NOS inhibition restores renal responses to atrial distension during pregnancy

Nitric oxide (NO) biosynthesis increases during pregnancy and has been shown to suppress baroreceptor activity. The renal response to a simulated increase in circulating blood volume (atrial distension) is also attenuated at this time. We hypothesized that blocking NO biosynthesis during pregnancy would restore the renal response. Female rats were implanted with indwelling intracardiac balloons and central venous cannulas. After recovery, they were mated, and on day 14 of pregnancy, osmotic minipumps containing the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) or its inactive enantiomer N(G)-nitro-D-arginine methyl ester (D-NAME) (120 mg/2 ml at 10 microg/min) were implanted. In response to atrial distension (1 h), urine output increased in the D- and L-NAME-treated virgin rats. During pregnancy (day 20), this response was attenuated in the D-NAME-treated, but not the L-NAME-treated, animals, i.e., after a simulated increase in circulating blood volume, inhibition of NO biosynthesis restored the renal response of pregnant rats to that seen in virgin animals. We conclude that, during normal pregnancy, increased NO biosynthesis blunts the reflex renal response to atrial distension.     

Effect of nitric oxide synthase inhibition on cardiovascular and hormonal regulation during pregnancy in the rat

Recent studies have shown that nitric oxide (NO) biosynthesis increases in pregnancy and that inhibition of nitric oxide synthase (NOS) induces some pathological processes characteristic of preeclampsia. The current project sought to study the effect of the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 10 microg x min(-1), sc for 7 days) on plasma volume, plasma atrial natriuretic factor (ANF), plasma endothelin-1 (ET), and plasma renin activity (PRA) during gestation in conscious rats. NOS inhibition caused mean arterial pressure to increase in both virgin and 21-day pregnant rats. Plasma volume fell in the pregnant rats [L-NAME, 4.5 +/- 0.3 mL x 100 g(-1) body wt. (n = 7) vs. D-NAME, 6.8 +/- 0.2 mL x 100 g(-1) body wt. (n = 10); P < 0.05] but not in the virgin rats [L-NAME, 4.3 +/- 0.1 mL x 100 g(-1) body wt. (n = 6) vs. D-NAME, 4.8 +/- 0.2 mL x 100 g(-1) body wt. (n = 8)]. There was no effect of NOS inhibition on plasma ANF levels or PRA in either the virgin or pregnant rats. However, L-NAME did decrease plasma ET levels in the pregnant rats [L-NAME, 19.6 +/- 1.6 pg x mL(-1) (n = 8) vs. D-NAME, 11.6 +/- 2.5 pg x mL(-1) (n = 9); P < 0.05]. Our results confirm that NO is involved in cardiovascular homeostasis in pregnancy; NOS inhibition selectively reduces plasma volume in pregnant rats, thus mimicking a major pathophysiological perturbation of preeclampsia. However, it does not induce the hormonal changes characteristic of preeclampsia, namely the decrease in PRA and increase in plasma ET and ANF levels.     

Pregnancy-associated reduction in vascular protein kinase C activity rebounds during inhibition of NO synthesis

Vascular reactivity has been shown to be reduced during pregnancy and to be enhanced during chronic inhibition of nitric oxide (NO) synthesis in pregnant rats; however, the cellular mechanisms involved are unclear. The purpose of this study was to investigate whether the pregnancy-induced changes in vascular reactivity are associated with changes in the amount and/or activity of vascular protein kinase C (PKC). Active stress as well as the amount and activity of PKC was measured in deendothelialized thoracic aortic strips from virgin and pregnant rats untreated or treated with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). In virgin rats, the PKC activator phorbol 12,13-dibutyrate (PDBu, 10(-6) M) and the alpha-adrenergic agonist phenylephrine (Phe, 10(-5) M) caused significant increases in active stress and PKC activity that were inhibited by the PKC inhibitors staurosporine and calphostin C. Western blot analysis in aortic strips of virgin rats showed significant amount of the alpha-PKC isoform. Both PDBu and Phe caused significant translocation of alpha-PKC from the cytosolic to the particulate fraction. Compared with virgin rats, the PDBu- and Phe-stimulated active stress and PKC activity as well as the amount and the PDBu- and Phe-induced translocation of alpha-PKC were significantly reduced in late pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. The PDBu- and Phe-induced changes in active stress and the amount, distribution, and activity of alpha-PKC in virgin rats treated with L-NAME were not significantly different from that in virgin rats, whereas the changes in pregnant rats treated with L-NAME + the NO synthase substrate L-arginine were not significantly different from that in pregnant rats. These results provide evidence that a PKC-mediated contractile pathway in vascular smooth muscle is reduced during pregnancy and significantly enhanced during chronic inhibition of NO synthesis. The results suggest that one possible mechanism of the pregnancy-associated changes in vascular reactivity may involve changes in the amount and activity of the alpha-PKC isoform.     

