bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular pH modulates cytosolic free magnesium in cultured chicken heart cells
Authors: Freudenrich C. C. , Murphy E. , Levy L. A. , London R. E. , Lieberman M.
Published: December 2017

To assess the role of pH in cellular Mg homeostasis, cytosolic pH (pHi) was manipulated by the NH4Cl prepulse technique; pHi, cytosolic Mg2+ (Mgi), and cytosolic Ca2+ (Cai) were measured fluorometrically in single cultured embryonic chicken heart cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), FURAPTRA, and fura-2, respectively. The basal values obtained were as follows: pHi = 7.21 +/- 0.10 (n = 7), [Mg]i = 0.51 +/- 0.08 mM (n = 9), [Ca]i = 126 +/- 15 nM (n = 7). When cells were perfused with 10 mM NH4Cl solution for 5 min, a transient alkalinization (0.53 U) of the cytosol was accompanied by a transient decrease (0.12 mM) in [Mg]i and a transient increase (59 nM) in [Ca]i; these changes approached control levels within 5 min. Upon removal of NH4Cl, a transient acidification (0.89 U) of the cytosol was accompanied by a transient increase (0.10 mM) in [Mg]i and a transient increase (125 nM) in [Ca]i; again, these changes returned toward control levels within 5 min. No significant changes in total cell Mg or Ca were observed during these manipulations. NH4Cl-evoked changes in [Mg]i were not altered significantly by either Mg-free or Ca-free conditions. Changes in [Mg]i were inversely correlated with changes in pHi and were not secondary to changes in [Ca]i. The results suggest that pHi modulates Mgi, probably by affecting cytosolic Mg binding and/or the transport of Mg across subcellular organelles.
Nitrovasodilators relax arterial smooth muscle by decreasing [Ca2+]i and uncoupling stress from myosin phosphorylation
Authors: McDaniel N. L. , Chen X. L. , Singer H. A. , Murphy R. A. , Rembold C. M.
Published: December 2017

Elevations in guanosine 3',5'-cyclic monophosphate concentration ([cGMP]) are proposed to induce arterial smooth muscle relaxation by either 1) decreasing myoplasmic [Ca2+] ([Ca2+]i), 2) decreasing the [Ca2+]i sensitivity of phosphorylation, or 3) uncoupling force from myosin phosphorylation. We evaluated the importance of each of these mechanisms by measuring changes in [cGMP], aequorin- and fura-2-estimated [Ca2+]i, myosin light chain phosphorylation, and stress in histamine-stimulated swine carotid arteries. In tissues submaximally stimulated with 3 microM histamine, nitroprusside (NP) induced a proportional decrease in myoplasmic [Ca2+] and myosin phosphorylation, suggesting that the relaxation was at least partially induced by decreases in [Ca2+]i without a change in the [Ca2+]i sensitivity of phosphorylation. In tissues maximally stimulated with 10 microM histamine, NP and nitroglycerin produced significant relaxations that were not associated with significant sustained reductions in [Ca2+]i or myosin phosphorylation. With both submaximal and maximal histamine stimulation, nitrovasodilators produced more substantial relaxation than that expected from the nitrovasodilator-induced reduction in myosin phosphorylation. These results suggest that nitrovasodilators relax histamine-stimulated swine arterial smooth muscle by at least two mechanisms: 1) reducing [Ca2+]i, an effect observed in submaximally stimulated tissues, and 2) uncoupling of stress from myosin phosphorylation.
Ca(2+)-activated K+ channels in pregnant rat myometrium: modulation by a beta-adrenergic agent
Authors: Anwer K. , Toro L. , Oberti C. , Stefani E. , Sanborn B. M.
Published: December 2017

The properties of Ca(2+)-activated K+ currents and channels were characterized in pregnant rat myometrium in whole cell and cell-attached patches and in lipid bilayers. Membrane depolarization of cultured myometrial cells from a holding potential of -50 to +70 mV in 10-mV steps under voltage-clamp conditions (whole cell mode) activated K+ outward currents (IK). At +70 mV, in the presence of 0.2 mM external Ca2+, the amplitude and activation time constant of IK were 15.0 +/- 2.1 microA/microF and 1.5 +/- 0.2 ms, respectively. Addition of 1 microM A23187 to the external solution increased the current from a control value of 16.0 +/- 2.0 to 67.9 +/- 9.1 microA/microF. Charybdotoxin, a blocker of Ca(2+)-activated K (KCa) channels, and a low concentration of tetraethylammonium chloride (TEA; 1 mM) decreased the amplitude of IK by 47 and 62%, respectively. In cell-attached patches from these cells, 1 microM A23187 increased the open time probability of a 143 +/- 6.0 pS K+ channel. Incorporation of plasma membrane vesicles from pregnant myometrium into lipid bilayers resulted in one predominant type of K+ channel. The unitary conductance of the K+ channel was 326 +/- 9.0 pS in symmetrical 450 mM KCl. The channel activation was both voltage and Ca2+ dependent. TEA inhibited the channel activity with a dissociation constant (Kd) of 378 +/- 10 microM at -60 mV or 1,477 +/- 80 microM at +60 mV. The whole cell currents were found to be stimulated by isoproterenol, a beta-adrenergic agent.(ABSTRACT TRUNCATED AT 250 WORDS)
Differences in gap junction channels between cardiac myocytes, fibroblasts, and heterologous pairs
Authors: Rook M. B. , van Ginneken A. C. , de Jonge B. , el Aoumari A. , Gros D. , Jongsma H. J.
Published: December 2017

