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Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. Although β2 adrenergic agonists fail to provide adequate tocolysis, the expression of the β3 adrenergic receptor in myometrium and its unique signaling suggest a role for β3 agonist in the management of preterm labor. Western blot analysis showed that the β3 adrenergic receptor expression increased in human pregnancy myometrium compared to nonpregnant tissues (p 0.05). The addition of the β3 agonist mirabegron in the tissue bath relaxed oxytocin contracted myometrium with an EC50 of 41.5 µM. Relaxation was partially blocked by the addition of the eNOS blocker Nω-nitro-L-arginine, or the large conductance potassium channel blocker paxilline. Combination of Nω-nitro-L-arginine and paxilline prevented mirabegron-mediated relaxation. Imaging revealed that the β3 adrenergic receptors are expressed by both myocyte and microvascular endothelial cells isolated from human myometrium. Nitric oxide production measured by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate revealed that mirabegron stimulated nitric oxide production in myometrial endothelial cells. These data suggest that both endothelial and smooth muscle cells contribute to relaxation through disparate signaling pathways. Repurposing of approved medications tested in human myometrium as uterine tocolytics can advance prevention of preterm birth. These data argue that further examination of β3 adrenergic receptor signaling in myometrium may reveal mirabegron as a useful tocolytic in combination tocolysis regimens.

Approximately 10% of US births deliver preterm before 37 weeks of completed gestation. Premature infants are at risk for life-long debilitating morbidities and death, and spontaneous preterm labour explains 50% of preterm births. In all cases existing treatments are ineffective, and none are FDA approved. The mechanisms that initiate preterm labour are not well understood but may result from dysfunctional regulation of quiescence mechanisms. Human pregnancy is accompanied by large increases in blood flow, and the uterus must enlarge by orders of magnitude to accommodate the growing fetus. This mechanical strain suggests that stretch-activated channels may constitute a mechanism to explain gestational quiescence. Here we identify for the first time that Piezo1, a mechanosensitive cation channel, is present in the uterine smooth muscle and microvascular endothelium of pregnant myometrium. Piezo is downregulated during preterm labour, and stimulation of myometrial Piezo1 in an organ bath with the agonist Yoda1 relaxes the tissue in a dose-dependent fashion. Further, stimulation of Piezo1 while inhibiting protein kinase A, AKT, or endothelial nitric oxide synthase mutes the negative inotropic effects of Piezo1 activation, intimating that actions on the myocyte and endothelial nitric oxide signalling contribute to Piezo1-mediated contractile dynamics. Taken together, these data highlight the importance of stretch-activated channels in pregnancy maintenance and parturition, and identify Piezo1 as a tocolytic target of interest. KEY POINTS: Spontaneous preterm labour is a serious obstetric dilemma without a known cause or effective treatments. Piezo1 is a stretch-activated channel important to muscle contractile dynamics. Piezo1 is present in the myometrium and is dysregulated in women who experience preterm labour. Activation of Piezo1 by the agonist Yoda1 relaxes the myometrium in a dose-dependent fashion, indicating that Piezo1 modulation may have therapeutic benefits to treat preterm labour.

Currently available tocolytics are ineffective at significantly delaying preterm birth. This is due in part to our failure to better understand the mechanisms that drive spontaneous preterm labor (sPTL). Cyclic nucleotides are not the primary contributors to myometrial quiescence, but instead nitric oxide (NO)-mediated protein S-nitrosation (SNO) is integral to the relaxation of the tissue. Connexin-43 (Cx43), a myometrial "contractile-associated protein" that functions as either a gap junction channel or an hemichannel (HC), was the focus of this study. Protein analysis determined that Cx43 is downregulated in sPTL myometrium. Furthermore, Cx43 is S-nitrosated by NO, which correlates with an increase of phosphorylated Cx43 at serine 368 (Cx43-pS368 -gap junction inhibition) as well as an increase in the HC open-state probability (quiescence). Pharmacologic inhibition of Cx43 with 18β-glycyrrhetinic acid (18β-GA) exhibits a negative inotropic effect on the myometrium in a dose-dependent manner, as does administration of nebivolol, an NO synthase activator that increases total protein SNOs. When 18β-GA and nebivolol were coadministered at their IC50 values, the effect on contractile dynamics was additive and all but eliminated contractions. The development of new tocolytics demands a better understanding of the underlying mechanisms of sPTL. Here it has been shown that 18β-GA and nebivolol leverage dysregulated pathways in the myometrium, resulting in a novel approach for the treatment of sPTL. SIGNIFICANCE STATEMENT: Although there are many known causes of preterm labor (PTL), the mechanisms of "spontaneous" PTL (sPTL) remain obfuscated, which is why treating this condition is so challenging. Here we have identified that connexin-43 (Cx43), an important contractile-associated protein, is dysregulated in sPTL myometrium and that the pharmacologic inhibition of Cx43 and its S-nitrosation with 18β-glycyrrhetinic acid and nebivolol, respectively, significantly blunts contraction in human myometrial tissue, presenting a novel approach to tocolysis that leverages maladjusted pathways in women who experience sPTL.

A wealth of evidence supports the broad therapeutic potential of NF‐κB and EZH2 inhibitors as adjuvants for breast cancer treatment. We contribute to this knowledge by elucidating, for the first time, unique regulatory crosstalk between EZH2, NF‐κB and the NF‐κB interacting long non‐coding RNA (NKILA). We define a novel signaling loop encompassing canonical and non‐canonical actions of EZH2 on the regulation of NF‐κB/NKILA homeostasis, with relevance to breast cancer treatment. We applied a respective silencing approach in non‐transformed breast epithelial cells, triple negative MDA‐MB‐231 cells and hormone responsive MCF‐7 cells, and measured changes in EZH2/NF‐κB/NKILA levels to confirm their interdependence. We demonstrate cell line‐specific fluctuations in these factors that functionally contribute to epithelial‐to‐mesenchymal transition (EMT) remodelling and cell fate response. EZH2 inhibition attenuates MDA‐MB‐231 cell motility and CDK4‐mediated MCF‐7 cell cycle regulation, while inducing global H3K27 methylation and an EMT phenotype in non‐transformed cells. Notably, these events are mediated by a cell‐context dependent gain or loss of NKILA and NF‐κB. Depletion of NF‐κB in non‐transformed cells enhances their sensitivity to growth factor signaling and suggests a role for the host microenvironment milieu in regulating EZH2/NF‐κB/NKILA homeostasis. Taken together, this knowledge critically informs the delivery and assessment of EZH2 inhibitors in breast cancer.

An examination of the origins of medical approaches to breast cancer marks this disease as one of the most difficult to manage. As the early identification, diagnosis and treatment of breast cancer evolve, we will move to a time when each patient and their cancer can be assessed to determine unique patient-specific (personalized) approaches to therapy. Humans have attempted to manage breast cancer for millennia. Even today, the disease claims thousands of lives each year. In light of the increasingly sophisticated understanding of cancer diagnosis and treatment, together with our ultimate failure to offer a cure in the most difficult cases, it is instructive to reflect on the beginnings of our understanding.

