Search

Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. [PAX7.sup.+] satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 [PAX7.sup.+] satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human [PAX7.sup.+] satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for [PAX7.sup.+] cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human [PAX7.sup.+] satellite cells with Sleeping Beauty transposon-mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these [PAX7.sup.+] satellite cells have potential to restore gene function in muscular dystrophies., Introduction Satellite cells are the proper stem cells of the adult skeletal muscle. Like other stem cell types, these cells are maintained over very long time periods (1, 2). Despite [...]

The coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 [alpha] (PGC-1[alpha]) coordinates a broad set of transcriptional programs that regulate the response of skeletal muscle to exercise. However, the complete transcriptional network controlled by PGC-la has not been described. In this study, we used a qPCR-based screen of all known transcriptional components (Quanttrx) to identify transcription factors that are quantitatively regulated by PGC-la in cultured skeletal muscle cells. This analysis identified hypoxia-inducible factor 2 [alpha] (HIF2[alpha]) as a major PGC-1[alpha] target in skeletal muscle that is positively regulated by both exercise and [beta]-adrenergic signaling. This transcriptional regulation of HIF2[alpha] is completely dependent on the PGC-1[alpha]/ERR[alpha] complex and is further modulated by the action of SIRT1. Transcriptional profiling of HIF2[alpha] target genes in primary myotubes suggested an unexpected role for HIF2[alpha] in the regulation of muscle fiber types, specifically enhancing the expression of a slow twitch gene program. The PGC-1[alpha]-mediated switch to slow, oxidative fibers in vitro is dependent on HIF2[alpha], and mice with a muscle-specific knockout of HIF2[alpha] increase the expression of genes and proteins characteristic of a fast-twitch fiber-type switch. These data indicate that HIF2[alpha] acts downstream of PGC-1[alpha] as a key regulator of a muscle fiber-type program and the adaptive response to exercise. doi: 10.1073/pnas.1016089107

Atherosclerosis is a chronic inflammation disease characterized by acidic micromilieu and the accumulation of numerous bioactive lipid mediators, such as lysophosphatidic acid (LPA) and prostaglandins, in the atherosclerotic lesion. Chronic acidification induced various effects on vascular smooth muscle cells, but the molecular mechanisms underlying these effects remain unknown. In this study, we examine the role of proton-sensing ovarian cancer G protein-coupled receptor 1 (OGRI) in extracellular acidification-induced regulation of cyclooxygenase (COX)-2 induction, PGI2 production, MAPK phosphatase (MKP)-1 expression, and plasminogen activator inhibitor (PAI)-1 expression and proliferation in human aortic smooth muscle cells (AoSMCs). Experiments with knockdown with small interfering RNA specific to OGR1 and specific inhibitors for G proteins showed that acidificationinduced COX-2 expression, [PGI.sub.2] production, and MKP-1 expression, but not PAL1 expression and inhibition of proliferation, were dependent on OGR1 and mainly mediated by [G.sub.q/11] protein. LPA remarkably enhanced, through the [LPA.sub.1] receptor/[G.sub.i] protein, the OGR1-mediated vascular actions to acidic pH. In conclusion, acidic pH-induced vascular actions of AoSMCs can be dissected to OGR1-dependent and -independent pathways: COX-2 expression, [PGI.sub.2] production, and MKP-I expression are mediated by OGR1, but PAI-1 expression and inhibition of proliferation are not. LPA, which is usually thought to be a proatherogenic lipid mediator, may exert antiatherogenic actions under acidic micromilieu through cross-talk between [LPA.sub.1]/[G.sub.i] protein and OGR1/[G.sub.q/11] protein. acidic micromilieu; vascular action; lysophosphatidic acid receptor doi: 10.1152/ajpheart.00977.2009.

Plasma membrane large-conductance [Ca.sup.2+]-activated [K.sup.+] ([BK.sub.ca]) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate ([IP.sub.3]) receptors ([IP.sub.3]Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied [BK.sub.ca] channel regulation by [IP.sub.3] and [IP.sub.3]Rs in rat and mouse cerebral artery smooth muscle cells. [IP.sub.3] activated BiOta channels both in intact cells and in excised inside-out membrane patches. [IP.sub.3] caused concentration-dependent [BK.sub.ca] channel activation with an apparent dissociation constant ([K.sub.d]) of ~4 [micro]M at physiological voltage (-40 mV) and intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]; 10 [micro]M). [IP.sub.3] also caused a leftward-shift in [BK.sub.ca] channel apparent [Ca.sup.2+] sensitivity and reduced the [K.sub.d] for free [[[Ca.sup.2+]].sub.i] from ~20 to 12 [micro]M, but did not alter the slope or maximal [P.sub.o]. BAPTA, a fast [Ca.sup.2+] buffer, or an elevation in extracellular [Ca.sup.2+] concentration did not alter [IP.sub.3]-induced [BK.sub.ca] channel activation. Heparin, an [IP.sub.3]R inhibitor, and a monoclonal type 1 [IP.sub.3]R ([IP.sub.3]R1) antibody blocked [IP.sub.3]-induced [BK.sub.ca] channel activation. Adenophostin A, an [IP.sub.3]R agonist, also activated [BK.sub.ca] channels. [IP.sub.3] activated [BK.sub.ca] channels in inside-out patches from wild-type ([IP.sub.3][R1.sup.+/+]) mouse arterial smooth muscle cells, but had no effect on [BK.sub.ca] channels of [IP.sub.3]Rl-deficient ([IP.sub.3][R1.sup.-/-]) mice. Immunofluorescence resonance energy transfer microscopy indicated that [IP.sub.3]R1 is located in close spatial proximity to [BK.sub.ca] [alpha] subunits. The [IP.sub.3]R1 monoclonal antibody coimmunoprecipitated [IP.sub.3]R1 and [BK.sub.ca] channel [alpha] and [beta]1 subunits from cerebral arteries. In summary, data indicate that [IP.sub.3]R1 activation elevates [BK.sub.ca] channel apparent [Ca.sup.2+] sensitivity through local molecular coupling in arterial smooth muscle cells. doi/10.1085/jgp.201010453

Amino acids modulate mRNA translation through the 70 kDa ribosomal protein S6 kinase (S6K1) and the general control nondepressible 2 protein kinase (GCN2)/eukaryotic initiation factor 2[alpha] elF2[alpha] pathways. The aim of the present study was therefore to explore the signaling cascades potentially modulated by methionine availability in quail muscle QM7 myoblasts using media providing all other amino acids. Methionine deprivation caused a lower S6K1 phosphorylation compared with control (Ctl) cells. Supplying the methionine-deprived media with L- and DL-methionine isomers restored S6K1 phosphorylation to the levels observed in Ctl cells. Methionine also regulated downstream S6K1 targets (i.e. ribosomal protein S6 and eukaryotic elongation factor 2), modulated translation preinitiation complex (PIC) assembly, and stimulated protein synthesis. Replacing the lacking methionine with D-methionine or its hydroxyanalog [2-hydroxy- (4methylthio) butanoic acid] did not restore S6K1 activation or protein synthesis. Conversely, the S6K1 pathway was activated by a methionine precursor, the ketoanalog of methionine. Methionine availability regulated the GCN2/eIF2[alpha] pathway. However, our results indicate that methionine deprivation led to lower protein synthesis without activating eIF2[alpha] phosphorylation, a process known to limit the formation of the 43S PIC. Using the amino acid alcohol methioninol did not decrease S6K1 phosphorylation or activity and did not alter the regulation of protein synthesis by methionine. These findings suggest that methionine exerts an effect on S6K1 signaling and protein synthesis in avian QM7 myoblasts through a mechanism partly independent of the global regulation via tRNA charging. J. Nutr. 140:1539-1545, 2010. doi: 10.3945/jn.110.122663.

