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The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes. [ABSTRACT FROM AUTHOR]

Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with hyperresponsiveness of airway smooth muscle (ASM). Chronic exposure to cigarette smoke (CS) leads to airway inflammation and remodelling. However, the direct effect of gaseous CS or CS extract (CSE) on human airway smooth muscle cell (hASMC) function remains poorly understood. This study investigated the acute effect of CS/CSE on calcium homeostasis, a key regulator of ASM physiology and pathophysiology. Primary hASMC were isolated from non-smoking donor lungs, and subjected to Ca2+ imaging studies. We found that both CS, and CSE, rapidly elevated cytosolic Ca2+ in hASMC through stimulation of plasmalemmal Ca2+ influx, but excluded store-operated and L-type Ca2+ channels as mediators of this effect. Using a specific pharmacological inhibitor, or shRNA-driven knockdown, we established that both CS and CSE stimulated Ca2+ influx in hASMC through the neurogenic pain receptor channel, transient receptor potential ankyrin 1 (TRPA1). CS/CSE-dependent, TRPA1-mediated Ca2+ influx led to myosin light-chain phosphorylation, a key process regulating ASM contractility. We conclude that TRPA1 is likely an important link between CS/CSE exposure and airway hyperresponsiveness, and speculate that acute CS/CSE-induced Ca2+ influx could lead to exacerbated ASM contraction and potentially initiate further chronic pathological effects of tobacco smoke. [ABSTRACT FROM AUTHOR]

Methionine demethylation during metabolism generates homocysteine (Hcy) and its remethylation requires folate and cobalamin. Elevated Hcy concentrations are associated with vascular-related complications of pregnancy, including increased vascular stiffness, predictive of clinical vascular disease. Maternal and fetal total Hcy (tHcy) concentrations are positively related, yet the influence of Hcy on fetoplacental vascular function in normal pregnancy has not been examined. We hypothesized that Hcy alters fetoplacental vascular characteristics with influences on fetal growth outcomes. We investigated (1) placental chorionic plate artery distensibility and neonatal blood pressure in relation to umbilical plasma tHcy; (2) relationships between cord venous (CV) and cord arterial (CA) plasma tHcy, folate, and cobalamin concentrations; and (3) tHcy associations with birth weight and anthropometric measurements of body size as indices of fetal growth in normal pregnancies with appropriate weight-for-gestational age newborns. Maternal plasma tHcy, folate, and cobalamin concentrations were consistent with published data. Placental chorionic plate artery distensibility index (β; measure of vessel stiffness) was inversely related to CA tHcy, yet neonatal blood pressure was not significantly affected. CV and CA tHcy concentrations were positively related and CV tHcy negatively related to CV cobalamin but not folate. CV tHcy concentration positively related to birth weight, corrected birth weight percentile, length, head circumference, and mid-arm circumference of newborns. CV cobalamin was inversely related to fetal growth indices but not to folate concentration. Our study demonstrates a potential relationship between fetal tHcy and placental artery distensibility, placing clinical relevance to cobalamin in influencing Hcy concentration and maintaining low vascular resistance to facilitate nutrient exchange favorable to fetal growth. [ABSTRACT FROM AUTHOR]

Abdominal uterine electromyograms (uEMG) studies have focused on uterine contractions to describe the evolution of uterine activity and preterm birth (PTB) prediction. Stationary, non-contracting uEMG has not been studied. The aim of the study was to investigate the recurring patterns in stationary uEMG, their relationship with gestation age and PTB, and PTB predictivity. A public database of 300 (38 PTB) three-channel (S1-S3) uEMG recordings of 30 min, collected between 22 and 35 weeks' gestation, was used. Motion and labour contraction-free intervals in uEMG were identified as 5-min weak-sense stationarity intervals in 268 (34 PTB) recordings. Sample entropy (SampEn), percentage recurrence (PR), percentage determinism (PD), entropy (ER), and maximum length ( L) of recurrence were calculated and analysed according to the time to delivery and PTB. Random time series were generated by random shuffle (RS) of actual data. Recurrence was present in actual data ( p < 0.001) but not RS. In S3, PR ( p < 0.005), PD ( p < 0.01), ER ( p < 0.005), and L ( p < 0.05) were higher, and SampEn lower ( p < 0.005) in PTB. Recurrence indices increased (all p < 0.001) and SampEn decreased ( p < 0.01) with decreasing time to delivery, suggesting increasingly regular and recurring patterns with gestation progression. All indices predicted PTB with AUC ≥0.62 ( p < 0.05). Recurring patterns in stationary non-contracting uEMG were associated with time to delivery but were relatively poor predictors of PTB. [ABSTRACT FROM AUTHOR]

