Reductions In Uterine Perfusion Pressure Rats Research Articles

Agonistic Autoantibodies to the Angiotensin II Type I Receptor Cause Pathophysiologic Characteristics of Preeclampsia

Preeclampsia (PE), new-onset hypertension with proteinuria during pregnancy, is associated with increased reactive oxygen species, the vasoactive peptide endothelin-1 (ET-1), T and B lymphocytes, soluble antiangiogenic factors sFlt-1 and sEndoglin (sFlt-1 and sEng), and agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA). One important area of investigation for our laboratory was to determine what role AT1-AA plays in the pathophysiology associated with PE. To achieve this goal, we examined the effect of AT1-AA suppression on hypertension in response to placental ischemia as well as the effect of AT1-AA on increased blood pressure, ET-1, reactive oxygen species, and sFlt-1 in normal pregnant rats (NP). We demonstrated reductions in uterine perfusion pressure (RUPP) to be a stimulus for AT1-AA during pregnancy. We utilized the technique of B-cell depletion to suppress circulating AT1-AA in RUPP rats and found that AT1-AA suppression in RUPP rats was associated with lower blood pressure and ET-1 activation. To determine a role for AT1-AA to mediate hypertension during pregnancy, we infused purified rat AT1-AA (1:50) into NP rats, and analyzed blood pressure and soluble factors. We consistently found that AT1-AA infused rats had significantly increased AT1-AA and blood pressure above NP rats. This hypertension was associated with significantly increased ET-1 in renal cortices (11-fold) and placenta (4-fold), and there was an approximately 2- to 3-fold increase in placental oxidative stress. Furthermore, antiangiogenic factors sFlt-1 and sEng were significantly increased in the AT1-AA induced hypertensive group compared with the NP controls. Collectively, these data indicated an important role for AT1-AA stimulated in response to placental ischemia that caused hypertension during pregnancy.

Hypertension in response to chronic reductions in uterine perfusion in pregnant rats: effect of tumor necrosis factor-alpha blockade.

Reductions in uterine perfusion pressure (RUPP) in pregnant rats is associated with increased tumor necrosis factor-alpha (TNF-alpha). This study was designed to determine the role of endogenous TNF-alpha in mediating changes in arterial pressure and endothelin-1 (ET-1) in RUPP rats. To achieve this goal we examined the effect of RUPP in the presence and absence of a TNF-alpha-soluble receptor, etanerecept (0.4 mg/kg). Mean arterial pressure increased from 102+/-1 mm Hg in normal pregnant (NP) rats to 134+/-3 mm Hg (P<0.05) in RUPP rats. Serum TNF-alpha increased to 40+/-7.6 pg/mL in RUPP rats (n=24) versus 14.8+/-3.3 pg/mL (n=16; P<0.05) in NP rats. Administration of etanerecept decreased TNF-alpha in RUPP rats (n=20) to 17.2+/-3 pg/mL and mean arterial pressure to 118+/-2 mm Hg (P<0.05). Tissue ET-1 decreased in etanerecept-treated RUPP rats compared with control RUPP rats. The direct effect of TNF-alpha blockade on endothelial activation in response to placental ischemia was examined in human umbilical vein endothelial cells. ET-1 secreted from human umbilical vein endothelial cells treated with RUPP serum was 59.2+16 pg/mg and decreased when etanerecept was added to the medium with RUPP serum (7.60+/-0.77 pg/mg), as well as in response to serum from etanerecept-treated RUPP rats (7.30+/-0.55 pg/mg; P<0.001). ET-1 secreted from human umbilical vein endothelial cells was 15.6+/-2 pg/mg when treated with NP serum. These data support the hypothesis that endogenous TNF-alpha is an important stimulus for ET-1 in response to placental ischemia and is important in mediating endothelial cell activation and hypertension during pregnancy.

Autoantibodies to the angiotensin type I receptor in response to placental ischemia and tumor necrosis factor alpha in pregnant rats.

Circulating factors, such as agonistic autoantibodies to the angiotensin II type 1 (AT1) receptor (AT1-AAs), and inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), are suggested to be important links between placental ischemia and hypertension in preeclamptic women. The purpose of this study was to determine the role of placental ischemia and TNF-alpha in stimulating the AT1-AA and the importance of AT1 receptor activation in mediating hypertension during reductions in uterine perfusion pressure (RUPP) and chronic TNF-alpha excess in pregnant rats. Increased mean arterial pressure in RUPP pregnant rats (122+/-1 mm Hg RUPP versus 101+/-1 mm Hg normal pregnant [NP]; P<0.001) was associated with increased circulating TNF-alpha (RUPP 48+/-13 pg/mL versus N 8+/-1 pg/mL; P<0.05) and AT1-AA (RUPP 15.3+/-1.6 U versus NP 0.6+/-0.3 U; P<0.001). Moreover, TNF-alpha-induced hypertension (97+/-2 to 112+/-2 mm Hg; P<0.05) in pregnant rats was associated with AT1-AA production (TNF-alpha rats 9.2+/-2.3 U versus NP rats 1.0+/-0.8 U; P<0.05). To determine the importance of AT1 receptor activation in mediating hypertension in RUPP- and TNF-alpha-treated rats, we administered an AT1 receptor antagonist to RUPP-, TNF-alpha-treated, and NP rats. Blood pressure responses were attenuated in RUPP rats (Delta 32 mm Hg versus Delta 20 mm Hg, NP; P<0.001), as well as in TNF-alpha-treated rats (Delta 10 mm Hg versus Delta 5 mm Hg, NP; P<0.05). Collectively, these data indicate that placental ischemia and TNF-alpha are important stimuli of AT1-AA, and activation of the AT1 receptor appears to, in part, mediate hypertension produced by RUPP and TNF-alpha in pregnant rats.

