bluuebunny/crossref_metadata_2025 huggingface dataset:
Short-chain fatty acids inhibit cAMP-mediated chloride secretion in rat colon
Authors: Dagher P. C. , Egnor R. W. , Taglietta-Kohlbrecher A. , Charney A. N.
Published: December 2017

Butyrate stimulates salt absorption in mammalian colon. We examined whether butyrate also affects Cl- secretion. Mucosal segments of distal colon of male Sprague-Dawley rats and T84 cells were studied in Ussing chambers. In control colon, 1 mM dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) increased short-circuit current (Isc) and serosal-to-mucosal Cl- flux (JsmCl) by 3.2 +/- 0.8 and 2.9 +/- 0.8 mueq.cm-2.h-1, respectively. Mucosal or serosal 25 mM butyrate prevented DBcAMP-induced increases in Isc and JsmCl. Four and eight millimolar butyrate caused half-maximal inhibition of the increases in JsmCl and Isc, respectively. Butyrate also inhibited basal JsmCl (by 2.0 +/- 0.4 mueq.cm-2.h-1) but not carbachol-mediated Cl- secretion. The relative inhibitory potency at 25 mM of other short-chain fatty acids (SCFA) paralleled their degree of cellular metabolism: butyrate > acetate = propionate > isobutyrate. At 25 mM, all SCFA reduced mucosal intracellular pH (pHi) transiently by 0.1 pH unit. In intact T84 cells, 50 mM butyrate inhibited the DBcAMP-induced rise in Isc by 55%. In T84 cells with nystatin-permeabilized basolateral membranes, butyrate inhibited the increase in Isc by 82%. We conclude that butyrate inhibits basal and cAMP-mediated Cl- secretion by a mechanism independent of pHi, possibly located at the apical membrane.
A role for a background sodium current in spontaneous action potentials and secretion from rat lactotrophs
Authors: Sankaranarayanan S. , Simasko S. M.
Published: December 2017

We have used the perforated-patch variation of whole cell patch-clamp techniques, measurements of cytosolic calcium with use of fura 2, and secretion measurements with use of the reverse-hemolytic plaque assay to address the role of depolarizing background currents in maintaining spontaneous action potentials and spontaneous secretion from rat lactotrophs in primary culture. Replacement of bath sodium with tris(hydroxymethyl)aminomethane or N-methyl-D-glucamine caused a dramatic hyperpolarization of the cells, a cessation of spontaneous action potentials, and an increase in input resistance of cells. Tetrodotoxin had no effect on spontaneous action potentials, and removal of bath calcium stopped spiking but did not hyperpolarize the cells. The hyperpolarization in response to removal of bath sodium was associated with a decrease in cytosolic calcium levels. Finally, removal of bath sodium caused a decrease in spontaneous secretion of prolactin from lactotrophs. These data suggest that a background sodium current is essential to drive the membrane to threshold for firing spontaneous calcium-dependent action potentials in lactotrophs. This, in turn, results in elevated intracellular calcium, which supports spontaneous secretion of prolactin from these cells.
Oxygen dependence of respiration in coupled and uncoupled endothelial cells
Authors: Steinlechner-Maran R. , Eberl T. , Kunc M. , Margreiter R. , Gnaiger E.
Published: December 2017

We studied the oxygen dependence of respiration in cultured human umbilical vein endothelial cells by use of high-resolution respirometry. The rate of oxygen consumption varied from 30 to 50 pmol O2.s-1.(10(6) cells)-1 over a sixfold range of cell densities. Respiration was stimulated up to 3.5-fold by uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone or 2,4-dinitrophenol, and the PO2 at half-maximal respiration (P50) increased from 0.05 to 0.12 kPa (0.3 to 0.9 Torr) with respiratory rate. P50 decreased to a minimum of 0.02 kPa when uncoupled cells were inhibited to control levels. Differences in cell size explained a variation of approximately 0.015 kPa in P50 at similar respiratory rates per cell. Oxygen diffusion to mitochondria contributed maximally 30% to the regulation of P50 in coupled cells, as deduced from the shallow slope of the flux dependence of P50 in uncoupled-inhibited cells compared with the slope in coupled cells. Therefore 70% of the flux dependence of P50 in coupled cells was caused by changes in metabolic state, which correlated with respiratory rate.
An electrophysiological study of neurons in the horizontal limb of the diagonal band of Broca
Authors: Easaw J. C. , Petrov T. , Jhamandas J. H.
Published: December 2017

We examined the morphological and electrophysiological properties of neurons within the horizontal limb of the diagonal band of Broca (hDBB) and investigated the role of excitatory amino acid mediated synaptic transmission in this region. Whole cell patch-clamp recordings were obtained from hDBB neurons in rat forebrain slices. The hDBB cells examined in this study display a morphological and electrophysiological profile that is consistent with the type B, noncholinergic cell type. Cable analysis reveals that hDBB neurons are electrotonically compact and may therefore function as efficient relays for transmission of inputs to other forebrain target sites. Application of agonists for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate, N-methyl-D-aspartate (NMDA), and metabotropic receptors all evoke inward currents in hDBB neurons. Pharmacological analyses of synaptic events indicate that evoked excitatory postsynaptic currents (EPSC) are either mediated by non-NMDA receptors alone or a combination of non-NMDA and NMDA receptors. In some neurons, the metabotropic receptor agonist, 1-aminocyclopentane-trans-1, 3-dicarboxylic acid, reduced EPSC amplitude without altering postsynaptic input conductance, thus suggesting a presynaptic locus of action. The electrical and pharmacological properties described for hDBB neurons may be physiologically relevant for the effective transmission of excitatory synaptic inputs to sites that receive projections from the hDBB.
Muscarinic activation and calcium permeation of nonselective cation currents in airway myocytes
Authors: Fleischmann B. K. , Wang Y. X. , Kotlikoff M. I.
Published: December 2017

