Virgin Rats Research Articles

Evidence against the Hypothesis that Endothelial Endothelin B Receptor Expression Is Regulated by Relaxin and Pregnancy

The endothelial endothelin B (ET(B)) receptor subtype is critical for renal vasodilation induced by relaxin in nonpregnant rats and during pregnancy (the latter via endogenous circulating relaxin). Here we tested whether expression of vascular ET(B) receptor protein is regulated by relaxin. Small renal arteries were harvested from virgin and midterm pregnant rats as well as nonpregnant rats that were administered recombinant human relaxin (rhRLX) at 4 mug/h or vehicle for 5 d or 4-6 h. Small renal arteries dissected from additional virgin rats were incubated in vitro with rhRLX or vehicle for 3 h at 37 C. ET(B) expression was also evaluated in cultured human endothelial cells: aortic, coronary, umbilical vein, and dermal microvascular endothelial cells. Cells were incubated for 4, 8, or 24 h with rhRLX (5, 1, or 0.1 ng/ml) or vehicle. ET(B) protein expression in arteries and cells was evaluated by Western analysis. No regulation of ET(B) expression was observed in small renal arteries in any of the experimental protocols, nor was there an increase in the vasorelaxation response to ET-3 in small renal arteries incubated in vitro with rhRLX. rhRLX only sporadically altered ET(B) expression in human coronary artery endothelial cells and human umbilical vein endothelial cells at certain time points or doses, and no regulation was observed in human aortic endothelial cells or human dermal microvascular endothelial cells. These results suggest that regulation of ET(B) receptor protein has little or no role in relaxin stimulation of the endothelial ET(B)/nitric oxide vasodilatory pathway.

Effect of the chemical form of supranutritional selenium on selenium load and selenoprotein activities in virgin, pregnant, and lactating rats1

Virgin, pregnant, and lactating rats were used to assess the influence of selenomethionine and selenocystine, fed at four to seven times the daily Se requirement (supranutritional), on Se load and selenoprotein activities. Female Sprague Dawley rats (n = 48; age = 13 wk), reared on a low-Se torula yeast diet, were assigned to one of three reproductive states (n = 16 per reproductive state) to occur simultaneously: virgin, pregnant, and lactating. Once reproductive state was achieved, rats were fed (ad libitum) either l-selenomethionine (n = 24) or L-selenocystine (n = 24) diets providing 2.0 microg Se/g of diet (as-fed basis) for 18 d, and then killed. Lactating rats consuming selenomethionine had the greatest Se concentration in the brain, with pregnant rats being intermediate, and virgin rats having the least (P < 0.02). When selenocystine was fed, the concentration of Se in the brain was greater (P = 0.008) in lactating rats, but not different (P = 0.34) between pregnant and virgin rats. Selenium concentrations in the heart, liver, lung, muscle, spleen, plasma, placenta, uterus, and fetus were greatest (P < 0.001) in rats consuming selenomethionine. Brain, kidney, and liver thioredoxin reductase, and brain, erythrocyte, kidney, and liver glutathione peroxidase activities did not differ (P = 0.13 to P = 0.85) between Se treatments. Lactating rats exhibited the greatest (P < 0.006) Se concentration in the heart, lung, muscle, plasma, and spleen compared with pregnant and virgin rats. Thioredoxin reductase was greatest (P < 0.004) in the brain of pregnant rats, greatest (P < 0.004) in the liver of lactating rats, and greater (P < 0.03) in the kidney of lactating and pregnant vs. virgin rats. Regardless of reproductive state, supranutritional Se (2.0 microg/g of diet) fed as selenocystine resulted in less Se load, and when fed as selenomethionine, was equally available for thioredoxin reductase synthesis as the Se in selenocystine. Independent of dietary Se chemical form, thioredoxin reductase activity was responsive to reproductive state.

