bluuebunny/crossref_metadata_2025 huggingface dataset:
cAMP-activated Cl channels in CFTR-transfected cystic fibrosis pancreatic epithelial cells
Authors: Cliff W. H. , Schoumacher R. A. , Frizzell R. A.
Published: December 2017

Retrovirus-mediated transfection of cDNA for the cystic fibrosis (CF) gene into the CF pancreatic cell line, CFPAC-1, confers adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of Cl conductance. We used patch-clamp techniques to identify the single-channel basis of this conductance pathway and to study its properties. Forskolin or cAMP activated Cl channels with a conductance of 9 +/- 1 pS in 26 of 62 cell-attached patches of cystic fibrosis transmembrane conductance regulator (CFTR)-transfected CFPAC-1 cells. The current-voltage (I-V) relation showed slight outward rectification (chord conductance of 10 +/- 2 pS at +80 mV vs. 7 +/- 1 pS at -80mV) with high Cl concentrations (170 mM) in the pipette solution. Channel kinetics were voltage sensitive, with longer openings at positive clamp voltages. Channel properties were unaffected by the substitution of N-methyl-D-glucamine for pipette Na or by the addition of disulfonic stilbenes (100 microM DNDS or DIDS) to the pipette. The channels usually inactivated within seconds of patch excision, but in three of nine patches, activity could be maintained by addition of the catalytic subunit of protein kinase A and ATP. With equal Cl concentrations on both membrane surfaces, the single-channel I-V relation was linear, suggesting that the outward rectification of the cell-attached channel is due to a pipette-to-cell Cl gradient. Anion substitution on the extracellular side of the membrane indicates a halide permselectivity of Br approximately Cl greater than I.(ABSTRACT TRUNCATED AT 250 WORDS)
Active transport of dopamine in human placental brush-border membrane vesicles
Authors: Ramamoorthy S. , Leibach F. H. , Mahesh V. B. , Ganapathy V.
Published: December 2017

Brush-border membrane vesicles isolated from normal human term placentas were found to accumulate dopamine against a concentration gradient when an inwardly directed NaCl gradient was imposed across the membrane. The activity of the transport system was obligatorily dependent on Na+ as well as Cl-. Intravesicular H+ and intravesicular K+ stimulated the transport activity. The system possessed high affinity for dopamine and norepinephrine, with considerably lower affinity for serotonin. The stoichiometry of Na(+)-Cl(-)- dopamine was 1:1:1. The system was electrogenic because the NaCl-dependent dopamine uptake was stimulated by an inside-negative membrane potential, and this characteristic was observed in the presence and in the absence of intravesicular K+. Kinetic analysis revealed that the uptake was due to a carrier-mediated component plus a diffusion/binding component. The apparent Michaelis-Menten constant for dopamine for the carrier-mediated component was 19 +/- 7 nM. The transport system was clearly distinct from the serotonin transporter. Analysis of the inhibition of dopamine uptake by various monoamine uptake inhibitors showed that the uptake occurred via a transport system that is similar to the neuronal norepinephrine transporter.
Activation of protein kinase C reduces L-type calcium channel activity of GH3 pituitary cells
Authors: Haymes A. A. , Kwan Y. W. , Arena J. P. , Kass R. S. , Hinkle P. M.
Published: December 2017

These studies describe the effect of protein kinase C (PKC) activation on the activity of voltage-sensitive L-type Ca2+ channels of GH3 pituitary cells. The rate of 45Ca2+ uptake was stimulated greater than 25-fold by depolarization in the presence of BAY K 8644; the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced this response by 70% in a concentration-dependent fashion. Phorbol 12,13-dibutyrate (PDBu) inhibited depolarization-induced 45Ca2+ uptake within 1 min and caused a nearly maximal reduction after 1 h; its effects were rapidly reversible. TPA decreased the high K(+)-stimulated increase in intracellular free calcium ion concentration ([Ca2+]i) from 8.5- to 3.2-fold by 5 min and to 2.0-fold after 18 h without altering the peak [Ca2+]i response to the peptide hormone TRH. Ca2+ channel current, measured directly using the whole cell configuration of the patch-clamp technique, declined an average of 6.4% over 5 min for control cells and 28.9% when TPA was added to the bathing medium for 5 min. Treatment with 100 nM TPA for 24 h dramatically reduced peak current without shifting the peak of the current-voltage relationship. The mean peak Ca2+ channel current was reduced from 423 to 128 pA, although a few cells seemed completely resistant. To determine whether the effects of phorbol esters were due to the activation of PKC we tested the potency of several drugs to inhibit L-channel activity and to shift the affinity of the epidermal growth factor (EGF) receptor, an established PKC response.(ABSTRACT TRUNCATED AT 250 WORDS)
ATP-sensitive potassium channel is essential to maintain basal coronary vascular tone in vivo
Authors: Samaha F. F. , Heineman F. W. , Ince C. , Fleming J. , Balaban R. S.
Published: December 2017