Differential [Ca2+]i signaling of vasoconstriction in mesenteric microvessels of normal and reduced uterine perfusion pregnant rats

Vascular resistance and blood pressure (BP) are reduced during late normal pregnancy (Norm-Preg). In contrast, studies in human preeclampsia and in animal models of hypertension in pregnancy (HTN-Preg) have suggested that localized reduction in uterine perfusion pressure (RUPP) in late pregnancy is associated with increased systemic vascular resistance and BP; however, the vascular mechanisms involved are unclear. Because Ca2+ is a major determinant of vascular contraction, we hypothesized that the intracellular free calcium concentration ([Ca2+]i) signaling of vasoconstriction is differentially regulated in systemic microvessels during normal and RUPP in late pregnancy. Pressurized mesenteric microvessels from Norm-Preg and RUPP rats were loaded with fura 2 in preparation for simultaneous measurement of diameter and [Ca2+]i (presented as fura 2 340/380 ratio). Basal [Ca2+]i was lower in RUPP (0.73 +/- 0.03) compared with Norm-Preg rats (0.82 +/- 0.03). Membrane depolarization by 96 mM KCl, phenylephrine (Phe, 10(-5) M), angiotensin II (ANG II, 10(-7) M), or endothelin-1 (ET-1, 10(-7) M) caused an initial peak followed by maintained vasoconstriction and [Ca2+]i. KCl caused similar peak vasoconstriction and [Ca2+]i in Norm-Preg (45.5 +/- 3.3 and 0.89 +/- 0.02%) and RUPP rats (46.3 +/- 2.1 and 0.87 +/- 0.01%). Maximum vasoconstriction to Phe, ANG II, and ET-1 was not significantly different between Norm-Preg (28.6 +/- 4.8, 32.5 +/- 6.3, and 40 +/- 4.6%, respectively) and RUPP rats (27.8 +/- 5.9, 34.4 +/- 4.3, and 38.8 +/- 4.1%, respectively). In contrast, the initial Phe-, ANG II-, and ET-1-induced 340/380 ratio ([Ca2+]i) was reduced in RUPP (0.83 +/- 0.02, 0.82 +/- 0.02, and 0.83 +/- 0.03, respectively) compared with Norm-Preg rats (0.95 +/- 0.04, 0.93 +/- 0.01, and 0.92 +/- 0.02, respectively). Also, the [Ca2+]i-vasoconstriction relationship was similar in KCl-treated but shifted to the left in Phe-, ANG II-, and ET-1-treated microvessels of RUPP compared with Norm-Preg rats. The lower agonist-induced [Ca2+]i signal of vasoconstriction and the leftward shift in the [Ca2+]i-vasoconstriction relationship in microvessels of RUPP compared with Norm-Preg rats suggest activation of [Ca2+]i sensitization pathway(s). The similarity in vasoconstriction in RUPP and Norm-Preg rats suggests that such a [Ca2+]i sensitization pathway(s) may also provide a feedback effect on Ca2+ mobilization/homeostatic mechanisms to protect against excessive vasoconstriction in systemic microvessels during RUPP in late pregnancy.

Endothelial cell activation in response to placental ischemia in pregnant rats is mediated by agonistic autoantibodies to the angiotensin type I receptor

The release of soluble factors such as endothelin‐1 (ET‐1) and agonistic autoantibodies to the angiotensin II (AngII) type I receptor (AT1‐AA) may be important links between maternal endothelial cell activation and placental ischemia during hypertension in preeclamptic women. We recently demonstrated a role for AT1 receptor activation in mediating ET‐1 production by endothelial cells exposed to serum from pregnant rats with reductions in uterine perfusion pressure (RUPP). The purpose of this study was to determine if activation of the AT1 receptor on endothelial cells in response to placental ischemia is mediated by the AT1‐AA. To achieve this goal, HUVECs were exposed to RUPP serum in the presence and absence of a blocking peptide corresponding to the AT1AA binding site. Arterial pressure was significantly higher in RUPP rats (135+/−2mmHg)) than in normal pregnant rats (106+/−1mmHg). Six hours after exposure to control RUPP serum (n=14), the ET‐1 concentration in cell culture supernatant was 45.8+/− 7.2 pg/ml as compared to 25.0+/− 2.8 pg/ml from cell supernatant exposed to serum from normal pregnant rats (n=17). Interestingly, the increase in ET‐1 production in response to RUPP serum was abolished (20.5+/−3.0 pg/ml; n=17) by pretreatment with the AT1AA blocking peptide. These data indicate that the AT1AA mediates the increase in ET‐1 production induced by serum from pregnant rats exposed to placental ischemia.

