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The skeletal muscle regulates glucose homeostasis. Here, the effects of vitamin A metabolites including retinoic acid (RA) alone, and in combination with insulin, on glucose utilization were investigated in rat L6 muscle cells during the differentiation process. L6 cells were treated with differentiation medium containing retinol, retinal, RA, and (or) insulin. The glucose levels and pH values in the medium were measured every 2 days. The expression levels of insulin signaling and glycogen synthesis proteins, as well as glycogen content were determined. Retinal and RA reduced the glucose content and pH levels in the medium of the L6 cells. RA acted synergistically with insulin to reduce glucose and pH levels in the medium. The RA- and insulin-mediated reduction of glucose in the medium only occurred when glucose levels were at or above 15 mmol/L. Insulin-induced phosphorylation of Akt Thr308 was further enhanced by RA treatment through the activation of retinoic acid receptor. RA acted synergistically with insulin to phosphorylate glycogen synthase kinase 3[beta], and dephosphorylate glycogen synthase (GS), which was associated with increases in the protein and mRNA levels of GS. Increases in glycogen content were induced by insulin, and was further enhanced in the presence of RA. We conclude that activation of the RA signaling pathway enhanced insulin-induced glucose utilization in differentiating L6 cells through increases in glycogenesis. Key words: L6 cell differentiation, glucose, retinoic acid, insulin, glycogen synthase. Le muscle squelettique regule l'homeostasie du glucose. Ici, les effets de metabolites de la vitamine A, dont l'acide retinoique (AR), combines a l'insuline sur l'utilisation du glucose ont ete examines dans les cellules musculaires de rat L6 durant le processus de differenciation. Les cellules L6 ont ete traitees avec un milieu de differenciation comportant du retinol, du retinal, de l'AR en presence ou non d'insuline. Les niveaux de glucose et le pH du milieu ont ete mesures tous les deux jours. Les niveaux d'expression des proteines de signalisation de l'insuline et de synthese du glycogene, et le contenu en glycogene ont ete determines. Le retinal et l'AR reduisaient le contenu en glucose et le pH du milieu des cellules L6. L'AR agissait en synergie avec l'insuline pour reduire les niveaux de glucose et le pH du milieu. La reduction du glucose du milieu mediee par l'AR et l'insuline survenait uniquement lorsque sa concentration etait de 15 mmol/L ou plus. La phosphorylation de la Thr308 de l'Akt induite par l'insuline etait accrue par le traitement a l'AR par l'intermediaire de l'activation du recepteur d'acide retinoique. L'AR agissait en synergie avec l'insuline pour phosphoryler la glycogene synthase kinase 3[beta] et dephosphoryler la glycogene synthase (GS), ce qui etait associe a une augmentation des niveaux de GS et de son ARNm. Le contenu en glycogene etait regule par l'insuline, et il etait accru de maniere plus importante par la presence d'AR. Les auteurs concluent que l'activation de la voie de signalisation de l'AR a augmente l'utilisation du glucose induite par l'insuline par les cellules L6 en differenciation par l'intermediaire de l'accroissement de la glycogenese. [Traduit par la Redaction] Mots-cles: differenciation des cellules L6, glucose, acide retinoique, insuline, glycogene synthase., Introduction In 2017, the International Diabetes Federation (IDF) estimated that the worldwide incidence of diabetes in adults (20-79 years) was 481 million (Cho et al. 2018). To prevent further increase [...]

The main goal of this study was to develop a straightforward and rapid microplate assay for measuring propidium iodide (PI) in C2C12 cells. The PI method has proven to be an efficient quantitative assay for analyzing cell viability through PI fluorescence analysis. Importantly, the protocol takes less than 30 min and the results are reproducible. C2C12 cells were exposed to an increasing concentration of palmitate for a period of 24 h to induce cell death, and the PI fluorescence increased in a concentration-dependent manner. Evaluation of mitochondrial function and the production of reactive oxygen species confirmed the deleterious effects of palmitate. Also, the microplate PI assay demonstrated high sensitivity, as indicated by the detection of modest fluctuations in cell viability in response to catalase overexpression in palmitate-treated cells. The microplate PI assay, therefore, offers an accurate method for use in in-vitro studies. Key words: cell death, propidium iodide, skeletal muscle cell. L'objectif principal de cette etude etait de developper un dosage simple et rapide sur microplaques pour mesurer la presence d'iodure de propidium (IP) dans les cellules C2C12. La methode utilisant l'IP s'avere etre un dosage quantitatif efficace pour analyser la viabilite cellulaire par l'analyse de la fluorescence de l'IP. Fait important, le protocole prend moins de 30 minutes a completer et les resultats sont reproductibles. Les cellules C2C12 ont ete exposees a des concentrations croissantes de palmitate pendant une periode de 24 h afin de provoquer la mort cellulaire; la fluorescence emanant de l'IP augmentait de maniere dependante de la concentration. L'evaluation de la fonction mitochondriale et la production de derives reactifs d'oxygene confirmaient les effets dommageables du traitement au palmitate. Le dosage sur microplaque demontrait aussi une haute sensibilite comme l'indiquait la detection de fluctuations modestes de la viabilite cellulaire en reponse a une surexpression de catalase par les cellules traitees au palmitate. Le dosage d'IP sur microplaques offre ainsi un methode fiable qui peut etre utilisee lors d'etudes in vitro. [Traduit par la Redaction] Mots-cles : mort cellulaire, iodure de propidium, cellule musculaire squelettique., Introduction Accurate and reliable methods for assessing cellular viability is a crucial requirement for in-vitro studies. Currently, there is a wide range of assays that can be used for estimating [...]