Glibenclamide, a known selective inhibitor of ATP-sensitive potassium channels, was infused into the coronary vasculature of anesthetized dogs and of isolated perfused rabbit hearts to assess the role of this channel in the maintenance of basal coronary resistance. Infusion of glibenclamide at a concentration of 55-80 microM in the dogs resulted in a twofold steady-state increase in coronary resistance with resultant tissue ischemia. Infusion of 1 microM glibenclamide in the isolated hearts resulted in a 67% increase in coronary resistance with resultant tissue ischemia. The ischemic changes were reversible upon removal of the drug. These findings indicate that the ATP-sensitive K+ channel plays a significant role in the maintenance of basal coronary resistance in vivo. Higher concentrations of glibenclamide (80-100 microM) in the in vivo dog heart consistently gave rise to an oscillating pattern of coronary flow. These oscillations were either eliminated or decreased in amplitude and frequency by the infusion of 8-phenyltheophylline, a specific competitive inhibitor of adenosine receptors. 31P-nuclear magnetic resonance spectroscopy performed at the peaks and troughs of these oscillations revealed oscillation of the phosphorylation potential at the same frequency. Thus adenosine release caused by tissue ischemia appears to play a major role in creating the oscillating pattern of coronary blood flow, that occurs during the inhibition of ATP-sensitive K+ channels by glibenclamide.
Immunocytochemical and functional characterization of Na+ conductance in adult alveolar pneumocytes
Authors: Matalon S. , Kirk K. L. , Bubien J. K. , Oh Y. , Hu P. , Yue G. , Shoemaker R. , Cragoe E. J. , Benos D. J.
Published: December 2017

The purpose of this study was to document the existence, assess the spatial localization, and characterize some of the transport properties of proteins antigenically related to epithelial Na+ channels in freshly isolated rabbit and rat alveolar type II (ATII) cells. ATII cells, isolated by elastase digestion of lung tissue and purified by density-gradient centrifugation, were incubated with polyclonal antibodies raised against Na+ channel protein purified from beef kidney papilla (NaAb), followed by a secondary antibody (goat antirabbit immunoglobulin G conjugated to fluorescein isothiocyanate). Rat ATII cells exhibited specific staining with NaAb at the level of the plasma membrane, which, in most cells, colocalized with that of the lectin Maclura pomiferra agglutinin, an apical surface marker. In Western blots, NaAb specifically recognized a 135 +/- 10-kDa protein in rat ATII membrane vesicles. When patch clamped in the whole cell mode using symmetrical solutions (150 mM Na+ glutamate), ATII cells exhibited outwardly rectified Na+ currents that were diminished by amiloride (10-100 microM) instilled into the bath solution. Ion substitution studies showed that the conductive pathways were three times more permeable to Na+ than K+. Amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride were equally effective in diminishing 22Na+ flux into rabbit and rat ATII cells (45% inhibition at 100 microM, with IC50 of approximately 1 microM for all inhibitors). Tetraethylammonium chloride (10 mM) or BaCl2 (2 mM), well-known K+ channel blockers, had no effect on 22Na+ uptake. These results indicate that ATII cells express an amiloride-sensitive Na+ conductance, probably a channel, with a lower affinity for amiloride and its structural analogues than the well-established amiloride-sensitive Na+ channels found in bovine renal papila and cultured amphibian A6 kidney cells.
Calibration of ion-selective microelectrodes
Authors: Croxton T. L. , Armstrong W. M.
Published: December 2017

Measurements of intracellular and extracellular ion activities with ion-selective microelectrodes generally involve calibration of the electrodes in solutions of known composition and fitting of the calibration data with a theoretical expression. The Nicolsky equation is frequently used to describe the variation of electrode potential with primary ion activity in the presence of a constant amount of interference. In this report, we review the estimation of primary ion activities in calibration solutions and discuss the practical use of the Nicolsky equation. We describe a specialized computer program, developed in this laboratory, for routine input and editing of calibration data, fitting of data with the Nicolsky equation, and calculation of experimental ion activities from the fitted curve. Earlier versions of this program have proven helpful in several investigations in this and in other laboratories.
Ca2+/calmodulin regulation of putrescine uptake in cultured gastrointestinal epithelial cells
Authors: Groblewski G. E. , Hargittai P. T. , Seidel E. R.
Published: December 2017

Regulation of putrescine uptake in a small intestinal crypt cell line, IEC-6 cells, was examined. Uptake of [14C]putrescine was measured throughout a normal growth curve and was found to be inversely related to growth. Kinetic analysis at low and high cell density revealed the inhibition of uptake in confluent cells was due to a five-fold reduction in Vmax of uptake, 199.5 vs. 43.1 pmol.10(5) cells-1.h-1, respectively. Three gastrointestinal hormones, gastrin, secretin, and cholecystokinin, produced partial inhibition of [14C]putrescine uptake. Conversely, treatment of quiescent cells with 5% fetal bovine serum to stimulate growth did not affect uptake. Influence of putrescine uptake on free ionized intracellular Ca2+ ([Ca2+]i) was measured by microspectrofluorometry using the Ca(2+)-sensitive fluoroprobe fura-2. Basal [Ca2+]i was calculated to be 112 nM and increased rapidly to 313 nM upon addition of 10 microM putrescine. Preventing the rise in [Ca2+]i using an intracellular Ca2+ buffer, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, decreased [14C]putrescine uptake to 29.5 +/- 5.3% of control values. 45Ca2+ flux experiments and measurement of transport in 0 Ca2+ and 0.5 mM EDTA suggested an intracellular source of calcium was mobilized during putrescineuptake. Finally, use of the putative calmodulin antagonist N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide caused a dose-dependent inhibition of [14C]putrescine uptake with 50% inhibitory concentration of approximately 7 microM. These data suggest that putrescine uptake in IEC-6 cells may be regulated by a Ca2+/calmodulin-dependent mechanism.
Secretion of K and Cl across colonic epithelium: cellular localization using electron microprobe analysis
Authors: Halm D. R. , Rick R.
Published: December 2017

