bluuebunny/crossref_metadata_2025 huggingface dataset:
bFGF and VEGF synergistically enhance endothelial cytoprotection via decay-accelerating factor induction
Authors: Mason Justin C. , Lidington Elaine A. , Ahmad Saifur R. , Haskard Dorian O.
Published: May 2013

The complement-regulatory protein decay-accelerating factor (DAF) can be upregulated on endothelial cells (EC) by protein kinase C (PKC)-dependent and -independent pathways. We hypothesized that basic fibroblast growth factor (bFGF) might induce EC DAF expression, providing a cytoprotective mechanism for angiogenic neovessels against complement-mediated injury. Incubation of umbilical vein, aortic, and dermal EC with bFGF or vascular endothelial growth factor (VEGF) significantly increased DAF expression. Growth factor-induced EC proliferation was inhibited by PKC antagonists. In contrast, although PKC antagonists inhibited VEGF-induced DAF expression, bFGF-induced DAF was unaffected. Investigation of mitogen-activated kinase (MAPK) pathways also revealed differences, with bFGF-induced DAF dependent on p44/42 and p38 MAPK and VEGF requiring activation of p38 MAPK alone. Upregulation of DAF by bFGF was functionally relevant, reducing C3 deposition on EC after complement activation by 60% and resulting in marked reduction in complement-mediated EC lysis. bFGF and VEGF were synergistic in terms of DAF expression, resulting in enhanced cytoprotection. These observations reveal parallel PKC-dependent and -independent pathways regulating complement activation during angiogenesis. Further elucidation of these pathways may provide important insights into innate cytoprotective mechanisms in endothelium.
Role of IGF system of mitogens in the induction of fibroblast proliferation by keloid-derived keratinocytes in vitro
Authors: Phan Toan-Thang , Lim Ivor Jiun , Bay Boon Huat , Qi Robert , Longaker Michael Thornton , Lee Seng-Teik , Huynh Hung
Published: May 2013

Keloids are proliferative dermal growths representing a pathological wound-healing response. We report high proliferation rates in normal (NF) and keloid-derived fibroblasts (KF) cocultured with keloid-derived keratinocytes (KK). IGF binding protein (IGFBP)-3 mRNA and secreted IGFBP-3 in conditioned media were increased in NF cocultured with KK compared with NF but markedly reduced in KF cocultured with KK or normal keratinocytes (NK). IGFBP-2 and IGFBP-4 mRNA levels were elevated, whereas IGFBP-5 mRNA was decreased in KF cocultured with KK or NK. Significant increases in IGFBP-2 and -4 mRNA in KF cocultured with KK did not correlate with protein secretion. Downstream IGF signaling cascade components, phospho-Raf, phospho-MEK1/2, phospho-MAPK, PI-3 kinase, phospho-Akt, and phospho-Elk-1, were elevated in KF cocultured with KK. Addition of recombinant human IGFBP-3 or antibodies against IGF-I or IGF-IR significantly inhibited proliferation of KF. The bioavailability of IGF-I may be related to the levels of IGFBP-3 produced, which in turn influences KF proliferation, suggesting that modulation of IGF-I, IGF-IR, and IGFBP-3, individually or in combination, may represent novel approaches to the treatment of keloids.
Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochromec
Authors: Yuan Qing , Ray Ramesh M. , Johnson Leonard R.
Published: May 2013

C1297, 2002; 10.1152/ajpcell.00351.2001.We have shown previously that depletion of polyamines delays apoptosis induced by camptothecin in rat intestinal epithelial cells (IEC-6). Mitochondria play an important role in the regulation of apoptosis in mammalian cells because apoptotic signals induce mitochondria to release cytochrome c. The latter interacts with Apaf-1 to activate caspase-9, which in turn activates downstream caspase-3. Bcl-2 family proteins are involved in the regulation of cytochrome c release from mitochondria. In this study, we examined the effects of polyamine depletion on the activation of the caspase cascade, release of cytochrome cfrom mitochondria, and expression and translocation of Bcl-2 family proteins. We inhibited ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, with α-difluoromethylornithine (DFMO) to deplete cells of polyamines. Depletion of polyamines prevented camptothecin-induced release of cytochrome c from mitochondria and decreased the activity of caspase-9 and caspase-3. The mitochondrial membrane potential was not disrupted when cytochrome c was released. Depletion of polyamines decreased translocation of Bax to mitochondria during apoptosis. The expression of antiapoptotic proteins Bcl-xL and Bcl-2 was increased in DFMO-treated cells. Caspase-8 activity and cleavage of Bid were decreased in cells depleted of polyamines. These results suggest that polyamine depletion prevents IEC-6 cells from apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c.
Mechanism of constrictive vascular remodeling by homocysteine: role of PPAR
Authors: Mujumdar Vibhas S. , Tummalapalli Chandra M. , Aru Giorgio M. , Tyagi Suresh C.
Published: May 2013