The interaction between vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) plays an important role in the modulation of vascular tone. There is, however, no information on whether direct physical communication regulates the intracellular calcium levels of human VECs (hVECs) and (or) human VSMCs (hVSMCs). Thus, the objective of the study is to verify whether co-culture of hVECs and hVSMCs modulates cytosolic ([[[Ca.sup.2+]].sub.c]) and nuclear calcium ([[[Ca.sup.2+]].sub.n]) levels via physical contact and (or) factors released by both cell types. Quantitative 3D confocal microscopy for [[[[Ca.sup.2+]].sub.c] and [[[[Ca.sup.2+]].sub.n] measurement was performed in cultured hVECs or hVSMCs or in co-culture of hVECs-hVSMCs. Our results show that: (1) physical contact between hVECs-hVECs or hVSMCs-hVSMCs does not affect [[[[Ca.sup.2+]].sub.c] and [[[[Ca.sup.2+]].sub.n] in these 2 cell types; (2) physical contact between hVECs and hVSMCs induces a significant increase only of [[[[Ca.sup.2+]].sub.n] of hVECs without affecting the level of [[[[Ca.sup.2+]].sub.c] and [[[Ca.sup.2+]].sub.n] of hVSMCs; and (3) preconditioned culture medium of hVECs or hVSMCs does not affect [[[[Ca.sup.2+]].sub.c] and [[[[Ca.sup.2+]].sub.n] of both types of cells. We concluded that physical contact between hVECs and hVSMCs only modulates [[[[Ca.sup.2+]].sub.n] in hVECs. The increase of [[[[Ca.sup.2+]].sub.n] in hVECs may modulate nuclear functions that are calcium dependent. Key words: vascular endothelial cells, vascular smooth muscle cells, co-culture, nuclear calcium, calcium release. L'interaction entre les cellules endotheliales vasculaires (CEV) et les cellules musculaires lisses vasculaires (CMLV) joue un role important dans la modulation du tonus vasculaire. Il n'existe cependant aucune information quant aux effets de leur communication physique directe sur la regulation des concentrations intracellulaires de calcium dans les CEV humaines (CEVh) ou les CMLV humaines (CMLVh). Par consequent, l'etude avait pour but de verifier si la co-culture de CEVh avec des CMLVh entraine une modulation des concentrations de calcium cytosolique ([[[[Ca.sup.2+]].sub.c]) et nucleaire ([[[[Ca.sup.2+]].sub.n]) par l'intermediaire d'un contact physique et (ou) de facteurs liberes par les deux types de cellules. A l'aide de la microscopie confocale quantitative en 3D, nous avons mesure les [[[[Ca.sup.2+]].sub.c] et les [[[[Ca.sup.2+]].sub.n] dans les CEVh et les CMLVh en culture ou en co-culture les unes avec les autres (CEVh-CMLVh). Nos resultats montrent les choses suivantes: (1) Le contact physique entre CEVh-CEVh ou CMLVh-CMLVh n'a pas d'effet sur les [[[[Ca.sup.2+]].sub.c] ni sur les [[[[Ca.sup.2+]].sub.n] dans ces deux types cellulaires; (2) Le contact physique entre CEVh-CMLVh entraine une augmentation marquee uniquement des [[[[Ca.sup.2+]].sub.n] dans les CEVh, sans affecter les [[[[Ca.sup.2+]].sub.c] ni les [[[[Ca.sup.2+]].sub.n] dans les CMLVh; et (3) Le milieu de culture preconditionne des CEVh ou des CMLVh n'a aucun effet sur les [[[[Ca.sup.2+]].sub.c] ni les [[[[Ca.sup.2+]].sub.n] pour les deux types de cellules. Nous en sommes arrives a la conclusion que le contact physique entre les CEVh et les CMLVh entraine une modulation uniquement des [[[[Ca.sup.2+]].sub.n] dans les CEVh. Cette augmentation des [[[[Ca.sup.2+]].sub.n] dans les CEVh pourrait entrainer une modulation de fonctions nucleaires qui dependent du calcium. [Traduit par la Redaction] Mots-cles: cellules endotheliales vasculaires, cellules musculaires lisses vasculaires, co-culture, calcium nucleaire, liberation de calcium., Introduction Physical contact between the same type of cells involves different types of junctions such as tight junctions (Alberts 1994), adherence junctions (Alberts 1994), platelet-endothelial cell adhesion molecule (DeLisser et [...]

Although plasma levels of lysophosphatidylglycerol (LPG) are increased in hypertension, its role in the pathogenesis of vascular defects is not clear. In view of the importance of [Ca.sup.2+] overload in causing vascular smooth muscle (VSM) dysfunction, the action of LPG on [[[Ca.sup.2+]].sub.i] in cultured A10 VSM cell line was examined by using Fura 2-AM acetoxymethyl ester technique. LPG was found to induce a concentration-dependent increase in [[[Ca.sup.2+]].sub.i] in VSM cells. This change was dependent both on the extracellular and intracellular [Ca.sup.2+] sources, as it was reduced by 30% by EGTA, an extracellular [Ca.sup.2+] chelator, and 70% by thapsigargin, a sarcoplasmic reticulum (SR) [Ca.sup.2+]-pump inhibitor. However the increase in [[[Ca.sup.2+]].sub.i] due to LPG was not altered by caffeine or ryanodine, which affect [Ca.sup.2+]-release through the ryanodine receptors in the SR. On the other hand, LPG-induced change in [[[Ca.sup.2+]].sub.i] was suppressed by 2-nitro-4-carboxyphenyl N,N- diphenylcarbamate, a phospholipase C (PLC) inhibitor, as well as by xestospongin and 2-aminoethoxydiphenyl borate, two inositol trisphosphate (IP3) receptor inhibitors in the SR. These observations support the view that LPG-induced increase in [[[Ca.sup.2+]].sub.i] in VSM cells is mainly a result of [Ca.sup.2+] release from [Ca.sup.2+] pool in the SR through PLC/IP3-sensitive signal transduction mechanism. Furthermore, it is suggested that the elevated level of LPG may induce intracellular [Ca.sup.2+] overload and thus play a critical role in the development of vascular abnormalities. Key words: lysophosphatidylglycerol, vascular smooth muscle cells, intracellular calcium, sarcoplasmic reticulum, vascular abnormalities. Meme si les taux plasmatiques de lysophosphatidylglycerol (LPG) augmentent en cas d'hypertension, son role dans la pathogenese des defectuosites vasculaires demeure a clarifier. Etant donne l'importance de la surcharge en ions [Ca.sup.2+] dans l'apparition d'un dysfonctionnement du muscle lisse vasculaire (MLV), nous avons etudie l'action du LPG sur les concentrations intracellulaires de [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) de lignees cellulaires A10 de MLV en culture a l'aide de la technique de Fura 2-AM acetoxymethyl ester. Nous avons observe que le LPG entraine une augmentation--proportionnelle a la concentration--de [[[Ca.sup.2+]].sub.i] dans les cellules de MLV. Cette variation etait dependante des sources extracellulaires comme intracellulaires de [Ca.sup.2+], car celles-ci diminuaient de 30 % avec l'EGTA, un chelateur du [Ca.sup.2+] extracellulaire, et de 70 % avec la thapsigargine, un inhibiteur de la pompe a [Ca.sup.2+] dans le reticulum sarcoplasmique (RS). Cependant, l'augmentation de [[[Ca.sup.2+]].sub.i] qu'entraine le LPG n'etait pas modifiee par la cafeine ni par la ryanodine, lesquelles ont des effets sur la liberation du [Ca.sup.2+] par l'intermediaire des recepteurs de la ryanodine dans le RS. Par contre, les variations de la [[[Ca.sup.2+]].sub.i] qu'entraine le LPG etaient inhibees par le 2-nitro- 4-carboxyphenyl N,N-diphenylcarbamate, un inhibiteur de la phospholipase C (PLC), ainsi que par la xestospongine et le 2 aminoethoxydiphenyl borate, deux inhibiteurs des recepteurs de l'inositol trisphosphate (IP3) dans le RS. Ces observations viennent appuyer la notion selon laquelle l'augmentation de [[[Ca.sup.2+]].sub.i] qu'entraine le LPG dans les cellules du MLV provient principalement de la liberation de [Ca.sup.2+] du<>de [Ca.sup.2+] dans le RS qui suit un mode d'action dependant de la transduction de signaux sensible au systeme PLC/IP3. De plus, il est pressenti que l'augmentation des taux de LPG pourrait entrainer une augmentation de la surcharge en ions [Ca.sup.2+] et, par le fait meme, jouer un role crucial dans la formation d'anomalies vasculaires. [Traduit par la Redaction] Mots-cles: lysophosphatidylglycerol, cellules musculaires lisses vasculaires, calcium intracellulaire, reticulum sarcoplasmique, anomalies vasculaires., Introduction Intracellular free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) plays an important role in the regulation of cardiovascular function, cell growth, and apoptosis under both physiological and pathological conditions (Dhalla et al. 1982; [...]