Electron microprobe analysis of quick-frozen distal colonic epithelium from guinea pig was used to locate the cells responding to secretory stimuli. Concentrations of Na, K, and Cl were similar for cells of surface and crypt in the unstimulated state, 8, 149, and 46 mmol/kg wet weight, respectively. Stimulation of either K and Cl secretion with prostaglandin E2 or K secretion alone with epinephrine increased Na to approximately 12 mmol/kg wet weight in crypt cells but not in surface cells or cells in the crypt neck. This result supports the location of ion secretory cells in the lower two-thirds of the crypt. In the vacuoles of crypt columnar cells, stimulation of KCl secretion decreased K, S, Mg, and Ca and increased Na and Cl, indicative of the concomitant release of vacuole contents. Mucin granules in crypt goblet cells contained more S and Mg than granules in surface goblet cells. These findings support the concept of differentiation in ion and macromolecular secretory function along the axis from crypt to surface epithelium.
Ca(2+)-activated nonselective cation channel in apical membrane of vestibular dark cells
Authors: Marcus D. C. , Takeuchi S. , Wangemann P.
Published: December 2017

Patch-clamp recordings were made on cell-attached and excised apical membrane from dark cells of the semicircular canal of the gerbil. These cells are thought to secrete K+ and absorb Na+ from the luminal fluid (endolymph). Single-channel events were identified as being equally conductive (27.6 +/- 0.4 pS; n = 48) for K+, Na+, Rb+, Li+, and Cs+ and 1.4 times more permeable to NH4+ but not permeable to Cl-, Ca2+, Ba2+, nor to N-methyl-D-glucamine. The channels displayed linear current-voltage relations that passed nearly through the origin (intercept: -2.6 +/- 0.5 mV; n = 48) when conductive monovalent cations were present on both sides of the membrane in equal concentrations. Channel activity required the presence of Ca2+ at the cytosolic face; there was no activity at less than or equal to 10(-7) M Ca2+ and full activity at greater than or equal to 10(-5) M Ca2+. Cell-attached recordings had a mean reversal voltage of -36.4 +/- 7.9 mV (n = 7), which was interpreted to reflect the intracellular potential of dark cells under the present conditions. We have identified a nonselective cation channel in the apical membrane of vestibular dark cells that might participate in K+ secretion or Na+ absorption under stimulated conditions, but the density appears to be insufficient to fully account for the transepithelial K+ flux.
Effects of myosin kinase inhibiting peptide on contractility and LC20 phosphorylation in skinned smooth muscle
Authors: Strauss J. D. , de Lanerolle P. , Paul R. J.
Published: December 2017

A peptide inhibitor, myosin kinase inhibitor (MKI), of myosin light chain kinase (MLCK) was tested for its effects on contractility and myosin light chain phosphorylation in Triton X-100 skinned guinea pig taenia coli. MKI is based on the amino acid sequence of the myosin light chain (residues 11-19 LC20) and is a competitive inhibitor [inhibitory constant (Ki) congruent to 10 microM] of purified MLCK with respect to myosin light chain (LC20). MKI inhibited unloaded shortening velocity (V(us)) and the calcium-sensitive ATPase activity of the skinned fibers but had no significant effect on steady-state isometric force or myosin light chain phosphorylation, as measured by IEF-polyacrylamide gel electrophoresis analysis. MKI had no significant effect on V(us) of thiophosphorylated fibers in the absence of calcium. MKI inhibited MLCK activity in protein extracts from taenia coli, as measured by radioactive phosphate incorporation into LC20. Surprisingly, MKI also inhibited the phosphatase activity of these same extracts. This peptide slowed the rate and extent of relaxation of calcium-contracted fibers and elicited a contraction in relaxed fibers. These results are consistent with the hypothesis that MKI may be a phosphatase inhibitor as well as an inhibitor of MLCK. Our data further suggest that the rate of phosphorylation-dephosphorylation turnover may be important in regulating V(us) in smooth muscle.
Gs and Gi protein subunits during cell differentiation in intestinal crypt-villus axis: regulation at the mRNA level
Authors: Couvineau A. , Darmoul D. , Blais A. , Rouyer-Fessard C. , Daviaud D. , Voisin T. , Paris H. , Rouot B. , Laburthe M.
Published: December 2017

The expression of subunits of the guanine nucleotide regulatory protein that mediates hormonal stimulation of adenylyl cyclase (Gs) and of the guanine nucleotide regulatory protein that mediates hormonal inhibition of adenylyl cyclase (Gi) was studied during cell migration and differentiation in the rat small intestine crypt-villus axis. Proliferative crypt cells were separated from nonproliferative mature villus cells and the following data were obtained: 1) alpha s subunits were more abundant in crypt cells than in villus cells as evidenced by cholera toxin-catalyzed [32P]NAD ribosylation and Western blotting of this relative molecular weight (M(r)) 42,000 protein; 2) alpha i2- and alpha i3-subunits (M(r) 40,000 and M(r) 41,000, respectively) were preferentially expressed in villus cells as evidenced by pertussis toxin-catalyzed [32P]NAD ribosylation and Western blotting (alpha i1-subunit was not detectable in intestinal epithelium by using these techniques); 3) Western blotting showed a higher expression of the common beta- (M(r) 36,000) subunit of G proteins in villus cells than in crypt cells; and 4) Northern blot analysis using an alpha s-subunit oligonucleotide probe showed a 1.9-kb mRNA that was more abundant in crypt cells than in villus cells. In contrast, alpha i2- and alpha i3-mRNA species (2.3 and 3.5 kb, respectively), analyzed by using specific cDNA probes, were much more abundant in villus cells than in crypt cells. Finally, two beta-subunit mRNA species of 3.3 and 1.8 kb were detectable in intestinal epithelial cells and were more abundant in villus cells than in crypt cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Kinetics of L-glutamate influx in single barnacle muscle fibers under 0-trans conditions
Authors: Horn L. W.
Published: December 2017

