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.

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.

Gasotransmitters in pregnancy: from conception to uterine involution.

Gasotransmitters are endogenous small gaseous messengers exemplified by nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S or sulfide). Gasotransmitters are implicated in myriad physiologic functions including many aspects of reproduction. Our objective was to comprehensively review basic mechanisms and functions of gasotransmitters during pregnancy from conception to uterine involution and highlight future research opportunities. We searched PubMed and Web of Science databases using combinations of keywords nitric oxide, carbon monoxide, sulfide, placenta, uterus, labor, and pregnancy. We included English language publications on human and animal studies from any date through August 2018 and retained basic and translational articles with relevant original findings. All gasotransmitters activate cGMP signaling. NO and sulfide also covalently modify target protein cysteines. Protein kinases and ion channels transduce gasotransmitter signals, and co-expressed gasotransmitters can be synergistic or antagonistic depending on cell type. Gasotransmitters influence tubal transit, placentation, cervical remodeling, and myometrial contractility. NO, CO, and sulfide dilate resistance vessels, suppress inflammation, and relax myometrium to promote uterine quiescence and normal placentation. Cervical remodeling and rupture of fetal membranes coincide with enhanced oxidation and altered gasotransmitter metabolism. Mechanisms mediating cellular and organismal changes in pregnancy due to gasotransmitters are largely unknown. Altered gasotransmitter signaling has been reported for preeclampsia, intrauterine growth restriction, premature rupture of membranes, and preterm labor. However, in most cases specific molecular changes are not yet characterized. Nonclassical signaling pathways and the crosstalk among gasotransmitters are emerging investigation topics.

Functional conservation between mammalian MGRN1 and plant LOG2 ubiquitin ligases.

Plant LOSS OF GDU 2 (LOG2) and Mammalian Mahogunin Ring Finger 1 (MGRN1) proteins are RING-type E3 ligases sharing similarity N-terminal to the RING domain. Deletion of this region disrupts the interaction of LOG2 with the plant membrane protein GLUTAMINE DUMPER1 (GDU1). Phylogenetic analysis identified two clades of LOG2/MGRN1-like proteins in vertebrates and plants. The ability of MGRN1 to functionally replace LOG2 was tested. MGRN1 ubiquitylates GDU1 in vitro and can partially substitute for LOG2 in the plant, partially restoring amino acid resistance to a GDU1-myc over-expression, log2-2 background. Altogether, these results suggest a conserved function for the N-terminal domain in evolution.

The ubiquitin E3 ligase LOSS OF GDU2 is required for GLUTAMINE DUMPER1-induced amino acid secretion in Arabidopsis.

Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.