Cultures of neonatal rat heart cells contain predominantly myocytes and fibroblastic cells. Most abundant are groups of synchronously contracting myocytes, which are electrically well coupled through large gap junctions. Cardiac fibroblasts may be electrically coupled to each other and to adjacent myocytes, be it with low intercellular conductances. Nevertheless, synchronously beating myocytes interconnected via a fibroblast were present, demonstrating that nonexcitable cardiac cells are capable of passive impulse conduction. In fibroblast pairs as well as in myocyte-fibroblast cell pairs, no sensitivity to junctional voltage could be detected when transjunctional conductance was > 1-2 nS. However, in pairs coupled by a conductance of < 1 nS, complex voltage-dependent gating was evident; gap junction channel open probability decreased with increasing junctional voltage but a nongated residual conductance remained at all voltages tested. Single gap junction channel conductance between fibroblasts was approximately 21 pS, very similar to an approximately 18-pS channel conductance that was found between myocytes next to the major conductance of 43 pS. Single-channel conductance in heterologous myocyte-fibroblast gap junctions was approximately 32 pS, which matches the theoretical value of 29 pS for gap junction channels composed of a fibroblast connexon and the major myocyte connexon. A site-directed antibody against rat heart gap junction protein connexin43 recognized gap junctions between neonatal cardiomyocytes, as demonstrated by immunocytochemical labeling. In contrast, junctions between fibroblasts showed no labeling, while in myocyte-fibroblast junctions labeling occasionally was present. Our results suggest the existence of two gap junction proteins between neonatal rat cardiocytes, connexin43 and another yet unidentified connexin. An alternative explanation (cell-specific regulation of the conductance of connexin43 channels) is discussed.
Localization of the CHIP28 water channel in rat kidney
Authors: Sabolic I. , Valenti G. , Verbavatz J. M. , Van Hoek A. N. , Verkman A. S. , Ausiello D. A. , Brown D.
Published: December 2017

CHIP28 is an integral membrane protein that has been identified as the erythrocyte water channel and that is also expressed in the kidney. Antibodies against erythrocyte CHIP28 were used to localize this protein along the rat urinary tubule. By Western blotting, CHIP28 was detected in kidney plasma membrane and endosome fractions. With the use of immunocytochemistry, CHIP28 was located in brush-border and basolateral plasma membranes of the proximal tubule. The initial S1 segment was weakly stained, but the S2 and S3 segments were heavily labeled. Subapical vesicles were also positive. Apical and basolateral membranes of the long thin descending limb were strongly labeled, but ascending thin and thick limbs of Henle and distal convoluted tubules were negative. Some vasa recta profiles in the medulla were positive. CHIP28 is, therefore, present in membranes with a high constitutive water permeability, where it probably acts as a transmembrane water-conducting channel. Finally, a weak staining of apical and basolateral membranes of cortical collecting duct principal cells was detectable, suggesting a potential relationship of CHIP28 to the vasopressin-sensitive water channel.
Extracellular matrix-stimulated phospholipase activation is mediated by beta 1-integrin
Authors: Cybulsky A. V. , Carbonetto S. , Cyr M. D. , McTavish A. J. , Huang Q.
Published: December 2017

beta 1-Integrins are major mediators of interactions between cells and extracellular matrix (ECM). Adhesion of rat glomerular epithelial cells (GEC) to collagen stimulated phospholipase C. As a result, 1,2-diacylglycerol (DAG) was increased, and inositol phospholipids were decreased in collagen-adherent cells, as compared with GEC adherent to plastic substrata. Adhesion to collagen also stimulated production of free arachidonic acid (the precursor for eicosanoids) due to metabolism of DAG through the DAG lipase pathway and due to phospholipase A2-induced hydrolysis of phospholipids. Phospholipase A2 appeared to be stimulated as a result of protein kinase C (PKC) activation, probably secondary to increased DAG. The collagen-induced increases in DAG and free arachidonic acid, as well as the decrease in inositol phospholipids, were partially inhibited by lowering extracellular Ca2+ concentration to 200 nM or less and by anti-beta 1-integrin antibody Fab. In contrast, anti-beta 1-integrin immunoglobulin G (IgG) enhanced collagen-mediated increases in DAG and arachidonic acid. Proliferation of GEC adherent to collagen was reduced in the presence of anti-beta 1-integrin IgG. The antiproliferative effect of anti-beta 1-IgG appeared to be mediated through PKC, since it was absent in PKC-depleted GEC. Immunoprecipitation with integrin subunit-specific antibodies demonstrated alpha 2 beta 1- and alpha 3 beta 1-integrins in GEC. Thus, in GEC, ECM induces activation of phospholipases C and A2, which is mediated, at least in part, by beta 1-integrins. Products of integrin-mediated phospholipase activation may modulate GEC proliferation.
Inhibition of slow-wave repolarization and Ca(2+)-activated K+ channels by quaternary ammonium ions
Authors: Carl A. , Frey B. W. , Ward S. M. , Sanders K. M. , Kenyon J. L.
Published: December 2017

We studied the effects of the K+ channel blocker tetrapentylammonium (TPeA) on the electrical activity of intact circular smooth muscle from canine colon. TPeA (10 and 20 microM) increased slow-wave duration and "locked" the membrane potential around -30 mV plateau potential after several minutes of application, suggesting that K+ channels are essential for termination of colonic slow waves. Repolarization and normal slow-wave activity resumed after 20-30 min of washout. The patch-clamp technique was used to study the block of large-conductance Ca(2+)-activated K+ channels (BK channels) by TPeA and tetraethylammonium (TEA) in excised and cell-attached patches from isolated colonic smooth muscle cells. Channel block was characterized by a voltage-dependent dissociation constant [Kd(V)] for the binding of TEA and TPeA to a blocking site located a fraction of the distance across the membrane field (delta). The extracellular TEA binding site had a Kd(0) of 0.33 mM and a delta of 0.23. The extracellular TPeA binding site had a Kd(0) of 2.2 mM but showed significantly less voltage dependence (delta = 0.02). The intracellular binding site for TEA was of low affinity [Kd(0) = 76 mM]. Intracellular TPeA was the most potent blocker of BK channel current [Kd(0) = 11.7 microM]. The voltage dependence of block by intracellular TPeA (delta = -0.21) was not significantly different from that of intracellular TEA (delta = -0.3). Internal TPeA (10 microM) also blocked a 70-pS K+ channel and a 23-pS K+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)
Localization of angiotensin peptide-forming enzymes of 3T3-F442A adipocytes
Authors: Saye J. A. , Ragsdale N. V. , Carey R. M. , Peach M. J.
Published: December 2017