Systemic hemodynamic and regional blood flow changes in response to chronic reductions in uterine perfusion pressure in pregnant rats

Preeclampsia (PE) is associated with increased total peripheral resistance (TPR), reduced cardiac output (CO), and diminished uterine and placental blood flow. We have developed an animal model that employs chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats to generate a "preeclamptic-like" state during late gestation that is characterized by hypertension, proteinuria, and endothelial dysfunction. Although this animal model has many characteristics of human PE, the systemic hemodynamic and regional changes in blood flow that occur in response to chronic RUPP remains unknown. Therefore, we hypothesized that RUPP would decrease uteroplacental blood flow and CO, and increase TPR. Mean arterial pressure (MAP), CO, cardiac index (CI), TPR, and regional blood flow to various tissues were measured using radiolabeled microspheres in the following two groups of conscious rats: normal pregnant rats (NP; n = 8) and RUPP rats (n = 8). MAP was increased (132 +/- 4 vs. 99 +/- 3 mmHg) in the RUPP rats compared with the NP dams. The hypertension in RUPP rats was associated with increased TPR (2.15 +/- 0.02 vs. 0.98 +/- 0.08 mmHg x ml(-1) x min(-1)) and decreased CI (246 +/- 20 vs. 348 +/- 19 ml x min(-1) x kg(-1), P < 0.002) when contrasted with NP dams. Furthermore, uterine (0.16 +/- 0.03 vs. 0.38 +/- 0.09 ml x min(-1) x g tissue(-1)) and placental blood flow (0.30 +/- 0.08 vs. 0.70 +/- 0.10 ml x min(-1) x g tissue(-1)) were decreased in RUPP compared with the NP dams. These data demonstrate that the RUPP model of pregnancy-induced hypertension has systemic hemodynamic and regional blood flow alterations that are strikingly similar to those observed in women with PE.

Effects of reduced uterine perfusion pressure on blood pressure and metabolic factors in the pregnant rat

Placental ischemia is thought to be the initiating factor in preeclampsia. Although elements of the metabolic syndrome are often present in preeclamptic pregnancies, it remains unclear whether these metabolic aberrations presage or result from placental ischemia. This purpose of this study was to examine metabolic factors in response to chronic reductions in uterine perfusion pressure (RUPP). We measured arterial pressure, fasting plasma concentrations of triglycerides (TG), glucose, resistin, insulin and glucose tolerance at day 19 of gestation. Mean arterial pressure increased (130 ± 2.1 vs. 100 ± 4.3 mm Hg) and fetal weight decreased (1.93 ± 0.08 vs. 2.19 ± 0.06 g) in RUPP rats compared to normal pregnant (NP) rats. Maternal fasting glucose (4.2 ± 0.3 vs. 3.1 ± 0.4 mmol/l; P = 0.05) tended to increase in RUPP compared to NP rats. Serum TGs (2.62 ± 0.29 vs. 2.45 ± 0.51 mmol/l), insulin (9.9 ± 0.7 vs. 8.5 ± 0.7 μU/ml), resistin (46.25 ± 4.19 vs. 49.71 ± 4.01 pg/ml), and glucose area under the curve (650 ± 35 vs. 570 ± 34 mmol/min/l) were not different between the RUPP and NP rats. Although these findings do not discount the hypothesis that pre-existing symptoms of the metabolic syndrome may contribute to the onset of preeclampsia in women, these data clearly show that pregnancy induced hypertension resulting from RUPP does not elicit manifestations of the metabolic syndrome in late gestation rats.

Agonistic autoantibodies to the AT1 receptor in rat models of preeclampsia: induced by chronic reductions in uterine perfusion pressure (RUPP) and low dose TNFalpha infusion

