bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of voltage-gated Ca2+ channels in rabbit ventricular myocytes expressing Ca2+ channel α1E cDNA
Authors: Tateyama Michihiro , Zong Shuqin , Tanabe Tsutomu , Ochi Rikuo
Published: December 2017

Using the whole-cell patch-clamp technique, we have studied the properties of α1ECa2+ channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current ( I Ca,L). Expression of green fluorescent protein significantly decreased the I Ca,L. By contrast, expression of α1E with β2b and α2/δ significantly increased the total Ca2+ current, and in these cells a Ca2+ antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni2+and was little affected by changing the charge carrier from Ca2+ to Ba2+ or by β-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of α1E did not generate T-like current in cardiac myocytes. On the other hand, expression of α1E decreased I Ca,L and slowed the I Ca,L inactivation. This inactivation slowing was attenuated by the β2b coexpression, suggesting that the α1E may slow the inactivation of I Ca,L by scrambling with α1C for intrinsic auxiliary β.
Gender-related distinctions in protein kinase C activity in rat vascular smooth muscle
Authors: Kanashiro Celia A. , Khalil Raouf A.
Published: December 2017

Gender differences in vascular reactivity have been suggested; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the gender differences in vascular reactivity reflect gender-related, possibly estrogen-mediated, distinctions in the expression and activity of specific protein kinase C (PKC) isoforms in vascular smooth muscle. Aortic strips were isolated from intact and gonadectomized male and female Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Isometric contraction was measured in endothelium-denuded aortic strips. PKC activity was measured in the cytosolic and particulate fractions, and the amount of PKC was measured using Western blots and isoform-specific anti-PKC antibodies. In intact male WKY rats, phenylephrine (Phe, 10−5 M) and phorbol 12,13-dibutyrate (PDBu, 10−6 M) stimulated contraction to 0.37 ± 0.02 and 0.42 ± 0.02 g/mg tissue wt, respectively. The basal particulate/cytosolic PKC activity ratio was 0.86 ± 0.06, and Western blots revealed α-, δ-, and ζ-PKC isoforms. Phe and PDBu increased PKC activity and caused significant translocation of α- and δ-PKC from the cytosolic to particulate fraction. In intact female WKY rats, basal PKC activity, the amount of α-, δ-, and ζ-PKC, the Phe- and PDBu-induced contraction, and PKC activity and translocation of α- and δ-PKC were significantly reduced compared with intact male WKY rats. The basal PKC activity, the amount of α-, δ-, and ζ-PKC, the Phe and PDBu contraction, and PKC activity and α- and δ-PKC translocation were greater in SHR than WKY rats. The reduction in Phe and PDBu contraction and PKC activity in intact females compared with intact males was greater in SHR (∼30%) than WKY rats (∼20%). Phe and PDBu contraction and PKC activity were not significantly different between castrated males and intact males but were greater in ovariectomized (OVX) females than intact females. Treatment of OVX females or castrated males with 17β-estradiol, but not 17α-estradiol, subcutaneous implants caused significant reduction in Phe and PDBu contraction and PKC activity that was greater in SHR than WKY rats. Phe and PDBu contraction and PKC activity in OVX females or castrated males treated with 17β-estradiol plus the estrogen receptor antagonist ICI-182,780 were not significantly different from untreated OVX females or castrated males. Thus a gender-related reduction in vascular smooth muscle contraction in female WKY rats with intact gonads compared with males is associated with reduction in the expression and activity of vascular α-, δ-, and ζ-PKC. The gender differences in vascular smooth muscle contraction and PKC activity are augmented in the SHR and are possibly mediated by estrogen.
Tissue distribution and subcellular localization of the ClC-5 chloride channel in rat intestinal cells
Authors: Vandewalle Alain , Cluzeaud Françoise , Peng Kou-Cheng , Bens Marcelle , Lüchow Anke , Günther Willy , Jentsch Thomas J.
Published: December 2017

ClC-5 is the Cl− channel that is mutated in Dent's disease, an X-chromosome-linked disease characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones. It is predominantly expressed in endocytically active renal proximal cells. We investigated whether this Cl− channel could also be expressed in intestinal tissues that have endocytotic machinery. ClC-5mRNA was detected in the rat duodenum, jejunum, ileum, and colon. Western blot analyses revealed the presence of the 83-kDa ClC-5 protein in these tissues. Indirect immunofluorescence studies showed that ClC-5 was mainly concentrated in the cytoplasm above the nuclei of enterocytes and colon cells. ClC-5 partially colocalized with the transcytosed polymeric immunoglobulin receptor but was not detectable together with the brush-border-anchored sucrase isomaltase. A subfractionation of vesicles obtained by differential centrifugation showed that ClC-5 is associated with the vacuolar 70-kDa H+-ATPase and the small GTPases rab4 and rab5a, two markers of early endosomes. Thus these results indicate that ClC-5 is present in the small intestine and colon of rats and suggest that it plays a role in the endocytotic pathways of intestinal cells.
bHLH transcription factor MyoD affects myosin heavy chain expression pattern in a muscle-specific fashion
Authors: Seward David J. , Haney John C. , Rudnicki Michael A. , Swoap Steven J.
Published: December 2017