We examined the activation and Ca2+ permeation of nonselective cation channels in voltage-clamped (nystatin), fura 2-loaded equine tracheal myocytes at 35 degrees C. Methacholine (50 microM) induced a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i) and a biphasic inward current consisting of a large, rapidly inactivating Ca(2+)-activated Cl current [ICl(Ca)] and a smaller, sustained nonselective cation current (Icat) ICl(Ca) but not Icat was activated by caffeine. Neither Icat nor the sustained rise in [Ca2+]i was blocked by nisoldipine, whereas both were rapidly blocked by Ni2+; Icat was determined to be Ca2+ permeant, since 1) a sustained elevation of [Ca2+]i occurred when Icat was activated, and blockade of Icat produced a rapid decline in [Ca2+]i; 2) increasing extracellular Ca2+ during Icat increased [Ca2+]i; 3) 110 mM extracellular Ca2+ shifted the reversal potential of Icat to 12 mV (Ca(2+)-to-Cs+ permeability ratio = 3.6); and 4) instantaneous voltage-clamp steps to negative potentials during Icat increased the current and [Ca2+]i, whereas depolarizing steps decreased the current and [Ca2+]i. The fraction of Icat carried by Ca2+ under physiological conditions was estimated to be 14% at -60 mV.
Reinterpretation of the RACTK1 K+ channel
Authors: Shmukler B. , Sun T. , Brugnara C. , Alper S. L.
Published: December 2017

The RACTK1 cDNA cloned from rabbit kidney cortical collecting duct cells was associated with inwardly rectifying pH-regulated K+ channel activity (M. Suzuki, K. Takahashi, M. [keda, H Hayakawa, A. Ogawa, Y. Kawaguchi, and O. Sakai. Nature Lond. 367: 642-645, 1994). The deduced amino acid sequence of the encoded novel polypeptide lacked the signature sequence of a K(+)-selective pore region but predicted a topography suggestive of the inward rectifier K+ channel family. In subsequent articles a RACTK1 epitope was immunolocalized to the apical surface of kidney collecting duct and to arteriolar smooth muscle [M. Suzuki, T. Takigawa, K. Kimura, C. Koseki, and M. Imai. Am. J. Physiol. 269 (Cell Physiol, 38): C496-C503, 1995], and apamin-sensitive K+ currents displaying Ca(2+)-dependent and voltage-independent activation accompanied stable heterologous overexpression of RACTK1 [M. Suzuki, M. Murata, M. Ikeda, T. Miyoshi, and M. Imai. Am. J. Physiol. 270 (Cell Physiol, 39): C964-C968, 1996]. We now report that the "RACTK1" open reading frame is a frame-shifted translation of the antisense strand of an Escherichia coli gene member of a coenzyme A transferase gene family. "RACTK1" mRNA was absent from tissues free of E. coli contamination, and the "RACTK1" gene was undetectable in Southern blots of human and rabbit genomic DNA. We conclude that the immunostaining patterns and Ca(2+)-activated K+ channel activity heretofore attributed to RACTK1 must be otherwise explained.
ADP-regenerating enzyme systems in mitochondria of guinea pig myometrium and heart
Authors: Clark J. F. , Kuznetsov A. V. , Radda G. K.
Published: December 2017

Any enzyme or enzyme system that produces ADP in proximity to the mitochondria may be capable of stimulating respiration. Hexokinase (HK), adenylate kinase (AK), and mitochondrial creatine kinase (Mi-CK) all catalyze reactions that produce ADP and thus may play a role in cellular nucleotide metabolism or control of mitochondrial oxidative phosphorylation. Respiratory characteristics and enzyme activities of mitochondria simultaneously isolated from heart and uterus of the gravid guinea pig were compared. The abilities of AMP, glucose, and creatine to stimulate mitochondrial respiration via AK, HK, and Mi-CK systems, respectively, were examined. Although the uterine Mi-CK activity is low compared with the values found in heart, the activities of HK and AK were significantly greater. Furthermore, the abilities of HK and AK to stimulate respiration (functional activity) were greater in the uterine mitochondria. Indeed, the activity of AK was sufficient to generate maximal (state 3) respiration. The apparent Michaelis constant (Km) for ADP to stimulate respiration in the isolated uterine mitochondria was significantly different from that of the heart mitochondria (9.6 +/- 0.9 and 5.1 +/- 1 microM ADP, respectively). It is concluded that uterine mitochondria can use HK and AK systems in addition to the CK system in enhancing local ADP concentration, which may aid in the mitochondrial responses to energetic demands.
Effect of acidosis on control of respiration in skeletal muscle
Authors: Harkema S. J. , Meyer R. A.
Published: December 2017

The relationships between oxygen consumption (Q(O2)) and calculated cytoplasmic ADP concentration ([ADP]) and the free energy of ATP hydrolysis (deltaG(ATP)) were examined in ex vivo arterially perfused cat soleus muscles during repetitive twitch stimulation under normocapnic (5% CO2) and hypercapnic (70% CO2) conditions. Hypercapnia decreased extra- and intracellular pH by over 0.5 but had no significant effect on Q(O2) or phosphocreatine (PCr)/ATP in muscles at rest. The maximum Q(O2) measured during stimulation and the rate constant for PCr recovery after stimulation both decreased during hypercapnic compared with normocapnic perfusion, but the estimated ATP/O2 was unchanged. The change in PCr and deltaG(ATP) with increasing Q(O2) was greater during hypercapnic compared with normocapnic stimulation, as expected from the decrease in maximum Q(O2). However, the relationships between Q(O2) and [ADP] and deltaG(ATP) were both shifted to the left during hypercapnia compared with normocapnia. The results show that changes in cytoplasmic adenine nucleotides and phosphate are not sufficient to explain the control of respiration in skeletal muscle. However, in the context of thermodynamic models of respiratory control, the results can be explained by increased intramitochondrial potential for ATP synthesis at low pH.
Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species
Authors: Nieminen A. L. , Byrne A. M. , Herman B. , Lemasters J. J.
Published: December 2017