To test the hypothesis that homocysteine induces constrictive vascular remodeling by inactivating peroxisome proliferator-activated receptor (PPAR), aortic endothelial cells (ECs) and smooth muscle cells (SMCs) were isolated. Collagen gels were prepared, and ECs or SMCs (105) or SMCs + ECs (104) were incorporated into the gels. To characterize PPAR, agonists of PPAR-α [ciprofibrate (CF)] and PPAR-γ [15-deoxy-12,14-prostaglandin J2 (PGJ2)] were used. To determine the role of disintegrin metalloproteinase (DMP), cardiac inhibitor of metalloproteinase (CIMP) was used in collagen gels. Gel diameter at 0 h was 14.1 ± 0.2 mm and was unchanged up to 24 h as measured by a digital micrometer. SMCs reduce gel diameter to 10.5 ± 0.4 mm at 24 h. Addition of homocysteine to SMCs reduces further the gel diameter to 8.0 ± 0.2 mm, suggesting that SMCs induce contraction and that the contraction is further enhanced by homocysteine. Addition of ECs and SMCs reduces gel diameter to 12.0 ± 0.3 mm, suggesting that ECs play a role in collagen contraction. Only PGJ2, not CF, inhibits SMC contraction. However, both PGJ2 and CF inhibit contraction of ECs and SMCs + ECs. Addition of anti-DMP blocks SMC- as well as homocysteine-mediated contraction. However, CIMP inhibits only homocysteine-mediated contraction. The results suggest that homocysteine may enhance vascular constrictive remodeling by inactivating PPAR-α and -γ in ECs and PPAR-γ in SMCs.
Transcriptional regulation of the type I myosin heavy chain promoter in inactive rat soleus
Authors: Huey K. A. , Roy R. R. , Haddad F. , Edgerton V. R. , Baldwin K. M.
Published: May 2013

Chronic muscle inactivity with spinal cord isolation (SI) decreases expression of slow type I myosin heavy chain (MHC) while increasing expression of the faster MHC isoforms, primarily IIx. The purpose of this study was to determine whether type I MHC downregulation in the soleus muscle of SI rats is regulated transcriptionally and to identify cis-acting elements or regions of the rat type I MHC gene promoter involved in this response. One week of SI significantly decreased in vivo activity of the −3500-, −408-, −299-, −215-, and −171-bp type I MHC promoters. The activity of all tested deletions of the type I MHC promoter, relative to the human skeletal α-actin promoter, were significantly reduced in the SI soleus, except activity of the −171-bp promoter, which increased. Mutation of the βe3 element (−214/−190 bp) in the −215- and −408-bp promoters and deletion of this element (−171-bp promoter) attenuated type I downregulation with SI. Gel mobility shift assays demonstrated a decrease in transcription enhancer factor-1 binding to the βe3 element with SI, despite an increase in total binding to this region. These results demonstrate that type I MHC downregulation with SI is transcriptionally regulated and suggest that interactions between transcription enhancer factor-1 and the βe3 element are likely involved in this response.
Nonselective cation conductance activated by muscarinic and purinergic receptors in rat spiral ganglion neurons
Authors: Ito Ken , Dulon Didier
Published: May 2013

The present study characterizes the ionic conductances activated by acetylcholine (ACh) and ATP, two candidate neuromodulators, in isolated spiral ganglion neurons (SGNs). Brief application (1 s) of ACh evoked in a dose-dependent manner (EC50 = 4.1 μM) a reversible inward current with a long latency (average 1.3 s), at holding potential ( V h) = −50 mV. This current was reversibly blocked by atropine and mimicked by muscarine. Application of ATP also evoked a reversible inward current at V h = −50 mV, but the current showed two components. A fast component with a short latency was largely reduced when N-methyl-d-glucamine (NMDG) replaced extracellular sodium, implying a P2X-like ionotropic conductance. The second component had a longer latency (average 1.1 s) and was presumably activated by metabotropic P2Y-like receptors. The second component of ATP-evoked current shared similar characteristics with the responses evoked by ACh: the current reversed near 0 mV, displayed inward rectification, could be carried by NMDG, and was insensitive to extracellular and intracellular calcium. This ACh-/ATP-evoked conductance was reversibly inhibited by preapplication of ionomycin. These results suggest that muscarinic receptors and purinergic metabotropic receptors activate a similar large nonselective cation conductance via a common intracellular pathway in SGNs, a candidate mechanism to regulate neuronal excitability of SGNs.
Loss of cell volume regulation during metabolic inhibition in renal epithelial cells (A6): role of intracellular pH
Authors: Smets Ilse , Ameloot Marcel , Steels Paul , Van Driessche Willy
Published: May 2013

In renal ischemia, tubular obstruction induced by swelling of epithelial cells might be an important mechanism for reduction of the glomerular filtration rate. We investigated ischemic cell swelling by examining volume regulation of A6 cells during metabolic inhibition (MI) induced by cyanide and 2-deoxyglucose. Changes in cell volume were monitored by recording cell thickness ( T c). Intracellular pH (pHc) measurements were performed with the pH-sensitive probe 5-chloromethyl-fluoresceine diacetate. T c measurements showed that MI increases cell volume. Cell swelling during MI is proportional to the rate of Na+ transport and is not followed by a volume regulatory response. Furthermore, MI prevents the regulatory volume decrease (RVD) elicited by a hyposmotic shock. MI induces a pronounced intracellular acidification that is conserved during a subsequent hypotonic shock. A transient acidification induced by a NH4Cl prepulse causes a marked delay of the RVD in response to a hypotonic shock. On the other hand, acute lowering of external pH to 5, simultaneously with the hypotonic shock, allowed the onset of RVD. However, this RVD was completely arrested ∼10 min after the initiation of the hyposmotic challenge. The inhibition of RVD appears to be related to the pronounced acidification that occurred within this time period. In contrast, when external pH was lowered 20 min before the hyposmotic shock, RVD was absent. These data suggest that internal acidification inhibits cellular volume regulation in A6 cells. Therefore, the intracellular acidification associated with MI might at least partly account for the failure of volume regulation in swollen epithelial cells.
Sphingosine 1-phosphate induces Ca2+ transients and cytoskeletal rearrangement in C2C12 myoblastic cells
Authors: Formigli Lucia , Francini Fabio , Meacci Elisabetta , Vassalli Massimo , Nosi Daniele , Quercioli Franco , Tiribilli Bruno , Bencini Chiara , Piperio Claudia , Bruni Paola , Orlandini Sandra Zecchi
Published: May 2013