We have demonstrated that angiotensinogen is synthesized by 3T3-F442A cells and is hydrolyzed to angiotensins I and II (ANG I and II) by this model adipocyte system. This study was designed to determine whether ANG I is generated by renin or some other enzyme and where the formation of ANG I and/or II occurs in 3T3-F442A cells. Renin mRNA was not detected by Northern blot analysis of poly(A)(+)-selected RNA from cultures of fully differentiated adipocytes nor by the more sensitive polymerase chain reaction, implying that renin is not synthesized in this model adipocyte system. Hydrolysis of angiotensinogen to ANG I and II was demonstrated to be associated with the cell but not the media. Inhibitors, including EDTA, aimed at inactivating enzymes belonging to the serine, acid, or aspartyl proteases, and metalloproteases were ineffective in preventing the formation of either ANG I or II. Therefore the model adipocyte 3T3-F442A cell system forms ANG I and II in the absence of renin and angiotensin-converting enzyme. The unidentified enzymes responsible for peptide formation are associated with the cell itself.
ASC system activity is altered by development of cell polarity in trophoblast from human placenta
Authors: Furesz T. C. , Smith C. H. , Moe A. J.
Published: December 2017

Pathways of neutral amino acid uptake were investigated in vitro during differentiation of primary cultures of trophoblast isolated from full-term human placentas and a clone (b30) of the BeWo cell line. Inhibition of initial alanine (0.1 microM) uptake by 2-(methylamino)isobutyric acid and unlabeled alanine revealed two Na(+)-dependent systems and one Na(+)-independent transporter. Characterization of these transporters, by selective inhibition, suggested system A, ASC, and L-like transporters. Concomitant with formation of microvillous membrane and syncytium, system ASC activity decreased from 16.1 +/- 2.8 pmol.mg DNA-1.min-1 at 24 h to 2.4 +/- 1.1 pmol.mg DNA-1.min-1 at 72 h. Na(+)-independent alanine uptake increased from 6.0 +/- 2.0 to 12.9 +/- 0.9 pmol.mg DNA-1.min-1 at 24 and 72 h, respectively. Similarly, alpha-(methylamino)isobutyric acid-insensitive, Na(+)-dependent activity in b30 cells (100 microM alanine) decreased from 6.5 +/- 1.6 to 1.2 +/- 1.2 nmol.mg DNA-1.min-1 for control and forskolin-treated cells, respectively. We conclude that membrane specialization accompanying fusion and differentiation of the cytotrophoblast to form syncytiotrophoblast results in a polarization of neutral amino acid transport systems.
Activation of epithelial Na+ channels by protein kinase A requires actin filaments
Authors: Prat A. G. , Bertorello A. M. , Ausiello D. A. , Cantiello H. F.
Published: December 2017

We have recently demonstrated a novel role for "short" actin filaments, a distinct species of polymerized actin different from either monomeric (G-actin) or long actin filaments (F-actin), in the activation of epithelial Na+ channels. In the present study, the role of actin in the activation of apical Na+ channels by the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) was investigated by patch-clamp techniques in A6 epithelial cells. In excised inside-out patches, addition of deoxyribonuclease I, which prevents actin polymerization, inhibited Na+ channel activation mediated by PKA. Disruption of endogenous actin filament organization with cytochalasin D for at least 1 h prevented the PKA-mediated activation of Na+ channels but not activation following the addition of actin to the cytosolic side of the patch. To assess the role of PKA on actin filament organization, actin was used as a substrate for the specific phosphorylation by the PKA. Actin was phosphorylated by PKA with an equilibrium stoichiometry of 2:1 mol PO4-actin monomer. Actin was phosphorylated in its monomeric form, but only poorly once polymerized. Furthermore, phosphorylated actin reduced the rate of actin polymerization. Thus actin allowed to polymerize for at least 1 h in the presence of PKA and ATP to obtain phosphorylated actin filaments induced Na+ channel activity in excised inside-out patches, in contrast to actin polymerized either in the absence of PKA or in the presence of PKA plus a PKA inhibitor (nonphosphorylated actin filaments). This was also confirmed by using purified phosphorylated G-actin incubated in a polymerizing buffer for at least 1 h at 37 degrees C. These data suggest that the form of actin required for Na+ channel activation (i.e., "short" actin filaments) may be favored by the phosphorylation of G-actin and may thus mediate or facilitate the activation of Na+ channels by PKA.
B-cell size influences glucose-stimulated insulin secretion
Authors: Giordano E. , Cirulli V. , Bosco D. , Rouiller D. , Halban P. , Meda P.
Published: December 2017

To determine whether the heterogeneous B-cell response to glucose is related to a different metabolic handling of this sugar, we have compared rat B-cells differing in their redox response to glucose stimulation. To this end, a population of B-cells showing increased NAD(P)H autofluorescence after a 15-min exposure to 16.7 mM glucose was sorted from a population of B-cells that, under the very same conditions, failed to show detectable changes in basal NADP(H) autofluorescence. Insulin secretion was evaluated by a reverse hemolytic plaque assay in these two populations, referred to as high and low NAD(P)H, respectively. After a 30-min stimulation by 16.7 mM glucose, both populations comprised secreting B-cells and B-cells that did not release detectable amounts of insulin. However, the percentage of secreting B-cells and total insulin output were larger (P < 0.01-0.02) in the high- (77.9 +/- 6.5% and 141.7 +/- 27.4 microns2 x 10(3)) than in the low-NAD(P)H population (58.7 +/- 6% and 92.7 +/- 20.5 microns2 x 10(3)). The high-NAD(P)H population also comprised B-cells that, on average, had a larger (P < 0.001) profile area (142.9 +/- 2.3 microns2) than the B-cells of the low-NAD(P)H population (118.6 +/- 1.5 microns2). Glucose-induced insulin secretion was similar in the high- and low-NAD(P)H group when cells of similar sizes were compared and increased similarly in the two populations as a function of B-cell size. Analysis of variance revealed that insulin secretion was influenced (P < 0.005) by the size of B-cells and not by their NAD(P)H level.(ABSTRACT TRUNCATED AT 250 WORDS)
Bradykinin inhibition of EGF- and PDGF-induced DNA synthesis in human fibroblasts
Authors: McAllister B. S. , Leeb-Lundberg F. , Olson M. S.
Published: December 2017

