Control Pregnant Rats Research Articles

Maternal–fetal transport and disposition of copper, iron, molybdenum, selenium and zinc in experimentally induced diabetic rats

Objective. To assess maternal–fetal status of essential trace elements such as copper, iron, molybdenum, selenium and zinc, in experimentally induced diabetic and control pregnant rats, and to correlate the findings with those observed in human diabetic pregnancies. Fetal–maternal ratios of the elements and Cu:Zn and Cu:Fe ratios were also computed in control and study groups.Methods. Diabetes was experimentally induced in pregnant Sprague Dawley rats by injection of streptozotocin. A cocktail of essential trace elements along with antipyrine as internal reference marker were then injected intra-peritoneally to diabetic and matched control pregnant rats on the 20th day of pregnancy. Maternal and fetal blood and tissue samples were collected after sacrificing the animals at 30- and 60-minutes following cocktail injection. Concentrations of trace elements and antipyrine in various blood and tissue samples were then determined by atomic absorption spectrophotometry and colorimetry, respectively.Results. Concentrations of Cu, Fe, Mo, Se, Zn, and antipyrine averaged 2907.0 ± 212.0 μg/L, 3950.0 ± 766.0 μg/L, 15.8 ± 1.7 μg/L, 74.8 ± 6.5 μg/L, 726.4 ± 67.4 μg/L, and 170.5 ± 8.2 mg/L, respectively, in maternal blood in control pregnant rats (n = 5) at day 20 in the 30-minute study phase, while in the diabetic group (n = 5) the values of the various trace element concentrations and antipyrine averaged 2875.0 ± 225.0 μg/L, 5875.0 ± 688.0 μg/L, 21.2 ± 2.1 μg/L, 116.0 ± 3.6 μg/L, 753.0 ± 71.3 μg/L, and 171.7 ± 4.2 mg/L, respectively. Unpaired student's t-test showed that Fe and Se levels were significantly higher (p < 0.05) in the diabetic pregnant rats compared to controls. Cu, Mo and Zn values, however, were not significantly different (p > 0.05) between the two groups. Cu:Zn and Cu:Fe ratios showed varying differences between maternal and fetal samples in the control and study groups.Conclusions. Considering the disparity of results in pregnant diabetic rats and pregnant diabetic women, we urge exercising caution when comparing data from animal studies to human situations.

The Hypo-Osmolality of Pregnancy Does Not Alter Baroreceptor Responses to Acute Changes in Osmolality

Reducing extracellular osmolality by 10-12 mOsm/L reduces baroreceptor firing in vitro in tissues from male rats. Pregnancy is associated with a similar reduction in plasma osmolality (pOsm) as well as with alterations in baroreceptor firing and reduced baroreceptor reflex activity. Mechanisms for reduced baroreflex activity are not yet understood, but they have important implications for maintenance of cardiovascular homeostasis in the pregnant female. Thus, this study was designed to test whether changes in plasma osmolality alter baroreceptor discharge during pregnancy. Late-gestation pregnant and virgin control rats were anesthetized, femoral vessels were cannulated for measurement of arterial pressure and drug infusion, and the aortic depressor nerve, containing baroreceptor afferents, was isolated and prepared for recording. Plasma osmolality was measured before and 30 min after graded intraperitoneal injections of NaCl (50-1500 mOsm/L). Arterial pressure, heart rate, and aortic depressor nerve activity (ADNA) were measured continuously before and after injections. A 50 mOsm/L NaCl injection significantly decreased pOsm but did not alter ADNA in either group. Likewise, 1200 mOsm/L NaCl injections significantly increased pOsm and had no effect on ADNA in either group. The 1500 mOsm salt load significantly increased pOsm and ADNA in pregnant rats, and in virgin animals, it increased pOsm but evoked a paradoxical decrease in ADNA. The authors' studies indicate that pOsm has minimal effects on baroreceptor activity, which are not significantly different during gestation. Their findings suggest that in vitro data from male rats indicating a correlation between pOsm and baroreceptor discharge are not applicable in anesthetized intact female animals.