Effects of previous strength training can be long-lived, even after prolonged subsequent inactivity, and retraining is facilitated by a previous training episode. Traditionally, such 'muscle memory' has been attributed to neural factors in the absence of any identified local memory mechanism in the muscle tissue. We have used in vivo imaging techniques to study live myonuclei belonging to distinct muscle fibers and observe that new myonuclei are added before any major increase in size during overload. The old and newly acquired nuclei are retained during severe atrophy caused by subsequent denervation lasting for a considerable period of the animal's lifespan. The myonuclei seem to be protected from the high apoptotic activity found in inactive muscle tissue. A hypertrophy episode leading to a lasting elevated number of myonuclei retarded disuse atrophy, and the nuclei could serve as a cell biological substrate for such memory. Because the ability to create myonuclei is impaired in the elderly, individuals may benefit from strength training at an early age, and because anabolic steroids facilitate more myonuclei, nuclear permanency may also have implications for exclusion periods after a doping offense. muscle memory | muscle nuclei | muscle atrophy | muscle hypertrophy | apoptosis doi/ 10.1073/pnas.0913935107

Insulin-like growth factors (IGFs) stimulate myoblast proliferation and differentiation. It remains elusive how these mutually exclusive cellular responses are elicited by the same growth factor. Here we report that whereas IGF promotes myoblast differentiation under normoxia, it stimulates proliferation under hypoxia. Hypoxia activates the HIF-1 transcriptional program and knockdown of HIF-1[alpha] changes the mitogenic action of IGF into myogenic action under hypoxia. Conversely, overexpression of HIF-1[alpha] abolishes the myogenic effect of IGF under normoxia. Under normoxia, IGF activates the Akt-mTOR, p38, and Erk1/2 MAPK pathways. Hypoxia suppresses basal and IGF-induced Akt-mTOR and p38 activity, whereas it enhances and prolongs IGF-induced Erk1/2 activation in a HIF-1--dependent fashion. Activation of Akt-mTOR and p38 promotes myogenesis, and p38 also inhibits proliferation. Activation of Erk stimulates myoblast proliferation but inhibits differentiation. These results suggest that hypoxia converts the myogenic action of IGFs into mitogenic action by differentially regulating multiple signaling pathways via HIF-1-dependent mechanisms. Our findings provide a mechanistic explanation for the paradoxical actions of IGFs during myogenesis and reveal a novel mechanism by which cells sense and integrate growth factor signals and oxygen availability in their microenvironments. Akt | hypoxia-inducible factor | muscle cells | MAPK | mTOR doi/10.1073/pnas.0909570107

Contractile forces generated by the actomyosin cytoskeleton within individual cells collectively generate tissue-level force during epithelial morphogenesis. During Drosophila mesoderm invagination, pulsed actomyosin meshwork contractions and a ratchet-like stabilization of cell shape drive apical constriction. Here, we investigate how contractile forces are integrated across the tissue. Reducing adherens junction (AJ) levels or ablating actomyosin meshworks causes tissue-wide epithelial tears, which release tension that is predominantly oriented along the anterior--posterior (a-p) embryonic axis. Epithelial tears allow cells normally elongated along the a-p axis to constrict isotropically, which suggests that apical constriction generates anisotropic epithelial tension that feeds back to control cell shape. Epithelial tension requires the transcription factor Twist, which stabilizes apical myosin II, promoting the formation of a supracellular actomyosin meshwork in which radial actomyosin fibers are joined end-to-end at spot AJs. Thus, pulsed actomyosin contractions require a supracellular, tensile meshwork to transmit cellular forces to the tissue level during morphogenesis. doi/10.1083/jcb.200910099

There is now a significant interest in being able to locate single cells within geometrically defined regions of a microfluidic chip and to gain intracellular access through the local electroporation of the cell membrane. This paper describes the microfabricafion of electroporation devices which can enable the regional electroporation of adult ventricular myocytes, in order to lower the local electrical resistance of the cell membrane. Initially three different devices, designed to suit the characteristic geometry of the cardiomyocyte, were investigated (all three designs serve to focus the electric field to selected regions of the cell). We demonstrate that one of these three devices revealed the sequence of cellular responses to field strengths of increasing magnitudes, namely, cell contraction, hypercontraction, and lysis. This same device required a reduced threshold voltage for each of these events, including in particular membrane breakdown. We were not only able to show the gradual regional increase in the electric conductivity of the cell membrane but were also able to avoid changes in the local intra- and extracellular pH (by preventing the local generation of protons at the electrode surface, as a consequence of the reduced threshold voltage). The paper provides evidence for new slyategies for achieving robust and reproducible regional electroporation, a technique which, in future, may be used for the insertion of large molecular weight molecules (including genes) as well as for on-chip voltage clamping of the primary adult cardiomyocyte. 10.1021/ac901886j

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 intermuscular adipose tissue (IMAT) is a depot of adipocytes located between muscle bundles. Several investigations have recently been carried out to define the phenotype, the functional characteristics, and the origin of the adipocytes present in this depot. Among the different mechanisms that could be responsible for the accumulation of fat in this site, the dysdifferentiation of muscle-derived stem cells or other mesenchymal progenitors has been postulated, turning them into cells with an adipocyte phenotype. In particular, muscle satellite cells (SCs), a heterogeneous stem cell population characterized by plasticity and self-renewal that allow muscular growth and regeneration, can acquire features of adipocytes, including the abilities to express adipocyte-specific genes and accumulate lipids. Failure to express the transcription factors that direct mesenchymal precursors into fully differentiated functionally specialized cells may be responsible for their phenotypic switch into the adipogenic lineage. We proved that human SCs also possess a clear adipogenic potential that could explain the presence of mature adipocytes within skeletal muscle. This occurs under some pathological conditions (i.e., primary myodystrophies, obesity, hyperglycemia, high plasma free fatty acids, hypoxia, etc.) or as a consequence of thiazolidinedione treatment or simply because of a sedentary lifestyle or during aging. Several pathways and factors (PPARs, WNT growth factors, myokines, GEF-GAP-Rho, [p66.sup.shc], mitochondrial ROS production, PKC[beta]) could be implicated in the adipogenic conversion of SCs. The understanding of the molecular pathways that regulate muscle-to-fat conversion and SC behavior could explain the increase in IMAT depots that characterize many metabolic diseases and age-related sarcopenia. muscle satellite cells; mesenchymal stem cells; myogenesis; adipogenesis doi: 10.1152/ajpendo.00229.2009.

Trabeculae carneae are the smallest naturally arising collections of linearly arranged myocytes in the heart. They are the preparation of choice for studies of function of intact myocardium in vitro. In vivo, trabeculae are unique in receiving oxygen from two independent sources: the coronary circulation and the surrounding ventricular blood. Because oxygen partial pressure ([PO.sub.2]) in the coronary arterioles is identical in specimens from both ventricles, whereas that ofventricular blood is 2.5-fold higher in the left ventricle than in the right ventricle, trabeculae represent a 'natural laboratory' in which to examine the influence of'extravascular' [PO.sub.2] on the extent of capillarization of myocardial tissue. We exploit this advantage to test four hypotheses. (1) In trabeculae from either ventricle, a peripheral annulus of cells is devoid of capillaries. (2) Hence, sufficiently small trabeculae from either ventricle are totally devoid of capillaries. (3) The capillary-to-myocyte ratios in specimens from either ventricle are identical to those of their respective walls. (4) Capillary-to-myocyte ratios are comparable in specimens from either ventricle, reflecting equivalent energy demands in vivo, driven by identical contractile frequencies and comparable wall stresses. We applied confocal fluorescent imaging to trabeculae in cross section, subsequently using semi-automated se[G.sub.m]entation techniques to distinguish capillaries from myocytes. We quantified the capillary-to- myocyte ratios of trabeculae from both ventricles and compared them to those determined for the ventricular free walls and septum. Quantitative interpretation was furthered by mathematical modeling, using both the classical solution to the diffusion equation for elliptical cross sections, and a novel approach applicable to cross sections of arbitrary shape containing arbitrary disposition of capillaries and non-respiring collagen cords.