A strong correlative pattern between MyoD gene expression and myosin heavy chain IIB (MHC IIB) gene expression exists. To test whether this correlative relationship is causative, MHC gene expression in muscles from MyoD(−/−) mice was analyzed. The MHC IIB gene was not detectable in the MyoD(−/−) diaphragm, whereas the MHC IIB protein made up 10.0 ± 1.7% of the MHC protein pool in the wild-type (WT) mouse diaphragm. Furthermore, the MHC IIA protein was not detectable in the MyoD(−/−) biceps brachii, and the MHC IIB protein was overexpressed in the masseter. To examine whether MyoD is required for the upregulation of the MHC IIB gene within slow muscle after disuse, MyoD(−/−) and WT hindlimb musculature was unweighted. MyoD(−/−) exhibited a diminished response in the upregulation of the MHC IIB mRNA within the soleus muscle as a result of the hindlimb unweighting. Collectively, these data suggest that MyoD plays a role in the MHC profile in a muscle-specific fashion.
Effects of aging on actin sliding speed on myosin from single skeletal muscle cells of mice, rats, and humans
Authors: Höök Peter , Sriramoju Vidyasagar , Larsson Lars
Published: December 2017

The effects of aging on the mechanical properties of myosin were measured in 87 fibers from muscles of humans ( n = 40), rats ( n = 21), and mice ( n = 26) using a single fiber in vitro motility assay. Irrespective of species, an 18–25% aging-related slowing in the speed of actin filaments was observed from 62 single fibers expressing the slow (type I) β-myosin heavy chain isoform. The mechanisms underlying the aging-related slowing of motility speed remain unknown, but it is suggested that posttranslational modifications of myosin by oxidative stress, glycation, or nitration play an important role. The aging-related slowing in the speed of actin filaments propelled by the type I myosin was confirmed in three mammalian species with an ∼3,400-fold difference in body size. Motility speed from human myosin was 3-fold slower than from myosin of the ∼3,400-fold smaller mouse and approximately twofold slower when compared with the ∼130-fold smaller rat, irrespective of age. A strong correlation was observed between the log values of actin sliding speed and body mass, suggesting that the effects of scaling is, at least in part, due to altered functional properties of the motor protein itself.
PLD pathway involved in carbachol-induced Cl− secretion: possible role of TNF-α
Authors: Oprins Judith C. J. , van der Burg Claudia , Meijer Helen P. , Munnik Teun , Groot Jack A.
Published: December 2017

In a previous study, it was found that exposure to tumor necrosis factor-α (TNF-α) potentiated the electrophysiological response to carbachol in a time-dependent and cycloheximide-sensitive manner. It was deduced that the potentiation could be due to protein kinase C activity because of increased 1,2-diacylglycerol. It was also observed that propranolol could decrease the electrophysiological response to carbachol (Oprins JC, Meijer HP, and Groot JA. Am J Physiol Cell Physiol 278: C463–C472, 2000). The aim of the present study was to investigate whether the phospholipase D (PLD) pathway plays a role in the carbachol response and the potentiating effect of TNF-α. The transphosphatidylation reaction in the presence of the primary alcohol 1-butanol [leading to stable phosphatidylbutanol (Pbut) formation] was used to measure activity of PLD. The phosphatidic acid (PA) levels were also measured. Muscarinic stimulation resulted in an increased formation of Pbut and PA. TNF-α decreased levels of PA.
Caveolin-1 expression sensitizes fibroblastic and epithelial cells to apoptotic stimulation
Authors: Liu Jun , Lee Peiyee , Galbiati Ferruccio , Kitsis Richard N. , Lisanti Michael P.
Published: December 2017

The potential role of caveolin-1 in apoptosis remains controversial. Here, we investigate whether caveolin-1 expression is proapoptotic or antiapoptotic using a well-defined antisense approach. We show that NIH/3T3 cells harboring antisense caveolin-1 are resistant to staurosporine-induced apoptosis, as assessed using cell morphology, DNA content, caspase 3 activation, and focal adhesion kinase cleavage. Importantly, sensitivity to apoptosis is recovered when caveolin-1 levels are restored. Conversely, recombinant stable expression of caveolin-1 in T24 bladder carcinoma cells sensitizes these cells to caspase 3 activation. Consistent with the observations using NIH/3T3 cells, downregulation of caveolin-1 in T24 cells substantially diminishes caspase 3-like activity. Loss of sensitivity to apoptotic stimulation is recovered by inhibition of the phosphatidylinositol 3-kinase pathway using LY-294002, suggesting a possible mechanism for the sensitizing effect of caveolin-1. Thus our results suggest that caveolin-1 may act as a coupling or sensitizing factor in signaling apoptotic cell death in both fibroblastic (NIH/3T3) and epithelial (T24) cells.
Cloning and functional expression of a liver isoform of the small conductance Ca2+-activated K+ channel SK3
Authors: Barfod Elisabeth T. , Moore Ann L. , Lidofsky Steven D.
Published: December 2017