Bradykinin exhibits proliferative influences in several types of cells; however, in the present study, bradykinin did not promote DNA synthesis but actually inhibited the DNA synthesis induced by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in human gingival fibroblasts (HGF). This dose-dependent inhibitory effect was a specific intracellular interaction in that increasing concentrations of EGF did not counteract the inhibitory actions of bradykinin when added at 100 nM. The phosphoinositide-calcium signaling cascade is a likely point of interaction for the inhibitory influences of bradykinin; however, no interactions between bradykinin and EGF were observed with the generation of inositol phosphates or intracellular calcium fluxes. The inhibitory influences of bradykinin do not appear to be the result of a transmodulation of the EGF receptor, since EGF-mediated autophosphorylation was not negatively affected by bradykinin. Bradykinin-stimulated prostaglandin E2 (PGE2) release was potentiated by EGF, and, in the presence of indomethacin, the inhibition of the EGF-induced DNA synthesis by bradykinin was minimized. The results presented demonstrate that bradykinin can inhibit EGF- and PDGF-induced DNA synthesis and suggest that PGE2 synthesis is responsible for the observed bradykinin inhibition of EGF-induced DNA synthesis.
External Ca effect on water permeability, regulatory volume decrease, and extracellular space in barnacle muscle cells
Authors: Berman D. M. , Pena-Rasgado C. , Holmgren M. , Hawkins P. , Rasgado-Flores H.
Published: December 2017

The effect of extracellular Ca2+ (Cao) on sarcolemmal hydraulic water permeability (L'p), regulatory volume decrease (RVD), and extracellular space (ECS) was studied in barnacle muscle cells. Absence or presence of Cao had no effect on L'p [0 Cao = 2.762 +/- 0.098 x 10(-5), and 11 mM Cao = 2.720 +/- 0.222 x 10(-5) cm.kg.s-1 x osmol x 1-kgH2O-1]. Likewise, cells exposed to anisosmotic media (for < 30 min) behaved as osmometers in 0 and 11 mM Cao, showing similar slopes and intercepts in van't Hoff plots. At longer incubation times, however, hyposmotic conditions promoted a Cao-dependent RVD. The relationship between Cao and the percentage of cells responding with RVD to a hyposmotic challenge was sigmoidal (half-maximal Cao = 4.83 mM). The mean rate of RVD (40 nl/min) was independent of the level of swelling in response to hyposmotic challenges. However, the magnitude of RVD increased with larger hyposmotic challenges. Both the presence of Cao and hypotonicity reduced the "apparent" ECS by 47 +/- 6 and 39 +/- 6%, respectively. Three-dimensional reconstruction of autoradiographs of the cells was made to interpret these results.
Functional CFTR in endosomal compartment of CFTR-expressing fibroblasts and T84 cells
Authors: Biwersi J. , Verkman A. S.
Published: December 2017

It was proposed that the cystic fibrosis transmembrane conductance regulator (CFTR) functions in the endosomal compartment as a adenosine 3',5'-cyclic monophosphate (cAMP)-regulated Cl channel that regulates endosomal acidification (J. Barasch, B. Kiss, A. Prince, L. Saiman, D. Gruenert, and A. Al-Awqati, Nature Lond. 352: 70-73, 1991). This hypothesis was tested in stably transfected Swiss 3T3 fibroblasts expressing CFTR or delta F508 CFTR and in T84 epithelial cells that normally express CFTR. In fibroblasts, the time course of pH in individual endosomes was measured by quantitative image analysis after 1 min pulse labeling with 2 microM carboxyfluorescein (Cf)-tetramethylrhodamine-transferrin (K. Zen, J. Biwersi, N. Periasamy, and A. S. Verkman. J. Cell Biol. 119: 99-110, 1992). Average endosomal pH reached 6.20 +/- 0.07 (SE) after 15 min in the mock-transfected cells with a half time of approximately 3 min; pH was slightly lower (5.97 +/- 0.06) in the CFTR-expressing fibroblasts. The difference did not result from a subpopulation of highly acidic endosomes. Forskolin (10 microM) increased average pH to 6.62 +/- 0.03 and abolished the difference. For determination of Cl conductance, endosomes in fibroblasts and T84 cells were labeled with Cf-dextran (5 mg/ml); dissipation of the endosomal pH gradient was measured in response to rapid addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP; 20 microM). Because the proton flux across the endosomal membrane is limited by the movement of K and Cl, the rate of alkalinization (dpH/dt) after CCCP addition provided a measure of endosomal Cl conductance. In CFTR-expressing fibroblasts, forskolin (10 microM) increased dpH/dt 1.6 +/- 0.2-fold (n = 14).(ABSTRACT TRUNCATED AT 250 WORDS)
Immunolocalization of anion exchanger AE2 and cation exchanger NHE-1 in distinct adjacent cells of gastric mucosa
Authors: Stuart-Tilley A. , Sardet C. , Pouyssegur J. , Schwartz M. A. , Brown D. , Alper S. L.
Published: December 2017