L-type [Ca.sup.2+] currents conducted by [Ca.sub.v] 1.2 channels initiate excitation-contraction coupling in cardiac myocytes. Intracellular [Mg.sup.2+] ([Mg.sub.i]) inhibits the ionic current of [Ca.sub.v] l.2 channels. Because [Mg.sub.i] is altered in ischemia and heart failure, its regulation of [Ca.sub.v] 1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of [Mg.sub.i] on voltage-dependent inactivation (VDI) of [Ca.sub.v] l.2 channels using [Na.sup.+] as permeant ion to eliminate the effects of permeant divalent cations that engage the [Ca.sup.2+]-dependent inactivation process. We confirmed that increased [Mg.sub.i] reduces peak ionic currents and increases VDI of [Ca.sub.v] 1.2 channels in ventricular myocytes and in transfected cells when measured with [Na.sup.+] as permeant ion. The increased rate and extent of VDI caused by increased [Mg.sub.i] were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of [Mg.sub.i] to the EF-hand ([K.sub.D] [approximately equal to] 0.9 mM) near the resting level of Mgi in ventricular myocytes. VDI was more rapid for L-type [Ca.sup.2+] currents in ventricular myocytes than for [Ca.sub.v] 1.2 channels in transfected cells. Coexpression of [Ca.sub.v] [[beta].sub.2b] subunits and formation of an autoinhibitory complex of truncated [Ca.sub.v] 1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the [Ca.sub.v] 1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mgi binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of [Ca.sub.v] 1.2 channels by [Mg.sub.i], the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

Membrane depolarization activates voltage-dependent [Ca.sup.2+] channels (VDCCs) inducing [Ca.sup.2+] release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced [Ca.sup.2+] release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local [Ca.sup.2+] release, leading to a significant increase in the activity of [Ca.sup.2+] sparks and contraction in airway SMCs. The increased [Ca.sup.2+] sparks are independent of VDCCs and the associated extracellular [Ca.sup.2+] influx. This format of local [Ca.sup.2+] release results from a direct activation of G protein-coupled, [M.sub.3] muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate ([IP.sub.3]), inducing initial [Ca.sup.2+] release through [IP.sub.3] receptors and then further [Ca.sup.2+] release via RyR2 due to a local [Ca.sup.2+]-induced [Ca.sup.2+] release process. These findings demonstrate an important mechanism for [Ca.sup.2+] signaling and attendant physiological function in SMCs. muscarinic receptors | ryanodine receptors

Increased plasma levels of lactosylceramide (LacCer) have been associated with cardiovascular disease. However, it is largely unknown whether LacCer directly contributes to dysfunction of smooth muscle cells (SMCs), a key event in vascular lesion formation. In the present study, we determined the effects and potential mechanisms of LacCer on cell migration and proliferation in human aortic SMCs (AoSMCs). Cell migration and proliferation were determined by a modified Boyden chamber assay and nonradioactive colorimetric (MTS) assay, respectively. We found that LacCer significantly induced AoSMC migration and proliferation in a concentration- and time-dependent manner. In addition, LacCer significantly upregulated the expression of PDGFR-B, integrins ([[alpha].sub.v] and [[beta].sub.3]), and matrix metalloproteinases (matrix metalloproteinase-1 and -2) at both mRNA and protein levels, as determined by real-time PCR and Western blot analyses, respectively. Furthermore, LacCer increased superoxide anion production and the transient phosphorylation of ERK1/2 in AoSMCs, as determined by dihydroethidium staining and immunoassay, respectively. Accordingly, LacCer-induced cell migration and proliferation were effectively blocked by antioxidants (seleno-L-methionine and Mn tetrakis porphyrin) and by a specific ERK1/2 inhibitor. Thus, LacCer promotes cell migration and proliferation through oxidative stress and activation of ERK1/2 in AoSMCs. These findings demonstrate the functional role of LacCer in the vascular disease pathogenesis. vascular smooth muscle cell; cell proliferation; oxidative stress; extracellular signal-regulated kinase 1/2; antioxidant; vascular disease

Airway inflammation leads to increased intracellular [Ca.sup.2+] ([[[Ca.sup.2+].sub.i]) levels in airway smooth muscle (ASM) cells. Sarcoplasmic reticulum [Ca.sup.2+] release and reuptake are key components of ASM [[[Ca.sup.2+]].sub.i] regulation. [Ca.sup.2+] reuptake occurs via sarcoendoplasmic reticulum [Ca.sup.2+] ATPase (SERCA) and is regulated by the inhibitory protein phospholamban (PLB) in many cell types. In human ASM, we tested the hypothesis that inflammation increases PLB, thus inhibiting SERCA function, and leading to maintained [[[Ca.sup.2+]].sub.i] levels. Surprisingly, we found that human ASM does not express PLB protein (although mRNA is detectable). Overnight exposure to the proinflammatory cytokines TNF[alpha] and IL-13 did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (via CaMKII) occurs in human ASM. In fura-2-loaded human ASM cells, we found that the CaMKII antagonist KN-93 significantly slowed the rate of fall of [[Ca.sup.2+]].sub.i] transients induced by ACh or bradykinin (in zero extracellular [Ca.sup.2+]), suggesting a role for CaMKII-mediated SERCA regulation. SERCA expression was decreased by cytokine exposure, and the rate of fall of [[[Ca.sup.2+]].sub.i] transients was slowed in cells exposed to TNF[alpha] and IL-13. Cytokine effects on [Ca.sup.2+] reuptake were unaffected by additional exposure to KN-93. These data indicate that in human ASM, SERCA is regulated by mechanisms such as CaMKII and that airway inflammation maintains [[[Ca.sup.2+]].sub.i] levels by decreasing SERCA expression and slowing [Ca.sup.2+] reuptake. sarcoendoplasmic reticulum [Ca.sup.2+] ATPase; calmodulin; calmodulin kinase; tumor necrosis factor-[alpha]; interleukin-13

EGF receptors (EGFRs) are increased in airway smooth muscle in asthma, which may contribute to both their hyperproliferation and hypercontractility. Lysophosphatidic acid (LPA) is a candidate pathological agent in asthma and other airway diseases, and LPA upregulates EGFRs in human airway smooth muscle (HASM) cells. We tested whether therapeutic glucocorticoids and/or [[beta].sub.2]-adrenergic receptor ([[beta].sub.2]AR) agonists also alter EGFR binding in HASM cells. Exposure to glucocorticoids for 24 h induced a twofold increase in EGFR binding similar to that with LPA; fluticasone was markedly more potent than dexamethasone. The increase in EGFR binding by glucocorticoids required 24-h exposure, consistent with transcription-mediated effects. Although the increase in EGFR binding was blocked by the protein synthesis inhibitor cycloheximide for LPA, fluticasone, and dexamethasone, only LPA induced a significant increase in EGFR protein expression detected by immunoblotting. In contrast to the increased binding induced by the glucocorticoids, the [[beta].sub.2]AR agonists isoproterenol, albuterol, and salmeterol all induced a decrease in EGFR binding. [[beta].sub.2]AR agonist effects were multiphasic, with an initial decline at 2-4 h that reversed by 6 h and a second, somewhat greater decrease by 18-24 h. In cells pretreated with glucocorticoids, the decreases in EGFR binding by subsequent [[beta].sub.2]AR treatment were not statistically significant; glucocorticoid upregulation of EGFRs also prevented further increases by LPA. Similar increases by glucocorticolds and decreases by [[beta].sub.2]AR agonists were found in HFL-1 human lung fibroblasts. These complex and opposing effects of clinically relevant glucocorticoids and [[beta].sub.2]AR agonists on airway mesenchymal cell EGFRs likely contribute to their overall therapeutic profile in the diseased airway. lysophosphatidic acid; lung fibroblasts; combination therapy

Although ozone enhances leukocyte function and recruitment in airways, the direct effect of ozone in modulating structural cell-derived inflammatory mediators remains unknown. Using a coculture model comprised of differentiated human airway epithelial cells (NHBE) and smooth muscle cells (ASM), we postulate that ozone regulates IL-6 secretion in basal and cytokine-primed structural cells. Air-liquid interface (ALI) cultures of NHBE cells underwent differentiation as determined by mucin secretion, transepithelial electrical resistance (TEER), and ultrastructure parameters. Whereas TNF enhanced basal secretion of IL-6 (57 [+ or -] 3%), ozone exposure at 0.6 ppm for 6 h augmented IL-6 levels in basal (41 [+ or -] 3%) and TNF- (50 [+ or -] 5%) primed cocultures compared with that derived from NHBE or ASM monolayers alone. Levels of [PGE.sub.2], 6-keto-[PGF.sub.1[alpha]], [PGF.sub.2[alpha]], and thromboxane [B.sub.2] (Tx[B.sub.2]) levels in basal and TNF-primed cocultures revealed that ozone selectively enhanced [PGE.sub.2] production in TNF- (6 [+ or -] 3-fold) primed cocultures, with little effect (P > 0.05) on diluent-treated cultures. In accordance with ozone-induced increases in [PGE.sub.2] levels, cyclooxygenase inhibition with indomethacin partially abolished IL-6 secretion. Surprisingly, indomethacin had little effect on constitutive secretion of IL-6 in cocultures, whereas indomethacin completely restored ozone-mediated TEER reduction in TNF-primed cocultures. Collectively, our data for the first time suggest a dual role of ozone in modulating IL-6 secretion and TEER outcomes in a [PGE.sub.2]-dependent (in presence of TNF stimulus) and -independent manner (in absence of cytokine stimulus). asthma; airway remodeling; inflammation; cytokines; chronic obstructive pulmonary disease