Effect of cardiac receptor stimulation on renal vascular resistance in the pregnant rat

Stimulation of cardiac receptors (CR) evokes blunted reflex reductions in mean arterial pressure (MAP) in pregnant compared with virgin rats. Because CR-mediated sympathoinhibition has preferential effects on the kidney, we tested whether, during pregnancy, renal vascular resistance (RVR) changes less in response to CR stimulation and investigated possible mechanisms. MAP, right atrial pressure, renal sympathetic nerve activity (RSNA), renal blood flow (RBF), and RVR were measured in anesthetized animals in response to CR stimulation by graded atrial injections of saline. Baseline MAP and RVR and reflex changes in these variables during CR stimulation were reduced in late-pregnant vs. virgin rats (P<0.05). Reflex changes in RSNA were attenuated in pregnant rats, but changes in RBF as a function of RSNA were similar in both groups. ANG II AT(1)-receptor blockade increased basal RBF more in virgin rats (P<0.05), but between-group differences in reflex changes in MAP, RSNA, and RVR were maintained after AT(1) blockade. Thus during CR simulation, reflex changes in RVR were reduced in pregnant versus virgin rats. This difference does not appear to involve differential effects of ANG II.     

Ca(2+)](i) signaling in renal arterial smooth muscle cells of pregnant rat is enhanced during inhibition of NOS

Vascular resistance and arterial pressure are reduced during normal pregnancy, but dangerously elevated during pregnancy-induced hypertension (PIH), and changes in nitric oxide (NO) synthesis have been hypothesized as one potential cause. In support of this hypothesis, chronic inhibition of NO synthesis in pregnant rats has been shown to cause significant increases in renal vascular resistance and hypertension; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the pregnancy-associated changes in renal vascular resistance reflect changes in contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) of renal arterial smooth muscle. Smooth muscle cells were isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 4 mg. kg(-1). day(-1) for 5 days), then loaded with fura 2. In cells of virgin rats incubated in Hanks' solution (1 mM Ca(2+)), the basal [Ca(2+)](i) was 86 +/- 6 nM. Phenylephrine (Phe, 10(-5) M) caused a transient increase in [Ca(2+)](i) to 417 +/- 11 nM and maintained an increase to 183 +/- 8 nM and 32 +/- 3% cell contraction. Membrane depolarization by 51 mM KCl, which stimulates Ca(2+) entry from the extracellular space, caused maintained increase in [Ca(2+)](i) to 292 +/- 12 nM and 31 +/- 2% contraction. The maintained Phe- and KCl-induced [Ca(2+)](i) and contractions were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. Phe- and KCl-induced contraction and [Ca(2+)](i) were not significantly different between untreated and L-NAME-treated virgin rats or between untreated and L-NAME + L-arginine treated pregnant rats. In Ca(2+)-free Hanks', application of Phe or caffeine (10 mM), to stimulate Ca(2+) release from the intracellular stores, caused a transient increase in [Ca(2+)](i) and a small cell contraction that were not significantly different among the different groups. Thus renal interlobular smooth muscle of normal pregnant rats exhibits reduction in [Ca(2+)](i) signaling that involves Ca(2+) entry from the extracellular space but not Ca(2+) release from the intracellular stores. The reduced renal smooth muscle cell contraction and [Ca(2+)](i) in pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy, whereas the enhanced cell contraction and [Ca(2+)](i) during inhibition of NO synthesis in pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.     