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349–357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.
Role of phosphoinositide 3-kinase in the nonselective cation channel activation by endothelin-1/endothelinBreceptor
Authors: Kawanabe Yoshifumi , Hashimoto Nobuo , Masaki Tomoh
Published: May 2013

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) in Chinese hamster ovarian cells expressing endothelinB receptor (CHO-ETBR). These channels can be discriminated using the Ca2+ channel blockers, LOE 908 and SK&F 96365. LOE 908 is a blocker of NSCC-1 and NSCC-2, whereas SK&F 96365 is a blocker of NSCC-2. In this study, we investigated the possible role of phosphoinositide 3-kinase (PI3K) in the ET-1-induced activation of NSCCs in CHO-ETBR using wortmannin and LY-294002, inhibitors of PI3K. ET-1-induced Ca2+ influx was partially inhibited in CHO-ETBR pretreated with wortmannin or LY-294002. In contrast, addition of wortmannin or LY-294002 after stimulation with ET-1 did not suppress Ca2+ influx. The Ca2+ channels activated by ET-1 in wortmannin- or LY-294002-treated CHO-ETBR were sensitive to LOE 908 and resistant to SK&F 96365. In conclusion, NSCC-2 is stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated independently of the PI3K pathway. Moreover, PI3K seems to be required for the initiation of the Ca2+ entry through NSCC-2 but not for its maintenance.
Transgenic livers expressing mitochondrial and cytosolic CK: mitochondrial CK modulates free ADP levels
Authors: Askenasy Nadir , Koretsky Alan P.
Published: May 2013

The function of creatine kinase (CK) and its effect on phosphorus metabolites was studied in livers of transgenic mice expressing human ubiquitous mitochondrial CK (CK-Mit) and rat brain CK (CK-B) isoenzymes and their combination.31P NMR spectroscopy and saturation transfer were recorded in livers of anesthetized mice to measure high-energy phosphates and hepatic CK activity. CK reaction velocity was related to total enzyme activity irrespective of the isoenzyme expressed, and it increased with increasing concentrations of creatine (Cr). The fluxes mediated by both isoenzymes in both directions (phosphocreatine or ATP synthesis) were equal. Over a 20-fold increase in CK-Mit activity (28–560 μmol · g wet wt−1 · min−1), the fraction of phosphorylated Cr increased 1.6-fold. Hepatic free ADP concentrations calculated by assuming equilibrium of the CK-catalyzed reaction in vivo decreased from 84 ± 9 to 38 ± 4 nmol/g wet wt. Calculated free ADP levels in mice expressing high levels of CK-B (920–1,635 μmol · g wet wt−1 · min−1) were 52 ± 6 nmol/g wet wt. Mice expressing both isoenzymes had calculated free ADP levels of 36 ± 4 nmol/g wet wt. These findings indicate that CK-Mit catalyzes its reaction equally well in both directions and can lower hepatic apparent free ADP concentrations.
Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0
Authors: Cooper Gordon J. , Fong Peying
Published: May 2013

During maturation of oocytes, Cl− conductance ( G Cl) oscillates and intracellular pH (pHi) increases. Elevating pHi permits the protein synthesis essential to maturation. To examine whether changes in G Cl and pHi are coupled, the Cl− channel ClC-0 was heterologously expressed. Overexpressing ClC-0 elevates pHi, decreases intracellular Cl− concentration ([Cl−]i), and reduces volume. Acute acidification with butyrate does not activate acid extrusion in ClC-0-expressing or control oocytes. The ClC-0-induced pHichange increases after overnight incubation at extracellular pH 8.5 but is unaltered after incubation at extracellular pH 6.5. Membrane depolarization did not change pHi. In contrast, hyperpolarization elevates pHi. Thus neither membrane depolarization nor acute activation of acid extrusion accounts for the ClC-0-dependent alkalinization. Overnight incubation in low extracellular Cl− concentration increases pHiand decreases [Cl−]i in control and ClC-0 expressing oocytes, with the effect greater in the latter. Incubation in hypotonic, low extracellular Cl− solutions prevented pHi elevation, although the decrease in [Cl−]i persisted. Taken together, our observations suggest that KCl loss leads to oocyte shrinkage, which transiently activates acid extrusion. In conclusion, expressing ClC-0 in oocytes increases pHi and decreases [Cl−]i. These parameters are coupled via shrinkage activation of proton extrusion. Normal, cyclical changes of oocyte G Cl may exert an effect on pHi via shrinkage, thus inducing meiotic maturation.
Nicotinic acetylcholine receptor α7 regulates cAMP signal within lipid rafts
Authors: Oshikawa Jin , Toya Yoshiyuki , Fujita Takayuki , Egawa Masato , Kawabe Junichi , Umemura Satoshi , Ishikawa Yoshihiro
Published: May 2013