Tert-butyl hydroperoxide (t-BuOOH) induces the mitochondrial permeability transition (MPT) in hepatocytes, leading to cell death. Using confocal microscopy, we visualized pyridine nucleotide oxidation and reactive oxygen species (ROS) formation induced by t-BuOOH. Reduced mitochondrial pyridine nucleotides (NADH and NADPH) were imaged by autofluorescence. Mitochondrial membrane potential, ROS, onset of MPT, and cell death were monitored with tetramethylrhodamine methyl ester (TMRM), dichlorofluorescin, calcein, and propidium iodide, respectively. t-BuOOH rapidly oxidized mitochondrial NAD(P)H. Oxidation was biphasic, and the second slower phase occurred during mitochondrial ROS generation. Subsequently, MPT took place, mitochondria depolarized, and cells died. beta-Hydroxybutyrate, which reduces mitochondrial NAD+, delayed cell killing, but lactate, which reduces cytosolic NAD+, did not. Trifluoperazine, which inhibits MPT, did not block the initial oxidation of NAD(P)H but prevented the second phase of oxidation, partially blocked ROS formation, and preserved cell viability. The antioxidants, deferoxamine and diphenylphenylenediamine, also prevented the second phase of NAD(P)H oxidation. They also blocked ROS formation nearly completely and stopped cell killing. Both antioxidants also prevented the mitochondrial permeability transition and subsequent mitochondrial depolarization. In conclusion, NAD(P)H oxidation and ROS formation are critical events promoting MPT in oxidative injury and death of hepatocytes.
Evidence for a calmodulin-dependent phospholipase A2 that inhibits Na-K-ATPase
Authors: Okafor M. C. , Schiebinger R. J. , Yingst D. R.
Published: December 2017

We tested whether calnaktin, a proposed Ca/calmodulin (CaM)-dependent protein that inhibits the Na-K-ATPase, was a kinase, a phosphatase, a phospholipase A2 (PLA2), or a Ca-dependent protease. Human red blood cell membranes were extracted to remove associated calmodulin but to retain the proposed endogenous calnaktin. Exclusively cytoplasmic proteins and cofactors were presumably absent. In these membranes, free Ca inhibited the Na-K-ATPase with an inhibition constant (K[i]) of > or = 9 microM at a Na concentration of 18 mM. Addition of 100 nM CaM decreased the Ki to < 2 microM and increased the percent inhibition at 2 microM free Ca from 18 +/- 1 to 68 +/- 2%. The inhibitory effect of Ca/CaM was reversible, indicating that calnaktin is not a protease. Neither staurosporine (500 nM), 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpipera zin e (5 microM), nor genistein (100 microM) diminished Ca/CaM inhibition of the Na-K-ATPase. Thus there is no evidence that this protein is a kinase. Likewise, the phosphatase inhibitors microcystin (1 microM) and okadaic acid (10 microM) had no effect. PLA2 inhibitors arachidonyl trifluoromethyl ketone (AACOCF3), parabromophenacyl bromide (pBPB), and quinacrine all abolished Ca/CaM inhibition of the Na-K-ATPase. Ca/CaM also increased PLA2 activity, as reflected by an increase in the slope of fluorescence signal of 10-pyrene phosphatidylcholine, a substrate for PLA2. This Ca/CaM-induced change in slope was inhibited by both pBPB and AACOCF3. These data suggest that human red cell membranes contain a form of PLA2 that is activated by Ca/CaM and that this enzyme may mediate Ca/CaM inhibition of the Na-K-ATPase.
Cloning of a bovine renal epithelial Na+ channel subunit
Authors: Fuller C. M. , Awayda M. S. , Arrate M. P. , Bradford A. L. , Morris R. G. , Canessa C. M. , Rossier B. C. , Benos D. J.
Published: December 2017

Pages C641-C654: C. M. Fuller, M. S. Awayda, M. P. Arrate, A. L. Bradford, R. G. Morris, C. M. Canessa, B. C. Rossier, and D. J. Benos. “Cloning of a bovine renal epithelial Na+ channel subunit.” Page C642: Fig. 1 contains two errors in the published sequence of the cDNA agr-bENaC clone presented. A severe COOH compression at nucleotide positions 1750 and 1760 resulted in a double frameshift in the COOH-terminal portion of the sequence. Correction of the nucleotide sequence causes the termination codon to fall at position 1951 (as opposed to position 2092 as previously published), predicting a translated polypeptide of 650 amino acids as opposed to 697 residues as previously reported. This shortened protein has a calculated molecuar mass of 73.4 kDa, although it is observed to migrate with a relative molecular weight of ap80,000 on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The overall homology of the nucleotide sequence with the rat and human agr-ENaC clones is slightly increased by this sequence change to 80 and 84% identities, respectively. In the COOH-terminal region, the homology increases to 53% identity from 43% identity for the rat clone and to 64% identity from 51% identity for the human sequence. A revised nucleotide and amino acid sequence is given in the revised Fig. 1. The sites of the COOH insertion are underlined and the altered amino acid sequence is given in bold. However, this sequence revision does not affect the conclusions of this or subsequent papers from our laboratory concerning this cDNA clone. The amended sequence has been deposited with GenBank (accession no. U14944). The authors apologize for any inconvenience caused by this error.

