vasorelaxant, is unchanged in tissues of pregnant rats and increased (p < 0.05) in those of post partum animals. In the presence of indomethacin (10 jJ-moI/L) the three groups of tissues show a decreased potency. The effects of phenylephrine on aortic rings of both nonpregnant and pregnant rats are markedly increased in the presence of N9-monomethyl-L-arginine. Indeed, the concentration producing 50% of the maximum response of phenylephrine decreases (p < 0.001) from 50.7 to 8.02, from 93.8 to 37.6, and from 60.4 to 5.97 nmol/L with the use of N9-monomethyl-L-arginine (0.1 mmol/L) in rings from nonpregnant, pregnant, and postpartum rats, respectively. Simultaneously, the maximum response to phenylephrine increases markedly in the three groups of tissues. In the presence of N9-monomethyl-L-arginine, indomethacin does not influence the response to phenylephrine. Our results do not support the possible involvement of an endogenous vasodilator (prostaglandin-like or endothelium-derived) in the blunted responses to vasoconstrictors during pregnancy. (AM J OSSTET GVNECOL 1992;166:684-92.)
Key words: Pregnancy, vasoconstriction, endothelium-derived relaxation, prostaglandins
From the Vascular and Perinatal Pharmacology Laboratory, Research Center, Hopital Ste-Justine, and the Department of Obstetrics and Gynecology, Faculty of Medicine, Universite de Montreal. Supported fry Medical Research Council of Canada Grant No. MA9302, Inter-Service Club Council (establishment grant), and the Fonds de la Recherche en Sante du Quebec (Senior Scholarship to j.S.L.). Reported in part in abstract form in the Proceedings of the Seventh World Congress of Hypertension in Pregnancy, Bologna, Italy October 7-10, 1990. Received for publication May 29, 1991; revised September 23, 1991; accepted October 14, 1991. Reprint requests: Jean St-Louis, PhD, Centre de Recherche, Hopital Ste-Justine, 3175 Chemin Cote Ste-Catherine, Montreal, Quebec, Canada H3T 1C5. 611134366
In recent years, it has become clear that the endothelial lining of blood vessels performs many physiologic functions. Such an influence was first demonstrated by Furchgott and Zawadsky, I accounting for the vascular smooth muscle-relaxing effect of acetylcholine. It has also become evident that endothelium-dependent relaxation modulates the effects of vasoconstrictor agents· and mediates flow-dependent relaxation.' Some inhibitors of this endothelium-dependent relaxation have become available recently as, for example, Ng-monomethyl-L-arginine and L-argininosuccinic acid! Under physiologic conditions, such as pregnancy,
Volume 166 Number 2
blood pressure decreases,5. 6 possibly as a consequence of the decreased sensitivity of blood vessels to vasoconstrictor agents. This reduced sensitivity has been noted in many tissues and in some vascular beds of pregnant rats such as aorta,7 portal vein," caudal artery,9 and pulmonarylO and mesenteric S beds. This effect is not specific for a given agent, because it was observed with several vasoconstrictors, such as angiotensin, vasopressin, norepinephrine, etc." This decreased sensitivity of blood vessels of pregnant rats is similar in magnitude to the blunted pressor responses to vasoconstrictors observed in conscious pregnant rats. II . 13 The vasoconstrictor effect of many agents is reported to be diminished in the presence of the endothelium in isolated vessels. 14 Because we have recently reported that the sensitivity of mesenteric artery rings of both nonpregnant and pregnant rats to a-adrenergic agents is dependent on ·the functional presence of the endothelium,15 we undertook the present experiments to further investigate the involvement of the endogenous vasorelaxant influence of the vessel wall in the blunted responses to vasoconstrictors at the end of pregnancy and in the postpartum period in the rat. In these experiments, we look particularly at endothelium-derived relaxing factor (EDRF) and arachidonate derivatives through the cyclooxygenase pathway. The end of pregnancy (twenty-first day) and postpartum period were chosen because, under these conditions, important changes occur in vascular reactivity.7 Methods