Reduced Activity of the Noradrenergic System in the Paraventricular Nucleus at the End of Pregnancy: Implications for Stress Hyporesponsiveness

We investigated whether changes in noradrenaline neurotransmission in the hypothalamus could explain the hyporesponsiveness of the hypothalamic-pituitary-adrenal (HPA) axis in late pregnancy. Noradrenaline release within the hypothalamic paraventricular nucleus in response to swim stress, as estimated by microdialysis and high-performance liquid chromatography, was lower in 20-day pregnant rats compared to virgin rats. Driving a central noradrenergic pathway using intravenous cholecystokinin increased adrenocorticotropic hormone (ACTH) secretion in virgin rats, but the response was significantly less in 16-day and 20-day pregnant rats. Thus, the activity of noradrenergic inputs to the paraventricular nucleus and the HPA axis is attenuated in late pregnancy. The sensitivity of the HPA axis to noradrenaline in pregnancy was investigated by intracerebroventricular administration of an alpha1-receptor antagonist, benoxathian, before and during exposure to swim stress. In virgin rats, benoxathian increased basal and stress-induced ACTH secretion, but in late pregnant rats the benoxathian effects were attenuated, indicating reduced sensitivity of the HPA axis to noradrenaline neurotransmission and/or the inability of the system to become disinhibited at this time. alpha1A-adrenoreceptor mRNA expression in the parvocellular and magnocellular paraventricular nucleus, measured by in situ hybridisation, was decreased in late pregnant compared to virgin rats. Additionally, blocking endogenous opioid inhibition with naloxone pretreatment restored the ACTH secretory response to cholecystokinin in pregnant rats. Thus, in late pregnancy, there is reduced noradrenergic input to the paraventricular nucleus and reduced alpha1A-receptor expression in the paraventricular nucleus, both of which may contribute to the reduced responsiveness of the HPA axis in pregnancy.

Reduced Hypothalamic Vasopressin Secretion Underlies Attenuated Adrenocorticotropin Stress Responses in Pregnant Rats

We sought to explain decreased ACTH secretory responses to stress in pregnant rats by investigating hypothalamic CRH and vasopressin secretion and actions on anterior pituitary corticotrophs. In late pregnancy median eminence, CRH content was reduced (by 12%). Anterior pituitary proopiomelanocortin mRNA expression, measured by in situ hybridization but not radioimmunoassayed ACTH content, was also reduced (by 45% on d 21); CRH receptor (CRHR)1 mRNA expression was unaltered in pregnancy, but V1b receptor mRNA expression was reduced (by 19%). ACTH secretory responses, measured in jugular blood, to CRH (200 ng/kg iv) or vasopressin (1.7 microg/kg, iv) were reduced on d 21 vs. virgins (49% and 44%), but the response to combined CRH and vasopressin injection was intact. Either antalarmin (CRHR1 antagonist; 20 mg/kg ip) or dP(Tyr(Me)2),Arg-NH2(9))AVP (V1a/b antagonist; 10 microg/kg, iv) pretreatment reduced the ACTH secretory response to forced swimming (90 sec) in virgin rats (by 57% and 40%), but only antalarmin was effective in pregnant rats (53% decrease). In vitro, measuring ACTH secretion from acutely dispersed anterior pituitary cells showed increased corticotroph sensitivity in pregnancy to CRH and to CRH augmentation by vasopressin, attributable to increased intracellular cAMP action. Hence, in late pregnancy, reduced anterior pituitary CRHR1 or V1b receptor expression did not impair corticotroph responses to CRH or vasopressin. Rather, diminished secretagogue secretion in vivo accounts for reduced action of stress levels of exogenous CRH or vasopressin alone; the late pregnancy attenuated ACTH secretory response to swim stress is deduced to be due to reduced vasopressin release by parvocellular paraventricular nuclei neurones.

Morphometric Analysis of Follicular Growth and Biochemical Constituents in Albino Rats Exposed to Mancozeb

Mancozeb, an ethylenebisdithiocarbamate (EBDC), was administered orally at a dose of 700 mg/kg body weight/day to female virgin rats for 5, 10, 20, and 30 days to examine the effect on ovarian follicular development. No significant change occurred in the number of estrous cycles and the duration of proestrus, estrus, and metestrus, but mancozeb treatment for 5 days significantly increased the duration of diestrus. Mancozeb treatment for 10 days significantly increased the number of estrous cycle and the duration of estrus, with a concomitant significant increase in diestrus, but no change in proestrus and metestrus. With mancozeb treatment for 20 and 30 days, the number of estrous cycles and duration of proestrus, estrus, and metestrus were significantly decreased, with a concomitant significant increase in the duration of diestrus. Exposure of rats to mancozeb for 5 days resulted in a significant decrease in stage II and the total number of healthy follicles but no change in atretic follicles. Mancozeb treatment for 10 days resulted in a significant decrease in stages I, II, and IV and in the total number of healthy follicles. A significant increase in atretic follicles was found in rats treated with mancozeb for 20 and 30 days. No significant change occurred in body and organ weights in any group, but the thyroid weight of 20 and 30 days mancozeb-treated rats was significantly increased. The level of protein in the ovary was significantly decreased, but no change was found in the uterus and liver of mancozeb-treated animals. The level of glycogen was significantly decreased in the ovary and the uterus with mancozeb treatment, but not in the liver. With mancozeb treatment, the levels of phospholipids and neutral lipids were significantly increased in the liver but significantly decreased in the uterus. The change in the biochemical constituents of ovary, uterus, and liver was duration dependent. The results of the study thus indicate a marked disruption of the estrous cycle, pathological changes in the gonads, and organ-specific biochemical changes in rats after exposure to mancozeb.