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Shrinkage-induced activation of Na+/H+ exchange: role of cell density and myosin light chain phosphorylation
Authors: Shrode L. D. , Klein J. D. , Douglas P. B. , O'Neill W. C. , Putnam R. W.
Published: December 2017

Previously, we suggested that myosin light chain kinase (MLCK) is involved in shrinkage-induced activation of the Na+/H+ exchanger in rat astrocytes. Here we have studied the effects of hyperosmotic exposure in C6 glioma cells, a common model for astrocytes. Shrinkage-induced activation of the Na+/H+ exchanger in C6 cells is directly proportional to the degree of shrinkage, results in an alkaline shift in the pK' of the exchanger, is dependent on ATP, and is inhibited by ML-7 (an MLCK inhibitor) and by various calmodulin inhibitors. Cell shrinkage also results in increased phosphorylation of myosin light chain (MLC). Interestingly, shrinkage-induced activation of the exchanger does not occur in subconfluent C6 cells. However, phosphorylation of MLC still occurs in subconfluent cultures of C6 cells on shrinkage, suggesting that the lack of activation in these cells occurs at a point between MLC phosphorylation and Na+/H+ exchange activation. The lack of activation of Na+/H+ exchange in subconfluent C6 cells can be utilized to further elucidate the shrinkage-induced activation pathway.
Sarco(endo)plasmic reticulum Ca2+ pump isoform SERCA3 is more resistant than SERCA2b to peroxide
Authors: Grover A. K. , Samson S. E. , Misquitta C. M.
Published: December 2017

Sarco(endo)plasmic reticulum Ca2+ pumps are encoded by genes SERCA1, SERCA2, and SERCA3. Most tissues express SERCA2 Ca2+ pumps (splice SERCA2b) which are inactivated by reactive oxygen. In contrast, SERCA3 is expressed in tissues such as tracheal epithelium, mast cells, lymphoid cells, and aortic endothelium, which are frequently exposed to oxidative stress. Therefore, we compared SERCA3 and SERCA2b proteins for their sensitivity to oxidation. We isolated microsomes from HEK-293 cells overexpressing SERCA3 or SERCA2b. We incubated the microsomes with different concentrations of hydrogen peroxide and then determined Ca2+ pump activities in them in the following three assay systems: ATP-dependent oxalate-stimulated azide-insensitive 45Ca2+ uptake by the microsomal vesicles, Ca(2+)-Mg(2+)-ATPase, and Ca(2+)-dependent acylphosphate formation. Peroxide inhibited the pump activities in microsomes with half-maximal inhibitory concentration (IC50) values of 69 +/- 14, 66 +/- 13, and 84 +/- 15 microM for the 45Ca2+ uptake, Ca(2+)-Mg(2+)-ATPase, and the acylphosphate formation reactions, respectively. However, for microsomes from SERCA3-expressing cells, the corresponding values of IC50 for peroxide were 274 +/- 47, 857 +/- 110, and 746 +/- 40 microM. Thus, in each assay system, the resistance to inactivation by peroxide was significantly (P < 0.05) higher for the SERCA3 protein than for SERCA2b. The SERCA3 resistance to oxidants may aid the cells expressing it to function during exposure to oxidative stress.
Immuno and functional characterization of CFTR in submandibular and pancreatic acinar and duct cells
Authors: Zeng W. , Lee M. G. , Yan M. , Diaz J. , Benjamin I. , Marino C. R. , Kopito R. , Freedman S. , Cotton C. , Muallem S. , Thomas P.
Published: December 2017

Cystic fibrosis results from defective Cl- channel activity mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) gene product. In the gastrointestinal tract this is manifested in abnormal salivary secretion and pancreatic insufficiency. This is generally attributed to defective Cl- transport by the ductal system of the glands. We provide the first immunocytochemical and functional evidence for expression of CFTR protein and Cl- current in rat and mouse submandibular gland (SMG) and pancreatic acinar cells, a site proximal to the ductal system of these secretory glands. Monoclonal and polyclonal antibodies recognizing COOH-terminal epitopes of CFTR show that duct and acinar cells from the two glands express CFTR in the luminal membrane. Specificity of the polyclonal antibody was verified by absence of staining in duct and acinar cells of the SMG of cf-/cf- and delta F/delta F mice. Identification of CFTR in acinar cells was aided by demonstrating coexpression of CFTR and type 3 inositol 1,4,5-trisphosphate receptors in the luminal pole of acini and absence of type 3 inositol 1,4,5-trisphosphate receptors in ducts. Electrophysiological characterization in single SMG duct and acinar cells shows the presence of a protein kinase A-activated, voltage- and time-independent, ohmic Cl- current and absence of repolarization-dependent tail currents, all of which are kinetic properties of the CFTR-dependent Cl- channel. In addition, the channel was activated by the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate and the benzimidazalone NS-004. Channels activated by all activators were inhibited by glibenclamide and a known inhibitory antiserum [anti-CFTR-(505-511)]. Combined immunologic, functional, and pharmacological evidence allows us to conclude that acinar cells of the SMG and pancreas express functional CFTR-dependent Cl- channels. Because this site is proximal to the duct, modification of activity of this channel in acinar cells is likely to contribute to abnormal salivary secretion and pancreatic insufficiency typical of cystic fibrosis.
M2 receptor activation of nonselective cation channels in smooth muscle cells: calcium and Gi/G(o) requirements
Authors: Wang Y. X. , Fleischmann B. K. , Kotlikoff M. I.
Published: December 2017