Small conductance Ca2+-activated K+(SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC50 630 nM) and were blocked by the SK channel inhibitor apamin (IC50 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than −40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.
Activation of K+ channels induces apoptosis in vascular smooth muscle cells
Authors: Krick Stefanie , Platoshyn Oleksandr , Sweeney Michele , Kim Hyong , Yuan Jason X.-J.
Published: December 2017

Intracellular K+ plays an important role in controlling the cytoplasmic ion homeostasis for maintaining cell volume and inhibiting apoptotic enzymes in the cytosol and nucleus. Cytoplasmic K+ concentration is mainly regulated by K+ uptake via Na+-K+-ATPase and K+ efflux through K+ channels in the plasma membrane. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore that dissipates the H+ gradient across the inner membrane of mitochondria, induces apoptosis in many cell types. In rat and human pulmonary artery smooth muscle cells (PASMC), FCCP opened the large-conductance, voltage- and Ca2+-sensitive K+ (maxi-K) channels, increased K+ currents through maxi-K channels [ I K(Ca)], and induced apoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM) decreased I K(Ca) by blocking the sarcolemmal maxi-K channels and inhibited the FCCP-induced apoptosis in PASMC cultured in media containing serum and growth factors. Furthermore, inhibition of K+ efflux by raising extracellular K+ concentration from 5 to 40 mM also attenuated PASMC apoptosis induced by FCCP and the K+ ionophore valinomycin. These results suggest that FCCP-mediated apoptosis in PASMC is partially due to an increase of maxi-K channel activity. The resultant K+ loss through opened maxi-K channels may serve as a trigger for cell shrinkage and caspase activation, which are major characteristics of apoptosis in pulmonary vascular smooth muscle cells.
Increased functional cell surface expression of CFTR and ΔF508-CFTR by the anthracycline doxorubicin
Authors: Maitra Rangan , Shaw Collin M. , Stanton Bruce A. , Hamilton Joshua W.
Published: December 2017

Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, ΔF508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, ΔF508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 μM) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased ΔF508-CFTR cell surface expression and ΔF508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF.
Nongenomic effect of testosterone on chloride secretion in cultured rat efferent duct epithelia
Authors: Leung G. P. H. , Cheng-Chew S. B. , Wong P. Y. D.
Published: December 2017

Short-circuit current ( I sc) technique was used to investigate the role of testosterone in the regulation of chloride secretion in cultured rat efferent duct epithelia. Among the steroids tested, only testosterone, and to a lesser extent, 5α-dihydrotestosterone (5α-DHT), reduced the basal and forskolin-induced I sc in cultured rat efferent duct epithelia when added to the apical bathing solution. Indomethacin, a 3α-hydroxysteroid dehydrogenase, did not affect the inhibitory effect of 5α-DHT. The effect of testosterone occurred within 10–20 s upon application and was dose dependent with apparent IC50 value of 1 μM. The effect was abolished by removal of Cl− but not HCO[Formula: see text] from the normal Krebs-Henseleit solution, suggesting that testosterone mainly inhibited Cl− secretion. The efferent duct was found to be most sensitive to testosterone, while the caput and the cauda epididymidis were only mildly sensitive. Cyproterone acetate, a steroidal antiandrogen, or flutamide, a nonsteroidal antiandrogen, did not block the effect of testosterone on the forskolin-induced I sc, nor did protein synthesis inhibitors, cycloheximide, or actinomycin D. However, pertussis toxin, a Gi protein inhibitor, attenuated the inhibition of forskolin-induced I sc by testosterone. Testosterone caused a dose-dependent inhibition of forskolin-induced rise in cAMP in efferent duct cells. It is suggested that the rapid effect of testosterone was mediated through a membrane receptor that is negatively coupled to adenylate cyclase via Gi protein. The role of nongenomic action of testosterone in the regulation of electrolyte and fluid transport in the efferent duct is discussed.
Nifedipine-activated Ca2+ permeability in newborn rat cortical collecting duct cells in primary culture
Authors: Valencia Laura , Bidet Michel , Martial Sonia , Sanchez Elsa , Melendez Estela , Tauc Michel , Poujeol Chantal , Martin Dolores , Namorado Maria Del Carmen , Reyes Jose Luis , Poujeol Philippe
Published: December 2017

To characterize Ca2+ transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca2+concentration ([Ca2+]i) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 μM) produced an increase in [Ca2+]i from 87.6 ± 3.3 nM to 389.9 ± 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca2+]i in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca2+]i. Experiments in the presence of EGTA showed that external Ca2+ was required for the nifedipine effect, while lanthanum (20 μM), gadolinium (100 μM), and diltiazem (20 μM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K+ channels were not involved in the nifedipine-induced [Ca2+]i rise. H2O2also triggered [Ca2+]i rise. However, nifedipine-induced [Ca2+]i increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca2+transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca2+ channel of capacitive type (either transient receptor potential or leak channel).
Soluble heparin-binding peptides regulate chemokinesis and cell adhesive forces
Authors: Chon John H , Chaikof Elliot L.
Published: December 2017

