Cystathionine γ-lyase promotes estrogen-stimulated uterine artery blood flow via glutathione homeostasis.

During pregnancy, estrogen (E2) stimulates uterine artery blood flow (UBF) by enhancing nitric oxide (NO)-dependent vasodilation. Cystathionine γ-lyase (CSE) promotes vascular NO signaling by producing hydrogen sulfide (H2S) and by maintaining the ratio of reduced-to-oxidized intracellular glutathione (GSH/GSSG) through l-cysteine production. Because redox homeostasis can influence NO signaling, we hypothesized that CSE mediates E2 stimulation of UBF by modulating local intracellular cysteine metabolism and GSH/GSSG levels to promote redox homeostasis. Using non-pregnant ovariectomized WT and CSE-null (CSE KO) mice, we performed micro-ultrasound of mouse uterine and renal arteries to assess changes in blood flow upon exogenous E2 stimulation. We quantified serum and uterine artery NO metabolites (NOx), serum amino acids, and uterine and renal artery GSH/GSSG. WT and CSE KO mice exhibited similar baseline uterine and renal blood flow. Unlike WT, CSE KO mice did not exhibit expected E2 stimulation of UBF. Renal blood flow was E2-insensitive for both genotypes. While serum and uterine artery NOx were similar between genotypes at baseline, E2 decreased NOx in CSE KO serum. Cysteine was also lower in CSE KO serum, while citrulline and homocysteine levels were elevated. E2 and CSE deletion additively decreased GSH/GSSG in uterine arteries. In contrast, renal artery GSH/GSSG was insensitive to E2 or CSE deletion. Together, these findings suggest that CSE maintenance of uterine artery GSH/GSSG facilitates nitrergic signaling in uterine arteries and is required for normal E2 stimulation of UBF. These data have implications for pregnancy pathophysiology and the selective hormone responses of specific vascular beds.

Estrogen Regulates Local Cysteine Metabolism in Mouse Myometrium.

Sulfur amino acid metabolism influences reproductive physiology, and transsulfuration in particular may be critical for normal cellular function. The sex hormone estrogen (E2) modulates gene expression and redox balance in some tissues by inducing the transsulfuration enzymes cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE). The role of sex hormones in sulfur amino acid metabolism by uterine smooth muscle is not known. Here, we show that CBS and CSE proteins increase in the mouse myometrium during estrus and diestrus, respectively, suggesting that E2 reciprocally regulates myometrial CBS and CSE expression. In ovariectomized mice, exogenous E2 upregulates CBS and downregulates CSE levels. E2 promotes CBS mRNA and protein expression but attenuates CSE protein expression without affecting CSE mRNA. This pattern of E2-stimulated changes in transsulfuration enzyme expression is specific to the uterine smooth muscle. E2 does not change vaginal or cervical expression of CBS or CSE significantly, and E2 decreases expression of CSE in the liver without affecting CBS. E2 also downregulates myometrial cysteinesulfinic acid decarboxylase (CSAD) and decreases myometrial biochemical synthesis of the gaso-transmitter hydrogen sulfide (H2S). These findings suggest that myometrial sulfur amino acid metabolism may regulate uterine redox homeostasis, with implications for the source and metabolism of myometrial cysteine in high E2 states such as estrus and pregnancy.

Effects of resveratrol or estradiol on postexercise endothelial function in estrogen-deficient postmenopausal women.