Triton X-100 (TX-100) is a nonionic detergent frequently used at millimolar concentrations to disrupt cell membranes and solubilize proteins. At low micromolar concentrations, TX-100 has been reported to inhibit the function of potassium channels. Here, we have used electrophysiological and functional techniques to examine the effects of TX-100 on another class of ion channels, L-type voltage-operated calcium channels (VOCCs). TX-100 (30 nmol-L-1 to 3 µmol x [L.sup.-1]) caused reversible concentration-dependent inhibition of recombinant L-type VOCC ([Ca.sub.V] 1.2) currents and of native L-type VOCC currents recorded from rat vascular smooth muscle cells and cardiac myocytes, and murine and human pancreatic β-cells. In functional studies, TX-100 (165 nmol x [L.sup.-1] to 3.4 µmol x [L.sup.-1]) caused concentration-dependent relaxation of rat isolated mesenteric resistance arteries prestimulated with phenylephrine or KCl. This effect was independent of the endothelium. TX-100 (1.6 µmol x [L.sup.-1]) inhibited depolarization-induced exocytosis in both murine and human isolated pancreatic β-cells. These data indicate that at concentrations within the nanomolar to low micromolar range, TX-100 significantly inhibits L-type VOCC activity in a number of cell types, an effect paralleled by inhibition of cell functions dependent upon activation of these channels. This inhibition occurs at concentrations below those used to solubilize proteins and may compromise the use of solutions containing TX-100 in bioassays. Key words: Triton X-100, voltage-operated calcium channel, vascular smooth muscle, cardiac myocyte, pancreatic β- cell. Le Triton X-100 (TX-100) est un detergent non ionique frequemment utilise a des concentrations millimolaires afin de desorganiser les membranes cellulaires et solubiliser les proteines. On rapporte qu'a de faibles concentrations millimolaires, le TX-100 inhibe la fonction des canaux potassiques. Nous avons utilise ici des techniques electro-physiologiques et fonctionnelles afin d'examiner les effets du TX-100 surun autre type de canaux ioniques, les canaux calciques dependants du voltage (CCDV) de type L. Le TX-100 (30 nmol x [L.sup.-1] -3 [micron]mol x [L.sup.-1]) provoquait une inhibition reversible et dependante de la concentration des courants du CCDV de type L recombinant [Ca.sub.V] 1.2 et des courants du CCDV de type L sauvage, enregistres a partir de cellules vasculaires musculaires lisses et de myocytes cardiaques ainsi que de cellules β du pancreas de souris et humaines. Lors des etudes fonctionnelles, le TX-100 (165 nmol x [L.sup.-1] - 3.4 [micron]mol x [L.sup.-1]) provoquait une relaxation dependante de la concentration des arteres mesenteriques isolees de rat, stimulees prealablement a la phenylephrine ou au KCl. Cet effet etait independant de l'endothelium. Le TX-100 (1.6 [micron]mol x [L.sup.-1]) inhibait l'exocytose induite par la depolarisation chez les cellules p du pancreas de souris et humaines. Ces donnees indiquent qu'a des concentrations allant du nanomolaire au micromolaire faible, le TX-100 inhibe significativement l'activite des CCDV de type L chez plusieurs cellules, un effet qui coincide avec l'inhibition des fonctions cellulaires dependantes de l'activation de ces canaux. Cette inhibition survient a des concentrations inferieures a celles utilisees pour solubiliser les proteines, ce qui peut compromettre l'utilisation de solutions contenant du TX-100 dans les tests biologiques. [Traduit par la Redaction] Mots-cles: Triton X-100, canal calcique dependant du voltage, muscle vasculaire lisse, myocytes cardiaques, cellules β du pancreas., Introduction Triton X-100 (TX-100; [C.sub.14][H.sub.22]O[([C.sub.2][H.sub.4]O).sub.n]) is a nonionic detergent commonly used at millimolar concentrations for the purification of membrane proteins (Brutsaert et al. 1988; Mink et al. 2005; Maurice et [...]

9:00 VIBRIO CHOLERA GHOST ENHANCE CHLAMYDIA-SPECIFIC IMMUNE RESPONSES VIA THP-1 CELL ACTIVATION **, April Stevens (1), (2), Roshan Pais (2), Francis Eko (2), Qing He (1) and Godwin Ananaba (1), [...]

The results of our study show that whole-cell and nuclear levels of NADPH oxidase-1 (NOX1) are similar in human vascular endothelial cells (hVECs) and smooth muscle cells (hVSMCs), but lower in human endocardial endothelial cells (hEECs). NOX2 levels were higher in hVECs and lower in hVSMCs. NOX3 levels were the same in hVECs and hVSMCs, but lower in hEECs. NOX4 levels were similar in all of the cell types. NOX4 levels were higher in hVECs than in hVSMCs. NOX5 was also present throughout the 3 cell types, including their nuclei, in the following order: hEECs > hVSMCs > hVECs. The level of basal reactive oxygen species (ROS) was highest in hVECs and lowest in hVSMCs. However, the [Ca.sup.2+] level was highest in hVSMCs and lowest in hVECs. These findings suggest that all types of NOXs exist in hEECs, hVECs, and hVSMCs, although their density and distribution are cell-type dependent. The density of the different NOXs correlated with the ROS level, but not with the [Ca.sup.2+] level. In conclusion, NOXs, including NOX3, exist in cardiovascular cells and their nuclei. The nucleus is a major source of ROS generation. The nuclear NOXs may contribute to ROS and [Ca.sup.2+] homeostasis, which may affect cell remodeling, including the formation of nuclear T-tubules in vascular diseases and aging. Key words: NOX, [Ca.sup.2+], ROS, human endocardial endothelial cells, human vascular endothelial cells, human vascular smooth muscle cells, nuclear GPCR, nuclear [Ca.sup.2+], confocal microscopy, free radicals. Nos resultats ont montre que les niveaux de NADPH oxydase-1 (NOX1) dans la cellule entiere par rapport au noyau sont similaires chez les cellules endotheliales vasculaires (hCEV) et les cellules de muscle lisse vasculaires (hCMLV) humaines, mais plus faibles chez les cellules endotheliales endocardiques (hCEE) humaines. Le niveau de NOX2 etait plus eleve chez les hCEV et plus faible chez les hCMLV. Le niveau de NOX3 etait plus eleve chez les hCEV et plus faible chez les hCEE. Les niveaux de NOX4 etaient similaires chez tous les types de cellules. Ces niveaux etaient plus eleves chez les hCEV que chez les hCMLV. NOX5 etait aussi presente dans les trois types cellulaires incluant dans leur noyau selon l'ordre suivant : hCEE > hCMLV > hCEV. Le niveau basal d'ERO le plus eleve se trouvait chez les hCEVet le plus faible chez les hCMLV. Cependant, le niveau de [Ca.sup.2+] le plus eleve se trouvait chez les hCMLV et le plus faible chez les hCEV. Ces donnees suggerent que tous les types de NOX existent chez les hCEE, les hCEV et les hCMLV, toutefois, leur densite et leur distribution dependent du type cellulaire. La densite des differentes NOX correlait avec le niveau d'ERO mais pas avec le niveau de [Ca.sup.2+]. En conclusion, les NOX, incluant NOX3 existent chez les cellules cardiovasculaires et dans leurs noyaux. Le noyau est la principale source d'ERO. Les NOX nucleaires peuvent contribuer a l'homeostasie des ERO et du [Ca.sup.2+], ce qui peut affecter le remodelage cellulaire incluant la formation des tubules T nucleaires dans les maladies vasculaires et lors du vieillissement. [Traduit par la Redaction] Mots-cles : NOX, [Ca.sup.2+], ERO, cellules endotheliales endocardiques humaines, cellules endotheliales vasculaires humaines, cellules de muscle lisse vasculaires humaines, GPCR nucleaire, [Ca.sup.2+] nucleaire, microscopie confocale, radicaux libres., Introduction In the vasculature, superoxide and other reactive oxygen species (ROS) can be derived from several sources (Lassegue and Clempus 2003; Bedard and Krause 2007; Williams and Griendling 2007; Petry [...]