The ability of a soluble heparin-binding peptide sequence derived from fibronectin to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells was assessed using a novel glass microsphere centrifugation assay and automated time-lapse fluorescence videomicroscopy, respectively. Treatment of cells grown on fibronectin-coated substrates with the soluble heparin-binding peptide resulted in the disassembly of focal adhesions, as assessed by immunohistochemical staining. These observations were consistent with an observed dose-dependent two- to fivefold reduction in cell-substrate adhesive strength ( P < 0.001) and a biphasic effect on migration speed ( P < 0.05). Moreover, heparin-binding peptides induced a twofold reduction ( P < 0.01) in two-dimensional cell dispersion in the presence of a non-heparin-binding growth factor, platelet-derived growth factor-AB (PDGF-AB). Heparin-binding peptides were unable to mediate these effects when cells were grown on substrates lacking a heparin-binding domain. These data support the notion that competitive interactions between cell surface heparan sulfates with heparin-binding peptides may modulate chemokinetic cell migration behavior and other adhesion-related processes.
Hypoxic vasodilation in porcine coronary artery is preferentially inhibited by organ culture
Authors: Thorne George D. , Shimizu Shunichi , Paul Richard J.
Published: December 2017

Hypoxia (95% N2-5% CO2) elicits an endothelium-independent relaxation (45–80%) in freshly dissected porcine coronary arteries. Paired artery rings cultured at 37°C in sterile DMEM (pH ∼7.4) for 24 h contracted normally to KCl or 1 μM U-46619. However, relaxation in response to hypoxia was sharply attenuated compared with control (fresh arteries or those stored at 4°C for 24 h). Hypoxic vasorelaxation in organ cultured vessels was reduced at both high and low stimulation, indicating that both Ca2+-independent and Ca2+-dependent components are altered. In contrast, relaxation to G-kinase (sodium nitroprusside) or A-kinase (forskolin and isoproterenol) activation was not significantly affected by organ culture. Additionally, there was no difference in relaxation after washout of the stimulus, indicating that the inhibition is specific to acute hypoxia-induced relaxation. Simultaneous force and intracellular calcium concentration ([Ca2+]i) measurements indicate the reduction in [Ca2+]i concomitant with hypoxia at low stimulus levels in these tissue is abolished by culture. Our results indicate that organ culture at 37°C specifically attenuates hypoxic relaxation in vascular smooth muscle by altering dynamics of [Ca2+]i handling and decreasing a Ca2+-independent component of relaxation. Thus organ culture can be a novel tool for investigating the mechanisms of hypoxia-induced vasodilation.
Regulation by GDI of RhoA/Rho-kinase-induced Ca2+sensitization of smooth muscle myosin II
Authors: Gong Ming Cui , Gorenne Isabelle , Read Paul , Jia Taiping , Nakamoto Robert K. , Somlyo Avril V. , Somlyo Andrew P.
Published: December 2017

We characterized the role of guanine nucleotide dissociation inhibitor (GDI) in RhoA/Rho-kinase-mediated Ca2+ sensitization of smooth muscle. Endogenous contents (∼2–4 μM) of RhoA and RhoGDI were near stoichiometric, whereas a supraphysiological GDI concentration was required to relax Ca2+ sensitization of force by GTP and guanosine 5′- O-(3-thiotriphosphate) (GTPγS). GDI also inhibited Ca2+ sensitization by GTP · G14V RhoA, by α-adrenergic and muscarinic agonists, and extracted RhoA from membranes. GTPγS translocated Rho-kinase to a Triton X-114-extractable membrane fraction. GTP · G14V RhoA complexed with GDI also induced Ca2+ sensitization, probably through in vivo dissociation of GTP · RhoA from the complex, because it was reversed by addition of excess GDI. GDI did not inhibit Ca2+ sensitization by phorbol ester. Constitutively active Cdc42 and Rac1 inhibited Ca2+ sensitization by GTP · G14V RhoA. We conclude that 1) the most likely in vivo function of GDI is to prevent perpetual “recycling” of GDP · RhoA to GTP · RhoA; 2) nucleotide exchange (GTP for GDP) on complexed GDP · RhoA/GDI can precede translocation of RhoA to the membrane; 3) activation of Rho-kinase exposes a hydrophobic domain; and 4) Cdc42 and Rac1 can inhibit Ca2+ sensitization by activated GTP · RhoA.
Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters
Authors: Nakamura Tomoe Y. , Iwata Yuko , Sampaolesi Maurilio , Hanada Hironori , Saito Naohiro , Artman Michael , Coetzee William A. , Shigekawa Munekazu
Published: December 2017

Deficiency of δ-sarcoglycan (δ-SG), a component of the dystrophin-glycoprotein complex, causes cardiomyopathy and skeletal muscle dystrophy in Bio14.6 hamsters. Using cultured myotubes prepared from skeletal muscle of normal and Bio14.6 hamsters (J2N-k strain), we investigated the possibility that the δ-SG deficiency may lead to alterations in ionic conductances, which may ultimately lead to myocyte damage. In cell-attached patches (with Ba2+ as the charge carrier), an ∼20-pS channel was observed in both control and Bio14.6 myotubes. This channel is also permeable to K+ and Na+ but not to Cl−. Channel activity was increased by pressure-induced stretch and was reduced by GdCl3 (>5 μM). The basal open probability of this channel was fourfold higher in Bio14.6 myotubes, with longer open and shorter closed times. This was mimicked by depolymerization of the actin cytoskeleton. In intact Bio14.6 myotubes, the unidirectional basal Ca2+ influx was enhanced compared with control. This Ca2+ influx was sensitive to GdCl3, signifying that stretch-activated cation channels may have been responsible for Ca2+ influx in Bio14.6 hamster myotubes. These results suggest a possible mechanism by which cell damage might occur in this animal model of muscular dystrophy.
Direct in situ reverse transcriptase-polymerase chain reaction
Authors: Kher Rajesh , Bacallao Robert
Published: December 2017

