bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of colonic ion transport by GRP. I. GRP stimulates transepithelial K and Na secretion
Authors: Traynor T. R. , O'Grady S. M.
Published: December 2017

Regulation of electrolyte transport across porcine distal colon epithelium by gastrin-releasing peptide (GRP) was examined using mucosal sheets mounted in Ussing chambers. Serosal GRP produced a biphasic response consisting of a transient increase in short-circuit current (ISC) followed by a long-lasting decrease. Indomethacin and tetrodotoxin inhibited the ISC increase without affecting the secondary decrease. Addition of GRP to the mucosal solution produced a decrease in ISC similar to that observed with serosal treatment, but no transient increase in ISC was observed. GRP and bombesin (50% effective concentrations of 26 and 30 nM, respectively) were more effective than neuromedin B in decreasing the ISC, and the GRP receptor antagonist [D-Phe(6)]bombesin(6-13)-O-methyl produced a sixfold dextral shift in the GRP concentration-response relationship. The GRP-stimulated decrease was reduced in the absence of Cl and by serosal bumetanide. Flux measurements showed that GRP increased Rb and Na secretion while having no effect on transepithelial Cl transport. Phosphoinositide turnover was increased by GRP, suggesting that the ion transport changes may be mediated by intracellular Ca concentration. The results of this study demonstrate that GRP stimulates K and Na secretion across the porcine distal colon epithelium and that these processes are dependent, in part, on a bumetanide-sensitive transport pathway located in the basolateral membrane.
cAMP-independent regulation of CFTR by the actin cytoskeleton
Authors: Prat A. G. , Xiao Y. -F. , Ausiello D. A. , Cantiello H. F.
Published: December 2017

Pages C1552<C1561: A. G. Prat, Y.-F. Xiao, D. A. Ausiello, and H. F. Cantiello. “cAMP-independent regulation of CFTR by the actin cytoskeleton.” Page C1558, Fig. 8A: units on the y-axis of the bar graph should have been 10 times larger. The corrected figure appears below.

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Doxorubicin selection for MDR1/P-glycoprotein reduces swelling-activated K+ and Cl- currents in MES-SA cells
Authors: Luckie D. B. , Krouse M. E. , Law T. C. , Sikic B. I. , Wine J. J.
Published: December 2017

To test the hypothesis that P-glycoprotein enhances swelling currents through regulation of volume-sensitive Cl- channels [recently termed VSOAC (volume-sensitive osmolyte and anion channel)], a human uterine sarcoma cell line (MES-SA) and its doxorubicin-selected counterpart (Dx5) were studied. P-glycoprotein mRNA and protein levels were detected only in Dx5 cells. However, whole cell patch-clamp experiments showed that swollen Dx5 cells (n = 5) produced smaller VSOAC currents than MES-SA cells (n = 4; 106 +/- 26 pA/pF vs. 232 +/- 76 pA/pF at 90 mV). In radioisotopic efflux experiments, both swelling-activated 125I (Cl-) currents (n = 15) and 86Rb (K+) currents (n = 8) were found to be two-to fourfold smaller in the Dx5 (high P-glycoprotein) cells. Inhibitors of P-glycoprotein showed no specificity for the doxorubicin-selected cells (Dx5). Dideoxyforskolin (100 microM) blocked swelling-activated 125I efflux equally in both cell lines, whereas 100 microM verapamil had no effect. Thus, in this cell line, selection for P-glycoprotein expression is associated with reduced swelling currents. These findings suggest that P-glycoprotein expression does not directly facilitate VSOAC.
Evidence for receptor-mediated mineralocorticoid action in rat osteoblastic cells
Authors: Agarwal M. K. , Mirshahi F. , Mirshahi M. , Bracq S. , Chentoufi J. , Hott M. , Jullienne A. , Marie P. J.
Published: December 2017

Aldosterone significantly enhanced the proliferation of osteoblastic cells from rat calvaria, and this effect was inhibited by RU 26752 and ZK 91587, two antagonists specific to the mineralocorticoid receptor (MCR). In addition, aldosterone inhibited the activity of alkaline phosphatase, a marker of the osteoblastic phenotype, and this effect was also reversed by RU 26752. Cytoplasmic staining of MCR was observed in rat calvaria osteoblasts incubated with a specific polyclonal antiserum raised against rat kidney MCR. This anti-MCR immunoglobulin G immunoprecipitated and macroaggregated the MCR-[3H]RU 26752 complex in osteoblastic cytosol. A single 98-kDa band was observed when osteoblastic cytosol was analyzed by Western blotting with anti-MCR serum. The 98-kDa band was also obtained after autoradiography of irradiated osteoblastic cytosol-[3H]R 5020 complex, and this was abolished in the presence of RU 26752. A p26MR probe, specific to COOH-terminal end of MCR, hybridized with the predicted product after amplification of total cell RNA by polymerase chain reaction technique. Furthermore, hybridization of poly(A)+ mRNA from at calvaria osteoblastic cells with the p26MR probe revealed a major band of approximately 4.2 kb. Collectively, our studies demonstrate the existence of a functional MCR in rat calvaria osteoblasts.
Potential-dependent block of human delayed rectifier K+ channels by internal Na+
Authors: Johansson S. , Sundgren A. K. , Kahl U.
Published: December 2017