The dependence of L-glutamate influx on extracellular Na and L-glutamate concentrations was determined using internally dialyzed single muscle fibers of Balanus nubilus. Internal Na and glutamate concentrations were held at zero, and the cell membrane potential was constant. Flux activation curves for external glutamate were measured for five different external Na concentrations, and flux activation curves for external Na were measured independently for three different external glutamate concentrations. An analysis of alternative kinetic models for the transporter mechanism was made and led to the conclusions that under 0-trans conditions the Na:glutamate stoichiometry is 1:1, that glutamate first binding to the external transporter binding site is the preferred order under most extracellular conditions, and that the Na:glutamate coupling is too tight to permit measurable Na-independent glutamate uptake by the transporter.
Determination of Na(+)-K(+)-ATPase alpha- and beta-isoforms and kinetic properties in mammalian liver
Authors: Sun Y. , Ball W. J.
Published: December 2017

While Western blot analysis clearly revealed the presence of the alpha- and beta-subunits of Na(+)-K(+)-ATPase in a variety of rat tissues, beta was not readily detectable in liver. This observation was consistent with a previous report indicating that Na(+)-K(+)-ATPase immunoprecipitated from rat liver gives no clear evidence for the presence of a beta-subunit (Hubert et al. Biochemistry 25: 4156-4163, 1986). However, Western blot analysis of density gradient-purified lamb and rat liver microsomes showed the presence of a protein with an approximate molecular mass of 42 kDa that was immunoreactive with beta-specific polyclonal antibodies as well as beta-directed monoclonal antibodies. Deglycosylation of this protein by N-glycosidase F generated a core protein (beta c, M(r) approximately 32,000) that had the identical electrophoretic mobility as the beta c protein of the purified kidney enzyme. Isoform-specific monoclonal and synthetic peptide-directed polyclonal antibodies were used to demonstrate the presence of only the alpha 1- and beta 1-proteins in the liver and the presence of beta 2 in rat brain. Functional studies then showed that although both rat and lamb liver enzymes had sensitivities to cardiac glycoside inhibition similar to that of their corresponding kidney enzyme, the lamb liver enzyme had higher affinities for Na+, K+, and ATP than the kidney enzyme.
Regulation of alpha 1-beta 3-NA(+)-K(+)-ATPase isozyme during meiotic maturation of Xenopus laevis oocytes
Authors: Pralong-Zamofing D. , Yi Q. H. , Schmalzing G. , Good P. , Geering K.
Published: December 2017

During progesterone-induced maturation of Xenopus oocytes, the transport and ouabain binding capacity of Na(+)-K(+)-ATPase at the plasma membrane is completely downregulated. To elucidate the mechanism and the physiological significance of this process, we have followed the fate of oocyte alpha-beta 3-Na(+)-K(+)-ATPase complexes during meiotic maturation and early embryonic development. An immunocytochemical follow-up of the catalytic alpha-subunit, ouabain binding studies, cell surface iodination, and oocyte cell fractionation combined with immunochemical subunit detection provides evidence that following progesterone treatment Na(+)-K(+)-ATPase molecules are retrieved from the oocyte plasma membrane. The enzyme complexes are recovered in an active form in an intracellular compartment in both in vitro and in vivo matured eggs. Exogenous Xenopus alpha 1- and beta 1-complexes expressed in the oocyte from injected cRNAs are regulated by progesterone similar to endogenous Na(+)-K(+)-ATPase complexes. Finally, active Na(+)-K+ pumps internalized during oocyte maturation appear to be redistributed to plasma membrane fractions during blastula formation in Xenopus embryos. In conclusion, our data suggest that endocytosis of alpha 1- and beta 3-complexes during meiotic maturation of Xenopus oocytes is responsible for downregulation of Na(+)-K(+)-ATPase activity and results in an intracellular pool of functional enzymes, which might be reexpressed during early development in response to physiological needs.
Protein kinase C does not participate in carbachol's secretory action in T84 cells
Authors: Lindeman R. P. , Chase H. S.
Published: December 2017

We investigated the role of protein kinase C (PKC) in mediating carbachol's stimulation of transepithelial Cl- secretion in T84 cells. Direct PKC activation with phorbol 12-myristate 13-acetate (PMA) stimulated transepithelial Cl- transport (measured as the short-circuit current), demonstrating that PKC could interact with the secretory apparatus. Carbachol stimulated PKC activity, suggesting that the enzyme might participate in the hormone's action. Diacylglycerol metabolism inhibitors (DMIs), known to augment hormone-stimulated increases in diacylglycerol levels, potentiated the short-circuit current response to carbachol. The effect of DMIs was not due to amplification of carbachol-induced increases in PKC activity, however; PKC activity during carbachol stimulation was no higher in the presence of DMIs than in their absence. Augmentation of carbachol's action by DMIs appeared to be due to the direct activation of PKC which, like PMA, stimulated the Cl- conductance of the apical membrane (GCl). The effects of DMIs and carbachol on GCl were additive. Carbachol itself stimulated GCl but not by activating PKC; staurosporine did not blunt the effect of carbachol on GCl. Nor did staurosporine reduce the effect of carbachol on transepithelial Cl- secretion. These observations demonstrate that PKC does not participate in the secretory action of carbachol in T84 cells and suggest that direct PKC activation with DMIs and PMA stimulates an apical pool of PKC that is not accessible to carbachol applied to the basolateral membrane.
Metabolic substrates can alter postischemic recovery in preconditioned ischemic heart
Authors: Fralix T. A. , Steenbergen C. , London R. E. , Murphy E.
Published: December 2017