Metformin is known to improve insulin sensitivity in part via a rise in AMP-activated protein kinase (AMPK) activity and alterations in muscle metabolism. However, a full understanding of how metformin alters AMPK-[[alpha].sub.1] vs. AMPK-[[alpha].sub.2] activation remains unknown. To study this question, L6 skeletal muscle cells were treated with or without RNAi oligonucleotide sequences to downregulate AMPK-[[alpha].sub.1] or AMPK-[[alpha].sub.2] protein expression and incubated with or without 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribofuranoside (AICAR) or metformin and/or insulin. In contrast to AICAR, which preferentially activated AMPK-[[alpha].sub.2], metformin preferentially activated AMPK-[[alpha].sub.1] in a dose- and time-dependent manner. Metformin increased (P < 0.05) glucose uptake and plasma membrane (PM) Glut4 in a dose- and time-dependent manner. Metformin significantly reduced palmitate uptake (P < 0.05) and oxidation (P < 0.05), and this was accompanied by a similar decrease (P < 0.05) in PM CD36 content but with no change in acetyl-CoA carboxylase (ACC) phosphorylation (P > 0.05). AICAR and metformin similarly increased (P < 0.05) nuclear silent mating-type information regulator 2 homolog 1 (SIRT1) activity. Downregulation of AMPK-[[alpha].sub.1] completely prevented the metformin-induced reduction in palmitate uptake and oxidation but only partially reduced the metformin-induced increase in glucose uptake. Downregulation of AMPK-[[alpha].sub.2] had no effect on metformin-induced glucose uptake, palmitate uptake, and oxidation. The increase in SIRT1 activity induced by metformin was not affected by downregulation of either AMPK-[[alpha].sub.1] or AMPK-[[alpha].sub.2]. Our data indicate that, in muscle cells, the inhibitory effects of metformin on fatty acid metabolism occur via preferential phosphorylation of AMPK-[[alpha].sub.1], and the data indicate that cross talk between AMPK and SIRT1 does not favor either AMPK isozyme. AMP-activated protein kinase; fatty acid; glucose uptake; 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribofuranoside, silent mating-type information regulator 2 homolog 1; fatty acid transporter/CD36; Glut4 doi: 10.1152/ajpcell.00279.2010.

Regulator of G protein signaling 4 (RGS4) regulates the strength and duration of G protein signaling and plays an important role in smooth muscle contraction, cardiac development, and psychiatric disorders. Little is known about the posttranscriptional regulation of RGS4 expression. We cloned the full-length cDNA of rabbit RGS4, which contains a long 3'-untranslated region (UTR) with several AU-rich elements (AREs). We determined whether RGS4 mRNA stability is mediated by the RNA-binding protein human antigen R (HuR) and contributes to IL-1[beta]-induced upregulation of RGS4 expression. We show that IL-1[beta] treatment in colonic smooth muscle cells doubled the half-life of RGS4 mRNA. Addition of RGS4 3'-UTR to the downstream of Renilla luciferase reporter induced dramatic reduction in the enzyme activity and mRNA expression of luciferase, which was attenuated by the site-directed mutation of the two 3'-most ARE sites. IL-1[beta] increased luciferase mRNA stability in a UTR-dependent manner. Knockdown of HuR significantly aggravated UTR-mediated instability of luciferase and inhibited IL-1[beta]-induced upregulation of RGS4 mRNA. In addition, IL-1[beta] increased cytosolic translocation and RGS4 mRNA binding of HuR. These findings suggest that 3'-most ARE sites within RGS4 3'-UTR are essential for the instability of RGS4 mRNA and IL-1[beta] promotes the stability of RGS4 mRNA through HuR. regulators of G protein signaling; Interleukin; AU-rich element doi: 10.1152/ajpcell.00093.2010.

Recent animal studies have reported that exercise pressor reflex (EPR)-mediated increases in blood pressure are exaggerated in hypertensive (HTN) rodents. Whether these findings can be extended to human hypertension remains unclear. Mean arterial pressure (MAP), muscle sympathetic nerve activity (MSNA), and venous metabolites were measured in normotensive (NTN; n = 23; 60 [+ or -] 1 yr) and HTN 01 = 15; 63 [+ or -] 1 yr) subjects at baseline, and during static handgrip at 30 and 40% maximal voluntary contraction (MVC) followed by a period of post-exercise ischemia (PEI) to isolate the metabolic component of the EPR. Changes in MAP from baseline were augmented in HTN subjects during both 30 and 40% MVC handgrip (P < 0.05 for both), and these group differences were maintained during PEI (30% PEI trial: [DELTA]15 [+ or -] 2 NTN vs. [DELTA]19 [+ or -] 2 HTN mmHg; 40% PEI trial: [DELTA]16 [+ or -] 1 NTN vs. [DELTA]23 [+ or -] 2 HTN mmHg; P < 0.05 for both). Similarly, in HTN subjects, MSNA burst frequency was greater during 30 and 40% MVC handgrip (P < 0.05 for both), and these differences were maintained during PEI [30% PEI trial: 35 [+ or -] 2 (NTN) vs. 44 [+ or -] 2 (HTN) bursts/min; 40% PEI trial: 36 [+ or -] 2 (NTN) vs. 48 [+ or -] 2 (HTN) bursts/min; P < 0.05 for both]. No group differences in metabolites were observed. MAP and MSNA responses to a cold pressor test were not different between groups, suggesting no group differences in generalized sympathetic responsiveness. In summary, compared with NTN subjects, HTN adults exhibit exaggerated sympathetic and pressor responses to handgrip exercise that are maintained during PEI, indicating that activation of the metabolic component of the EPR is augmented in older HTN humans. blood pressure regulation; exercise pressor reflex; muscle sympathetic nerve activity; static exercise; hypertension doi: 10.1152/aioheart.00556.2010.

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that cannonical transient receptor potential channel TRPCI and stromal interaction molecule 1 (STIM1) mediate the sustained component of capacitative [Ca.sup.2+] entry (CCE), but the molecular candidate(s) that mediate the transient component of CCE remain unknown. The aim of the present study was to examine whether Orai1 mediates the transient component of CCE through activation of STIM1 in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]].sub.i]). The transient but not the sustained rise in [[Ca.sup.2+].sub.i] was partially inhibited by nifedipine. The nifedipine-insensitive transient rise in [[Ca.sup.2+].sub.i] and the increase in [Mn.sup.2+] quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 small interfering (si)RNA. Moreover, overexpression of STIM1 enhanced the rise in [[Ca.sup.2+].sub.i] and the increase in [Mn.sup.2+] quench of fura-2 fluorescence caused by CPA, and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to coimmunoprecipitate with STIM1, and the precipitation level of Orai1 was increased in cells subjected to store-depletion. Immunostaining revealed colocalization of Orai1 and STIM1 proteins, and the colocalization of these proteins was more apparent after store-depletion. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel as a result of STIM1 activation in mouse PASMCs. stromal interaction molecule 1; store-depletion; intracellular [Ca.sup.2+] concentration doi: 10.1152/ajpcell.00548.2009.