Hypertension Produced by Reductions in Uterine Perfusion in the Pregnant Rat

Inflammatory cytokines such as tumor necrosis factor (TNF)-alpha are elevated in preeclamptic women and are thought to be an important link between placental ischemia and endothelial dysfunction. The purpose of this study was to determine the role of TNF in mediating hypertension in response to chronic reductions in uterine perfusion (RUPPs) in pregnant rats. Arterial pressure was significantly higher in RUPP rats (138+/-1 mm Hg) than in pregnant rats (107+/-1 mm Hg). Serum TNF-alpha levels in the RUPP rats were 17+/-4 pg/mL compared with 8+/-1 pg/mL in normal pregnant rats. To determine the long-term effects of a 2- to 3-fold elevation in plasma TNF-alpha on renal and systemic hemodynamics in pregnant rats, we infused TNF-alpha for 5 days at a rate of 50 ng/d during days 14 to 19 of gestation in pregnant rats. Serum levels were 7+/-2 pg/mL in the control pregnant rats and 14+/-2 pg/mL in the TNF-alpha-treated pregnant rats. Mean arterial pressure was higher in the TNF-alpha-treated pregnant rats (123+/-3 mm Hg) compared with pregnant controls (96+/-3 mm Hg) at day 19 of gestation. TNF-alpha increased renal vascular resistance in pregnant rats by 182%. Renal plasma flow was 5.4+/-1.2 mL/min in the TNF-alpha-treated group and 9.2+/-1.6 mL/min in the control group. Glomerular filtration rate was 1.7+/-0.4 mL/min in the TNF-alpha-treated group and 2.6+/-0.4 mL/min in the control group. In summary, these data suggest that TNF-alpha may play an important role in mediating the increased arterial pressure in response to chronic RUPPs in pregnant rats.

Modulation of lipid metabolism by n−3 polyunsaturated fatty acids in gestational diabetic rats and their macrosomic offspring

The time course of changes in lipid metabolism by dietary n-3 PUFAs (polyunsaturated fatty acids) in streptozotocin-induced diabetic rats during pregnancy (days 12 and 21) and their macrosomic offspring at birth (day 0) and through adulthood (days 60 and 90) was studied with respect to adipose tissue, liver and serum lipid concentrations, and fatty acid composition. Glucose and insulin levels were also assessed in order to characterize the diabetic state of macrosomic offspring. Pregnant diabetic and control rats were fed either an Isio-4 or EPAX diet (enriched with n-3 PUFA). The same diets were also consumed by pups at weaning. Compared with control rats, during pregnancy diabetic rats had a significant elevation in liver and serum triacylglycerol (triglyceride) and cholesterol concentrations. At birth, macrosomic pups had higher serum insulin and glucose levels than control pups. The macrosomic rats maintained accelerated postnatal growth combined with high adipose tissue weight and lipid content through the first 12 weeks of age. The macrosomic pups from diabetic rats fed the Isio-4 diet also showed a significant enhancement in liver and serum triacylglycerol and cholesterol levels at birth and during adulthood. Feeding the EPAX diet to diabetic mothers as well as their macrosomic pups increased serum and liver levels of EPA (eicospentaenoic acid) and DHA (docosahexaenoic acid) with a reduction in arachidonic acid. The EPAX diet induced a significant decrease in liver and serum triacylglycerol and cholesterol concentrations in mothers during pregnancy and in their macrosomic pups during adulthood. Since the EPAX diet improves lipid anomalies considerably in diabetic mothers and their macrosomic offspring, it may prevent long-term metabolic abnormalities associated with macrosomia.

Low-Protein Diets Reduce PKAα Expression in Islets from Pregnant Rats

We investigated the effect of protein restriction on insulin secretion and the expression of protein kinase (PK)Aalpha and PKCalpha in islets from control and pregnant rats. Adult control nonpregnant (CN) and control pregnant (CP) rats were fed a normal-protein diet (17%), whereas low-protein nonpregnant (LPN) and low-protein pregnant (LPP) rats were fed a low-protein diet (6%) for 15 d. In the presence of 2.8 and 8.3 mmol glucose/L, insulin secretion by islets of CP rats was higher than that by islets of CN rats. Compared with the CN groups, insulin secretion by islets of LPN rats was lower with 8.3 but not with 2.8 mmol glucose/L. The insulin secretion by islets of LPP rats was higher than by LPN rats at both glucose concentrations. IBMX (1 mmol/L), a phosphodiesterase inhibitor, increased insulin secretion by islets from pregnant rats, and this effect was greater in islets of CP rats than in LPP rats. Forskolin (0.01-100 micromol/L), a stimulator of adenylyl cyclase, increased insulin secretion only in islets of CN and CP rats, with a higher 50% effective concentration in islets of CP rats compared with CN rats. The insulin secretion induced by phorbol 12-myristate 13-acetate (a stimulator of PKC) was higher in islets of LPN and LPP rats than in the respective controls, especially at 8.3 mmol glucose/L. PKAalpha, but not PKCalpha, expression was lower in islets of rats fed low protein than in the controls, regardless of the physiological status of the rats. All endocrine cells of the islets, including beta-cells, expressed the PKAalpha isoform. The cytoplasmic distribution of this enzyme in beta-cells was not modified by pregnancy and/or protein restriction. In conclusion, our results indicate that the response of islets from rats fed low protein during pregnancy is similar to that of control rats, at least for physiologic glucose concentration. However, the decreased response to IBMX and forskolin indicates decreased production and/or sensitivity to cAMP; this was associated with a decrease in PKA expression, which may result in lower PKA activity.