The gastric mucosa secretes both protons and bicarbonate. The molecular identity of the H(+)-K(+)-ATPase, which mediates acid secretion, has long been known, but the other components of the secretory machinery and their cellular disposition are less well characterized. This study identifies and localizes in rat and rabbit gastric mucosa a chloride-bicarbonate exchanger protein and a Na(+)-H+ exchanger protein. The previously described band 3-related protein of the parietal cell has been identified by isoform-specific antibodies as anion exchanger (AE) 2 and localized to the basolateral membranes of the parietal cells. The Na(+)-H+ exchanger protein NHE-1 was located in the basolateral membranes of the mucous neck cells, interdigitated between the parietal cells of the gastric glands and in the basolateral membranes of the surface mucous cells. Neither transporter protein was abundantly expressed deep in the gland, where most of the pepsinogen cells reside. Carbonic anhydrase II (CA II) was expressed at higher abundance in the surface mucous cells and mucous neck cells, which expressed NHE-1, than in the parietal cells, which expressed AE2. The morphological evidence identified AE2 as a major parietal cell anion exchanger, whereas NHE-1 and CA II colocalized in mucous neck, chief, and surface mucous cells. We propose that all three of these cell types contribute to gastric bicarbonate secretion.
Primary structure and functional properties of an epithelial K channel
Authors: Zhou H. , Tate S. S. , Palmer L. G.
Published: December 2017

Expression cloning in Xenopus oocytes was used to identify a clone for a renal K channel. The clone, named ROMK2, was obtained from a cDNA library constructed in the plasmid vector pSPORT using size-selected poly(A)+ RNA from whole rat kidney. ROMK2 consists of 1,837 nucleotides, with an open reading frame of 1,116 bases predicted to code for a 372-amino acid peptide. The clone appears to be a splice variant of a recently reported K channel (ROMK1) from rat renal outer medulla (Ho, K.H., C.G. Nichols, W.J. Lederer, J. Lytton, P.M. Vassilev, M.V. Kanazirska, and S.C. Hebert. Nature Lond. 362: 31-37, 1993). Northern blot analysis indicates that ROMK2 is expressed in renal cortex, medulla, and papilla. Expression in other tissues appears to be much lower. The functional properties of the channel as measured in Xenopus oocytes indicate its close relationship to ROMK1 and more distant relationship to the inward rectifier K channel (IRK1) (Kubo, Y, T.J. Baldwin, Y. N. Jan, and L. Y. Jan. Nature Lond. 362: 127-133, 1993). The inward conductance of the channel is a saturable function of external K, with a half-maximal conductance at <5 mM. The selectivity sequence for ion permeability based on reversal potential measurements was K > Rb > NH4 > Na, Li. The conductance to Rb was only one-half that to K. Extracellular Ba2+ and Cs+ blocked the channel in a voltage-dependent manner. The high sensitivity of Cs+ block to voltage is consistent with the channel's operating as a multi-ion pore. The channel was blocked by high concentrations (100 microM) of glibenclamide. It did not appear to be blocked by extracellular Na+ or tetraethyl-ammonium ion. Patch-clamp measurements indicated a single-channel conductance of 30 pS in the presence of 110 mM K and high open probability that was weakly dependent on voltage. This channel may be involved in maintaining the membrane potential of renal cells and/or mediating renal K secretion.
Glucocorticoids regulate the transcription of Na(+)-K(+)-ATPase genes in the infant rat kidney
Authors: Wang Z. M. , Yasui M. , Celsi G.
Published: December 2017

Glucocorticoids modulate the maturation of Na(+)-K(+)-ATPase mRNA in a tissue- and age-dependent manner. In this study, we report the effect of glucocorticoids on Na(+)-K(+)-ATPase gene transcription in the infant rat kidney. Ten-day-old rats were treated with one intraperitoneal injection of betamethasone. In glucocorticoid-treated rats, there was a significant increase in renal cortical alpha 1- and beta 1-mRNAs (3.08 +/- 0.34- and 4.06 +/- 0.10-fold). Pretreatment with cycloheximide, an inhibitor of protein synthesis, did not abolish the increase in alpha 1- and beta 1-mRNA after glucocorticoids. The alpha 1- and beta 1-gene transcription rates were significantly increased in nuclei isolated from kidneys of glucocorticoid-treated rat (2.16 +/- 0.05- and 3.12 +/- 0.50-fold). Interaction between nuclear proteins and Na(+)-K(+)-ATPase alpha 1-promoter was studied by gel retardation assay. Nuclear protein from glucocorticoid-treated rats retarded a fragment of alpha 1-promoter that includes a half-consensus glucocorticoid response element (GRE) at position -750 bp but did not retard a fragment including a half-consensus GRE at position -481. Retardation of alpha 1-promoter was inhibited by incubation with molar excess of GRE or with a monoclonal antibody against glucocorticoid receptor. We conclude that in the infant kidney, glucocorticoids directly stimulate the transcription of alpha 1- and beta 1-Na(+)-K(+)-ATPase subunits. It is likely that the binding of glucocorticoid receptor to alpha 1-Na(+)-K(+)-ATPase promoter requires the presence of an auxiliary factor.
Isometric, shortening, and lengthening contractions of muscle fiber segments from adult and old mice
Authors: Brooks S. V. , Faulkner J. A.
Published: December 2017

In old animals, skeletal muscle force decreases during both isometric and shortening contractions. In contrast, force during lengthening appears to be unaffected by aging. We hypothesized that with aging single permeabilized muscle fibers would demonstrate the same impairments in force as are observed for whole muscles. For single permeabilized fibers from extensor digitorum longus muscles of adult and old mice, forces were measured during isometric, shortening, and lengthening contractions performed at 15 degrees C. Maximum isometric forces normalized for fiber area were not different for fibers from adult and old mice. During submaximal isometric contractions a decreased calcium sensitivity resulted in lower forces for fibers from old compared with adult mice. In contrast to a lack of difference in forces developed by fibers from old and adult mice during shortening contractions, during lengthening contractions fibers from old mice developed forces approximately 30% higher than those of adult mice. We conclude that the impairments in force of whole muscles with aging are not the result of impairments in intrinsic force-generating capacity of cross bridges, but changes do occur in single permeabilized muscle fibers of old mice that result in higher forces during stretch.
ATP is not required for anion current activated by cell swelling in multidrug-resistant lung cancer cells
Authors: Jirsch J. D. , Loe D. W. , Cole S. P. , Deeley R. G. , Fedida D.
Published: December 2017