Preterm birth before 37 weeks of completed gestation results in numerous health consequences for the foetus. Preterm labour leads to preterm birth in over 50% of cases, and no FDA‐approved treatment can prevent labour or help a foetus remain in the womb until term. Examination of nitric oxide mediated relaxation signaling in the uterine smooth muscle reveals a role for protein S‐nitrosation. The recent discovery of upregulated S‐nitrosoglutathione reductase (GSNOR) in spontaneously preterm labouring women has emphasized the need to explore the function of S‐nitrosation regulation in the maintenance of uterine quiescence. Here we have examined the ability of nebivolol to relax uterine smooth muscle and tested recent claims that nebivolol is a GSNOR inhibitor. In uterine smooth muscle strips from both mouse and human, nebivolol relaxes oxytocin‐induced contractions in a dose dependent manner. Our data indicates that nebivolol has no effect on GSNOR activity, nor does nebivolol inhibit thioredoxin reductase, two of the major protein denitrosylases. The ability of nebivolol to relax uterine smooth muscle is likely the combined effects of increased nitric oxide synthase activity and β3‐adregnegic stimulation.

This chapter considers that role of S-nitrosoglutathione (GSNO) in the critical regulation of the actions of nitric oxide in a mammalian system. GSNO can transfer its NO moiety to a cysteine thiol, resulting in the posttranslational modification of S-nitrosation of a target thiol. The posttranslational modification of proteins has long been recognized as a key regulator of cellular function. S-Nitrosation in the cell is highly dependent on the availability of intracellular GSNO. As GSNO concentrations increase, so do levels of total protein S-nitrosation. S-Nitrosoglutathione reductase utilizes the coenzyme NADH to carry out a 2e- reduction of GSNO to generate glutathione sulfinamide before it is further reduced back to glutathione by glutathione reductase. The aberrant expression of this enzyme is associated with disease. In fact, the deletion of the GSNO reductase gene increases both the levels of GSNO and total protein S-nitrosation in vivo.

Tocolytics show limited efficacy to prevent preterm delivery. In uterine smooth muscle cGMP accumulation following addition of nitric oxide (NO) has little effect on relaxation suggesting a role for protein S-nitrosation. In human myometrial tissues from women in labor at term (TL), or spontaneously in labor preterm (sPTL), direct stimulation of soluble guanylyl cyclase (sGC) fails to relax myometrium, while the same treatment relaxes vascular smooth muscle completely. Unlike term myometrium, effects of NO are not only blunted in sPTL, but global protein S-nitrosation is also diminished, suggesting a dysfunctional response to NO-mediated protein S-nitrosation. Examination of the enzymatic regulator of endogenous S-nitrosoglutathione availability, S-nitrosoglutathione reductase, reveals increased expression of the reductase in preterm myometrium associated with decreased total protein S-nitrosation. Blockade of S-nitrosoglutathione reductase relaxes sPTL tissue. Addition of NO donor to the actin motility assay attenuates force. Failure of sGC activation to mediate relaxation in sPTL tissues, together with the ability of NO to relax TL, but not sPTL myometrium, suggests a unique pathway for NO-mediated relaxation in myometrium. Our results suggest that examining the action of S-nitrosation on critical contraction associated proteins central to the regulation of uterine smooth muscle contraction can reveal new tocolytic targets.

The data described in this article is the subject of an article in the American Journal of Physiology: Cell Physiology, titled “The Human Uterine Smooth Muscle S-nitrosoproteome Fingerprint in Pregnancy, Labor, and Preterm Labor” (doi:10.1152/ajpcell.00198.2013) (Ulrich et al., 2013) [1]. The data described is a large scale mass spectrometry data set that defines the human uterine smooth muscle S-nitrosoproteome differences among laboring, non-laboring, preterm laboring tissue after treatment with S-nitrosoglutathione.

Mechanical strain associated with the expanding uterus correlates with increased preterm birth rates. Mechanical signals result in a cascading network of protein phosphorylation events. These signals direct cellular activities and may lead to changes in contractile phenotype and calcium signaling. In this study, the complete phospho-proteome of uterine smooth muscle cells subjected to mechanical strain for 5 min was compared to un-strained controls. Statistically significant, differential phosphorylation events were annotated by Ingenuity Pathway Analysis to elucidate mechanically induced phosphorylation networks. Mechanical strain leads to the direct activation of ERK1/2, HSPB1, and MYL9, in addition to phosphorylation of PAK2, vimentin, DOCK1, PPP1R12A, and PTPN11 at previously unannotated sites. These results suggest a novel network reaction to mechanical strain and reveal proteins that participate in the activation of contractile mechanisms leading to preterm labor.

S-nitrosoglutathione reductase (GSNOR), or ADH5, is an enzyme in the alcohol dehydrogenase (ADH) family. It is unique when compared to other ADH enzymes in that primary short-chain alcohols are not its principle substrate. GSNOR metabolizes S-nitrosoglutathione (GSNO), S-hydroxymethylglutathione (the spontaneous adduct of formaldehyde and glutathione), and some alcohols. GSNOR modulates reactive nitric oxide (•NO) availability in the cell by catalyzing the breakdown of GSNO, and indirectly regulates S-nitrosothiols (RSNOs) through GSNO-mediated protein S-nitrosation. The dysregulation of GSNOR can significantly alter cellular homeostasis, leading to disease. GSNOR plays an important regulatory role in smooth muscle relaxation, immune function, inflammation, neuronal development, and cancer progression, among many other processes. In recent years, the therapeutic inhibition of GSNOR has been investigated to treat asthma, cystic fibrosis and interstitial lung disease (ILD). The direct action of •NO on cellular pathways, as well as the important regulatory role of protein S-nitrosation, are closely tied to GSNOR regulation and define this enzyme as an important therapeutic target.

The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K+ current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K+ currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 μM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 μM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.

Focal adhesions are integrin-rich microdomains that structurally link the cytoskeleton to the extracellular matrix and transmit mechanical signals. In the pregnant uterus, increases in integrin expression and activation are thought to be critical for the formation of the mechanical syncytium required for labor. The aim of this study was to determine which integrins are upregulated and localized to focal adhesions in pregnant human myometrium. We used quantitative polymerase chain reaction, Western blotting, and confocal microscopy to determine the expression levels and colocalization with focal adhesion proteins. We observed increases in several integrin transcripts in pregnant myometrium. At the protein level, integrins such as α5-integrin (ITGA5), ITGA7, ITGAV, and ITGB3 were significantly increased during pregnancy. The integrins ITGA3, ITGA5, ITGA7, and ITGB1 colocalized with focal adhesion proteins in term human myometrium. These data suggest that integrins α3β1, α5β1, and α7β1 are the most likely candidates to transmit mechanical signals from the extracellular matrix through focal adhesions in pregnant human myometrium.