Role of Endothelin in Mediating Tumor Necrosis Factor-Induced Hypertension in Pregnant Rats

Hypertension during preeclampsia is associated with an increase in plasma levels of tumor necrosis factor (TNF)-alpha, a cytokine known to contribute to endothelial dysfunction. Recently, our laboratory reported that a 2-fold increase in plasma TNF-alpha produces hypertension in pregnant rats. Endothelin is also elevated in preeclampsia and endothelin synthesis is enhanced by TNF-alpha. The purpose of this study was to determine the role of endothlelin in mediating TNF-alpha-induced hypertension in pregnant rats. To achieve this goal, TNF-alpha (50 ng/d for 5 days) was infused into control pregnant rats and pregnant rats treated with an endothelin receptor A antagonist, ABT 627 (5 mg/kg per day for 5 days). At day 19 of gestation, arterial pressure was measured and aorta, kidneys, and placentas were harvested. Infusion of TNF-alpha into pregnant rats increased plasma concentration of TNF-alpha (13.5+/-0.8 to 28.0+/-3.7 pg/mL) and arterial pressure (101+/-2 to 122+/-1 mm Hg). The increase in arterial pressure was associated with an increase in preproendothelin mRNA expression in placenta, aorta, and kidneys measured by real-time polymerase chain reaction (PCR). Pretreatment with the endothelin receptor A antagonist completely abolished the blood pressure response to TNF-alpha in pregnant rats (105+/-1 versus 97+/-2 mm Hg). In sharp contrast, the ETA receptor antagonist had no effect on arterial pressure in normal pregnant rats (97+/-2 versus 101+/-2 mm Hg). Moreover, chronic infusion of TNF-alpha had no significant effect on arterial pressure or renal preproendothelin levels in virgin rats. These results suggest an important role for endothelin in mediating TNF-alpha-induced hypertension in pregnant rats.

Pregnancy and social stress in female rats: Influences on blood leukocytes and corticosterone concentrations

The consequences of pregnancy and social stress on blood immune cells and on plasma corticosterone concentrations were assessed in Long Evans rats. Normal pregnancy in control females was characterized by a progressive increase in corticosterone concentration and increasing numbers of granulocytes. In contrast, CD4 T, CD8 T, and B cell numbers as well as the proliferative response of lymphocytes decreased as pregnancy progressed. Stress was induced in pregnant females by social confrontation for 2 h daily with a female resident opponent over a period of 2 months. Corticosterone concentrations were substantially higher in pregnant stressed than in pregnant control rats. Furthermore, the numbers of monocytes, NK and B cells were lower in stressed females, and there was a strong trend towards suppressed lymphocyte proliferation. Interestingly, pregnant females did not show granulocytosis in response to the stressor. In sum, the social stress paradigm in females appears to be a good model for the investigation of the interactions between stress, pregnancy and the immune system. It also provides an excellent platform for studies on prenatal stress under relatively naturalistic conditions.