In situ hybridization has been used for localization of specific nucleic acid sequences at the cellular level despite providing relatively low-detection sensitivity. In situ reverse transcriptase-polymerase chain reactions (RT-PCR) enhance sensitivity and thus enable localization of low-abundance mRNA in a cell. However, the available methods are fraught with problems of nonspecific amplifications as a result of mispriming and/or amplification from partially digested residual genomic DNA in tissue. Herein, we demonstrate that nonspecific background amplification can be eliminated by pretreatment of samples with restriction enzymes before DNase I digestion. Primers tagged with a far-red shifted fluorescent dye such as Cy5 in PCR reactions allow identification of target mRNA by fluorescence microscopy. These novel modifications lead to increased specificity and rapid in situ detection of cellular mRNA and thus may be used for pathological diagnosis.
Regulation of PGE2 and PGI2 release from human umbilical vein endothelial cells by actin cytoskeleton
Authors: Sawyer Sara J. , Norvell Suzanne M. , Ponik Suzanne M. , Pavalko Fredrick M.
Published: December 2017

Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin E2 (PGE2) and a 3.4- to 6.5-fold increase in prostacyclin (PGI2) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of PGE2 1.7- to 1.9-fold and PGI2 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of PGE2 and PGI2 from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more PGE2 and 3.8-fold more PGI2 released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more PGE2 and 11.2-fold more PGI2 released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC.
Ca2+ influx and cAMP elevation overcame botulinum toxin A but not tetanus toxin inhibition of insulin exocytosis
Authors: Huang Xiaohang , Kang You-Hou , Pasyk Ewa A. , Sheu Laura , Wheeler Michael B. , Trimble William S. , Salapatek Annemarie , Gaisano Herbert Y.
Published: December 2017

Previous reports showed that cleavage of vesicle-associated membrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting β-cells inhibited Ca2+-evoked insulin secretion. In these reports, the soluble N-ethylmaleimide-sensitive factor attachment protein target receptor proteins might have formed complexes, which preclude full accessibility of the putative sites for neurotoxin cleavage. In this work, VAMP-2 and SNAP-25 were effectively cleaved before they formed toxin-insensitive complexes by transient transfection of insulinoma HIT or INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A (BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca2+ (glucose or KCl)-evoked insulin release proportionate to the transfection efficiency (40–50%) and an accumulation of insulin granules. With the use of patch-clamp capacitance measurements, Ca2+-evoked exocytosis by membrane depolarization to −10 mV was abolished by TeTx (6% of control) but only moderately inhibited by BoNT/A (30% of control). Depolarization to 0 mV to maximize Ca2+ influx partially overcame BoNT/A (50% of control) but not TeTx inhibition. Of note, cAMP activation potentiated Ca2+-evoked secretion by 129% in control cells but only 55% in BoNT/A-transfected cells and had negligible effects in TeTx-transfected cells. These results indicate that, whereas VAMP-2 is absolutely necessary for insulin exocytosis, the effects of SNAP-25 depletion on exocytosis, perhaps on insulin granule pool priming or mobilization steps, could be partially reversed by higher levels of Ca2+ or cAMP potentiation.
Preservation of metabolic reserves and function after storage of myocytes in hypothermic UW solution
Authors: Hegge Julia O. , Southard James H. , Haworth Robert A.
Published: December 2017

Isolated rat myocytes cold stored anaerobically up to 24 h in University of Wisconsin solution lost 95% of their ATP and 100% of their glycogen. They underwent contracture when rewarmed in a Krebs-Henseleit (KH) medium that contained Ca unless Ca addition was delayed. In the latter case, cell function, measured by stimulation-induced cell shortening, was surprisingly well retained. Aerobically stored cells were resistant to Ca on rewarming, although 96% of glycogen was still lost, along with 46% of ATP. Cells that were incubated for 48 h aerobically with the substrates glucose and pyruvate at pH 6.2 retained 77% of their ATP and 59% of their glycogen, with good cell morphology. At pH 6.2, the demand for ATP was only 55% of that at pH 7.4. However, after rewarming, these cells functioned no better than anaerobically stored cells, although their inotropic response to isoproterenol was improved. We conclude that 1) aerobic conditions with substrates at low pH preserve myocyte metabolic reserves well for 48 h, partly by reducing the demand for ATP; 2) rewarming conditions are critical for anaerobically stored cells with metabolic stores that are severely depleted; and 3) unloaded cell function is surprisingly insensitive to a period of severe metabolic deprivation.
Gap junction assembly: PTX-sensitive G proteins regulate the distribution of connexin43 within cells
Authors: Lampe Paul D. , Qiu Qiu , Meyer Rita A. , TenBroek Erica M. , Walseth Timothy F. , Starich Todd A. , Grunenwald Haiying L. , Johnson Ross G.
Published: December 2017