Neuronal nicotinic acetylcholine receptors (nAChRs) are made of multiple subunits with diversified functions. The nAChR α7-subunit has a property of high Ca2+ permeability and may have specific functions and localization within the plasma membrane as a signal transduction molecule. In PC-12 cells, fractionation by sucrose gradient centrifugation revealed that nAChRα7 existed in low-density, cholesterol-enriched plasma membrane microdomains known as lipid rafts where flotillin also exists. In contrast, nAChR α5- and β2-subunits were located in high-density fractions, out of the lipid rafts. Type 6 adenylyl cyclase (AC6), a calcium-inhibitable isoform, was also found in lipid rafts and was coimmunoprecipitated with nAChRα7. Cholesterol depletion from plasma membranes with methyl-β-cyclodextrin redistributed nAChRα7 and AC6 diffusely within plasma membranes. Nicotine stimulation reduced forskolin-stimulated AC activity by 35%, and this inhibition was negated by either treatment with α-bungarotoxin, a specific antagonist of nAChRα7, or cholesterol depletion from plasma membranes. The effect of cholesterol depletion was negated by the addition of cholesterol. These data suggest that nAChRα7 has a specific membrane localization relative to other nAChR subunits and that lipid rafts are necessary to localize nAChRα7 with AC within plasma membranes. In addition, nAChRα7 may regulate the AC activity via Ca2+ within lipid rafts.
Phosphatidylinositol 3-kinase-dependent, MEK- independent proliferation in response to CaR activation
Authors: Bilderback Tim R. , Lee Fred , Auersperg Nelly , Rodland Karin D.
Published: May 2013

Although ovarian surface epithelial (OSE) cells are responsible for the majority of ovarian tumors, we know relatively little about the pathway(s) that is responsible for regulating their proliferation. We found that phosphatidylinositol 3-kinase (PI3K) is activated in OSE cells in response to elevated extracellular calcium, and the PI3K inhibitors wortmannin and LY-294002 inhibited extracellular signal-regulated kinase (ERK) activation by ∼75%, similar to effects of the mitogen-activated protein kinase/ERK kinase inhibitor PD-98059. However, in assays of proliferation, we found that PD-98059 inhibited proliferation by ∼50%, whereas wortmannin inhibited >90% of the proliferative response to elevated calcium. Expression of a dominant negative PI3K totally inhibited ERK activation in response to calcium. These results demonstrate that ERK activation cannot account for the full proliferative effect of elevated calcium in OSE cells and suggest the presence of an ERK-independent, PI3K-dependent component in the proliferative response.
An extracellular sulfhydryl group modulates background Na+ conductance and cytosolic Ca2+ in pituitary cells
Authors: Fonteriz Rosalba I. , Villalobos Carlos , Garcı́a-Sancho Javier
Published: May 2013

Treatment of GH3 pituitary cells with p-chloromercurybenzenesulfonate (PCMBS) increased the cytosolic Ca2+ concentration ([Ca2+]i). This effect was reversed by dithiothreitol and blocked by L-type Ca2+ channel antagonists or Na+ removal. PCMBS increased membrane conductance and depolarized the plasma membrane. Apart from minor effects on K+ and Ca2+ channels, PCMBS increased (6 times at −80 mV) an inward Na+ current whose properties were similar to those of a background Na+conductance (BNC) described previously, necessary for generation of spontaneous electrical activity. In rat lactotropes and somatotropes in primary culture, PCMBS also produced a Na+-dependent [Ca2+]i increase, whereas little or no effect was observed in thyrotropes, corticotropes, and gonadotropes. The Na+ conductance elicited by PCMBS in somatotropes seemed to be the same as that stimulated by the hypothalamic growth hormone (GH)-releasing hormone, which regulates membrane excitability and GH secretion. The BNC studied here could play a physiological role, regulating excitability and spontaneous activity, and explains satisfactorily the [Ca2+]i-increasing actions of the mercurials reported previously in several excitable tissues.
Permeant but not impermeant divalent cations enhance activation of nondesensitizing α7 nicotinic receptors
Authors: Eddins Donnie , Lyford Lisa K. , Lee Jung Weon , Desai Sanjay A. , Rosenberg Robert L.
Published: May 2013

Neuronal α7 nicotinic acetylcholine receptors (nAChRs) are permeable to Ca2+ and other divalent cations. We characterized the modulation of the pharmacological properties of nondesensitizing mutant (L247T and S240T/L247T) α7 nAChRs by permeant (Ca2+, Ba2+, and Sr2+) and impermeant (Cd2+ and Zn2+) divalent cations. α7 receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh, whereas impermeant divalent cations decreased potency and maximal efficacy. The antagonist dihydro-β-erythroidine (DHβE) was a strong partial agonist of L247T and S240T/L247T α7 receptors in the presence of divalent cations but was a weak partial agonist in the presence of impermeant divalent cations. Mutation of the “intermediate ring” glutamates (E237A) in L247T α7 nAChRs eliminated Ca2+conductance but did not alter the Ca2+-dependent increase in ACh potency, suggesting that site(s) required for modulation are on the extracellular side of the intermediate ring. The difference between permeant and impermeant divalent cations suggests that sites within the pore are important for modulation by divalent cations.
Pore formation is not associated with macroscopic redistribution of P2X7 receptors
Authors: Smart Megan L. , Panchal Rekha G. , Bowser David N. , Williams David A. , Petrou Steven
Published: May 2013

The present study examines whether changes in P2X7 purinergic receptor density precede formation of the cytolytic pore characteristic of this receptor. We fused P2X7 receptors with enhanced green fluorescent protein (EGFP) at the amino or carboxy termini (EGFP-P2X7 and P2X7-EGFP). Electrophysiological characterization in Xenopus oocytes revealed wild-type responses to ATP for GFP-tagged receptors. However, differences in sensitivity to ATP were apparent with the P2X7-EGFP receptor displaying a threefold reduction in ATP sensitivity compared with control. Ethidium ion uptake was used to measure cytolytic pore formation. Comparison of tagged receptors with wild type in HEK-293 and COS-7 cells showed there was no significant difference in ethidium ion uptake, suggesting that fusions with EGFP did not interfere with cytolytic pore formation. Confocal microscopy confirmed that tagged receptors localized to the plasmalemma. Simultaneous monitoring of EGFP and ethidium ion fluorescence revealed that changes in receptor distribution do not precede pore formation. We conclude that it is unlikely that large scale changes in P2X7 receptor density precede pore formation.
Rabbit retinal neurons and glia express a variety of ENaC/DEG subunits
Authors: Brockway L. M. , Zhou Z.-H. , Bubien J. K. , Jovov B. , Benos D. J. , Keyser K. T.
Published: May 2013