Decreased contraction-stimulated glucose transport in isolated epitrochlearis muscles of pregnant rats

Late pregnancy is characterized by insulin resistance for glucose transport in skeletal muscle. The main purpose of this study was to investigate the effect of late pregnancy on contraction-stimulated glucose transport in isolated rat skeletal muscle after in vitro electrical stimulation. Isolated epitrochlearis muscles of 19-day pregnant and aged-matched nonpregnant control rats were studied. One muscle from each rat was stimulated to contract, and the contralateral muscle served as a resting control. Tension developed during contractile activity, 3-O-methylglucose (3-MG) transport rate, and glycogen concentration were determined. Epitrochlearis muscles from other rats were used to measure insulin-stimulated 3-MG transport. There was no detectable difference between the nonpregnant and pregnant groups for contractile performance (peak tension, total tension, or fatigue). Pregnancy was not associated with significant changes in muscle glycogen concentration (resting or after contractile activity) or the contraction-stimulated decrement in glycogen concentration. For muscles from pregnant vs. nonpregnant groups, there was a 22% reduction (P < or = 0.05) in contraction-stimulated glucose transport, a 28% decrease (P < or = 0.05) in insulin-stimulated glucose transport, and unchanged basal glucose transport. In conclusion, isolated epitrochlearis muscles from pregnant vs. nonpregnant rats had a relative decrement in contraction-stimulated glucose transport that was similar to the relative decline in insulin-stimulated glucose transport. The decrement in contraction-stimulated glucose transport was not attributable to pregnancy-related changes in tension development or glycogen levels. The similar relative decline in insulin- and contraction-stimulated glucose transport raises the possibility that pregnancy impairs a distal process that is common to mechanisms whereby each stimulus activates glucose transport.

Long-lasting effects of serotonin deficiency on differentiating peptidergic neurons in the rat suprachiasmatic nucleus

Serotonin (5-HT, 5-hydroxytryptamine) is known to be an inductor of the brain development [Whitaker-Azmitia, P.M., Druse, M., Walker, P., Lauder, J.M., 1996. Serotonin as a developmental signal. Behav. Brain Res. 73, 19–29; Ugrumov, M.V., 1997. Hypothalamic monoaminergic systems in ontogenesis: development and functional significance. Int. J. Dev. Biol. 41, 809–816]. This study was aimed to test whether it provides long-lasting effects on the differentiating vasoactive intestinal polypeptide (VIP) and vasopressin (VP) neurons of the suprachiasmatic nucleus (SCN) in rats. To this aim, 5-HT was depleted in fetal brain by daily injections of p-chlorophenylalanine (pCPA), an inhibitor of 5-HT synthesis, to pregnant rats from the 13th to the 21st day of gestation. Pregnant rats injected with saline served as controls. The offsprings (males) of pCPA-treated and control pregnant rats were maintained after birth for two months under normal laboratory conditions. Then, the SCN was processed for immunocytochemistry of VIP and VP and in situ hybridization of appropriate mRNAs. There were no differences in concentrations of VIP and VP mRNAs in the SCN in adult offsprings of the 5-HT-depleted pregnant rats compared to the controls. Moreover, 5-HT deficiency did not induce any change in size of VIP-immunoreactive (IR) and VP-IR neurons. Conversely, both the numbers of VIP- and VP-immunoreactive neurons and concentrations of the peptides in cell bodies increased significantly. It is concluded that 5-HT provides long-lasting effects on differentiating VIP and VP neurons in the SCN resulting in attenuated release rather than elevated synthesis of both peptides in adulthood.

L -Arginine Attenuates Hypertension in Pregnant Rats With Reduced Uterine Perfusion Pressure

A chronic reduction in uterine perfusion pressure in the pregnant rat is associated with significant elevations in mean arterial pressure, proteinuria, and reductions in kidney function as is chronic nitric oxide blockade, suggesting that nitric oxide deficiency may contribute to the clinical manifestations of preeclampsia. The purpose of this study was to determine whether supplementation with L-arginine, the precursor for nitric oxide, attenuates the hypertension produced in response to a chronic reduction in uterine perfusion pressure in the pregnant rat. Reduced uterine perfusion was initiated at day 14 of gestation with arterial pressure determined at day 19 of gestation in conscious, chronically instrumented rats. Arterial pressure was significantly elevated in pregnant rats with chronic reductions in uterine perfusion as compared with pregnant control rats (132+/-2 versus 109+/-2 mm Hg, P<0.01, respectively). Treatment with L-arginine (2%) in the drinking water was initiated at day 10 of gestation. l-arginine supplementation resulted in a significant decrease in arterial pressure in both pregnant rats with reduced uterine perfusion pressure (113+/-2 mm Hg treated, P<0.01 versus untreated pregnant with reduced uterine perfusion pressure) and pregnant control (97+/-3 mm Hg treated, P<0.01 versus untreated pregnant) rats. However, supplementation with L-arginine decreased blood pressure by 19 mm Hg in pregnant with reduced uterine perfusion pressure (untreated versus treated) as compared with 12 mm Hg in pregnant (untreated versus treated) rats. Thus, these results suggest that l-arginine supplementation may be beneficial in attenuating the hypertension in preeclampsia.

