bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of MARCKS in regulating endothelial cell proliferation
Authors: Zhao Ying , Neltner Bonnie S. , Davis Harold W.
Published: December 2017

Myristoylated alanine-rich C kinase substrate (MARCKS), as a specific protein kinase C (PKC) substrate, mediates PKC signaling through its phosphorylation and subsequent modification of its association with filamentous actin (F-actin) and calmodulin (CaM). PKC has long been implicated in cell proliferation, and recent studies have suggested that MARCKS may function as a cell growth suppressor. Therefore, in the present study, we investigated MARCKS protein expression, distribution, and phosphorylation in preconfluent and confluent bovine pulmonary microvascular endothelial cells (BPMEC) in the presence or absence of the vascular endothelial growth factor (VEGF). In addition, we examined functional alterations of MARCKS in these cells by studying the association of MARCKS with F-actin and CaM-dependent myosin light chain (MLC) phosphorylation. Our results indicate that MARCKS protein is downregulated during BPMEC proliferation. Decreased MARCKS association with F-actin, increased actin polymerization, and CaM-dependent MLC phosphorylation appear to mediate cell shape changes and motility during BPMEC growth. In contrast, VEGF stimulated MARCKS phosphorylation without alteration of protein expression during BPMEC proliferation, which may result in reduced interaction between MARCKS and actin or CaM, leading to actin reorganization and MLC phosphorylation. Our data suggest a regulatory role of MARCKS during endothelial cell proliferation.
Cation transport and cell volume changes in maturing rat reticulocytes
Authors: Mairbäurl Heimo , Schulz Susanne , Hoffman Joseph F.
Published: December 2017

During maturation, reticulocytes lose membrane material, including transporters, and this is accompanied by a loss of cell water and volume. Here we determined a possible role of ion transport in adjusting cell volume during maturation. Reticulocytes and red blood cells of different ages were prepared from erythropoietin-treated rats by density gradient fractionation. Cell volume and ion transport were measured in freshly prepared cells and in reticulocytes during in vitro maturation. Reticulocytes had an increased K content and cell volume, whereas intracellular Na was decreased. All parameters approached whole blood values after 2 days in culture. Na-K pump was elevated in reticulocytes and decreased during maturation. Na-K-2Cl cotransport (NKCC) activity was lower in reticulocytes and was activated 8- and 20-fold by shrinkage and okadaic acid, respectively, whereas stimulation was barely detectable in high-buoyant density red blood cells. The ouabain- and bumetanide-insensitive Na flux in reticulocytes decreased on maturation. Most of it was inhibited by amiloride, indicating the presence of Na/proton exchange. Our results show that, although the Na-K-pump activity in reticulocytes is very much increased, the enhanced capacity of NKCC is essentially cryptic until stimulated. Both types of capacities (activities) decrease during maturation, indicating a possible loss of transport protein. The decrease was constrained to the period of reticulocyte maturation. Loss of transport capacity appears to exceed the loss of membrane surface area. Reticulocyte age-related changes in the net electrochemical driving force indicate that the increasing NKCC activity might contribute to the reduction in cell water.
Stimulation of unitary T-type Ca2+ channel currents by calmodulin-dependent protein kinase II
Authors: Barrett Paula Q. , Lu Hong-Kai , Colbran Roger , Czernik Andrew , Pancrazio Joseph J.
Published: December 2017

The effect of Ca2+/calmodulin-dependent protein kinase II (CaMKII) stimulation on unitary low voltage-activated (LVA) T-type Ca2+ channel currents in isolated bovine adrenal glomerulosa (AG) cells was measured using the patch-clamp technique. In cell-attached and inside-out patches, LVA channel activity was identified by voltage-dependent inactivation and a single-channel conductance of ∼9 pS in 110 mM BaCl2 or CaCl2. In the cell-attached patch, elevation of bath Ca2+ from 150 nM to 1 μM raised intracellular Ca2+ in K+-depolarized (140 mM) cells and evoked an increase in the LVA Ca2+ channel probability of opening ( NP o) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank sweeps, a rise in the frequency of channel opening in nonblank sweeps, and a 30% reduction in first latency. No apparent changes in the single-channel open-time distribution, burst lengths, or openings/burst were apparent. Preincubation of AG cells with lipophilic or peptide inhibitors of CaMKII in the cell-attached or excised (inside-out) configurations prevented the rise in NP oelicited by elevated Ca2+ concentration. Furthermore, administration of a mutant recombinant CaMKIIα exhibiting cofactor-independent activity in the absence of elevated Ca2+ produced a threefold elevation in LVA channel NP o. These data indicate that CaMKII activity is both necessary and sufficient for LVA channel activation by Ca2+.
β-Amyloid-induced migration of monocytes across human brain endothelial cells involves RAGE and PECAM-1
Authors: Giri Ranjit , Shen Yamin , Stins Monique , Du Yan Shi , Schmidt Ann Marie , Stern David , Kim Kwang-Sik , Zlokovic Berislav , Kalra Vijay K.
Published: December 2017