Regular exercise enhances endothelial function in older men, but not consistently in estrogen-deficient postmenopausal women. Estradiol treatment improves basal endothelial function and restores improvements in endothelial function (flow-mediated dilation, FMD) to aerobic exercise training in postmenopausal women; however, estradiol treatment is controversial. Resveratrol, an estrogen receptor ligand, enhances exercise training effects on cardiovascular function and nitric oxide (NO) release in animal models, but impairs exercise training effects in men. We conducted a randomized cross-over, double-blinded, placebo-controlled pilot study to determine whether acute (single dose) resveratrol (250-mg tablet) or estradiol (0.05 mg/day transdermal patch) treatment enhances FMD at rest and after a single bout of moderate-intensity aerobic exercise in healthy estrogen-deficient postmenopausal women (n = 15, 58.1 ± 3.2 yr). FMD was measured before and after (30, 60, and 120 min) a 40-min bout of moderate-intensity treadmill exercise (60-75% peak heart rate) under the respective conditions (separated by 1-2 wk). FMD was higher (P < 0.05) before exercise and at all post-exercise time points in the resveratrol and estradiol conditions compared to placebo. FMD was increased from baseline by 120 min postexercise in the estradiol condition (P < 0.001), but not resveratrol or PL conditions. Consistent with our previous findings, estradiol also enhances endothelial function in response to acute endurance exercise. Although resveratrol improved basal FMD, there was no apparent enhancement of FMD to acute exercise and, therefore, may not act as an estradiol mimetic.NEW & NOTEWORTHY The benefits of endurance exercise training on endothelial function are diminished in estrogen-deficient postmenopausal women, but estradiol treatment appears to restore improvements in endothelial function in this group. We show that basal endothelial function is enhanced with both acute estradiol and resveratrol treatments in estrogen-deficient postmenopausal women, but endothelial function is only enhanced following acute endurance exercise with estradiol treatment.

Effect of neuronal nitric oxide synthase serine-1412 phosphorylation on hypothalamic-pituitary-ovarian function and leptin response.

Hypothalamic neuronal nitric oxide synthase (nNOS) potentiates adult female fertility in rodents by stimulating gonadotropin releasing hormone (GnRH) secretion, which in turn promotes luteinizing hormone (LH) release and ovulation. The mechanism of hypothalamic nNOS activation is not clear but could be via nNOS serine1412 (S1412) phosphorylation, which increases nNOS activity and physiologic NO effects in other organ systems. In female rodents, hypothalamic nNOS S1412 phosphorylation reportedly increases during proestrus or upon acute leptin exposure during diestrus. To determine if nNOS S1412 regulates female reproduction in mice, we compared the reproductive anatomy, estrous cycle duration and phase proportion, and fecundity of wild-type and nNOS serine1412➔alanine (nNOSS1412A) knock-in female mice. We also measured hypothalamic GnRH and serum LH, follicle stimulating hormone (FSH), estradiol, and progesterone in diestrus mice after intraperitoneal leptin injection. Organ weights and histology were not different by genotype. Ovarian primordial follicles, antral follicles, and corpora lutea were similar for wild-type and nNOSS1412A mice. Likewise, estrous cycle duration and phase length were not different, and fecundity was unremarkable. There were no differences among genotypes for LH, FSH, estradiol, or progesterone. In contrast to prior studies, our work suggests that nNOS S1412 phosphorylation is dispensable for normal hypothalamic-pituitary-ovarian function and regular estrous cycling. These findings have important implications for current models of fertility regulation by nNOS phosphorylation.

Protein kinase A facilitates relaxation of mouse ileum via phosphorylation of neuronal nitric oxide synthase.