Cells expressing connexin43 are able to upregulate gap junction (GJ) communication by enhancing the assembly of new GJs, apparently through increased connexin trafficking. Because G proteins are known to regulate different aspects of protein trafficking, we examined the effects of pertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJ assembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicated by a reduction in dye transfer. Electron microscopy also revealed a 60% decrease in the number of GJ channels per cell interface. Importantly, PTX blocked the twofold enhancement in GJ assembly found in the presence of low-density lipoprotein. Two Giαproteins (Giα2 and Giα3), which have been implicated in the control of membrane trafficking, reacted with PTX in ADP-ribosylation studies. PTX and/or the trafficking inhibitors, brefeldin A and monensin, inhibited GJ assembly to comparable degrees. In addition, assays for GJ hemichannels demonstrated reduced plasma membrane levels of connexin43 following PTX treatment. These results suggest that PTX-sensitive G proteins regulate connexin43 trafficking, and, as a result of inhibition with PTX, the number of plasma membrane hemichannels available for GJ assembly is reduced.
cGMP-mediated inhibition of cardiac L-type Ca2+current by a monoclonal antibody against the M2 ACh receptor
Authors: Nascimento J. H. M. , Sallé L. , Hoebeke J. , Argibay J. , Peineau N.
Published: December 2017

The effects of a monoclonal antibody (B8E5) directed against the second extracellular loop of the muscarinic M2receptor were studied on the L-type Ca2+ currents ( I Ca,L) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoproterenol (ISO)-stimulated I Ca,L but did not significantly affect basal I Ca,L. Atropine blocked the inhibitory effect of B8E5. The electrophysiological parameters of ISO-stimulated I Ca,L were not modified in presence of B8E5. Inhibition of I Ca,L by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hydrolysis-resistant cAMP analog (8-bromoadenosine 3′,5′-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 was mimicked by 8-bromoguanosine 3′,5′-cyclic monophosphate, a potent stimulator of cGMP-dependent protein kinase, and prevented by a selective inhibitor of nitric oxide-sensitive guanylyl cyclase {1 H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one}. These results indicate that the antibody B8E5 inhibits the β-adrenergic-stimulated I Ca,L through activation of the M2 muscarinic receptor and further suggest that the antibody acts not via the classical pathway of decreasing intracellular cAMP, but rather by increasing cGMP.
Expression of endoplasmic reticulum stress proteins during skeletal muscle disuse atrophy
Authors: Hunter R. Bridge , Mitchell-Felton Heather , Essig David A. , Kandarian Susan C.
Published: December 2017

Disuse atrophy of skeletal muscle leads to an upregulation of genes encoding sarcoplasmic reticulum (SR) calcium-handling proteins. Because many of the proteins that are induced with endoplasmic reticulum (ER) stress are ER calcium-handling proteins, we sought to determine whether soleus muscle atrophy was associated with a prototypical ER stress response. Seven days of rat hindlimb unloading did not alter expression of ubiquitous ER stress proteins such as Grp78, calreticulin, and CHOP/GADD-153, nor other proteins that have been shown to be activated by ER stressors such as vinculin, the type I d- myo-inositol 1,4,5-trisphosphate receptor, or protein kinase R, a eukaryotic initiation factor 2α kinase. On the other hand, expression of heme oxygenase-1 (HO-1), an antioxidant ER stress protein, was significantly increased 2.2-fold. In addition, unloading led to an increase in calsequestrin, the muscle-specific SR calcium-binding protein, at both the mRNA (68%) and protein (24%) levels. Although disuse atrophy is associated with a significant remodeling of muscle-specific proteins controlling SR calcium flux, it is not characterized by a prototypical ER stress response. However, the upregulation of HO-1 may indicate ER adaptation to oxidative stress during muscle unloading.
Oxygen sensitivity in the sheep adrenal medulla: role of SK channels
Authors: Keating Damien J. , Rychkov Grigori Y. , Roberts Michael L.
Published: December 2017

The hypoxia-evoked secretion of catecholamines from the noninnervated fetal adrenal gland is essential for surviving intrauterine hypoxemia. The ion channels responsible for the initial depolarization that leads to catecholamine secretion have not been identified. Patch-clamp studies of adrenal chromaffin cells isolated from fetal and adult sheep revealed the presence of a Ca2+-dependent K+ current that was reduced by hypoxia. Apamin, a blocker of small-conductance K+ (SK) channels, reduced the Ca2+-dependent K+current, and the sensitivity of the channels to apamin indicated that the channels involved were of the SK2 subtype. In the presence of apamin, the hypoxia-evoked change in K+ currents was largely eliminated. Both hypoxia and apamin blocked a K+current responsible for maintaining the resting potential of the cell, and the depolarization resulting from both led to an influx of Ca2+. Simultaneous application of hypoxia and apamin did not potentiate the increase in cytosolic Ca2+ concentration beyond that seen with either agent alone. Similar results were seen with curare, another blocker of SK channels. These results indicate that closure of SK2 channels would be the initiating event in the hypoxia-evoked catecholamine secretion in the adrenal medulla.
Evidence for functional role of εPKC isozyme in the regulation of cardiac Na+ channels
Authors: Xiao Guang-Qian , Qu Yongxia , Sun Zhou-Qian , Mochly-Rosen Daria , Boutjdir Mohamed
Published: December 2017

