bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of kinins on cultured arterial smooth muscle
Authors: Dixon B. S. , Breckon R. , Fortune J. , Vavrek R. J. , Stewart J. M. , Marzec-Calvert R. , Linas S. L.
Published: December 2017

The present study uses various kinin agonists and antagonists to examine the cellular mechanisms of bradykinin's actions on intracellular calcium, prostaglandins, and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cultured arterial smooth muscle cells (casmc) obtained from rat mesenteric arteries. Exposure to bradykinin produced a rapid release of calcium (peak less than or equal to 20 s) from intracellular stores and an increase in prostaglandin (PG) E2 and cAMP production in casmc. Compared with bradykinin, the bradykinin B1-agonist [des-Arg9]BK produced only a small increase in intracellular calcium. The bradykinin-mediated increase in intracellular calcium was competitively blocked by the B2 receptor antagonist [D-Arg-O-Hyp3-Thi5,8-D-Phe7]BK (B4307) but not the B1-antagonist ([des-Arg9-Leu8]BK). In addition, the similarity of the dose-response curves for the bradykinin-mediated increase in Ca2+, PGE2, and cAMP (half-maximal stimulation of 12, 11, and 13 nM, respectively) and the ability of the B2-antagonist (B4307) to block each of these effects of bradykinin suggest that all three effects are mediated by the same bradykinin (B2) receptor. Further studies revealed that increases in intracellular calcium are necessary for the bradykinin-mediated increase in PGE2 formation and the subsequent PGE2-dependent formation of cAMP. Taken together, these results suggest that bradykinin acts via a B2-receptor on arterial smooth muscle cells to release calcium from intracellular stores, leading to increases in PGE2 production and the PGE2-dependent activation of adenylate cyclase.
FITC-dextran as a probe for endosome function and localization in kidney
Authors: Lencer W. I. , Weyer P. , Verkman A. S. , Ausiello D. A. , Brown D.
Published: December 2017

Fluorescein isothiocyanate (FITC)-labeled endosomes were localized in kidney epithelial cells after tissue fixation and sectioning, and specific membrane transport properties of isolated endocytic vesicles were measured using the same probe. Rats were infused intravenously with 10 kDa FITC-dextran, and kidneys were fixed with paraformaldehyde lysine periodate. FITC-labeled vesicles were visualized in semithin (1 micron) frozen sections of excised tissue by epifluorescent microscopy and by electron microscopy after a photoconversion reaction. Most FITC-labeled endosomes were apically located in epithelial cells lining the urinary tubules. By immunocytochemistry the anti-lysosomal glycoprotein LGP 120 was absent from most of the FITC-labeled vesicles, although some colocalization was noted. The limiting membrane of FITC-labeled endosomes contained a vacuolar proton pump (pHmin = 6.23 +/- 0.033) and a water channel (osmotic water permeability coefficient, Pf = 0.052 +/- 0.005 cm/s) and was highly permeable to ethylene glycol and urea but relatively impermeable to glucose. Methods allowing the attribution of specific membrane functions to vesicles that can be visualized in the apical endocytic pathway of epithelial cells should be of general use for the study of endocytic pathways in a variety of systems.
Regulation of oxidative phosphorylation in the mammalian cell
Authors: Balaban R. S.
Published: December 2017

The cell is capable of maintaining a steady-state flux of energy from mitochondrial oxidative phosphorylation, producing ATP, to the cytosolic adenosinetriphosphatases (ATPases), performing work. Considerable effort has been devoted to investigating the individual mechanisms involved in these two processes. However, less effort has been directed toward learning how these reactions of energy metabolism interact through the cytosol to maintain the observed steady state in the intact cell. The "classical" model for the cytosolic interaction of these two processes involves the feedback of ATP hydrolysis products, ADP and Pi, from the ATPases to oxidative phosphorylation. This model is based on data from isolated mitochondria in which the rate of oxidative phosphorylation is controlled by the concentration of ADP and Pi. Yet, recent data from intact tissues with high oxidative phosphorylation capacities (i.e., heart, brain, and kidney) indicate that the cytosolic concentration of ADP and Pi do not change significantly with work. These data imply that this simple feedback model is not adequate to explain the regulation of energy metabolism in these tissues. Other sites within the oxidative phosphorylation process must be playing a regulatory role or the kinetics of ATP synthesis must be very different than currently believed to establish the steady state. This review covers the potential sites within oxidative phosphorylation which may be regulated through cytosolic transducers to result in the necessary feedback network regulating the steady-state flow of energy in the cell. These sites will include substrate delivery to the cytochrome chain, the processes involved in the phosphorylation of ADP to ATP, and the delivery of oxygen.
A nonselective cation channel in rat liver cells is activated by membrane stretch
Authors: Bear C. E.
Published: December 2017

