bluuebunny/crossref_metadata_2025 huggingface dataset:
Coordinate changes in C protein and myosin expression during skeletal muscle hypertrophy
Authors: McCormick K. M. , Baldwin K. M. , Schachat F.
Published: December 2017

In this study, two new C protein isoforms in adult rat skeletal muscle were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These isoforms migrated between previously identified fast (Cf) and slow (Cs) C protein isoforms; hence they were named intermediate C proteins (Ci1 and Ci2). Cyanogen bromide peptide mapping and Western blotting showed that the intermediate isoforms were more similar to Cs than Cf. The distribution of specific C protein and myosin heavy chain (MHC) isoforms was highly correlated in several hindlimb muscles, suggesting that the expression of these two thick-filament proteins is coordinated. This notion was tested by determining whether specific C protein and MHC isoforms change in parallel during muscle hypertrophy. Eight weeks after ablation of its synergists, the overloaded plantaris muscle showed significant increases in type IIa MHC and intermediate C protein, with corresponding decreases in type IIb MHC and Cf protein. These results indicate that C protein expression is linked to MHC expression during plantaris muscle hypertrophy.
Hypertonic upregulation of amino acid transport system A in vascular smooth muscle cells
Authors: Chen J. G. , Klus L. R. , Steenbergen D. K. , Kempson S. A.
Published: December 2017

The A10 line of vascular smooth muscle cells has Na+ dependent transport systems for alanine, proline, and Pi, whereas uptake of leucine, myo-inositol and D-glucose is Na+ independent. When A10 cells were incubated for 4 h in medium made hypertonic by addition of sucrose, there was a marked increase in Na(+)-dependent transport of alanine and proline but no change in Na(+)-dependent Pi uptake or Na(+)-independent uptake of leucine and inositol. Intracellular alanine content was increased 61% by the hypertonic treatment. Other nonpenetrating solutes, such as cellobiose and mannitol, reproduced the effect of sucrose, but urea, a penetrating solute, did not. Studies with 2-(methylamino)-isobutyric acid revealed that the upregulation by hypertonicity involved only system A. Increases in alanine and proline uptake also occurred after incubating the cells in isotonic medium containing 0.1 mM ouabain, suggesting that an increase in intracellular Na+ may be part of the intracellular signal for upregulation of system A. Hypertonic upregulation of Na(+)-dependent alanine transport occurred also in primary cultures of vascular smooth muscle cells. The response was blocked by actinomycin D and cycloheximide, indicating that gene transcription and protein synthesis play important roles in the mechanism leading to increased alanine uptake. We conclude that vascular smooth muscle cells, during prolonged hypertonic stress, activate system A and accumulate specific neutral amino acids which may act as organic osmolytes to help maintain normal cell volume.
Intercellular communication between mouse Leydig cells
Authors: Varanda W. A. , de Carvalho A. C.
Published: December 2017

In this paper we describe the basic features of gap junctions in pairs of Leydig cells mechanically dissociated from mouse testes, studied with the double whole cell patch-clamp technique. These cells are extensively coupled with regard to dye injection and electrophysiological measurements. The mean junctional conductance (gj) measured in 61 pairs of cells was 10.6 +/- 1.5 (SE) nS. In most pairs gj was voltage dependent when transjunctional voltage exceeded +/- 50 mV. On imposition of a voltage gradient across the junction the transjunctional current decayed exponentially to a lower level, with a time constant of 3.3 s at 50 mV and 430 ms at 100 mV. As in other systems, octanol (600 microM final concentration) uncoupled the cells within approximately 2 min. In a few cell pairs, gj was low enough to permit recording of single channel currents without the use of uncoupling agents. Single channel conductance fluctuations measured using pipettes containing potassium aspartate were distributed mainly around three peaks, at 21, 39, and 60 pS, suggesting the presence of channels formed by connexin 43. Western blots of Percoll gradient purified Leydig cells using specific antibodies indicate that connexin 43 is indeed expressed in these cells, whereas connexin 26 and connexin 32 are not.
Characterization of gap junctions between pairs of Leydig cells from mouse testis
Authors: Perez-Armendariz E. M. , Romano M. C. , Luna J. , Miranda C. , Bennett M. V. , Moreno A. P.
Published: December 2017

Leydig cells are coupled in vivo by numerous gap junctions. In vivo and in vitro cells were immunolabeled by connexin 43 (Cx43) but not by Cx26 or Cx32 antibodies; immunoblotting confirmed specificity of Cx43 labeling. Pairs of Leydig cells dissociated from mouse testis were studied by dual whole cell voltage clamp, and a high incidence of dye (n = 20) and electrical coupling (n = 60; > 90%) was found. Coupling coefficients were near 1 and junctional conductance (gj) averaged 7.2 +/- 1.2 nS (SE, n = 40). Large transjunctional voltage (Vj) decreased gj; currents decayed exponentially with time constants of seconds that decreased at greater Vj. The residual conductance at large Vj was at least approximately 40% of the initial conductance. Exposure of cell pairs to saline solutions saturated with CO2 (n = 15) or containing 2 mM halothane (n = 15) or 3.5 mM heptanol (n = 15) rapidly and reversibly reduced gj. In eight cell pairs, gating of single junctional channels was observed during halothane-induced reduction in gj. Most gating events at Vj < 40 mV were fit by a Gaussian distribution with a mean of approximately 100 pS. With Vj > 40 mV, smaller transitions of approximately 30 pS were also recorded, and the frequency and duration of the approximately 100-pS transitions decreased. Also, approximately 70-pS transitions between 30- and 100-pS conductances were observed in the absence of 70-pS transitions to or from the baseline, indicating that the 30-pS conductance was a substate induced by large Vj.
Mammalian alpha 1-subunit of Na(+)-K(+)-ATPase does not need its amino terminus to maintain cell viability
Authors: Shanbaky N. M. , Pressley T. A.
Published: December 2017