The delayed rectifier K+ currents in differentiated human SH-SY5Y neuroblastoma cells were characterized with tight-seal recording techniques. Activation and inactivation parameters were measured. At high positive potentials, the current showed a marked rectification, causing a region of negative slope conductance in the current vs. potential curve. The rectification depended markedly on the pipette Na+ concentration. Without Na+, no rectification was observed, whereas with high Na+ (20-60 mM), a marked rectification was always observed. Tail current measurements showed a fast ( < 400 microseconds) block of K+ currents in the presence of internal Na+. With 60 mM Na+ in the pipette 8% of the K+ current was blocked at 0 mV, 27% at +20 mV, and 82% at +100 mV. Similar degrees of block were often seen with 30 mM Na+ in the pipette. The submembrane Na+ concentration in intact cells was estimated, on the basis of the reversal of Na+ current, to be approximately 15 mM. Single-channel K+ currents, in the cell-attached configuration, showed a conductance of approximately 20 pS at 40-60 mV above rest but showed rectification at high potentials.
Expression of inositol 1,4,5-trisphosphate receptors changes the Ca2+ signal of Xenopus oocytes
Authors: DeLisle S. , Blondel O. , Longo F. J. , Schnabel W. E. , Bell G. I. , Welsh M. J.
Published: December 2017

The receptors for the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] form a family of closely related proteins that play an important role in regulating the free intracellular Ca2+ concentration. To test the hypothesis that changing the expression level of Ins(1,4,5)P3 receptors could alter the Ins(1,4,5)P3-mediated Ca2+ signal, we overexpressed Ins(1,4,5)P3 receptor type 1 (InsP3R-1) or type 3 (InsP3R-3) in Xenopus laevis oocytes. Expression of InsP3R-1 increased the velocity of the propagating waves of intracellular Ca2+ release but did not affect the Ins(1,4,5)P3-induced entry of extracellular Ca2+ across the plasma membrane. In contrast, expression of intracellular Ca2+ but markedly increased the magnitude and duration of Ca2+ influx. Immunolocalization studied revealed InsP3R-3 at the endoplasmic reticulum, with a relatively stronger signal at or near the plasma membrane. The results suggest that changing the expression level of an InsP3R can alter the Ins(1,4,5)P3-mediated Ca2+ signal and that InsP3R-1 and InsP3R-3 may have different biological functions.
Calphostin C, a widely used protein kinase C inhibitor, directly and potently blocks L-type Ca channels
Authors: Hartzell H. C. , Rinderknecht A.
Published: December 2017

Calphostin C is a widely used inhibitor of protein kinase C; in the past 4 years at least 350 articles have been published using this drug as a selective inhibitor of protein kinase C. In this paper, we show that calphostin C also potently inhibits cardiac L-type Ca channels by a mechanism that does not involve changes in adenosine 3',5'-cyclic monophosphate levels or dephosphorylation. The inhibition requires illumination by visible light during exposure to calphostin C. The Ca current (ICa) that remains after partial inhibition of ICa has the same voltage-dependent characteristics as the control current.
Pathways for K+ transport across the bovine articular chondrocyte membrane and their sensitivity to cell volume
Authors: Hall A. C. , Starks I. , Shoults C. L. , Rashidbigi S.
Published: December 2017

The contributions of various K+ transport pathways in bovine chondrocytes isolated from articular cartilage and their responses to changes in cell volume have been studied. K+(86Rb+) uptake mediated by the Na(+)-K(+) pump and Na(+)-K(+)-2Cl- cotransporter were stimulated by cell shrinkage, the latter as part of the regulatory volume increases (RVI) response, the former as an indirect effect resulting from the rise in intracellular Na+ concentration during RVI. For both transporters, there was an increase in the maximum velocity with no detectable effect on the Michaelis constant. There was no evidence for volume-sensitive K+ transport mediated by the K(+)-Cl- cotransporter, or Ca(2+)-activated K+ channels. However, chondrocyte swelling stimulated a ouabain- and bumetanide-insensitive K+ flux sensitive to pimozide and other drugs, which exhibited some of the properties of the relatively nonspecific volume-sensitive "osmolyte" channel described in other cell types.
Dynamics of cortical granule exocytosis at fertilization in living mouse eggs
Authors: Tahara M. , Tasaka K. , Masumoto N. , Mammoto A. , Ikebuchi Y. , Miyake A.
Published: December 2017

Sperm-egg fusion induces an intracellular free calcium concentration ([Ca2+]i) increase and exocytosis of cortical granules (CGs). Recently we used an impermeable fluorescent membrane probe, 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), to develop a method to evaluate the kinetics of exocytosis in single living cells. In this study we used digital imaging and confocal laser scanning microscopy to evaluate CG exocytosis in living mouse eggs with TMA-DPH. Time-related changes of CG exocytosis were estimated as the percent increase of TMA-DPH fluorescence. The increase of fluorescence in the egg started after sperm attachment, continued at an almost uniform rate, and ceased at 45-60 min. Whereas the [Ca2+]i increase at fertilization was transient or oscillatory, exocytosis was not always induced concomitantly with each [Ca2+]i peak. Next we used this method to determine some intracellular mediators of exocytosis in the egg. An intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and a microfilament inhibitor, cytochalasin B, blocked sperm-induced exocytosis. A guanosine 5'-triphosphate-binding protein activator, AlF4-, induced exocytosis. These results suggest that [Ca2+]i, microfilament, and guanosine 5'-triphosphate-binding proteins may be involved in CG exocytosis. In conclusion, this method has significant advantages for studying exocytosis in living eggs.
Simultaneous measurement of intracellular pH and Ca2+ in insulin-secreting cells by spectral imaging microscopy
Authors: Martinez-Zaguilan R. , Gurule M. W. , Lynch R. M.
Published: December 2017