During whole cell recording with 4 mM ATP and 0.1 mM GTP in the pipette, outwardly rectifying Cl- currents (155 +/- 20.5 pA/pF) were repetitively activated on reduction of bath solution osmolarity from 290 mosM (control) to 210 mosM. These currents were sensitive to 0.1-1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Omission of ATP from the pipette solution reduced the current magnitude to 42.7 +/- 9.5 pA/pF and prevented repetitive activation. More hyposmotic solutions (160 mosM) usually elicited current repetitively despite an ATP-free pipette solution. In cells depleted of ATP (to < 5% of control) by preincubation with 2-deoxyglucose (10 mM) and rotenone (100 nM), hyposmotic solutions failed to activate significant current. Cell volume increased to 230 +/- 18% of control (19.1 +/- 1.2 microns) in 210 mosM bath (normal cells) but only to 114 +/- 13% of control in ATP-depleted cells exposed to 160 mosM solution. This failure of ATP-depleted cells to swell in hypotonic external solutions was reversed by overnight pretreatment with cytochalasin D (2 micrograms/ml; n = 6) but not by colchicine (250 microM; n = 8). In outside-out patches of membrane dialyzed with zero ATP and excised from swollen cells, we observed sustained activation of a 53-pS outwardly rectifying channel (chord conductance, +100 mV; open probability approximately 1.0). In cell-attached patches from normal and ATP-depleted cells, we activated similar channels by suction. ATP does not appear to be an absolute requirement for the activation of this Cl- channel in H69AR cells but may be essential for the normal volume response and channel activation mediated through cytoskeletal elements within cells.
Clenbuterol, a beta 2-agonist, retards wasting and loss of contractility in irradiated dystrophic mdx muscle
Authors: Zeman R. J. , Zhang Y. , Etlinger J. D.
Published: December 2017

Treatment with the adrenergic beta 2-receptor agonist clenbuterol prevented, in dystrophic muscle from mdx mice, a pronounced loss of contractile strength that is observed after blockade of muscle regeneration with gamma irradiation. In addition, muscle mass and myosin content were greater (62-109%) in irradiated hindlimbs from clenbuterol-treated mdx mice, whereas the effects of the beta 2-agonist were relatively smaller (12-21%) in the nonirradiated hindlimbs. Together, these results suggest that beta 2-agonists can antagonize degenerative processes occurring in muscle fibers lacking dystrophin.
Subcellular distribution of dystrophin in isolated adult and neonatal cardiac myocytes
Authors: Frank J. S. , Mottino G. , Chen F. , Peri V. , Holland P. , Tuana B. S.
Published: December 2017

The subcellular localization of dystrophin was examined in adult rabbit and rat cardiac myocytes with immunofluorescence and at higher resolution with immunogold. The aim was to resolve the conflicting reports on the presence of dystrophin in the transverse tubules (T tubules) of cardiac muscle and to determine its distribution in neonatal myocytes before and during the development of the T tubules. Dystrophin was localized to the peripheral sarcolemma and the T-tubular membrane and was absent from the intercalated disk membranes. In addition, dystrophin localization was followed with immunofluorescence in developing rabbit myocytes at 4 days, 1 wk, and 1 mo after birth. At 4 days of age, T tubules are absent and dystrophin was localized only in the peripheral sarcolemma. Dystrophin was present in the developing T tubules at 1 wk and 1 mo. These results imply that dystrophin is expressed in the T tubules as soon as they develop and confirm the different distribution of dystrophin in the T tubules of cardiac and skeletal muscle.
Bioelectric characteristics of exon 10 insertional cystic fibrosis mouse: comparison with humans
Authors: Smith S. N. , Steel D. M. , Middleton P. G. , Munkonge F. M. , Geddes D. M. , Caplen N. J. , Porteous D. J. , Dorin J. R. , Alton E. W.
Published: December 2017

Two important issues that can be addressed by animal models are disease pathogenesis and the testing of new treatments, including gene therapy. How closely these models mimic the relevant disorder in humans will determine their usefulness. This study examines how closely the characteristic bioelectric features of cystic fibrosis (CF) are reproduced in the airways and intestinal tract of the exon 10 insertional mutant mouse (cf/cf). In agreement with CF subjects these cf/cf mutant mice demonstrate the following: 1) reduced adenosine 3',5'-cyclic monophosphate-related chloride secretion throughout the respiratory and intestinal tracts both in vivo and in vitro, 2) calcium-related chloride secretion that is preserved in the airways but reduced in the intestine, and 3) a more negative nasal potential difference and increased amiloride response compared with wild-type animals, likely to relate to increased sodium absorption. In contrast to humans, sodium absorption is not increased in the small intestine and is reduced in the trachea of the cf/cf mice. We conclude that the majority of the salient electrophysiological features of CF required for studies of pathogenesis or testing of new treatments are present in these cf/cf mice.
G protein-mediated suppression of L-type Ca2+ current by interleukin-1 beta in cultured rat ventricular myocytes
Authors: Liu S. , Schreur K. D.
Published: December 2017