The amino terminus of the catalytic alpha-subunit from Na(+)-K(+)-adenosinetriphosphatase (ATPase) is not conserved among the various isoforms and species sequenced to date, yet it always includes a lysine-rich motif. To investigate the functional role played by this highly charged region, we altered the amino terminus of rat alpha 1 and evaluated the ability of the mutant to sustain cell viability. Nucleotide sequence encoding a 10-amino acid epitope from the human c-myc-oncogene product was substituted for the wild-type sequence encoding the first 31 amino acids of alpha 1. The chimeric cDNA containing the myc substitution was then introduced into ouabain-sensitive monkey kidney cells. Selection in ouabain produced viable colonies, suggesting that the introduced mutant was functional and conferred the ouabain-resistant phenotype of rats, despite the removal of the highly charged region from its amino terminus. Subsequent enzymatic analysis confirmed the presence of low-affinity binding sites for ouabain in the recipient colonies, and immunoblotting revealed the myc epitope on the expressed polypeptides in a membrane fraction. These results suggest that the first 31 amino acids are not required for function of alpha 1 and that the posttranslational cleavage associated with the amino terminus is unnecessary.
Delayed activation of single mechanosensitive channels in Lymnaea neurons
Authors: Small D. L. , Morris C. E.
Published: December 2017

Some stretch-activated (SA) channels challenged with suction jumps exhibit adaptation, a dynamic behavior that can be overlooked because of its mechanical fragility. In previous studies of neuronal SA K channels, we detected no adaptation, but the protocols used were not designed to detect dynamics. Here, we reproduce the adaptation seen by others in Xenopus SA cationic (Cat) channels but show that, with the same protocol, no adaptation occurs with SA K channels. Instead, SA K channels exhibit a different dynamic behavior, delayed activation. Lymnaea SA K channels subjected to pressure jumps responded after a 1- to 4-s delay with a gradual, rather than abrupt, onset of activation. The delay was pressure dependent and was longer for patches from older cultured neurons. Delayed responses were fragile like SA Cat channel adaptation; they disappeared with repeated stimuli. Cytochalasin D decreased the delay and increased the stretch activation of SA K channels. Unlike SA Cat channel adaptation, which occurs only at hyperpolarized potentials, SA K channel delay was not voltage dependent. We note that once SA Cat and SA K channels are "stripped" of their fragile (cytoskeleton-dependent?) dynamics, however, their gating behaviors show little fundamental difference; both are stretch activatable and have a higher open probability at depolarized potentials.
Elevated extracellular Mg2+ increases Mg2+ buffering through a Ca-dependent mechanism in cardiomyocytes
Authors: Koss K. L. , Grubbs R. D.
Published: December 2017

To investigate the physiological basis for the pharmacological action of extracellular magnesium (Mg2+O), cultured ventricular myocytes were exposed to either 0.8 mM (physiological) or 5.0 mM Mg2+ (therapeutic concentration) in the presence and absence of metabolic inhibitors. Metabolic inhibition, induced with 1 mM iodoacetate and 1 mM NaCN, was used to liberate Mg2+ from MgATP into the myoplasm, permitting examination of the role of elevated Mg2+O on myoplasmic Mg2+ buffering. The increase in Mg2+ activity observed in the presence of 5 mM Mg2+O was diminished compared with that observed in cells exposed to 0.8 mM Mg2+O. Furthermore, the increase in myoplasmic Mg2+ activity observed in the presence of 5 mM Mg2+O was identical to that reported previously in the absence of extracellular Ca2+. Fura 2 measurements of Ca2+ activity in these experimental conditions suggested that the Ca2+ permeability of cells conditions suggested that the Ca2+ permeability of cells exposed to 5 mM Mg2+ was less than that observed for cells exposed to 0.8 mM Mg2+. Using the Mg2+ buffer coefficient to quantitate cellular Mg2+ buffering, we observed a 63% increase in Mg2+ buffering in cells exposed to 5 mM Mg2+ compared with cells exposed to 0.8 mM Mg2+. This study demonstrates that elevated Mg2+O alters cardiomyocyte myoplasmic Mg2+ activity as the result of increased Mg2+ buffering through a Ca(2+)-sensitive mechanism.
Role of G proteins in shear stress-mediated nitric oxide production by endothelial cells
Authors: Kuchan M. J. , Jo H. , Frangos J. A.
Published: December 2017

Exposure of cultured endothelial cells to shear stress resulting from well-defined fluid flow stimulates the production of nitric oxide (NO). We have established that an initial burst in production is followed by sustained steady-state NO production. The signal transduction events leading to this stimulation are not well understood. In the present study, we examined the role of regulatory guanine nucleotide binding proteins (G proteins) in shear stress-mediated NO production. In endothelial cells not exposed to shear stress, AIF4-, a general activator of G proteins, markedly elevated the production of guanosine 3',5'-cyclic monophosphate (cGMP). Pretreatment with NO synthase inhibitor N omega-nitro-L-arginine completely blocked this stimulation. Incubation with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a general G protein inhibitor, blocked the flow-mediated burst in cGMP production in a dose-dependent manner. Likewise, GDP beta S inhibited NOx (NO2 + NO3) production for the 1st h. However, inhibition was not detectable between 1 and 3 h. Pertussis toxin (PTx) had no effect on the shear response at any time point. The burst in NO production caused by a change in shear stress appears to be dependent on a PTx-refractory G protein. Sustained shear-mediated production is independent of G protein activation.
Disruptive effects of protein on performance of liquid membrane-based ion-selective microelectrodes
Authors: Coombs H. V. , Miller A. J. , Sanders D.
Published: December 2017