Recent studies have found that the IgG fraction from preeclamptic women contains an angiotensin-1 receptor autoantibody (AT1R-AA) that stimulates the angiotensin II type I (AT1) receptor, suggesting that these antibodies could contribute to the pathogenesis of preeclampsia. Therefore, the purpose of this study was to determine whether pregnant rats with chronic reductions in uterine perfusion pressure (RUPP) or low-dose TNFa infusion, develop AT1R-AA. AT1R-AA were detected by the chronotropic responses to AT1 receptor–mediated stimulation of cultured neonatal rat cardiomyocytes coupled with receptor-specific antagonists. Mean arterial pressure (MAP) was significantly higher in RUPP rats (137±1mmHg)) than in normal pregnant (NP) rats (107±1mmHg). The hypertension in the RUPP rats was associated with significant elevations in agonistic antibodies capable of activating the angiotensin AT1 receptor (RUPP, 15.3±1.6 vs. NP, 0.6±0.3 Δbeats/min). Pregnant rats with low dose TNFa infusion (5 days at a rate of 50 ng/d during days 14 to 19 of gestation in pregnant rats) had higher blood pressures (125 + 1mm Hg) than NP rats (101+2mm Hg) also develop AT1R-AA(9.2±2.3 vs. 1.0±0.8 Δbeats/min). Low dose TNFa in virgin rats did not increase MAP or develop AT1R-AA. We also investigated TNFa expression in the placentas of rats with RUPP and could show that TNFa expression is upregulated (p<0.05) Serum TNFa levels were nearly 3fold higher in the RUPP rats compared with NP (p<0.05). In summary, we found that AT1R-AA are present in two different animal models for preeclampsia: the chronic reductions in uterine perfusion pressure (RUPP) and low dose TNFa infusion during pregnancy. We also found that TNFa in the placenta and in the serum is upregulated in the RUPP model. These findings suggest that the generation of AT1R-AA is a secondary event to reduction in placental perfusion leading to chronic inflammation.

Hypertension Produced by Reductions in Uterine Perfusion in the Pregnant Rat

The purpose of this study was to determine the role of interleukin (IL) 6 in mediating the increase in arterial pressure (AP) in response to chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats. AP was higher in RUPP rats (138+/-1 mm Hg) than in normal pregnant (NP) rats (104+/-1 mm Hg). Serum IL-6 levels in the RUPP rats were 104.5+/-28.6 pg/mL as compared with 36.6+/-7.4 pg/mL in NP rats. To determine the long-term effects of a 2- to 3-fold elevation in plasma IL-6 on renal function and AP in pregnant rats, we infused IL-6 for 5 days (2.5 ng/day) in NP rats starting at day 14 of gestation. Five days later, serum IL-6 levels were 55.5+/-6.5 pg/mL in the control NP rats and 157.0+/-36.1 pg/mL in the IL-6-treated NP rats. AP was higher in the IL-6-treated NP rats (115+/-3 mm Hg) as compared with NP controls (101+/-1 mm Hg) at day 19 of gestation. Renal plasma flow and GFR were lower in the IL-6-treated NP rats than in the NP group. IL-6 increased plasma renin activity but did not affect endothelin in IL-6-treated NP rats. In contrast to the NP rats, IL-6 had no effect on AP or renal hemodynamics in virgin rats. In summary, these data indicate that plasma IL-6 is elevated in response to chronic reductions in uterine perfusion in pregnant rats and that a comparable elevation in plasma IL-6 increases AP and reduces renal function in pregnant rats.

Hypertension in response to chronic reductions in uterine perfusion in pregnant rats: Effect of IL‐6 blockade.

Placental ischemia plays an important role in the pathogenesis of hypertension in preeclamptic women. Increases in inflammatory cytokines have been proposed to be an important link between placental ischemia, endothelial dysfunction, and hypertension. Our laboratory has developed a novel disease model in which hypertension develops in response to Reductions in Uterine Perfusion Pressure (RUPP) induced during late gestation in the pregnant rat. Mean arterial pressure increases from 105 + 1 mmHg in normal pregnant rats to 129 + 2 mm Hg in RUPP rats. The increase in blood pressure is associated with enhanced cytokine production. The purpose of this study was to determine the role of IL-6 in mediating the increase in blood pressure in response to reductions in uterine perfusion. IL-6 levels increased from 67 + 14 pg/ml in normal pregnant rats to 121 + 13 pg/ml in RUPP rats. Intraperitoneal injections of a monoclonal IL- 6 antibody (10 ug/kg/day) to normal pregnant and RUPP rats were performed on day 17 or day 18 of gestation. Circulating IL-6 levels decreased in RUPP rats from 121 + 13 pg/ml to 66 + 15 pg/ml in those receiving injections on day 17 and to 70.1+ 8.9 pg/ml in those receiving the monoclonal IL-6 antibody on day 18. Monoclonal IL-6 antibody administration had no effect on mean arterial pressure in RUPP rats (129+ 2 mmHg vs 130 + 2 mmHg). In summary, although the increase in blood pressure in response to reductions in uterine perfusion in pregnant rats is associated with increases in plasma IL-6 levels, lowering circulating IL-6 levels in RUPP rat with a monoclonal IL-6 antibody had no effect on arterial pressure.