Here we provide a protocol for the directed differentiation of hEPI-NCSC into midbrain dopaminergic neurons, which degenerate in Parkinson's disease. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. The experimental design is distinctly different from conventional protocols for embryonic stem cells and induced pluripotent stem (iPS) cells. It includes pre-differentiation of the multipotent hEPI-NCSC into neural stem cell-like cells, followed by ventralizing, patterning, continued exposure to the TGFβ receptor inhibitor, SB431542, and at later stages of differentiation the presence of the WNT inhibitor, IWP-4. All cells expressed A9 midbrain dopaminergic neuron progenitor markers with gene expression levels comparable to those in normal human substantia nigra. The current study shows for the first time that virtually homogeneous populations of dopaminergic neurons can be derived ex vivo from somatic stem cells without the need for purification, with useful timeliness and high efficacy. This novel development is an important first step towards the establishment of fully functional dopaminergic neurons from an ontologically relevant stem cell type, hEPI-NCSC. [ABSTRACT FROM AUTHOR]

Posttranslational modifications (PTMs) of proteins by phosphorylation are a well-established mechanism by which their activities can be regulated to affect cellular physiology. However, it is becoming increasingly evident that PTMs of proteins by acetylation of lysine residues is also a key effecter in regulating their functional abilities. The best characterized case of this is the epigenetic effects of histone acetyltransferases and deacetylases on gene expression via modulation of nuclear histone acetylation and chromatin remodeling. However, recent published evidence now strongly implicates an important role for nonhistone acetylation in regulating cellular function. In this review, we have considered the potential for regulating myometrial activity not only by epigenetic mechanisms but also by nonepigenetic protein acetylation processes that could directly affect the contractile machinery within these smooth muscle cells. [ABSTRACT FROM AUTHOR]

Objective: Two-pore domain K+ channels (K2P), an emerging K+ channel subfamily, contribute to setting membrane potential in both electrically excitable and nonexcitable cells and, as such, influence cellularfunction. The multinucleate syncytiotrophoblast of human placenta, formedfrom thefusion of mononucleate cytotrophoblast cells, is a transporting epithelium whosefunction likely depends on the activity of K+ channels. We have therefore investigated the gene expression of two members of this family, TASK and TREK, in cultured human cytotrophoblast cells, and have also investigated protein expression in cytotrophoblast cells and placenta.Methods: We used reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry to investigate the gene and protein expression of TASK and TREK isoforms in both isolated cytotrophoblast cells and term placental tissue.Results: In cytotrophoblast cells, mRNAs encoding TASK1, 2, 4, 5, and TREK1 were detected, whereas weak or no signals were observedfor TASK3 and TREK2. Western blottingfor TASK1 in cytotrophoblast cells gave two bands of approximately 78 and 150 kd; TREK1 gave bands of approximately 90 and 130 kd. TASK1 immunofluorescence in placenta colocalized with cytokeratin-7, a trophoblast specific marker. TREK1 predominantly stained cells around the villous perimeter and this staining was colocalized with propidium iodide nuclear staining.Conclusion: Human cytotrophoblast cellsfrom term placenta are a site of expression for various K2p genes, two of which, namely, TASK1 and TREK1, are transcribed into protein. [ABSTRACT FROM PUBLISHER]