The effects of addition of protein [bovine serum albumin (BSA)] to the calibration solutions of various liquid membrane-based ion-selective microelectrodes have been assessed. Protein at typically cytosolic levels of 100 mg/ml had no effect on the response of Na(+)-, K(+)-, Mg(2+)-, Ca(2+)-, H(+)-, NO3(-)-, and Cl(-)- selective microelectrodes at ion concentrations well above the respective detection limits. By contrast, this level of protein resulted in a pronounced increase in the detection limits of Na(+)-, K(+)-, Mg(2+)-, Ca2+, and Cl(-)-selective microelectrodes; H+ and NO3- microelectrodes were largely unaffected by the protein. The effect of BSA on detection limit is independent of pH and ionic strength, nor is it due to hysteresis, to binding of ions to the protein, or to the generation of junction potentials. More extensive studies on Na(+)-selective microelectrodes revealed that other proteins and large molecules (e.g., polyethylene glycol) also increase detection limits, with the extent of this effect apparently correlated positively with the molecular weight. Because biological macromolecules at physiological concentrations can influence the properties of alkali- and alkali earth cation-selective microelectrodes, care must be taken if the use of these electrodes entails cytosolic measurements close to their detection limits.
Control of intracellular pH during regulatory volume decrease in HL-60 cells
Authors: Hallows K. R. , Restrepo D. , Knauf P. A.
Published: December 2017

Intracellular pH (pHi) homeostasis was investigated in human promyelocytic leukemic HL-60 cells as they undergo regulatory volume decrease (RVD) in hypotonic media to determine how well pHi is regulated and which transport systems are involved. Cells suspended in hypotonic (50-60% of isotonic) media undergo a small (< 0.2 pH units), but significant (P < 0.05), intracellular acidification within 5 min. However, after 30 min of RVD, pHi is not significantly different from the initial pHi in 20 mM HCO3- medium and is significantly higher in HCO3(-)-free medium. Experiments performed in media with or without 150 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and HCO3- demonstrate that the anion exchanger (AE) mediates a net Cl- influx, with compensating HCO3- efflux, during RVD. To determine which transport systems are involved in counteracting this tendency toward acidification, we measured transport rates and examined the effect of transport system inhibitors on pHi. We found that inhibition of Na+/H+ exchange (NHE) with 12.5 microM ethylisoproplamiloride (EIPA) causes pHi to fall significantly by the end of 30 min of RVD. As assessed by EIPA-sensitive 22Na+ uptake measurements, NHE, largely dormant under resting isotonic conditions, becomes significantly activated by the end of 30 min of RVD, despite recovery of pHi and cell volume to near-normal levels. Thus a shift in the normal pHi dependence and/or volume dependence of NHE activity must occur during RVD under hypotonic conditions. In contrast, H(+)-monocarboxylate cotransport appears to play only a supportive role in pH regulation during RVD, as indicated by lack of stimulation of [14C]lactate efflux during RVD.
Na(+)-coupled alanine transport in LLC-PK1 cells
Authors: Kimmich G. A. , Randles J. , Wilson J.
Published: December 2017

Transport of alanine (Ala) was characterized in LLC-PK1 renal epithelial cells. Transport capability for Ala falls by 75% in postconfluent cultures, while Na(+)-coupled alpha-methylglucoside (AMG) transport rises more than fourfold during the same interval. The kinetics of Ala transport were characterized in ATP-depleted cells to allow experimental imposition of changes in Na+ gradient and control of membrane potential across the plasma membrane. At 100 microM Ala and 135 mM Na+, > 98% of the unidirectional Ala influx is dependent on the presence of Na+ in cells from postconfluent cultures. Li+ is only 1% as effective as Na+, and other monovalent cations are ineffective in supporting Ala uptake. alpha-(Methylamino)isobutyric acid (MeAIB; 5 mM) causes only a small inhibition (approximately 10%) of 100 microM Ala influx. The low selectivity for Li+; low sensitivity to competition by MeAIB or aminoisobutyric acid; pronounced inhibition by serine, homoserine, cysteine, homocysteine and threonine; moderate inhibition by valine, isoleucine, proline and histidine; and lack of inhibition by lysine, arginine, and aspartate are more consistent with those characteristics reported for entry via the ASC amino acid transport system rather than those associated with the A system. Alanine influx exhibits a hyperbolic relationship with increasing Ala or Na+ concentration. Kinetic analysis suggests a single transport pathway with a Michaelis constant (Km) for alanine of 380 microM (when Na+ is 135 mM), apparent Km for Na+ of 32 mM (with 100 microM Ala), and a maximum velocity of 7 nmol.min-1.mg cell protein-1. An interior-negative diffusion potential induces a similar enhancement of [14C]alanine or [14C]tetraphenylphosphonium influx (approximately 40%). In contrast, AMG influx is enhanced by a factor of 2.2 under the same conditions. AMG uptake also shows a sigmoidal relationship with Na+ concentration. Hill coefficients are 1.56 for AMG and 1.0 for alanine. Direct measurement of Na(+)-Ala coupling stoichiometry yields a value of 1.01 +/- 0.07. Under the same conditions, Na(+)-AMG coupling stoichiometry is 2.1 +/- 0.25. The difference in coupling stoichiometries provides an explanation for differences in intensity of interaction between Na(+)-coupled transport systems for sugars and amino acids.
Insulin activates furosemide-sensitive K+ and Cl- uptake system in BC3H1 cells
Authors: Weil-Maslansky E. , Gutman Y. , Sasson S.
Published: December 2017