Alterations in Perivascular Sympathetic and Nitrergic Innervation Function Induced by Late Pregnancy in Rat Mesenteric Arteries

Background and Purpose

We investigated whether pregnancy was associated with changed function in components of perivascular mesenteric innervation and the mechanism/s involved.

Experimental Approach

We used superior mesenteric arteries from female Sprague-Dawley rats divided into two groups: control rats (in oestrous phase) and pregnant rats (20 days of pregnancy). Modifications in the vasoconstrictor response to electrical field stimulation (EFS) were analysed in the presence/absence of phentolamine (alpha-adrenoceptor antagonist) or L-NAME (nitric oxide synthase-NOS- non-specific inhibitor). Vasomotor responses to noradrenaline (NA), and to NO donor DEA-NO were studied, NA and NO release measured and neuronal NOS (nNOS) expression/activation analysed.

Key Results

EFS induced a lower frequency-dependent contraction in pregnant than in control rats. Phentolamine decreased EFS-induced vasoconstriction in segments from both experimental groups, but to a greater extent in control rats. EFS-induced vasoconstriction was increased by L-NAME in arteries from both experimental groups. This increase was greater in segments from pregnant rats. Pregnancy decreased NA release while increasing NO release. nNOS expression was not modified but nNOS activation was increased by pregnancy. Pregnancy decreased NA-induced vasoconstriction response and did not modify DEA-NO-induced vasodilation response.

Conclusions and Implications

Neural control of mesenteric vasomotor tone was altered by pregnancy. Diminished sympathetic and enhanced nitrergic components both contributed to the decreased vasoconstriction response to EFS during pregnancy. All these changes indicate the selective participation of sympathetic and nitrergic innervations in vascular adaptations produced during pregnancy.     

Interleukin-6 impairs endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of pregnant rats

IL-6 is elevated in plasma of preeclamptic women, and twofold elevation of plasma IL-6 increases vascular resistance and arterial pressure in pregnant rats, suggesting a role of the cytokine in hypertension of pregnancy. However, whether the hemodynamic effects of IL-6 reflect direct effects of the cytokine on the mechanisms of vascular contraction/relaxation is unclear. The purpose of this study was to test the hypothesis that IL-6 directly impairs endothelium-dependent relaxation and enhances vascular contraction in systemic vessels of pregnant rats. Active stress was measured in aortic strips isolated from virgin and late pregnant Sprague-Dawley rats and then nontreated or treated for 1 h with IL-6 (10 pg/ml to 10 ng/ml). In endothelium-intact vascular strips, phenylephrine (Phe, 10(-5) M) caused an increase in active stress that was smaller in pregnant (4.2 +/- 0.3) than virgin rats (5.1 +/- 0.3 x 10(4) N/m(2)). IL-6 (1,000 pg/ml) caused enhancement of Phe contraction that was greater in pregnant (10.6 +/- 0.7) than virgin rats (7.5 +/- 0.4 x 10(4) N/m(2)). ACh and bradykinin caused relaxation of Phe contraction and increases in vascular nitrite production that were greater in pregnant than virgin rats. IL-6 caused reductions in ACh- and bradykinin-induced vascular relaxation and nitrite production that were more prominent in pregnant than virgin rats. Incubation of endothelium-intact strips in the presence of N(omega)-nitro-L-arginine methyl ester (10(-4) M) to inhibit nitric oxide (NO) synthase, or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10(-5) M) to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in nontreated but to a lesser extent in IL-6-treated vessels, particularly those of pregnant rats. Removal of the endothelium enhanced Phe-induced stress in nontreated but not IL-6-treated vessels, particularly those of pregnant rats. In endothelium-denuded strips, relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was not different between nontreated and IL-6-treated vessels of virgin or pregnant rats. Thus IL-6 inhibits endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of virgin and pregnant rats. The greater IL-6-induced inhibition of vascular relaxation and enhancement of contraction in systemic vessels of pregnant rats supports a direct role for IL-6 as one possible mediator of the increased vascular resistance associated with hypertension of pregnancy.     