Objective Preterm labor is defined as labor prior to 37 weeks of gestation. There are no FDA approved drugs available to treat spontaneous preterm labor. Uterine smooth muscle is unique in that it relaxes in a cGMP-independent manner in the presence of nitric oxide (NO). However, NO-mediated relaxation in tissues from mothers who deliver preterm is blunted. S-nitrosation of cysteines via NO acts as an important mediator in a number of disease states. Our analysis of the myometrial S-nitrosoproteome has revealed that several smooth muscle contractile proteins are differentially S-nitrosated based upon the state of labor in women. An important regulator of protein S-nitrosation is the availability of S-nitrosoglutathione (GSNO), an endogenously expressed NO donor. The enzyme GSNO reductase (GSNOR) regulates GSNO levels in smooth muscle. We performed experiments to test the hypotheses that GSNOR activity is altered in the myometrium from women in preterm labor and that inhibition of GSNOR attenuates myometrial contraction and restores quiescence in spontaneous preterm labor. Methods Experiments were performed in the Myometrial Research Laboratory at UNR Med. Uterine biopsies were obtained with IRB approval and written informed consent from mothers undergoing Cesarean section and transported to the laboratory in physiological buffer for immediate processing. GSNOR expression was measured by western blot and normalized to GAPDH expression. GSNOR activity was measured by the decrease in absorbance at 340 nm via conversion of NADH to NAD+ by GSNOR in the presence of GSNO. Eight (8) nM N6022, a potent and specific inhibitor of GSNOR, was used to verify GSNOR specific activity. Human myometrial tissue was dissected and six uterine strips from each patient were mounted in tissue baths and tested for their ability to contract in response to KCl prior to addition of test agents. Results Human preterm myometrium fails to relax to NO addition whether in the presence or absence of oxytocin stimulation. GSNOR expression is increased in myometrial tissue from women in spontaneous preterm labor compared to term laboring controls. GSNOR enzymatic activity was also increased in protein lysates from myometrial tissue from patients undergoing spontaneous preterm labor than those in labor at term. A potent and selective inhibitor of GSNOR (N6022) decreases the peak force of contraction in guinea pig myometrium and restores sensitivity to NO. Conclusions GSNOR is more highly expressed in preterm laboring myometrium. Preterm laboring myometrium exhibits a blunted response to NO and the GSNOR inhibitor N6022 restores sensitivity to relaxation by NO. Our data suggests that increased GSNOR activity in spontaneous preterm labor contributes to a preterm contractile phenotype through the enzymatic degradation of endogenous GSNO. Funding Source(s) NIH 1U54GM 104944

The notion that breast cancers can survive in an individual patient in a dormant state only to grow as metastatic disease in the future, is in our view incontrovertibly established. Convincing too is the evidence that surgery to remove the primary tumor often terminates dormancy resulting in accelerated relapses. Accepting that many deaths due to breast cancer might be averted were we to understand the cellular mechanisms underlying escape from dormancy, we have examined the extracellular signals produced by breast cancers derived from women with metastatic breast disease. In this perspective, we explore the role of extracellular nucleotide signaling that we have proposed constitutes a pathological axis from the transformed tumor cell to the endothelium in the service of intravasation, dissemination, extravasation and angiogenesis. A role for the dinucleotide kinase NM23/NDPK (nucleoside diphosphate kinase) secreted by breast tumor cells in the generation of signals that stimulate vascular leakiness, anti-thrombosis, endothelial migration and growth, constitutes a mechanistic basis for escape from latency and offers putative therapeutic targets for breast cancer management not previously appreciated.

Rates of premature birth are alarming and threaten societies and healthcare systems worldwide. Premature labor results in premature birth in over 50% of cases. Preterm birth accounts for three-quarters of infant morbidity and mortality. Children that survive birth before 34 weeks gestation often face life-long disability. Current treatments for preterm labor are wanting. No treatment has been found to be generally effective and none are systematically evaluated beyond 48 h. New approaches to the treatment of preterm labor are desperately needed. Recent studies from our laboratory suggest that the uterine muscle is a unique compartment with regulation of uterine relaxation unlike that of other smooth muscles. Here we discuss recent evidence that the mechanically activated 2-pore potassium channel, TREK-1, may contribute to contraction-relaxation signaling in uterine smooth muscle and that TREK-1 gene variants associated with human labor and preterm labor may lead to a better understanding of preterm labor and its possible prevention.

Background: Human breast carcinoma cells secrete an adenosine 5′-diphosphate transphosphorylase (sNDPK) known to induce endothelial cell tubulogenesis in a P2Y receptor-dependent manner. We examined sNDPK secretion and its effects on human endothelial cells. Methods: Nucleoside diphosphate kinase (NDPK) secretion was measured by western blot and enzyme-linked immunosorbent assay, while transphosphorylase activity was measured using the luciferin-luciferase ATP assay. Activation of MAPK was determined by western blot analysis, immunofluorescence and endothelial cell proliferation and migration. Results: A panel of breast cancer cell lines with origin as ductal carcinoma, adenocarcinoma or medullary carcinoma, secrete sNDPK-A/B. Addition of purified NDPK-B to endothelial cultures activated VEGFR-2 and Erk1/2, both of which were blocked by inhibitors of NDPK and P2Y receptors. Activation of VEGFR-2 and ErK1/2 by 2-methylthio-ATP (2MeS-ATP) was blocked by pretreatment with the P2Y1-specific antagonist MRS2179, the proto-oncogene non-receptor tyrosine kinase (Src) inhibitor PP2 or the VEGFR-2 antagonist SU1498. Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor. Treatment of endothelial cells with either NDPK-B or 2MeS-ATP induced migration, blocked by P2Y1, Src or VEGFR-2 antagonists. Conclusion: sNDPK supports angiogenesis. Understanding the mechanism of action of sNDPK and P2Y1 nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

There is strong evidence implicating exosomes, cell-derived nanoscale vesicles, and their protein cargo in facilitating dialogue between primary tumor cells and the premetastatic niche. Identified by our lab as one such protein, secreted nucleoside diphosphate kinase (NM23/NDPK) has been shown to activate endothelial remodeling in support of proangiogenic and prometastatic events. We have previously demonstrated that triple-negative breast cancer (MDA-MB-231) cells elaborate exosomes enriched with NDPK transphosphorylase activity and target the lung as a future site of metastasis. Using in vitro and in vivo approaches, we interrogate the functional role of NDPK in exosome-mediated lung remodeling. In cell-based assays, we demonstrate that MDA-MB-231 exosomes stimulate migration of pulmonary vascular endothelial cells and enhance monolayer permeability. These effects are reversed following treatment with an inhibitor of NDPK activity or an antagonist to the purinergic P2Y1 receptor. Further, we show that immune-compromised mice receiving intravenous injections of MDA-MB-231 exosomes exhibit enhanced pulmonary vascular leakage, whereas treatment with an inhibitor of NDPK attenuates this effect. Using tandem mass tag labeling and mass spectrometry, we profiled proteomic changes to the lung and identified the purinergic signaling pathway as among those significantly affected by exosome treatment. These results elucidate the functional role of exosomal NDPK in compromising vascular integrity and transforming the pulmonary landscape to support colonization by circulating breast cancer cells. Citation Format: Suzann Duan, Senny Nordmeier, Aidan E. Byrnes, Miles A. Brown, Iain L. Buxton. Triple-negative breast cancer cells alter the pulmonary landscape to favor metastasis via exosome-mediated release of NM23 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 35.