The mechanisms that contribute to myocardial cell injury are not well understood. Furthermore, the ability of reperfusion conditions to modify ischemic injury is unclear. Recent studies have indicated that glucose utilization may improve ionic homeostasis. Because considerable derangement of ion concentrations occurs during ischemia, glucose utilization may be beneficial when stimulated during the reperfusion period. The effects of glycolytic vs. mitochondrial substrates on postischemic contractile function, high-energy phosphates and ion balance (intracellular Ca2+ and pH) were determined. Reperfusion conditions were compared in the "preconditioned ischemic" heart where baseline contractile recovery during reperfusion with glucose as the sole exogenous substrate was 74 +/- 5% (n = 10). Contractile recovery was determined for reperfusion with pyruvate (14 +/- 2%, n = 10), pyruvate+glucose (23 +/- 4%, n = 10), deoxyglucose+acetate (25 +/- 4%, n = 10), and lactate+glucose (60 +/- 11%, n = 10). Contractile dysfunction could not be attributed to differences in high-energy phosphate contents. Elevated levels of intracellular Ca2+ during reperfusion were, however, correlated with poor contractile function. After 20 min of reperfusion, the mean time-averaged intracellular Ca2+ values, measured with 19F-nuclear magnetic resonance of 5-fluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded hearts, were 994 +/- 110 nM (glucose, n = 5), 2,270 +/- 494 nM (pyruvate, n = 5), 2,671 +/- 419 nM (pyruvate+glucose, n = 5), 2,382 +/- 480 nM (deoxyglucose+acetate, n = 5), and 1,019 +/- 33 nM (lactate+glucose, n = 5). These results are consistent with a beneficial role for glucose utilization during reperfusion, where enhanced recovery of contractile function and ionic homeostasis were observed.
Gastric H(+)-K(+)-ATPase activity is inhibited by reduction of disulfide bonds in beta-subunit
Authors: Chow D. C. , Browning C. M. , Forte J. G.
Published: December 2017

H(+)-K(+)-ATPase activity of rabbit isolated gastric microsomes was irreversibly inactivated by reducing agents, such as 2-mercaptoethanol and dithiothreitol. Similar to what has been observed for Na(+)-K(+)-ATPase, high concentrations of reagents, at moderately elevated temperatures, were required to inactivate H(+)-K(+)-ATPase, suggesting relative inaccessibility of the responsible disulfide bonds. Resistance against inactivation was conferred by monovalent cation activators of K(+)-stimulated ATPase and p-nitro-phenylphosphatase. The effectiveness of K+ congeners in protecting the enzyme was similar in sequence (Tl+ greater than K+ greater than Rb+) and concentration to their respective affinities for stimulating enzymatic activity, suggesting that the K(+)-bound form of the enzyme is more resistant to reduction than the free enzyme. Furthermore, Na+ antagonized the protective effect of K+. Labeling studies using fluorescein-maleimide indicated that 60-70% of the cysteine residues in the beta-subunit are in the oxidized form. Coupled with primary sequence data, this suggests that three disulfide bonds are present in the native beta-subunit. In contrast, less than 10% of the cysteine residues in the alpha-subunit are in the oxidized form. Kinetic studies showed that the 2-mercaptoethanol-induced loss of H(+)-K(+)-ATPase activity was correlated with a reduction of disulfide groups in the beta-subunit, while there was no significant change in the alpha-subunit. We conclude that reduction of disulfide bonds irreversibly inhibits H(+)-K(+)-ATPase activity, binding of K+ to the enzyme confers a resistance to disulfide bond reduction, and the responsible disulfide bonds are present in the beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
Ammoniagenesis in LLC-PK1 cultures: role of transamination
Authors: Gstraunthaler G. , Landauer F. , Pfaller W.
Published: December 2017

The LLC-PK1 renal epithelial cell line has been used as a model system to study renal ammoniagenesis and its regulation by metabolic acidosis in vitro. Experiments were performed on confluent LLC-PK1 epithelia grown for 10-14 days in conventional monolayer technique. After the medium pH was changed from 7.6 to 7.0 for 24-72 h by lowering the bicarbonate concentration in culture medium, LLC-PK1 cells responded with an adaptive increase in glutamine consumption and ammonia production. The rates of glutamine uptake and ammonia generation displayed a ratio of 1:1, i.e., 1 mol ammonia was produced per mole of glutamine consumed. Glutamine consumption and ammonia formation were paralleled by an equimolar production of L-alanine, indicating that transamination appears to be the main ammoniagenic pathway in LLC-PK1 cells. Analysis of the key enzymes of renal ammoniagenesis, phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GDH), revealed no changes in enzyme activities up to 72 h of adaptation. Alanine aminotransferase (ALT) activity in LLC-PK1 cells also remained unchanged during the adaptation period. Because transamination seems to play a crucial role in channeling the metabolic flux in LLC-PK1 ammoniagenesis, experiments were performed in which transamination was inhibited by (aminooxy)acetate (AOA). After incubation of control and pH 7.0-adapted LLC-PK1 cultures for 24-72 h in 0.2 mM AOA, no alanine production was found, but 2 mol of ammonia were formed per mole of glutamine consumed, again, without adaptive changes in PDG and GDH activities.(ABSTRACT TRUNCATED AT 250 WORDS)
Time course of sodium-induced Na(+)-K(+)-ATPase recruitment in rabbit cortical collecting tubule
Authors: Coutry N. , Blot-Chabaud M. , Mateo P. , Bonvalet J. P. , Farman N.
Published: December 2017