A 16-pS channel was studied using patch-clamp electrophysiology in freshly dissociated rat liver cells and rat hepatoma cells. The channel was found to be cation selective and permeable to Na+, K+, and Ca2+. Its gating was unaffected by addition of the calcium ionophore A23187 (5 microM) in the presence of extracellular Ca2+ (2 mM). Ca2+ channel blockers, nifedipine, verapamil, and lanthanum, failed to inhibit the channel. The channel was activated by stretch, applied as suction to the interior of the patch pipette, and by cell swelling, induced by hypotonic shock or organic solute uptake (10 mM L-alanine). Channel activation by cell swelling was transient, lasting approximately 1 min. An elevation in cytosolic Ca2+ was evoked by hypotonic shock, as measured using the fluorescent indicator indo-1/AM. This change in intracellular Ca2+ concentration was dependent on extracellular Ca2+. Inasmuch as the time course for this response corresponded to that of channel activation, it is likely that hypotonic shock stimulated Ca2+ influx through the stretch-activated channel. To determine the role for Ca2+ influx in regulatory volume decrease (RVD), cell volume changes after hypotonic shock were studied using a Coulter counter. RVD was slightly but significantly inhibited by depletion of extracellular Ca2+. On the basis of these results it is proposed that stretch-activated channels in liver cells permit the transient influx of Ca2+, which in turn acts to trigger changes in ion conductance or cytoskeletal components involved in cell volume regulation.
pH regulatory transport systems in a smooth muscle-like cell line
Authors: Putnam R. W.
Published: December 2017

The membrane transport systems responsible for pH regulation in BC3H-1 cells were studied using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). In nominally CO2-free Na N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (NHB) recovery from acidification after an NH4Cl pulse was reversibly inhibited by 1 mM amiloride or by Na-free solutions. On exposure to 5% CO2-HCO3 (external pH constant at 7.4), BC3H-1 cells alkalinized by approximately 0.3-0.4 pH unit. This CO2-induced alkalinization was unaffected by 1 mM amiloride, markedly reduced by 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and inhibited by Na-free solutions. On readdition of Na, cells rapidly alkalinized, even in the presence of 1 mM amiloride. Exposure to Cl-free CO2-HCO3 solutions caused a rapid alkalinization of nearly 1 pH unit that was abolished by SITS, largely independent of Na, unaffected by amiloride, and unchanged by membrane depolarization in high external K solutions. CO2-induced alkalinization was slowed by approximately 75% after prolonged exposure of cells to Cl-free NHB, but a distinct recovery from acidification remained in these Cl-depleted cells. This recovery was Na-dependent, SITS-inhibitable, and unaffected by depolarization in high-K solutions. In the presence of CO2, the acidification seen in response to NH4Cl-induced alkalinization was reduced 50% by 0.5 mM SITS. These data suggest that the regulation of pH in BC3H-1 cells is mediated by at least three transport systems: 1) Na-H exchange; 2) Cl-HCO3 exchange; and 3) electroneutral (Na + HCO3)-Cl exchange.(ABSTRACT TRUNCATED AT 250 WORDS)
Histamine receptors in human fibroblasts: inositol phosphates, Ca2+, and cell growth
Authors: Johnson C. L. , Johnson C. G. , Bazan E. , Garver D. , Gruenstein E. , Ahluwalia M.
Published: December 2017

Histamine stimulated inositol phosphate formation by human skin fibroblasts. The effect of histamine was reduced but still readily apparent in the absence of extracellular Ca2+. Histamine caused a transient increase in intracellular free Ca2+ as detected by indo-1 and fura-2 fluorescence studies on cell populations and on individual cells. Similar increases were observed in the absence of extracellular Ca2+, indicating that the effect was primarily due to mobilization of Ca2+ from intracellular stores, presumably by inositol trisphosphate (IP3). The effects of histamine on phosphoinositide metabolism and intracellular Ca2+ were inhibited by pretreatment of the cells with phorbol esters, suggesting that the histamine receptor in fibroblasts is subject to feedback regulation by protein kinase C. Histamine inhibited the incorporation of [3H]-thymidine into DNA. The effects of histamine on inositol phosphate formation, intracellular Ca2+, and thymidine incorporation were blocked by the H1 receptor antagonist mepyramine. Our results indicate that human skin fibroblasts have H1 receptors coupled to the formation of inositol phosphates and mobilization of intracellular Ca2+. We suggest that this H1 receptor also mediates a block of the cell cycle and that histamine may play a physiological role in the regulation of fibroblast proliferation.
A thermodynamic model of hemoglobin suitable for physiological applications
Authors: Yoshida T. , Dembo M.
Published: December 2017