Measurements of simultaneous force and intracellular Ca concentration ([Ca]) in rat uterine smooth muscle have been made to elucidate the mechanisms involved when force produced spontaneously, by high-K depolarization or carbachol is altered by a change of intracellular pH (pH). Rises in force and [Ca] were closely correlated for all forms of contraction, with the Ca transient peaking before force. In spontaneously active preparations, alkalinization significantly increased, and acidification decreased, force and [Ca]. Inhibition of the sarcoplasmic reticulum ATPase (cyclopiazonic acid) did not affect these changes, whereas removal of external Ca abolished both responses, suggesting that the effect of pH is on Ca entry. Alkalinization caused a prolongation of the action potential complex, associated with a potentiation of contractile activity. Acidification produced hyperpolarization and abolition of action potentials and spontaneous activity, but did not prevent brief applications of carbachol or high-K from producing depolarization and increasing force, suggesting no impairment of the mechanism of generation of the action potential. For depolarized preparations, acidification increased tonic force and [Ca]; the increase in the calcium signal persisted in zero-external calcium. In the presence of carbachol, acidification transiently increased force and [Ca], followed by a reduction in both. It is concluded that changes in pH act at more than one step in excitation-contraction coupling and that changes in [Ca] can account for most of the changes in uterine force. [ABSTRACT FROM AUTHOR]

Although intracelluar pH (pH) is expected to change during inhibition of oxidative phosphorylation and to affect force, there have been no simultaneous measurements of its effect on pH and force in smooth muscle. Therefore, we have investigated the relationship between force and pH in strips of longitudinal rat myometrium, loaded with the pH-sensitive indicator carboxy-SNARF and simultaneously measured tension. The application of cyanide produced an abolition of spontaneous contractions and a rapid initial fall in pH. In 9/19 preparations pH then started to return to resting values but there was no corresponding restoration of force. Cyanide and weak base were simultaneously added to uterine preparations to prevent any acidification; force still fell. Addition of cyanide to depolarized preparations also produced an acidification and a fall in force. Depolarization of preparations in which spontaneous force had been abolished by cyanide often produced a transient rise in force, despite further acidification of the cytoplasm. Cyanide produced an acidification in zero Ca-containing solution, similar to that in the presence of Ca indicating little role for changes in [Ca] in producing the acidifiction. It is concluded that cyanide decreases pH and force in the uterus, but that there is not a simple relationship between the two. [ABSTRACT FROM AUTHOR]

The contractile pattern of uterine smooth muscle is markedly altered by even modest manipulations of intracellular pH (pH); an acidification can abolish contractions, while alkalinization increases contractility. As at the end of gestation the uterus changes from being relatively quiescent to highly contractile, we investigated whether pH changes with pregnancy in women. Isolated tissue was loaded with the pH-sensitive fluorophore, carboxy-SNARF. We found a significant alkalinization over the last few weeks of pregnancy. Such an increase in pH will increase contractility of the uterus, and may therefore contribute to the mechanisms ensuring strong and efficient contractions occur during labour. [ABSTRACT FROM AUTHOR]

Changes in intracellular pH (pH) are thought to produce large changes in force production in the uterus. There have however, been no simultaneous measurements of pH and force in the uterus and therefore no direct information is available about the relation between the two. We have used carboxy-SNARF (a pH-sensitive fluorophore) in small strips of longitudinal myometrium and obtained simultaneous measurements of pH and force. SNARF did not alter contractile function, and continuous measurements of pH could be made for 2 hours. The mean resting pH (7.16) was similar to that reported previously. Application of weak bases rapidly raised pH, in a concentration-dependent manner, followed by a gradual restoration of pH to resting levels. Alkalinization greatly increased the frequency of contractions, often accompanied by a small increase in their amplitude. Removal of base produced a rebound acidification which transiently abolished contractions. Direct acidification of the cytoplasm, by application of weak acid, also abolished contractions. However the alkalinization which accompanied removal of acid, produced variable effects on force. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the editor discusses various reports within the issue on topics including the pregnancy in terms of progressive changes of tissue remodeling under localized inflammatory control,, mechanisms for the timing of parturition, and instances of pre-term birth (PTB).