The L-type [Ca.sup.2+] channel expressed in gastrointestinal smooth muscle is mechanosensitive. Direct membrane stretch and shear stress result in increased [Ca.sup.2+] entry into the cell. The mechanism for mechanosensitivity is not known, and mechanosensitivity is not dependent on an intact cytoskeleton. The aim of this study was to determine whether L-type [Ca.sup.2+] channel mechanosensitivity is dependent on tension in the lipid bilayer in human jejunal circular layer myocytes. Whole cell currents were recorded in the amphotericin-perforated-patch configuration, and lysophosphatidyl choline (LPC), lysophosphatidic acid (LPA), and choline were used to alter differentially the tension in the lipid bilayer. Shear stress (perfusion at 10 ml/min) was used to mechanostimulate L-type [Ca.sup.2+] channels. The increase in L-type [Ca.sup.2+] current induced by shear stress was greater in the presence of LPC (large head-to-tail proportions), but not LPA or choline, than in the control perfusion. The increased peak [Ca.sup.2+] current also did not return to baseline levels as in control conditions. Furthermore, steady-state inactivation kinetics were altered in the presence of LPC, leading to a change in window current. These findings suggest that changes in tension in the plasmalemmal membrane can be transmitted to the mechanosensitive L-type [Ca.sup.2+] channel, leading to altered activity and [Ca.sup.2+] entry in the human jejunal circular layer myocyte. calcium channel; gastrointestinal; lipid bilayer tension; shear stress; lysophosphatidic acid

Extracellular inorganic pyrophosphate ([PP.sub.i]) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ectonucleotide pyrophosphatase/phosphodiesterases (eNPPs) generate [PP.sub.i] via the hydrolysis of ATP released into extracellular compartments by poorly understood mechanisms. Here we report that cultured vascular smooth muscle cells (VSMC) from rat aorta generate extracellular [PP.sub.i] via an autocrine mechanism that involves ATP release tightly coupled to eNPP activity. The nucleotide analog [beta], [gamma]-methylene ATP (MeATP or AMPPCP) was used to selectively suppress ATP metabolism by eNPPs but not the CD39-type ecto-ATPases. In the absence of MeATP, VSMC generated extracellular [PP.sub.i] to accumulate [greater than or equal to] 600 nM within 2 h while steadily maintaining extracellular ATP at 1 nM. Conversely, the presence of MeATP completely suppressed [PP.sub.i] accumulation while increasing ATP accumulation. Probenecid, which inhibits [PP.sub.i] efflux dependent on ANK, a putative [PP.sub.i] transporter or transport regulator, reduced extracellular [PP.sub.i] accumulation by approximately twofold. This indicates that autocrine ATP release coupled to eNPP activity comprises [greater than or equal to] 50% of the extracellular PP,-generating capacity of VSMC. The accumulation of extracellular [PP.sub.i] and ATP was markedly attenuated by reduced temperature but was insensitive to brefeldin A, which suppresses constitutive exocytosis of Golgi-derived secretory vesicles. The magnitude of extracellular [PP.sub.i] accumulation in VSMC cultures increased with time postplating, suggesting that ATP release coupled to [PP.sub.i] generation is upregulated as cultured VSMC undergo contact-inhibition of proliferation or deposit extracellular matrix. adenosine 5'-triphosphate release; vascular calcification; ectonucleotide pyrophosphatase; ectoadenosine 5'-triphosphatase

Human ether-a-go-go-related gene (HERG) potassium channel acts as a delayed rectifier in cardiac myocytes and is an important target for both pro-and antiarrhythmic drugs. Many drugs have been pulled from the market for unintended HERG block causing arrhythmias. Conversely, recent evidence has shown that HERG plays a role in cell proliferation and is overexpressed both in multiple tumor cell lines and in primary tumor cells, which makes HERG an attractive target for cancer treatment. Therefore, a drug that can block HERG but that does not induce cardiac arrhythmias would have great therapeutic potential. Roscovitine is a cyclin-dependent kinase (CDK) inhibitor that is in phase II clinical trials as an anticancer agent. In the present study we show that R-roscovitine blocks HERG potassium current (human embryonic kidney-293 cells stably expressing HERG) at clinically relevant concentrations. The block ([IC.sub.50] = 27 [micro]M) was rapid ([tau] = 20 ms) and reversible ([tau] = 25 ms) and increased with channel activation, which supports an open channel mechanism. Kinetic study of wild-type and inactivation mutant HERG channels supported block of activated channels by roscovitine with relatively little effect on either closed or inactivated channels. A HERG gating model reproduced all roscovitine effects. Our model of open channel block by roscovitine may offer an explanation of the lack of arrhythmias in clinical trials using roscovitine, which suggests the utility of a dual CDK/HERG channel block as an adjuvant cancer therapy. S620T mutant; open channel block; terfenadine; human ether-a-go-go-related gene model

NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of [H.sub.2][O.sub.2]. Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-[[beta].sub.1] activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future. NADPH oxidase; serum response factor; myocardin

A number of recent studies have demonstrated the effectiveness of atomic force microscopy (AFM) for characterization of cellular stress-relaxation behavior. However, this technique's recent development creates considerable need for exploration of appropriate mechanical models for analysis of the resultant data and of the roles of various cytoskeletal components responsible for governing stress-relaxation behavior. The viscoelastic properties of vascular smooth muscle cells (VSMCs) are of particular interest due to their role in the development of vascular diseases, including atherosclerosis and restenosis. Various cytoskeletal agents, including cytochalasin D, jasplakinolide, paclitaxel, and nocodazole, were used to alter the cytoskeletal architecture of the VSMCs. Stress-relaxation experiments were performed on the VSMCs using AFM. The quasilinear viscoelastic (QLV) reduced-relaxation function, as well as a simple power-law model, and the standard linear solid (SLS) model, were fitted to the resultant stress-relaxation data. Actin depolymerization via cytochalasin D resulted in significant increases in both rate of relaxation and percentage of relaxation; actin stabilization via jasplakinolide did not affect stress-relaxation behavior. Microtubule depolymerization via nocodazole resulted in nonsignificant increases in rate and percentage of relaxation, while microtubule stabilization via paclitaxel caused significant decreases in both rate and percentage of relaxation. Both the QLV reduced-relaxation function and the power-law model provided excellent fits to the data ([R.sup.2]=0.98), while the SLS model was less adequate ([R.sup.2] =0.91). Data from the current study indicate the important role of not only actin, but also microtubules, in governing VSMC viscoelastic behavior. Excellent fits to the data show potential for future use of both the QLV reduced-relaxation function and power-law models in conjunction with AFM stress-relaxation experiments. [DOI: 10.1115/1.3049860] Keywords: stress-relaxation, atomic force microscopy, vascular smooth muscle cells

This paper explores the conditions under which nonmyocytes, when electrically coupled to myocytes, act as a passive load during the depolarization phase. Using theoretical arguments and numerical simulations in a tissue incorporating fibroblasts, the passive load approximation is shown to be accurate at low coupling conductances ( Index Terms--Computer modeling, coupling, electrophysioiogy, fibroblast, impulse propagation.

Homocysteine (HCY) activated mitochondrial matrix metalloproteinase-9 and led to cardiomyocyte dysfunction, in part, by inducing mitochondrial permeability (MPT). Treatment with MK-801 [N-methyl-D-aspartate (NMDA) receptor antagonist] ameliorated the HCY-induced decrease in myocyte contractility. However, the role of cardiomyocyte NMDA-receptor 1 (R1) activation in hyperhomocysteinemia (HHCY) leading to myocyte dysfunction was not well understood. We tested the hypothesis that the cardiac-specific deletion of NMDA-R1 mitigated the HCY-induced decrease in myocyte contraction, in part, by decreasing nitric oxide (NO). Cardiomyocyte-specific knockout of NMDA-R1 was generated using cre/lox technology. NMDA-R1 expression was detected by Western blot and confocal microscopy. MPT was determined using a spectrophotometer. Myocyte contractility and calcium transients were studied using the IonOptix video-edge detection system and fura 2-AM loading. We observed that HHCY induced NO production by agonizing NMDA-R1. HHCY induced the MPT by agonizing NMDA-R1. HHCY caused a decrease in myocyte contractile performance, maximal rate of contraction and relaxation, and prolonged the time to 90% peak shortening and 90% relaxation by agonizing NMDA-R1. HHCY decreased contraction amplitude with the increase in calcium concentration. The recovery of calcium transient was prolonged in HHCY mouse myocyte by agonizing NMDA-R1. It was suggested that HHCY increased mitochondrial NO levels and induced MPT, leading to the decline in myocyte mechanical function by agonizing NMDA-R1. mitochondrial matrix metalloproteinase; myocyte mechanics; calcium transient; mitochondfial permeability; N-methyl-D-aspartate receptor 1

Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-[beta]1 (TGF-[beta]1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-[beta]1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-[beta]1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-[beta]1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-[beta]1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.