In patients with amyloid β-related cerebrovascular disorders, e.g., Alzheimer's disease, one finds increased deposition of amyloid peptide (Aβ) and increased presence of monocyte/microglia cells in the brain. However, relatively little is known of the role of Aβ in the trafficking of monocytes across the blood-brain barrier (BBB). Our studies show that interaction of Aβ1–40 with monolayer of human brain endothelial cells results in augmented adhesion and transendothelial migration of monocytic cells (THP-1 and HL-60) and peripheral blood monocytes. The Aβ-mediated migration of monocytes was inhibited by antibody to Aβ receptor (RAGE) and platelet endothelial cell adhesion molecule (PECAM-1). Additionally, Aβ-induced transendothelial migration of monocytes were inhibited by protein kinase C inhibitor and augmented by phosphatase inhibitor. We conclude that interaction of Aβ with RAGE expressed on brain endothelial cells initiates cellular signaling leading to the transendothelial migration of monocytes. We suggest that increased diapedesis of monocytes across the BBB in response to Aβ present either in the peripheral circulation or in the brain parenchyma may play a role in the pathophysiology of Aβ-related vascular disorder.
Formation of sarcomeres in developing myotubes: role of mechanical stretch and contractile activation
Authors: De Deyne Patrick G.
Published: December 2017

In a series of experiments, cultured myotubes were exposed to passive stretch or pharmacological agents that block contractile activation. Under these experimental conditions, the formation of Z lines and A bands (morphological structures, resulting from the specific structural alignment of sarcomeric proteins, necessary for contraction) was assessed by immunofluorescence. The addition of an antagonist of the voltage-gated Na+ channels [tetrodotoxin (TTX)] for 2 days in developing rat myotube cultures led to a nearly total absence of Z lines and A bands. When contractile activation was allowed to resume for 2 days, the Z lines and A bands reappeared in a significant way. The appearance of Z lines or A bands could not be inhibited nor facilitated by the application of a uniaxial passive stretch. Electrical stimulation of the cultures increased sarcomere assembly significantly. Antagonists of L-type Ca2+ channels (verapamil, nifedipine) combined with electrical stimulation led to the absence of Z lines and A bands to the same degree as the TTX treatment. Western blot analysis did not show a major change in the amount of sarcomeric α-actinin nor a shift in myosin heavy chain phenotype as a result of a 2-day passive stretch or TTX treatment. Results of experiments suggest that temporal Ca2+ transients play an important factor in the assembly and maintenance of sarcomeric structures during muscle fiber development.
Shear stress induces iNOS expression in cultured smooth muscle cells: role of oxidative stress
Authors: Gosgnach Willy , Messika-Zeitoun David , Gonzalez Walter , Philipe Monique , Michel Jean-Baptiste
Published: December 2017

After deendothelialization, the most luminal smooth muscle cells of the neointima are in contact with blood flow and express inducible nitric oxide synthase (iNOS) in vivo. We hypothesized that shear stress may be a stimulus for this iNOS overexpression. We have thus submitted smooth muscle cells to laminar shear and measured the iNOS expression. Shear stress (20 dyn/cm2) induced iNOS mRNA and protein expression, whereas brain NOS mRNA expression was decreased. Conversely, nitrite production was increased. This production was blocked by a selective iNOS inhibitor. Pyrrolidine dithiocarbamate, an antioxidant molecule, and BXT-51072, a gluthation peroxidase mimic, both inhibited the shear-induced iNOS expression. Shear stress also increased the expression of both membrane subunits of NADPH oxidase p22phox and Mox-1. Shear stress activated the redox-sensitive nuclear translocation of the transcription nuclear factor-κB (NF-κB) and stimulated the degradation of both cytosolic inhibitors κB α and β. These results show that shear stress can induce iNOS expression and nitrite production in smooth muscle cells and suggest that this regulation is probably mediated by oxidative stress-induced NF-κB activation.
Transepithelial resistance can be regulated by the intestinal brush-border Na+/H+ exchanger NHE3
Authors: Turner Jerrold R. , Black Eric D. , Ward Jeff , Tse Chung-Ming , Uchwat Frederick A. , Alli Halima A. , Donowitz Mark , Madara James L. , Angle Jason M.
Published: December 2017

Initiation of intestinal Na+-glucose cotransport results in transient cell swelling and sustained increases in tight junction permeability. Since Na+/H+ exchange has been implicated in volume regulation after physiological cell swelling, we hypothesized that Na+/H+ exchange might also be required for Na+-glucose cotransport-dependent tight junction regulation. In Caco-2 monolayers with active Na+-glucose cotransport, inhibition of Na+/H+ exchange with 200 μM 5-( N, N-dimethyl)- amiloride induced 36 ± 2% increases in transepithelial resistance (TER). Evaluation using multiple Na+/H+ exchange inhibitors showed that inhibition of the Na+/H+ exchanger 3 (NHE3) isoform was most closely related to TER increases. TER increases due to NHE3 inhibition were related to cytoplasmic acidification because cytoplasmic alkalinization with 5 mM NH4Cl prevented both cytoplasmic acidification and TER increases. However, NHE3 inhibition did not affect TER when Na+-glucose cotransport was inhibited. Myosin II regulatory light chain (MLC) phosphorylation decreased up to 43 ± 5% after inhibition of Na+/H+ exchange, similar to previous studies that associate decreased MLC phosphorylation with increased TER after inhibition of Na+-glucose cotransport. However, NHE3 inhibitors did not diminish Na+-glucose cotransport. These data demonstrate that inhibition of NHE3 results in decreased MLC phosphorylation and increased TER and suggest that NHE3 may participate in the signaling pathway of Na+-glucose cotransport-dependent tight junction regulation.
Severe hypoxemia in the absence of blood loss causes a gender dimorphic immune response
Authors: Knöferl Markus W. , Jarrar Doraid , Schwacha Martin G. , Angele Martin K. , Cioffi William G. , Bland Kirby I. , Chaudry Irshad H.
Published: December 2017