Ten-week-old female Sprague-Dawley rats, obtained from Charles River Canada (St-Constant, Quebec), were mated with males of the same age. The morning on which vaginal smears were found to contain spermatozoa was labeled day 1 of gestation. The experiments were performed on the twenty-first day of pregnancy and between 18 and 36 hours after parturition (post partum). Unmated, age-matched (13 to 14-weekold) females served as controls. After the rats were decapitated, the thoracic aorta was rapidly removed and placed in cold Krebs solution. The aortic tube was freed from surrounding tissues and cut transversally to obtain rings 3 to 4 mm in axial length. Small hooks, made with 30-gauge needles, were carefully inserted into each ring to suspend them vertically in IS-ml glassjacketed organ baths. The tissues were attached to isometric force-displacement transducers (FT-03, Grass Instruments, Quincy, Mass.) connected to a #7 Grass Polygraph. They were stretched to a passive tension of 2.0 gm, which, in preliminary experiments (Parent and St-Louis, unpublished data), was found to be optimal for aortic rings of both nonpregnant and pregnant rats. The Krebs solution used in these experiments contained in millimoles per liter: sodium chloride, 118; sodium bicarbonate, 25; potassium chloride, 4.7; cal-
Vasodilation in blunted responses in pregnant rats
685
cium chloride, 2.5; magnesium sulfate, 1.18; potassium phosphate, 1.18; and dextrose, 5.5. The pH was 7.4 at 37° C, and the solution was bubbled with a mixture of 95% oxygen: 5% carbon dioxide. After 1 hour of equilibration, phenylephrine (1 jLmoIlL) was added to the bathing solution to measure tissue reactivity. At the plateau response of phenylephrine, the functional integrity of the endothelium was assessed by the application of acetylcholine (0.1 mmoIlL). This response was quantified as the percent relaxation of phenylephrine-induced contraction (i.e., relaxation from the plateau response to phenylephrine). After full relaxation (45 to 50 minutes), the different experimental protocols were done. In the first protocol, we measured the response to acetylcholine, an endothelium-dependent vasorelaxant, in aortic rings of nonpregnant, pregnant, and post partum rats. A cumulative concentration-response curve to phenylephrine (1 nmoIlL to 10 jLmoIlL) was charted for the tissues of each group of rats. The rings were then washed to remove phenylephrine by frequent changes in bathing fluid, and 75 to 90 minutes later a cumulative concentration-response curve to acetylcholine (l0 nmoIlL to 100 jLmol/L) was obtained after precontracting rings with phenylephrine. The phenylephrine concentration used to precontract the tissues was 1.0 jLmoIlL, which produces an increase in tissue tension of about 80% to 90% of maximum response in rings of the different groups. A similar protocol was used for a second set of tissues, except that the curve to acetylcholine was replaced by a cumulative concentration-response curve to sodium nitroprusside (l nmoIlL to 10 jLmoIlL). In a second protocol, a cumulative concentrationresponse curve to phenylephrine was first measured in the absence and then, on the same tissue, in the presence of Ng-monomethyl-L-arginine (0.10 mmol/L) or indomethacin (10 jLmol/L) to block the influence of EDRF and of endogenous derivatives of arachidonic acid, respectively. Similar groups of rats were used (nonpregnant, pregnant, and post partum). Each experimental concentration-response curve was analyzed as previously7.8 by microcomputer using the ALLFIT program to determine the concentration producing 50% of the maximum response and the maximum asymptote of the response. Statistical comparisons were made on the mean negative logarithm of the concentration producing 50% of the maximum response and mean maximum asymptote for each experimental group. Analysis of variance was performed, followed by the Dunnett test to compare each pregnant and post partum group with the nonpregnant controls. The paired t test was also performed to compare the concentration-response curve in the absence and in the presence of inhibitors in each experimental group. Analysis of variance with a two-factor mixed design and
686
St-Louis and Sicotte
February 1992 Am J Obstet Gynecol
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Fig. 1. Concentration-response curves to phenylephrine (left) and acetylcholine (right) in aortic rings of nonpregnant (jilled circles), pregnant (open circles), and post partum (open squares) rats. Symbols and bars represent the means ± SE of number of measurements shown in Table I. Abscissa indicates molar concentration of agonists in logarithm and ordinate, contractile responses in grams for phenylephrine (left panel), and the percent relaxation from precontracted level (phenylephrine, 1 fLmollL) for acetylcholine (right panel).