The impact of reduced uterine perfusion pressure on ascorbic acid status in pregnant rats

Reduced placental perfusion is a central component of many pathological pregnancies. Using a rat model in which chronic reductions in uterine perfusion pressure (RUPP) had been surgically produced, we have shown that resistance sized mesenteric arteries from pregnant RUPP rats exhibit a significant increase in myogenicity and decreased responsiveness to the endothelium-dependent vasodilator methylcholine compared to controls. Thus, suggesting that endothelium dependent vasodilation is altered in the model. (FASEB J 2004, A475.5). Such vascular changes may be associated with oxidative stress increases due to decreased ascorbic acid (AA). Pregnant 10–12 week old Sprague-Dawley rats underwent RUPP or a SHAM operation (n=8, 9) on day 14 of gestation, and experiments were carried out on day 20. Blood pressure, maternal plasma, fetal number and weight were collected upon termination. Plasma AA was measured by HPLC with UV detection. RUPP resulted in statistically significant maternal hypertension (p=0.009), and reduced fetal number (p=0.02) and weight (p=0.04). Significant decreases were seen in plasma AA levels from RUPP compared to SHAM rats (14.2±1.6 vs. 22.14±1.4 respectively; p=0.007). These results support the hypothesis that RUPP results in decreased plasma AA. This decrease likely contributes to the maternal pathophysiology of RUPP, and potentially related human diseases, such as preeclampsia.

Enhanced Endothelin Synthesis by Endothelial Cells Exposed to Sera From Pregnant Rats With Decreased Uterine Perfusion

The initiating event in preeclampsia is thought be to reduced uteroplacental perfusion. Although we have reported previously that chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats results in hypertension and enhanced endothelin production, the factors linking placental ischemia and endothelial cell activation remain unclear. The purpose of this study was to determine the role of angiotensin II type-1 (AT1) receptor activation on endothelin production induced by serum from pregnant rats exposed to reductions in uterine perfusion. To achieve this goal, human umbilical vein endothelial cells were exposed to sera collected from RUPP rats or normal pregnant rats. Arterial pressure was significantly higher in RUPP rats (135+/-2 mm Hg) than in pregnant rats (106+/-1 mm Hg). Six hours after exposure to RUPP serum (n=17), cell media endothelin concentration was 18.4+/-2.7 pg/mL as compared with 9.22+/-1.3 pg/mL from cells exposed to serum from normal pregnant rats (n=9). Eighteen hours after exposure to RUPP serum (n=7), endothelin concentration was 30.5+/-3.8 pg/mL as compared with 12.8+/-5.3 pg/mL from cells exposed to normal pregnant rat serum (n=6). In contrast, serum from RUPP rats did not increase endothelin production in human umbilical vein endothelial cells pretreated with an AT1 receptor antagonist, losartan (15 micromol/L). Eighteen hours after exposure to RUPP serum and losartan (n=14), endothelin concentration was 21.3+/-2.2 pg/mL as compared with 16.4+/-3.3 pg/mL from cells exposed to normal pregnant rat serum and losartan (n=10). These data indicate that serum from pregnant rats exposed to reductions in uterine perfusion enhances endothelin production by endothelial cells via by AT1 receptor activation.

Cytochrome P-450 inhibition attenuates hypertension induced by reductions in uterine perfusion pressure in pregnant rats.

The present study tested the hypothesis that cytochrome P-450 (CYP) metabolites of arachidonic acid (AA) are involved in mediating hypertension and renal vasoconstriction during chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats. 1-aminobenzotriazole (ABT), a CYP enzyme inhibitor (25 mg/kg per day), or vehicle (saline 0.9%) was administered for 7 days to normal pregnant (NP) rats and to pregnant rats with chronic RUPP. RUPP rats infused with vehicle showed significantly (P<0.01) higher mean arterial pressure (MAP) (130+/-2 versus 106+/-1 mm Hg), renal vascular resistance (RVR) (22.6+/-1.8 versus 16.3+/-1.1 mm Hg/mL per minute) and lower (P<0.05) glomerular filtration rate (GFR) (1.6+/-0.1 versus 2.3+/-0.1 mL/min) than NP rats. ABT decreased (P<0.01) MAP in RUPP rats (111+/-1 mm Hg), whereas it had no effect in NP rats (108+/-2 mm Hg). CYP inhibition also attenuated the differences in renal hemodynamics observed between NP and RUPP rats. After treatment with ABT, RVR and GFR were similar in RUPP rats (19.3+/-1.5 mm Hg/mL per minute and 2.0+/-0.2 mL/min, respectively) and NP rats (16.3+/-2.4 mm Hg/mL per minute and 2.4+/-0.2 mL/min). The effects of CYP enzymes inhibitor in RUPP rats were associated with a reduction (P<0.05) of 20-HETE formation (32%) and a decreased (P<0.05) expression (33%) of CYP4A protein in renal cortex. In contrast, renal epoxygenase activity did not change in these animals. These results suggest that 20-HETE contributes to hypertension and renal vasoconstriction induced by chronic RUPP in pregnant rats.