Insulin augments the activity of Na(+)-K(+)-adenosinetriphosphatase (ATPase) in skeletal muscles. This study shows that when furosemide- and bumetanide-inhibitable 86Rb+ uptake is measured in the skeletal muscle-like BC3H1 cell line, insulin and insulin-like growth factor I (IGF-I) activate a loop diuretic-sensitive K+ and Cl- transport system but have no effect on Na(+)-K(+)-ATPase. The insulin-stimulated K+ transport system is extracellular Na+ concentration ([Na+]o) independent and extracellular Cl- concentration ([Cl-]o) dependent. Na(+)-independent K(+)-Cl- cotransport systems have been identified in other cells, but their sensitivity to insulin or growth factors has not been described. The affinities of the insulin-stimulated K+ uptake in BC3H1 cells for K+ (0.9 +/- 0.1 mM) and loop diuretics (5.9 x 10(-7) and 10(-7) M for furosemide and bumetanide, respectively) are higher than those of K(+)-Cl- cotransporters in other cells. Thus the insulin-stimulated K+ and Cl- transport system in BC3H1 seems kinetically different from K(+)-Cl- cotransporters in other cells. Insulin and IGF-I may activate a unique K(+)-Cl- cotransporter or activate a [Na+]o-independent K(+)-Cl- cotransport mode of Na(+)-K(+)-Cl- cotransporter in BC3H1 cells.
Na(+)-dependent and Na(+)-independent systems of choline transport by plasma membrane vesicles of A549 cell line
Authors: Kleinzeller A. , Dodia C. , Chander A. , Fisher A. B.
Published: December 2017

Membrane vesicles of A549 lung cells accumulate choline by two pathways: the Na(+)-independent uphill uptake of choline [Michaelis-Menten constant (Km) approximately 44 microM; steady-state gradient approximately 45 at 5 microM external choline] is dependent on a transmembrane H+ gradient, is relatively insensitive to hemicholinium-3, is amiloride sensitive, and is abolished by valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The Na(+)-dependent active choline uptake (Km approximately 4 microM, inhibitor constant for hemicholinium-3 approximately 0.1 microM), is specific for Na+, is amiloride and FCCP sensitive, and is electrogenic: the overshoot using K(+)-loaded vesicles and NaCl gradient was increased by valinomycin. The time of the overshoot peak, T was approximately 90 s in a NaSCN medium (or in presence of other lipid-soluble anions), a value close to that for alpha-aminoisobutyrate as substrate (T = approximately 1.5 min). T was lengthened in NaCl medium to approximately 10 min, and the overshoot was abolished by impermeant anions. External Cl- is not required for the choline uptake: valinomycin produced an overshoot in the presence of only impermeant anions, with T approximately 90 s. Most of the above properties are shared by the high-affinity Na(+)-dependent choline transport in synaptosomes. The characteristics of the Na(+)-dependent choline uptake by membrane vesicles of A549 cells are consistent with an electrogenic choline(+)-Na+ cotransport, with the rate-limiting anion (e.g., Cl-) influx balancing the positive charges transferred into the vesicles. The data are also consistent with an involvement of an amiloride-sensitive choline+/H+ antiport (or choline(+)-OH- symport) in the low- and high-affinity choline uptake pathways.
Fast sodium currents induced by serum in human uterine leiomyosarcoma cells
Authors: Kusaka M. , Sperelakis N.
Published: December 2017

We previously demonstrated that fast Na+ channels are expressed in uterine smooth muscle cells during pregnancy [Y. Ohya, and N. Sperelakis. Am. J. Physiol. 257 (Cell Physiol. 26): C408-C412, 1989; Y. Inoue, and N. Sperelakis. Am. J. Physiol. 260 (Cell Physiol. 29): C658-C663, 1991]. In the present study, we investigated the possible expression of fast Na+ channels and resultant fast Na+ current [INa(f)] using whole cell voltage clamp in cultured uterine leiomyosarcoma cells (SK-UT-1B), which are derived from human myometrium. The expression of INa(f) was dependent on the concentration of fetal bovine serum (FBS) in the culture medium. The mean current densities of peak INa(f) in cells incubated with 0, 1, 5, and 10% FBS were (in pA/pF) 1.3 +/- 0.5, 1.6 +/- 0.9, 9.8 +/- 2.4, and 9.8 +/- 1.7, respectively. Two types of INa(f) were identified based on different time courses of current decay. Both types of current were equally tetrodotoxin sensitive (half-maximal inhibitory concentration approximately 50 nM) and had similar electrical properties. These characteristics are consistent with those in the pregnant myometrial cells, yet the current density in the sarcoma cells is much higher. These findings suggest that one or more factors in serum induce INa(f) in the uterine sarcoma cells, and this cell line could be a good model for the study of the fast Na+ channels in uterine smooth muscle.
Effects of prostaglandin E2 on amiloride-blockable Na+ channels in a distal nephron cell line (A6)
Authors: Kokko K. E. , Matsumoto P. S. , Ling B. N. , Eaton D. C.
Published: December 2017

We studied the mechanisms by which prostaglandin E2 (PGE2) regulates amiloride-blockable 4-pS Na+ channels in A6 distal nephron cells. With each apical cell-attached patch acting as its own control, acute (3-6 min) basolateral, but not apical, exposure to 1 microM PGE2 inhibited Na+ channel activity by decreasing the open probability (Po). This PGE2-induced inhibition was attenuated by 30 min pretreatment with the protein kinase C (PKC) antagonists 1 microM staurosporine or 100 microM D-sphingosine but was insensitive to pertussis toxin (PTX). Furthermore, the time course for channel inhibition by acute PGE2 correlated with a transient increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels. In contrast, after chronic (10-50 min) exposure of A6 cells to 1 microM basolateral PGE2, channel activity was stimulated compared with controls. This stimulation was due to an increase in the number of apical Na+ channels, similar to the effect of maneuvers that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels in A6 cells (22). Indeed, chronic exposure to basolateral PGE2 correlated with a sustained increase in cAMP levels. In conclusion, 1) the regulation of apical 4-pS highly selective Na+ channel activity by basolateral PGE2 is a complicated biphasic process, which includes inhibition by acute PGE2 and stimulation by chronic PGE2 exposure; 2) acute PGE2 promotes a transient generation of IP3 which activates Ca(2+)-dependent PKC and promotes a decrease in Po; 3) chronic PGE2 promotes a sustained generation of cAMP that leads to an increase in channel density; and 4) both the acute and chronic effects of PGE2 on Na+ channels are PTX-insensitive processes.
Calcium-activated sodium and chloride fluxes modulate platelet volume: role of Ca2+ stores
Authors: Fine B. P. , Marques E. S. , Hansen K. A.
Published: December 2017