Although elevated plasma prorenin levels are commonly found in diabetic patients and correlate with microvascular complications, the pathological role of these increases, if any, remains unclear. Prorenin/ renin binding to the prorenin/renin receptor [(p)RR] enhances the efficiency of angiotensinogen cleavage by renin and unmasks prorenin catalytic activity. We asked whether plasma prorenin could be activated in local vascular tissue through receptor binding. Immunohistochemical staining showing localization of the (p)RR in the aorta to vascular smooth muscle cells (VSMCs). After cultured rat VSMCs were incubated with [10.sup.-7] M inactive prorenin, cultured supernatant acquired the ability to generate ANG I from angiotensinogen, indicating that prorenin had been activated. Activated prorenin facilitated angiotensin generation in cultured VSMCs when exogenous angiotensinogen was added. Small interfering RNA (siRNA) against the (p)RR blocked this activation and subsequent angiotensin generation. Prorenin alone induced dose- and time-dependent increases in mRNA and protein for the profibrotic molecule plasminogen activator inhibitor (PAI)-1, effects that were blocked by siRNA, but not by the ANG II receptor antagonist saralasin. When inactive prorenin and angiotensinogen were incubated with cells, PAI-1 mRNA increased a striking 54-fold, 8-fold higher than the increase seen with prorenin alone. PAI-1 protein increased 2.75-fold. These effects were blocked by treatment with siRNA + saralasin. We conclude that prorenin at high concentration binds the (p)RR on VSMCs and is activated. This activation leads to increased expression of PAI-1 via ANG II-independent and -dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may contribute to the progression of fibrotic disease. diabetes; fibrosis; renin-angiotensin system

Insulin causes translocation of glucose transporter 4 (GLUT4) to the membrane of muscle and fat cells, a process requiting Akt activation. Two Rab-GTPase-activating proteins (Rab-GAP), AS160 and TBC1D1, were identified as Akt substrates. AS160 phosphorylation is required for insulin-stimulated GLUT4 translocation, but the participation of TBC1D1 on muscle cell GLUT4 is unknown. Moreover, there is controversy as to the AS160/ TBC1D1 target Rabs in fat and muscle cells, and Rab effectors are unknown. Here we examined the effect of knockdown of AS160, TBC1D1, and Rabs 8A, 8B, 10, and 14 (in vitro substrates of AS160 and TBC1D1 Rab-GAP activities) on insulin-induced GLUT4 translocation in L6 muscle cells. Silencing AS160 or TBC1D1 increased surface GLUT4 in unstimulated cells but did not prevent insulin-induced GLUT4 translocation. Knockdown of Rab8A and Rab14, but not of Rab8B or Rab10, inhibited insulin-induced GLUT4 translocation. Furthermore, silencing Rab8A or Rab14 but not Rab8B or Rab10 restored the basal-state intracellular retention of GLUT4 impaired by AS 160 or TBC1D1 knockdown. Lastly, overexpression of a fragment of myosin Vb, a recently identified Rab8A-interacting protein, inhibited insulin-induced GLUT4 translocation and altered the subcellular distribution of GTP-loaded Rab8A. These results support a model whereby AS160, Rab8A, and myosin Vb are required for insulin-induced GLUT4 translocation in muscle cells, potentially as part of a linear signaling cascade. glucose transporter 4; insulin signaling; Rab guanosine 5'-triphosphatases; Rab-guanosine 5'-triphosphatase-activating protein; myosin Vb

Plasma C-reactive protein (CRP) concentration is a biomarker of systemic atherosclerosis and may also be associated with vein graft disease. It remains unclear whether CRP is also an important modulator of biological events in the vessel wall. We hypothesized that CRP influences vein graft healing by stimulating smooth muscle cells (SMCs) to undergo a phenotypic switch. Distribution of CRP was examined by immunohistochemistry in prebypass human saphenous veins (HSVs, n = 21) and failing vein grafts (n = 18, 25-4,400 days postoperatively). Quiescent HSV SMCs were stimulated with human CRP (5-50 [micro]g/ml). SMC migration was assessed in modified Boyden chambers with platelet-derived growth factor (PDGF)-BB (5-10 ng/ ml) as the chemoattractant. SMC viability and proliferation were assessed by trypan blue exclusion and reduction of Alamar Blue substrate, respectively. Expression of PDGF ligand and receptor (PDGFR) genes was examined at RNA and protein levels after 24-72 h of CRP exposure. CRP staining was present in 13 of 18 diseased vein grafts, where it localized to the deep media and adventitia, but it was minimally detectable in most prebypass veins. SMCs pretreated with CRP demonstrated a dose-dependent increase in migration to PDGF-BB (P = 0.02), which was inhibited by a PDGF-neutralizing antibody. SMCs treated with CRP showed a dose-dependent increase in PDGFR[beta] expression and phosphorylation after 24-48 h. Exogenous CRP had no effect on SMC viability or proliferation. These data suggest that CRP is detectable within the wall of most diseased vein grafts, where it may exert local effects. Clinically relevant levels of CRP can stimulate SMC migration by a mechanism that may involve upregulation and activation of PDGFR[beta]. C-reactive protein chemistry; vascular smooth muscle cytology; cultured cells; biological markers; metabolism

Colon muscle strips and cells from female patients with slow-transit constipation (STC) exhibit impaired motility, signal transduction abnormalities characterized by downregulation of [G.sub.q/11] and upregulation of [G.sub.s] proteins, decreased cyclooxygenase (COX)-1 and thromboxane (Tx)[B.sub.2] levels, increased COX-2 and [PGE.sub.2] levels, and overexpression of progesterone receptors (PGR). Progesterone ([P.sub.4]) treatment of normal cells reproduced these motility and signal transduction abnormalities. The purpose of the study was to examine whether overexpression of PGR-B reproduces these abnormalities by rendering the cells more sensitive to physiological concentrations of [P.sub.4]. Cultured human colon muscle was transfected with a plasmid DNA expressing PGR-B. The mRNAs of PGR, COX-l, COX-2, and [G.sub.q/11] were determined by quantitative real-time PCR. Their protein expression was determined by Western blot, and prostaglandins were measured by radioimmunoassay. Cultured muscle cells maintained their phenotypic features determined with myosin light chain (MLC) and h-caldesmon antibodies. Control and transfected muscle cells responded to [10.sup.-6] M [P.sub.4]. In contrast, muscle cells transfected with PGR-B responded to lower [P.sub.4] concentration ([10.sup.-7] M). This [P.sub.4] concentration reduced MLC phosphorylation induced by CCK-8 ([10.sup.-8] M), downregulated [G.sub.q/11], and decreased COX-1 and Tx[B.sub.2] levels. It upregulated [G.sub.s] proteins. It also increased COX-2 and [PGE.sub.2] levels. We conclude that overexpression of PGR-B renders the cells more sensitive to physiological concentrations of [P.sub.4]. These results are consistent with the hypothesis that overexpression of PGR-B contributes to the motility and signal transduction abnormalities observed in female patients with STC and normal serum levels of [P.sub.4]. G proteins; cyclooxygenase enzymes; prostaglandins

The role of PKC in the regulation of store-operated [Ca.sup.2+] entry (SOCE) is rather controversial. Here, we used [Ca.sup.2+]-imaging, biochemical, pharmacological, and molecular techniques to test if [Ca.sup.2+]-independent [PLA.sub.2][beta] ([iPLA.sub.2][beta]), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular [Ca.sup.2+] stores and triggered [Ca.sup.2+] influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of [iPLA.sub.2][beta] and [Ca.sup.2+] entry that were absent in SMCs in which [iPLA.sub.2][beta] was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced [Ca.sup.2+] entry only in proliferating SMCs, which correlates with the expression of the specific PKC-[epsilon] isoform. Molecular downregulation of PKC-[epsilon] impaired TG- and OAG-induced [Ca.sup.2+] influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of [Ca.sup.2+] stores as well as direct PKC-[epsilon]-dependent activation of [iPLA.sub.2][beta], resulting in a complex regulation of SOCE in proliferating and confluent SMCs. protein kinase C-[epsilon]; [Ca.sup.2+]-independent phospholipase [A.sub.2]; diacylglycerol; smooth muscle cells

Mechanisms that control vascular smooth muscle cell (SMC) differentiation are poorly understood. We identify Pitx2 as a previously unknown homeodomain transcription factor that is rapidly induced in an in vitro model of SMC differentiation from multipotent stem cells. Pitx2 induces expression of multiple SMC differentiation marker genes by binding to a TAATC(C/T) cis-element, by interacting with serum response factor, and by increasing histone acetylation levels within the promoters of SMC differentiation marker genes. Suppression of Pitx2 reduces expression of SMC differentiation marker genes in the early stages of SMC differentiation in vitro, whereas Prx1, another homeodomain protein, regulates SMC differentiation marker genes in fully differentiated SMCs. Pitx2, but not Prx1, knockout mouse embryos exhibit impaired induction of SMC differentiation markers in the dorsal aorta and branchial arch arteries. Our results demonstrate that Pitx2 functions to regulate the early stages of SMC differentiation.