Described is a microscopic spectral imaging approach to monitor pH and Ca2+ simultaneously from combined spectra of multiple ion indicators. Emitted light from a cell is focused onto a grating spectrograph and spectra are imaged with a cooled charge-coupled device camera. The combined spectral output of fura 2 and SNARF-1 was analyzed to follow changes in intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) simultaneously and to correct the Ca2+ signal for concurrent changes in pHi. Responses of individual hamster insulinoma (HIT-T15) cells to effectors of ion homeostasis were heterogeneous. Treatment with NH4Cl increased pHi and transiently increased [Ca2+]i. Removal of NH4Cl induced cytosolic acidification concomitant with either no change or sustained increases in [Ca2-]i. Glucose treatment generally resulted in rapid and sustained increases in both [Ca2+]i and pHi but also heterogeneous pHi and [Ca2+]i responses. Corrections of the fura 2 signal for pH were important for following Ca2+ transitions elicited by NH4Cl but were less important for glucose-induced responses. The spectral imaging microscope provides a sensitive method for simultaneous measurements of pHi and [Ca2+]i in single cells.
MDR1/P-glycoprotein function. II. Effect of hypotonicity and inhibitors on Cl- efflux and volume regulation
Authors: Weaver J. L. , Aszalos A. , McKinney L.
Published: December 2017

Resistance to anti-tumor drugs can be mediated by overexpression of the multidrug resistance 1 (MDR1) protein (P-glycoprotein). In three MDR1-transfected cell lines (Gill et al. Cell 71: 23-32, 1992; Altenberg et al. Cancer Res. 54: 618-622, 1994), a hypotonic stress-induced Cl- current has been demonstrated that can be inhibited by MDR1 substrates and Cl- channel blockers. We tested the hypothesis that MDR1 expression confers additional Cl- conductance by measuring regulatory volume decrease (RVD) in four pairs of isogenic cell lines and 36Cl efflux in two cell lines with and without hypotonic stress. The kinetics of RVD and response to Cl- channel blockers were indistinguishable in MDR and parental cells. Additionally, no significant difference was seen between 36Cl efflux rate constants under hypotonic conditions between NIH/3T3 and L1210 parental and MDR cells. We conclude that, in intact cells, the expression of MDR1 does not alter the rate of volume regulation or the rate 36Cl efflux under hypotonic conditions between parental and MDR cells.
Is gamma-actin a regulator of amino acid transport?
Authors: Lin G. , McCormick J. I. , Johnstone R. M.
Published: December 2017

A mutated yeast cell line incapable of growth in minimal medium with proline as the sole nitrogen source was restored to normal growth by transfection with a cDNA from mouse Ehrlich cells. The cloned cDNA (E51) was found to be 90% homologous to gamma-actin. Immediately after transfection with E51 cDNA, both alpha-aminoisobutyric acid (AIB) and proline uptake in the mutated yeast were increased, particularly at pH 5. The expression of the same E51 cDNA also enhanced amino acid uptake in Xenopus laevis oocytes after injection into the Xenopus nuclei. A mutated mammalian lymphocyte cell line (GF-17), deficient in system A transport, also showed increased Na(+)-dependent transport after transfection with E51 cDNA. Whereas the mock transfected GF-17 cells failed to grow in the selection medium, the transfectants with E51 cDNA grew better than the untransfected cells. The data are consistent with the conclusion that expression of E51 cDNA can modify inactive, endogenous amino acid transporters, permitting substantial amino acid uptake in cells deficient in amino acid transporter(s) and permitting rapid cell growth. The data suggest that the gamma-actin-like protein coded for by E51 cDNA may play a significant regulatory role in amino acid transport.
Angiotensin II stimulates sodium-dependent proton extrusion in perfused ferret heart
Authors: Grace A. A. , Metcalfe J. C. , Weissberg P. L. , Bethell H. W. , Vandenberg J. I.
Published: December 2017

The Na+/H+ antiport and Na(+)-HCO3- coinflux carrier contribute to recovery from intracellular acidosis in cardiac tissue. The effects of angiotensin II (10(-12)-10(-6) M) on H+ fluxes after intracellular acid loading and during reperfusion after myocardial ischemia have been investigated in the isovolumic, Langendorff-perfused ferret heart. Intracellular pH (pHi) was estimated using 31P nuclear magnetic resonance (NMR) spectroscopy from the chemical shift of intracellular deoxyglucose-6-phosphate or inorganic phosphate. Angiotensin II produced concentration-dependent stimulation (maximum at 10(-6) M: 67%) of 5-(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive Na(+)-dependent of H+ efflux consistent with stimulation of the Na+/H+ antiport. Half-maximal stimulation of H+ efflux occurred at approximately 10(-9) M, which is close to the dissociation constant of the cardiac angiotensin AT1 receptor. Stimulation via this receptor was confirmed with the nonpeptide AT1 receptor blocker, GR-117289. Angiotensin II had less pronounced effects on HCO3(-)-dependent pHi recovery after acid loading with no effect on pHi recovery after intracellular alkalosis. During reperfusion, angiotensin II significantly increased H+ extrusion but impaired contractile recovery. The results support the hypothesis that angiotensin II facilitates H+ extrusion in the heart. This may help maintain physiological homeostasis, but the hypothesized obligated Na+ influx could exacerbate cellular dysfunction during reperfusion.
Mechanism of human sperm activation by extracellular ATP
Authors: Foresta C. , Rossato M. , Chiozzi P. , Di Virgilio F.
Published: December 2017

We have identified the mechanism whereby extracellular ATP (ATPe) triggers the acrosome reaction in human spermatozoa. This nucleotide opens a ligand-gated ion channel expressed on the sperm plasma membrane. ATPe threshold and 50% effective concentration calculated on the total added ATPe are 0.1 and 2 mM, respectively, corresponding to a free ATP concentration (ATP4-) of 3 and 200 microM, respectively. The ATPe-gated channel is selective for monovalent cations (Na+, choline, and methylglucamine), whereas on the contrary, permeability to Ca2+ is negligible. Isosmolar replacement of extracellular Na+ with sucrose fully blocked ATPe-dependent sperm activation, thus suggesting a mandatory role for Na+ influx. These results show that human sperm express an ATPe-gated Na+ channel that might have an important role in sperm activation before egg fertilization.
Role of large Ca(2+)-activated K+ channels in regulation of mesangial contraction by nitroprusside and ANP
Authors: Stockand J. D. , Sansom S. C.
Published: December 2017