Some members of the epithelial Na+ channel/degenerin (ENaC/DEG) family of ion channels have been detected in mammalian brain. Therefore, we examined the RNA and protein expression of these channels in another part of the central nervous system, the rabbit retina. We next sought to demonstrate physiological evidence for an amiloride-sensitive current in Müller glia, which, on the basis of a previous study, are thought to express α-ENaC (Golestaneh N, de Kozak Y, Klein C, and Mirshahi M. Glia 33: 160–168, 2001). RT-PCR of retinal RNA revealed the presence of α-, β-, γ-, and δ-ENaC as well as acid-sensing ion channel (ASIC)1, ASIC2, ASIC3, and ASIC4. Immunohistochemical localization with antibodies against α-ENaC and β-ENaC showed labeling in Müller cells and neurons, respectively. The presence of α-ENaC, β-ENaC, and ASIC1 was detected by Western blotting. Cultured Müller cells were whole cell patch clamped. These cells exhibited an inward Na+ current that was blocked by amiloride. These data demonstrate for the first time both the expression of a variety of ENaC and ASIC subunits in the rabbit retina as well as distinct cellular expression patterns of specific subunits in neurons and glia.
Autologous nitric oxide protects mouse and human keratinocytes from ultraviolet B radiation-induced apoptosis
Authors: Weller Richard , Schwentker Ann , Billiar Timothy R. , Vodovotz Yoram
Published: May 2013

Nitric oxide (NO) can either prevent or promote apoptosis, depending on cell type. In the present study, we tested the hypothesis that NO suppresses ultraviolet B radiation (UVB)-induced keratinocyte apoptosis both in vitro and in vivo. Irradiation with UVB or addition of the NO synthase (NOS) inhibitor N G-nitro-l-arginine methyl ester (l-NAME) increased apoptosis in the human keratinocyte cell line CCD 1106 KERTr, and apoptosis was greater when the two agents were given in combination. Addition of the chemical NO donor S-nitroso- N-acetyl-penicillamine (SNAP) immediately after UVB completely abrogated the rise in apoptosis induced by l-NAME. An adenoviral vector expressing human inducible NOS (AdiNOS) also reduced keratinocyte death after UVB. Caspase-3 activity, an indicator of apoptosis, doubled in keratinocytes incubated with l-NAME compared with the inactive isomer, d-NAME, and was reduced by SNAP. Apoptosis was also increased on addition of 1,H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. Mice null for endothelial NOS (eNOS) exhibited significantly higher apoptosis than wild-type mice both in the dermis and epidermis, whereas mice null for inducible NOS (iNOS) exhibited more apoptosis than wild-type mice only in the dermis. These results demonstrate an antiapoptotic role for NO in keratinocytes, mediated by cGMP, and indicate an antiapoptotic role for both eNOS and iNOS in skin damage induced by UVB.
A negatively charged residue in the outer mouth of rat sodium channel determines the gating kinetics of the channel
Authors: Zhang Zhao , Xu Yanfang , Dong Pei Hong , Sharma Dipika , Chiamvimonvat Nipavan
Published: May 2013

Previous studies using combined techniques of site-directed mutagenesis and electrophysiology of voltage-gated Na+ channels have demonstrated that there are significant overlaps in the regions that are important for the two fundamental properties of the channels, namely gating and permeation. We have previously shown that a pore-lining residue, W402 in S5-S6 region (P loop) in domain I of the μ1 skeletal muscle Na+channel, was important in the gating of the channel. Here, we determined the role of an adjacent pore-lining negatively charged residue (E403) in channel gating. Charge neutralization or substitution with positively charged side chain at this position resulted in a marked delay in the rate of recovery from slow inactivation. Indeed, the fast inactivation process appeared intact. Restoration of the negatively charged side chain with a sulfhydryl modifier, MTS-ethylsulfonate, resulted in a reactivation profile from a slow-inactivated state, which was indistinguishable from that of the wild-type channels. We propose an additional functional role for the negatively charged residue. Assuming no major changes in the pore structure induced by the mutations, the negatively charged residue E403 may work in concert with other pore regions during recovery from slow inactivation of the channel. Our data represent the first report indicating the role of negative charge in the slow inactivation of the voltage-gated Na+ channel.
Colocalization but differential regulation of neuronal NO synthase and nicotinic acetylcholine receptor in C2C12 myotubes
Authors: Ebert Jutta G. , Zelenka Marek , Gath Ingolf , Gödtel-Armbrust Ute , Förstermann Ulrich
Published: May 2013

In mammalian skeletal muscle, neuronal-type nitric oxide synthase (nNOS) is found to be enriched at neuromuscular endplates. Here we demonstrate the colocalization of the nicotinic acetylcholine receptor (nAChR, stained with α-bungarotoxin) and nNOS (stained with a specific antibody) in murine C2C12 myotubes. However, coimmunoprecipitation experiments demonstrated no evidence for a direct protein-protein association between the nAChR and nNOS in C2C12myotubes. An antibody to the α1-subunit of the nAChR did not coprecipitate nNOS, and an nNOS-specific antibody did not precipitate the α1-subunit of the nAChR. Treatment of mice with bacterial LPS downregulated the expression of nNOS in skeletal muscle, and treatment of C2C12 cells with bacterial LPS and interferon-γ markedly decreased nNOS mRNA and protein expression. In contrast, mRNA and protein of the nAChR (α-, γ-, and ε-subunits) remained unchanged at the mRNA and protein levels. These data demonstrate that nNOS and the nAChR are colocalized in murine skeletal muscle and C2C12 cells but differ in their expressional regulation.
Identification of domains that control the heteromeric assembly of Kir5.1/Kir4.0 potassium channels
Authors: Konstas Angelos-Aristeidis , Korbmacher Christoph , Tucker Stephen J.
Published: May 2013