Mitochondrial biogenesis is involved in the control of cell metabolism, signal transduction, and regulation of mitochondrial reactive oxygen species (ROS) production. Despite the central role of mitochondria in cellular aging and endothelial physiology, there are no studies extant investigating agerelated alterations in mitochondrial biogenesis in blood vessels. Electronmicroscopy and confocal microscopy (en face Mitotracker staining) revealed that in aortas of F344 rats, a decline in mitochondrial biogenesis occurs with aging. In aged vessels, the expression of the mitochondrial biogenesis factors (including mitochondrial transcription factor A and peroxisome proliferator-activated receptor-[gamma]/coactivator-1) was decreased. The vascular expression of complex I, III, and IV significantly declined with age, whereas aging did not alter the expression of complex II and V. Cytochrome c oxidase (COX) expression/activity exhibited the greatest age-related decline, which was associated with increased mitochondrial ROS production in the aged vessels. In cultured coronary arterial endothelial cells, a partial knockdown of COX significantly increased mitochondrial ROS production. In conclusion, vascular aging is characterized by a decline in mitochondrial mass in the endothelial cells and an altered expression of components of the mitochondrial electron transport chain likely due to a dysregulation of mitochondrial biogenesis factors. We posit that impaired mitochondrial biogenesis and downregulation of COX may contribute to the increased mitochondrial oxidative stress in aged endothelial cells. vascular senescence

Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (I[C.sub.50] = 26.1 [+ or -] 0.2 and 27.5 [+ or -] 0.5 [micro]M for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (I[C.sub.50] = 32.3 [+ or -] 0.9 and 31.6 [+ or -] 1.3 [micro]M for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the [Mg.sup.2+]-ATPase activity of the unphosphorylated (I[C.sub.50] = 12.6 [+ or -] 1.6 and 4.3 [+ or -] 0.5 [micro]M for gizzard and bovine stomach, respectively) and phosphorylated (I[C.sub.50] = 15.0 [+ or -] 0.6 [micro]M for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the [Mg.sup.2+]-ATPase activities of gizzard myosin II fragments, heavy meromyosin (I[C.sub.50] = 14.4 [+ or -] 1.6 [micro]M) and subfragment 1 (I[C.sub.50] = 5.5 [+ or -] 0.4 [micro]M), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II. Boyden chamber; fluorescence resonance energy transfer; ATPase; in vitro motility assay; electron microscopy

Postangioplasty and in-stent restenosis remain ominous problems in percutaneous coronary intervention where good animal models of restenosis proneness and resistance are needed. We accidentally discovered that the carotid arteries (CAs) of the Harlan and Sasco substrains of Sprague-Dawley rats display drastically different restenosis phenotypes following balloon-induced endothelial denudation. When subjected to balloon injury, Sasco CAs exhibited significantly larger neointimal mass than did Harlan CAs at both days 14 and 32, as evidenced by a higher intima-to-media ratio and a greater number of intimal cells in Sasco CAs. This was due to a greater cell proliferation and to a less vigorous apoptosis of Sasco neointima, as assessed by 5-bromo-2'-deoxyuridine and terminal deoxynucleotidyl transferase-deoxyuridine nick-end labeling staining, respectively. At a cellular level, whereas vascular smooth muscle cells (VSMCs) isolated from Sasco and Harlan CAs were identical in morphology and in propensity to migrate, Sasco VSMCs proliferated more robustly and died far less, suggesting that under the exact same microenvironment, Sasco and Harlan VSMCs respond to growth and noxious stimuli in a drastically different fashion and that Sasco's significantly more robust neointimal proliferation after vascular injury in vivo can be accounted for by these intrinsic differences in VSMCs of these substrains in vitro. Sasco and Harlan Sprague-Dawley rats as well as VSMCs from these rats will prove to be powerful tools to study genes involved in the pathogenesis of restenosis. Harlan rats; Sasco rats; carotid artery injury; vascular smooth muscle cells; terminal deoxynucleotidyl transferase-deoxyuridine nick-end labeling; 5-bromo-2'-deoxyuridine

Surfactant protein D (SP-D) is a constituent of the innate immune system that plays a role in the host defense against lung pathogens and in modulating inflammatory responses. While SP-D has been detected in extrapulmonary tissues, little is known about its expression and function in the vasculature. Immunostaining of human coronary artery tissue sections demonstrated immunoreactive SP-D protein in smooth muscle cells (SMCs) and endothelial cells. SP-D was also detected in isolated human coronary artery SMCs (HCASMCs) by PCR and immunoblot analysis. Treatment of HCASMCs with endotoxin (LPS) stimulated the release of IL-8, a proinflammatory cytokine. This release was inhibited >70% by recombinant SP-D. Overexpression of SP-D by adenoviral-mediated gene transfer in HCASMCs inhibited both LPS-and TNF-[alpha]-induced IL-8 release. Overexpression of SP-D also enhanced uptake of Chlamydia pneumoniae elementary bodies into HCASMCs while attenuating IL-8 production induced by bacterial exposure. Both LPS and TNF-[alpha] increased SP-D mRNA levels by five-to eightfold in HCASMCs, suggesting that inflammatory mediators upregulate the expression of SP-D. In conclusion, SP-D is expressed in human coronary arteries and functions as an anti-inflammatory protein in HCASMCs. SP-D may also participate in the host defense against pathogens that invade the vascular wall. inflammation; Chlamydia pneumoniae; innate immunity; interleukin-8

The numerous nonmyocytes present within the myocardium may establish electrical connections with myocytes through gap junctions, formed naturally or as a result of a cell therapy. The strength of the coupling and its potential impact on action potential characteristics and conduction are not well understood. This study used computer simulation to investigate the load-induced electrophysiological consequences of the coupling of myocytes with fibroblasts, where the fibroblast resting potential, density, distribution, and coupling strength were varied. Conduction velocity (CV), upstroke velocity, and action potential duration (APD) were analyzed for longitudinal and transverse impulse propagation in a two-dimensional microstructure tissue model, developed to represent a monolayer culture of cardiac cells covered by a layer of fibroblasts. The results show that 1) at weak coupling (8 nS), all of the effects saturated; and 4) APD at 90% repolarization was usually prolonged by 0-20 ms (up to 60-80 ms for high fibroblast density and coupling) by the coupling to fibroblasts. The changes in APD depended on the fibroblast resting potential. This complex, coupling-dependent interaction of fibroblast and myocytes also has relevance to the integration of other nonmyocytes in the heart, such as those used in cellular therapies. computer modeling; electrophysiology; nonmyocytes; gap junctions; fibrosis

This study determined the role of a slowly inactivating component of sodium current ([I.sub.Na]), late [I.sub.Na], to induce delayed afterdepolarizations (DADs) and triggered activity. We hypothesized that an increase of late [I.sub.Na] may induce not only early afterdepolarizations (EADs), but also intracellular calcium overload and DADs. Guinea pig atrial myocytes were studied using the whole cell patch-clamp technique. Anemone toxin II (ATX-II) (5-10 nmol/l) was used to enhance late [I.sub.Na]. Ranolazine (10 [micro]mol/l) and TTX (2 [micro]mol/l) were applied to block ATX-II-induced late [I.sub.Na]. ATX-II prolonged action potential duration and induced EADs. In the continuous presence of ATX-II, following the appearance of EADs, both DADs and sustained triggered activity occurred. Triggered activity was abolished and DADs were reduced by either ranolazine or TTX. Consistent with induction of DADs, ATX-II induced the transient inward current ([I.sub.Ti]). The amplitude of [I.sub.Ti] was significantly reduced by ranolazine. ATX-II induced only EADs, but no DADs, in the presence of the sodium-calcium exchange inhibitor KB-R7943 or the sarcoplasmic reticulum calcium release channel inhibitor ryanodine, or when the calcium chelator EGTA or BAPTA was included in the pipette solution. In conclusion, an increase of late [I.sub.Na], in addition to inducing EADs, can cause cellular calcium overload and induce DADs and sustained triggered activity in atrial myocytes. The data reveal that an increase of late [I.sub.Na] is a novel mechanism for initiation of atrial arrhythmic activity. action potential; calcium overload; transient inward current