An increase in cytosolic ionized Ca2+ concentration ([Ca2+]i) initiates volume changes in various types of cells. In response to increases in [Ca2+]i most cell types contract by efflux of K+ and Cl-, whereas platelets expand in response to rises in [Ca2+]i. This study examined the importance of the source of Ca2+, the flux of ions responsible for the volume change, and the role of Ca(2+)-dependent protein kinases in regulating these ionic fluxes. The baseline platelet volume was independent of extracellular Ca2+ but when stimulated by the Ca2+ ionophore A-23187 (50 nM) the volume increased in both the presence and absence of extracellular Ca2+ (1.18 +/- 0.08 vs. 0.83 +/- 0.06 fl, respectively). The increased volume was caused by the gain of Na+ and Cl-. Na+ entered through both conductive and nonconductive (Na+/H+ exchange) pathways, whereas the influx of Cl- was conductive and inhibited by the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The Ca(2+)-induced volume change was blocked by both calmodulin and protein kinase inhibitors. Thus the activation of Ca(2+)-dependent protein kinases promotes platelet swelling by stimulating Na+ and Cl- influx.
Permeability properties of the mammalian bladder apical membrane
Authors: Chang A. , Hammond T. G. , Sun T. T. , Zeidel M. L.
Published: December 2017

The luminal surface of mammalian bladder is exposed to urine with a composition widely different from that of plasma that bathes the basolateral surface of epithelium. Therefore we predict that the bladder permeability barrier, which is likely located in the apical membrane (AM), will exhibit low permeabilities to water, urea, NH3, H+, and small nonelectrolytes. AM surface area increases as the bladder fills with urine and decreases during emptying, a process that involves cyclical endocytosis and reinsertion of membrane from a pool of AM endosomes (AME). Rigid-appearing plaques composed of three proteins, uroplakins, have been identified and occupy 70-90% of AM surface area. To determine permeability properties of the AM permeability barrier, we purified AME and measured their permeabilities. Rabbit urinary bladders were removed, and their apical surface was exposed to carboxyfluorescein (CF) or horseradish peroxidase (HRP). Exposure to hypotonic and then isotonic basolateral solutions induced endocytosis of luminal CF or HRP into AME. Electron microscopy of bladders after this treatment revealed HRP entrapped within AME bordered by plaques. AME were purified by differential and sucrose-gradient centrifugation, and CF-containing AME were purified 17.0 +/- 3-fold (SD) with respect to homogenate. Analysis of purified AME by flow cytometry showed that > 95% of vesicles contained CF entrapped from luminal solution and were selectively labeled with anti-uroplakin antibody. AME osmotic water permeability averaged 2.3 +/- 0.66 x 10(-4) cm/s and exhibited a high activation energy, indicating that AM contains no water channels. Permeability to urea and NH3 averaged 7.8 +/- 3.7 x 10(-7) and 1.5 +/- 0.3 x 10(-3) cm/s, respectively, which are exceptionally low and similar to permeabilities of other water-tight membranes, including toad urinary bladder and gastric mucosa. AME behaved as a single population in all permeability studies, which will permit future characterization of protein and lipid structure responsible for these unique permeability properties.
Antidiuretic hormone and exocytosis: lessons from neurosecretion
Authors: Hays R. M. , Franki N. , Simon H. , Gao Y.
Published: December 2017

Many cells, both single and epithelial, are programmed for exocytosis. In most cases, the contents of cytoplasmic vesicles are delivered rapidly and directly to the extracellular fluid. The process has been intensively studied in the chromaffin cell and the nerve terminal, where, as in other cells, exocytosis is under a complex type of cytoskeletal control. An array of vesicle-associated proteins mediates attachment of the vesicles to the cytoskeleton, their release, and their fusion with the plasma membrane. Two functional pools of vesicles, the releasable and reserve pool, carry out immediate and long-term secretory activity. Some of the mediators of neurotransmitter vesicle fusion, originally thought to be restricted to neurosecretory cells, have now been found in nonneuronal cells. The mammalian collecting duct and the amphibian bladder are also engaged in exocytosis. In both epithelia, antidiuretic hormone (ADH) induces the transfer of water channels from cytoplasmic vesicles to the apical cell membrane. The process is slower than in the nerve terminal and ends with channel placement rather than the extrusion of vesicular contents. Nevertheless, there are several respects in which cytoskeletal control, vesicle positioning in the cell, docking, and fusion may prove to resemble the events in neurosecretion. This review begins with a survey of cytoskeletal structure and function in the erythrocyte, the chromaffin cell, and the nerve terminal and then presents current studies of ADH-induced exocytosis, emphasizing common themes in cytoskeletal control.
IKir regulation in murine macrophages: whole cell and perforated patch studies
Authors: Judge S. I. , Montcalm-Mazzilli E. , Gallin E. K.
Published: December 2017