In cortical collecting tubules (CCD) of aldosterone-repleted rabbit kidney, an increase in intracellular sodium concentration (Nai) induces the recruitment and/or activation of latent Na(+)-K(+)-ATPase pumps (Blot-Chabaud et al., J. Biol. Chem. 265: 11676-11681, 1990). The present study was addressed to determine the time course of this Nai-dependent pump recruitment and to examine some of the factors possibly involved in this phenomenon. CCD from adrenalectomized rabbits complemented with aldosterone and dexamethasone were incubated at 4 degrees C either in a K(+)-free saline solution (Na(+)-loaded CCD) or in a sucrose solution (control CCD) and then rewarmed for various time periods to allow pump recruitment to occur. The number of pumps in the membrane was determined by specific [3H]ouabain binding; Nai was measured using 22Na. A rise in Nai induced a threefold increase in the number of basolateral pumps, which was fully achieved within 1-2 min. This pump recruitment was reversible within 15 min after restoration of low Nai. It was unaffected by inhibitors of cytoskeleton and Ca2+ ionophore A 23187. The blocker of the Na(+)-H+ antiporter, amiloride, did not prevent it. The protein kinase C activator, phorbol 12-myristate 13-acetate, did not induce it in the absence of Na+. We conclude that Nai is a major determinant of pump recruitment and/or activation, which occurs over a very short period of time. It may constitute a rapid adaptative response to an increase in the cell Na+ load.
Differences in regulation between nuclear and cytoplasmic Ca2+ in cultured smooth muscle cells
Authors: Himpens B. , De Smedt H. , Droogmans G. , Casteels R.
Published: December 2017

The free Ca2+ concentrations in the nucleus ([Ca2+]n) and cytoplasm ([Ca2+]c) of cultured smooth muscle cells were estimated using the fluorescent dye indo-1 and the ACAS 570 confocal laser microscope. In resting DDT1MF2 smooth muscle cells [Ca2+]n was found to be lower than [Ca2+]c. Both values increased transiently in response to histamine (100 microM), but during this stimulation [Ca2+]n exceeded [Ca2+]c. Maximal increase of [Ca2+]n was observed in the center of the nucleus, and a maximal increase of [Ca2+]c was observed in the immediate vicinity of the plasma membrane. A similar response was obtained with other agonists, such as carbachol or ATP. Comparable results with ATP were obtained in cultured aorta cells. The differential rise of [Ca2+]n over [Ca2+]c in DDT1MF2 cells did not occur during either spontaneous release of Ca2+ or Ca2+ release induced by caffeine (7.5 mM). The differential rise during histamine stimulation was abolished by the presence of the intercalating substance ethidium bromide. Thapsigargin, a presumed specific inhibitor of the endoplasmic reticulum Ca(2+)-Mg(2+)-adenosine-triphosphatase, abolished the Ca2+ gradient between nucleus and cytosol at rest. During subsequent histamine stimulation the Ca2+ increase was largely blocked in both compartments and attained similar levels. We propose that the lower value of [Ca2+]n at rest is dependent on an active Ca2+ extrusion system. The differential rise of [Ca2+]n over [Ca2+]c during agonist stimulation can be explained by an influx of Ca2+ from perinuclear stores and/or by a release of intranuclear Ca2+ possibly mediated by a process dependent on the inositol lipid metabolism.
Altered kinetics of AMP deaminase by myosin binding
Authors: Rundell K. W. , Tullson P. C. , Terjung R. L.
Published: December 2017

AMP deaminase catalyzes the deamination of AMP to inosine 5'-monophosphate (IMP) and ammonia. Factors controlling the enzyme in muscle can rapidly promote high rates of IMP formation when ATP utilization exceeds supply. We evaluated whether binding of AMP deaminase to myosin, which occurs during intense contraction conditions, alters the kinetic behavior of the enzyme. Reaction kinetics of myosin-bound and free AMP deaminase were evaluated. Reaction kinetics of the free enzyme yielded a near-linear double-reciprocal plot with an expected Km of approximately 1 mM AMP concentration (AMP). In contrast, reaction kinetics of AMP deaminase became bimodal when bound to myosin. At [AMP] less than 0.15 mM, a high-affinity Km (0.05-0.10 mM) with maximal velocity approximately 20% that of free enzyme was evident. At [AMP] greater than 0.15 mM, the Km and maximal velocity values were similar to that of the free enzyme. The 10- to 20-fold higher affinity Km would allow for a higher rate of AMP deamination at the low [AMP] found physiologically. AMP deaminase binding to myosin also induced a marked resistance to orthophosphate inhibition (10 mM) in the presence of 50 microM ADP. Results were similar for purified preparations of AMP deaminase bound to myosin subfragment 2 and crude extracts obtained from contracting muscle. Our results add further support to the hypothesis that AMP deaminase binding to myosin serves an important role in control of enzyme activity in contracting muscle.
Differential modulation of a sodium conductance in skeletal muscle by intracellular and extracellular fatty acids
Authors: Wieland S. J. , Fletcher J. E. , Gong Q. H.
Published: December 2017

Voltage-activated sodium channels of cultured skeletal muscle show diametrically divergent responses to intracellular vs. extracellular exposure to free fatty acids. Intracellular exposure to 1-20 microM arachidonic acid increased the magnitude of voltage-activated sodium currents, but not potassium currents, in whole cell recordings of human primary muscle cells and in the C2C12 mouse cell line. Oleic and stearic acids also stimulated increased sodium currents. In contrast, extracellular exposure to 5-10 microM arachidonic acid reversibly inhibited inward currents. Externally applied oleic acid was a less effective inhibitor, and stearic acid (up to 20 microM) produced no inhibition. The difference in sodium current responses to intracellular vs. extracellular exposure indicates that fatty acids can modulate skeletal muscle sodium channel function by at least two different pathways.
Acceleration of growth of cultured cardiomyocytes and translocation of protein kinase C
Authors: Allo S. N. , Carl L. L. , Morgan H. E.
Published: December 2017