Enhanced thromboxane synthesis during chronic reductions in uterine perfusion pressure in pregnant rats

Background The purpose of this study was to determine the role of thromboxane A 2 (TXA 2) in a conscious, chronically instrumented rat model of pregnancy-induced hypertension (PIH) produced by chronic reductions in uterine perfusion pressure (RUPP). Methods Mean arterial pressure (MAP), glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and 24-h urinary excretion of TXB 2 (metabolite of TXA 2) were determined in normal pregnant rats and RUPP pregnant rats. Results At day 20 of pregnancy, RUPP rats showed a significantly ( P < .05) higher MAP (125 ± 3 mm Hg v 100 ± 2 mm Hg) as compared with normal pregnant controls. The elevation in arterial pressure in RUPP group was associated with a marked increase ( P < .05) in the urinary concentration of TXB 2 compared with normal pregnant group (3663 ± 488 v 2646 ± 257 pg/24 h). Baseline GFR (1.74 ± 0.13 v 2.40 ± 0.20 mL/min, respectively, P < .05) and ERPF (5.13 ± 0.44 v 6.44 ± 0.58 mL/min, respectively) were decreased in RUPP rats relative to pregnant controls. Infusion of a TX receptor antagonist, SQ 29,548 (2 mg/kg bolus plus 2 mg/kg per h infusion) had no significant effect on increased MAP in RUPP pregnant rats. Similarly, ERPF and GFR did not change during acute blockade of TXA 2 receptors in this group. Conclusion These findings suggest that enhanced production of TXA 2 does not play a major role in mediating the hypertension and renal vasoconstriction produced by chronic RUPP in pregnant rats.

Role of CYP450 metabolites of arachidonic acid during pregnancy induced hypertension in rats

We have reported that chronic reductions in uterine perfusion pressure in pregnant rats lead to a hypertensive state closely resembles preeclampsia in women. The increased arterial pressure in these rats is associated with renal vasoconstriction, proteinuria and endothelial dysfunction. Alterations in renal metabolism of CYP450 metabolites of arachidonic have been reported in different models of hypertension and in women with pregnancy-induced hypertension. Therefore, the purpose of this study was to examine the contribution of CYP450 derived metabolites to the alterations in renal function and arterial pressure (AP) observed in a rat model of preeclampsia induced by long-term reductions in uterine perfusion pressure (RUPP). ABT, a suicide substrate inhibitor of CYP 450 enzymes (25 mg/kg/day) or vehicle (saline 0.9%) were administered during 7 days to normal and RUPP pregnant rats. RUPP rats infused with vehicle showed significantly (p<0.05) higher AP (132±1 vs 107±1 mmHg), renal vascular resistance (RVR) (24±3 vs 16±1 mmHg/mlmin-1) and lower GFR (1.7±0.2 vs 2.2±0.1 ml/min) than normal pregnant controls. ABT decreased (p<0.05) AP in RUPP rats (114±3 vs 132±1 mmHg) but had no effect in normal pregnant rats (109±3 vs 108±1 mmHg). ABT also normalized the alterations in RVR (18±2 vs 16±1 mmHg/mlmin-1) and in GFR (2.0±0.2 vs 2.2±0.1 ml/min) observed in RUPP pregnant rats as compared with normal pregnant rats. CYP450 derived metabolites synthesis was measured in renal cortex of these animals. Cortical homogenates (1mg/ml protein) were incubated with 14C-AA (7μM) in the presence of NADPH (1mM) for 30 min at 370C in potassium phosphate buffer (1 ml). After acidic lipid extraction, eicosanoids were separated using reverse-phase HPLC and conversion monitored by an on-line radiodetector. We found that this dose of ABT was effective in inhibiting (p<0.05) renal CYP450 derived metabolites synthesis by 50%. These results indicate that CYP450-AA metabolites of arachidonic acid play an important role in hypertension and renal vasoconstriction during chronic RUPP in pregnant rats.

Low-Salt Diet Enhances Vascular Reactivity and Ca 2+ Entry in Pregnant Rats With Normal and Reduced Uterine Perfusion Pressure