A gender dimorphic immune response has been observed after trauma and severe hemorrhage, a condition believed to be associated with tissue hypoxia. Although studies have shown that hypoxemia per se in males causes a systemic inflammatory response, it is unclear if the inflammatory response to hypoxemia exhibits gender dimorphic characteristics. To study this, male and female C3H/HeN mice in the proestrus state of the estrous cycle were subjected to hypoxemia (95% N2-5% O2) or sham hypoxemia (room air) for 60 min. Later (2 h), plasma interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels were determined along with splenic immune responses. Plasma IL-6 and TNF-α concentrations after hypoxemia were significantly increased in males but not in females. Splenocyte proliferation was depressed in males after hypoxemia but not in females. A shift toward an immunosuppressive Th-2 cytokine profile was observed in males after hypoxemia [decreased interferon-γ (Th-1) and increased IL-10 (Th-2)], whereas no such shift was observed in females. Splenic macrophage IL-6, IL-10, and IL-12 production were suppressed in males after hypoxemia; however, such suppression was not observed in females. These findings therefore indicate that a gender dimorphic immune response also exists after hypoxemia in the absence of blood loss and tissue trauma, similar to trauma-hemorrhage. Furthermore, because no systemic inflammatory response or alterations in T lymphocyte or macrophage functions are observed in proestrus females but such parameters are markedly altered after severe hypoxemia in males, these studies indicate that proestrus females can tolerate hypoxemia better than males.
Serine phosphorylation of p60 tumor necrosis factor receptor by PKC-δ in TNF-α-activated neutrophils
Authors: Kilpatrick Laurie E. , Song Young-Han , Rossi Michael W. , Korchak Helen M.
Published: December 2017

Tumor necrosis factor-α (TNF-α) triggers degranulation and oxygen radical release in adherent neutrophils. The p60TNF receptor (p60TNFR) is responsible for proinflammatory signaling, and protein kinase C (PKC) is a candidate for the regulation of p60TNFR. Both TNF-α and the PKC-activator phorbol 12-myristate 13-acetate triggered phosphorylation of p60TNFR. Receptor phosphorylation was on both serine and threonine but not on tyrosine residues. The PKC-δ isotype is a candidate enzyme for serine phosphorylation of p60TNFR. Staurosporine and the PKC-δ inhibitor rottlerin inhibited TNF-α-triggered serine but not threonine phosphorylation. Serine phosphorylation was associated with receptor desensitization, as inhibition of PKC resulted in enhanced degranulation (elastase release). After neutrophil activation, PKC-δ was the only PKC isotype that associated with p60TNFR within the correct time frame for receptor phosphorylation. In vitro, only PKC-δ, but not the α-, βI-, βII-, or ζ-isotypes, was competent to phosphorylate the receptor, indicating that p60TNFR is a direct substrate for PKC-δ. These findings suggest a selective role for PKC-δ in negative regulation of the p60TNFR and of TNF-α-induced signaling.
Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells
Authors: Janiak Robert , Wilson Sean M. , Montague Stephen , Hume Joseph R.
Published: December 2017

To examine the nature of inositol 1,4,5-trisphosphate (IP3)-sensitive and ryanodine (Ryn)-sensitive Ca2+ stores in isolated canine pulmonary arterial smooth cells (PASMC), agonist-induced changes in global intracellular Ca2+ concentration ([Ca2+]i) were measured using fura 2-AM fluorescence. Properties of elementary local Ca2+release events were characterized using fluo 3-AM or fluo 4-AM, in combination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca2+ stores with Ryn (300 μM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-induced intracellular Ca2+ transients but had little or no effect on the initial IP3-mediated intracellular Ca2+transient induced by ANG II (1 μM). Cyclopiazonic acid (CPA; 10 μM) abolished IP3-induced intracellular Ca2+ transients but failed to attenuate the initial Caf-induced intracellular Ca2+ transient. These results suggest that in canine PASMC, IP3-, and Ryn-sensitive Ca2+ stores are organized into spatially distinct compartments while similar experiments in canine renal arterial smooth muscle cells (RASMC) reveal that these Ca2+ stores are spatially conjoined. In PASMC, spontaneous local intracellular Ca2+ transients sensitive to modulation by Caf and Ryn were detected, exhibiting spatial-temporal characteristics similar to those previously described for “Ca2+ sparks” in cardiac and other types of smooth muscle cells. After depletion of Ryn-sensitive Ca2+ stores, ANG II (8 nM) induced slow, sustained [Ca2+]i increases originating at sites near the cell surface, which were abolished by depleting IP3stores. Discrete quantal-like events expected due to the coordinated opening of IP3 receptor clusters (“Ca2+puffs”) were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca2+ stores and elementary Ca2+ release events in isolated PASMC.
A role for PYK2 in regulation of ERK1/2 MAP kinases and PI 3-kinase by ANG II in vascular smooth muscle
Authors: Rocic Petra , Govindarajan Geetha , Sabri Abdelkarim , Lucchesi Pamela A.
Published: December 2017