Muscarinic stimulation of fura 2-loaded smooth muscle cells evoked a rapidly inactivating Ca(2+)-activated Cl- current [ICl(Ca)] and a sustained nonselective cation current (Icat) as well as a transient (delta Ca(tran)) and a sustained (delta Ca(sus)) elevation of cytosolic Ca2+ concentration ([Ca2+]i). Caffeine and inositol 1,4,5-trisphosphate induced delta Ca(tran) and ICl(Ca) but not Icat or delta Ca(sus). M2 receptor antagonism blocked muscarinic activation of Icat and delta Ca(sus) but not ICl(Ca) and delta Ca(tran). M3 antagonism blocked activation of ICl(Ca) and Icat and a rise in [Ca2+]i, but application of caffeine with methacholine restored Icat and delta Ca(sus). After depletion of intracellular Ca2+ stores, methacholine failed to induce Icat or a [Ca2+]i increase and, in pertussis toxin-treated cells, ICl(Ca) and delta Ca(tran) but not Icat or delta Ca(sus) were evoked. Anti-G alpha i-1/G alpha i-2 antibodies and anti-G alpha i-3/ G(o) alpha antibodies blocked Icat but did not affect ICl(Ca). Anti-Gq alpha/ G alpha 11 antibodies greatly inhibited ICl(Ca) but did not affect Icat. Activation of M2 receptors leads to the opening of nonselective cation channels through Gi/G(o) proteins in smooth muscle cells, resulting in a sustained rise in [Ca2+]i. Arise in [Ca2+]i is necessary but not sufficient for activation of nonselective cation channels.
Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting
Authors: Allen D. L. , Linderman J. K. , Roy R. R. , Bigbee A. J. , Grindeland R. E. , Mukku V. , Edgerton V. R.
Published: December 2017

The role of apoptosis in the elimination of myonuclei during hindlimb unloading-induced atrophy and the inhibition of apoptosis in the prevention of muscle atrophy were examined. The number of nuclei demonstrating double-stranded DNA fragmentation seen by terminal deoxynucleotidyl transferase (TDT) histochemical staining, an indicator of apoptosis, was significantly increased after 14 days of suspension. Double staining with TDT and antilaminin immunohistochemistry revealed that some TDT-positive nuclei were within the fiber lamina and were most likely myonuclei. The number of fibers containing morphologically abnormal nuclei was also significantly greater in suspended compared with control rats. Combined treatment with growth hormone and insulin-like growth factor I (GH/ IGF-I) and resistance exercise attenuated the increase in TDT-positive nuclei (approximately 26%, P > 0.05) and significantly decreased the number of fibers with morphologically abnormal nuclei. The data suggest that 1) “programmed nuclear death” contributes to the elimination of myonuclei and/or satellite cells from atrophying fibers, and 2) GH/IGF-I administration plus muscle loading ameliorates the apoptosis associated with hindlimb unloading.
Protein kinase and Ca2+ modulation of myo-inositol transport in cultured retinal pigment epithelial cells
Authors: Karihaloo A. , Kato K. , Greene D. A. , Thomas T. P.
Published: December 2017

The acute regulation of inwardly directed Na(+)-myo-inositol (MI) cotransporter activity and basal and volume-sensitive MI efflux by protein kinases C (PKC) and A (PKA), cytosolic Ca2+, and phosphoinositide (PI) turnover were characterized in cultured human retinal pigment epithelial cells using 2-[3H]MI and liquid scintillation spectrometry. Kinetic analysis revealed two distinct Na(+)-MI cotransporter components differing in apparent Michaelis constant and maximal velocity. Composite Na(+)-MI cotransport activity was stimulated by PKA activation, the muscarinic agonist carbachol, and the Ca2+ ionophore A-23187 and was inhibited by PKC activation. PKC activation also increased MI efflux, but only the volume-sensitive component, whereas PKA activation increased both basal and volume-sensitive MI efflux. These studies implicate PKC as a negative modulator of MI content through Na(+)-MI cotransport inhibition and potentiation of volume-sensitive MI efflux. PKA is a positive modulator of both Na(+)-MI cotransport and basal and volume-sensitive MI efflux. Cytosolic Ca2+ release through receptor-mediated PI hydrolysis may facilitate Na(+)-MI cotransport activity.
Determinants of work produced by skeletal muscle: potential limitations of activation and relaxation
Authors: Caiozzo V. J. , Baldwin K. M.
Published: December 2017

The objective of this study was to estimate the limitations imposed by the kinetics of activation and relaxation on the ability of slow skeletal muscle to produce mechanical work. These estimates were made by the following methods: 1) using the work loop technique and measuring the actual mechanical work (WA) produced by rat soleus muscles (n = 6) at four different frequencies (0.5, 1, 2, and 4 Hz) and seven different amplitudes of length change (1, 2, 3, 4, 5, 6, and 7 mm); 2) determining the force-velocity relationships of the soleus muscles and using this data to quantify the theoretical mechanical work (WT) that could be produced under the work loop conditions described above; and 3) subtracting WA from WT. The difference between WT and WA was interpreted to represent limitations imposed by activation and relaxation. Under certain conditions (high frequency, small strain), factors controlling the kinetics of activation and relaxation reduced the mechanical work of the soleus muscle by approximately 60%. Hence, activation and relaxation collectively represent important factors limiting the production of mechanical work by slow skeletal muscle.
Effects of PKCα activation on Ca2+ pump and KCa channel in deoxygenated sickle cells
Authors: Fathallah Hassana , Sauvage Monique , Romero Jose R. , Canessa Mitzy , Giraud Françoise
Published: December 2017

We have previously shown that a pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), reduced deoxygenation-induced K+loss and Ca2+ uptake and prevented cell dehydration in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 1999–2007, 1995). The present study explores the detailed mechanism of this PMA-induced inhibition. The main findings are, first, the detection of PKCα and PKCζ in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The α-isoform only is translocated to the membrane and activated by PMA and by elevation of cytosolic Ca2+. Second, PMA is demonstrated to activate Ca2+ efflux in deoxygenated SS cells by a direct stimulation of the Ca2+ pump. PMA, moreover, inhibits deoxygenation-induced, charybdotoxin-sensitive K+ efflux in SS cells. This inhibition is partly indirect and explained by the reduced deoxygenation-induced rise in cytosolic Ca2+ resulting from Ca2+ pump stimulation. However, a significant inhibition of the Ca2+-activated K+ channels (KCa channels) by PMA can also be demonstrated when the channels are activated by Ca2+ plus ionophore, under conditions in which the Ca2+ pump is operating near its maximal extrusion rate, but swamped by Ca2+ plus ionophore. The data thus suggest a PKCα-mediated phosphorylation both of the Ca2+ pump and of the KCa channel or an auxiliary protein.
Ryanodine receptor expression is associated with intracellular Ca2+ release in rat parotid acinar cells
Authors: Zhang Xuejun , Wen Jiayu , Bidasee Keshore R. , Besch Henry R. , Rubin Ronald P.
Published: December 2017