Heteromultimerization between different inwardly rectifying (Kir) potassium channel subunits is an important mechanism for the generation of functional diversity. However, little is known about the mechanisms that control this process and that prevent promiscuous interactions in cells that express many different Kir subunits. In this study, we have examined the heteromeric assembly of Kir5.1 with other Kir subunits and have shown that this subunit exhibits a highly selective interaction with members of the Kir4.0 subfamily and does not physically associate with other Kir subunits such as Kir1.1, Kir2.1, and Kir6.2. Furthermore, we have identified regions within the Kir4.1 subunit that appear to govern the specificity of this interaction. These results help us to understand the mechanisms that control Kir subunit recognition and assembly and how cells can express many different Kir channels while maintaining distinct subpopulations of homo- and heteromeric channels within the cell.
Translocation of telokin by cGMP signaling in smooth muscle cells
Authors: Komatsu Satoshi , Miyazaki Koji , Tuft Richard A. , Ikebe Mitsuo
Published: May 2013

Telokin is an acidic protein with a sequence identical to the COOH-terminal domain of myosin light chain kinase (MLCK) produced by an alternate promoter of the MLCK gene. Although it is abundantly expressed in smooth muscle, its physiological function is not understood. In the present study, we attempted to clarify the function of telokin by analyzing its spatial and temporal localization in living single smooth muscle cells. Primary cultured smooth muscle cells were transfected with green fluorescent protein (GFP)-tagged telokin. The telokin-GFP localized mostly diffusely in cytosol. Stimulation with both sodium nitroprusside (SNP) and 8-bromo-cyclic GMP induced translocation of GFP-tagged telokin to near plasma membrane in living single smooth muscle cells. The translocation was slow, and it took more than 10 min at room temperature. Mutation of the phosphorylation sites of telokin (S13A, S19A, and S13A/S19A) significantly attenuated SNP-induced translocation. Both KT-5823 (cGMP-dependent protein kinase inhibitor) and PD-98059 (mitogen-activated protein kinase inhibitor) diminished the telokin-GFP translocation. These results suggest that telokin changes its intracellular localization because of phosphorylation at Ser13 and/or Ser19 via the cGMP-signaling pathway.
Regulation of apoptosis in the endocardial cushions of the developing chick heart
Authors: Keyes William M. , Sanders Esmond J.
Published: May 2013

During the early stages of heart development, there are two main foci of cell death: outflow tract (OT) and atrioventricular (AV) endocardial cushions. These tissues contribute to the septa and valves of the mature heart and receive cell populations from neural crest (NC) cell migration and epicardial cell invasion. We examined embryonic chick hearts for expression, in the cushions, of bcl-2 family members, caspase-9, and the caspase substrate poly(ADP-ribose) polymerase. Antiapoptotic bcl-2 is expressed heavily in the OT and AV regions throughout embryonic days (ED) 4–7, with a decrease in levels at ED 4 and 5 in OT and AV cushions, respectively. Proapoptotic bax predominantly associated with the prongs of the NC-derived aorticopulmonary (AP) septum but was expressed throughout the AV cushions. Proapoptotic bak also associated with the prongs of the AP septum in the OT, while protein levels were upregulated at ED 4–5 and 4–6 in OT and AV cushions, respectively. Bid expression showed a similar time course. We found the 10-kDa cleavage fragment of active caspase-9 at ED 4–8 and 5–8 in OT and AV cushions, respectively, and the 24-kDa cleavage fragment of poly(ADP-ribose) polymerase throughout ED 3–8 and 7–8 in OT and AV cushions, respectively. Caspase-3 cleavage occurred throughout the time period examined. Using cushion cell cultures, we found that inhibitors of caspases-3 and -9 and a universal caspase inhibitor significantly reduced apoptosis, as did retroviral overexpression of bcl-2 using an RCAS expression vector. Premigratory NC cells were fluorescently labeled in vivo with 1,1-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine. Subsequent nuclear staining of cushion cells with 4,6-diamidino-2-phenylindole revealed the presence of apoptotic nuclei in the NC cells in the OT cushions and in the prongs of the AP septum. These results demonstrate a developmentally regulated role for the bcl-2 and the caspase families of molecules in the endocardial cushions of the developing heart and lend support to the possibility that some of the dying cells in the cushions are derived from the NC.
Nuclear CaMKII inhibits neuronal differentiation of PC12 cells without affecting MAPK or CREB activation
Authors: Kutcher Louis W. , Beauman Shirelyn R. , Gruenstein Eric I. , Kaetzel Marcia A. , Dedman John R.
Published: May 2013