Table I. Sensitivity and responsiveness to phenylephrine and acetylcholine in aortic rings of nonpregnant, pregnant and postpartum rats Acetylcholine
Phenylephrine
Group
n
Negative log of concentration producing 50% of maximal response
Nonpregnant Pregnant Postpartum
20 36 16
7.26 ± 0.06 6.96 ± 0.08* 6.85 ± 0.07*
50% of maximal response (nmol/ L)
54.9 110 141
Maximum response (gm)
Negative log of concentration producing 50% of maximal response
50% of maximal response (ILmol/ L)
Maximum response (%)
2.22 ± 0.10 2.04 ± 0.05* 2.38 ± 0.10*
6.60 ± 0.09 6.68 ± 0.08 6.57 ± 0.11
0.25 0.21 0.27
71 ± 4 64 ± 4 78 ± 3
n, number of tissues tested.
*p < 0.01, evaluated by the
Dunnett test.
repeated measure of one factor l6 were also used for the indomethacin and Ng-monomethyl-L-arginine results. The agents used were L-phenylephrine hydrochloride and acetylcholine chloride obtained from Sigma Chemical Co. (St. Louis, Mo.), sodium nitroprusside from Fisher Scientific (Montreal, Quebec), Ng-monomethyl-L-arginine monoacetate from Calbiochem Biochemicals (San Diego, Calif.), and indomethacin lactose from Merck Frosst (Pointe-Claire, Quebec).
Results The effects of phenylephrine and acetylcholine on aortic rings from nonpregnant, pregnant, and post partum rats are reported in Fig. I and Table I. The responses to phenylephrine were markedly decreased in pregnant and post partum tissues, as shown by the rightward shift of their concentration-response curves in comparison with the controls (Fig. I); the maximum responses were only slightly modified. These results, reported in Table I, show that the concentration elic-
Vasodilation in blunted responses in pregnant rats
Volume 166 Number 2
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Fig. 2. Concentration-response curves to phenylephrine (left) and sodium nitroprusside (right) in aortic rings of nonpregnant (filled circles), pregnant (open circles), and post partum (open squares) rats. Symbols and bars represent means ± SE of number of measurements shown in Table II. Abscissa indicates molar concentration of agonists in logarithm and ordinate, contractile responses in grams for phenylephrine (left panel), and percent relaxation from precontracted level (phenylephrine, I j..lmoI/L) for sodium nitroprusside (right panel).
Table II. Sensitivity and responsiveness to phenylephrine and sodium nitroprusside in aortic rings of nonpregnant, pregnant, and postpartum rats Phenylephrine
Group
n
Negative log of concentration producing 50% of maximal response
Nonpregnant Pregnant Postpartum
23 20 20
7.29 ± 0.06 7.14 ± 0.04* 6.56 ± 0.05*
50% of maximal response (nmoll L)
51.7 72.7 183
Sodium nitroprusside
Maximum response (gm)
Negative log of concentration producing 50% of maximal response
50% of maximal response (nmoll L)
Maximum response (%)
2.13 ± 0.09 2.25 ± 0.08t 1.83 ± 0.07*
7.52 ± 0.10 7.38 ± 0.06 7.79 ± 0.09t
30.0 42.2 16.4
101.7 ± l.l 101.9 ± 0.8 104.9 ± l.l
n, number of tissues tested.
*p < 0.01, evaluated by the Dunnett test. tp < 0.05, evaluated by the Dunnett test.
iting 50% maXImum response of phenylephrine was significantly (p < 0.01) increased by a factor of 2 and 2.6 in (ring of) pregnant and postpartum rats, respectively, compared with nonpregnant rats, whereas the maximum response was significantly (p < 0.01) decreased in pregnant and elevated in postpartum animals. The vasorelaxant responses to acetylcholine were similar in both groups of experimental tissues relative to nonpregnant rats, in terms of concentration producing 50% maximum response and maximum response.