BACKGROUND AND PURPOSE: The enteric neurotransmitter nitric oxide (NO) regulates gastrointestinal motility by relaxing smooth muscle. Pharmacological cAMP induction also relaxes gastrointestinal smooth muscle, but it is uncertain whether cAMP augments or suppresses enteric NO signalling. In other organ systems, cAMP can increase neuronal NO production by stimulating protein kinase A (PKA) to phosphorylate neuronal NOS (nNOS) Serine-1412 (S1412). We hypothesized that cAMP also increases nNOS S1412 phosphorylation by PKA in enteric neurons to augment nitrergic relaxation of mouse ileum. EXPERIMENTAL APPROACH: We measured contractile force and nNOS S1412 phosphorylation in ileal rings suspended in an organ bath. We used forskolin to induce cAMP-dependent relaxation of wild type, nNOSS1412A knock-in and nNOSα-null ileal rings in the presence or absence of PKA, protein kinase B (Akt) and NOS inhibitors. KEY RESULTS: Forskolin stimulated phosphorylation of nNOS S1412 in mouse ileum. Forskolin relaxed nNOSα-null and nNOSS1412A ileal rings less than wild-type ileal rings. PKA inhibition blocked forskolin-induced nNOS phosphorylation and attenuated relaxation of wild type but not nNOSS1412A ileum. Akt inhibition did not alter nNOS phosphorylation with forskolin but did attenuate relaxation of wild type and nNOSS1412A . NOS inhibition with L-NAME eliminated the effects of PKA and Akt inhibitors on relaxation. CONCLUSION AND IMPLICATIONS: PKA phosphorylation of nNOS S1412 augments forskolin-induced nitrergic ileal relaxation. The relationship between cAMP/PKA and NO is therefore synergistic in enteric nitrergic neurons. Because NO regulates gut motility, selective modulation of enteric neuronal cAMP synthesis may be useful for the treatment of gastrointestinal motility disorders.

Adiponectin links maternal metabolism to uterine contractility.

Adiponectin is secreted by adipose tissue and promotes insulin sensitivity. Low circulating adiponectin is associated with increased risk for preterm labor, but the influence of adiponectin on uterine myometrial physiology is unknown. We hypothesized that adiponectin receptors (AdipoRs) decrease myometrial contractility via AMPK to promote uterine quiescence in pregnancy. Using quantitative RT-PCR, we found that nonpregnant or pregnant human and mouse myometrium express AdipoR1 and AdipoR2 mRNAs. We confirmed AdipoR2 protein expression in human and mouse myometrium, with increased abundance in late mouse pregnancy. Both recombinant adiponectin and a pharmacologic AdipoR agonist, AdipoRon, potently inhibited uterine myometrial strip contractions in physiologic organ bath. The relaxation was independent of contractile stimulus (oxytocin, KCl, U46619). AdipoR agonists increased AMPK phosphorylation in pregnant mouse myometrium, and the direct AMPK activator A769662 also relaxed myometrial strips. However, the AMPK inhibitor dorsomorphin (compound C) blocked AMPK phosphorylation but did not abolish relaxation with either AdipoRon or A769662. In summary, adiponectin inhibits myometrial contractility consistent with the possibility that it is a previously unrecognized link between maternal metabolism and pregnancy maintenance. We also identify a separate role for AMPK regulating myometrial contractions that may influence labor onset.-Vyas, V., Guerra, D. D., Bok, R., Powell, T., Jansson, T., Hurt, K. J. Adiponectin links maternal metabolism to uterine contractility.

Akt phosphorylation of neuronal nitric oxide synthase regulates gastrointestinal motility in mouse ileum.

Nitric oxide (NO) is a major inhibitory neurotransmitter that mediates nonadrenergic noncholinergic (NANC) signaling. Neuronal NO synthase (nNOS) is activated by Ca2+/calmodulin to produce NO, which causes smooth muscle relaxation to regulate physiologic tone. nNOS serine1412 (S1412) phosphorylation may reduce the activating Ca2+ requirement and sustain NO production. We developed and characterized a nonphosphorylatable nNOSS1412A knock-in mouse and evaluated its enteric neurotransmission and gastrointestinal (GI) motility to understand the physiologic significance of nNOS S1412 phosphorylation. Electrical field stimulation (EFS) of wild-type (WT) mouse ileum induced nNOS S1412 phosphorylation that was blocked by tetrodotoxin and by inhibitors of the protein kinase Akt but not by PKA inhibitors. Low-frequency depolarization increased nNOS S1412 phosphorylation and relaxed WT ileum but only partially relaxed nNOSS1412A ileum. At higher frequencies, nNOS S1412 had no effect. nNOSS1412A ileum expressed less phosphodiesterase-5 and was more sensitive to relaxation by exogenous NO. Under non-NANC conditions, peristalsis and segmentation were faster in the nNOSS1412A ileum. Together these findings show that neuronal depolarization stimulates enteric nNOS phosphorylation by Akt to promote normal GI motility. Thus, phosphorylation of nNOS S1412 is a significant regulatory mechanism for nitrergic neurotransmission in the gut.