Long-term effects of repeated pregnancies (multiparity) on blood pressure regulation.

Pregnancy is associated with profound alterations in the cardiovascular system, the long-term effects of which are unknown. Human epidemiological studies suggest that multiparity (multiple pregnancies) increases the risk of cardiovascular disease. The mechanisms underlying these findings remain to be elucidated. The objective of this study was to determine the long-term effects of parity on cardiovascular regulation. Pressor responses to phenylephrine (PE) and acute stress were compared in conscious age-matched repeatedly breed (RB) and virgin rats. Vascular compliance and reactivity of isolated resistance-sized mesenteric arteries were studies using pressure and wire myograph. We found that both exogenous PE and acute stress elicited greater pressor responses in RB than in aged-matched virgins. Pressure and wire myography also revealed that small mesenteric arteries from RB rats were less compliant than those from virgins (RB: 0.24+/-0.04 microm mm Hg(-1), n=6 vs. virgins: 0.63+/-0.06 microm mm Hg(-1), n=6; p< or =0.05) and were more sensitive to PE (EC(50) RB: 1.58+/-0.08 x 10(-6) M, n=10 vs. virgins: 2.05+/-0.09 x 10(-6) M, n=14; p< or =0.05). Removal of the endothelium abolished the difference in sensitivity. More specifically, the augmented vascular response of RB was both nitric oxide (NO) and cyclooxygenase dependent. By contrast, there was no difference in methacholine-induced vasodilation of phenylephrine-preconstricted vessels. Our results suggest that repeated pregnancies induce long-term alterations in cardiovascular regulation due to changes in vascular compliance and endothelium-dependent vasoconstriction. We propose that such changes might influence the risk for cardiovascular disease in multiparous women.

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.

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.

Nitric oxide synthase isoforms in the rat kidney during pregnancy.

Nitric oxide mediates renal vasodilation and hyperfiltration during pregnancy in conscious rats through the endothelin B (ETB) receptor subtype. We tested the hypothesis that immunoreactive levels of endothelial nitric oxide synthase (eNOS) would be greater in the kidneys of midterm pregnant rats compared with virgin rats. We studied midterm pregnancy because renal plasma flow and glomerular filtration rate are maximal at this gestational stage. Western analysis was used to determine the level of eNOS in the three major zones of the kidney-inner medulla, outer medulla, and cortex-and in isolated small renal arteries, and in purified renal microvessels from the cortex. There were no significant differences in eNOS expression between virgin and midterm pregnant rats in any of those renal tissues, regardless of whether immunoreactivity was expressed as arbitrary densitometry units, as "microg placental equivalents" interpolated from the linear portion of a dose-response curve of placental villous protein (2.5-30 microg, positive control) run concurrently on each gel, or normalized for beta-actin. We also investigated other NOS isoforms. In particular, immunoreactive neuronal NOS (nNOS) was detectable in the inner and outer medulla, but it was not significantly different between groups. nNOS immunoreactivity was below the level of detection in the cortex, but mRNA expression was not significantly different between pregnant and virgin rats by reverse transcriptase polymerase chain reaction. Our results suggest that an increase in eNOS isoform mass does not contribute to the endothelin and NO-dependent renal vasodilation in rat gestation.