Fig. 2 shows the results of an identical protocol with sodium nitroprusside, an endothelium-independent vasorelaxant. The responses to phenylephrine were again decreased in pregnant and post partum tissues in comparison with nonpregnant controls (Table II). The sensitivity of the vasorelaxant response to sodium nitroprusside was increased in tissues of post partum rats compared with their nonpregnant counterparts but not modified in the pregnant rats (Fig. 2, Table II). In contrast to acetylcholine, sodium nitroprusside fully re-
688
St-Louis and Sicotte
--
February 1992 Am J Obstet Gynecol
2,0
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LOG [PEl (M) Fig. 3. Concentration-response curves to phenylephrine in absence (filled circles) and presence (open circles) of indomethacin (10 fLmol/L) in aortic rings of nonpregnant (left), pregnant, (middle), and post partum (right) rats. Symbols and bars represent means ± SE of number of measurements shown in Table III. Abscissa indicates molar concentration of phenylephrine in logarithm and ordinate, contractile responses in grams.
laxed the tissues from the precontracted level induced by 1.0 /-l-mollL phenylephrine. The maximum responses to sodium nitroprusside were identical in all groups of tissues and were not statistically different from 100% relaxation. Fig. 3 shows the effect of 10 mollL indomethacin on concentration-response curves to phenylephrine. We have previously observed, by mass spectroscopy coupled to gas chromatography, that this concentration of indomethacin (10 /-l-moIlL) totally inhibited the output of prostaglandin material in experimental conditions similar to the ones used here (Falardeau and St-Louis, unpublished data). In the present experiments, indomethacin decreased the responsiveness of nonpregnant and post partum aortic rings to phenylephrine (Fig. 3, left and right panels, respectively), as illustrated by the rightward shift of the concentration-response curves and the depression of the maximum response in post partum rats. On the other hand, it did not substantially affect the response to phenylephrine in tissues from pregnant rats (Fig. 3, middle panel). Significant decreases in the negative logarithm of the concentration required for 50% maximum response were observed in tissues of all groups (Table III), but the magnitude of this effect was much less in pregnant rats. The maximum responses were decreased in nonpregnant and postpartum tissues (p < 0.001), suggesting that an arachidonic acid derivative was secreted on stimulation
with phenylephrine and that it exerted contractile properties on the aortic rings. In a similar protocol we tested the blockade of EDRF liberation by Ng-monomethyl-L-arginine. Fig. 4 shows that Ng-monomethyl-L-arginine (0.1 mmoll L) substantially increased the responses of the aortic rings in the three groups of animals. This concentration of Ngmonomethyl-L-arginine (0.1 mmollL) was shown to almost totally abolish the vasorelaxant response to carbachol in phenylephrine-precontracted aortic rings of the rat (St-Louis and Sicotte, preliminary experiments). This was manifested by both an augmented potency (leftward shift of the concentration-response curves) and increased maximum responses. These effects were highly significant (Table IV) for the three groups of tissues, but the increase in potency was of a smaller magnitude in pregnant animals than in nonpregnant and post partum rats, whereas the enhancement of the maximum response was similar in all three tissue types. This is demonstrated by smaller increases in the negative logarithm of the concentration producing 50% maximum response values in pregnant than in nonpregnant and post partum tissues, which are respectively 0.39 ± 0.04, 0.80 ± 0.09, and 1.05 ± 0.10 units of the negative logarithm of the concentration producing 50% maximum response (all p < 0.001). These observations suggest that, in intact aortic rings of the rat, the effect of phenylephrine is
Vasodilation in blunted responses in pregnant rats
Volume 166 Number 2
689
Table III. Effect of indomethacin on sensitivity and responsiveness to phenylephrine in aortic rings of nonpregnant, pregnant, and postpartum rats Control
Indomethacin
Group
n
Negative log of concentration producing 50% of maximal response (nmoIlL)*
Nonpregnant
20
7.36 ± 0.06 (43.7)
1.77 ± 0.09
Pregnant
24
6.92 ± 0.05 (121)t
2.29 ± 0.08t
Postpartum
18
7.09 ± 0.08 (81.6)t
1.69 ± 0.07
Maximal response (gm)
Negative log of concentration producing 50% of maximal response (nmoll L)*
Maximal response (%)
7.06 ± 0.08 (86.8) p
nism of activation of latent recombinant transforming growth factor ~ 1 by plasmin. J Cell Bioi 1990; 110: 1361-7. 24. Sha X, Brunner AM, Purchio AF, Gentry LE. Transforming growth factor ~ 1: importance of glycosylation and acidic proteases for processing and secretion. Mol Endocrinol 1989;3: 1090-8. 25. Sipes NJ, Lyons RM, Moses HL. Isolation and characterization of Kirsten murine sarcoma virus-transformed mouse keratinocytes resistant to transforming growth factor~. Mol Carcino 1990;3:12-9.