The patch-clamp method, in conjunction with measurements of cell contraction, was employed to investigate activation by guanosine 3',5'-cyclic monophosphate (cGMP) and guanylyl cyclase-stimulating vasodilators of large Ca(2+)-activated K+ channels (BKCa) in human glomerular mesangial cells (MC). In cell-attached patches, with physiological NaCl bathing solutions, BKCa was activated transiently by nitroprusside [NP; a nitric oxide (NO) donor], atrial natriuretic peptide (ANP), and dibutyryl cGMP (DBcGMP), reaching peak responses between 10 and 60 s and decreasing to near baseline activity within the next 120 s. In the presence of LY-83583, a specific inhibitor of guanylyl cyclase, BKCa was activated on cell by DBcGMP but not by NP or ANP. In all cases, the increase in channel activity coincided with a decrease in channel amplitude, indicating that the membrane potential was approaching equilibrium potential as BKCa was activated. If membrane potential was maintained depolarized with 140 mM KCl in the bathing solution, DBcGMP induced a sustained activation of BKCa. In the continued presence of DBcGMP, BKCa was further activated when 100 nM angiotensin II (ANG II) was added to the bathing solution. Experiments were performed to determine the role of BKCa in the regulation by vasorelaxants of mesangial contraction measured as percent maximal and defined by reduction in length induced by replacing 135 mM bath NaCl with KCl. Contraction by ANG II (100 nM = 60.5%) was attenuated by NP (100 microM), ANP (1.0 microM), and DBcGMP (10 microM) in the absence, but not the presence, of iberiotoxin, a specific inhibitor of BKCa. These results indicate that guanylyl cyclase-stimulating vasorelaxants counteract ANG II-induced contraction of MC, in part, by repolarizing the membrane through activation of BKCa channels.
Modal gating in neuronal and skeletal muscle ryanodine-sensitive Ca2+ release channels
Authors: Armisen R. , Sierralta J. , Velez P. , Naranjo D. , Suarez-Isla B. A.
Published: December 2017

The bursting behavior of ryanodine-sensitive single Ca2+ release channels present in chicken cerebellum endoplasmic reticulum (ER), rat hippocampus ER, and frog and rabbit skeletal muscle sarcoplasmic reticulum was established. Unconditional dwell time distributions fitted by the maximum likelihood method reveal at least three open and closed exponential components. Trains of low open probability (P(o)) bursts were interspersed with trains of high P(o) bursts (> or = 0.8) in all the ryanodine receptor isotypes tested. The gating kinetics of the Ca2+ release channels were defined in long recordings by analyzing burst sequences and gamma distributions of average intraburst open (T(o)) and closed times (Tc). The gamma distributions of T(o) had two gamma components, suggesting the existence of two distinct burst types. In contrast, the gamma distributions of Tc had only one component. The correlation between consecutive burst pairs was defined in terms of T(o) and then statistically tested by 2 x 2 matrix contingency analysis. The probability that the ubiquitous sequential burst pattern was generated by random occurrence was < 0.01 (two-tailed Fisher's exact test). Temporal correlations were observed in all ryanodine receptor isotypes under a variety of experimental conditions. These data strongly suggest that single Ca2+ release channels switch slowly between modes of gating. We propose that the effects of agonists of Ca2+ release channels such as Ca2+ itself can be explained as concentration-dependent changes in the availability of each mode.
FK-506 and rapamycin but not cyclosporin inhibit aldosterone-stimulated sodium transport in A6 cells
Authors: Rokaw M. D. , West M. E. , Palevsky P. M. , Johnson J. P.
Published: December 2017

The immunosuppressants cyclosporin A (CyA), FK-506, and rapamycin (RAP) have multiple actions on target cells that appear to be mediated by interaction of drug-binding protein complexes. Both FK-506 and CyA, but not RAP, inhibit the Ca2(+)-dependent phosphatase, calcineurin, and in so doing have been found to inhibit Na(+)-K(+)-ATPase activity in various nephron segments. Of interest, FK-506 and RAP, but not CyA, are bound by the steroid receptor-associated FK-506-binding heat shock protein of 56 kDa, HSP56. To determine the physiological effect of this interaction on a steroid-mediated phenomenon, the effect of these agents on steroid-mediated Na+ transport in A6 cells was investigated. Aldosterone stimulation of Na+ transport and Na(+)-K(+)-ATPase activity are significantly inhibited by prolonged incubation with FK-506 and RAP. Although CyA inhibits basal Na(+)-K(+)-ATPase activity, it has no effect on aldosterone-induced Na+ transport or the aldosterone-induced increase in Na(+)-K(+)-ATPase activity. FK-506 inhibits the aldosterone-induced synthesis of G alpha i-3 protein but has no effect on glucocorticoid receptor number as quantified by Western blotting. The results suggest that FK-506 and RAP inhibit steroid-mediated Na+ transport at some pretranslational site. The common interaction of these agents with the steroid receptor-associated HSP56 might account for these findings.
Altered sodium current response to intracellular fatty acids in halothane-hypersensitive skeletal muscle
Authors: Wieland S. J. , Gong Q. H. , Fletcher J. E. , Rosenberg H.
Published: December 2017