The ryanodine receptor mediates intracellular Ca2+ mobilization in muscle and nerve, but its physiological role in nonexcitable cells is less well defined. Like adenosine 3′,5′-cyclic monophosphate and inositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3–5 μM) and ADP (1–25 μM) produced a concentration-dependent rise in cytosolic Ca2+ in permeabilized rat parotid acinar cells. Adenosine and AMP were less effective. Ryanodine markedly depressed the Ca2+-mobilizing action of the adenine nucleotides and forskolin in permeabilized cells and was likewise effective in depressing the action of forskolin in intact cells. Cyclic ADP-ribose-evoked Ca2+ release was enhanced by calmodulin and depressed by W-7, a calmodulin inhibitor. A fluorescently labeled ligand, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression and distribution of ryanodine receptors. In addition, ryanodine receptor expression was detected in rat parotid cells with a sequence highly homologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of a ryanodine-sensitive intracellular Ca2+ store in rat parotid cells that shares many of the characteristics of stores in muscle and nerve and may mediate Ca2+-induced Ca2+ release or a modified form of this process.
Neuronal NOS gene expression in gastrointestinal myenteric neurons and smooth muscle cells
Authors: Chakder Sushanta , Bandyopadhyay Alok , Rattan Satish
Published: December 2017

Nitric oxide synthase (NOS) has been characterized in different tissues, and its localization has been suggested in different neuronal tissues, including the myenteric neurons and other nonneuronal cells. The present study examined the distribution of the neuronal NOS (nNOS) mRNA in different tissues of the opossum gastrointestinal tract, internal anal sphincter (IAS) smooth muscle cells, and myenteric neurons using slot-blot and Northern blot hybridization techniques with a specific rat brain nNOS cDNA probe. Significant levels of nNOS gene expression were found in both smooth muscle cells and myenteric neurons of the opossum IAS. This finding was confirmed by reverse transcriptase-polymerase chain reaction analysis of the RNA obtained from cultured opossum IAS smooth muscle cells and myenteric neurons and also from human intestinal smooth muscle and neuroblastoma cell lines. Pyloric sphincter had the highest level of nNOS gene expression compared with other gastrointestinal tissues. There was no significant difference in the nNOS gene expression between other sphincteric and nonsphincteric tissues examined. The present study shows the presence of nNOS gene expression in both neurons and smooth muscle cells. The higher levels of nNOS gene expression in the pyloric sphincter compared with other tissues may have pathophysiological significance in some disease conditions.
Interferon-γ induces a decrease in the intracellular calcium pump in a human salivary gland cell line
Authors: Meehan Sean , Wu Ava J. , Kang Elaine C. , Sakai Takayuki , Ambudkar Indu S.
Published: December 2017

Interferon-γ (IFN-γ) ± tumor necrosis factor-α (TNF-α) induces antiproliferation and intracellular Ca2+ store depletion in a human submandibular ductal cell line (HSG), which can be reversed on cytokine removal [A. J. Wu, G. C. Chen, B. J. Baum, and I. S. Ambudkar. Am. J. Physiol. 270 ( Cell Physiol. 39): C514–C521, 1996]. Here we have examined a possible mechanism for the IFN-γ-induced intracellular Ca2+store depletion. There was a time-dependent decrease in thapsigargin-dependent internal Ca2+ release after exposure of the cells to the cytokines. The intracellular Ca2+ pump [sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)] protein in lysates and membranes of cells treated with IFN-γ ± TNF-α, but not with TNF-α alone, showed a similar time-dependent decrease (examined using a SERCA2 antibody). Removal of the cytokines, which resulted in recovery of cell growth and refill of internal Ca2+ stores, also increased the level of SERCA protein. The decrease in SERCA is not a result of decreased cell proliferation, since thapsigargin, 2,5-di-( t-butyl)-1,4-hydroquinone, or serum-free growth conditions induced antiproliferative effects on HSG cells without any corresponding decrease in SERCA. We suggest that the IFN-γ-induced decrease in the level of SERCA accounts for the depleted state of internal Ca2+stores in cytokine-treated HSG cells. These data suggest a novel mechanism for the inhibition of HSG cell growth by IFN-γ.
Inducible nitric oxide synthase augments injury elicited by oxidative stress in rat cardiac myocytes
Authors: Igarashi Junsuke , Nishida Masashi , Hoshida Shiro , Yamashita Nobushige , Kosaka Hiroaki , Hori Masatsugu , Kuzuya Tsunehiko , Tada Michihiko
Published: December 2017

The effects of nitric oxide (NO) produced by cardiac inducible NO synthase (iNOS) on myocardial injury after oxidative stress were examined. Interleukin-1β induced cultured rat neonatal cardiac myocytes to express iNOS. After induction of iNOS,l-arginine enhanced NO production in a concentration-dependent manner. Glutathione peroxidase (GPX) activity in myocytes was attenuated by elevated iNOS activity and by an NO donor, S-nitroso- N-acetyl-penicillamine (SNAP). Although NO production by iNOS did not induce myocardial injury, NO augmented release of lactate dehydrogenase from myocyte cultures after addition of H2O2(0.1 mM, 1 h). Inhibition of iNOS with Nω-nitro-l-arginine methyl ester ameliorated the effects of NO-enhancing treatments on myocardial injury and GPX activity. SNAP augmented the myocardial injury induced by H2O2. Inhibition of GPX activity with antisense oligodeoxyribonucleotide for GPX mRNA increased myocardial injury by H2O2. Results suggest that the induction of cardiac iNOS promotes myocardial injury due to oxidative stress via inactivation of the intrinsic antioxidant enzyme, GPX.
Suppression of cAMP by phosphoinositol/Ca2+ pathway in the cardiac κ-opioid receptor
Authors: Zhang Wei-Min , Wong Tak-Ming
Published: December 2017