Previous studies have reported that the inwardly rectifying K+ conductance (GKir) in macrophages is modulated by intracellular perfusion with inositol 1,4,5-triphosphate (InsP3), inositol 1,3,4,5-tetrakisphosphate (InsP4), or GTP analogues and by exposing cells to macrophage-specific colony-stimulating factor (CSF) I. This study uses both conventional whole cell and amphotericin B perforated patch studies to investigate GKir modulation in thioglycollate-elicited mouse peritoneal macrophages (MO). Under whole cell recording conditions with 150 mM Cl- in the patch pipette, GKir decreased within 25 min. The GKir decrease was slowed by exchanging glutamate for Cl- as the major anion in the pipette or by adding guanosine 5'-O-(2-thiodiphosphate) (50 nM) or ATP (0.5 mM) to the pipette. Addition of InsP3 or InsP4 to the pipette had no effect on the magnitude of GKir or its rate of decrease but activated an outward current in the voltage range of +60 to +120 mV in 57% of the cells studied. Thus in murine MO GKir may be modulated by G proteins but is unaffected by inositol phosphate metabolites, which have been reported to enhance GKir in phorbol 12-myristate 13-acetate (PMA)-differentiated HL-60 cells. In contrast to whole cell studies, perforated patch recordings of murine MO GKir were stable for > 1 h. Perforated patch studies demonstrated that murine MO also differ from PMA-differentiated HL-60 cells in that CSF I had no effect on GKir. Additionally, arachidonic acid, PMA, and H2O2, agents implicated in macrophage activation, did not modulate GKir. We conclude that GKir regulation in murine MO differs from that reported in PMA-differentiated HL-60 cells and that although our data suggest that GKir is modulated by G protein(s), they differ from the G proteins involved in MO responses to CSF I and the other agents tested.
Osmotic activation of a Na(+)-dependent Cl-/HCO3- exchanger
Authors: Reusch H. P. , Lowe J. , Ives H. E.
Published: December 2017

In many systems, osmotically induced cell shrinkage activates the Na+/H+ exchanger. To assess the role of H(+)-extruding transporters in the response to osmotic shrinkage in vascular smooth muscle (VSM) and Chinese hamster ovary (CHO) cells, intracellular pH (pHi) was measured with 2',7'-bis(carboxy-ethyl)-5(6)- carboxyfluorescein-acetoxymethyl ester (BCECF-AM) after exposing cells to hypertonic medium. In nominally HCO(3-)-free medium, addition of 200 mM sucrose caused pHi to increase 0.33 pH unit on average in VSM cells but only 0.13 pH unit in CHO cells. Permeant solutes failed to increase pHi significantly. Cytochalasin B (1-20 microM), colchicine (1-10 microM), Ca2+ removal, and downregulation of protein kinase C activity did not affect osmotic activation of H+ extrusion in either cell type. Additional work was carried out to determine why osmotic activation of H+ extrusion was less in CHO than in VSM cells. In CHO cells, the osmotically induced delta pHi was only weakly sensitive to amiloride, suggesting that osmotic forces may activate an H+ transport system other than Na+/H+ exchange. In the presence of 10 mM HCO3-, osmotically induced delta pHi decreased by 60% in VSM cells but increased by 50% in CHO cells compared with the delta pHi in HCO(3-)-free medium. Lastly, removal of extracellular Cl- did not affect osmotically induced delta pHi in VSM cells but completely abolished the response in CHO cells. We conclude that in VSM cells osmotically induced changes in pHi are mediated by Na+/H+ exchange, whereas in CHO cells they are most likely mediated by a Na(+)-dependent Cl-/HCO3- exchanger.
Corticoid-induced differentiation of amiloride-blockable active Na+ transport across larval bullfrog skin in vitro
Authors: Takada M. , Yai H. , Takayama-Arita K.
Published: December 2017

The hormone-induced differentiation of an active Na+ transport across larval bullfrog skin during metamorphosis was investigated in vitro and in vivo. In in vitro experiments, EDTA-treated larval dorsal skin from which apical cells were removed was used. Even in the absence of thyroid hormone, corticoids induced the differentiation. Although aldosterone was the most potent hormone, hydrocortisone or corticosterone was also effective. Prolactin inhibited the corticoid-induced differentiation. The differentiation of the transport system coincided almost exactly with the appearance of adult features of the epidermis, namely, the epidermis at 7 days carried the human blood group antigen A, a specific molecular marker of adult-type bullfrog epidermis. The transport system appeared to develop in cells that had been newly generated from basal cells. On the contrary, in in vivo experiments, the effect of amiloride on the short-circuit current of the skin of tadpoles raised in the presence of aldosterone was very small, suggesting that a mechanism exists to inhibit the ability of aldosterone to induce the differentiation of the transport system in vivo.
Sarcoplasmic reticulum gene expression in pressure overload-induced cardiac hypertrophy in rabbit
Authors: Matsui H. , MacLennan D. H. , Alpert N. R. , Periasamy M.
Published: December 2017

Pressure overload (PO)-induced cardiac hypertrophy in rabbits has been utilized extensively to study alterations in systolic and diastolic functions of the heart. In earlier studies we showed that the levels of mRNA encoding two important sarcoplasmic reticulum (SR) proteins, the cardiac/slow-twitch muscle Ca(2+)-ATPase (SERCA2a) and phospholamban, were decreased in PO rabbit hearts. In this study, we analyzed the expression of the Ca(2+)-release channel (ryanodine receptor), calsequestrin, SERCA2a, and phospholamban in PO-induced cardiac hypertrophy after 2, 4, 8, and 16 days of pulmonary artery banding. Northern blot and slot blot analyses showed that the steady-state level of mRNA encoding the cardiac ryanodine receptor, SERCA2a, and phospholamban was decreased significantly as early as 2 days after PO. In 16-day PO hearts, SERCA2a mRNA was reduced to 7.9 +/- 3.4% (P < 0.05), phospholamban mRNA was reduced to 15.9 +/- 6.5% (P < 0.05), and ryanodine receptor mRNA was reduced to 49.2 +/- 23.6% (P < 0.05). In this study, calsequestrin mRNA levels were also reduced to 29.9 +/- 15.2% by day 16 (P < 0.05). ATP-dependent Ca2+ uptake was reduced to 78% (P < 0.05); in contrast, the steady-state formation of ATPase phosphoenzyme was reduced to 81% of control (P < 0.05) and Ca(2+)-ATPase protein was reduced to 78% of control (P < 0.05) in crude SR vesicles or total muscle homogenate obtained from 16-day PO hearts. On the basis of these data, we propose that decreases in the expression of SR proteins may contribute to dysfunctions seen in systolic and diastolic properties of the hypertrophied myocardium.
Regulatory volume decrease in frog retinal pigment epithelium
Authors: Adorante J. S.
Published: December 2017