Ca2+/calmodulin-regulated protein kinase II (CaMKII) mediates many cellular events. The four CaMKII isoforms have numerous splice variants, three of which contain nuclear localization signals. Little is known about the role of nuclear localized CaMKII in neuronal development. To study this process, PC12 cells were transfected to produce CaMKII targeted to either the cytoplasm or the nucleus and then treated with nerve growth factor (NGF). NGF triggers a signaling cascade (MAPK) that results in the differentiation of PC12 cells into a neuronal phenotype, marked by neurite outgrowth. The present study found that cells expressing nuclear targeted CaMKII failed to grow neurites, whereas cells expressing cytoplasmic CaMKII readily produced neurites. Inhibition of neuronal differentiation by nuclear CaMKII was independent of MAPK signaling, as sustained Erk phosphorylation was not affected. Phosphorylation of CREB was also unaffected. Thus nuclear CaMKII modifies neuronal differentiation by a mechanism independent of MAPK and CREB activation.
AE4 is a DIDS-sensitive Cl−/HCO 3 − exchanger in the basolateral membrane of the renal CCD and the SMG duct
Authors: Ko Shigeru B. H. , Luo Xiang , Hager Henrik , Rojek Alexandra , Choi Joo Young , Licht Christoph , Suzuki Makoto , Muallem Shmuel , Nielsen Søren , Ishibashi Kenichi
Published: May 2013

The renal cortical collecting duct (CCD) plays an important role in systemic acid-base homeostasis. The β-intercalated cells secrete most of the HCO[Formula: see text], which is mediated by a luminal, DIDS-insensitive, Cl−/HCO[Formula: see text] exchange. The identity of the luminal exchanger is a matter of debate. Anion exchanger isoform 4 (AE4) cloned from the rabbit kidney was proposed to perform this function (Tsuganezawa H et al. J Biol Chem 276: 8180–8189, 2001). By contrast, it was proposed (Royaux IE et al. Proc Natl Acad Sci USA 98: 4221–4226, 2001) that pendrin accomplishes this function in the mouse CCD. In the present work, we cloned, localized, and characterized the function of the rat AE4. Northern blot and RT-PCR showed high levels of AE4 mRNA in the CCD. Expression in HEK-293 and LLC-PK1 cells showed that AE4 is targeted to the plasma membrane. Measurement of intracellular pH (pHi) revealed that AE4 indeed functions as a Cl−/HCO[Formula: see text] exchanger. However, AE4 activity was inhibited by DIDS. Immunolocalization revealed species-specific expression of AE4. In the rat and mouse CCD and the mouse SMG duct AE4 was in the basolateral membrane. By contrast, in the rabbit, AE4 was in the luminal and lateral membranes. In both, the rat and rabbit CCD AE4 was in α-intercalated cells. Importantly, localization of AE4 was not affected by the systemic acid-base status of the rats. Therefore, we conclude that expression and possibly function of AE4 is species specific. In the rat and mouse AE4 functions as a Cl−/HCO[Formula: see text] exchanger in the basolateral membrane of α-intercalated cells and may participate in HCO[Formula: see text] absorption. In the rabbit AE4 may contribute to HCO[Formula: see text] secretion.
Identification of an apical sorting determinant in the cytoplasmic tail of megalin
Authors: Takeda Tetsuro , Yamazaki Hajime , Farquhar Marilyn G.
Published: May 2013

Megalin is the main endocytic receptor of the proximal tubule and is responsible for reabsorption of many filtered proteins. In contrast to other members of the low-density lipoprotein (LDL) receptor gene family, it is expressed on the apical plasma membrane (PM) of polarized epithelial cells. To identify megalin's apical sorting signal, we generated deletion mutants and chimeric minireceptors composed of complementary regions of megalin and LDL receptor-related protein (LRP) and assessed the distribution of the mutants in Madin-Darby canine kidney (MDCK) cells by immunofluorescence and cell surface biotinylation. Megalin and LRP minireceptors are correctly targeted to the apical and basolateral PM, respectively, of MDCK cells. We found that the information that directs apical sorting is present in the cytoplasmic tail (CT) of megalin, which contains three NPXY motifs, YXXØ, SH3, and dileucine motifs, and a PDZ-binding motif at its COOH terminus. Deletion analysis established that amino acids 107–136 of the megalin-CT containing the second NPXY-like motif are critical for apical sorting and targeting, whereas the regions containing the first and third NPXY motifs are required for efficient endocytosis. We conclude that the megalin-CT contains a novel apical sorting determinant and that cytoplasmic sorting machinery exists in MDCK cells for some apical transmembrane proteins.
Microtubule-dependent PKC-α localization in A7r5 smooth muscle cells
Authors: Dykes A. C. , Fultz M. E. , Norton M. L. , Wright G. L.
Published: May 2013

Using laser scanning confocal, fluorescence resonance energy transfer (FRET), and atomic force (AFM) microscopy, we investigated association of protein kinase C (PKC)-α with microtubules during stimulus-induced relocalization in A7r5 smooth muscle cells. Confocal microscopy with standard immunostaining techniques confirmed earlier observations that colchicine disruption of microtubules blocked PKC-α localization in the perinuclear region of the cell caused by phorbol 12,13-dibutyrate (PDBu; 10–6M). Dual immunostaining suggested colocalization of PKC-α and β-tubulin in both unstimulated and PDBu-treated cells. This finding was verified by FRET microscopy, which indicated that association of PKC-α was heterogeneous in distribution and confined primarily to microtubules in the perinuclear region. FRET analysis further showed that association between the molecules was not lost during colchicine-induced dissolution of microtubules, suggesting formation of tubulin-PKC-α complexes in the cytosol. Confocal imaging indicated that perinuclear microtubular structure was more highly sensitive to colchicine dissolution than other regions of the cell. Topographic imaging of fixed cells by AFM indicated a well-defined elevated structure surrounding the nucleus that was absent in colchicine-treated cells. It was calculated that the volume of the nuclear sleevelike structure of microtubules increased approximately fivefold in PDBu-treated cells, suggesting a probable increase in microtubular mass. In light of PKC-α localization, increased colchicine sensitivity, and their volume change in stimulated cells, the results suggest that perinuclear microtubules form a specialized structure that may be more dynamically robust than in other regions of the cell. PKC-α could contribute to this dynamic activity. Alternatively, perinuclear microtubules could act as a scaffold for regulatory molecule interaction at the cell center.
Identification of the G protein-activating sequence of the single-transmembrane natriuretic peptide receptor C (NPR-C)
Authors: Zhou Huiping , Murthy Karnam S.
Published: May 2013