Investigation of the role of individual protein kinase C (PKC) isozymes in the regulation of Na+ channels has been largely limited by the lack of isozyme-selective modulators. Here we used a novel peptide-specific activator (εV1–7) of εPKC and other peptide isozyme-specific inhibitors in addition to the general PKC activator phorbol 12-myristate 13-acetate (PMA) to dissect the role of individual PKCs in the regulation of the human cardiac Na+ channel hH1, heterologously expressed in Xenopus oocytes. Peptides were injected individually or in combination into the oocyte. Whole cell Na+ current ( I Na) was recorded using two-electrode voltage clamp. εV1–7 (100 nM) and PMA (100 nM) inhibited I Na by 31 ± 5% and 44 ± 8% (at −20 mV), respectively. These effects were not seen with the scrambled peptide for εV1–7 (100 nM) or the PMA analog 4α-phorbol 12,13-didecanoate (100 nM). However, εV1–7- and PMA-induced I Na inhibition was abolished by εV1–2, a peptide-specific antagonist of εPKC. Furthermore, PMA-induced I Na inhibition was not altered by 100 nM peptide-specific inhibitors for α-, β-, δ-, or ηPKC. PMA and εV1–7 induced translocation of εPKC from soluble to particulate fraction in Xenopus oocytes. This translocation was antagonized by εV1–2. In native rat ventricular myocytes, PMA and εV1–7 also inhibited I Na; this inhibition was antagonized by εV1–2. In conclusion, the results provide evidence for selective regulation of cardiac Na+channels by εPKC isozyme.
Bcl-2 protects lymphoma cells from apoptosis but not growth arrest promoted by cAMP and dexamethasone
Authors: Zhang Lingzhi , Insel Paul A.
Published: December 2017

Glucocorticoids or increases in cellular cAMP promote apoptosis in many cell types, including murine S49 cells. We examined the impact of Bcl-2, an antiapoptotic protein, on S49 cell growth and death promoted by the glucocorticoid dexamethasone or agents that increase cAMP: isoproterenol (a β-adrenergic agonist) + 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) and forskolin (diterpene). These agents promoted apoptosis (i.e., increased expression of annexin V) of wild-type (WT) S49 cells, but Bcl-2-overexpressing S49 cells were protected from this response. Bcl-2 overexpression did not protect cells from G1 growth arrest but did allow cells to grow longer in culture and protected cells from culture-dependent necrosis. Commitment to and reversal from apoptosis vs. G1 growth arrest by isoproterenol + 3-isobutyl-1-methylxanthine showed different kinetics. Although both processes required several hours to develop, removal of agonists readily reversed growth arrest, but not apoptosis. Thus commitment to apoptosis is less reversible than G1 growth arrest. The findings also indicate that glucocorticoid- and cAMP-mediated G1 growth arrest is unaffected by Bcl-2 overexpression, even though increased Bcl-2 allows these lymphoma cells to resist necrosis and apoptosis.
c-Src and HSP72 interact in ATP-depleted renal epithelial cells
Authors: Wang Y.-H. , Li F. , Schwartz J. H. , Flint P. J. , Borkan S. C.
Published: December 2017

Disruption of cell contact sites during ischemia contributes to the loss of organ function in acute renal failure. Because prior heat stress protects cell contact sites in ATP-depleted renal epithelial cells in vitro, we hypothesized that heat shock protein 72 (HSP72), the major inducible cytoprotectant in mammalian cells, interacts with protein kinases that regulate cell-cell and cell-matrix interactions. ATP depletion increased the content of Tyr416 Src, the activated form of this kinase. c-Src activation was associated with an increase in the tyrosine phosphorylation state of β-catenin, paxillin, and vinculin, three c-Src substrate proteins that localize to and regulate cell contact sites. Prior heat stress inhibited c-Src activation and decreased the degree of tyrosine phosphorylation of all three Src substrates during ATP depletion and/or early recovery. HSP72 coimmunoprecipitated with c-Src only in cells subjected to heat stress. ATP depletion markedly increased the interaction between HSP72 and c-Src, supporting the hypothesis that HSP72 regulates Src kinase activity. These results suggest that alterations in the tyrosine phosphorylation state of proteins located at the cell-cell and cell-matrix interface mediate, at least in part, the functional state of these structures during ATP depletion and may be modulated by interactions between HSP72 and c-Src.
Mechanisms of modulation of neuronal nicotinic receptors by substance P and OAG
Authors: Andoh Tomio , Itoh Hideki , Watanabe Itaru , Sasaki Toshio , Higashi Tomoko
Published: December 2017