P2Y purine nucleotide receptors (P2YRs) promote endothelial cell tubulogenesis through breast cancer cell-secreted nucleoside diphosphate kinase (NDPK). We tested the hypothesis that activated P2Y(1) receptors transactivate vascular endothelial growth factor receptor (VEGFR-2) in angiogenic signaling. P2Y(1)R stimulation (10 microM 2-methyl-thio-ATP (2MS-ATP)) of angiogenesis is suppressed by the VEGFR-2 tyrosine kinase inhibitor, SU1498 (1 microM). Phosphorylation of VEGFR-2 by 0.0262 or 2.62 nM VEGF was comparable with 0.01 or 10 microM 2MS-ATP stimulation of the P2Y(1)R. 2MS-ATP, and VEGF stimulation increased tyrosine phosphorylation at tyr1175. 2MS-ATP (0.1-10 microM) also stimulated EC tubulogenesis in a dose-dependent manner. The addition of sub-maximal VEGF (70 pM) in the presence of increasing concentrations of 2MS-ATP yielded additive effects at 2MS-ATP concentrations3 microM, whereas producing saturated and less than additive effects ator =3 microM. We propose that the VEGF receptor can be activated in the absence of VEGF, and that the P2YR-VEGFR2 interaction and resulting signal transduction is a critical determinant of vascular homoeostasis and tumour-mediated angiogenesis.

MDA-MB-435S human breast cancer cells (435S) secrete nucleoside diphosphate kinase (NDPK) that supports metastases and is inhibited by epigallocatechin gallate (EGCG) and ellagic acid (EA). We hypothesise that 435S cell-secreted NDPK-B supports tumour formation by modulating ATP levels locally to activate endothelial cell (EC) P2Y receptor-mediated angiogenesis. Epigallocatechin gallate (IC50=8-10 microM) and EA (IC50=2-3 microM) suppressed 435S cell growth, but had less effect on human CD31+ EC growth. Epigallocatechin gallate (IC50=11 microM) and EA (IC50=1 microM) also prevented CD31+ EC tubulogenesis on Matrigeltrade mark. 435S cell-conditioned media induced tubulogenesis in a cell number, time, and nucleotide-dependent manner. Ellagic acid (1 microM), but not equimolar EGCG, reduced cell number-dependent angiogenesis. P2Y 1 receptor activation by NDPK-generated nucleotide (100 microM ATP) or by 10 microM 2-methyl-thio-ATP (2MS-ATP) promoted tubulogenesis on collagen and was blocked by the P2Y 1 antagonist MRS2179 (10 microM). Physiological amounts of purified as well as 435S cell-secreted NDPK also promoted angiogenesis that was attenuated by NDPK depletion or 10 microM MRS2179, indicating a P2Y 1 receptor-mediated pathway. These results support the notion that secreted NDPK mediates angiogenesis via P2Y receptor signalling and suggests that novel inhibitors of NDPK may be useful as therapeutics.

TREK-1, an outward-rectifying potassium channel activated by stretch, is found in the myometrium of pregnant women. Decreased expression of TREK-1 near term suggests that TREK-1 may contribute to uterine quiescence during gestation. Five alternatively spliced TREK-1 variants were identified in the myometrium of mothers who delivered spontaneously preterm (

Nucleoside Diphosphate Kinase (NDPK), described as NM23 a metastasis suppressor, is found in the culture medium of cancer cells lines suggesting that the kinase may have an extracellular role. We propose that extracellular NM23 released from breast cancers in vivo stimulates tumor cell migration, proliferation and endothelial cell angiogenesis in support of metastasis development.NM23 in the bloodstream of immunocompromised mice carrying human triple-negative breast cancers or in breast cancer patients was measured by ELISA. Primary and metastatic tumor development, the impact of blockade of NM23 and/or its stimulation of nucleotide receptors were measured using in vivo imaging. NM23 expression data in the Curtis breast dataset was examined to test our hypothesis that NM23 may play a mechanistic role in breast cancer development.SCID mice carrying metastatic MDA-MB-231Luc+ triple-negative human breast tumor cells elaborate NM23 into the circulation correlated with primary tumor growth. Treatment of mice with the NM23 inhibitor ellagic acid (EA) or the purinergic receptor antagonist MRS2179 slowed primary tumor growth. At 16 weeks following implantation, lung metastases were reduced in mice treated with EA, MRS2179 or the combination. Expression of NM23 in the Curtis breast dataset confirmed a likely role for NM23 in tumor metastasis.Extracellular NM23 may constitute both a biomarker and a therapeutic target in the management of breast cancer.

Endothelial cell apoptosis is intimately involved in the balance between blood vessel growth and regression and is promoted by numerous stimuli including angiostatin and endostatin, reactive oxygen species (ROS) released during inflammatory processes, and chronic use of drugs of abuse such as cocaine. Apoptosis is characterized by many biological signalling events, including the activation of caspases. Caveolar domains have been hypothesized to mediate apoptotic signalling. We have addressed this hypothesis in cardiac endothelial cells and here we show that caspase-3 proenzyme (32 kDa) and its activated counterpart (17 kDa) co-purify with low-density, caveolin-enriched microdomains and that caspase-3 can be localized with caveolae in intact cells using fluorescent microscopy. Disruption of caveolae results in temporal and spatial changes in enzyme activity. While caspase-3 has been associated with mitochondrial, cytosolic, and high-density regions, the co-purification of activated caspase-3 and caveolar domains reported here suggests the possibility that sarcolemmal caspase-3 may be targeted to plasma-membrane associated substrates.