Phorbol 12-myristate 13-acetate (PMA), norepinephrine (NE), and contraction stimulate cardiomyocyte growth (increased protein content). Differences exist in the time course and extent of protein and RNA accumulation. Cells plated at 4 x 10(6) cells/60-mm dish and arrested with 50 mM KCl demonstrated no significant growth. Treatment with PMA stimulated growth to a maximum of 17% at 48 h. In contrast, maximal stimulation of growth was 36% at 48 h and 31% at 72 h for contracting and NE-treated cells, respectively. Maximal stimulation of the capacity for protein synthesis (RNA content) was 32% for PMA-treated cells at 24 h compared with 59% and 77% for NE-treated and contracting cells, respectively, at 72 h. In support of a primary role for altered capacity in the regulation of protein synthesis, there was a significant correlation (r = 0.84) between RNA and protein contents that was independent of the stimulus used. Angiotensin II increased RNA content by 28% at 48 h but had no effect on growth up to 72 h. Growth stimulation and increased nuclear protein kinase C (PKC) activity were induced by contraction, NE, and PMA treatment and were inhibited by staurosporine (a PKC inhibitor), suggestive of a central role for PKC.
Hypoxia induces glucose transporter expression in endothelial cells
Authors: Loike J. D. , Cao L. , Brett J. , Ogawa S. , Silverstein S. C. , Stern D.
Published: December 2017

Endothelial cells in various tissues of the body are often exposed to hypoxic conditions. To examine the effects of sustained hypoxia on energy metabolism in endothelial cells, we have maintained bovine aortic and human umbilical vein endothelial cells in an atmosphere containing low oxygen concentrations (14 mmHg) for up to 96 h. We report here that endothelial cells maintained under these conditions upregulate their glucose transport activity, consume more glucose, and produce greater amounts of lactic acid than normoxic cells. Upregulation of glucose transport activity by hypoxic endothelial cells required several hours to occur, was associated with increased expression of mRNA and protein for the erythroid/brain form of the facilitative glucose transporter, and was not due to depletion of glucose from the medium. Prolonged treatment of endothelial cells with inhibitors or uncouplers of oxidative phosphorylation (antimycin, azide, dinitrophenol) under normoxic conditions also upregulated glucose transporter expression. These results suggest that reduced rates of oxidative metabolism may represent an important signal for cells to adapt metabolically to hypoxia. Furthermore, in our examination of endothelial cell energy metabolism, we discovered that endothelial cells contain phosphocreatine and express both the brain and muscle isozymes of creatine kinase.
ANP-(7-23) stimulates a DHP-sensitive Ca2+ conductance and reduces cellular cAMP via a cGMP-independent mechanism
Authors: Isales C. M. , Lewicki J. A. , Nee J. J. , Barrett P. Q.
Published: December 2017

Atrial natriuretic peptide (ANP) potently inhibits aldosterone secretion from the adrenal glomerulosa cell. In many tissues ANP action is associated with an increase in cellular guanosine 3',5'-cyclic monophosphate (cGMP) mediated through binding of the peptide to one of its receptors [ANP-A(R1)]. However, in the adrenal glomerulosa cell, the physiological significance of this rise in cGMP content has been contested. In an effort to determine whether non-cyclase-containing ANP receptors, such as ANP-C(R2), are linked to any of the events triggered by ANP binding, we utilized a truncated ANP analogue, ANP-(7-23), which at low doses exhibits selectivity for the ANP-C(R2) receptor. With the use of bovine adrenal glomerulosa cells, low concentrations (1 nM) of ANP-(7-23) failed to stimulate cGMP production, did not lower cytosolic calcium in the presence of low K+, and did not inhibit aldosterone secretion. At 1 nM, however, the analogue decreased cellular adenosine 3',5'-cyclic monophosphate content [8.27 +/- 0.51 vs. 6.74 +/- 0.09 (SE) pmol/10(6) cells; P less than 0.02] and, only in the presence of high extracellular [K+], increased cytosolic calcium. This ANP-induced rise in cytosolic calcium was abolished by the addition of a low dose (30 nM) of the dihydropyridine nitrendipine. ANP-(7-23) when utilized at a higher concentration (500 nM) lost its selectivity for the ANP-R2 receptor and increased cellular cGMP content (control, 0.27 +/- 0.02 vs. 500 nM ANP-(7-23), 0.448 +/- 0.02 pmol/10(6) cells; P less than 0.01). At 500 nM, ANP-(7-23) also inhibited aldosterone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
Amphibian ryanodine receptor isoforms are related to those of mammalian skeletal or cardiac muscle
Authors: Lai F. A. , Liu Q. Y. , Xu L. , el-Hashem A. , Kramarcy N. R. , Sealock R. , Meissner G.
Published: December 2017

The ryanodine receptor (RyR)-Ca2+ release channels of frog skeletal muscle have been purified as 30S protein complexes comprised of two high molecular weight polypeptides. The upper and lower bands of the frog doublet comigrated on sodium dodecyl sulfate polyacylamide gels with the mammalian skeletal and cardiac RyR polypeptides, respectively. Immunoblot analysis showed that a polyclonal antiserum to the rat skeletal RyR preferentially cross-reacted with the upper band, whereas monoclonal antibodies to the canine cardiac RyR preferentially cross-reacted with the lower band of the frog receptor doublet. Immunoprecipitation studies indicated the presence of two homooligomer 30S RyR complexes comprised of either the lower or upper polypeptide band of the frog doublet, and immunocytochemical staining revealed their colocalization in frog gastrocnemius muscle. After planar lipid bilayer reconstitution of the 30S frog RyR, single-channel currents were observed that exhibited a Na+ and Ca2+ conductance and pharmacological characteristics similar to those of the mammalian skeletal and cardiac Ca2+ release channels. These results suggest that amphibian skeletal muscle expresses two distinct RyR isoforms that share epitopes in common with the mammalian skeletal or cardiac RyR.
Maturational changes in respiratory control through creatine kinase in heart in vivo
Authors: Portman M. A. , Ning X. H.
Published: December 2017