To measure changes in cell water during cell volume regulation, retinal pigment epithelial cells were loaded with tetramethylammonium (TMA). Regulatory volume decrease (RVD) in TMA-loaded retinal pigment epithelial (RPE) cells was measured using double-barreled K(+)-specific microelectrodes. Hyposmotic removal of 12.5 mM NaCl from the apical bath caused bullfrog RPE cells to rapidly swell by approximately 10% and to recover to control level within 10-15 min. Hyposmotic RVD was inhibited by 5 mM basal but not apical BaCl2. Raising K+ in the basal bath from 2 to 12 mM also inhibited RVD. Hyposmotic swelling was accompanied by an increase in the ratio of apical to basolateral membrane resistance (Ra/Rb). The swelling-induced increase in Ra/Rb was inhibited by 5 mM BaCl2. Together, the above findings suggest that hyposmotic swelling enhances basolateral K+ conductance such that K+ and presumably anion efflux mediate net solute and water loss during RVD. RPE cells can also regulate their volume when swollen in isosmotic Ringer solution under certain conditions. When urea or apical HCO3- was used to induce cell swelling, RPE cells underwent an RVD. In contrast, isosmotic elevation of apical K+ from 2 to 5 mM resulted in an increase in RPE cell volume with no subsequent RVD. Thus the method used to swell RPE cells is an important determinant of RVD. Because changes in RPE cell volume in vivo may alter the volume and composition of the extracellular (subretinal) space surrounding the photoreceptors, isosmotic volume regulation may play an important physiological role in maintaining the integrity and health of the neural retina under normal and pathophysiological conditions.
Iodoacetate-induced skeletal muscle contracture: changes in ADP, calcium, phosphate, and pH
Authors: Ruff R. L. , Weissman J.
Published: December 2017

The effects of iodoacetic acid (IAA) and ischemic contraction were studied in rat extensor digitorum longus muscles. Ischemic stimulation of IAA-treated muscles produced contracture. We measured total muscle water content, distribution of water between intracellular and extracellular spaces, creatine concentration ([Cr]), creatine phosphate concentration ([PCr]), [ATP], [Pi], intracellular pH, and intracellular Ca2+ concentration ([Ca2+]i) at the onset of contracture. [ADP] was calculated from the equilibrium of the creatine kinase reaction using the measured values of [ATP], [PCr], [Cr], and pH. At the onset of contracture there was a 75% reduction of [PCr], a 12-fold increase in [ADP], and an 11-fold increase in [Ca2+]i compared with unstimulated IAA-treated muscles. [ATP] was not depleted at contracture compared with unstimulated IAA-treated muscles, and [Pi] increased less in muscles at contracture compared with stimulated control muscles. The persistent tension in contractures probably resulted from increased [Ca2+]i combined with increased myofibrillar Ca2+ sensitivity due to elevated [ADP] and relatively reduced intracellular acidification and [Pi].
Subcellular localization of PEPCK and metabolism of gluconeogenic substrains of renal cell lines
Authors: Holcomb T. , Curthoys N. P. , Gstraunthaler G.
Published: December 2017

The two gluconeogenic substrains of renal epithelial cells, LLC-PK1-FBPase+ and OKGNG+, have been shown to differ markedly in their metabolism of lactate and pyruvate. OKGNG+ cells consumed lactate as well as pyruvate at high rates in contrast to LLC-PK1-FBPase+ cells, which failed to take up or utilize lactate. (Aminooxy)acetate (AOA), an inhibitor of transamination reactions, was used to further delineate these differences. Lactate consumption of OKGNG+ cells was significantly inhibited by AOA, whereas pyruvate consumption by LLC-PK1-FBPase+ cells was slightly stimulated. Growth of OKGNG+ cultures, however, could be achieved on lactate in the presence of AOA. From these results it was concluded that the cell strains might differ in the subcellular distribution of phosphoenolpyruvate carboxykinase (PEPCK). LLC-PK1-FBPase+ cells may express both mitochondrial and cytosolic PEPCK isoenzymes, whereas OKGNG+ cells express only the mitochondrial isoenzyme. This was tested by directly assaying PEPCK activity in subcellular fractions of the cells. In OKGNG+ cells PEPCK activity fractionated with the mitochondrial marker glutamate dehydrogenase; however, in LLC-PK1-FBPase+ cells two-thirds of PEPCK activity was found in the cytosol. In LLC-PK1-FBPase+ cells, PEPCK activity increased twofold on incubation in acidic culture medium (pH 6.9) for 18 h, in contrast to the PEPCK activity in OKGNG+ cells. Northern blot analysis using cDNA probes specific for the mitochondrial and cytosolic PEPCK mRNAs confirmed the enzyme activity data. In LLC-PK1-FBPase+ cells strong expression of cytosolic PEPCK mRNA was observed, whereas in OKGNG+ cells only very low levels could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)
Acute cell volume changes in anisotonic media affect F-actin content of HL-60 cells
Authors: Hallows K. R. , Packman C. H. , Knauf P. A.
Published: December 2017