The effect and possible signal transduction pathway of interleukin-1 beta (IL-1 beta) on the L-type Ca2+ current (ICa,L) in cultured adult rat ventricular myocytes were examined using whole cell patch-clamp techniques. When myocytes were internally dialyzed with a solution containing GTP, IL-1 beta caused a concentration-dependent decrease in the peak ICa,L (Ba2+ as the charge carrier). IL-1 beta did not significantly alter the voltage dependence of the peak ICa,L nor the steady-state inactivation and activation, but did slightly slow the rate of inactivation. In myocytes dialyzed with solutions without GTP or including guanosine 5'-O-(2-thiodiphosphate) to replace GTP, IL-1 beta had no effect on ICa,L. In contrast, when guanosine 5'-O-(3-thiotriphosphate) was used to replace GTP, the suppression of ICa,L induced by IL-1 beta remained. Preincubation of myocytes with pertussis toxin (PTX), which completely abolished the acetylcholine effect on isoproterenol-stimulated ICa,L, had no effect on the inhibitory action of IL-1 beta on ICa,L. We conclude that in cultured rat ventricular myocytes, IL-1 beta suppresses ICa,L via a PTX-insensitive G protein.
Shrinkage-induced activation of Na+/H+ exchange in primary rat astrocytes: role of myosin light-chain kinase
Authors: Shrode L. D. , Klein J. D. , O'Neill W. C. , Putnam R. W.
Published: December 2017

Primary rat astrocytes exposed to hyperosmotic solutions undergo Na(+)-dependent amiloride-sensitive alkalinization of 0.36 U [measured with the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein], suggesting that shrinkage-induced alkalinization is due to activation of Na+/H+ exchange (NHE). Alkalinization is maintained for at least 20 min, and is readily reversible and ATP dependent. Hyperosmotic solutions produced no increase of intracellular Ca2+ or adenosine 3',5'-cyclic monophosphate (cAMP). Loading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a Ca2+ chelator, or depleting cells of protein kinase C (PKC) had no effect on activation of NHE. Thus shrinkage-induced activation of NHE does not involve cAMP, Ca2+, or PKC. However, ML-7, an inhibitor of myosin light-chain kinase (MLCK), inhibited shrinkage-induced activation with a half-maximal inhibition of 56 microM. This activation was also inhibited by 500 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, 100 microM chlorpromazine, and 50 microM trifluoperazine, all calmodulin inhibitors. Shrinkage increased the phosphorylation of an 18-kDa protein that colocalizes with myosin light chain. Our data suggest that shrinkage-induced activation of NHE in astrocytes occurs via a novel pathway involving activation of calmodulin-dependent MLCK and phosphorylation of myosin light chain.
Effect of Mg2+ and ATP on depolarization-induced Ca2+ release in isolated triads
Authors: Ritucci N. A. , Corbett A. M.
Published: December 2017

The effect of different free Mg2+ and ATP concentrations on depolarization-induced Ca2+ release in isolated skeletal muscle triadic vesicles was examined by simultaneously monitoring direct effects on ryanodine receptors from either isolated or coupled terminal cisternae. Free Mg2+ was increased to concentrations of 11-14 microM, 81 microM, 175-181 microM, and 1 mM while total ATP concentration was kept constant or MgATP concentration was kept constant. We observed the following. 1) Increasing MgATP reduces the measurable Ca2+ release from isolated vesicles by stimulating the Ca(2+)-ATPase in the terminal cisternae. 2) Half-maximal inhibition of functionally coupled ryanodine receptors during depolarization-induced Ca2+ release is observed at 1 mM Mg2+, whereas half-maximal inhibition of the nondepolarized ryanodine receptor is seen at 75 microM Mg2+ at the same free ATP and MgATP concentrations. 3) Two separate time constants for Ca2+ release were obtained for nondepolarized ryanodine receptors with free Mg2+ at 14 microM and free ATP at 6.1 mM; this may represent triadic ryanodine receptors vs. isolated terminal cisternae ryanodine receptors.
Ion microscopy evidence that La3+ releases Ca2+ from Golgi complex in LLC-PK1 cells
Authors: Zha X. , Morrison G. H.
Published: December 2017

The effect of La3+ on LLC-PK1 cells was investigated by ion microscopy, a mass spectrometry-based technique with a spatial resolution of approximately 0.5 micron. Cells were incubated with LaCl3 for 10 min. (1 mM) or 30 min (0.1 mM), and intracellular calcium distributions were measured with a Cameca IMS-3f ion microscope in cryogenically prepared cells. Compared with control cells, La3+ reduced total calcium in the Golgi complex by > 100 microM in both treatments, whereas other cellular regions, such as the nucleus and cytoplasm, remained largely unchanged. These two treatments were repeated on cells that were preincubated with 1 mM ouabain. The presence of ouabain in the medium increased the loss of calcium from the Golgi by about fourfold compared with the treatments without ouabain. The La3+ effect, therefore, was amplified by ouabain-induced Na+ loading, indicating a possible involvement of a Na+/La3+ exchanger. La3+ was detected within cells and its influx was facilitated by Na+ loading. These results suggest that La3+ may affect cellular calcium homeostasis by actions other than as a simple Ca2+ antagonist.
Calcium currents in postinfarction rat cardiac myocytes
Authors: Zhang X. Q. , Moore R. L. , Tillotson D. L. , Cheung J. Y.
Published: December 2017