Motor innervation in the canine rectoanal region was examined in isolated strips of the circular muscle layer. Contractile responses to electrical field stimulation began at lower frequencies and were more persistent in the internal anal sphincter (IAS) than in the rectum. Motor innervation to the IAS was almost exclusively sympathetic, since it was blocked by guanethidine (Guan 3 μM) while the response in the proximal rectum was approximately 50% muscarinic, and sensitive to the M3 selective antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 0.1 μM) and 50% tachykinergic, and sensitive to the neurokinin 2 (NK2) receptor antagonist GR 94800 (1 μM). From IAS to rectum there was a gradual shift in the relative contribution of intrinsic and extrinsic neural innervation. Responses to exogenously applied transmitters exhibited a similar pattern to that observed with motor innervation. Norepinephrine (NE) was most potent in the IAS and acetylcholine (ACh) and NK-A were most potent in the proximal rectum. The responses were inhibited by prazosin, 4-DAMP and GR 94800 respectively. A gradient in the density of adrenergic α1, muscarinic and NK2 receptors also existed from IAS to rectum as determined by measuring the binding of [3H]-prazosin, [3H]-quinuclidinyl benzilate ([3H]-QNB and [3H]-SR-48968 to smooth muscle membranes. In summary, these data suggest that the shift in motor innervation in the rectoanal region is achieved in part by changes in receptor populations available for activation by sympathetic and enteric motor neurons. British Journal of Pharmacology (2002) 137, 1321–1329. doi:10.1038/sj.bjp.0704987

Studies of endothelial cell signaling involving cholesterol-rich domains require large numbers of cells of bona fide origin. The growth of any cell in culture, particularly for extended periods, results in an altered phenotype that could include changes in the properties of caveolae and lipid raft structures. While continuously propagated cells are used to study specific questions because their origin is known and because proteins of interest are still expressed, such reasoning is an oversimplification and can lead to findings that are descriptive of the cell's adaptation to culture rather than its original phenotype. We are particularly cognizant of this concern as we examine caveolar signaling domains in endothelial cells. Here we present a reproducible method for the isolation and characterization of large numbers of bona fide endothelial cells suitable for studies of the regulation of receptor signal transduction. Digestion of guinea pig hearts with collagenase results in the liberation of cells that adhere to collagen-coated plastic and express platelet-endothelial cell adhesion molecule 1 (PECAM-1) and binding sites for Ulex europaeus agglutinin 1 (UEA-1) that permit segregation of cells using fluorescence-activated cell sorting. Growth of cells over 7 doublings results in enrichment in the expression of both PECAM-1 and UEA-1 and retention of functional low-density lipoprotein receptor. The ability of cells to differentiate into endothelial tubes at any stage during their characterization up to 20 doublings in culture suggests that this method can be employed to generate endothelial cells that are minimally altered from their site of origin.

The vasodilatory effects of nucleotides in the guinea-pig thoracic aorta were examined to determine the relationship between molecular expression and function of P2Y receptors. In aortic rings precontracted with norepinephrine, vasodilatory responses to purine nucleotides exhibited a rank-order of potency of 2-methylthio-ATP>ADP>ATP. Responses to UTP, but not UDP suggested a functional role for P2Y4 but not P2Y6 receptors. Aortic endothelial cells express at least four P2Y receptors; P2Y1, P2Y2, P2Y4 and P2Y6. In primary culture, these cells exhibit desensitizing transient calcium responses characteristic of P2Y1, P2Y2 and P2Y4, but not P2Y6 receptors. UDP had no effect on endothelial cell calcium. The pyrimidinergic receptor agonist UTP is capable of eliciting robust vasodilation in aortic rings and causing calcium responses in cultured guineapig aortic endothelial cells. These responses are equivalent to the maximum responses observed to ATP and ADP. Measurement of intracellular calcium release in response to ATP and 2-methylthio-ATP were similar, however only the 2-methylthio-ATP response was sensitive to the P2Y1 antagonist N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179). In aortic rings, vasodilatory responses to 2-methylthio-ATP, ATP and ADP were all blocked by pre-incubation of tissues with MRS2179. MRS2179 pretreatment had no effect of the ability of UTP to cause relaxation of norepinephrine responses in aortic rings or the ability of UTP to cause calcium release in aortic endothelial cells. We demonstrate robust effects of purine and pyrimidine nucleotides in guineapig aorta and provide functional and biochemical evidence that MRS2179 is a selective P2Y1 antagonist.

Functional interactions between primary tumor cells and their vascular targets orchestrate the formation of a metastatic milieu enriched with oxygen and metabolic substrates. These interactions have been largely attributed to the activation of VEGF receptor pathways through localized tumor-mediated release of VEGF, in addition to indirect receptor phosphorylation. However, pro-angiogenic events are known to occur in the absence of VEGF and alternative pathways resulting in dual activation of neovascularization and enhanced vascular permeability are of growing interest. Our lab has previously shown that triple-negative human breast cancer (MDA-MB-231) cells elaborate exosomes carrying NM23, a nucleoside diphosphate kinase (eNDPK) that is suggested to act as an upstream mediator of vasodilation and pro-angiogenic activity through its action of elevating extracellular ADP/ATP levels. To further elucidate the role of eNDPK in breast cancer signaling and metastasis, we show that 231-exosomes induce a pro-angiogenic phenotype in both human umbilical vein and murine pulmonary endothelial cells. Treatment with an NDPK-specific inhibitor and an antagonist to the ADP/ATP-activated P2Y1 receptor results in amelioration of pro-angiogenic characteristics. Further, we report the development and application of a novel murine implant model to demonstrate increased specificity of 231 exosome targeting to pulmonary endothelial cells over unrelated cell lines. These results suggest the involvement of exosomes and eNDPK in pro-angiogenic communication between metastatic breast cancer cells and their vascular targets. Exosomes elaborated by these cells specifically target pulmonary endothelium in vivo, mirroring the organotropic nature of breast cancer metastasis. Implication of eNDPK in angiogenesis and metastasis may challenge current and invasive treatment methods in favor of developing novel small molecule inhibitors of eNDPK signaling. Lastly, eNDPK can be explored as a potential biomarker for early detection of metastatic breast cancer as it uniquely appears in the serum of patients with breast cancer and not in the serum of individuals with unrelated pathologies. Citation Format: Suzann Duan, Senny Nordmeier, Iain L. Buxton. Exosomal NM23 facilitates pro-angiogenic communication between triple-negative breast cancer cells and their vascular targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1807. doi:10.1158/1538-7445.AM2017-1807

In myometrial strips from near-term non-labouring human uterus, addition of oxytocin (OT) evoked dose-dependent (10 – 3000 nM) phasic contractions that were antagonized by atosiban (1 μM) and relaxed by addition of the nitric oxide donor S-nitroso L-cysteine (Cys-NO). In near-term labouring myometrium, however, addition of OT was ineffective at raising additional tone. In both labouring and non-labouring tissue, Cys-NO mediated relaxation of spontaneous or OT-induced contractions (IC50=1 μM) was unaffected by prior addition of the guanylyl cyclase (GC) inhibitors ODQ (1H-[1,2,4]oxadiazolo[4,3,-α]quinoxalin-1-one; 1 μM), or methylene blue (MB; 10 μM). Elevation of intracellular cyclic GMP accompanying 30 μM Cys-NO addition in non-labouring tissue (7.5 fold) or in labouring tissues (2.5 fold) was completely blocked in tissues that had been pre-treated with ODQ or MB. Charybdotoxin (ChTx), iberiotoxin (IbTx) and kaliotoxin (KalTx) all shifted the Cys-NO inhibition curve to the right and reduced the degree of relaxation produced by maximal Cys-NO treatment (100 μM in non-labouring tissue; in labouring tissue, KalTx prevented Cys-NO mediated relaxation in both stimulated and unstimulated tissue. Addition of the NO-donor S-nitroso N-acetyl penicillamine (SNAP) produced a dose-dependent relaxation of pregnant myometrium while 3-morpholinosyndonimine (SIN-1) did not. The failure of SIN-1 to relax OT-induced contractions was not due to a failure of the donor to stimulate myometrial GC. We demonstrate that despite the ability of NO to stimulate myometrial GC in pregnant uterine muscle, relaxations are independent of cyclic GMP action. Effects of K+-channel inhibitors suggests that NO-induced relaxation in human uterine smooth muscle may be subserved by direct or indirect activation of one or more calcium-activated K+-channels. British Journal of Pharmacology (2001) 134, 206–214; doi:10.1038/sj.bjp.0704226