Pregnancy alters cardiac receptor afferent discharge in rats

Reflex effects of cardiac receptor (CR) stimulation are attenuated in pregnant rats. We tested whether CR afferent discharge is reduced during pregnancy by measuring single fiber activity in response to increases in right atrial pressure (RAP) in anesthetized pregnant and virgin rats with sinoaortic denervation. Single fiber activity was isolated from fine filaments of the right cervical vagus nerve. Changes in CR discharge, RAP, and arterial pressure were recorded in response to atrial saline injections (25-300 microl). Resting RAP was similar between groups, and spontaneous CR discharge was similar in pregnant rats (1.95+/-0.21 Hz) and in low-frequency (LF) receptors in virgin rats (1.30+/-0.2 Hz). In virgin, but not pregnant rats, a subset (24%) of CR had higher-frequency (HF) spontaneous discharge (9.91+/-1.19 Hz). During stimulation, the level of RAP above which CR firing increased was significantly higher in pregnant rats, but CR activity was clustered into an LF discharge range. Thus gestation appears to reduce the activity of CR afferents, possibly by increasing stimulus threshold or by selective inactivation of a subset of HF discharging receptors.     

Suppression of endotoxin-induced fever in near-term pregnant rats is mediated by brain nitric oxide

Over the last three decades, experiments in several mammalian species have shown that the febrile response to bacterial endotoxin is attenuated late in pregnancy. More recent evidence has established that the expression of nitric oxide synthase (NOS) enzymes is increased in the brain late in pregnancy. The current study investigated the possible role of brain nitric oxide in mediating the phenomenon of fever suppression. Core body temperature (Tb) of near-term pregnant rats (day 19 and 20) was measured following inhibition of brain NOS and intraperitoneal injection of LPS (50 microg/kg); they were compared with both day 15 pregnant and virgin animals. Intracerebroventricular injection with an inhibitor of NOS, NG-monomethyl-L-arginine citrate (L-NMMA; 280 microg), in near-term pregnant rats restored the febrile response to LPS. As expected, near-term dams that received intracerebroventricular vehicle + IP LPS did not increase Tb, in contrast to the 1.0 +/- 0.2 degrees C rise in Tb in dams treated with ICV L-NMMA + IP LPS (P < 0.01). In virgin females and day 15 pregnant controls receiving this treatment, the increases in Tb were 1.5 +/- 0.3 degrees C and 1.6 +/- 0.4 degrees C, respectively. Thus, blockade of brain NOS restored the febrile response to LPS in near-term dams; at 5 h postinjection, Tb was 60-70% of that observed in virgins and day 15 pregnant animals. Intracerebroventricular L-NMMA alone did not induce a significant change in Tb in any group. These results suggest that the mechanism underlying the suppression of the febrile response in near-term pregnancy is mediated by nitric oxide signaling in the brain.     

Right atrial dimension-pressure relation during volume expansion is unaltered by pregnancy in the rat

Blood volume expands significantly during pregnancy, but afferent signals from cardiac receptors are reduced. In addition, during exogenous volume expansion, right atrial pressure (RAP) increases more for equivalent volumes in pregnant animals, implying reduced atrial compliance. To examine possible gestational alterations in atrial dimension during volume expansion, we compared the effects of volume expansion on RAP and right atrial dimension (RAD) in pregnant vs. virgin rats. Anesthetized animals were ventilated and catheterized for measurement of arterial pressure and RAP and for drug infusion. Through a parasternal incision, ultrasonic crystals were glued to the medial and lateral surfaces of the right atrium for measurement of RAD. Plasma volume and hematocrit were determined before experimentation. RAP, RAD, and arterial pressure were recorded at baseline and during progressive volume expansion (6% dextran, 60% of initial blood volume). Baseline RAP was similar in the two groups: 2.82 +/- 0.40 and 2.72 +/- 0.47 mmHg in pregnant and virgin rats, respectively. Basal RAD was significantly larger in pregnant than in virgin rats: 4.36 +/- 0.66 vs. 3.36 +/- 0.48 mm. Despite increased basal RAD in pregnant rats, the slope of the RAD-RAP relation during volume expansion was similar in the two groups. Results indicate that resting RAD is increased in pregnant rats and that the change in dimension during volume loads is similar to that in virgin rats. Thus, during pregnancy, the right atrium appears to accommodate the increased blood volume, and reduced afferent signaling most likely is due to mechanisms other than mechanical alterations of the atrium by expanded volume.     