February 1992 Am J Obstet Gynecol
26. Valverius EM, Walker-Jones D, Bates SE, et at. Production of and responsiveness to transforming growth factor-~ in normal and oncogene-transformed human mammary epithelial cells. Cancer Res 1989;49:6269-74. 27. Hubbs AF, Hahn FF, Thomassen DG. Increased resistance to transforming growth factor ~ accompanied neoplastic progression of rat tracheal epithelial cells. Carcinogenesis 1989;10;1599-605.
Prostaglandin- or endothelium-mediated vasodilation is not involved in the blunted responses of blood vessels to vasoconstrictors in pregnant rats Jean St-Louis, PhD, and Benoit Sicotte, BSc Montreal, Quebec, Canada Pregnancy is associated with decreases of blood pressure and vascular sensitivity to vasopressor agents. We have hypothesized that the increased liberation of endogenous vasodilator(s) by the vascular endothelium or other structures could mediate these blunted responses. Thoracic aorta rings of nonpregnant, 21 days pregnant, and first day post partum rats respond similarly to acetylcholine, an endothelium-dependent vasorelaxant. In contrast, the potency of the response to sodium nitroprusside, an endothelium-independent vasorelaxant, is unchanged in tissues of pregnant rats and increased (p < 0.05) in those of post partum animals. In the presence of indomethacin (10 jJ-moI/L) the three groups of tissues show a decreased potency. The effects of phenylephrine on aortic rings of both nonpregnant and pregnant rats are markedly increased in the presence of N9-monomethyl-L-arginine. Indeed, the concentration producing 50% of the maximum response of phenylephrine decreases (p < 0.001) from 50.7 to 8.02, from 93.8 to 37.6, and from 60.4 to 5.97 nmol/L with the use of N9-monomethyl-L-arginine (0.1 mmol/L) in rings from nonpregnant, pregnant, and postpartum rats, respectively. Simultaneously, the maximum response to phenylephrine increases markedly in the three groups of tissues. In the presence of N9-monomethyl-L-arginine, indomethacin does not influence the response to phenylephrine. Our results do not support the possible involvement of an endogenous vasodilator (prostaglandin-like or endothelium-derived) in the blunted responses to vasoconstrictors during pregnancy. (AM J OSSTET GVNECOL 1992;166:684-92.)
Key words: Pregnancy, vasoconstriction, endothelium-derived relaxation, prostaglandins
From the Vascular and Perinatal Pharmacology Laboratory, Research Center, Hopital Ste-Justine, and the Department of Obstetrics and Gynecology, Faculty of Medicine, Universite de Montreal. Supported fry Medical Research Council of Canada Grant No. MA9302, Inter-Service Club Council (establishment grant), and the Fonds de la Recherche en Sante du Quebec (Senior Scholarship to j.S.L.). Reported in part in abstract form in the Proceedings of the Seventh World Congress of Hypertension in Pregnancy, Bologna, Italy October 7-10, 1990. Received for publication May 29, 1991; revised September 23, 1991; accepted October 14, 1991. Reprint requests: Jean St-Louis, PhD, Centre de Recherche, Hopital Ste-Justine, 3175 Chemin Cote Ste-Catherine, Montreal, Quebec, Canada H3T 1C5. 611134366
In recent years, it has become clear that the endothelial lining of blood vessels performs many physiologic functions. Such an influence was first demonstrated by Furchgott and Zawadsky, I accounting for the vascular smooth muscle-relaxing effect of acetylcholine. It has also become evident that endothelium-dependent relaxation modulates the effects of vasoconstrictor agents· and mediates flow-dependent relaxation.' Some inhibitors of this endothelium-dependent relaxation have become available recently as, for example, Ng-monomethyl-L-arginine and L-argininosuccinic acid! Under physiologic conditions, such as pregnancy,
Volume 166 Number 2
blood pressure decreases,5. 6 possibly as a consequence of the decreased sensitivity of blood vessels to vasoconstrictor agents. This reduced sensitivity has been noted in many tissues and in some vascular beds of pregnant rats such as aorta,7 portal vein," caudal artery,9 and pulmonarylO and mesenteric S beds. This effect is not specific for a given agent, because it was observed with several vasoconstrictors, such as angiotensin, vasopressin, norepinephrine, etc." This decreased sensitivity of blood vessels of pregnant rats is similar in magnitude to the blunted pressor responses to vasoconstrictors observed in conscious pregnant rats. II . 13 The vasoconstrictor effect of many agents is reported to be diminished in the presence of the endothelium in isolated vessels. 14 Because we have recently reported that the sensitivity of mesenteric artery rings of both nonpregnant and pregnant rats to a-adrenergic agents is dependent on ·the functional presence of the endothelium,15 we undertook the present experiments to further investigate the involvement of the endogenous vasorelaxant influence of the vessel wall in the blunted responses to vasoconstrictors at the end of pregnancy and in the postpartum period in the rat. In these experiments, we look particularly at endothelium-derived relaxing factor (EDRF) and arachidonate derivatives through the cyclooxygenase pathway. The end of pregnancy (twenty-first day) and postpartum period were chosen because, under these conditions, important changes occur in vascular reactivity.7 Methods