Preterm labor (PTL) is defined as uterine irritability accompanied by cervical dilation and/or effecement that occurs before 37 weeks gestation. In most cases, PTL becomes preterm delivery (PTD), accounting for 8% to 10% of births in the United States. Fetuses born before 37 weeks' gestation are at risk for a multitude of health and developmental problems. Most perinatal morbidity and mortality in the United States are caused by PTL. It is a costly problem, in both monetary and human terms. Although some risk factors have been identified, they by no means identify, in advance, every case of PTL and PTD. Despite the understandable emphasis on attempts to find and test risk factors that predict PTL, the ultimate benefit--preventing PTD--will come only from an understanding of the physiologic mechanisms of parturition and how to halt those processes when they occur too early. This article reviews current approaches to preventing PTD, describes the biology of myometrial contraction, and discusses recent progress from several laboratories including the authors' that may shed light on approaches to inhibit uterine contractility in the setting of PTL.

See related article, pages 185–192 In response to hemodynamic overload, the heart undergoes a complex adaptive remodeling process that involves cardiac myocyte hypertrophy, transformation of fibroblast into myofibroblast, high-level expression of extracellular matrix (ECM) proteins, interstitial fibrosis, and cell death.1 Differentiation of cardiac fibroblasts into myofibroblasts is critical to the production and deposition of collagens and plays a decisive role in myocardial fibrosis and morphological alterations during the progression of adaptive myocardial hypertrophy to decompensation and heart failure.1,2 Among the plethora of identified fibrogenic factors, transforming growth factor (TGF)-β3 plays a fundamental role in hypertrophic and fibrotic remodeling of the heart, where it regulates cardiomyocyte growth, fibroblast activation, and ECM deposition.4,5 The expression of ventricular TGF-β mRNA and protein is increased in numerous models of pathological cardiac hypertrophy and in cardiac cells in response to putative hypertrophic stimuli.6,7 In vitro, TGF-β activates myofibroblast transformation and increases ECM production.7 Blockade of TGF-β signaling is predicted to blunt fibrosis.5 Recent studies from Chen et al8 convincingly demonstrated that disruption of TGF-β signaling by inducible dominant-negative mutation of the TGF-β receptor type II (TβRII) gene significantly reduced the pressure overload–induced myofibroblast transformation and interstitial fibrosis in mouse heart. Thus, there is considerable interest in understanding how signaling by TGF-β receptors is transduced and how the inevitable damage produced could be mitigated. Of the 3 isoforms of TGF-β expressed in mammals,9,10 TGF-β1 is expressed in the adult heart, where it is secreted by cardiomyocytes and myofibroblasts and retained in significant amounts in ECM as a latent cytokine. Recent advances have led to clear description of downstream of TGF-β signal transduction pathways initiated by binding of TGF-β to membrane-bound heteromeric receptor kinases (TβRI and TβRII) that transduce intracellular signals via both Smad and non-Smad pathways …

We evaluated the effect of the nitric oxide (NO) donor CysNO (S-nitroso-L-cysteine) and endogenous NO upon spontaneous contractility in non-pregnant cynomolgus monkeys. We also assessed the role of intracellular guanosine 3′,5′-cyclic monophosphate ([cyclic GMP]i) as a second messenger for NO in monkey uterine smooth muscle. CysNO reduced spontaneous contractility by 84% (P

Molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor at term are incompletely known. More than 10% of babies born worldwide are premature and 1,000,000 die annually. Preterm labor results in preterm delivery in 50% of cases in the United States explaining 75% of fetal morbidity and mortality. There is no Food and Drug Administration-approved treatment to prevent preterm delivery. Nitric oxide-mediated relaxation of human uterine smooth muscle is independent of global elevation of cGMP following activation of soluble guanylyl cyclase. S-nitrosation is a likely mechanism to explain cGMP-independent relaxation to nitric oxide and may reveal S-nitrosated proteins as new therapeutic targets for the treatment of preterm labor. Employing S-nitrosoglutathione as an nitric oxide donor, we identified 110 proteins that are S-nitrosated in 1 or more states of human pregnancy. Using area under the curve of extracted ion chromatograms as well as normalized spectral counts to quantify relative expression levels for 62 of these proteins, we show that 26 proteins demonstrate statistically significant S-nitrosation differences in myometrium from spontaneously laboring preterm patients compared with nonlaboring patients. We identified proteins that were up- S-nitrosated as well as proteins that were down- S-nitrosated in preterm laboring tissues. Identification and relative quantification of the S-nitrosoproteome provide a fingerprint of proteins that can form the basis of hypothesis-directed efforts to understand the regulation of uterine contraction-relaxation and the development of new treatment for preterm labor.

Previous pharmacological and immunohistochemical studies have suggested that nitric oxide (NO) is a mediator of enteric neuromuscular transmission in the canine proximal colon. The present study demonstrates the release of nitric oxide (NO) and/or its oxidation products (collectively termed NOx) following stimulation of intrinsic neurons with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). Strips of muscularis externa, including the ganglionated myenteric and submucosal plexuses were suspended under tension in modified Krebs solution. DMPP (1-100 microM) produced concentration-dependent inhibition of circular muscle contractile activity and this effect was antagonized by L-nitroarginine methyl ester or L-nitroarginine (100 microM). Tetrodotoxin (TTX, 1 microM) significantly reduced mechanical responses to DMPP (10 microM) but had no effect on responses to high concentrations of DMPP (100 microM). Aliquots of the bathing medium were assayed for NOx after regeneration of NO from NO2- and/or NO3-. NO was measured as chemiluminescence produced by reaction with ozone. Detection was linear over the range 6-25,000 pmol of added NO2- or NO3-. In the absence of drugs, a basal release of 1113.5 +/- 100.4 pmol NOx/g tissue (n = 27) was detected after a 30-min incubation period. DMPP (1-100 microM) stimulated a concentration-dependent increase in NOx to 5099.9 +/- 430 pmol/g per 30 min (n = 17) at 100 microM. NOx release was inhibited by an inhibitor of nitric oxide synthase activity (N(G)-monomethyl-L-arginine) or by reduction in extracellular Ca2+ concentration. DMPP-stimulated NOx accumulation was significantly reduced, but not abolished by TTX (1 microM). These results provide further evidence that NO is released following stimulation of intrinsic neurons in canine proximal colon and support previous suggestions that nicotinic agonists may directly stimulate terminals of NO-releasing neurons.