Insulin‐Induced Hypertension, l‐Arginine, and Endothelial Nitric Oxide Synthase in Pregnant Rats

Pregnancy‐induced hypertension in rats with chronic exogenous hyperinsulinemia is associated with reduced urinary excretion of nitric oxide metabolites. We tested the hypothesis that there are perturbations of endothelial nitric oxide synthase in their kidneys. We studied three groups of rats: control pregnant rats (n = 6); pregnant rats with hyperinsulinemia by subcutaneous sustained‐release insulin pellet (n = 5); and hyperinsulinemic pregnant rats treated with l‐arginine 2 gL in drinking water (n = 5). By the end of pregnancy blood pressure was 78 ± 12 mm Hg in controls, 119 ± 15 mm Hg in hyperinsulinemic rats, and 77 ± 8 mm Hg in l‐arginine‐treated hyperinsulinemic rats, p < 0.007. Serum creatinine was 0.4 mg/dl in controls, 0.6 mg/dl in hyperinsulinemic rats, and 0.5 mg/dl in l‐arginine‐treated rats, p < 0.05. Corresponding urinary excretion of nitric oxide metabolites was 2.1 ± 0.5, 1.2 ± 0.2, and 1.5 ± 0.2 µmols/mg creatinine, p < 0.01. Expression of endothelial nitric oxide synthase protein in kidneys by Western blot was not different between controls and hyperinsulinemic rats, 5.6 ± 2.4 and 5.8 ± 3.4 OD × mm2, but was nearly doubled in l‐arginine‐treated rats, 10.8 ± 2.3, p < 0.03. Thus, the salutary effect of l‐arginine on hyperinsulinemic pregnancy‐induced hypertension (PIH) may be mediated, in part, by endothelial nitric oxide synthase in their kidneys.

Enhanced [Ca2+]i in renal arterial smooth muscle cells of pregnant rats with reduced uterine perfusion pressure.

Reduction of uterine perfusion pressure (RUPP) during late pregnancy has been suggested to trigger increases in renal vascular resistance and lead to hypertension of pregnancy. We investigated whether the increased renal vascular resistance associated with RUPP in late pregnancy reflects increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and contraction of renal arterial smooth muscle. Single smooth muscle cells were isolated from renal interlobular arteries of normal pregnant Sprague-Dawley rats and a rat model of RUPP during late pregnancy. The cells were loaded with fura 2 and both cell length and [Ca(2+)](i) were measured. In cells of normal pregnant rats incubated in Hanks' solution (1 mM Ca(2+)), ANG II (10(-7) M) caused an initial increase in [Ca(2+)](i) to 414 +/- 13 nM, a maintained increase to 149 +/- 8 nM, and 21 +/- 1% cell contraction. In RUPP rats, the initial ANG II-induced [Ca(2+)](i) (431 +/- 18 nM) was not different from pregnant rats, but both the maintained [Ca(2+)](i) (225 +/- 9 nM) and cell contraction (48 +/- 2%) were increased. Membrane depolarization by 51 mM KCl and the Ca(2+) channel agonist BAY K 8644 (10(-6) M), which stimulate Ca(2+) entry from the extracellular space, caused maintained increases in [Ca(2+)](i) and cell contraction that were greater in RUPP rats than control pregnant rats. In Ca(2+)-free (2 mM EGTA) Hanks' solution, the ANG II- and caffeine (10 mM)-induced [Ca(2+)](i) transient and cell contraction were not different between normal pregnant and RUPP rats, suggesting no difference in Ca(2+) release from the intracellular stores. The enhanced maintained ANG II-, KCl- and BAY K 8644-induced [Ca(2+)](i) and cell contraction in RUPP rats compared with normal pregnant rats suggest enhanced Ca(2+) entry mechanisms of smooth muscle contraction in resistance renal arteries and may explain the increased renal vascular resistance associated with hypertension of pregnancy.

Impaired lipoprotein metabolism in obese offspring of streptozotocin-induced diabetic rats.