Susceptibility of Rats to Mammary Gland Carcinogenesis by the Food-Derived Carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[4,5- b]Pyridine (PhIP) Varies with Age and Is Associated with the Induction of Differential Gene Expression

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, induces mammary gland cancer when administered to adolescent female rats (43-day-old). In contrast, mature virgin rats (150-day-old) were resistant to mammary carcinogenesis by PhIP. To explore the possible mechanisms for the age-related differences in susceptibility, PhIP-DNA adduct levels, mutations, and gene expression were examined in glands from 43-day and 150-day-old PhIP-treated rats. In rats of different ages, PhIP-DNA adduct levels detected by the (32)P-post-labeling assay and mutant frequency measured in the lacI reporter gene of Big Blue rats were not statistically different. PhIP-DNA adduct levels, adduct removal, and mutation burden did not appear to account for the variation in carcinogen susceptibility with age. However, cDNA microarray analysis indicated that PhIP treatment differentially altered the profile of gene expression in glands from 43-day-old and 150-day-old rats. In 150-day-old rats, PhIP enhanced the expression of genes associated with differentiation (eg, beta-casein, kappa-casein, whey acidic protein) and induced morphological differentiation. In contrast, in 43-day-old rats, PhIP inhibited the expression of differentiation genes and enhanced cellular proliferation. From 3 hours to 6 weeks after PhIP dosing, the number of clones showing altered expression declined more than 50% in 150-day-old rats but increased fourfold in 43-day-old rats (29 clones versus 194, respectively) suggesting that PhIP induced a cascade of gene expression alterations only in susceptible rats. Genes showing altered expression specifically in 43-day-old rats included the Ras superfamily genes and genes associated with protein synthesis/degradation (lysosomal proteins, heat shock proteins, and proteasomes). The microarray data support the notion that the mechanism of age-dependent susceptibility to mammary gland cancer is largely associated with differential responses in expression of genes involved in cellular differentiation, proliferation, and protein homeostasis.

Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression through estrogen receptor-alpha activation via the mitogen-activated protein kinase pathway.

IntroductionPregnancy protects against breast cancer development in humans and rats. Parous rats have persistently reduced circulating levels of growth hormone, which may affect the activity of the growth hormone/insulin-like growth factor (IGF)-I axis. We investigated the effects of IGF-I on parity-associated protection against mammary cancer.MethodsThree groups of rats were evaluated in the present study: IGF-I-treated parous rats; parous rats that did not receive IGF-I treatment; and age-matched virgin animals, which also did not receive IGF-I treatment. Approximately 60 days after N-methyl-N-nitrosourea injection, IGF-I treatment was discontinued and all of the animal groups were implanted with a silastic capsule containing 17β-estradiol and progesterone. The 17β-estradiol plus progesterone treatment continued for 135 days, after which the animals were killed.ResultsIGF-I treatment of parous rats increased mammary tumor incidence to 83%, as compared with 16% in parous rats treated with 17β-estradiol plus progesterone only. Tumor incidence and average number of tumors per animal did not differ between IGF-I-treated parous rats and age-matched virgin rats. At the time of N-methyl-N-nitrosourea exposure, DNA content was lowest but the α-lactalbumin concentration highest in the mammary glands of untreated parous rats in comparison with age-matched virgin and IGF-I-treated parous rats. The protein levels of estrogen receptor-α in the mammary gland was significantly higher in the age-matched virgin animals than in untreated parous and IGF-I-treated parous rats. Phosphorylation (activation) of the extracellular signal-regulated kinase-1/2 (ERK1/2) and expression of the progesterone receptor were both increased in IGF-I-treated parous rats, as compared with those in untreated parous and age-matched virgin rats. Expressions of cyclin D1 and transforming growth factor-β3 in the mammary gland were lower in the age-matched virgin rats than in the untreated parous and IGF-I-treated parous rats.ConclusionWe argue that tumor initiation (transformation and fixation of mutations) may be similar in parous and age-matched virgin animals, suggesting that the main differences in tumor formation lie in differences in tumor progression caused by the altered hormonal environment associated with parity. Furthermore, we provide evidence supporting the notion that tumor growth promotion seen in IGF-I-treated parous rats is caused by activation of estrogen receptor-α via the Raf/Ras/mitogen-activated protein kinase cascade.

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.