Salt moderation is often recommended to prevent excessive increases in blood pressure during pregnancy, particularly in women who are prone to pregnancy-induced hypertension; however, the vascular effects of low dietary salt intake during pregnancy are unclear. We investigated whether a low-salt diet during pregnancy alters the mechanisms of vascular smooth muscle contraction. Active stress and (45)Ca(2+) influx were measured in endothelium-denuded aortic strips of virgin and normal pregnant Sprague-Dawley rats and a hypertensive pregnant rat model produced by reduction in uterine perfusion pressure (RUPP), fed either a normal-sodium (NS, 1% NaCl) or low-sodium diet (LS, 0.2% NaCl) for 7 days. The mean arterial pressure was as follows: virgin/NS 108 +/- 8, virgin/LS 117 +/- 7, pregnant/NS 102 +/- 3, pregnant/LS 117 +/- 4, RUPP/NS 119 +/- 3, and RUPP/LS 133 +/- 6 mm Hg. Phenylephrine (Phe) caused concentration-dependent increases in active stress and (45)Ca(2+) influx that were greater in RUPP rats than in normal pregnant or virgin rats and were enhanced in pregnant/LS and RUPP/LS compared with pregnant/NS and RUPP/NS, respectively. High KCl (16 to 96 mmol/L), which stimulates Ca(2+) entry from the extracellular space, also caused increases in active stress that were greater in RUPP than in normal pregnant, in pregnant/LS than in pregnant/NS, and in RUPP/LS than in RUPP/NS rats. The Phe-induced (45)Ca(2+) influx--active stress relation was greater in RUPP/NS than in pregnant/NS and was enhanced in pregnant/LS and RUPP/LS compared with pregnant/NS and RUPP/NS, respectively. In Ca(2+)-free (2 mmol/L ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetra-acetic acid) Krebs, stimulation of intracellular Ca(2+) release by Phe (10(-5) mol/L) or caffeine (25 mmol/L) caused a transient contraction that was not significantly different in all groups of rats. Thus, a low-salt diet in pregnant and RUPP rats is associated with increases in vascular reactivity that involves Ca(2+) entry from the extracellular space but not Ca(2+) release from the intracellular stores. The enhancement of the Phe-induced Ca(2+) influx--active stress relation in pregnant and RUPP rats on a low-salt diet suggests activation of other vascular contraction mechanisms in addition to Ca(2+) entry. Although it is difficult to extrapolate the experimental data in rats to clinical data in women, the increased vascular reactivity and Ca(2+) entry and the possible enhancement of additional vascular contraction mechanisms with a low-salt diet suggest that reduction of dietary salt intake should be carefully monitored during pregnancy and pregnancy-induced hypertension.

High-salt diet enhances vascular reactivity in pregnant rats with normal and reduced uterine perfusion pressure.

High-salt (HS) diet is often associated with increased vascular resistance and arterial pressure; however, the effects of HS intake on the vascular control mechanisms of arterial pressure during pregnancy are unclear. We investigated whether a HS diet during pregnancy is associated with increases in vascular reactivity. Active stress was measured in aortic strips of virgin and normal pregnant Sprague-Dawley rats and a hypertensive pregnant rat model produced by reduction in uterine perfusion pressure (RUPP), fed either normal-sodium (NS, 1%) or HS diet (8%) for 7 days. In endothelium-intact strips, phenylephrine (Phe) caused a concentration-dependent contraction that was greater in RUPP rats than in normal pregnant or virgin rats and was significantly enhanced in pregnant/HS and RUPP/HS rats compared with pregnant/NS and RUPP/NS rats, respectively. Removal of the endothelium enhanced the Phe-induced stress slightly in virgin rats and significantly in pregnant/NS but not in pregnant/HS, RUPP/NS, or RUPP/HS. In endothelium-intact strips, acetylcholine (ACh) caused a concentration-dependent relaxation that was reduced in RUPP/NS (max, 31%) compared with pregnant/NS rats (max, 75%). ACh relaxation was further reduced in pregnant/HS rats compared with pregnant/NS rats and in RUPP/HS rats compared with RUPP/NS rats. Pretreatment of endothelium-intact strips with N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) mol/L), to inhibit NO synthase, or with 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10(-6) mol/L), to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced contraction in pregnant/NS rats but not in pregnant/HS, RUPP/NS, or RUPP/HS rats. Basal and ACh-induced nitrite/nitrate production from aortic strips showed significant reduction in pregnant/HS rats compared with pregnant/NS rats but not in RUPP/HS rats compared with RUPP/NS rats. Sodium nitroprusside, an exogenous NO donor, caused relaxation of Phe contraction that was similar in virgin or pregnant rats on an NS or HS diet but was significantly reduced in RUPP/HS rats (ED(50) 6x10(-8) mol/L) compared with RUPP/NS rats (ED(50) 6x10(-9) mol/L). Thus, a HS diet in normal pregnant and RUPP rats is associated with increases in vascular reactivity. The enhanced vascular reactivity with the HS diet is possibly related to abnormalities in NO synthesis/release from the endothelium in normal pregnant rats and an additional decrease in the sensitivity of the smooth muscle to relaxation by NO in pregnant rats with reduced uterine perfusion pressure.

Enhanced [Ca 2+ ] I and Contractility of Renal Arterial Smooth Muscle in a Rat Model of Hypertension Produced by Reduction of Uterine Perfusion Pressure in Late Pregnancy