Myocytes isolated from rat hearts that have suffered 35% myocardial infarction (MI) 3 wk prior have lower peak cytosolic Ca2+ concentration ([Ca2+]i) during contraction compared with Sham myocytes, a difference that is amplified by isoproterenol or high extracellular Ca2+ concentration ([Ca2+]o). To evaluate whether reduced [Ca2+]i in MI myocytes is due to decreased Ca2+ entry, we measured [3H]PN-200-110 [dihydropyridine (DHP)] binding and whole cell Ca2+ current (ICa). DHP binding decreased in both sarcolemmal vesicles and intact myocytes from hearts 3 wk after MI. In contrast, ICa was not different between Sham and MI myocytes incubated at 1.8 mM [Ca2+]o. At 5.0 mM [Ca2+]o, ICa increased similarly in Sham and MI myocytes. Steady-state voltage dependence of activation and inactivation were similar between Sham and MI myocytes, as were the fast- and slow-inactivation time constants. Isoproterenol (1 microM) significantly increased ICa in Sham but not in MI myocytes. Forskolin (10 microM) dibutyryl adenosine 3',5'-cyclic monophosphate (5 mM) significantly increased ICa in MI myocytes; the magnitude of ICa increase was similar to that observed in Sham myocytes. We conclude that 1) decreased systolic [Ca2+]i in MI myocytes was not due to reduced Ca2+ entry via L-type Ca2+ channels; 2) discrepancy between DHP binding (decrease) and ICa (no change) results may be explained by higher channel availability and/or increased long-opening modes (mode 2) in MI myocytes; 3) reduction in isoproterenol-induced [Ca2+]i increase in MI myocytes was partly due to decreased ICa, resulting in less Ca2+ release from sarcoplasmic reticulum; 4) the adenylate cyclase-protein kinase A signal-transduction pathway functioned normally in MI myocytes; and 5) decreased beta-adrenergic responsiveness in MI myocytes was likely due to altered coupling by G proteins. d
Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies
Authors: Lytle C. , Xu J. C. , Biemesderfer D. , Forbush B.
Published: December 2017

The Na-K-Cl cotransporter (NKCC) is present in most animal cells where it functions in cell volume homeostasis and epithelial salt transport. We developed six monoclonal antibodies (designated T4, T8, T9, T10, T12, and T14) against a fusion protein fragment encompassing the carboxy-terminal 310 amino acids of the human colonic NKCC. These T antibodies selectively recognized putative NKCC proteins in a diverse variety of animal tissues. Western blot analysis of membranes isolated from 23 types of cells identified single bands of immunoreactive protein ranging in mass from 146 to 205 kDa. The amount of immunoreactive protein detected in these cells correlated with loop diuretic binding site density. Proteins identified previously as Na-K-Cl cotransporters by loop diuretic photoaffinity labeling were mutually recognized by multiple T antibodies. Most of the T antibodies effectively immunoprecipitated the denatured form of the NKCC protein. Immunocytochemical studies on the rabbit parotid gland demonstrated that NKCC is restricted to the basolateral margin of the acinar cells and absent from the ducts, in accord with the central role of Na-K-Cl cotransport in chloride secretion. In the rabbit kidney, NKCC was localized to the apical membrane of thick ascending limb cells, consistent with its role in chloride reabsorption.
Flow- and bradykinin-induced nitric oxide production by endothelial cells is independent of membrane potential
Authors: Gooch K. J. , Frangos J. A.
Published: December 2017

The objective of this study was to evaluate the role transmembrane potential plays in flow-induced nitric oxide (NO) production in endothelial cells (EC). NO production was monitored by measuring intracellular guanosine 3',5'-cyclic monophosphate (cGMP) and extracellular nitrite plus nitrate (NOx). Primary human umbilical vein endothelial cells (HUVEC) were exposed to laminar flow (22 dyn/cm2) of medium with 5.4 mM KCl (control medium) with or without 3 mM tetraethylammonium chloride (TEA) or 90 mM KCl (K(+)-rich medium). Bradykinin (BK) was added to time-matched stationary cultures to give a final concentration of 5 nM. With control medium, 30 s, 2 min, and 3 h of treatment with flow or 2 min of treatment with BK resulted in an approximately threefold increase in cGMP over stationary cultures. Depolarization with KCl or TEA did not influence cGMP production in flow-treated or stationary cultures. Flow of either control or potassium-rich medium resulted in an approximately 10-fold increase in average NOx production rate over 3 h compared with stationary cultures. Taken together these data indicate that neither membrane hyperpolarization nor normal membrane potential is necessary for flow- or BK-induced NO production by HUVEC.
ANP-stimulated cGMP egression in renal principal cells: abrogation of polarity by SV40 large T
Authors: Millul V. , Prie D. , Geniteau-Legendre M. , Verpont M. C. , Baudouin B. , Ronco P. M.
Published: December 2017

Egression of atrial natriuretic peptide (ANP)-stimulated guanosine 3', 5'-cyclic monophosphate (cGMP) was compared with that of isoproterenol-stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) in a rabbit collecting duct cell line transformed with a temperature-sensitive strain of simian virus 40 (SV40). At 39.5 degrees C (inactivated large T), cells exhibit major features of principal cells, whereas at 33 degrees C (functional large T) they lose most of their specific properties. When cells were grown on plastic at 39.5 degrees C, both cyclic nucleotides were predominantly released extracellularly via probenecid-sensitive carriers. Probenecid (3mM) reduced the ratios of extracellular cGMP and cAMP by 84 and 70%, respectively. The amount of extracellular cGMP or cAMP ws linearly correlated with the time integral of the intracellular cyclic nucleotide, suggesting first-order kinetics. The apparent first-order rate constant (k) was sixfold greater for cGMP (0.139 +/- 0.037 min-1, n = 3 experiments) than for cAMP (0.022 +/- 0.003(-1), n = 3 experiments). 3-Isobutyl-1-methylxanthine markedly inhibited extrusion of cGMP (k = 0.022 +/- 0.003 min-1), whereas that of cAMP was unchanged. When cells were grown on filters at 39.5 degrees C, both nucleotides were predominantly released in the apical medium but with a greater polarity for cGMP (83 +/- 4%, n = 6 experiments) than for cAMP (60 +/- 6%, n = 3 experiments) and a prevailing apical localization of the probenecid-sensitive carrier. Activation of SV40 large T at 33 degrees C did not alter cyclic nucleotide transport characteristics but abolished the polarity of probenecid-sensitive cyclic nucleotide extrusion. These results suggest a physiological role for luminal cGMP in the rabbit collecting duct and a specific effect of large T on the probenecid-sensitive carrier polarity.