Muscle contraction stimulates glucose uptake acutely to increase energy supply, but suitable cellular models that faithfully reproduce this complex phenomenon are lacking. To this end, we have developed a cellular model of contracting [C.sub.2][C.sub.12] myotubes overexpressing GLUT4 with an exofacial mycepitope tag (GLUT4myc) and explored stimulation of GLUT4 traffic by physiologically relevant agents. Carbachol (an acetylcholine receptor agonist) induced a gain in cell surface GLUT4myc that was mediated by nicotinic acetylcholine receptors. Carbachol also activated AMPK, and this response was sensitive to the contractile myosin ATPase inhibitor N-benzyl-p-toluenesulfonamide. The gain in surface GLUT4myc elicited by carbachol or by the AMPK activator 5-amino-4-carboxamide-1 [beta]-ribose was sensitive to chemical inhibition of AMPK activity by compound C and partially reduced by siRNA-mediated knockdown of AMPK catalytic subunits or LKB1. In addition, the carbachol-induced gain in cell surface GLUT4myc was partially sensitive to chelation of intracellular calcium with BAPTA-AM. However, the carbachol-induced gain in cell surface GLUT4myc was not sensitive to the CaMKK inhibitor STO-609 despite expression of both isoforms of this enzyme and a rise in cytosolic calcium by carbachol. Therefore, separate AMPK- and calcium-dependent signals contribute to mobilizing GLUT4 in response to carbachol, providing an in vitro cell model that recapitulates the two major signals whereby acute contraction regulates glucose uptake in skeletal muscle. This system will be ideal to further analyze the underlying molecular events of contraction-regulated GLUT4 traffic. carbachol; glucose transporter 4; adenosine 5'-monophosphate-activated protein kinase; acetylcholine doi:10.1152/ajpendo.00773.2009.

One proposed explanation for the V[O.sub.2] slow component is that lower-threshold motor units may fatigue and develop little or no tension but continue to use [O.sub.2], thereby resulting in a dissociation of cellular respiration from force generation. The present study used intact isolated single myocytes with differing fatigue resistance profiles to investigate the relationship between fatigue, tension development, and aerobic metabolism. Single Xenopus skeletal muscle myofibers were allocated to a fast-fatiguing (FF) or a slow-fatiguing (SF) group, based on the contraction frequency required to elicit a fall in tension to 60% of peak. Phosphorescence quenching of a porphyrin compound was used to determine [DELTA] intracellular [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]; a proxy for V[O.sub.2]), and developed isometric tension was monitored to allow calculation of the time-integrated tension (TxT). Although peak [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]: was not different between groups (P = 0.36), peak tension was lower (P < 0.05) in SF vs. FF (1.97 [+ or -] 0. 17 V vs. 2.73 [+ or -] 0.30 V, respectively) and time to 60% of peak tension was significantly longer in SF vs. FF (242 [+ or -] 10 s vs. 203 [+ or -] l0 s, respectively). Before fatigue, both[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and TxT rose proportionally with contraction frequency in SF and FF, resulting in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]/TxT being identical between groups. At fatigue, TxT fell dramatically in both groups, but [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] decreased proportionately only in the FF group, resulting in an increase in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]/TxT in the SF group relative to the prefatigue condition. These data show that more fatigue-resistant fibers maintain aerobic metabolism as they fatigue, resulting in an increased [O.sub.2] cost of contractions that could contribute to the Vo2 slow component seen in whole body exercise. oxidative phosphorylation; skeletal muscle; fiber type; oxygen uptake doi: 10.1152/ajpregu.00715.2009.

Electroporation is a technique used in vitro, ex vivo, and in vivo to permeabilize cell membranes. The effect on the tissue describes a continuum ranging from mild perturbations to massive tissue damage. Thus care should be taken when choosing pulses for a given application. Here the effects of electroporation paradigms ranging from severe to very gentle permeabilization were investigated on soleus, mainly composed of slow-twitch fibers, and extensor digitorum longus (EDL) and tibialis anterior (TA), almost exclusively composed of fast-twitch fibers. Five key physiological parameters were studied: force, muscle [Na.sup.+], [K.sup.+], and [Ca.sup.2+] content, and plasma lactate dehydrogenase activity. Four-week-old Wistar rats were anesthetized, and the lower part of the hind leg was electroporated. Blood samples were collected from the tail vein, and at the times indicated animals were killed and TA, EDL, and soleus muscles were collected for analysis of force and ion contents. Muscles were given eight high-voltage pulses of 100-[micro]s duration (8HV) at varying field intensity, one short high-voltage pulse combined with one long low-voltage pulse (HVLV), or eight medium-voltage pulses of 20-ms duration (8MV). Intensity of the electrical field strength was determinant for the degree of changes observed in the muscle. Field strengths below 300 V/cm did not give rise to measurable changes, whereas 8HV pulses at high field intensities (1,200 V/cm) caused severe and long-lasting damage to the muscle. Interestingly, the damage was more pronounced in EDL and TA compared with soleus, possibly because of the difference in fiber type composition. HVLV only caused temporary changes, with force and ion content being normalized by 4 h, suggesting that this pulse combination may be useful for the introduction of ions and molecules (e.g., DNA) into muscle cells. electropermeabilization; extensor digitorum longus; tibialis anterior; soleus; fiber type doi: 10.1152/ajpregu.00692.2009.

Nitric oxide signaling has an important role in regulating pulmonary development and function. Expression of soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase I (PKGI), both critical mediators of nitric oxide (NO) signaling, is diminished in the injured newborn lung through unknown mechanisms. Recent studies suggest that excessive transforming growth factor-[beta] (TGF-[beta]) activity inhibits injured newborn lung development. To explore mechanisms that regulate pulmonary NO signaling, we tested whether TGF-[beta] decreases sGC and PKGI expression in the injured developing lung and pulmonary vascular smooth muscle cells (SMC). We found that chronic oxygen-induced lung injury decreased pulmonary sGC[[alpha].sub.1] and PKGI immunoreactivity in mouse pups and that exposure to a TGF-[beta]-neutralizing antibody prevented this reduction of sGC and PKGI protein expression. In addition, TGF-[[beta].sub.1] decreased expression of NO signaling enzymes in freshly isolated pulmonary microvascular SMC/ myofibroblasts, suggesting that TGF-[beta] has a direct role in modulating NO signaling in the pup lung. Moreover, TGF-[[beta].sub.1] decreased sGC and PKGI expression in pulmonary artery and aortic SMC from adult rats and mice, suggesting a general role for TGF-[beta] in modulating NO signaling in vascular SMC. Although other cytokines decrease sGC mRNA stability, TGF-[beta] did not modulate sGC[[alpha].sub.1] or PKGI[beta] mRNA turnover in vascular SMC. These studies indicate for the first time that TGF-[beta] decreases NO signaling enzyme expression in the injured developing lung and pulmonary vascular SMC. Moreover, they suggest that TGF-[beta]-neutralizing molecules might counteract the effects of injury on NO signaling in the newborn lung. soluble guanylate cyclase; cGMP-dependent protein kinase I; bronchopulmonary dysplasia; hyperoxia doi:10.1152/ajplung.00181.2009.