Recent studies suggest members of the degenerin (DEG)/epithelial [Na.sup.+] channel (ENaC)/acid-sensing ion channel (ASIC) protein family play an important role in vascular smooth muscle cell (VSMC) migration. In a previous investigation, we found suppression of a certain DEG/ENaC/ASIC member, ASIC2, increased VSMC chemotactic migration, raising the possibility that ASIC2 may play an inhibitory role. Because ASIC2 protein was retained in the cytoplasm, we reasoned increasing surface expression of ASIC2 might unmask the inhibitory role of ASIC2 in VSMC migration so we could test the hypothesis that ASIC2 inhibits VSMC migration. Therefore, we used the chemical chaperone glycerol to enhance ASIC2 expression. Glycerol 1) increased cytoplasm ASIC2 expression, 2) permitted detection of ASIC2 at the cell surface, and 3) inhibited platelet-derived growth factor (PDGF)-bb mediated VSMC migration. Furthermore, ASIC2 silencing completely abolished the inhibitory effect of glycerol on migration, suggesting upregulation of ASIC2 is responsible for glycerol-induced inhibition of VSMC migration. Because other investigators have shown that glycerol regulates ENaC/ASIC via interactions with a certain heat shock protein, heat shock protein 70 (Hsc70), we wanted to determine the importance of Hsc70 on ASIC2 expression in VSMCs. We found that Hsc70 silencing increases ASIC2 cell surface expression and inhibits VSMC migration, which is abolished by cosilencing ASIC2. These data demonstrate that Hsc70 inhibits ASIC2 expression, and, when the inhibitory effect of Hsc70 is removed, ASIC2 expression increases, resulting in reduced VSMC migration. Because VSMC migration contributes to vasculogenesis and remodeling following vascular injury, our findings raise the possibility that ASIC2-Hsc70 interactions may play a role in these processes. vascular smooth muscle cell migration; ion channel; degenerin proteins; acid-sensing ion channel; heat shock protein 70; interference ribonucleic acid

We assessed the roles of the protein kinase C (PKC) and the tyrosine kinase (TK) signaling pathways in regulating capacitative calcium entry (CCE) in human pulmonary artery smooth muscle cells (PASMCs) and investigated the effects of intravenous anesthetics (midazolam, propofol, thiopental, ketamine, etomidate, morphine, and fentanyl) on CCE in human PASMCs. Fura-2-loaded human PASMCs were placed in a dish (37[degrees]C) on an inverted fluorescence microscope. Intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) was measured as the 340/380 fluorescence ratio in individual PASMCs. Thapsigargin, a sarcoplasmic reticulum [Ca.sup.2+]-adenosine triphosphatase inhibitor, was used to deplete intracellular [Ca.sup.2+] stores after removing extracellular [Ca.sup.2+]. CCE was then activated by restoring extracellular [Ca.sup.2+] (2.2 mM). The effects of PKC activation and inhibition, TK inhibition, and the intravenous anesthetics on CCE were assessed. Thapsigargin caused a transient increase in [[[Ca.sup.2+]].sub.i]. Restoring extracellular [Ca.sup.2+] caused a rapid peak increase in [[[Ca.sup.2+]].sub.i], followed by a sustained increase in [[[Ca.sup.2+]].sub.i]; i.e., CCE was stimulated in human PASMCs. PKC activation attenuated (P < 0.05), whereas PKC inhibition potentiated (P < 0.05), both peak and sustained CCE. TK inhibition attenuated (P < 0.05) both peak and sustained CCE. Midazolam, propofol, and thiopental each attenuated (P < 0.05) both peak and sustained CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. Our results suggest that CCE in human PASMCs is influenced by both the TK and PKC signaling pathways. Midazolam, propofol, and thiopental each attenuated CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. protein kinase C; tyrosine kinase

Ornithine decarboxylase (ODC) is the first and rate-controlling enzyme in the synthesis of polyamines, which are essential for normal cell growth. We have previously demonstrated that IL-4 and IL-13 can stimulate rat aortic smooth muscle cell (RASMC) proliferation. The objective of this study was to determine whether IL-4 and IL-13 induce cell proliferation by upregulating ODC expression in RASMC. The results revealed that incubation of RASMC with IL-4 and IL-13 for 24 h caused four- to fivefold induction of ODC catalytic activity. The increased ODC catalytic activity was attributed to the increased expression of ODC mRNA. Moreover, these observations were paralleled by increased production of polyamines. We further investigated the signal transduction pathways responsible for ODC induction by IL-4 and IL-13. The data illustrated that PD-98059, a MEK (MAPK kinase) inhibitor, LY-294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and H-89, a protein kinase A (PKA) inhibitor, substantially decreased the induction of ODC catalytic activity and ODC mRNA expression induced by IL-4 and IL-13, suggesting positive regulation of the ODC gene by ERK, PI3K, and PKA pathways. Interestingly, dexamethasone, a known inhibitor of cell proliferation, completely abrogated the response of RASMC to IL-4 and IL-13. Furthermore, the inhibition of ODC by these inhibitors led to the reduced production of polyamines and decreased DNA synthesis as monitored by [[sup.3] H] thymidine incorporation. Our data indicate that upregulation of ODC by IL-4 and IL-13 might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth. cytokines; rat aortic smooth muscle cells; cell proliferation; polyamines; ornithine decarboxylase

Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of G protein-coupled P2Y purinergic receptors. We have previously shown that ATP stimulates a transient activation of protein kinase A (PKA), which, together with the established mitogenic signaling of purinergic receptors, promotes proliferation of VSMC (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449-C456, 2004). We also have shown that PKA can phosphorylate [beta]-catenin at two novel sites (Ser552 and Ser675) in vitro and in overexpression cell models (Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. J Biol Chem 281: 9971-9976, 2006). [beta]-Catenin promotes cell proliferation by activation of a family of T-cell factor (TCF) transcription factors, which drive the transcription of genes implicated in cell cycle progression including cyclin D1. In the present study, using the phosphospecific antibodies against phosphoSer552 or phospho-Ser675 sites of [beta]-catenin, we show that ATP can stimulate PKA-dependent phosphorylation of endogenous [beta]-catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA-dependent stimulation of TCF transcriptional activity through an increased association of phosphorylated (by PKA) [beta]-catenin with TCF-4. Using the PKA inhibitor PKI or dominant negative TCF-4 mutant, we show that ATP-induced cyclin D1 promoter activation, cyclin D1 protein expression, and proliferation of VSMC are all dependent on PKA and TCF activities. In conclusion, we show a novel mode of regulation of endogenous [beta]-catenin through its phosphorylation by PKA, and we demonstrate the importance of this mechanism for ATP-induced proliferation of VSMC. purinergic; protein kinase A; cyclin D1

Syncoilin is a striated muscle-specific intermediate filament-like protein, which is part of the dystrophin-associated protein complex (DPC) at the sarcolemma and provides a link between the extracellular matrix and the cytoskeleton through its interaction with [alpha]-dystrohrevin and desmin. Its upregulation in various neuromuscular diseases suggests that syncoilin may play a role in human myopathies. To study the functional role of syncoilin in cardiac and skeletal muscle in vivo, we generated syncoilin-deficient ([syncoilin.sup.-/-]) mice. Our detailed analysis of these mice up to 2 yr of age revealed that syncoilin is entirely dispensable for cardiac and skeletal muscle development and maintenance of cellular structure but is required for efficient lateral force transmission during skeletal muscle contraction. Notably, [syncoilin.sup.-/-] skeletal muscle generates less maximal isometric stress than wild-type (WT) muscle but is as equally susceptible to eccentric contraction-induced injury as WT muscle. This suggests that syncoilin may play a supportive role for desmin in the efficient coupling of mechanical stress between the myofibril and fiber exterior. It is possible that the reduction in isometric stress production may predispose the syncoilin skeletal muscle to a dystrophic condition. intermediate filament; sarcomere; mutant mouse