Biopsies of human skeletal muscle were analyzed by an in vitro contracture test (IVCT) for responsiveness to a halothane challenge: noncontracting (nonresponsive; IVCT-) and contracting (IVCT+). A muscle biopsy that is IVCT+ indicates potential malignant hyperthermia (MH) susceptibility. Primary cultures were grown from portions of the skeletal muscle biopsies, and voltage-activated currents were measured by whole cell recording in the presence or absence of 2-5 microM intracellular arachidonic or oleic acids. In untreated IVCT- cells, Na+ currents were predominantly tetrodotoxin (TTX) insensitive, indicating that most of the current was carried through the embryonic SkM2 isoform of the Na+ channel. Inclusion of fatty acids in the recording pipette of IVCT- cells produced an increase in voltage-activated Na+ currents during 20 min of recording. Approximately 70% of currents in fatty acid-treated cells were TTX sensitive, indicating activation of the adult SkM1 isoform of the Na+ channel. In contrast to IVCT- cells, IVCT+ cells expressed Na+ currents that were predominantly TTX sensitive even in the absence of added fatty acid, thus showing a relatively large baseline functional expression of SkM1 channels. Addition of fatty acids to the recording pipette produced little further change in the magnitude or TTX sensitivity of the whole cell currents in IVCT+ cells, suggesting altered functional regulation of Na+ channels in MH muscle.
Functional analysis of a genetic defect of copper transport (Menkes disease) in different cell lines
Authors: Qian Y. , Tiffany-Castiglioni E. , Harris E. D.
Published: December 2017

To define the function of the Cu-transporting ATPase in Menkes disease, Menkes and normal fibroblasts were incubated with 67Cu before and after brief exposure to -SH reagents, p-chloromercuribenzoate (PCMB) and dithiothreitol (DTT). Accumulation and retention were compared among these cells, BeWo cells, and rat C6 glioma cells similarly treated. The Michaelis constant for influx of 67Cu into normal and Menkes fibroblasts was practically the same (0.21 +/- 0.07 vs. 0.24 +/- 0.06 microM). The PCMB treatment stimulated 67Cu accumulation in C6 cells, inhibited accumulation in normal and Menkes fibroblasts, and did not affect BeWo cells. DTT stimulated 67Cu uptake in all cells but BeWo cells. DTT treatment after PCMB further enhanced 67Cu accumulation in normal fibroblasts and C6 cells but had no enhancing effect on Menkes fibroblasts or BeWo cells. Menkes fibroblasts and BeWo cells released 67Cu at rates considerably slower than normal fibroblasts (0.06 and 0.09 vs. 0.22%/min, respectively). The PCMB blocked 67Cu release from normal fibroblasts but did not affect Menkes fibroblasts or BeWo cells. Reverse transcription-polymerase chain reaction analysis of total RNA from BeWo cells failed to show a predicted 943-base pair fragment representing a partial transcript of the Menkes factor. The fragment was present in extracts from normal fibroblasts. We conclude that the mechanism underlying Cu homeostasis varies among different cell types. As exemplified by BeWo and Menkes cells, failure to efflux Cu ions may be linked with the failure to express a functional Cu-transporting ATPase, namely, the Menkes protein.
Regulation of ion channels in smooth muscles by calcium
Authors: Carl A. , Lee H. K. , Sanders K. M.
Published: December 2017

Intracellular Ca2+ concentration ([Ca2+]i) plays a central role in regulating tone and contractility of smooth muscle cells. In contrast to the “classic” model of electromechanical coupling where membrane potential determines [Ca2+]i, it is now well established that [Ca2+]i in turn may also affect membrane potential by modulating open probabilities of ion channels. Activation by [Ca2+]i of large-conductance K+ channels, Cl- channels, and nonselective cation channels has been described, as well as block of delayed rectifier K+ channels by [Ca2+]i and [Ca2+]i-induced inactivation of Ca2+ channels. Therefore, a network consisting of positive- and negative-feedback loops regulates [Ca2+]i as well as membrane potential. In this context, we review the properties of Ca(2+)-dependent ion channels and their functional role in vascular and visceral smooth muscles. Any alteration of the “Ca2+ sensitivity” of ion channels is expected to have a profound effect on the reciprocal relationship between membrane potential and [Ca2+]i. Already several molecular factors determining Ca2+ sensitivity of Ca(2+)-activated K+ channels have been identified. We provide a working definition for Ca2+ sensitivity.
Changes in action potentials and ion currents in long-term cultured neonatal rat ventricular cells
Authors: Guo W. , Kamiya K. , Cheng J. , Toyama J.
Published: December 2017

A primary culture of neonatal ventricular myocytes isolated from day-old rats was established for investigating the changes in action potentials and ion currents over long periods. Cells at days 5 and 15 in culture were studied. These changes in vitro were compared with those in situ derived from the age-matched freshly isolated cells. During primary culture, quiescent cells demonstrated shortening of action potential durations (APD) resembling the developmental changes observed in situ. The beating cultured cells were not associated with APD shortening. Despite constant current amplitudes, the densities of Ca2+ currents (ICa) decreased in the quiescent cultures at later ages as a result of cell enlargement. ICa densities were maintained in the beating cultured and freshly isolated cells. Acceleration in the inactivation of ICa was observed during developments both in vitro and in situ. In addition, the densities of transient outward currents (Ito) tripled and doubled in the quiescent and beating cells during 15-day cultures. However, Ito in beating cultured cells made less contribution to APD in contrast to the quiescent cultured and freshly isolated myocytes. These findings demonstrate that electrophysiological properties differ between two types of long-term cultured cells. ICa densities remained constant in the beating cultures, suggesting that cell beating may be required for the maintenance of ICa density in developing cardiomyocytes.
Differential targeting of facilitative glucose transporters in polarized epithelial cells
Authors: Pascoe W. S. , Inukai K. , Oka Y. , Slot J. W. , James D. E.
Published: December 2017