Increased Activity of cGMP-Specific Phosphodiesterase (PDE5) Contributes to Resistance to Atrial Natriuretic Peptide Natriuresis in the Pregnant Rat

Increased cGMP-specific phosphodiesterase (PDE5) activity in renal inner medullary collecting duct (IMCD) cells contributes to resistance to atrial natriuretic peptide (ANP) and the excessive sodium retention seen in experimental nephrotic syndrome and liver cirrhosis. Normal pregnancy is also accompanied by sodium retention and plasma volume expansion, and pregnant rats are resistant to the natriuretic action of ANP. The authors investigated a possible role of increased renal PDE5 activity in the physiologic sodium retention of normal rat pregnancy. The natriuresis and increased urinary cGMP excretion (U(cGMP)V) evoked by acute volume expansion (a measure of renal responsiveness to endogeneous ANP) was blunted in 16-d pregnant versus virgin rats, despite equivalent increases in circulating ANP in pregnants and virgins. The ANP-dependent cGMP accumulation in isolated IMCD cells from pregnants was blunted versus virgins and restored by the PDE5-selective antagonist DMPPO (10(-7) mol/L). PDE5 activity in vitro and PDE5 protein abundance in IMCD were greater in pregnants. Four days postpartum, volume expansion natriuresis, U(cGMP)V, and PDE5 protein levels in IMCD cell homogenates had returned to virgin values. These results demonstrate that normal rat pregnancy leads to in vivo and in vitro renal resistance to ANP, in association with heightened activity of the cGMP-specific PDE5 in IMCD. This may contribute to the physiologic sodium retention of normal pregnancy.

The monoamine reuptake inhibitor milnacipran does not affect nociception to acute visceral distension in rats.

The role of antidepressants in the treatment of visceral pain has not been extensively examined. Milnacipran, an antidepressant that inhibits monoamine reuptake, is widely used in the treatment of depression and fibromyalgia. In this study, we sought to determine the activity of milnacipran against acute visceral nociception. Female virgin rats were studied 7 days after bilateral ovariectomy. For uterine cervical distension (UCD), two metal rods were inserted into the cervical osses under general anesthesia for manual distension. Colorectal distension (CRD) was performed by insertion of a balloon catheter into the descending colon and rectum, followed by manual inflation. Two electrodes were inserted into the rectus abdominus muscle for recording UCD- or CRD-induced reflex contraction, which was quantified by electromyography (EMG). A dose response for milnacipran, administered intrathecally or i.v., was obtained for UCD and CRD stimulation. Milnacipran failed to inhibit the UCD-induced EMG response, whether administered i.v. or intrathecally. Similarly, i.v. milnacipran, administered either acutely or chronically, failed to inhibit the CRD-induced EMG response. CRD and UCD are well established animal models for the study of acute visceral pain. Milnacipran, although it provides some unique advantages compared with other antidepressants, is unlikely to produce analgesia after acute administration in the setting of acute visceral pain. Neither intrathecal nor i.v. milnacipran, a monoamine reuptake inhibitor, inhibits an acute visceral pain response induced by colorectal or uterine cervical distension.

The effect of pregnancy and the oestrus cycle on purinergic and cholinergic responses of the rat urinary bladder

The urinary bladder undergoes plastic changes during physiological alterations such as pregnancy. This study has shown that bladders from pregnant rats weighed three times more than bladders from virgin rats. Each milligram of detrusor muscle from pregnant rats contracted more strongly to nerve stimulation (150% greater) and agonists (50% greater or more) compared to detrusor from virgin rats at any stage during the oestrus cycle. The purinergic component of nerve-mediated responses altered during the oestrus cycle, being greatest during oestrus (oestrogen and progesterone fall rapidly) and dioestrus (low oestrogen and progesterone), smaller during pregnancy and even smaller during pro-oestrus (high oestrogen and progesterone); in contrast the cholinergic component remained relatively unchanged. In conclusion, during pregnancy the detrusor muscle generates larger contractions compared to virgin detrusor muscle, probably due to hormonal influences on smooth muscle contraction mechanisms. As agonist responses were unchanged during the oestrus cycle, changes in the purinergic component of nerve stimulation was not due to altered P2 receptor expression but possibly to an increase in ATP release or a reduction in breakdown. The hormonal effect may have implications for the treatment of bladder disorders due to alterations in hormones, such as stress incontinence in post-menopausal women.