The time course of changes in lipoprotein metabolism of obese offspring of mildly diabetic rats was studied with respect to serum lipoprotein composition as well as LCAT and tissue lipoprotein lipase (LPL) activities. Mild hyperglycemia in pregnant rats was induced by intraperitoneal injection of streptozotocin on day 5 of gestation. Control pregnant rats were injected with citrate buffer. At birth, obese pups had higher serum glucose, insulin, and lipoprotein (VLDL, LDL-HDL1, HDL(2-3)) levels than control pups. After 1 mon of life, all of these parameters in obese rats became similar to those of controls. However, LCAT, adipose tissue LPL, and hepatic triacylglycerol lipase activities were high. At 2 mon of age, VLDL-TAG levels were higher in obese females than in controls. By the age of 3 mon, obese offspring had developed insulin resistance with hyperglycemia, hyperinsulinemia, and higher serum lipoprotein concentrations. Indeed, qualitative abnormalities of lipoproteins were seen and were typical of obese and diabetic human beings. Fetal hyperinsulinemia should be considered as a risk factor for later metabolic diseases, including dyslipoproteinemia.

Age-related changes in fatty acids in obese offspring of streptozotocin-induced diabetic rats.

The long-term effects of fetal hyperinsulinemia, time course of changes in liver and very-low-density lipoprotein (VLDL) lipid levels and fatty acid compositions were investigated in obese offspring of streptozotocin-induced mildly diabetic rats. Mild hyperglycemia in pregnant rats was induced by intraperitoneal injection of streptozotocin on day 5 of gestation. Control pregnant rats were injected with citrate buffer. Liver and VLDL lipids and fatty acids were analyzed in offspring at different ages. At birth, obese pups had higher VLDL triglyceride levels, saturated fatty acids, and C20:4n-6. They also had lower C18:2n-6 proportions in VLDL triglycerides, phospholipids, and cholesteryl esters than controls pups. In 1-month-old male and female obese rats, VLDL and liver lipid amounts were similar to those in their respective controls; however, high levels of C18:2n-6 and C20:4n-6 were noted in liver and VLDL lipids. At the age of 2 months, liver and VLDL triglyceride levels were higher in obese females than in control females. Fatty acid abnormalities seen in obese rats included low C18:3n-3 and high C22:6n-3 proportions in liver triglycerides and phospholipids. At the age of 3 months, obese rats, both males and females, compared with control animals, had higher VLDL and hepatic lipids with reduced C20:4n-6 levels and polyunsaturated/saturated fatty acids ratios in hepatic and VLDL triglycerides and phospholipids. Fetal obesity, associated with alterations in VLDL lipid fatty acid composition, represents an important risk factor for adult obesity and diabetes.

Reduced endothelial NO-cGMP vascular relaxation pathway during TNF-α-induced hypertension in pregnant rats

Placental ischemia during pregnancy is thought to release cytokines such as tumor necrosis factor-alpha (TNF-alpha), which may contribute to the increased vascular resistance associated with pregnancy-induced hypertension. We have reported that a chronic twofold elevation in plasma TNF-alpha increases blood pressure in pregnant but not in virgin rats; however, the vascular mechanisms are unclear. We tested the hypothesis that increasing plasma TNF-alpha during pregnancy impairs endothelium-dependent vascular relaxation and enhances vascular reactivity. Active stress was measured in aortic strips of virgin and late-pregnant Sprague-Dawley rats untreated or infused with TNF-alpha (200 ng x kg(-1) x day(-1) for 5 days) to increase plasma level twofold. Phenylephrine (Phe) increased active stress to a maximum of 4.2 +/- 0.4 x 10(3) and 9.9 +/- 0.7 x 10(3) N/m2 in control pregnant and TNF-alpha-infused pregnant rats, respectively. Removal of the endothelium enhanced Phe-induced stress in control but not in TNF-alpha-infused pregnant rats. In endothelium-intact strips, ACh caused greater relaxation of Phe contraction in control than in TNF-alpha-infused pregnant rats. Basal and ACh-induced nitrite/nitrate production was less in TNF-alpha-infused than in control pregnant rats. Pretreatment of vascular strips with 100 microM N(G)-nitro-L-arginine methyl ester, to inhibit nitric oxide (NO) synthase, or 1 microM 1H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one, to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in control but not in TNF-alpha-infused pregnant rats. Phe contraction and ACh relaxation were not significantly different between control and TNF-alpha-infused virgin rats. Thus an endothelium-dependent NO-cGMP-mediated vascular relaxation pathway is inhibited in late-pregnant rats infused with TNF-alpha. The results support a role for TNF-alpha as one possible mediator of the increased vascular resistance associated with pregnancy-induced hypertension.