Store-operated channels (SOC) and store-operated [Ca.sup.2+] entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type [Ca.sup.2+] ([Ca.sup.2+.sub.L]) channels are thought to be fully responsible for agonist-induced [Ca.sup.2+] influx and vasoconstriction. We present evidence that SOC and their activation via a [Ca.sup.2+] -independent phospholipase [A.sub.2] ([iPLA.sub.2])-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular [Ca.sup.2+] in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) [Ca.sup.2+] and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a [Ca.sup.2.sub.L+] inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and [Ca.sup.2.sub.L+] channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of [iPLA.sub.2][beta] totally inhibited PE-induced [Ca.sup.2+] influx and constriction in cerebral, mesenteric, and carotid arteries, whereas [K.sup.+]-induced [Ca.sup.2+] influx and vasoconstriction mediated by [Ca.sup.2+.sub.L] channels were not affected. Thus [iPLA.sub.2]-dependent activation of SOC is crucial for agonist-induced [Ca.sup.2+] influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of [Ca.sup.2+] stores, nonselective SOC are activated via an [iPLA.sub.2]-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of [Ca.sup.2+.sub.L] channels and lead to [Ca.sup.2+] entry and vasoconstriction. store-operated calcium entry; smooth muscle cells; constriction

We have previously shown that interleukin (IL)-1[beta], transforming growth factor (TGF)-[[beta].sub.1], or bradykinin (BK) impair cAMP generation in response to prostacyclin analogs in human pulmonary artery smooth muscle (PASM), suggesting that inflammation can impair the effects of prostacyclin analogs on PASM in pulmonary hypertension. Here we explored the biochemical mechanisms involved. We found that IL-1[beta], BK, and TGF-[[beta].sub.1] reduced adenylyl cyclase isoform 1, 2, and 4 mRNA, increased G[[alpha].sub.i] protein levels, and reduced prostacyclin receptor (IP receptor) mRNA expression. In contrast, G[[alpha].sub.s] protein levels were unchanged. Protein kinase A (PKA) (H-89, KT-2750, PKIm) and p38 mitogen-activated protein (MAP) kinase (SB-202190) inhibitors attenuated these effects, but protein kinase C (bisindolylmaleide) or phosphoinositol 3-kinase (LY-294002) inhibitors did not. Fluorescent kemptide assay and Western blotting confirmed that PKA and p38 MAP kinase were activated by 1L-1[beta], BK, and TGF-[[beta].sub.1]. These studies suggest that IL-1[beta] BK, and TGF-[[beta].sub.1] impair IP receptor-mediated cAMP accumulation by multiple effects on different components of the signaling pathway and that these effects are PKA and p38 MAP kinase dependent. pulmonary artery smooth muscle; kinases; adenosine 3',5'-cyclic monophosphate; prostacyclin receptor

Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors ([NK.sub.1] R, [NK.sub.2]R, and [NK.sub.3]R) classically couple to [G.sub.q] protein-mediated inositol 1,4,5-trisphosphate ([IP.sub.3]) synthesis and liberation of intracellular [Ca.sup.2+], which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-l-based lentivirus transduction system. Specific NKR agonists {[NK.sub.1]R, [[Sar.sup.9],Met[([O.sub.2]).sup.11]] substance P; [NK.sub.2]R, [beta]-[Ala.sup.8]]-neurokinin A(4-10); [NK.sub.3]R, senktide} stimulated inositol phosphate synthesis and increased intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+].sub.i]]) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists ([NK.sub.1]R, L-732138; [NK.sub.2]R, GR-159897, [NK.sub.3]R, SB-222200). The initial transient and sustained phases of increased [[[Ca.sup.2+]].sub.i] were predominantly inhibited by the [IP.sub.3] receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated [Ca.sup.2+] channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that [IP.sub.3] levels are the primary mediators of NKR-stimulated initial [[[Ca.sup.2+]].sub.i] increases, whereas store-operated [Ca.sup.2+] channels mediate the sustained phase of the [[[Ca.sup.2+]].sub.i] increase. intracellular calcium: ryanodine: actin: myosin; lentivirus

The present experiments were performed to establish a method for simultaneous monitoring of excitation and contraction in isolated smooth muscle ceils. The smooth muscle cells were dissociated from the colons of Wistar rats by enzymatic digestion. All the experiments were performed on mixtures of circular and longitudinal cells. In a first set of experiments, focal extracellular potentials (FEPs) and transmembrane action potentials (APs) were simultaneously recorded from the cells by use of extracellular and intracellular pipettes, respectively. In a second set of experiments, cellular contraction induced by chemical stimulation was monitored simultaneous with the FEP recordings. The FEPs had spike and plateau amplitudes of 44.5 [+ or -] 2.3 and 8.9 [+ or -] 0.7 mV, respectively, and reproduced the general morphology of gastrointestinal APs. The parallel mechanical measurements from the rat colonic cells showed that they shortened with an average peak contraction of 8.8 [+ or -] 1.4 [micro]m and an average contraction velocity of 8.2 [+ or -] 0.9 [micro]m/s, to develop an average peak force of 1.2 [+ or -] 0.2 [micro]N, and generated an average peak power of 36 [+ or -] 15 pW. Simultaneous monitoring of FEPs and cellular contraction demonstrates correlations between the electrical and mechanical events taking place in the investigated cells. isolated cells; local extracellular potential; cellular contraction

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of [alpha]-galactosidase A that affects males and shows disease expression in heterozygotes. The characteristic progressive renal insufficiency, cardiac involvement, and neuropathology usually are ascribed to globotriaosylceramide accumulation in the endothelium. However, no direct correlation exists between lipid storage and clinical manifestations, and treatment of patients with recombinant enzymes does not reverse several key signs despite clearance of lipid from the endothelium. We therefore investigated the possibility that globotriaosylceramide metabolites are a missing link in the pathogenesis. We report that deacylated globotriaosylceramide, globotriaosylsphingosine, and a minor additional metabolite are dramatically increased in plasma of classically affected male Fabry patients and plasma and tissues of Fabry mice. Plasma globotriaosylceramide levels are reduced by therapy. We show that globotriaosylsphingosine is an inhibitor of [alpha]-galactosidase A activity. Furthermore, exposure of smooth muscle cells, but not fibroblasts, to globotriaosylsphingosine at concentrations observed in plasma of patients promotes proliferation. The increased intima-media thickness in Fabry patients therefore may be related to the presence of this metabolite. Our findings suggest that measurement of circulating globotriaosylsphingosine will be useful to monitor Fabry disease and may contribute to a better understanding of the disorder. [alpha]-galactosidase A | globotriaosylceramide | smooth muscle cell | lysoglycolipids

Prolonged exposure to decreased oxygen tension causes contraction and proliferation of pulmonary arterial smooth muscle cells (PASMCs) and pulmonary hypertension. Hypoxia-induced inhibition of voltage-gated [K.sup.+] ([K.sub.v]) channels may contribute to the development of pulmonary hypertension by increasing intracellular calcium concentration ([[Ca.sup.2+]][sub.i]). The peptide endothelin-1 (ET-1) has been implicated in the development of pulmonary hypertension and acutely decreases [K.sub.v] channel activity. ET-1 also activates several transcription factors, although whether ET-1 alters [K.sub.v] channel expression is unclear. The hypoxic induction of ET-1 is regulated by the transcription factor hypoxia-inducible factor-1 (HIF1), which we demonstrated to regulate hypoxia-induced decreases in [K.sub.v] channel activity. In this study, we tested the hypothesis that HIF-l-dependent increases in ET-1 lead to decreased [K.sub.v] channel expression and subsequent elevation in [[Ca.sup.2+]][sub.i]. Resting [[Ca.sup.2+]][sub.i] and [K.sub.v] channel expression were measured in cells exposed to control (18% 02, 5% C[O.sub.2]) and hypoxic (4% [O.sub.2], 5% C[O.sub.2]) conditions. Hypoxia caused a decrease in expression of [K.sub.v]l.5 and [K.sub.v]2.1 and a significant increase in resting [[Ca.sup.2+]][sub.i]. The increase in [[Ca.sup.2+]][sub.i] was reduced by nifedipine, an inhibitor of voltage-dependent calcium channels, and removal of extracellular calcium. Treatment with BQ123, an ET-1 receptor inhibitor, prevented the hypoxia-induced decrease in [K.sub.v] channel expression and blunted the hypoxia-induced increase in [[Ca.sup.2+]][sub.i] in PASMCs, whereas ET-1 mimicked the effects of hypoxia. Both hypoxia and overexpression of HIF-1 under normoxic conditions increased ET-1 expression. These results suggest that the inhibition of [K.sub.v] channel expression and rise in [[Ca.sup.2+]][sub.i] during chronic hypoxia may be the result of HIF-1-dependent induction of ET-1. hypoxia-inducible factor-l; pulmonary hypertension