Release of oxytocin in the hypothalamic paraventricular nucleus, but not central amygdala or lateral septum in lactating residents and virgin intruders during maternal defence

In lactating rats, the neuroendocrine responses of the oxytocinergic system and the hypothalamo–pituitary–adrenal axis to various kinds of stressors are attenuated. In this study, using intracerebral microdialysis in combination with a highly sensitive radioimmunoassay, we characterised oxytocin (OXT) release within the paraventricular nucleus (PVN), the central amygdala (CeA), and the medio-lateral septum (mS) before, during and after a psycho-social stressor (the maternal defence test) in both the virgin intruder and the lactating resident rat (day 3 of lactation). Within the PVN, local OXT release was found to increase significantly in virgin intruders during exposure to the resident (2.1-fold, P<0.05), as well as in lactating residents when exposed to the virgin intruder, though to a lesser extent when compared with basal levels (1.7-fold, P<0.05). In contrast, OXT release remained unchanged within the CeA and the mS of both virgin intruders and lactating residents. Release of OXT under basal conditions was clearly above the detection limit of the radioimmunoassay, and did not differ between lactating and virgin rats in any of the brain regions studied. Our study also demonstrates that recent surgery or ongoing intracerebral microdialysis does not affect the behavioural performance of the intruders or residents when comparing dialysed and non-dialysed rats. The results indicate that exposure to the maternal defence test is a relevant stressor for the brain OXT system which becomes activated in both intruder and resident rats, although to varying degrees depending upon their reproductive status and in a region-dependent manner. The behavioural and/or neuroendocrine functions of intra-PVN released OXT during this psycho-social challenge remain to be clarified.

Maternal memory in adult, nulliparous rats: Effects of testing interval on the retention of maternal behavior

The retention of maternal behavior (i.e., maternal memory) was measured in adult, nulliparous rats induced to respond maternally by continuous exposure to foster pups. Specifically, the effects of the interval duration between the initial induction and the reinduction of maternal behavior were determined. Intact virgin rats were first exposed to foster young to induce maternal behavior. During the initial induction phase, females were required to be fully maternal on 2 consecutive test days. Animals were then assigned to one of three interval groups (10, 20, or 40 days). After being isolated from rat pups for these designated periods, females in each group were tested again for their latencies to induce maternal behavior. Whereas the initial median latencies to display full maternal behavior ranged from 4.5 to 5 days for each group, upon retesting, median latencies for each group declined to 1 to 4 days. The greatest reduction in latency was present in the 10-day group (80%), and the smallest reduction was detected in the 40-day group (20%). A significant negative linear correlation was found between test interval and percentage reduction in behavioral latency. Based upon this relationship and under these test conditions, "maternal memory" in the adult, nulliparous rat would be expected to be nondetectable after about an interval of 50 days between tests. The pattern of maternal memory acquisition and loss appears similar to that reported in parous animals. The present study highlights similarities and possible differences underlying the establishment of the retention of maternal behavior (i.e., maternal memory).

Enhanced Pulmonary Inflammatory Response to Inhaled Endotoxin in Pregnant Rats

Evidence suggests that pregnant animals are more sensitive than nonpregnant animals to the systemic administration of endotoxin. Studies were undertaken to assess whether an enhanced sensitivity of the pulmonary system to aerosolized endotoxin might exist during pregnancy. Pregnant Sprague-Dawley female rats (17 d of gestation) or age-matched virgin female rats were exposed to air or endotoxin (lipopolysaccharide) by inhalation for 3 h. At 18 h following exposure to endotoxin, lactate dehydrogenase activity levels in bronchoalveolar lavage (BAL) fluid samples from pregnant rats were 1.5-fold greater than those from endotoxin-exposed virgin rats. BAL polymorphonuclear leukocyte (PMN) numbers were also approximately twofold greater in pregnant rats than in virgins following the inhalation of endotoxin. The increases in BAL PMNs in pregnant rats following endotoxin exposure were observed just following exposure to endotoxin as well as at 18 h following exposure. These results indicate that an increased pulmonary inflammatory response to inhaled endotoxin occurs during pregnancy in rats. Additional findings suggest that these pregnancy-linked pulmonary responses to endotoxin cannot be explained by the following potential mechanisms: changes in the inhaled dose of endotoxin, or alterations in the responsiveness of alveolar macrophages to endotoxin. To our knowledge this is the first study that has evaluated pulmonary responses to inhaled endotoxin during pregnancy. Our finding that pregnancy is associated with an increased lung inflammatory response to aerosolized endotoxin raises the possibility that there may be a generalized enhancement of pulmonary responses to inhaled toxic agents during pregnancy.

Induction of maternal behavior in adult female rats following chronic morphine exposure during puberty.