Acetylcholine-Induced Vasodilation in the Uterine Vascular Bed of Pregnant Rats with Adriamycin-Induced Nephrosis

Objective: This project was designed to study endothelium-dependent vasodilation in the uterine vascular bed during experimentally induced preeclampsia in rats. Methods: Uterine vascular beds were isolated from nonpregnant and pregnant rats with or without treatment with adriamycin (ADR) and perfused with physiological solution. Thereafter, vasodilator responses to acetylcholine were recorded. Records: Pregnant ADR-treated rats displayed symptoms of preeclampsia including hypertension and proteinuria. Blood pressure was 110.0 ± 4.7 mm Hg (n = 5) in control pregnant rats and 136.0 ± 5.3 mm Hg (n = 5) in ADR-treated pregnant rats, and urinary protein concentrations were 0.35 mg/ml (n = 5) and 13.2 ± 3.6 mg/ml (n = 9), respectively. Both blood pressure and proteinuria values were significantly (p < 0.05) different between controls and ADR-treated rats. However, acetylcholine-induced dose-dependent vasodilator responses in the vascular beds were not significantly different between the pregnant and nonpregnant rats. Although acetylcholine-induced vasodilation was significantly reduced by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) in both groups, the residual response to acetylcholine was not affected by indomethacin, suggesting that prostanoids were not involved in this response. The L-NAME-resistant component, endothelium-derived hyperpolarizing factor (EDHF), was greater in ADR-treated uterine beds than in those of the controls, indicating a significant contribution from EDHF in these vessels. In the presence of an elevated external potassium ion concentration, acetylcholine produced similar vasodilator responses, indicating that the release of nitric oxide was not impaired. Conclusion: These results indicate that endothelium-dependent vasodilation was not impaired in this model of preeclampsia.

Influence of prenatal alcohol exposure on myocardial contractile function in adult rat hearts: role of intracellular calcium and apoptosis.

To assess the teratogenic action of ethanol on cardiac contractile function in offspring exposed to ethanol in utero, pregnant Sprague-Dawley rats were fed with ethanol during gestation. Left-ventricular papillary muscles and myocytes were isolated from the offspring of the ethanol-ingesting and control pregnant rats. Mechanical parameters measured were peak tension development (PTD, indicating the myocardial force-generating capacity), peak cell shortening (PS), time-to-PTD/PS (TPT/TPS), time-to-90% relaxation/re-lengthening (RT(90)/TR(90)), and maximal velocities of contraction/shortening and relaxation/re-lengthening (+/- VT and +/- dL/dt). Intracellular Ca(2+) levels and apoptosis were evaluated with fura-2 fluorescent dye and Caspase-3 activation assay, respectively. Offspring of the ethanol group displayed decreased heart weight associated with comparable body, liver and kidney weight, and papillary muscle weight/size, compared to the control group. However, prenatal ethanol exposure depressed myocardial PTD and +/- VT. The myocardium from the ethanol group also exhibited slightly but significantly shortened TPT, accompanied with normal RT(90). Muscles from both groups exhibited comparable responses to post-rest potentiation, increasing extracellular Ca(2+) concentration, noradrenaline and acute ethanol challenge. Ventricular myocytes from both the control and ethanol groups possessed similar PS, TPS, TR(90) and +/- dL/dt. Both resting and peak intracellular Ca(2+) levels were elevated in myocytes from the ethanol group. Additionally, acute ethanol application depressed caffeine-induced intracellular Ca(2+) rise in myocytes from both groups. Myocytes from the ethanol group displayed an enhanced Caspase-3 activation, compared to control myocytes. These results suggest that prenatal ethanol exposure alters myocardial contractile function and may contribute to the development of postnatal cardiac dysfunction through, in part, increased intracellular Ca(2+) loading and apoptosis.

Increased serum angiotensin-converting enzyme activity and plasma angiotensin II levels during pregnancy and postpartum in the diabetic rat.