To determine whether the phosphoinositol/Ca2+ pathway interacts with the adenylate cyclase/adenosine 3′,5′-cyclic monophosphate (cAMP) pathway in the cardiac κ-receptor, the effects of U-50488, a specific κ-receptor agonist, on the intracellular Ca2+ concentration ([Ca2+]i) and forskolin-induced accumulation of cAMP in rat ventricular myocytes were determined after interference of the phosphoinositol/Ca2+ pathway. U-50488 suppressed the forskolin-induced accumulation of cAMP and elevated [Ca2+]i, which were blocked by norbinaltorphimine, a specific κ-receptor antagonist, and pertussis toxin. The effects of U-50488 were qualitatively similar to those of A-23187, a Ca2+ ionophore, but opposite to those of 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM), a [Ca2+]ichelator. Abolition of U-50488-induced elevation of [Ca2+]iby BAPTA-AM also abolished the effect of U-50488 on forskolin-induced accumulation of cAMP. Inhibition of the phospholipase C by specific inhibitors, U-73122 and neomycin, abolished the effects of U-50488 on both [Ca2+]iand forskolin-induced accumulation of cAMP. The results showed for the first time that κ-receptor stimulation may suppress cAMP accumulation via activation of the phosphoinositol/Ca2+ pathway in the rat heart.
Cl− currents activated via purinergic receptors inXenopus follicles
Authors: Arellano Rogelio O. , Garay Edith , Miledi Ricardo
Published: December 2017

Ionic currents elicited via purinergic receptors located in the membrane of Xenopus follicles were studied using electrophysiological techniques. Follicles responded to ATP-activating inward currents with a fast time course ( F in). In Ringer solution, reversal potential ( E rev) of F in was −22 mV, which did not change with external substitutions of Na+ or K+, whereas solutions containing 50 or 5% of normal Cl−concentration shifted E rev to about +4 and +60 mV, respectively, and decreased F in amplitude, indicating that F in was carried by Cl−. F in had an onset delay of ∼400 ms, measured by application of a brief jet of ATP from a micropipette positioned near the follicle (50 μm). F in was inhibited by 50% in follicles pretreated with pertussis toxin. This suggests a G protein-mediated receptor channel pathway. F in was mimicked by 2-MeSATP and UTP, the potency order (half-maximal effective concentration) was 2-MeSATP (194 nM) > UTP (454 nM) > ATP (1,086 nM). All agonists generated Cl− currents and displayed cross-inhibition on the others. F in activation by acetylcholine also cross-inhibited F in-ATP responses, suggesting that all act on a common channel-activation pathway.
Voltage-gated Ca2+ currents in rat gastric enterochromaffin-like cells
Authors: Bufler J. , Choi G. C. , Franke C. , Schepp W. , Prinz C.
Published: December 2017

Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric mucosa that maintain a negative membrane potential of about −50 mV, largely due to voltage-gated K+ currents [D. F. Loo, G. Sachs, and C. Prinz. Am. J. Physiol.270 ( Gastrointest Liver Physiol. 33): G739–G745, 1996]. The current study investigated the presence of voltage-gated Ca2+channels in single ECL cells. ECL cells were isolated from rat fundic mucosa by elutriation, density gradient centrifugation, and primary culture to a purity >90%. Voltage-gated Ca2+ currents were measured in single ECL cells using the whole cell configuration of the patch-clamp technique. Depolarization-activated currents were recorded in the presence of Na+ or K+ blocking solutions and addition of 20 mM extracellular Ca2+. ECL cells showed inward currents in response to voltage steps that were activated at a test potential of around −20 mV with maximal inward currents observed at +20 mV and 20 mM extracellular Ca2+. The inactivation rate of the current decreased with increasingly negative holding potentials and was totally abolished at a holding potential of −30 mV. Addition of extracellular 20 mM Ba2+ instead of 20 mM Ca2+ increased the depolarization-induced current and decreased the inactivation rate. The inward current was fully inhibited by the specific L-type Ca2+ channel inhibitor verapamil (0.2 mM) and was augmented by the L-type Ca2+ channel activator BAY K 8644 (0.07 mM). We conclude that depolarization activates high-voltage-activated Ca2+channels in ECL cells. Activation characteristics, Ba2+ effects, and pharmacological results imply the presence of L-type Ca2+ channels, whereas inactivation kinetics suggest the presence of additional N-type channels in rat gastric ECL cells.
Functional expression of a truncated Ca2+-activated Cl− channel and activation by phorbol ester
Authors: Ji Hong-Long , Duvall Michael D. , Patton Holly K. , Satterfield Cynthia Lyn , Fuller Catherine M. , Benos Dale J.
Published: December 2017

We have isolated a niflumic acid-insensitive, Ca2+-activated Cl− channel (CaCC) from bovine trachea that migrates at 38 kDa (p38) on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, a cloned CaCC isolated from a tracheal cDNA expression library by screening with an antibody raised against p38 has a primary cDNA transcript of 2712 base pairs that codes for a 100-kDa protein and is not susceptible to dithiothreitol reduction. To test the hypothesis that the functional channel may be a much smaller posttranslationally processed form of the 100-kDa protein, we generated a mutant construct (CaCCX, 42.5-kDa protein) truncated at the NH2 and COOH termini. The whole cell currents of wild-type (wt) CaCC and CaCCX expressed in Xenopus oocytes were 10-fold higher than those of water-injected oocytes and were further increased by ionomycin or A-23187 and inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and dithiothreitol. Whole cell currents in wtCaCC- and CaCCX-expressing oocytes could also be activated by phorbol 12-myristate 13-acetate and could be inhibited by chelerythrine chloride, suggesting that the cloned CaCC is regulated by protein kinase C. These results suggest that a smaller form of the full-length CaCC can form a functional channel.
Effect of expressing the water channel aquaporin-1 on the CO2 permeability ofXenopus oocytes
Authors: Nakhoul Nazih L. , Davis Bruce A. , Romero Michael F. , Boron Walter F.
Published: December 2017