We have examined the intracellular localization of five facilitative glucose transporter proteins, one endogenous (GLUT-1) and four exogenous (GLUT-2, -3, -4, and -5), in polarized epithelial cells. GLUT-2, -3, -4, and -5 were stably transfected into Madin-Darby canine kidney (MDCK) cells, and peptide-specific antibodies were used to establish their distribution by immunofluorescence and immunoelectron-microscopic techniques. GLUT-1 and -2 were predominantly targeted to the basolateral domain of the cell, whereas GLUT-3 and -5 were targeted to the apical plasma membrane. The insulin-regulatable glucose transporter GLUT-4 was found in intracellular tubulovesicular structures beneath the surface of the cell. Vectorial 2-deoxy-D-glucose uptake measurements revealed that approximately 95% of glucose entry into wild-type MDCK cells occurs via the basolateral membranes. In GLUT-3-transfected cells, however, apical glucose uptake increased to approximately 55%; this was not observed in cells expressing the other GLUT isoforms. The discrete and differential intracellular localizations of the various GLUTs, in addition to the high level of sequence homology and predicted secondary structure similarity, render the GLUT family ideal for the study of intrinsic targeting motifs involved in the establishment and maintenance of cellular polarity.
Muscle function during jumping in frogs. I. Sarcomere length change, EMG pattern, and jumping performance
Authors: Lutz G. J. , Rome L. C.
Published: December 2017

We determined the influence of temperature on muscle function during jumping to better understand how the frog muscular system is designed to generate a high level of mechanical power. Maximal jumping performance and the in vivo operating conditions of the semimembranosus muscle (SM), a hip extensor, were measured and related to the mechanical properties of the isolated SM in the accompanying paper [Muscle function during jumping in frogs. II. Mechanical properties of muscle: implication for system design. Am. J. Physiol. 271 (Cell Physiol. 40): C571-C578, 1996]. Reducing temperature from 25 to 15 degrees C caused a 1.75-fold decline in peak mechanical power generation and a proportional decline in aerial jump distance. The hip and knee joint excursions were nearly the same at both temperatures. Accordingly, sarcomeres shortened over the same range (2.4 to 1.9 microns) at both temperatures, corresponding to myofilament overlap at least 90% of maximal. At the low temperature, however, movements were made more slowly. Angular velocities were 1.2- to 1.4-fold lower, and ground contact time was increased by 1.33-fold at 15 degrees C. Average shortening velocity of the SM was only 1.2-fold lower at 15 degrees C than at 25 degrees C. The low Q10 of velocity is in agreement with that predicted for muscles shortening against an inertial load.
Muscle function during jumping in frogs. II. Mechanical properties of muscle: implications for system design
Authors: Lutz G. J. , Rome L. C.
Published: December 2017

We characterized the design of the frog muscular system for jumping by comparing the properties of isolated muscle with the operating conditions of muscle measured during maximal jumps. During jumping, the semimembranosus muscle (SM) shortened with a V/Vmax (where V is shortening velocity and Vmax is maximal shortening velocity) where 90 and 100% of maximal power would be generated at 15 and 25 degrees C, respectively. To assess the level of activation during jumping, the SM was driven through the in vivo length change and stimulus conditions while the resulting force was measured. The force generated under the in vivo conditions at both temperatures was at least 90% of the force generated at that same V under maximally activated conditions. Thus the SM was nearly maximally activated, and shortening deactivation was minimal. The initial sarcomere length and duration of the stimulus before shortening were important factors that minimized shortening deactivation during jumping. Thus the frog muscular system appears to be designed to meet the three necessary conditions for maximal power generation during jumping: optimal myofilament overlap, optimal V/Vmax, and maximal activation.
CFTR chloride channel activation by genistein: the role of serine/threonine protein phosphatases
Authors: Reenstra W. W. , Yurko-Mauro K. , Dam A. , Raman S. , Shorten S.
Published: December 2017

We have previously shown [B. Illek, H. Fischer, G. F. Santos, J. H. Widdicombe, T. E. Machen, and W. W. Reenstra, Am. J. Physiol. 268 (Cell Physiol. 37): C886-C893, 1995] that genistein, a tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in NIH/3T3 cells that have been stably transfected with an expression vector for the CFTR (NIH-CFTR cells). In this study, we present evidence suggesting that both genistein and the serine/threonine protein phosphatase (PPase) inhibitor calyculin A activate the CFTR by inhibiting PPase activity. As measured by 125I efflux, genistein and calyculin A stimulate the CFTR to approximately 50% of the maximal activity with forskolin. Neither agonist increases CFTR activity at saturating forskolin concentrations, but genistein and calyculin A have an additive effect on CFTR activity. Forskolin, but neither genistein nor calyculin A, stimulates protein kinase A(PKA) activity. The PKA inhibitor H-89 inhibits CFTR activation and in vivo phosphorylation by all three agonists. Proteolytic digestion of in vivo phosphorylated CFTR suggests that the CFTR is phosphorylated on the same sites during stimulation with genistein and forskolin but on different sites stimulation with calyculin A. The data suggest that genistein and calyculin A inhibit different PPase activities, allowing CFTR phosphorylation and partial stimulation, by a basal PKA activity.
Optimization of a mammalian expression system for the measurement of sodium channel gating currents
Authors: Sheets M. F. , Kyle J. W. , Krueger S. , Hanck D. A.
Published: December 2017