Role of neuronal nitric oxide synthase in mediating renal hemodynamic changes during pregnancy

Renal plasma flow (RPF) and glomerular filtration rate (GFR) are markedly increased during pregnancy. We recently reported that the renal hemodynamic changes observed during pregnancy in rats are associated with enhanced renal protein expression of neuronal nitric oxide synthase (nNOS). The purpose of this study was to determine the role of nNOS in mediating renal hemodynamic changes observed during pregnancy. To achieve this goal, we examined the effects of the nNOS inhibitor 7-nitroindazole (7-NI) on kidney function in normal conscious, chronically instrumented virgin (n = 6) and pregnant rats (n = 9) at day 16 of gestation. Infusion of 7-NI had no effect on RPF (4.7 +/- 0.7 vs. 4.8 +/- 0.9 ml/min), GFR (2.2 +/- 0.2 vs. 2.5 +/- 0.4 ml/min), or mean arterial pressure (MAP; 127 +/- 7 vs. 129 +/- 10 mmHg) in virgin rats. In contrast, 7-NI infused into pregnant rats decreased RPF (8.9 +/- 1.6 vs. 6.5 +/- 1.4 ml/min) and GFR (4.4 +/- 0.7 vs. 3.3 +/- 0.7 ml/min) while having no effect on MAP (123 +/- 4 vs. 123 +/- 3 mmHg). In summary, inhibition of nNOS in pregnant rats at midgestation results in significant decreases in RPF and GFR. nNOS inhibition in virgin rats had no effect on renal hemodynamics. These data suggest that nNOS may play a role in mediating the renal hemodynamic changes that occur during pregnancy.     

TNF-alpha enhances contraction and inhibits endothelial NO-cGMP relaxation in systemic vessels of pregnant rats

Tumor necrosis factor-alpha (TNF-alpha) is elevated in the plasma of preeclamptic women and may have a role in pregnancy-induced hypertension. However, whether the hemodynamic effects of TNF-alpha reflect the direct effects on vascular reactivity is unclear. We tested the hypothesis that TNF-alpha impairs endothelium-dependent relaxation and enhances vascular contraction in systemic vessels of pregnant rats. We measured isometric contraction in aortic strips isolated from virgin and pregnant Sprague-Dawley rats (nontreated vs. treated for 2 h with 10-1,000 pg/ml TNF-alpha). In endothelium-intact vascular strips, TNF-alpha caused greater enhancement of phenylephrine (Phe) contraction in pregnant than virgin rats. TNF-alpha caused significant inhibition of ACh- and bradykinin-induced vascular relaxation and nitrite/nitrate production that were more prominent in pregnant than virgin rats. N(G)-nitro-L-arginine methyl ester [L-NAME, 100 microM, an inhibitor of nitric oxide (NO) synthase] or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 1 microM, an inhibitor of cGMP production in smooth muscle) inhibited ACh relaxation and enhanced Phe contraction in nontreated but to a lesser extent in TNF-alpha-treated vessels, particularly those of pregnant rats. Endothelium removal enhanced Phe contraction in nontreated but not TNF-alpha-treated vessels, especially those of pregnant rats. Relaxation of Phe contraction with the NO donor sodium nitroprusside was not different between nontreated and TNF-alpha-treated vessels. Thus TNF-alpha enhances vascular contraction and inhibits endothelium-dependent NO-cGMP-mediated vascular relaxation in systemic vessels, particularly those of pregnant rats. The results support a direct role for TNF-alpha as a possible mediator of increased vascular resistance associated with pregnancy-induced hypertension.     