The peripubertal period in the female rat is the time when the stimulatory effects of opioids on prolactin (PRL) secretion develop. In the adult rat, the administration of chronic high-dose morphine has been shown to attenuate the ability of opiates to stimulate PRL secretion. One function of PRL in adult virgin rats is the induction of maternal behavior. The present study examined whether chronic high-dose morphine exposure during the peripubertal period alters PRL-mediated induction of maternal behavior in adult female rats. Two groups of juvenile female rats were administered increasing doses of morphine or vehicle (s.c.) from age 30 to 50 days. As adults, these females either remained intact, or were ovariectomized and treated with a PRL-dependent, steroid hormone regimen that stimulates a rapid onset of maternal behavior. All females were then exposed daily to rat foster pups to determine whether peripubertal morphine exposure affected their latencies to induce maternal behavior. Morphine treatment resulted in a delay in vaginal opening and a temporary reduction in the rate of weight gain; however, the rate of onset of maternal behavior was unaffected by peripubertal morphine treatment. Thus, chronic morphine exposure in the pubertal female did not impact the expression of pup-induced maternal care.

Role of differentiation in carcinogenesis and cancer prevention

Ductal carcinoma is the malignant disease most frequently diagnosed in American women. This cancer type originates in Stem Cells 1, epithelial cells that are the main components of the undifferentiated mammary gland. The susceptibility of Stem Cells 1 to become transformed has been attributed to their high rate of cell proliferation, their ability to bind and activate the carcinogen and their low DNA reparative activity. Epidemiological evidence indicates that a woman's lifetime risk for developing breast cancer is decreased by a full-term pregnancy at a young age. The protection conferred by pregnancy is the result of the differentiation of the organ. The reproduction of the hormonal milieu of an early full-term pregnancy by administration of human chorionic gonadotropin (hCG) to virgin rats induces lobular development, completing the cycle of differentiation of the breast that converts the highly susceptible Stem Cell 1 into a resistant Stem Cell 2 through a process of induction of a specific genomic signature in the mammary epithelium. Both pregnancy and hCG exert a protective effect on the mammary gland by reducing the incidence of 7,12-dimethyl-benz(a)anthracene (DMBA)-induced carcinomas. Although differentiation significantly reduces cell proliferation in the mammary epithelium, the differentiated Stem Cell 2 remains capable of responding with proliferation to given stimuli, such as a new pregnancy; it is also capable of metabolizing the carcinogen and of repairing DNA damage more efficiently than the Stem Cell 1. The basic biological concept is that the conversion of the susceptible Stem Cell 1 to a refractory Stem Cell 2 after pregnancy or hCG treatment of virgin rats results from the differentiation of the mammary gland, a phenomenon manifested at morphological, cell kinetic and functional levels. Morphological changes consist of a progressive branching of the mammary parenchyma and lobule formation, which in turn result in a reduction in cell growth fraction and lengthening of the cell cycle. The functional changes comprise increased synthesis of inhibin, β-casein and other milk-related bioactive peptides. In addition, pregnancy or hCG also increases the expression of programmed cell death genes, including TRPM2, ICE, p53, c-myc, and bcl-XS, inducing as well apoptosis, and downregulation of cyclins. Programmed cell death genes were activated through a p53-dependent process, modulated by c-myc, and with partial dependence on the bcl-2 family-related genes. Data generated using the cDNA microarray techniques have allowed us to demonstrated that while lobular development regressed after the cessation of pregnancy or hormone administration, programmed cell death genes remained activated, and new sets of genes reached a peak of maximal expression while others became downregulated, creating a genomic signature that is specific for both pregnancy and hCG treatment, but significantly different from those induced by the ovarian steroid hormones estrogen and progesterone. These mechanisms play a role in the protection exerted by hCG from chemically induced carcinogenesis, and might be even involved in the lifetime reduction in breast cancer risk induced in women by full-term and multiple pregnancies. In addition, hCG inhibits the progression of DMBA-induced mammary tumors on the early phases of tumor progression (i.e. intraductal proliferation, in situ carcinomas and invasive carcinomas). These observations led us to infer that hCG, like pregnancy, induces early genomic changes that lead the mammary gland to full differentiation, and that these changes result in a permanently imprinted genomic signature that regulates the long-lasting refractoriness of the mammary gland to carcinogenesis. The permanence of these changes makes them ideal surrogate markers of hCG effect in the evaluation of this hormone as a breast cancer preventive agent.