Substance P is known to modulate neuronal nicotinic acetylcholine receptors (nAChRs) in the sympathetic nervous system. There are two conflicting proposals for the mechanism of this effect, an indirect action mediated by protein kinase C (PKC) and a direct interaction with receptor subunits. We studied the mechanisms of this effect in PC-12 cells. Substance P enhanced the decay of the nicotine-induced whole cell current. This effect was fast in its onset and was not antagonized by guanosine 5′- O-(2-thiodiphosphate), a G protein blocker, or staurosporine, a nonselective PKC blocker. Staurosporine failed to reverse the inhibition by 1-oleoyl-2-acetyl- sn-glycerol (OAG), a synthetic diacylglycerol analog known to activate PKC. The inhibitory effects of the peptide and OAG were preserved in excised patches, but substance P applied to the extra patch membrane was ineffective in the cell-attached patch configuration. We conclude that substance P modulates neuronal nAChRs most likely by direct interactions with the receptors but independently from activation of PKC or G proteins and that PKC does not participate in modulation by OAG.
Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice
Authors: He L. , Chen J. , Dinger B. , Sanders K. , Sundar K. , Hoidal J. , Fidone S.
Published: December 2017

Various heme-containing proteins have been proposed as primary molecular O2 sensors for hypoxia-sensitive type I cells in the mammalian carotid body. One set of data in particular supports the involvement of a cytochrome b NADPH oxidase that is commonly found in neutrophils. Subunits of this enzyme have been immunocytochemically localized in type I cells, and diphenyleneiodonium, an inhibitor of the oxidase, increases carotid body chemoreceptor activity. The present study evaluated immunocytochemical and functional properties of carotid bodies from normal mice and from mice with a disrupted gp91 phagocytic oxidase (gp91 phox ) DNA sequence gene knockout (KO), a gene that codes for a subunit of the neutrophilic form of NADPH oxidase. Immunostaining for tyrosine hydroxylase, a signature marker antigen for type I cells, was found in groups or lobules of cells displaying morphological features typical of the O2-sensitive cells in other species, and the incidence of tyrosine hydroxylase-immunopositive cells was similar in carotid bodies from both strains of mice. Studies of whole cell K+currents also revealed identical current-voltage relationships and current depression by hypoxia in type I cells dissociated from normal vs. KO animals. Likewise, hypoxia-evoked increases in intracellular Ca2+ concentration were not significantly different for normal and KO type I cells. The whole organ response to hypoxia was evaluated in recordings of carotid sinus nerve activity in vitro. In these experiments, responses elicited by hypoxia and by the classic chemoreceptor stimulant nicotine were also indistinguishable in normal vs. KO preparations. Our data demonstrate that carotid body function remains intact after sequence disruption of the gp91 phox gene. These findings are not in accord with the hypothesis that the phagocytic form of NADPH oxidase acts as a primary O2 sensor in arterial chemoreception.
Role of hepatic portal osmoreception in the control of ADH release.
Authors: Chwalbińska-Moneta J
Published: December 2017

Dogs were prepared for experiments by chronic implantation of catheters into the hepatic portal vein. Intraportal infusions of hypertonic saline, 5 ml/min, increased plasma antidiuretic hormones (rat bioassay) in absence of significant changes in systemic plasma osmolality. In the 5th min of 1.8, 2.7, or 3.6% saline infusion, plasma antidiuretic hormone (ADH) increased significantly from 3.2 +/- 0.7 to 14.3 +/- 3.6 (SE) microU/ml, from 2.3 +/- 0.4 to 42.0 +/- 7.6 microU/ml, and from 1.9 +/- 0.6 to 64.4 +/- 16.8 microU/ml, respectively. In control experiments plasma ADH did not change with isotonic saline infused intraportally or with 3.6% saline given into a systemic vein. The transsection of hepatic vagal afferent fibers abolished plasma ADH increase dependent on hypertonic intraportal infusion. The data support the existence of osmoreceptors within the portal vascular bed and indicate ADH involvement in the control of fluid balance by intrahepatic blood osmalality. The information on osmolality changes is neurally transmitted to the hypothalamus via hepatic vagal afferents. A demonstration of an increase in portal but not systemic blood osmolality following food intake suggests that intrahepatic osmoreception may be important in physiological regulation of water metabolism.
Hemodynamics and metabolism of the in vivo vascularly isolated canine pancreas.
Authors: Alteveer R J , Jaffe M J , Van Dam J
Published: December 2017

Surgical procedures are detailed that have yielded for the first time an in vivo vascularly isolated, autoperfused preparation of the entire pancreas in anesthetized dogs. Previous studies had isolated only part of the pancreas or had resorted to blood-flow techniques not requiring pooled pancreatic venous blood, necessary for metabolic studies of the organ. Pancreatic blood flow (48 ml/min), O2 uptake (180 mumol/min), glucose uptake (51.0 mumol/min), lactate output (6.6 mumol/min), net free fatty acid uptake (2.23 mumol/min), all per 100 g tissue, and various other measured and calculated hemodynamic and metabolic variables were determined on the preparation during control conditions. The stability of the preparation was verified by serial determinations of these parameters and of blood alpha-amylase and beta-glucuronidase levels from 1 to 2.5 h postsurgery. Metabolic rate and glucose uptake were both found to be much higher than in intestinal tissues and approached values characteristic of liver tissue.