Endogenous estrogen mediates vascular reactivity and distensibility in pregnant rat mesenteric arteries

The role of estrogen in the maternal systemic cardiovascular adaptations during pregnancy is still controversial. Female Sprague-Dawley rats were implanted at day 14 of pregnancy with either a 50-mg tamoxifen pellet (estrogen receptor blocker, n = 10) or placebo pellet (n = 10). Virgin female rats were a nonpregnant control (n = 7). At days 20-22 of pregnancy, resistance-sized mesenteric arteries were mounted onto a dual-chamber arteriograph system. Pregnancy significantly blunted the pressor response to phenylephrine [measurement of the effective concentration that yielded 50% maximum response (EC(50)) values were 1.5 +/- 0.22 vs. 0.69 +/- 0.16 microM (P < 0.05)] and enhanced vasodilation to ACh [EC(50) = 1.13 +/- 2.53 vs. 3.13 +/- 6.04 nM (P < 0.05)] compared with nonpregnant rats. However, tamoxifen treatment during pregnancy reversed these effects. Inhibition of nitric oxide (NO) synthase with N(G)-monomethyl-L-arginine (250 microM) shifted only the responses of the placebo-treated pregnant group to both phenylephrine and ACh. Arterial distensibility in the placebo-treated pregnant group was also significantly increased (P < 0.05) compared with nonpregnant and tamoxifen-treated pregnant animals. In summary, endogenous estrogen during pregnancy increases NO-dependent modulation of vessel tone and arterial distensibility.     

Differential metabolic response to 48 h food deprivation at different periods of pregnancy in the rat

Since during pregnancy the mother switches from an anabolic to a catabolic condition, the present study was addressed to determine the effect of 48 h food deprivation on days 7, 14 and 20 of pregnancy in the rat as compared to age matched virgin controls. Body weight, free of conceptus, decreased with food deprivation more in pregnant than in virgin rats, with fetal weight (day 20) also diminishing with maternal starvation. The decline of plasma glucose with food deprivation was greatest in 20 day pregnant rats. Insulin was highest in fed 14 day pregnant rats, and declined with food deprivation in all the groups, the effect being not significant in 7-day pregnant rats. Food deprivation increased plasma glycerol only in virgin and 20 day pregnant rats. Plasma NEFA and 3-hydroxybutyrate increased with food deprivation in all groups, the effect being highest in 20 day pregnant rats. Food deprivation decreased plasma triacylglycerols in 14 day pregnant rats but increased in 20 day pregnant rats. In 20-day fetuses, plasma levels of glucose, NEFA and triacylglycerols were lower than in their mothers when fed, and food deprivation caused a further decline in plasma glucose, whereas both NEFA and 3-hydroxybutyrate increased. Liver triacylglycerols concentration did not differ among the groups when fed, whereas food deprivation caused an increase in all pregnant rats and fetuses, the effect being highest in 20-day pregnant rats. Lipoprotein lipase (LPL) activity in adipose tissue was lower in 20 day pregnant rats than in any of the other groups when fed, and it decreased in all the groups with food deprivation, whereas in liver it was very low in all groups when fed and increased with food deprivation only in 20 day pregnant rats. A significant increase in liver LPL was found with food deprivation in 20 day fetuses, reaching higher values than their mothers. Thus, the response to food deprivation varies with the time of pregnancy, being lowest at mid pregnancy and greatest at late pregnancy, and although fetuses respond in the same direction as their mothers, they show a specific response in liver LPL activity.     

Effect of starvation on nutrition and insulin secretion in pregnant rats and their fetuses

Pregnancy is thought to create a metabolic condition of accelerated starvation. To clarify this idea, the effect of fasting on pregnant rats (day 21 of gestation) and their fetuses was examined. Although pregnancy significantly increased plasma insulin, plasma ketone body concentrations in fed pregnant rats were higher than those of age-matched fed virgin rats. After 48 hr fasting (i.e., fasting during days 19-21 of gestation), plasma insulin was markedly decreased in virgin rats compared with term pregnant rats, while ketone bodies were significantly higher in pregnant rats than in virgin rats. Body weight was lower in fetuses from fasted mothers than those from fed mothers. Starvation also markedly diminished the insulin response to glucose in isolated, perfused pancreases in both virgin and pregnant rats. The amount of insulin released during glucose stimulation was greater in pregnancy, and the inhibitory effect of 48 hr fasting on insulin release was greater in virgin rats than in pregnant rats. It is possible, therefore, that in term pregnant rats a decrease in insulin release caused by fasting may cause more profound catabolism than in nongravid rats.     