Exogenous nucleoside diphosphate kinase (eNDPK or NM23) has been shown to promote endothelial cell proliferation and migration and tumor-mediated angiogenesis. This is facilitated by its transphosphorylase activity, in which a gamma terminal phosphate group from a triphosphate nucleoside is transferred to a diphosphate nucleoside, resulting in elevated ATP levels. ATP can active purinergic receptors (P2Y1) on adjacent endothelial cells to promote angiogenesis. We previously reported that eNDPK was detected in conditioned media of breast cancer cells and in the serum of breast cancer patients (Yokdang, et al., 2011; Yokdang, et al., 2015). This suggests that eNDPK may have a role in tumorigenesis, but the mechanism by which eNDPK is released into the extracellular environment remains unknown. It is known that all cell types secrete small vesicles called exosomes, 30-100 nm wide vesicles of endocytic origin. We hypothesize that exosomal eNDPK secreted by breast cancer cells targets and promotes endothelial cell tubulogenesis and direct inhibition of eNDPK and P2Y1 activity attenuates angiogenesis. Exosomes were purified from human breast cancer cells, MDA-MB-231 (231) and non-tumorigenic human mammary epithelial cells (HME1) using ExoQuick TC. Transmission electron microscopy, mass spectrometry, and flow cytometry were performed to characterize the exosomes. Purified exosomes labeled with GFP-tagged tetraspanin (CD63) were introduced to human umbilical vein endothelial cells (HUVEC) to detect and measure tubulogenesis formation. We show that 231 secreted exosomes contain eNDPK and HUVEC cells treated with NDPK in the presence of ADP and GTP form tubules. In addition, fluorescently-labeled exosomes are associated with HUVEC tubules. The role of exosomal NDPK released by cancer cells in promoting angiogenesis and tumorigenesis may be used as a potential biomarker and target for treatment in breast cancer management. For future investigations of exosomal eNDPK, this study suggests that breast cancer exosomes promote endothelial tubulogenesis in a P2Y receptor-dependent manner. Citation Format: Senny Nordmeier, Suzann Duan, Iain L.O. Buxton. Purinergic regulation of tumor-mediated angiogenesis: A role for exosomal NM23. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3396.

Decades of research has established the idea that cancer cells of the primary tumor prime the metastatic microenvironment prior to establishing metastases at distant sites in the body. While this process is thought to facilitate neocolonization of dislodged primary tumor cells, the mechanisms by which cancer cells communicate to their remote targets remain largely unknown. Our lab has previously shown that triple negative breast cancer cells release exosomes carrying NM23, a nucleoside diphosphate kinase (eNDPK) implicated in promoting angiogenesis and pro-metastatic events extracellularly. To further elucidate the role of eNDPK in breast cancer signaling, we are developing a novel murine model that will allow us to examine its effects in vivo, as well as establish a timeline for the occurrence of metastases. To establish our metastasis model in mice, we first showed that injecting human MDA-MB-231 cells into the mammary fat pad results in the formation of a primary tumor and subsequent development of metastases in the lung. Treating mice with inhibitors of NDPK activity reduces the size of the primary tumor and prevents secondary tumor formation in the lung. To mimic the pulmonary tumor microenvironment, we have isolated endothelial cells from the lungs of 6-8 day old mouse pups using magnetic beads conjugated to the endothelial cell markers CD54, CD102, or CD106. Purity of endothelial cells was confirmed by immunofluorescence staining. In preparation for in vivo experiments that demonstrate eNDPK targeting to lung endothelial cells, we have 3D printed biocompatible polylactic acid (PLA) tubular inserts that are capable of supporting lung endothelial cells encompassed in a gelatinous cell scaffold. Inserts containing lung endothelial cells are surgically introduced into the subcutaneous tissue of adult SCID mice. Human MDA-MB-231 cells expressing GFP-tagged tetraspanin are then injected orthotopically into the mammary fat pads of mice. After a period of 7-21 days, the inserts are excised and analyzed for the presence of GFP-labeled exosomes containing eNDPK and reorganization of the insert microenvironment. Using this model for metastasis, we will be able to confirm the involvement of eNDPK in cell-cell communication between primary tumor cells and their targets in vivo. The significance of uncovering the mechanism(s) by which cancer cells metastasize is emphasized by the fact that recurrence of cancer at distant sites is associated with the most negative outcome in women diagnosed with breast cancer. Implication of eNDPK signaling in metastasis will lead to future research in developing novel small molecule inhibitors. Further, eNDPK can be used as a biomarker for the beginning stages of metastatic breast cancer, replacing current unreliable early detection methods and improving long-term survival outcomes. Citation Format: Suzann Duan, Senny Nordmeier, Iain L.O. Buxton. Exploring the metastatic potential of exosomal NM23 signaling using a triple negative breast cancer model in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4275.

Spontaneous preterm labor (PTL) is a uniquely human problem that results in preterm delivery of an underdeveloped fetus. The underlying cause remains elusive. The cost to societies in human suffering and treasure is enormous. The stretch-activated two pore potassium channel TREK-1 is up-regulated during gestation to term such that it may maintain uterine quiescence by hyperpolarizing the smooth muscle cell membrane. We have hypothesized that the human TREK-1 channel is involved in myometrial relaxation during pregnancy and that splice variants of the TREK-1 channel expressed in preterm myometrium are associated with preterm delivery by interaction with full-length TREK-1. We detected three wild-type human TREK-1 transcript isoforms in nonpregnant and pregnant human myometrium. Using RT-PCR, we identified five unique TREK-1 splice variants in myometrium from women in PTL. These myometrial TREK-1 variants lack either the pore or the transmembrane domains or both. In transiently transfected HEK293T cells, wild-type TREK-1 was predominantly expressed at the plasma membrane. However, individual splice variants were expressed uniformly throughout the cell. Wild-type TREK-1 was localized at the plasma membrane and cytoplasm close to the plasma membrane when coexpressed with each splice variant. Co-immunoprecipitation of FLAG epitope-tagged TREK-1 and six-His epitope-tagged splice variants using Ni bead columns successfully pulled down wild-type TREK-1. These results suggest that each of four TREK-1 splice variants interacts with full-length wild-type TREK-1 and that in vivo, such interactions may contribute to a PTL phenotype.