Abnormal vascular smooth muscle cell (VSMC) growth plays a key role in the pathogenesis of hypertension and atherosclerosis. Angiotensin II (ANG II) elicits a hypertrophic growth response characterized by an increase in protein synthesis without cell proliferation. The present study investigated the role of the nonreceptor tyrosine kinase PYK2 in the regulation of ANG II-induced signaling pathways that mediate VSMC growth. Using coimmunoprecipitation analysis, the role of PYK2 as an upstream regulator of both extracellular signal-related kinase (ERK) 1/2 mitogen-activated protein kinase and phosphatidylinositol 3-kinase (PI 3-kinase) pathways was examined in cultured rat aortic VSMC. ANG II (100 nM) promoted the formation of a complex between PYK2 and the ERK1/2 regulators Shc and Grb2. ANG II caused a rapid and Ca2+-dependent tyrosine phosphorylation of the adapter molecule p130Cas, which coimmunoprecipitated both PYK2 and PI 3-kinase in ANG II-treated VSMC. Complex formation between PI 3-kinase and p130Cas and PYK2 was associated with a rapid phosphorylation of the ribosomal p70S6 kinase in a Ca2+- and tyrosine kinase-dependent manner. These data suggest that PYK2 is an important regulator of multiple signaling pathways involved in ANG II-induced VSMC growth.
Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines
Authors: Dennis Robert G. , Kosnik Paul E. , Gilbert Mark E. , Faulkner John A.
Published: December 2017

The purpose of this study was to compare the excitability and contractility of three-dimensional skeletal muscle constructs, termed myooids, engineered from C2C12 myoblast and 10T½ fibroblast cell lines, primary muscle cultures from adult C3H mice, and neonatal and adult Sprague-Dawley rats. Myooids were 12 mm long, with diameters of 0.1–1 mm, were excitable by transverse electrical stimulation, and contracted to produce force. After ∼30 days in culture, myooid cross-sectional area, rheobase, chronaxie, resting baseline force, twitch force, time to peak tension, one-half relaxation time, and peak isometric force were measured. Specific force was calculated by dividing peak isometric force by cross-sectional area. The specific force generated by the myooids was 2–8% of that generated by skeletal muscles of control adult rodents. Myooids engineered from C2C12-10T½ cells exhibited greater rheobase, time to peak tension, and one-half relaxation time than myooids engineered from adult rodent cultures, and myooids from C2C12-10T½ and neonatal rat cells had greater resting baseline forces than myooids from adult rodent cultures.
IFN-γ + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38 mapk in a mouse macrophage cell line
Authors: Chan Edward D. , Riches David W. H.
Published: December 2017

Nitric oxide (NO·) produced by inducible nitric oxide synthase (iNOS) mediates a number of important physiological and pathophysiological processes. The objective of this investigation was to examine the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and NO· by interferon-γ (IFN-γ) + lipopolysaccharide (LPS) in macrophages using specific inhibitors and dominant inhibitory mutant proteins of the MAPK pathways. The signaling pathway utilized by IFN-γ in iNOS induction is well elucidated. To study signaling pathways that are restricted to the LPS-signaling arm, we used a subclone of the parental RAW 264.7 cell line that is unresponsive to IFN-γ alone with respect to iNOS induction. In this RAW 264.7γNO(−) subclone, IFN-γ and LPS are nevertheless required for synergistic activation of the iNOS promoter. We found that extracellular signal-regulated kinase (ERK) augmented and p38 mapk inhibited IFN-γ + LPS induction of iNOS. Dominant-negative MAPK kinase-4 inhibited iNOS promoter activation by IFN-γ + LPS, also implicating the c-Jun NH2-terminal kinase (JNK) pathway in mediating iNOS induction. Inhibition of the ERK pathway markedly reduced IFN-γ + LPS-induced tumor necrosis factor-α protein expression, providing a possible mechanism by which ERK augments iNOS expression. The inhibitory effect of p38 mapk appears more complex and may be due to the ability of p38 mapk to inhibit LPS-induced JNK activation. These results indicate that the MAPKs are important regulators of iNOS-NO· expression by IFN-γ + LPS.
Distinct PKC isozymes regulate bufalin-induced differentiation and apoptosis in human monocytic cells
Authors: Kurosawa Masahiro , Tani Yoshihiro , Nishimura Satoshi , Numazawa Satoshi , Yoshida Takemi
Published: December 2017

Bufalin, an Na+-K+-ATPase inhibitor, simultaneously induced cell differentiation and apoptosis in human monocytic leukemia THP-1 cells. In this study, we investigated the regulatory role of protein kinase C (PKC) isozymes in bufalin-induced cell differentiation and apoptosis. A PKC-specific but isozyme-nonselective inhibitor, Ro-31–8220, and a cPKC selective inhibitor, Gö-6976, caused significant attenuation of bufalin-induced interleukin-1β (IL-1β) gene expression, a mature monocytic marker, indicating that cPKC participates in the bufalin-induced cell differentiation. On the other hand, cPKCβ- and nPKCδ-defective THP-1/TPA cells displayed strong resistance to the bufalin-induced DNA ladder formation. Rottlerin, an nPKCδ-specific inhibitor, partially attenuated preapoptotic effects of bufalin, such as the limited proteolysis of nPKCδ and poly(ADP-ribose) polymerase and the cell staining by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, suggesting that nPKCδ is involved, at least in part, in bufalin-induced apoptosis. In contrast, Gö-6976 and rottlerin significantly augmented bufalin-induced apoptosis and differentiation, respectively. The findings suggest that bufalin-induced cell differentiation and apoptosis are interlinked and that distinct PKC isozymes are involved in the fate of the cell.
Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport
Authors: Kuang Kunyan , Li Yansui , Wen Quan , Wang Zheng , Li Jun , Yang Yingqing , Iserovich Pavel , Reinach Peter S. , Sparrow Janet , Diecke Friedrich P. J. , Fischbarg Jorge
Published: December 2017