Na+-Leak Channel, Non-Selective (NALCN) Regulates Myometrial Excitability and Facilitates Successful Parturition.

BACKGROUND/AIMS: Uterine contractility is controlled by electrical signals generated by myometrial smooth muscle cells. Because aberrant electrical signaling may cause inefficient uterine contractions and poor reproductive outcomes, there is great interest in defining the ion channels that regulate uterine excitability. In human myometrium, the Na+ leak channel, non-selective (NALCN) contributes to a gadolinium-sensitive, Na+-dependent leak current. The aim of this study was to determine the role of NALCN in regulating uterine excitability and examine its involvement in parturition. METHODS: Wildtype C57BL/6J mice underwent timed-mating and NALCN uterine expression was measured at several time points across pregnancy including pregnancy days 7, 10, 14, 18 and 19. Sharp electrode current clamp was used to measure uterine excitability at these same time points. To determine NALCN's contribution to myometrial excitability and pregnancy outcomes, we created smooth-muscle-specific NALCN knockout mice by crossing NALCNfx/fx mice with myosin heavy chain Cre (MHCCreeGFP) mice. Parturition outcomes were assessed by observation via surveillance video recording cre control, flox control, smNALCN+/-, and smNALCN-/- mice. Myometrial excitability was compared between pregnancy day 19 flox controls and smNALCN-/- mice. RESULTS: We found that in the mouse uterus, NALCN protein levels were high early in pregnancy, decreased in mid and late pregnancy, and then increased in labor and postpartum. Sharp electrode current clamp recordings of mouse longitudinal myometrial samples from pregnancy days 7, 10, 14, 18, and 19 revealed day-dependent increases in burst duration and interval and decreases in spike density. NALCN smooth muscle knockout mice had reduced myometrial excitability exemplified by shortened action potential bursts, and an increased rate of abnormal labor, including prolonged and dysfunctional labor. CONCLUSIONS: Together, our findings demonstrate that the Na+ conducting channel NALCN contributes to the myometrial action potential waveform and is important for successful labor outcomes.

Perinatal Endotoxemia Induces Sustained Hepatic COX-2 Expression through an NFκB-Dependent Mechanism.

BACKGROUND: Exposure to perinatal infection is associated with the multiple morbidities complicating preterm birth. How a relatively immature innate immune response contributes to this is unknown. OBJECTIVE: We sought to determine if the perinatal innate immune response to endotoxemia induces a unique pattern of cyclooxygenase-2 (COX-2) expression via an NFκB-dependent mechanism. METHODS: Hepatic and pulmonary COX-2 mRNA expression was assessed following perinatal (at embryonic days 15 and 19 and after birth) or adult endotoxemia. Hepatic NFκB activity was assessed by cytosolic inhibitory protein degradation and subunit nuclear translocation. Immunohistochemistry and isolated cell preparations determined hepatic macrophage COX-2 expression, and the effect of pharmacologic and genetic inhibition of NFκB activity was tested. RESULTS: Perinatal endotoxemia induced sustained hepatic macrophage COX-2 expression and NFκB activity compared to in exposed adults. Isolated hepatic macrophages and immunohistochemistry demonstrated enriched LPS-induced COX-2 expression that was sensitive to pharmacologic and genetic approaches to attenuate NFκB activity. Finally, pharmacologic inhibition of endotoxemia-induced NFκB activity in neonatal mice prevented hepatic NFκB activity and attenuated COX-2 expression. CONCLUSION: Our findings of sustained neonatal hepatic NFκB activity and COX-2 expression in response to endotoxemia support a robust perinatal innate immune response. This may represent a link between the innate immune response and the pathogenesis of diseases associated with preterm birth.