Distension of the uterus induces HspB1 expression in rat uterine smooth muscle

The uterine musculature, or myometrium, demonstrates tremendous plasticity during pregnancy under the influences of the endocrine environment and mechanical stresses. Expression of the small stress protein heat shock protein B1 (HspB1) has been reported to increase dramatically during late pregnancy, a period marked by myometrial hypertrophy caused by fetal growth-induced uterine distension. Thus, using unilaterally pregnant rat models and ovariectomized nonpregnant rats with uteri containing laminaria tents to induce uterine distension, we examined the effect of uterine distension on myometrial HspB1 expression. In unilaterally pregnant rats, HspB1 mRNA and Ser(15)-phosphorylated HspB1 (pSer(15) HspB1) protein expression were significantly elevated in distended gravid uterine horns at days 19 and 23 (labor) of gestation compared with nongravid horns. Similarly, pSer(15) HspB1 protein in situ was only readily detectable in the distended horns compared with the nongravid horns at days 19 and 23; however, pSer(15) HspB1 was primarily detectable in situ at day 19 in membrane-associated regions, while it had primarily a cytoplasmic localization in myometrial cells at day 23. HspB1 mRNA and pSer(15) HspB1 protein expression were also markedly increased in ovariectomized nonpregnant rat myometrium distended for 24 h with laminaria tents compared with empty horns. Therefore, uterine distension plays a major role in the stimulation of myometrial HspB1 expression, and increased expression of this small stress protein could be a mechanoadaptive response to the increasing uterine distension that occurs during pregnancy.     

Streptozotocin-Induced Diabetes Decreases Mammary Gland Lipoprotein Lipase Activity and Messenger Ribonucleic Acid in Pregnant and Nonpregnant Rats

Diabetes mellitus is associated with a reduction of lipoprotein lipase (LPL) activity in adipose tissue and development of hypertriglyceridemia. To determine how a condition of severe insulin deficiency affects mammary gland LPL activity and mRNA expression during late pregnancy, streptozotocin (STZ) treated (40 mg/kg) and non-treated (control) virgin and 20 day pregnant rats were studied. In control rats, both LPL activity and mRNA were higher in pregnant than in virgin rats. When compared to control rats, STZ-treated rats, either pregnant or virgin, showed decreased LPL activity and mRNA content. Furthermore, mammary gland LPL activity was linearly correlated with mRNA content, and either variable was linearly correlated with plasma insulin levels. Thus, insulin deficiency impairs the expression of LPL in mammary glands, revealing the role of insulin as a modulator of the enzyme at the mRNA expression level.     

Opposite metabolic response to fenofibrate treatment in pregnant and virgin rats.

The level of maternal circulating triglycerides during late pregnancy has been correlated to newborns' weight in humans. To investigate the response to fenofibrate, a hypotriglyceridemic agent, in pregnant rats, 0, 100, or 200 mg of fenofibrate/kg body weight as oral doses were given twice a day from day 16 of gestation and studied at day 20. Virgin rats were studied in parallel. Liver weight was higher in pregnant than in virgin rats, and either dose of fenofibrate increased this variable in both groups. The highest dose of fenofibrate decreased fetal weight. Although plasma triglycerides decreased during the first 2 days of fenofibrate treatment in pregnant rats, the effect disappeared on day 3, and plasma triglycerides were even enhanced at day 4. In virgin rats, fenofibrate decreased plasma triglycerides throughout the experiment. Plasma cholesterol levels in pregnant rats decreased during the first 3 days of treatment, and the effect disappeared on day 4, whereas in virgin rats, values remained decreased. Changes in plasma triglycerides paralleled those of VLDL triglycerides. In pregnant rats, VLDL cholesterol levels increased while LDL cholesterol decreased with the treatment, whereas in virgin rats, cholesterol levels decreased in all lipoprotein fractions. Only in virgin rats did liver triglyceride concentration increase with fenofibrate treatment. Lumbar adipose tissue LPL was lower in pregnant than in virgin rats, and fenofibrate treatment decreased this variable in both groups. Maternal fenofibrate treatment increased fetal plasma and liver triglyceride and cholesterol concentrations.It is proposed that the opposite effects of fenofibrate treatment in virgin and pregnant rats are a consequence of both the enhanced liver capability for VLDL triglyceride production and a rebound response to the drug in the latter.     