It is generally accepted that gases such as CO2 cross cell membranes by dissolving in the membrane lipid. No role for channels or pores in gas transport has ever been demonstrated. Here we ask whether expression of the water channel aquaporin-1 (AQP1) enhances the CO2 permeability of Xenopus oocytes. We expressed AQP1 in Xenopus oocytes by injecting AQP1 cRNA, and we assessed CO2permeability by using microelectrodes to monitor the changes in intracellular pH (pHi) produced by adding 1.5% CO2/10 mM[Formula: see text] to (or removing it from) the extracellular solution. Oocytes normally have an undetectably low level of carbonic anhydrase (CA), which eliminates the CO2 hydration reaction as a rate-limiting step. We found that expressing AQP1 (vs. injecting water) had no measurable effect on the rate of CO2-induced pHi changes in such low-CA oocytes: adding CO2 caused pHi to fall at a mean initial rate of 11.3 × 10−4 pH units/s in control oocytes and 13.3 × 10−4 pH units/s in oocytes expressing AQP1. When we injected oocytes with water, and a few days later with CA, the CO2-induced pHi changes in these water/CA oocytes were more than fourfold faster than in water-injected oocytes (acidification rate, 53 × 10−4 pH units/s). Ethoxzolamide (ETX; 10 μM), a membrane-permeant CA inhibitor, greatly slowed the pHi changes (16.5 × 10−4 pH units/s). When we injected oocytes with AQP1 cRNA and then CA, the CO2-induced pHi changes in these AQP1/CA oocytes were ∼40% faster than in the water/CA oocytes (75 × 10−4 pH units/s), and ETX reduced the rates substantially (14.7 × 10−4 pH units/s). Thus, in the presence of CA, AQP1 expression significantly increases the CO2 permeability of oocyte membranes. Possible explanations include 1) AQP1 expression alters the lipid composition of the cell membrane, 2) AQP1 expression causes overexpression of a native gas channel, and/or 3) AQP1 acts as a channel through which CO2 can permeate. Even if AQP1 should mediate a CO2 flux, it would remain to be determined whether this CO2movement is quantitatively important.
ATP stimulation of Na+/Ca2+exchange in cardiac sarcolemmal vesicles
Authors: Berberián Graciela , Hidalgo Cecilia , Dipolo Reinaldo , Beaugé Luis
Published: December 2017

In cardiac sarcolemmal vesicles, MgATP stimulates Na+/Ca2+exchange with the following characteristics: 1) increases 10-fold the apparent affinity for cytosolic Ca2+; 2) a Michaelis constant for ATP of ∼500 μM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory; 4) not observed in the presence of 20 μM eosin alone but reinstated when vanadate is added; 5) mimicked by adenosine 5′- O-(3-thiotriphosphate), without the need for vanadate, but not by β,γ-methyleneadenosine 5′-triphosphate; and 6) not affected by unspecific protein alkaline phosphatase but abolished by a phosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLC effect is counteracted by phosphatidylinositol. In addition, in the absence of ATP,l-α-phosphatidylinositol 4,5-bisphosphate (PIP2) was able to stimulate the exchanger activity in vesicles pretreated with PI-PLC. This MgATP stimulation is not related to phosphorylation of the carrier, whereas phosphorylation appeared in the phosphoinositides, mainly PIP2, that coimmunoprecipitate with the exchanger. Vesicles incubated with MgATP and no Ca2+ show a marked synthesis ofl-α-phosphatidylinositol 4-monophosphate (PIP) with little production of PIP2; in the presence of 1 μM Ca2+, the net synthesis of PIP is smaller, whereas that of PIP2increases ninefold. These results indicate that PIP2 is involved in the MgATP stimulation of the cardiac Na+/Ca2+exchanger through a fast phosphorylation chain: a Ca2+-independent PIP formation followed by a Ca2+-dependent synthesis of PIP2.
Relationship between paxillin and myosin phosphorylation during muscarinic stimulation of smooth muscle
Authors: Mehta Dolly , Wang Zhonglin , Wu Ming-Fang , Gunst Susan J.
Published: December 2017

The tyrosine phosphorylation of paxillin increases in association with force development during tracheal smooth muscle contraction, suggesting that paxillin plays a role in the contractile activation of smooth muscle [Z. L. Wang, F. M. Pavalko, and S. J. Gunst. Am. J. Physiol. 271 ( Cell Physiol. 40): C1594–C1602, 1996]. We compared the Ca2+ sensitivity of the tyrosine phosphorylation of paxillin and myosin light chain (MLC) phosphorylation in tracheal muscle and evaluated whether MLC phosphorylation is necessary to induce paxillin phosphorylation. Ca2+-depleted muscle strips were stimulated with 10−7–10−4M acetylcholine (ACh) in 0, 0.05, 0.1, or 0.5 mM extracellular Ca2+. In the absence of extracellular Ca2+, 10−4 M ACh induced a maximal increase in paxillin phosphorylation without increasing MLC phosphorylation or force. Increases in extracellular Ca2+ concentration did not further increase paxillin phosphorylation. However, during stimulation with 10−6 M ACh, paxillin phosphorylation increased with increases in extracellular Ca2+ concentration. We conclude that the tyrosine phosphorylation of paxillin can be stimulated by signaling pathways that do not depend on Ca2+ mobilization and that the activation of contractile proteins is not required to elicit paxillin phosphorylation.