Although Na+-K+-2Cl− cotransport has been demonstrated in cultured bovine corneal endothelial cells, its presence and role in the native tissue have been disputed. Using RT-PCR we have now identified a partial clone of the cotransporter protein in freshly dissected as well as in cultured corneal endothelial and epithelial cells. The deduced amino acid sequence of this protein segment is 99% identical to that of the bovine isoform (bNKCC1). [3H]bumetanide binding shows that the cotransporter sites are located in the basolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium. Immunocytochemistry confirms the basolateral location of the cotransporter. We calculate the turnover rate of the cotransporter to be 83 s−1. Transendothelial fluid transport, determined from deepithelialized rabbit corneal thickness measurements, is partially inhibited (30%) by bumetanide in a dose-dependent manner. Our results demonstrate that Na+-K+-2Cl− cotransporters are present in the basolateral domain of freshly dissected bovine corneal endothelial cells and contribute to fluid transport across corneal endothelial preparations.
Spatiotemporal expression of catenins, ZO-1, and occludin during early polarization of hepatic WIF-B9 cells
Authors: Decaens C. , Cassio D.
Published: December 2017

WIF-B9 is a suitable model for in vitro studies of hepatocyte polarity. To better understand polarity establishment, we have localized key proteins of the adhesion system, cytoskeleton, and tight junctions soon after plating, when most cells are isolated or in doublets. In isolated attached cells, only cytoskeletal proteins (tubulin, cytokeratins) displayed a precise localization. As soon as two cells formed a doublet, E-cadherin, α-, β-, and γ-catenins, and p120 protein were present at the doublet contiguous membrane. Actin, ezrin, and zonula occludens-1 (ZO-1) colocalized at this membrane, but not in all doublets: ezrin was present only at contiguous membrane expressing ZO-1, and ZO-1 was present only at membrane expressing actin. In contrast, occludin was spread throughout the doublet cytoplasm. With time in culture, these proteins localized transiently, as in cells expressing simple epithelial polarity, and finally, as in hepatocytes. We conclude that during WIF-B9 early polarization, key proteins are settled according to a hierarchy, as has been shown for Madin-Darby canine kidney cells. Cytoplasmic complexes of E-cadherin-catenin were detected during the whole polarization process; they were more abundant in fully polarized cells.
FPL-64176 modifies pore properties of L-type Ca2+channels
Authors: Fan Jing-Song , Yuan Yuhui , Palade Philip
Published: December 2017

In addition to its known effects on Ca2+and Ba2+ currents, the L-type Ca2+ channel agonist FPL-64176 was found to affect channel function in isolated rat ventricular myocytes in the absence of Ca2+, with other ions as current carriers through the channel. FPL-64176 induced Cd2+ current through the L-type Ca2+channel, suggesting that certain selectivity properties had changed, perhaps indicative of a small change in pore structure. FPL-64176 slightly but significantly decreased the effectiveness of Co2+ as a blocker of the channel. FPL-64176 also increased conductance through single L-type Ca2+ channels recorded in the cell-attached configuration, from 71.9 ± 11.6 to 94.1 ± 8.3 pS, with Na+ carrying the current at pH 9.0. At present it is uncertain whether FPL-64176 produces small alterations of a sole open state of the channel or whether it increases the prevalence of a second, higher conductance open state. These changes, particularly the conversion of Cd2+ from a pure blocker to a permeant ion, may be of eventual help in discriminating among different models for Ca2+ channel selectivity.
Urea sensitizes mIMCD3 cells to heat shock-induced apoptosis: protection by NaCl
Authors: Colmont Chantal , Michelet Stéphanie , Guivarc'h Dominique , Rousselet Germain
Published: December 2017

Urea, with NaCl, constitutes the osmotic gradient that allows water reabsorption in mammalian kidneys. Because NaCl induces heat shock proteins, we tested the responses to heat shock of mIMCD3 cells adapted to permissive urea and/or NaCl concentrations. We found that heat-induced cell death was stronger after adaptation to 250 mM urea. This effect was reversible, dose dependent, and, interestingly, blunted by 125 mM NaCl. Moreover, we have shown that urea-adapted cells engaged in an apoptotic pathway upon heat shock, as shown by DNA laddering. This sensitization is not linked to a defect in the heat shock response, because the induction of HSP70 was similar in isotonic and urea-adapted cells. Moreover, it is not linked to the presence of urea inside cells, because washing urea away did not restore heat resistance and because applying urea and heat shock at the same time did not lead to heat sensitivity. Together, these results suggest that urea modifies the heat shock response, leading to facilitated apoptosis.
Regulation of the human sodium-phosphate cotransporter NaPi-IIb gene promoter by epidermal growth factor
Authors: Xu Hua , Collins James F. , Bai Liqun , Kiela Pawel R. , Ghishan Fayez K.
Published: December 2017