Page C1160: K. R. Hallows, C. H. Packman, and P. A. Knauf.“Acute cell volume changes in anisotonic media affect F-actin content of HL-60 cells.” The acknowledgments should include the following sentence: K. R. Hallows is a student in the Medical Scientist Training Program funded by National Institute of General Medicine Sciences Grant T32 GM-07356.
Intracellular pH modulates the activity of chloride channels in isolated lacrimal gland acinar cells
Authors: Park K. , Brown P. D.
Published: December 2017

The effects of intracellular pH (pHi) on Ca(2+)-activated Cl- currents in rat lacrimal gland acinar cells were examined. Cl- currents were recorded by conventional whole cell patch-clamp methods using K(+)-free and Na(+)-free solutions. pHi was varied by using electrode solutions with pH at 6.8, 7.3, or 7.8, and Ca2+ activity was buffered at 100 nM with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Increasing pH from 6.8 to 7.8 was found to increase whole cell currents. The currents observed exhibited time-dependent activation at depolarizing potentials and time-dependent inactivation at hyperpolarizing potentials (pH 7.8). This behavior is characteristic of Ca(2+)-activated Cl- channels in lacrimal gland cells. The selectivity of the current was examined at pH 7.8 by removing Cl- from the bath solution. This maneuver caused a positive shift in the reversal potential, as expected for a Cl(-)-selective current. Thus increasing pHi appears to activate Ca(2+)-activated Cl- channels. The possibility that an increase in pHi may help sustain Cl- channel activity during secretory activity is discussed.
Creatine kinase is required for swelling-activated K-Cl cotransport in dog red blood cells
Authors: Colclasure G. C. , Parker J. C. , Dunham P. B.
Published: December 2017

K-Cl cotransport in resealed dog red cell ghosts requires the incorporation of creatine phosphate before resealing; incorporation of ATP has no effect [Colclasure and Parker. Am. J. Physiol. 265 (Cell Physiol. 34): C1648-C1652, 1993]. A role for creatine kinase (CK) in swelling-activated K-Cl cotransport was investigated. 2,4-Dinitrofluorobenzene (DNFB), an inhibitor of CK, inhibited K-Cl cotransport in intact red blood cells and resealed ghosts from DNFB-treated cells. Incorporation of exogenous CK into ghosts of DNFB-treated cells restored K-Cl cotransport. Therefore DNFB inhibits CK and not the cotransporter. Inhibition of native CK in ghosts by DNFB and the incorporation of CK into the ghosts were demonstrated in electrophoretic gels. In a dose-response experiment, approximately 770 molecules CK/ghost restored 50% of control cotransport. Since creatine phosphate is a substrate only for CK, CK provides ATP to a site inaccessible to cytoplasmic ATP. The nature of this site and its role in K-Cl cotransport are uncertain, but an essential function for CK is established.
Vesicle fusion proteins in rat inner medullary collecting duct and amphibian bladder
Authors: Franki N. , Macaluso F. , Gao Y. , Hays R. M.
Published: December 2017

The delivery of water channels to the apical membrane in response to antidiuretic hormone (ADH) requires the targeting of channel-containing vesicles to specific sites in the membrane, followed by fusion and exocytosis. A complex array of proteins is now believed to mediate targeting and fusion in eukaryotic cells. They include N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAP), and cellubrevin, a vesicle-associated protein present in the nerve terminal. We asked whether these proteins are in epithelial cells of rat inner medullary collecting duct (IMCD) and amphibian bladder. Immunoblots on both tissues showed the presence of NSF and alpha-SNAP. Cellubrevin was present in immunoblots of the IMCD, but not the bladder. Immunogold electron microscopy showed NSF, alpha-SNAP, and cellubrevin in rat IMCD cells, with vesicular labeling. In the bladder, NSF was seen on vesicles and aggrephores. We conclude that components of the vesicle-targeting and fusion systems are present in kidney and amphibian bladder and may mediate a wide variety of fusion events, including those initiated by ADH.
Increase in [Ca2+]i by CCh in adult rat sympathetic neurons are not dependent on intracellular Ca2+ pools
Authors: Foucart S. , Gibbons S. J. , Brorson J. R. , Miller R. J.
Published: December 2017

We have examined the effects of the muscarinic agonists, carbachol (CCh) and oxotremorine (Oxo), on the intracellular free Ca2+ concentration ([Ca2+]i) in acutely dissociated sympathetic neurons from adult rats using fura 2-based microfluorometry. The drugs increased [Ca2+]i by 86 +/- 7 and 38 +/- 10 nM for CCh and Oxo, respectively (both 10 microM). Basal [Ca2+]i was 52 +/- 3 nM. Depletion of the caffeine-sensitive Ca2+ store or blockade of the Ca(2+)-adenosinetriphosphatase with thapsigargin did not alter the effect of either agonist on the rise in [Ca2+]i. On the other hand, the omission of Ca2+ from the perfusion solution or the use of TA-3090, a Ca2+ channel antagonist, blocked the effects of CCh and Oxo. In whole cell current-clamp recordings, the muscarinic agonists elicited a depolarization and action potential firing, which probably explained the rise in [Ca2+]i observed with microfluorimetric recording. In addition to their direct effects on the [Ca2+]i, muscarinic agonists also reduced the rise in [Ca2+]i induced by a nicotinic agonist. This inhibitory effect, observed in 68% of cells that responded to the nicotinic agonist, was blocked by atropine and pertussis toxin, whereas the muscarinic agonist-induced increase in [Ca2+]i was blocked by atropine but was pertussis toxin insensitive. These results suggest that at least two muscarinic receptors are present on sympathetic neurons and that they mediate opposite effect on the fluctuation of [Ca2+]i.