We describe a new mammalian expression system that optimizes conditions for the measurement of Na channel gating currents (Ig). The small magnitude of Ig limits their study to preparations with high numbers of Na channels to improve signal-to-noise ratios. To increase Na channel Ig signals, single tsA201 cells (approximately 20 microns in diameter) were fused into large, multinucleated cells by treatment with polyethylene glycol. After being placed in cell culture for 48-72 h, fused tsA201 cells develop a spherical geometry with diameters up to 200 microns. Because of the large plasma membrane surface area, fused tsA201 cells are able to express high levels of Na channels after transient transfection with Na channel cDNAs using Lipofectamine. Typically, 5 days after transfection, fused tsA201 cells that are 60-100 microns in diameter are selected for voltage clamp with a large suction pipette (a pore size of 20-30 microns) that allows for both a low series resistance and internal perfusion. Approximately two-thirds of transfected fused tsA201 cells express Na current, with nearly one-third of transfected cells expressing sufficient numbers of Na channels to allow for the ready measurement of Ig. In addition to fused tsA201 cells being a preparation well suited for the study of Ig, they should also be useful for measurement of electrical signals from other voltage-gated channels and transporters that generate small electrical signals.
Mechanisms of recovery from mechanical injury of cultured rat hepatocytes
Authors: Sponsel H. T. , Guzelian P. S. , Brown S. E. , Breckon R. , Ray C. , Simon F. R. , Anderson R. J.
Published: December 2017

The mechanism(s) whereby hepatocytes restore denuded areas remains unknown. We therefore studied the recovery of denuded areas made in monolayers of primary cultures of rat hepatocytes. Minimal recovery occurred in cells plated on plastic. Plating on Matrigel produced modest recovery (25% at 24 h), whereas plating on a type I collagen substrate resulted in > 70% recovery at 24 h. The rate of recovery on collagen could be attenuated by a monoclonal antibody directed against the extracellular domain of the beta 1-integrin subunit. Monoclonal antibodies directed against CD44 (the hyaluron receptor) and E-cadherin did not influence the rate of recovery. Recovery could be stimulated, in a dose-dependent fashion, by epidermal and hepatocyte growth factors. The effects of epidermal and hepatocyte growth factors to promote recovery occurred in the absence of 5-bromo-2'-deoxyuridine uptake, suggesting a proliferation-independent mechanism. Transforming growth factor-beta 1 inhibited recovery. Exposure to selected cytokines (interleukins 1 and 2), an adenine nucleotide [adenosine 5'-O-(3-thiotriphosphate)], adenosine, pertussis toxin, and selected agents that bind to fibronectin and other matrix component adhesive sites (heparin and the RGD peptide) did not influence the rate of recovery of hepatocytes. However, the peptide DGEA, which can bind to collagen adhesive sites, attenuated recovery. These studies demonstrate that primary cultures of rat hepatocytes require a particular type of extracellular matrix to renew denuded areas and that the beta 1-integrin subunit may be involved in this recovery process. Hepatocyte recovery of denuded areas can be modulated by growth factors in both a stimulatory (epidermal and hepatocyte growth factors) and an inhibitory (transforming growth factor-beta 1) fashion.
Positive charge modifications alter the ability of XIP to inhibit the plasma membrane calcium pump
Authors: Xu W. , Gatto C. , Milanick M. A.
Published: December 2017

Exchange inhibitory peptide (XIP; RRLLFYKYVYKRYRAGKQRG) is the shortest peptide that inhibits the plasma membrane Ca pump at high Ca (A. Enyedi, T. Vorherr, P. James, D. J. McCormick, A. G. Filoteo, E. Carafoli, and J. T. Penniston, J. Biol. Chem. 264: 12313-12321, 1989). Sulfosuccinimidyl acetate (SNA)-modified XIP does not inhibit the Ca pump; SNA neutralizes the positive charge on Lys at positions 7, 11, and 17. Peptide 2CK-XIP (RRLLFYRYVYRCYCAGRQKG) inhibits the pump, but the iodoacetamido-modified peptide does not inhibit. Three peptide analogues, in which 7, 11, and 17 were Ala, Cys, or Lys, inhibited about as well as XIP. SNA modification of these analogues (each with 1 Lys) did not inhibit. SNA modification of 2CK-XIP results in a peptide that does not inhibit; thus position 19 is important. Our results suggest that it is critical that position 19 be positively charged, that positions 7, 11, and 17 are important contact points between XIP and the Ca pump (with at least one positively charged), and that, whereas it is not essential that residues 12 and 14 be positive, they cannot be negative.
Modulation of frog skeletal muscle Ca2+ release channel gating by anion channel blockers
Authors: Oba T. , Koshita M. , Van Helden D. F.
Published: December 2017

Effects of niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on frog skeletal muscle ryanodine receptors have been studied by incorporating sarcoplasmic reticulum vesicles into planar lipid bilayers. Niflumic acid increased the mean open probability (Po) at 10 microM and decreased Po at 100 microM with no change in open time constants, unitary conductance, and reversal potential. The Po was augmented by DIDS at 5-200 microM without affecting either unitary conductance or reversal potential. DIDS induced a new third open time constant, probably contributing to a long-lived open state. Channels modified by niflumic acid or DIDS still responded to Ca2+ release channel modulators. These results provide evidence that niflumic acid and DIDS modify the gating mechanism of ryanodine receptors without affecting binding sites to the modulators and the physical pathway of the conducting pore. p-Chloromercuriphenyl sulfonic acid (pCMPS) transiently increased the Po. The channel modified by DIDS responded to pCMPS, whereas that by ryanodine did not. The long open state of the channel induced by DIDS is produced by a quite different mechanism(s) from that by ryanodine. Contrary to cardiac ryanodine receptors, Po of skeletal muscle channels was independent of voltage after DIDS modification.
Metallothionein is a component of exocrine pancreas secretion: implications for zinc homeostasis
Authors: De Lisle R. C. , Sarras M. P. , Hidalgo J. , Andrews G. K.
Published: December 2017