The role of creatine kinase in regulation of myocardial respiration was studied in vivo as a function of maturation. Unidirectional creatine kinase flux (JCK), phosphocreatine to gamma-ATP, was measured in newborn lambs (age 3-9 days, n = 8) and mature sheep (age 30-60 days, n = 6) using 31P saturation transfer techniques, and total creatine kinase activity was measured using standard methods. Myocardial oxygen consumption (MVO2) was measured simultaneously via an extracorporeal shunt from the coronary sinus as cardiac work was increased via epinephrine (1-3 micrograms.kg-1.min-1). Findings were as follows: 1) baseline newborn JCK was markedly lower than in mature sheep despite higher levels of MVO2, and this could be related to a decrease in total creatine kinase activity; 2) JCK was substantially higher than the rate of ATP synthesis in both groups at baseline rates of oxygen consumption; and 3) JCK decreased significantly in newborns during increases in MVO2, whereas there was no change in flux rate in the mature sheep during even larger relative changes in work and oxygen consumption. These data imply that creatine kinase does not limit oxidative phosphorylation. However, this enzyme system probably maintains at least an indirect role in respiratory control that is a function of the myocardial developmental state.
Na+ alters the affinity for glucose and phosphate in rat renal brush-border membranes: a study of NMR relaxation rates
Authors: Barac-Nieto M. , Liu S. M. , Gupta R. K.
Published: December 2017

Transport of D-glucose (Glc) and Pi across renal microvilli brush-border membrane vesicles (BBMV) is coupled to Na+.Na+ may alter substrate binding and dissociation or the mobility of the translocator and its complexes. To evaluate substrate binding in NaCl or KCl solutions containing BBMV, we measured nuclear magnetic resonance transverse relaxation rates (1/T2) of [1-13C]Glc and 31P(i), as well as their temperature, magnetic field, and pH dependencies. The data indicate the following: 1) the alpha-anomer, but not the beta-anomer, of Glc binds to BBMV, more in the presence of Na+ than of K+; 2) interactions of P(i) with BBMV that increase its 1/T2 are significant in the presence of K+ but not of Na+; 3) temperature and magnetic field dependencies of the 1/T2 of P(i) bound to BBMV are consistent with more rapid exchange with free P(i) in the presence of Na+ than of K+; and 4) in the presence of Na+, [H+] promoted interactions of P(i) with BBMV that increase its 1/T2, probably by interfering with formation of rapidly dissociating Na(+)-P(i)-carrier complexes. In conclusion, Na+ promotes association of alpha-Glc to BBMV but promotes rapid exchange and dissociation of P(i) from BBMV. In both cases, Na+ effects coupling by altering the affinity of the cotransporters for the substrate.
Cystine dimethyl ester reduces the forces driving sodium-dependent transport in LLC-PK1 cells
Authors: Ben-Nun A. , Bashan N. , Potashnik R. , Cohen-Luria R. , Moran A.
Published: December 2017

Cystinosis is an inherited metabolic disease characterized by accumulation of lysosomal cystine and renal impairment. In an attempt to better understand the link between cystine accumulation and renal functions, we studied the effects of cystine loading on the Na(+)-H+ antiporter and the sodium pump in renal epithelial cells (LLC-PK1) in culture. Incubation of LLC-PK1 with 1 mM cystine dimethyl ester (CDME) for 48 h caused lysosomal cystine loading and reduced by 22 +/- 2% the maximal velocity of sodium-hydrogen antiport with no significant change in the affinity of sodium for the transporter. Rubidium influx decreased to 46 +/- 5% of control. Ouabain binding experiments revealed a 10% reduction in the number of Na(+)-K(+)-ATPase units in the intact cells. Na(+)-K(+)-ATPase activity in the particulate fraction of the cells homogenate declined to 50 +/- 7.5% of controls. No significant change was observed in the activity of ouabain-insensitive phosphatases. The intracellular concentration of sodium increased from 20.6 +/- 3.7 to 64.8 +/- 10 mM, and potassium concentration decreased from 103 +/- 6 to 80 +/- 13 mM. In addition to the observed reduction in the sodium gradient and in agreement with the reduction in the intracellular potassium concentration, the membrane potential changed from -80.8 +/- 7.5 to -69.9 +/- 7.0 mV. The results suggest that intracellular accumulation of cystine is associated with reduction in the number and the activity of membrane transporters. The consequence of the changes in the activity of Na(+)-K(+)-ATPase is a reduction in the electrochemical forces that drive transport in the renal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Conditional immortalization of bicarbonate-secreting intercalated cells from rabbit
Authors: Edwards J. C. , van Adelsberg J. , Rater M. , Herzlinger D. , Lebowitz J. , al-Awqati Q.
Published: December 2017

We have derived an immortalized cell line from primary cultures of bicarbonate-secreting intercalated cells from rabbit. Cells were transfected with a plasmid encoding a temperature-sensitive large T antigen of SV40 plus the neomycin resistance gene under the control of an SV40 promoter. Transfectants were selected for resistance to G418. One stably transfected clone, designated IC250, was subcloned to ensure clonality, and a subclone (clone C) was characterized in detail. The cells divide continuously at permissive temperature. At restrictive temperature, they cease dividing and assume morphological and transport properties of true bicarbonate-secreting intercalated cells. They express appropriate ultrastructural features, bind peanut lectin in an apical pattern, are rich in carbonic anhydrase, stain for proton-adenosinetriphosphatase in a basolateral pattern, and do not stain with antibodies to erythrocyte band 3. Most monolayers of transformed type B intercalated cells do not achieve a significant transepithelial resistance; those monolayers that are sufficiently electrically tight for electrophysiological studies are capable of chloride-dependent bicarbonate transport.