P147 Reduction of uterine perfusion pressure during late pregnancy has been suggested to trigger increases in renal vascular resistance and pregnancy-induced hypertension; however, the cellular mechanisms involved are unclear. We investigated whether reduction of uterine perfusion pressure in late pregnancy is associated with increased [Ca 2+ ] i and contractility of renal arterial smooth muscle. Single smooth muscle cells were isolated from renal interlobular arteries of late pregnant Sprague-Dawley rats and a hypertensive pregnant rat model produced by chronic reduction in uterine perfusion pressure (RUPP). The cells were loaded with fura-2 and cell length and [Ca 2+ ] i were measured. In cells of pregnant rats incubated in Hank’s solution (1 mmol/L Ca 2+ ), resting [Ca 2+ ] i was 62±3 nmol/L and cell length was 69±3 μm. In RUPP rats, resting [Ca 2+ ] i (101±3 nmol/L) was significantly greater and cell length (53±2 μm) was shorter than pregnant rats. In pregnant rats, angiotensin II (AII, 10 -7 mol/L) caused transient increase in [Ca 2+ ] i to 412±25 nmol/L followed by a maintained increase to 158±15 nmol/L, and 23±2% cell contraction. In RUPP rats, the AII-induced [Ca 2+ ] i transient (434±22 nmol/L) was not significantly different from pregnant rats, but the maintained [Ca 2+ ] i was significantly elevated to 198±7 nmol/L and cell contraction was increased to 33±3%. In pregnant rats, the Ca 2+ channel agonist Bay K8644 (1 μmol/L) caused maintained increase in [Ca 2+ ] i to 292±12 nmol/L and 31% contraction that were significantly enhanced in RUPP rats. In Ca 2+ -free (2 mmol/L EGTA) Hank’s, AII- and caffeine (10 mmol/L)-induced [Ca 2+ ] i transient and cell contraction were not significantly different between pregnant and RUPP rats suggesting no difference in Ca 2+ release from intracellular stores. The enhanced basal, maintained AII- and Bay K8644-induced [Ca 2+ ] i and cell contractility in RUPP rats suggest enhanced Ca 2+ entry mechanisms of smooth muscle contraction in resistance renal arteries and may, in part, explain the increased renal vascular resistance associated with pregnancy-induced hypertension.

Endothelin-a Receptor Blockade Attenuates the Hypertension in a Rat Model of Pregnancy-Induced Hypertension.

6 A rat model of pregnancy-induced hypertension (PIH) produced by chronic reductions in uterine perfusion pressure (RUPP) is associated with a significant elevation in mean arterial pressure and as previously reported by our laboratory, proteinuria and reductions in kidney function. The purpose of this study was to examine the role of endothelin in mediating the hypertension in response to chronic reductions in uterine perfusion pressure in conscious, chronically-instrumented pregnant rats. Mean arterial pressure in RUPP rats (n=7) was significantly higher (123.0 ± 0.8 vs 101.3 ± 1.5 mmHg) than control pregnant rats (n=8). Renal expression of preproendothelin was also significantly elevated (>45%, P 20%, 37.7 ± 2.4 vs 29.9 ± 2.8 densitometric units, respectively) of the RUPP animals as compared to normal pregnant animals. Chronic administration of the selective endothelin-A receptor antagonist (ABT-627, 5mg/kg/day for 10 days, n=9) markedly attenuated the increase in MAP observed in the RUPP animals (123.0 ± 0.8 mmHg in the RUPP animals vs. 103.3 ± 2.3 mmHg in RUPP animals with ET-A antagonist, P

Endothelin-a Receptor Blockade Attenuates the Hypertension in a Rat Model of Pregnancy-Induced Hypertension.

6 A rat model of pregnancy-induced hypertension (PIH) produced by chronic reductions in uterine perfusion pressure (RUPP) is associated with a significant elevation in mean arterial pressure and as previously reported by our laboratory, proteinuria and reductions in kidney function. The purpose of this study was to examine the role of endothelin in mediating the hypertension in response to chronic reductions in uterine perfusion pressure in conscious, chronically-instrumented pregnant rats. Mean arterial pressure in RUPP rats (n=7) was significantly higher (123.0 ± 0.8 vs 101.3 ± 1.5 mmHg) than control pregnant rats (n=8). Renal expression of preproendothelin was also significantly elevated (&gt;45%, P&lt;.05) in the medulla (44.1 ± 6.3 vs 24.0 ± 2.6 densitometric units, respectively) and in the cortex (&gt;20%, 37.7 ± 2.4 vs 29.9 ± 2.8 densitometric units, respectively) of the RUPP animals as compared to normal pregnant animals. Chronic administration of the selective endothelin-A receptor antagonist (ABT-627, 5mg/kg/day for 10 days, n=9) markedly attenuated the increase in MAP observed in the RUPP animals (123.0 ± 0.8 mmHg in the RUPP animals vs. 103.3 ± 2.3 mmHg in RUPP animals with ET-A antagonist, P&lt;.05). Endothelin-A receptor blockade had no significant effect on blood pressure in the normal pregnant animals (101.3 ± 1.5 mmHg in normal pregnant rats vs. 96.0 ± 0.9 mmHg in normal pregnant animals with ET-A antagonist, n=8). In summary, hypertension in the RUPP model of pregnancy-induced hypertension was associated with enhanced renal expression of preproendothelin. In addition, hypertension in the RUPP model of PIH was significantly attenuated by selective endothelin-A receptor blockade. These findings suggest that endothelin plays a major role in mediating the hypertension observed in this animal model of pregnancy-induced hypertension produced by chronic reductions in uterine perfusion pressure.