Using transgenic mice that overexpress metallothionein-I (MT-I) and zinc-induced normal and transgenic animals, we have explored the localization of MT in the pancreas. Light-level immunocytochemistry demonstrated MT in acinar cells but not islet cells. Immunolabeling also revealed the presence of MT in pancreatic ducts, suggesting that it is released from acinar cells. Ultrastructural immunolocalization showed that MT was cytoplasmic, and no MT immunoreactivity was detected in lumens of the vesicular secretory pathway. Secreted pancreatic juice was collected from pilocarpine-stimulated mice and assayed for MT by a 109Cd-labeled hemoglobin-exchange assay and by radioimmunoassay. Both methods revealed high (> 1,000 ng/ml) levels of MT in the stimulated secretion. The level of MT in pancreatic juice from transgenic mice was only slightly (2-fold) increased despite dramatic overexpression of MT-I in the pancreas (> 20-fold). In contrast, zinc induction of MT significantly increased MT by 5- to 10-fold in the pancreatic juice, in normal and transgenic mice. These data indicate that MT is released from pancreatic acinar cells but not by the classical vesicular secretory pathway. In addition, MT levels in pancreatic juice are regulated by zinc, suggesting a physiological role of the pancreas in metal homeostasis.
Inadequacy of high K+/nigericin for calibrating BCECF. I. Estimating steady-state intracellular pH
Authors: Boyarsky G. , Hanssen C. , Clyne L. A.
Published: December 2017

Intracellular pH (pHi) was measured in single vascular smooth muscle (VSM) cells, cultured from rabbit abdominal aorta, using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) on a microscope-based fluorescence system. Three lines of evidence are presented that using nigericin along with high external K+ to calibrate intracellular BCECF produces systematic errors in pHi. 1) The intrinsic buffering power (beta int), measured using weak bases (e.g., ammonium), was 2.5 times smaller than that measured using weak acids (e.g., propionic acid). This discrepancy became small if pHi had really been approximately 0.2 lower than what was estimated using nigericin-calibrated pHi values. 2) Total cellular buffering power (beta tot) in the presence of CO2/HCO-3 was measured and found to be much smaller than could account for the beta int, together with the contribution of CO2/HCO3 (beta CO2: assumed to be an open system buffer). If the true pHi values were approximately 0.2-0.4 lower than our nigericin-calibrated values, then the sum of beta int and beta CO2 equals beta tot. 3) A null technique was utilized for bracketing steady-state pHi; estimates of steady-state pHi using this null technique were approximately 0.2 lower than the high K+/nigericin-calibrated estimates. Four other cell types were examined: rat hepatocytes, rat corticotrophs, human keratinocytes, and rabbit fibroblasts. These other cells also displayed discrepancies between null and nigericin estimates of steady-state pHi, as well as differences between buffering power assessed using weak bases and acids. Finally, one potential source for these discrepancies is described: selecting an inappropriate external K+ to use with nigericin can produce systematic errors in pHi of approximately 0.1.
Characterization of inwardly rectifying potassium currents from dissociated rat taste receptor cells
Authors: Sun X. D. , Herness M. S.
Published: December 2017

Inwardly rectifying potassium currents were recorded from taste receptor cells dissociated from the rat tongue with the use of patch-clamp techniques in the whole cell configuration. These currents displayed strong inward rectification at potentials negative to the potassium reversal potential and little outward current at potentials positive to it. With elevations of external potassium concentration, the slope and chord conductance increased, activation shifted toward more depolarized potentials, and the reversal potential varied in a predicted Nernst relationship. These currents were insensitive to 4-aminopyridine and partially blocked by tetraethylammonium. Both barium and cesium gave more complete blocks with characteristic relief at more negative potentials. Inhibition with barium was more voltage sensitive than with cesium. These currents were unaffected by changes in external sodium. The high conductance at negative membrane potentials suggests these currents may contribute with other conductances to the resting potential of taste cells. They may also participate in yet unidentified processes of taste transduction, resulting in the early depolarization of the resting potential.
Potentiation by thyroxine of interferon-gamma-induced antiviral state requires PKA and PKC activities
Authors: Lin H. Y. , Thacorf H. R. , Davis F. B. , Davis P. J.
Published: December 2017

Added to HeLa cells previously exposed to recombinant human interferon (IFN)-gamma for 20 h, thyroid hormone [L-thyroxine (T4)] in physiological concentrations potentiates the antiviral action of IFN-gamma by more than 100-fold in 4 h. We examined protein kinase activities for their contributions to the mechanism of this posttranslational effect of thyroid hormone. Added concurrently with thyroid hormone, the protein kinase C (PKC) inhibitor CGP-41251 (5 nM) blocked T4 potentiation of IFN-gamma action. Coincubated with CGP-41251, phorbol 12-myristate 13-acetate (PMA) reversed the effect of the inhibitor on thyroid hormone action. U-73122 (10 nM), a phospholipase C inhibitor, also blocked hormone potentiation. KT-5720 (500 nM), a protein kinase A (PKA) inhibitor, completely inhibited the T4 effect, whereas 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) restored hormone action in the presence of KT-5720. In the absence of T4, 8-BrcAMP and PMA, added together to cells in the 4-h paradigm, fully reproduced hormone potentiation of the antiviral effect of IFN-gamma. Incubated individually with IFN-gamma-treated cells, the two agonists had no potentiating action. Thyroid hormone apparently must activate both PKA and PKC in the nongenomic pathway of IFN-gamma action to enhance antiviral activity in HeLa cells.