The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure, electrolyte balance and renal function in normal human pregnancy. The present study was designed to assess various components of the RAS and renal function during pregnancy and immediately after pregnancy in the streptozotocin (STZ)-diabetic rat. Pregnant Wistar rats were allocated to three groups: I-control, non-diabetic rats (n=24), II-STZ-diabetic rats (STZ 55 mg/kg body weight, i.v. on day 10 of pregnancy, n=24), III-diabetic rats, as above, treated with insulin (4 units/day, s.c. n=21). On days 17-18 of pregnancy, or within 24 hours after delivery, the rats were sacrificed and the various components of the RAS were determined. Urinary protein excretion (UP) and creatinine clearance(CCr) were greater in group II, four days after STZ, than in group I (UP: I-7.6+/-2.8, II-18.6+/-6.3 mg/24-hour, p<0.001, CCr: I-1.04+/-0.33, II-2.38+/-0.7 ml/minute, p<0.001). Mean (+/-SD) serum angiotensin-converting enzyme (ACE) activity and plasma angiotensin II(Ang II) levels at days 17-18 of pregnancy were greater in the untreated diabetic rats than in control pregnant rats (ACE: 163+/-18 vs. 111+/-21 nmol/ml/minute, p<0.001, Ang II: 115+/-45 vs. 43+/-10 pg/ml, p<0.005). Postpartum serum ACE activity and plasma Ang II levels were greater in group II (ACE: I-123+/-14, II-142+/-24, III-108+/-21 nmol/ml/minute, p<0.01, Ang II: I-56+/-38, II-148+/-62, III-38+/-17 pg/mI, p<0.001). ACE activity in the lung was greater, whereas the activity in the renal cortex was less, in group II than in group I. Kidney weight in untreated diabetic rats was greater than in the other two groups. Increased serum ACE activity during pregnancy and postpartum in the untreated diabetic rat is associated with enhanced serum Ang II levels, which may contribute to increased protein excretion and renal hypertrophy.

Reduced uterine perfusion pressure during pregnancy in the rat is associated with increases in arterial pressure and changes in renal nitric oxide.

A reduction in nitric oxide (NO) synthesis has been suggested to play a role in pregnancy-induced hypertension. We have recently reported that normal pregnancy in the rat is associated with significant increases in whole-body NO production and renal protein expression of neuronal and inducible NO synthase. The purpose of this study was to determine whether whole-body and renal NO production is reduced in a rat model of pregnancy-induced hypertension produced by chronically reducing uterine perfusion pressure starting at day 14 of gestation. Chronic reductions in uterine perfusion pressure resulted in increases in arterial pressure of 20 to 25 mm Hg, decreases in renal plasma flow (<23%) and glomerular filtration rate (<40%), but no difference in urinary nitrite/nitrate excretion relative to control pregnant rats. In contrast, reductions in uterine perfusion pressure in virgin rats resulted in no significant effects on arterial pressure. Renal endothelial (<4%) and inducible (<11%) NO synthase protein expression did not decrease significantly in the chronically reduced uterine perfusion pressure rats relative to normal pregnant rats; however, significant reductions in neuronal NO synthase were observed (<30%). The results of this study indicate that the reduction in renal hemodynamics and the increase in arterial pressure observed in response to chronic decreases in uterine perfusion pressure in pregnant rats are associated with no change in whole-body NO production and a decrease in renal protein expression of neuronal NO synthase.

Endothelin type a receptor blockade attenuates the hypertension in response to chronic reductions in uterine perfusion pressure.

A chronic reduction in uterine perfusion pressure in pregnant rats is associated with a significant elevation in mean arterial pressure (MAP) and reduction 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. MAP in pregnant rats with chronic reductions in uterine perfusion pressure (123.0+/-1.8 mm Hg) was significantly higher than that in control pregnant rats (101.3+/-4.0 mm Hg). Renal expression of preproendothelin mRNA as determined by ribonuclease protection assay was also significantly elevated in the medulla (>45%, P<0.05) and in the cortex (>22%, P:<0.05) of the pregnant rats with chronic reductions in uterine perfusion pressure compared with control pregnant rats. Chronic administration of the selective endothelin type A receptor antagonist (ABT-627, 5 mg/kg per day for 10 days) markedly attenuated the increase in MAP observed in the pregnant rats with chronic reductions in uterine perfusion pressure (103.3+/-5.6 mm Hg, plus endothelin antagonist; P<0.05). However, endothelin type A receptor blockade had no significant effect on blood pressure in the normal pregnant animals (96.0+/-2.7 mm Hg, plus endothelin antagonist). These findings suggest that endothelin plays a major role in mediating the hypertension produced by chronic reductions in uterine perfusion pressure in pregnant rats.