The intestinal sodium-phosphate cotransporter (NaPi-IIb) plays a major role in intestinal Pi absorption. Epidermal growth factor (EGF) is involved in the regulation of Pihomeostasis. However, the role of EGF in intestinal NaPi-IIb regulation is not clear. The current studies showed that EGF decreased NaPi-IIb mRNA abundance by 40–50% in both rat intestine and Caco-2 cells. To understand the mechanism of this regulation, we cloned the human NaPi-IIb gene and promoter region and studied the effect of EGF on NaPi-IIb gene transcription. The human NaPi-IIb gene has 12 exons and 11 introns. Two transcription initiation sites were identified by primer extension. Additionally, 2.8 kb of the 5′-flanking region of the gene was characterized as a functional promoter in human intestinal (Caco-2) and human lung (A549) cells. Additional studies showed that EGF inhibited promoter activity by 40–50% in Caco-2 cells and that actinomycin D treatment abolished this inhibition. EGF had no effect on promoter activity in lung (A549) cells. We conclude that the human NaPi-IIb gene promoter is functional in Caco-2 and A549 cells and that the gene is responsive to EGF by a transcriptionally mediated mechanism in intestinal cells.
Oxidant stress and endothelial cell dysfunction
Authors: Lum Hazel , Roebuck Kenneth A.
Published: December 2017

Reactive oxygen species (ROS) are generated at sites of inflammation and injury, and at low levels, ROS can function as signaling molecules participating as signaling intermediates in regulation of fundamental cell activities such as cell growth and cell adaptation responses, whereas at higher concentrations, ROS can cause cellular injury and death. The vascular endothelium, which regulates the passage of macromolecules and circulating cells from blood to tissues, is a major target of oxidant stress, playing a critical role in the pathophysiology of several vascular diseases and disorders. Specifically, oxidant stress increases vascular endothelial permeability and promotes leukocyte adhesion, which are coupled with alterations in endothelial signal transduction and redox-regulated transcription factors such as activator protein-1 and nuclear factor-κB. This review discusses recent findings on the cellular and molecular mechanisms by which ROS signal events leading to impairment of endothelial barrier function and promotion of leukocyte adhesion. Particular emphasis is placed on the regulation of cell-cell and cell-surface adhesion molecules, the actin cytoskeleton, key protein kinases, and signal transduction events.
Differential global gene expression in red and white skeletal muscle
Authors: Campbell W. G. , Gordon S. E. , Carlson C. J. , Pattison J. S. , Hamilton M. T. , Booth F. W.
Published: December 2017

The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of ∼3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30–35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.
Agouti regulates adipocyte transcription factors
Authors: Mynatt Randall L. , Stephens Jacqueline M.
Published: December 2017

Agouti is a secreted paracrine factor that regulates pigmentation in hair follicle melanocytes. Several dominant mutations cause ectopic expression of agouti, resulting in a phenotype characterized by yellow fur, adult-onset obesity and diabetes, increased linear growth and skeletal mass, and increased susceptibility to tumors. Humans also produce agouti protein, but the highest levels of agouti in humans are found in adipose tissue. To mimic the human agoutiexpression pattern in mice, transgenic mice (aP2-agouti) that express agouti in adipose tissue were generated. The transgenic mice develop a mild form of obesity, and they are sensitized to the action of insulin. We correlated the levels of specific regulators of insulin signaling and adipocyte differentiation with these phenotypic changes in adipose tissue. Signal transducers and activators of transcription (STAT)1, STAT3, and peroxisome proliferator-activated receptor (PPAR)-γ protein levels were elevated in the transgenic mice. Treatment of mature 3T3-L1 adipocytes recapitulated these effects. These data demonstrate that agouti has potent effects on adipose tissue. We hypothesize that agouti increases adiposity and promotes insulin sensitivity by acting directly on adipocytes via PPAR-γ.
Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity
Authors: Butler Peter J. , Norwich Gerard , Weinbaum Sheldon , Chien Shu
Published: December 2017

Blood flow-associated shear stress may modulate cellular processes through its action on the plasma membrane. We quantified the spatial and temporal aspects of the effects of shear stress (τ) on the lipid fluidity of 1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate [DiIC16(13)]-stained plasma membranes of bovine aortic endothelial cells in a flow chamber. A confocal microscope was used to determine the DiI diffusion coefficient ( D) by fluorescence recovery after photobleaching on cells under static conditions, after a step-τ of 10 or 20 dyn/cm2, and after the cessation of τ. The method allowed the measurements of D on the upstream and downstream sides of the cell taken midway between the respective cell borders and the nucleus. In <10 s after a step-τ of 10 dyn/cm2, D showed an upstream increase and a downstream decrease, and both changes disappeared rapidly. There was a secondary, larger increase in upstream D, which reached a peak at 7 min and decreased thereafter, despite the maintenance of τ. D returned to near control values within 5 s after cessation of τ. Downstream D showed little secondary changes throughout the 10-min shearing, as well as after its cessation. Further investigations into the early phase, with simultaneous measurements of upstream and downstream D, confirmed that a step-τ of 10 dyn/cm2 elicited a rapid (5-s) but transient increase in upstream D and a concurrent decrease in downstream D, yielding a significant difference between the two sites. A step-τ of 20 dyn/cm2 caused D to increase at both sites at 5 s, but by 30 s and 1 min the upstream D became significantly higher than the downstream D. These results demonstrate shear-induced changes in membrane fluidity that are time dependent and spatially heterogeneous. These changes in membrane fluidity may have important implications in shear-induced membrane protein modulation.
TNF-α inhibits flow and insulin signaling leading to NO production in aortic endothelial cells
Authors: Kim Francis , Gallis Byron , Corson Marshall A.
Published: December 2017