Rat natriuretic peptide clearance receptor (NPR-C) contains four sequences capable of inhibiting adenylyl cyclase. We have undertaken mutational and deletion studies on the intracellular domain of rat NPR-C to determine which of these sequences is functionally relevant. Nine mutant receptors were constructed by deletion of 11 or 28 COOH-terminal residues or by site-directed mutagenesis of basic residues in a 17-amino acid sequence, R469RNHQEESNIGKHRELR485, corresponding to the main active peptide. Substitution of arginine residues (R469R470) flanking the NH2 terminus abolished Gi1 and Gi2and PLC-β activities and inhibition of adenylyl cyclase. Substitution of one or two basic residues (H481 and/or R482or R485) in the COOH-terminal motif (H481RELR485) greatly decreased or abolished G protein and PLC-β activities and inhibition of adenylyl cyclase. This implies that sequences NH2-terminal to the motif or COOH-terminal to R470 could not sustain receptor activity in situ, although they exhibited activity when used as synthetic peptides. Deletion of the 11 COOH-terminal residues (E486to A496) suggested an autoinhibitory function for this sequence. We conclude that the 17-amino acid sequence (R469to R485) in the middle region of the intracellular domain of NPR-C is both necessary and sufficient for activation of G proteins and effector enzymes.
Structure-function relationships of AE2 regulation by Ca i 2 + -sensitive stimulators NH 4 + and hypertonicity
Authors: Chernova Marina N. , Stewart Andrew K. , Jiang Lianwei , Friedman David J. , Kunes Yune Z. , Alper Seth L.
Published: May 2013

We showed previously that the nonerythroid anion exchanger AE2 and the erythroid anion exchanger AE1 differ greatly in their regulation by acute changes in intracellular pH (pHi) and extracellular pH (pHo). We have now examined how AE2, but not AE1, is activated by two stimuli with opposing effects on oocyte pHi: an alkalinizing stimulus, hypertonicity, and an acidifying stimulus, NH[Formula: see text]. We find that both NH2-terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 are required for activation by either stimulus. Directed by initial deletion mutagenesis studies of the NH2-terminal cytoplasmic domain, an alanine scan of AE2 amino acids 336–347 identified residues whose individual mutation abolished or severely attenuated sensitivity to both or only one activating stimulus. Chelation of cytoplasmic Ca2+ (Ca[Formula: see text]) diminished or abolished AE2 stimulation by NH[Formula: see text] and by hypertonicity. Calmidazolium inhibited AE2 activity, but not that of AE1. AE2 was insensitive to many other modifiers of Ca2+ signaling. Unlike AE2 stimulation by NH[Formula: see text] and by hypertonicity, AE2 inhibition by calmidazolium required only AE2's COOH-terminal transmembrane domain.
Dystrophin-glycoprotein complex and Ras and Rho GTPase signaling are altered in muscle atrophy
Authors: Chockalingam Priya Sethu , Cholera Rushina , Oak Shilpa A. , Zheng Yi , Jarrett Harry W. , Thomason Donald B.
Published: May 2013

The dystrophin-glycoprotein complex (DGC) is a sarcolemmal complex whose defects cause muscular dystrophies. The normal function of this complex is not clear. We have proposed that this is a signal transduction complex, signaling normal interactions with matrix laminin, and that the response is normal growth and homeostasis. If so, the complex and its signaling should be altered in other physiological states such as atrophy. The amount of some of the DGC proteins, including dystrophin, β-dystroglycan, and α-sarcoglycan, is reduced significantly in rat skeletal muscle atrophy induced by tenotomy. Furthermore, H-Ras, RhoA, and Cdc42 decrease in expression levels and activities in muscle atrophy. When the small GTPases were assayed after laminin or β-dystroglycan depletion, H-Ras, Rac1, and Cdc42 activities were reduced, suggesting a physical linkage between the DGC and the GTPases. Dominant-negative Cdc42, introduced with a retroviral vector, resulted in fibers that appeared atrophic. These data support a putative role for the DGC in transduction of mechanical signals in muscle.
Cytoskeletal modulation of sodium current in human jejunal circular smooth muscle cells
Authors: Strege Peter R. , Holm Adrian N. , Rich Adam , Miller Steven M. , Ou Yijun , Sarr Michael G. , Farrugia Gianrico
Published: May 2013

A Na+ current is present in human jejunal circular smooth muscle cells. The aim of the present study was to determine the role of the cytoskeleton in the regulation of the Na+ current. Whole cell currents were recorded by using standard patch-clamp techniques with Cs+ in the pipette to block K+currents. Cytochalasin D and gelsolin were used to disrupt the actin cytoskeleton and phalloidin to stabilize it. Colchicine was used to disassemble the microtubule cytoskeleton (and intermediate filaments) and paclitaxel to stabilize it. Acrylamide was used to disrupt the intermediate filament cytoskeleton. Perfusion of the recording chamber at 10 ml/min increased peak Na+ current recorded from jejunal smooth muscle cells by 27 ± 3%. Cytochalasin D and gelsolin abolished the perfusion-induced increase in Na+current, whereas incubation with phalloidin, colchicine, paclitaxel, or acrylamide had no effect. In conclusion, the Na+ current expressed in human jejunal circular smooth muscle cells appears to be regulated by the cytoskeleton. An intact actin cytoskeleton is required for perfusion-induced activation of the Na+ current.