Effect of pregnancy and 5alpha-pregnan-3alpha-ol-20-one on atrial receptor afferent discharge in rats

The atrial volume reflex is attenuated in pregnancy. This may be mimicked by chronic administration of 5alpha-pregnan-3alpha-ol-20-one (pregnan). We investigated whether afferent output from sensory receptors may be suppressed at this time. Vagal afferent nerve activity was measured during discrete localized stimulation of the atrial volume receptors by inflation of a balloon at the superior vena caval-right atrial junction. The receptors were classified as high- (HF) or low- (LF) frequency subtypes on the basis of their response to graded atrial distension. Although both HF (regression coefficient = 0.50 +/- 0.11 Hz/microl, r(2) = 0.47, P < 0.001) and LF (regression coefficient = 0.03 +/- 0.05 Hz/microl, r(2) = 0.009, P = 0.613) subtypes could be identified in virgin rats, only LF (regression coefficient = 0.09 +/- 0.05 Hz/microl, r(2) = 0.044, P = 0.099) receptors were found in late-pregnant animals. Similarly, in virgin rats treated chronically with pregnan (500 microg/24 h for 2 days), only LF receptors were identified (regression coefficient = -0.004 +/- 0.078 Hz/microl, r(2) = 0.000, P = 0.962), whereas both subtypes were present in the vehicle-treated animals (HF regression coefficient = 0.626 +/- 0.255 Hz/microl, r(2) = 0.317, P = 0.029; LF regression coefficient = -0.012 +/- 0.071 Hz/microl, r(2) = 0.002, P = 0.866). By contrast, acute intracardiac pregnan (2.6 microg/kg) did not alter vagal afferent nerve activity. In conclusion, stretch-induced discharge of high-frequency atrial receptors is suppressed during pregnancy, whereas that of low-frequency receptors is preserved. This effect may be mimicked by chronic, but not acute, pregnan. We propose that, during pregnancy, pregnan alters the transducer properties of the atrial volume receptors, thus allowing blood volume to increase.     

Effect of 5 alpha-pregnan-3 alpha-ol-20-one on nitric oxide biosynthesis and plasma volume in rats

Plasma 5 alpha-pregnan-3 alpha-ol-20-one (pregnan) levels and nitric oxide (NO) biosynthesis increase during pregnancy. These factors have independently been implicated in the control of blood pressure and volume. We wished to determine whether pregnan might be responsible both for the increase in NO biosynthesis and for the increase in plasma volume observed during pregnancy. Virgin female Long-Evans rats were implanted with indwelling cannulas and maintained on a low nitrate/nitrite diet. After the rats recovered from surgery, 500 microg of pregnan or vehicle were given daily for 2 days. NO biosynthesis and plasma volume were measured in conscious animals before and after treatment. Pregnan caused a significant increase in NO biosynthesis (1.9 +/- 0.8 micromol/24 h, n = 10) compared with the vehicle-treated control group (0.3 +/- 0.4 micromol/24 h, n = 10, P < 0.05). Similarly, there was a significant increase in plasma volume in the pregnan-treated group (0.7 +/- 0.2 ml/100 g, n = 11) compared with the vehicle-treated control group (0.2 +/- 0.1 ml/100 g, n = 11, P < 0.05). These results confirm that pregnan can mimic pregnancy by its ability to increase both NO biosynthesis and plasma volume.