Endothelial cells release nitric oxide (NO) acutely in response to increased “flow” or fluid shear stress (FSS), and the increase in NO production is correlated with enhanced phosphorylation and activation of endothelial nitric oxide synthase (eNOS). Both vascular endothelial growth factor and FSS activate endothelial protein kinase B (PKB) by way of incompletely understood pathway(s), and, in turn, PKB phosphorylates eNOS at Ser-1179, causing its activation. In this study, we found that either FSS or insulin stimulated insulin receptor substrate-1 (IRS-1) tyrosine and serine phosphorylation and increased IRS-1-associated phosphatidylinositol 3-kinase activity, phosphorylation of PKB Ser-473, phosphorylation of eNOS Ser-1179, and NO production. Brief pretreatment of bovine aortic endothelial cells with tumor necrosis factor-α (TNF-α) inhibited the above described FSS- or insulin-stimulated protein phosphorylation events and almost totally inhibited FSS- or insulin-stimulated NO production. These data indicate that FSS and insulin regulate eNOS phosphorylation and NO production by overlapping mechanisms. This study suggests one potential mechanism for the development of endothelial dysfunction in disease states with alterations in insulin regulation and increased TNF-α levels.
Differential expression and alternative splicing of TRP channel genes in smooth muscles
Authors: Walker Rebecca L. , Hume Joseph R. , Horowitz Burton
Published: December 2017

Nonselective cation channels (NSCC) are targets of excitatory agonists in smooth muscle, representing the nonselective cation current I cat. Na+ influx through NSCC causes depolarizations and activates voltage-dependent Ca2+ channels, resulting in contraction. The molecular identity of I cat in smooth muscle has not been elucidated; however, products of the transient receptor potential (TRP) genes have characteristics similar to native I cat. We have determined the levels of TRP transcriptional expression in several murine and canine gastrointestinal and vascular smooth muscles and have analyzed the alternative processing of these transcripts. Of the seven TRP gene family members, transcripts for TRP4, TRP6, and TRP7 were detected in all murine and canine smooth muscle cell preparations. TRP3 was detected only in canine renal artery smooth muscle cells. The full-length cDNAs for TRP4, TRP6, and TRP7, as well as one splice variant of TRP4 and two splice variants of TRP7, were cloned from murine colonic smooth muscle. Quantitative RT-PCR determined the relative amounts of TRP4, TRP6, and TRP7 transcripts, as well as that of the splice variants, in several murine smooth muscles. TRP4 is the most highly expressed, while TRP6 and TRP7 are expressed at a lower level in the same tissues. Splice variants for TRP7, deleted for exons encoding amino acids including transmembrane segment S1, predominated in murine smooth muscles, while the full-length form of the transcript was expressed in canine smooth muscles.
Influence of caveolin-1 on cellular cholesterol efflux mediated by high-density lipoproteins
Authors: Frank Philippe G. , Galbiati Ferruccio , Volonte Daniela , Razani Babak , Cohen David E. , Marcel Yves L. , Lisanti Michael P.
Published: December 2017

Caveolin-1 is a principal structural component of caveolae membranes. These membrane microdomains participate in the regulation of signaling, transcytosis, and cholesterol homeostasis at the plasma membrane. In the present study, we determined the effect of caveolin-1 expression on cellular cholesterol efflux mediated by high-density lipoprotein (HDL). We evaluated this effect in parental NIH/3T3 cells as well as in two transformed NIH/3T3 cell lines in which caveolin-1 protein levels are dramatically downregulated. Compared with parental NIH/3T3 cells, these two transformed cell lines effluxed cholesterol more rapidly to HDL. In addition, NIH/3T3 cells harboring caveolin-1 antisense also effluxed cholesterol more rapidly to HDL. However, this effect was not due to changes in total cellular cholesterol content. We further showed that chronic HDL exposure reduced caveolin-1 protein expression in NIH/3T3 cells. HDL exposure also inhibited caveolin-1 promoter activity, suggesting a direct negative effect of HDL on caveolin-1 gene transcription. Moreover, we showed that HDL-induced downregulation of caveolin-1 prevents the uptake of oxidized low-density lipoprotein in human endothelial cells. These data suggest a novel proatherogenic role for caveolin-1, i.e., regarding the uptake and/or transcytosis of modified lipoproteins.
Cloning and functional characterization of a high-affinity Na+/dicarboxylate cotransporter from mouse brain
Authors: Pajor Ana M. , Gangula Rama , Yao Xiaozhou
Published: December 2017

