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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.

Cyclic nucleotides determine a spatiotemporally regulated system that modulates uterine myometrial contractility and parturition timing. Adenylate and guanylate cyclases synthesize cAMP and cGMP upon β-adrenergic, natriuretic peptide, and nitric oxide stimulation. Before parturition, cAMP suppresses contraction-associated protein synthesis via PKA, and cGMP activates hyperpolarizing K+ current via PKG. At term, cAMP stimulates contraction-associated protein expression via EPAC just as decreasing PKG attenuates cGMP tocolysis. The cAMP-specific phosphodiesterase 4 (PDE4) facilitates myometrial contraction by degrading cyclic nucleotides. The precise roles of cGMP on myometrial activation and the effect of other PDEs in parturition are not yet clear. Strategies to reduce preterm birth could benefit from a better understanding of the myometrial cyclic nucleotide system.

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.

Nitric oxide (NO) is a major inhibitory neurotransmitter that mediates nonadrenergic noncholinergic (NANC) signaling. Neuronal NO synthase (nNOS) is activated by Ca(2+)/calmodulin to produce NO, which causes smooth muscle relaxation to regulate physiologic tone. nNOS serine1412 (S1412) phosphorylation may reduce the activating Ca(2+) requirement and sustain NO production. We developed and characterized a nonphosphorylatable nNOS(S1412A) 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 nNOS(S1412A) ileum. At higher frequencies, nNOS S1412 had no effect. nNOS(S1412A) 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 nNOS(S1412A) 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.

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., Graphical abstract Image 1, Highlights • CSE-null mice exhibit abnormal estrogen augmentation of uterine artery blood flow. • Estrogen lowers uterine artery nitric oxide metabolites in CSE null mice. • CSE loss and estrogen additively impair uterine artery glutathione homeostasis. • Neither CSE loss nor estrogen influences renal artery blood flow or glutathione.

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, nNOS(S1412A) 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 nNOS(S1412A) ileal rings less than wild‐type ileal rings. PKA inhibition blocked forskolin‐induced nNOS phosphorylation and attenuated relaxation of wild type but not nNOS(S1412A) ileum. Akt inhibition did not alter nNOS phosphorylation with forskolin but did attenuate relaxation of wild type and nNOS(S1412A). 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.

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.

Purpose One third of the world’s population suffers from gastrointestinal (GI) dysmotility, a common feature of which is disrupted nitric oxide (NO) signaling and spastic contractility. In the healthy gut, neuronal NO synthase (nNOS) induces cGMP-dependent GI smooth muscle relaxation, which facilitates digestion. However, regulation of nNOS activity is poorly understood. PKA or Akt phosphorylation of nNOS S1412 enhances hypothalamic and penile NO synthesis. We hypothesized that nNOS S1412 phosphorylation promotes GI motility. Methods We quantified the contractile force of ileal segments from C57Bl/6 wildtype and nNOSS1412A mutant mice in an organ bath under non-adrenergic, non-cholinergic (NANC) conditions. Relaxation stimuli—neuronal depolarization (EFS; 20V, 2msec pulsewidth) or adenylate cyclase activation (FSK)—were applied to ileal segments in the presence of kinase, nNOS, or cGMP pathway inhibitors. We measured nNOS S1412 phosphorylation by western blot. We determined non-NANC ileal contraction velocity with the gastrointestinal motility monitor. Results EFS (1Hz) and FSK (300nM) increased nNOS S1412 phosphorylation by 310% (p Conclusions nNOS S1412 phosphorylation promotes GI motility by decreasing spastic contractility in murine ileal segments. Neuronal depolarization elicits Akt-dependent phosphorylation, while adenylate cyclase activation elicits PKA-dependent phosphorylation of nNOS. Common GI motility disorders like irritable bowel syndrome may benefit from therapies that upregulate nNOS S1412 phosphorylation.

Phosphorylation of the C-terminal Akt/PKA consensus site activates endothelial NOS (eNOS) and decreases its calcium/calmodulin requirement, but no definite physiological role for phosphorylation of the homologous site in neuronal NOS (nNOS) has been described. Therefore, we created a knock-in mouse in which nNOS Serine-1412 (S1412) is mutated to non-phosphorylatable alanine (nNOSS1412A). We tested whether nNOSS1412A ileal rings have altered nitrergic inhibitory non-adrenergic, non-cholinergic (NANC) signaling in a physiological organ bath. Under NANC conditions, electrical field stimulation (EFS; 20V, 2msec, 1Hz, 30sec train) relaxed WT to a greater degree than nNOSS1412A ileum (73 ± 20% inhibition vs 31 ± 17%; N=3). Similarly, pharmacological treatment with forskolin (FSK) also relaxed WT more effectively than nNOSS1412A ileum (IC50 95% CI 35-44nM; N=42 vs 78-107nM; N=29). Application of selective nNOS (NANT) or pan-NOS antagonists (L-NAME) reduced FSK dependent relaxation identically in WT and nNOSS1412A ilea (IC50 ~150nM for both genotypes), confirming that a portion of FSK relaxation was due to nNOS activity. Akt and PKA are putative nNOS S1412 kinases. While there was no difference between WT and nNOSS1412A relaxation with FSK using the Akt inhibitor MK-2206, PKA inhibitors (H-89, Myr-PKI, and Rp-cAMPs) only decreased FSK relaxation in WT ileum (~3-fold increase in IC50) but not in nNOSS1412A ileum, suggesting a critical link between PKA and nNOS. Together, our data identify a novel NANC neuroinhibitory mechanism in which PKA phosphorylation of nNOS S1412 leads to calcium independent neuronal NO production and smooth muscle relaxation. This previously unrecognized NO physiology might be important in other cell types and organ systems as well.