Vasomotion consists of cyclic arterial diameter variations induced by synchronous contractions and relaxations of smooth muscle cells. However, the arteries do not contract simultaneously on macroscopic distances, and a propagation of the contraction can be observed. In the present study, our aim was to investigate this propagation. We stimulated endothelium-denuded rat mesenteric arterial strips with phenylephrine (PE) to obtain vasomotion and observed that the contraction waves are linked to intercellular calcium waves. A velocity of -100 [micro]/s was measured for the two kinds of waves. To investigate the calcium wave propagation mechanisms, we used a method allowing a PE stimulation of a small area of the strip. No calcium propagation could be induced by this local stimulation when the strip was in its resting state. However, if a low PE concentration was added on the whole strip, local PE stimulations induced calcium waves, spreading over finite distances. The calcium wave velocity induced by local stimulation was identical to the velocity observed during vasomotion. This suggests that the propagation mechanisms are similar in the two cases. Using inhibitors of gap junctions and of voltage-operated calcium channels, we showed that the locally induced calcium propagation likely depends on the propagation of the smooth muscle cell depolarization. Finally, we proposed a model of the propagation mechanisms underlying these intercellular calcium waves. conducted vasomotor response; smooth muscle cell; rat mesenteric artery doi: 10.1152/ajpheart.00281.2009

The mechanisms by which mitochondria regulate the sustained phase of agonist-induced responses in cytosolic [Ca.sup.2+] concentration as an independent organelle in whole is not clear. By exposing to ethidium bromide and supplying pyruvate and uridine, we established mitochondrial DNA (mtDNA)-depleted rat airway smooth muscle cells (RASMCs) with maintained cellular energy. Upon an exposure to 2 [mu]M histamine, [[[Ca.sup.2+].sub.i] in control RASMCs increased to a peak followed by a plateau above baseline, whereas [[[Ca.sup.2+]].sub.i] in mtDNA-depleted RASMCs jumped to a peak and then declined to baseline without any plateau, mtDNA depletion apparently attenuated intracellular reactive oxygen species generation induced by histamine. By coexposure to 2 [mu]M histamine and 0.1 [mu]M exogenous [H.sub.2][O.sub.2], which did not affect [[[Ca.sup.2+]].sub.i] by itself, the above difference in [[[Ca.sup.2+]].sub.i] kinetics in mtDNA-depleted RASMCs was reversed. Intracellular [H.sub.2][O.sub.2] decomposition abolishes histamine-induced sustained elevation in [[[Ca.sup.2+]].sub.i] in RASMCs. Thus, mitochondria regulate agonist-induced sustained [[[Ca.sup.2+]].sub.i] elevation by a [H.sub.2][O.sub.2]-dependent mechanism. histamine; calcium; reactive oxygen species doi: 10.1152/ajplung.00134.2009

Am J Physiol Gastrointest Liver Physiol 298: G10-G13, 2010. First published November 5, 2009; doi: 10.1152/ajpgi.00426.2009.--How nerves transmit their signals to regulate activity of smooth muscle is of fundamental importance to autonomic and enteric physiology, clinical medicine, and therapeutics. A traditional view of neurotransmission to smooth muscles has been that motor nerve varicosities release neurotransmitters that act on receptors on smooth muscles to cause their contraction or relaxation via electromechanical and pharmacomechanical signaling pathways in the smooth muscle. In recent years, an old hypothesis that certain interstitial cells of Cajal (ICC) may transduce neural signals to smooth muscle cells has been resurrected. This later hypothesis is based on indirect evidence of closer proximity and presence of synapses between the nerve varicosities and ICC, gap junctions between ICC and smooth muscles, and presence of receptors and signaling pathways for the neurotransmitters and ICC. This indirect evidence is at best circumstantial. The direct evidence is based on the reports of loss of neurotransmission in mutant animals lacking ICC due to c-Kit receptor deficiency. However, a critical analysis of the recent data show that animals lacking ICC have normal cholinergic and purinergic neurotransmission and tachykinergic neurotransmission is actually increased. The status of nitrergic neurotransmission in c-Kit deficient animals has been controversial. However, reports suggest that 1) nitrergic neurotransmission in the internal anal sphincter does not require ICC and 2) the in vivo phenotype of ICC deficiency does not resemble nNOS deficiency. 3) The most recent report, in this issue of the Journal, concludes that impaired nitrergic neurotransmission may be due to smooth muscle defects associated with c-Kit receptor deficiency. bile gastritis; gastroparesis; intestinal phenotype of Ws/Ws; nitrergic neurotransmission

Am J Physiol Gastrointest Liver Physiol 298: G14--G24, 2010. First published October 22, 2009; doi: 10.1152/ajpgi.00266.2009.--To address the controversy surrounding the role of interstitial cells of Cajal (ICC) in nitrergic neurotransmission to gastrointestinal smooth muscle, circular smooth muscle from the lower esophageal sphincter (LES) of W/[W.sup.v] wild-type and mutant (ICC-deficient) mice were studied by using intracellular and tension recordings in vitro. Resting membrane potential was more negative, and the spontaneous unitary potentials diminished in mutant mice. In wild-type mice, nerve stimulation induced a biphasic inhibitory junction potential (IJP) consisting of a fast initial IJP followed by a long-lasting slow IJP (LSIJP). The IJP was markedly impaired in a significant proportion of mutant mice, whereas in others it was normal. Pharmacological studies in the mice with markedly impaired LIPs revealed that cholinergic and purinergic components of the nerve-mediated responses appeared intact. In wild-type mice, caffeine hyperpolarized smooth muscle cells, inhibited the initial fast IJP, and completely abolished the LSIJP. In mutant mice, caffeine depolarized smooth muscle cells and abolished the impaired LSIJP but did not affect the initial fast IJP. Immunohistochemical staining for c-Kit confirmed deficiency of ICC in mutant mice with a normal nitrergic IJP. Rings of LES circular smooth muscle from W/[W.sup.v] mutant mice generated significantly less spontaneous tone than controls. When tone was restored with carbachol, normal nitrergic LES relaxation was recorded. These data suggest that 1) there is significant variability in the generation of nitrergic neurotransmission in the LES of W/[W.sup.v] mutant mice, whereas purinergic and cholinergic neurotransmission are intact; 2) the altered nitrergic responses appear to be associated with abnormal [Ca.sup.2+]-dependent signaling initiated by spontaneous [Ca.sup.2+] release from sarcoplasmic reticulum in smooth muscle cells; and 3) c-Kit-positive ICC are not essential for nitrergic neurotransmission in mouse LES smooth muscle. smooth muscle; [Ca.sup.2+]-activated [Cl.sup.-] channels; sarcoplasmic reticulum; interstitial cells of Cajal

Am J Physiol Cell Physiol 298: C46-C55, 2010. First published November 4, 2009; doi:10.1152/ajpcell.00387.2009.--Acheron (Achn) was originally identified as novel gene that is induced when insect muscles become committed to die at the end of metamorphosis. In separate studies, we have demonstrated that Achn acts upstream of MyoD and is required by mammalian myoblasts to either differentiate or undergo apoptosis following loss of growth factors. In the present study we examined the role of Achn in regulating integrin-extracellular matrix interactions that are required for myogenesis. Both control [C.sub.2][C.sub.12] myoblasts and those engineered to express ectopic Achn expressed the fibronectin receptor integrin [[alpha].sub.5][[beta].sub.1] in the presence of growth factors and the laminin receptor [[alpha].sub.7][[beta].sub.1] following growth factor withdrawal. Expression of the laminin receptor was blocked in cells expressing either Achn antisense or an Achn deletion mutant that blocks differentiation. Control cells and those expressing ectopic Achn undergo sequential and transient increases in both substrate adhesion and migration before cell fusion. Blockade of Achn expression reduced these effects on laminin but not on fibronectin. Taken together, these data suggest that Achn may influence differentiation in part via its control of cell adhesion dynamics. [C.sub.2][C.sub.12]; differentiation; satellite cell; integrin signaling; skeletal muscle

An exercise-linked hormone, FNDC5/irisin, mediates the benefit of exercise in Alzheimer's disease models by enhancing synaptic plasticity and memory., Author(s): Xu Chen [sup.1] , Li Gan [sup.1] [sup.2] Author Affiliations: (1) Gladstone Institutes, Department of Neurology, University of California San Francisco, San Francisco CA, USA (2) Helen and Robert [...]