Ten-week-old female Sprague-Dawley rats, obtained from Charles River Canada (St-Constant, Quebec), were mated with males of the same age. The morning on which vaginal smears were found to contain spermatozoa was labeled day 1 of gestation. The experiments were performed on the twenty-first day of pregnancy and between 18 and 36 hours after parturition (post partum). Unmated, age-matched (13 to 14-weekold) females served as controls. After the rats were decapitated, the thoracic aorta was rapidly removed and placed in cold Krebs solution. The aortic tube was freed from surrounding tissues and cut transversally to obtain rings 3 to 4 mm in axial length. Small hooks, made with 30-gauge needles, were carefully inserted into each ring to suspend them vertically in IS-ml glassjacketed organ baths. The tissues were attached to isometric force-displacement transducers (FT-03, Grass Instruments, Quincy, Mass.) connected to a #7 Grass Polygraph. They were stretched to a passive tension of 2.0 gm, which, in preliminary experiments (Parent and St-Louis, unpublished data), was found to be optimal for aortic rings of both nonpregnant and pregnant rats. The Krebs solution used in these experiments contained in millimoles per liter: sodium chloride, 118; sodium bicarbonate, 25; potassium chloride, 4.7; cal-
Vasodilation in blunted responses in pregnant rats
685
cium chloride, 2.5; magnesium sulfate, 1.18; potassium phosphate, 1.18; and dextrose, 5.5. The pH was 7.4 at 37° C, and the solution was bubbled with a mixture of 95% oxygen: 5% carbon dioxide. After 1 hour of equilibration, phenylephrine (1 jLmoIlL) was added to the bathing solution to measure tissue reactivity. At the plateau response of phenylephrine, the functional integrity of the endothelium was assessed by the application of acetylcholine (0.1 mmoIlL). This response was quantified as the percent relaxation of phenylephrine-induced contraction (i.e., relaxation from the plateau response to phenylephrine). After full relaxation (45 to 50 minutes), the different experimental protocols were done. In the first protocol, we measured the response to acetylcholine, an endothelium-dependent vasorelaxant, in aortic rings of nonpregnant, pregnant, and post partum rats. A cumulative concentration-response curve to phenylephrine (1 nmoIlL to 10 jLmoIlL) was charted for the tissues of each group of rats. The rings were then washed to remove phenylephrine by frequent changes in bathing fluid, and 75 to 90 minutes later a cumulative concentration-response curve to acetylcholine (l0 nmoIlL to 100 jLmol/L) was obtained after precontracting rings with phenylephrine. The phenylephrine concentration used to precontract the tissues was 1.0 jLmoIlL, which produces an increase in tissue tension of about 80% to 90% of maximum response in rings of the different groups. A similar protocol was used for a second set of tissues, except that the curve to acetylcholine was replaced by a cumulative concentration-response curve to sodium nitroprusside (l nmoIlL to 10 jLmoIlL). In a second protocol, a cumulative concentrationresponse curve to phenylephrine was first measured in the absence and then, on the same tissue, in the presence of Ng-monomethyl-L-arginine (0.10 mmol/L) or indomethacin (10 jLmol/L) to block the influence of EDRF and of endogenous derivatives of arachidonic acid, respectively. Similar groups of rats were used (nonpregnant, pregnant, and post partum). Each experimental concentration-response curve was analyzed as previously7.8 by microcomputer using the ALLFIT program to determine the concentration producing 50% of the maximum response and the maximum asymptote of the response. Statistical comparisons were made on the mean negative logarithm of the concentration producing 50% of the maximum response and mean maximum asymptote for each experimental group. Analysis of variance was performed, followed by the Dunnett test to compare each pregnant and post partum group with the nonpregnant controls. The paired t test was also performed to compare the concentration-response curve in the absence and in the presence of inhibitors in each experimental group. Analysis of variance with a two-factor mixed design and
686
St-Louis and Sicotte
February 1992 Am J Obstet Gynecol
80,--------------------.---,
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-z
2
60
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LOG [ACH] (M)
LOG [PEl (M)
Fig. 1. Concentration-response curves to phenylephrine (left) and acetylcholine (right) in aortic rings of nonpregnant (jilled circles), pregnant (open circles), and post partum (open squares) rats. Symbols and bars represent the means ± SE of number of measurements shown in Table I. Abscissa indicates molar concentration of agonists in logarithm and ordinate, contractile responses in grams for phenylephrine (left panel), and the percent relaxation from precontracted level (phenylephrine, 1 fLmollL) for acetylcholine (right panel).