We have recently demonstrated the ability of neuropeptide Y to augment norepinephrine-sti,ulated contractions of rat tail artery. In [ 3 H]myo- inositol labeled segments of rat tail artery, addition of a concentration of neuropeptide Y (10 −8 M) known to potentiate contractile responses produced no effect on the basal accumulation of [ 3 H]inositol phosphates. Addition of norepinephrine (10 −7 M), however, resulted in the rapid accumulation of [ 3 H]inositol 1,4,5-trisphosphate to 325 ± 29 % of basal levels within 5 min. This effect of norepinephrine on [ 3 H]inositol 1,4,5-trisphosphate accumulation was mirrored by significant increases (274 ± 25 % of control) in total [ 3 H]inositol phosphates. When neuropeptide Y and norepinephrine were added together, the norepinephrine-stimulated increase in [ 3 H]inositol 1,4,5-trisphosphate was significantly augmented (467 ± 35 % of control) as was the increase measured in total [ 3 H]inositol phosphates (366 ± 21 % of control). We suggest that the ability of neuropeptide Y to augment norepinephrine-induced contraction of the rat tail artery is due, at least in part, to the ability of neuropeptide Y to increase norepinephrine-stimulated phospholipase C activity in vascular smooth muscle and may represent the mechanism of action of this co-neurotransmitter in vivo .

The molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor are not completely known. Nitric oxide relaxes uterine smooth muscle in a manner disparate from that for other smooth muscles because global elevation of cGMP after activation of soluble guanylyl cyclase does not relax the muscle. S-Nitrosylation, the covalent addition of an nitric oxide (NO) group to a cysteine thiol is a likely mechanism to explain the ability of NO to relax myometrium. This work is the first to describe the myometrial S-nitrosylproteome in both pregnant and nonpregnant tissue states. Using the guinea pig model, we show that specific sets of proteins involved in contraction and relaxation are S-nitrosylated in laboring and nonlaboring muscle and that many of these proteins are uniquely S-nitrosylated in only one state of the tissue. In particular, we show that S-nitrosylation of the intermediate filament protein desmin is significantly increased (5.7-fold, p < 0.005) in pregnancy and that this increase cannot be attributed solely to the increase in protein expression (1.8-fold, p < 0.005) that accompanies pregnancy. Elucidation of the myometrial S-nitrosylproteome provides a list of mechanistically important proteins that can constitute the basis of hypotheses formed to explain the regulation of uterine contraction/relaxation.

1. We have previously demonstrated that activation of M3 muscarinic receptors increases inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) accumulation in colonic smooth muscle. 2. In the present study, we demonstrate the existence of InsP3 and InsP4 binding sites in colonic circular smooth muscle by use of radioligand binding methods. Both [3H]-InsP3 and [3H]-InsP4 bound rapidly and reversibly to a single class of saturable sites in detergent-solubilized colonic membranes with affinities of 5.04 +/- 1.03 nM and 3.41 +/- 0.78 nM, respectively. The density of [3H]-InsP3 binding sites was 335.3 +/- 19.3 fmol mg-1 protein which was approximately 2.5 fold greater than that of [3H]-InsP4 sites (127.3 +/- 9.1 fmol mg-1 protein). 3. The two high affinity inositol phosphate binding sites exhibited markedly different pH optima for binding of each radioligand. At pH 9.0, specific [3H]-InsP3 binding was maximal, whereas [3H]-InsP4 binding was only 10% that of [3H]-InsP3. Conversely, at pH 5.0, [3H]-InsP4 binding was maximal, while [3H]-InsP3 binding was reduced to 15% of its binding at pH 9.0. 4. InsP3 was about 20 fold less potent (KI = 50.7 +/- 8.3 nM) than InsP4 in competing for [3H]-InsP4 binding sites and could compete for only 60% of [3H]-InsP4 specific binding. InsP4 was also capable of high affinity competition with [3H]-InsP3 binding (KI = 103.5 +/- 1.5 nM), and could compete for 100% of [3H]-InsP3 specific binding. 5. [3H]-InsP3 binding in subcellular fractions separated by discontinuous sucrose density gradients followed NADPH-cytochrome c reductase activity, suggesting an intracellular localization for the majority of InsP3 receptors in this tissue, whereas [3H]-InsP4 binding appeared to be equally distributed between plasma membrane and intracellular membrane populations.6. These results suggest the existence of distinct and specific InsP3 and InsP4 binding sites which may represent the physiological receptors for these second messengers; differences in the subcellular distribution of these receptors may contribute to differences in their putative physiological roles.

The effects of beta-adrenergic receptor stimulation on the electrical and mechanical activity of canine colonic circular muscles were compared with forskolin (Fsk), a known stimulator of adenylate cyclase. The actions of isoproterenol (Iso) were mediated by beta 2-receptors. Iso and Fsk increased intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels in both the presence and absence of acetylcholine (ACh), whereas ACh (0.3 microM) alone reduced cAMP levels. These agents caused inhibition of spontaneous and ACh-induced contractions. Inhibition was associated with a reduction in the amplitude and duration of electrical slow waves recorded near the submucosal border. Near the myenteric border, Iso and Fsk hyperpolarized the membrane by up to 30 mV and changed the pattern of electrical rhythmicity. These effects were mimicked by 8-bromo-cAMP (1-3 mM). Contractile inhibition with Fsk and Iso was associated with a decrease in the amplitude and duration of Ca2+ transients measured with fura-2 fluorescence. cAMP (10-300 microM) reduced the Ca2+ sensitivity of the contractile apparatus in muscles permeabilized with staphylococcal alpha-toxin. The actions of Iso appear linked to cAMP. We hypothesize that cAMP produces relaxation both by modulation of membrane ionic channels with a consequent decline in the entry of Ca2+ as well as through a decrease in the sensitivity of the contractile apparatus to Ca2+.

Nitric oxide relaxes myometrium in a cGMP-independent manner. Although cGMP activates its cognate kinase, this is not required for the inhibitory effect of nitric oxide. Thus, nitric oxide-mediated cGMP elevation does not enjoy the same set of substrates as it does in other smooth muscles. To further understand the regulation of relaxation of uterine muscle by cGMP, we have studied the actions of peptide-mediated cGMP action in guinea pig myometrium. We used both functional and biochemical studies of the action of the particulate guanylyl cyclase activator uroguanylin and its receptor, particulate guanylyl cyclase type C, to address the relationship between cGMP elevation acting in the membrane signaling domain to that of the nonmembrane region of the cell. Uroguanylin relaxed oxytocin-induced contractions in a dose-dependent fashion only in pregnant myometrium. Both relaxation and cGMP accumulation after uroguanylin stimulation were blocked by the putative particulate guanylyl cyclase type C inhibitors 2-chloro-ATP and isatin (1H-indole-2,3-dione), but not by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one (ODQ). Uroguanylin stimulated cGMP accumulation only in the pregnant myometrium. Caveolin-1 expression increased in pregnancy toward term. In the caveolin-1-containing membrane domain, uroguanylin, but not the nitric-oxide donor, led to the elevation of cGMP that was insensitive to ODQ. Particulate guanylyl cyclase C was expressed and prouroguanylin was detected in pregnant myometrium. We conclude that a uroguanylin–particulate cyclase-cGMP relaxation pathway is present and cGMP is compartmented in myometrium. The agonist-mediated selectivity of relaxation to cGMP is of fundamental pharmacological interest in understanding signal transduction in smooth muscle.