Kato K, Yamanouchi D, Esbona K, Kamiya K, Zhang F, Kent KC, Liu B. Caspase-mediated protein kinase C-5 cleavage is necessary for apoptosis of vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 297: H2253-H2261, 2009. First published October 16, 2009; doi: 10.1152/ajpheart.00274.2009.--Apoptotic death of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling and various vascular diseases. We have previously shown that protein kinase C-8 (PKC-[delta]) plays a critical role in SMC apoptosis. In this study, we tested the importance of PKC-[delta] proteolytic cleavage and tyrosine phosphorylation within the apoptosis pathway. Using hydrogen peroxide as a paradigm for oxidative stress, we showed that proteolytic cleavage of PKC-[delta] occurred in SMCs that underwent apoptosis, while tyrosine phosphorylation was detected only in necrotic cells. Furthermore, using a peptide (z-DIPD-fmk) that mimics the caspase-3 binding motif within the linker region of PKC-[delta], we were able to prevent the cleavage of PKC-[delta], as well as apoptosis. Inhibition of PKC-[delta] with rottlerin or small-interfering RNA diminished caspase-3 cleavage, caspase-3 activity, cleavage of poly (AD-Pribose) polymerase, cleavage of PKC-[delta], and DNA fragmentation, confirming the previously reported role of PKC-[delta] in initiation of apoptosis. In contrast, z-DIPD-fmk markedly diminished caspase-3 activity, cleavage of PKC-[delta], and DNA fragmentation without affecting cleavage of caspase-3 and poly (ADP-ribose) polymerase. Taken together, our data suggest that caspase-3-mediated PKC-[delta] cleavage underlies SMC apoptosis induced by oxidative stress, and that PKC-[delta] acts both upstream and downstream of caspase-3. oxidative stress; remodeling; phosphorylation; necrosis doi: 10.1152/ajpheart.00274.2009.

Frid MG, Li M, Gnanasekharan M, Burke DL, Fragoso M, Strassheim D, Sylman JL, Stenmark KR. Sustained hypoxia leads to the emergence of cells with enhanced growth, migratory, and promitogenic potentials within the distal pulmonary artery wall. Am J Physiol Lung Cell Mol Physiol 297: L1059-L1072, 2009. First published September 18, 2009; doi: 10.1152/ajplung.90611.2008.--All forms of chronic pulmonary hypertension (PH) are characterized by structural remodeling of the pulmonary artery (PA) media, a process previously attributed solely to changes in the phenotype of resident smooth muscle cells (SMC). However, recent experimental evidence in both systemic and pulmonary circulations suggests that other cell types, including circulating and local progenitors, contribute significantly to this process. The goal of this study was to determine if hypoxia-induced remodeling of distal PA (dPA) media involves the emergence of cells with phenotypic and functional characteristics distinct from those of resident dPA SMC and fibroblasts. In vivo, in contrast to the phenotypically uniform SMC composition of dPA media in control calves, the remodeled dPA media of neonatal calves with severe hypoxia-induced PH comprised cells exhibiting a distinct phenotype, including the expression of hematopoetic (CD45), leukocytic/monocytic (CD11b, CD14), progenitor (cKit), and motility-associated (S100A4) cell markers. Consistent with these in vivo observations, primary cell cultures isolated from dPA media of hypertensive calves yielded not only differentiated SMC, but also smaller, morphologically rhomboidal (thus termed here 'R') cells that transiently expressed CD11b, constitutively expressed the mesenchymal cell marker type I procollagen, expressed high mRNA levels of progenitor cell markers cKit, CD34, CD73, as well as for inflammatory mediators, IL-6 and MCP-1, and, with time in culture, gained expression of a myofibroblast marker, [alpha]-SM-actin. R cells exhibited highly augmented proliferative, migratory, invasive, and potent promitogenic capabilities, which were due, at least in part, to the production of PDGFs, SDF-1/CXCL12, and S100A4. These data suggest that the cellular mechanisms of dPA remodeling include the emergence of cells with phenotypic and functional characteristics markedly distinct from those of resident dPA cells. pulmonary hypertension; vascular remodeling; progenitor cells; inflammation; S100A4 doi: 10.1152/ajplung.90611.2008

Singh J, Mehendiratta V, Del Galdo F, Jimenez SA, Cohen S, DiMarino AJ, Rattan S. Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 297: G1206-G1213, 2009. First published September 24, 2009; doi: 10.1152/ajpgi.00286.2009.--Systemic sclerosis (SSc) IgGs affecting the [M.sub.3]-muscarinic receptor ([M.sub.3]-R) have been proposed to be responsible for the gastrointestinal (GI) dysmotility in this disease. However, the effect of SSc IgGs on smooth muscle cell (SMC) function has not been studied. We determined the effect of SSc IgGs on the muscarinic receptor activation by bethanechol (BeCh; methyl derivate of carbachol) in SMC and smooth muscle strips from rat internal anal sphincter. IgGs were purified from GI-symptomatic SSc patients and normal volunteers, with protein G-Sepharose columns. SMC lengths were determined via computerized digital micrometry. The presence of [M.sub.3]-R and IgG-[M.sub.3]-R complex was determined by Western blot. IgGs from SSc patients but not from normal volunteers caused significant and concentration-dependent inhibition of BeCh response (P < 0.05). The maximal shortening of 22.2 [+ or -] 1.2% caused by [10.sup.-4] M BeCh was significantly attenuated to 8.3 [+ or -] 1.2% by 1 mg/ml of SSc IgGs (P < 0.05). Experiments performed in smooth muscle strips revealed a similar effect of SSc IgG that was fully reversible. In contrast to the effect on BeCh, the SSc IgGs caused no significant effect (P > 0.05) on [K.sup.+] depolarization and [[alpha].sub.1]- adrenoceptor activation by phenylephrine. Western blot studies revealed the specific presence of SSc IgG-[M.sub.3]-R complex. SSc IgGs attenuated [M.sub.3]-R activation, which was reversible with antibody removal. These data suggest that SSc GI dysmotility may be caused by autoantibodies that inhibit the muscarinic neurotransmission. Future treatment of SSc patients may be directed at the removal or neutralization of these antibodies. systemic sclerosis; rectoanal function; muscarinic receptor; autoantibodies doi: 10.1152/ajpgi.00286.2009