Table I. Sensitivity and responsiveness to phenylephrine and acetylcholine in aortic rings of nonpregnant, pregnant and postpartum rats Acetylcholine
Phenylephrine
Group
n
Negative log of concentration producing 50% of maximal response
Nonpregnant Pregnant Postpartum
20 36 16
7.26 ± 0.06 6.96 ± 0.08* 6.85 ± 0.07*
50% of maximal response (nmol/ L)
54.9 110 141
Maximum response (gm)
Negative log of concentration producing 50% of maximal response
50% of maximal response (ILmol/ L)
Maximum response (%)
2.22 ± 0.10 2.04 ± 0.05* 2.38 ± 0.10*
6.60 ± 0.09 6.68 ± 0.08 6.57 ± 0.11
0.25 0.21 0.27
71 ± 4 64 ± 4 78 ± 3
n, number of tissues tested.
*p < 0.01, evaluated by the
Dunnett test.
repeated measure of one factor l6 were also used for the indomethacin and Ng-monomethyl-L-arginine results. The agents used were L-phenylephrine hydrochloride and acetylcholine chloride obtained from Sigma Chemical Co. (St. Louis, Mo.), sodium nitroprusside from Fisher Scientific (Montreal, Quebec), Ng-monomethyl-L-arginine monoacetate from Calbiochem Biochemicals (San Diego, Calif.), and indomethacin lactose from Merck Frosst (Pointe-Claire, Quebec).
Results The effects of phenylephrine and acetylcholine on aortic rings from nonpregnant, pregnant, and post partum rats are reported in Fig. I and Table I. The responses to phenylephrine were markedly decreased in pregnant and post partum tissues, as shown by the rightward shift of their concentration-response curves in comparison with the controls (Fig. I); the maximum responses were only slightly modified. These results, reported in Table I, show that the concentration elic-
Vasodilation in blunted responses in pregnant rats
Volume 166 Number 2
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Table II. Sensitivity and responsiveness to phenylephrine and sodium nitroprusside in aortic rings of nonpregnant, pregnant, and postpartum rats Phenylephrine
Group
n
Negative log of concentration producing 50% of maximal response
Nonpregnant Pregnant Postpartum
23 20 20
7.29 ± 0.06 7.14 ± 0.04* 6.56 ± 0.05*
50% of maximal response (nmoll L)
51.7 72.7 183
Sodium nitroprusside
Maximum response (gm)
Negative log of concentration producing 50% of maximal response
50% of maximal response (nmoll L)
Maximum response (%)
2.13 ± 0.09 2.25 ± 0.08t 1.83 ± 0.07*
7.52 ± 0.10 7.38 ± 0.06 7.79 ± 0.09t
30.0 42.2 16.4
101.7 ± l.l 101.9 ± 0.8 104.9 ± l.l
n, number of tissues tested.
*p < 0.01, evaluated by the Dunnett test. tp < 0.05, evaluated by the Dunnett test.
iting 50% maXImum response of phenylephrine was significantly (p < 0.01) increased by a factor of 2 and 2.6 in (ring of) pregnant and postpartum rats, respectively, compared with nonpregnant rats, whereas the maximum response was significantly (p < 0.01) decreased in pregnant and elevated in postpartum animals. The vasorelaxant responses to acetylcholine were similar in both groups of experimental tissues relative to nonpregnant rats, in terms of concentration producing 50% maximum response and maximum response.