Neurons contain a high-affinity Na+/dicarboxylate cotransporter for absorption of neurotransmitter precursor substrates, such as α-ketoglutarate and malate, which are subsequently metabolized to replenish pools of neurotransmitters, including glutamate. We have isolated the cDNA coding for a high-affinity Na+/dicarboxylate cotransporter from mouse brain, called mNaDC-3. The mRNA coding for mNaDC-3 is found in brain and choroid plexus as well as in kidney and liver. The mNaDC-3 transporter has a broad substrate specificity for dicarboxylates, including succinate, α-ketoglutarate, fumarate, malate, and dimethylsuccinate. The transport of citrate is relatively insensitive to pH, but the transport of succinate is inhibited by acidic pH. The Michaelis-Menten constant for succinate in mNaDC-3 is 140 μM in transport assays and 16 μM at −50 mV in two-electrode voltage clamp assays. Transport is dependent on sodium, although lithium can partially substitute for sodium. In conclusion, mNaDC-3 likely codes for the high-affinity Na+/dicarboxylate cotransporter in brain, and it has some unusual electrical properties compared with the other members of the family.
Overexpression of stomatin depresses GLUT-1 glucose transporter activity
Authors: Zhang Jin-Zhong , Abbud Wafa , Prohaska Rainer , Ismail-Beigi Faramarz
Published: December 2017

We showed previously that GLUT-1 glucose transporter is associated with stomatin (band 7.2b) in human red blood cell membranes and in Clone 9 cells. We show here that in a mixed population of stably transfected cells, overexpression of either murine or human stomatin resulted in 35–50% reduction in the basal rate of glucose transport. Moreover, there was a correlation between increased expression of stomatin and depression in the rate of glucose transport. In two clones chosen for further study, the ∼10% and ∼70% reduction in basal rate of glucose transport was associated with increases in stomatin mRNA and protein expression without a detectable change in GLUT-1 content in plasma membranes of either clone. In the clone overexpressing high levels of stomatin, immunoprecipitated GLUT-1 was associated with a large amount of stomatin as a coimmunoprecipitant. Employing extracts of cells overexpressing human stomatin, we found that stomatin bound to the glutathione- S-transferase (GST) fusion protein containing the COOH-terminal 42-amino acid segment of GLUT-1 but not to GST alone or a GST fusion protein containing the 66-amino acid central loop of GLUT-1. Rat stomatin cDNA was cloned by RT-PCR and found to be highly homologous to mouse (97%) and human (86%) stomatins. These results suggest that overexpression of stomatin results in a depression in the basal rate of glucose transport by decreasing the “intrinsic” activity of GLUT-1, probably through protein-protein interaction.
Role of vascular endothelial growth factor in regulation of physiological angiogenesis
Authors: Ferrara Napoleone
Published: December 2017

Evidence accumulating over the last decade has established the fundamental role of vascular endothelial growth factor (VEGF) as a key regulator of normal and abnormal angiogenesis. The biological effects of VEGF are mediated by two tyrosine kinase receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). The signaling and biological properties of these two receptors are strikingly different. VEGF is essential for early development of the vasculature to the extent that inactivation of even a single allele of the VEGF gene results in embryonic lethality. VEGF is also required for female reproductive functions and endochondral bone formation. Substantial evidence also implicates VEGF as an angiogenic mediator in tumors and intraocular neovascular syndromes, and numerous clinical trials are presently testing the hypothesis that inhibition of VEGF may have therapeutic value.
Vascular endothelial growth factor and vascular adjustments to perturbations in oxygen homeostasis
Authors: Dor Yuval , Porat Rinnat , Keshet Eli
Published: December 2017

Development of microvascular networks is set to meet the metabolic requirements of the tissue they perfuse. Accordingly, impairment of oxygen homeostasis, either due to increased oxygen consumption or as a result of blood vessel occlusion, triggers compensatory neovascularization. This feedback reaction is mediated by a hypoxia- and hypoglycemia-induced vascular endothelial growth factor (VEGF). VEGF accumulates under stress as a result of increased hypoxia-inducible factor-1α-mediated transcription, stabilization of the mRNA, and the function of a hypoxia-refractory internal ribosome entry site within its 5′-untranslated region. Matching of vascular density to the metabolic needs of the tissue may include a process of hyperoxia-induced vessel regression. Thus newly formed vascular networks may undergo a natural process of vascular pruning that takes place whenever VEGF, acting as a vascular survival factor, is downregulated below the level required to sustain immature vessels. Immature vessels are particularly vulnerable and are selectively obliterated upon withdrawal of VEGF. The plasticity window for vessel regression is determined by a delay in the recruitment of periendothelial cells to the preformed endothelial plexus. Thus fine-tuning of microvascular density takes place mostly in the newly formed plexus, but the mature system is refractory to episodic changes in tissue oxygenation. These regulatory links may malfunction in certain pathological settings.
Signaling mechanisms mediating vascular protective actions of vascular endothelial growth factor
Authors: Zachary Ian
Published: December 2017

Vascular endothelial growth factor (VEGF) is essential for angiogenesis in health and pathophysiology, and it is currently a major focus for drug targeting in the development of treatments for diverse human diseases. Recently, we proposed that VEGF could also play a role as a vascular protective factor in the adult vasculature and in disease. In this model, vascular protection is defined as a VEGF-induced enhancement of endothelial functions that mediate the inhibition of vascular smooth muscle cell proliferation, enhanced endothelial cell survival, suppression of thrombosis, and anti-inflammatory effects. A feature of this model is that protective effects of VEGF are essentially independent of angiogenesis or endothelial cell proliferation. VEGF-dependent cell survival and VEGF-induced synthesis of nitric oxide and prostacyclin are likely to be key mediators of a vascular protective effect. Vascular protection should help to improve insight into the underlying mechanisms of cardiovascular actions of VEGF and prove valuable for developing novel therapeutic approaches to cardiovascular disease.