Flynn RS, Mahavadi S, Murthy KS, Kellum JM, Kuemmerle JF. Insulin-like growth factor-binding protein-5 stimulates growth of human intestinal muscle cells by activation of G[alpha]i3. Am J Physiol Gastrointest Liver Physiol 297: G1232-G1238, 2009. First published October 1, 2009; doi: 10.1152/ajpgi.00323.2009.--In human intestinal smooth muscle cells, endogenous insulin-like growth factor-I (IGF-I) regulates growth and IGF-binding protein-5 (IGFBP-5) expression. The effects of IGF-I are facilitated by 1GFBP-5. We previously showed that IGFBP-5 acts independently of IGF-I in human intestinal muscle to stimulate proliferation and upregulate IGF-I production by activation of Erkl/2 and p38 MAPK. Thus a positive feedback loop exists between IGF-I and IGFBP-5, whereby both stimulate muscle growth and production of the other factor. In Crohn's disease, IGF-I and IGFBP-5 expression are increased and contribute to stricture formation through this effect on muscle growth. To determine the signaling pathways coupling IGFBP-5 to MAPK activation and growth, smooth muscle cells were isolated from muscularis propria of human intestine and placed into primary culture. Erkl/2 and p38 MAPK activation and type I collagen production were measured by immunoblot. Proliferation was measured by [[sup.3]H]thymidine incorporation. Activation of specific G proteins was measured by ELISA. AG1024, an IGF-I receptor tyrosine kinase inhibitor, was used to isolate the IGF-I-independent effects of IGFBP-5. IGFBP-5-induced phosphorylation of Erkl/2 and p38 MAPK and proliferation were abolished by pertussis toxin, implying the participation of Gi. IGFBP-5 specifically activated Gi3 but not other G proteins. Transfection of an inhibitory G[alpha]i minigene specifically inhibited MAPK activation, proliferation, and both collagen-I and IGF-I production. Our results indicate that endogenous IGFBP-5 activates Gi3 and regulates smooth muscle growth, IGF-I production, and collagen production via the [alpha]-subunit of Gi3, independently of IGF-I, in normal human intestinal muscle cells. smooth muscle cell; heterotrimeric G protein; p38 MAPK; Erkl/2 doi: 10.1152/ajpgi.00323.2009

Sundaram P, Pang Z, Miao M, Yu L, Wing SS. USP19-deubiquitinating enzyme regulates levels of major myofibrillar proteins in L6 muscle cells. Am J Physiol Endocrinol Metab 297: E1283-E1290, 2009. First published September 22, 2009; doi: 10.1152/ajpendo.00409.2009.--The ubiquitin-proteasome system plays an important role in the degradation of myofibrillar proteins that occurs in muscle wasting. Many studies have demonstrated the importance of enzymes mediating conjugation of ubiquitin. However, little is known about the role of deubiquitinating enzymes. We previously showed that the USP19- deubiquitinating enzyme is induced in atrophying skeletal muscle (Combaret L, Adegoke OA, Bedard N, Baracos V, Attaix D, Wing SS. Am J Physiol Endocrinol Metab 288: E693-E700, 2005). To further explore the role of USP 19, we used small interfering RNA (siRNA) in L6 muscle cells. Lowering USP19 by 70-90% in myotubes resulted in a 20% decrease in the rate of proteolysis and an 18% decrease in the rate of protein synthesis, with no net change in protein content. Despite the decrease in overall synthesis, there were ~1.5-fold increases in protein levels of myosin heavy chain (MHC), actin, and troponin T and a ~2.5-fold increase in tropomyosin. USP19 depletion also increased MHC and tropomyosin mRNA levels, suggesting that this effect is due to increased transcription. Consistent with this, USP19 depletion increased myogenin protein and mRNA levels approximately twofold. Lowering myogenin using siRNA prevented the increase in MHC and tropomyosin upon USP19 depletion, indicating that myogenin mediated the increase in myofibrillar proteins. Dexamethasone treatment lowered MHC and increased USP19. Depletion of USP19 reversed the dexamethasone suppression of MHC. These studies demonstrate that USP19 modulates transcription of major myofibrillar proteins and indicate that the ubiquitin system not only mediates the increased protein breakdown but is also involved in the decreased protein synthesis in atrophying skeletal muscle. ubiquitin-specific processing protease doi: 10.1152/ajpendo.00409.2009.

Akerud H, Ronquist G, Wiberg-Itzel E. Lactate distribution in culture medium of human myometrial biopsies incubated under different conditions. Am J Physiol Endocrinol Metab 297: E1414-E1419, 2009. First published October 13, 2009; doi: 10.1152/ajpendo.00458.2009.--It is generally believed that a relationship exists between muscle fatigue and intracellular accumulation of lactate. This reasoning is relevant to obstetrical issues. Myocytes in uterus work together during labor, and the contractions need to be strong and synchronized for a child to be delivered. At labor dystocia, the progress of labor becomes slow or arrested after a normal beginning. It has been described that, during labor dystocia, when the force of the contractions is low, the uterus is under hypoxia, and anaerobic conditions with high levels of lactate in amniotic fluid dominate. The purpose of this study was to examine whether myometrial cells are involved in the production of lactate in amniotic fluid and whether there are differences in production and distribution of lactate in cells incubated under aerobic and anaerobic conditions. We also wanted to elucidate the involvement of specific membrane-bound lactate carriers. Women undergoing elective caesarean section were included. Myometrial biopsies from uteri were collected and subjected to either immunohistochemistry to identify lactate carriers or in vitro experiments to analyze production of lactate. The presence of lactate carriers named monocarboxylate transporters 1 and 4 was verified. Myometrial cells produced lactate extracellularly, and the lactate carriers operated differently under anaerobic and aerobic conditions; while being mainly unidirectional under anaerobic conditions, they became bidirectional under aerobic conditions. Human myometrial cells produced and delivered lactate to the extracellular medium under both anaerobic and aerobic conditions. The delivery was mediated by lactate carriers. monocarboxylate transporter 1; monocarboxylate transporter 4; myometrial cells; uterus doi: 10.1152/ajpendo.00458.2009

The effect of constitutively activated proto-oncogene H-ras (H-rasQ61L) on the regulation of myosin heavy chain (MHC) promoter activities was investigated in rabbit satellite cell-derived muscle cell culture during the proliferation stage and early and later stages of differentiation, respectively. During proliferation, overexpression of H-rasQ61L did not affect basal level of activity of the slow MHCI/[beta] or the fast MHCIId/x promoter luciferase reporter gene construct in transient transfection assays. By contrast, H-rasQ61L affected both MHC promoter activities during differentiation, and this effect changes from inactivation after 2 days to activation after 4 days of differentiation. The activating effect of H-rasQ61L on both MHC promoters after 4 days of differentiation was significantly reduced by LY-294002, a specific inhibitor of the phosphoinositol-3-kinase (PI3K), a downstream target of Ras. Furthermore, the protein kinase Akt (protein kinase B), a downstream target of PI3k, was activated 4 days after initiation of differentiation in myotubes overexpressing H-rasQ61L. By contrast, inhibition of another Ras downstream pathway, mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated protein kinase 1/2 (MKK1/ 2-ERK1/2-MAPK), increased activities of both MHC promoters, indicating a suppressive role of this pathway. Moreover, the Ras-PI3K-Akt signaling pathway is involved in the activation of MHCI/[beta] and IId/x promoters in a later stage of differentiation of muscle cells, presumably by a known inhibiting effect of activated Akt on the MKK1/2-ERK1/2-MAPK pathway. The experiments demonstrate that during differentiation of muscle cells activated H-ras is an important regulator of MHC isoform promoter function with opposite effects during early and later stages. extracellular signal regulated protein kinase 1/2; mitogen-activated protein kinase; phosphosinositol-3-kinase; Akt; mammalian target of rapamycin doi: 10.1152/ajpcell.00567.2008