Fig. 2 shows the results of an identical protocol with sodium nitroprusside, an endothelium-independent vasorelaxant. The responses to phenylephrine were again decreased in pregnant and post partum tissues in comparison with nonpregnant controls (Table II). The sensitivity of the vasorelaxant response to sodium nitroprusside was increased in tissues of post partum rats compared with their nonpregnant counterparts but not modified in the pregnant rats (Fig. 2, Table II). In contrast to acetylcholine, sodium nitroprusside fully re-
688
St-Louis and Sicotte
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February 1992 Am J Obstet Gynecol
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LOG [PEl (M) Fig. 3. Concentration-response curves to phenylephrine in absence (filled circles) and presence (open circles) of indomethacin (10 fLmol/L) in aortic rings of nonpregnant (left), pregnant, (middle), and post partum (right) rats. Symbols and bars represent means ± SE of number of measurements shown in Table III. Abscissa indicates molar concentration of phenylephrine in logarithm and ordinate, contractile responses in grams.
laxed the tissues from the precontracted level induced by 1.0 /-l-mollL phenylephrine. The maximum responses to sodium nitroprusside were identical in all groups of tissues and were not statistically different from 100% relaxation. Fig. 3 shows the effect of 10 mollL indomethacin on concentration-response curves to phenylephrine. We have previously observed, by mass spectroscopy coupled to gas chromatography, that this concentration of indomethacin (10 /-l-moIlL) totally inhibited the output of prostaglandin material in experimental conditions similar to the ones used here (Falardeau and St-Louis, unpublished data). In the present experiments, indomethacin decreased the responsiveness of nonpregnant and post partum aortic rings to phenylephrine (Fig. 3, left and right panels, respectively), as illustrated by the rightward shift of the concentration-response curves and the depression of the maximum response in post partum rats. On the other hand, it did not substantially affect the response to phenylephrine in tissues from pregnant rats (Fig. 3, middle panel). Significant decreases in the negative logarithm of the concentration required for 50% maximum response were observed in tissues of all groups (Table III), but the magnitude of this effect was much less in pregnant rats. The maximum responses were decreased in nonpregnant and postpartum tissues (p < 0.001), suggesting that an arachidonic acid derivative was secreted on stimulation
with phenylephrine and that it exerted contractile properties on the aortic rings. In a similar protocol we tested the blockade of EDRF liberation by Ng-monomethyl-L-arginine. Fig. 4 shows that Ng-monomethyl-L-arginine (0.1 mmoll L) substantially increased the responses of the aortic rings in the three groups of animals. This concentration of Ngmonomethyl-L-arginine (0.1 mmollL) was shown to almost totally abolish the vasorelaxant response to carbachol in phenylephrine-precontracted aortic rings of the rat (St-Louis and Sicotte, preliminary experiments). This was manifested by both an augmented potency (leftward shift of the concentration-response curves) and increased maximum responses. These effects were highly significant (Table IV) for the three groups of tissues, but the increase in potency was of a smaller magnitude in pregnant animals than in nonpregnant and post partum rats, whereas the enhancement of the maximum response was similar in all three tissue types. This is demonstrated by smaller increases in the negative logarithm of the concentration producing 50% maximum response values in pregnant than in nonpregnant and post partum tissues, which are respectively 0.39 ± 0.04, 0.80 ± 0.09, and 1.05 ± 0.10 units of the negative logarithm of the concentration producing 50% maximum response (all p < 0.001). These observations suggest that, in intact aortic rings of the rat, the effect of phenylephrine is
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Table III. Effect of indomethacin on sensitivity and responsiveness to phenylephrine in aortic rings of nonpregnant, pregnant, and postpartum rats Control
Indomethacin
Group
n
Negative log of concentration producing 50% of maximal response (nmoIlL)*
Nonpregnant
20
7.36 ± 0.06 (43.7)
1.77 ± 0.09
Pregnant
24
6.92 ± 0.05 (121)t
2.29 ± 0.08t
Postpartum
18
7.09 ± 0.08 (81.6)t
1.69 ± 0.07
Maximal response (gm)
Negative log of concentration producing 50% of maximal response (nmoll L)*
Maximal response (%)
7.06 ± 0.08 (86.8) p