We propose a quantitative model of the thermodynamics of hemoglobin in contact with its five major ligands (O2, CO2, Cl-, 2,3-bisphosphoglycerate, and H+). Our model incorporates the two-state formalism of J. Monod, J. Wyman, and J.P. Changeux (J. Mol. Biol. 12: 88-118, 1965) for treatment of quanternary transitions and also the mean field formalism of K. Linderstrom-Lang (C. R. Trav. Lab. Carlsberg Ser. Chim. 15: 1-30, 1924) for treatment of electrostatic interactions. On the basis of this approach, we develop an algorithm for the efficient computation of observable quantities, such as the occupancy of various ligand binding sites, and an objective statistical procedure for determining both maximum likelihood values and confidence limits of all the intrinsic thermodynamic parameters of hemoglobin. Finally, we show that the predictions of our theory are in good agreement with independent experimental observations.
Irreversible inhibition of renal Na(+)-Pi cotransporter by alpha-bromophosphonoacetic acid
Authors: Szczepanska-Konkel M. , Hoppe A. , Lin J. T. , Dousa T. P.
Published: December 2017

We investigated the suitability of alpha-bromophosphonoacetic acid (alpha-BrPAA) to act as a possible irreversible inhibitor of Na(+)-dependent transport of Pi across renal brush-border membrane (BBM). When added directly into the Pi uptake medium, alpha-BrPAA causes specific, competitive [apparent inhibition constant (Ki) = 0.33 mM; no change in maximum velocity (Vmax)], and reversible (by washing) inhibition of Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by BBM vesicles (BBMV). Next, BBMV were preincubated with 5 mM alpha-BrPAA in alkaline (pH 9) medium for 30 min, then twice washed by 1:100 dilution and recentrifugation, and tested for transport and other properties. This preincubation of BBMV with alpha-BrPAA in alkaline medium resulted in a different type of inhibition [lower Vmax; no change in Michaelis constant (Km)] of the Na+ gradient-dependent uptake of 32Pi, whereas the uptakes of D-[3H]glucose and other solutes were not altered. This inhibition of Pi transport was not reversed by dilution and washing of BBMV. The BBMV Na(+)-dependent binding of [14C]phosphonoformic acid, but not of [3H]phlorizin, was decreased; activities of BBM marker enzymes were not changed. Results suggest that alpha-BrPAA binds onto the same locus on luminal surface of BBM on which Pi and Na+ bind and inhibits Na(+)-Pi cotransporter similar to phosphonoformic acid. Furthermore, after a 30-min incubation in alkaline medium, alpha-BrPAA apparently forms a more stable association with BBM in the vicinity of the Na(+)-Pi cotransporter. We thus suggest that alpha-BrPAA acts under these conditions as an apparently irreversible inhibitor of Na(+)-Pi cotransporter in BBM.(ABSTRACT TRUNCATED AT 250 WORDS)
Ligand binding and G protein coupling of muscarinic receptors in airway smooth muscle
Authors: Lucchesi P. A. , Scheid C. R. , Romano F. D. , Kargacin M. E. , Mullikin-Kilpatrick D. , Yamaguchi H. , Honeyman T. W.
Published: December 2017

Ligand binding properties of muscarinic receptors were examined in membranes and isolated cells prepared from bovine trachea. The binding of the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to both membranes and isolated cells was saturable, reversible, and of high affinity [dissociation constant (KD) = 100-200 pM]. The binding constants of three selective antagonists, pirenzepine, AF-DX 116, and 4-DAMP, were examined, and the results indicate that the smooth muscle cells contain at least two receptor subtypes. The majority of receptors exhibit binding constants for these selective antagonists similar to those of the M2-subtype. AF-DX 116 binding curves indicated the presence of a small population of receptors with binding constants similar to those reported for the M3-subtype. These findings suggest that the smooth muscle cells may contain both M2- and M3-receptors and are in agreement with evidence of the presence of mRNAs coding for these two subtypes in tracheal extracts (A. Maeda, T. Kubo, M. Mishina, and S. Numa. FEBS Lett. 239: 339-342, 1988). [3H]QNB displacement curves of the muscarinic agonist oxotremorine were best described as a sum of binding to high- and low-affinity sites with KD values of 3.8 nM and 2.2 microM. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) shifted the high-affinity sites to low affinity, suggesting that the high-affinity sites may represent receptors coupled to G proteins. Pertussis toxin catalyzed the ADP ribosylation of a 40- to 41-kDa protein band present in the membranes but had no significant effect on high-affinity agonist binding, suggesting that most of the receptors are coupled to G proteins in a toxin-insensitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Loop diuretic-sensitive potassium flux pathways of rat glomerular mesangial cells
Authors: Homma T. , Hoover R. L. , Harris R. C.
Published: December 2017

Glomerular mesangial cells are smooth muscle-like contractile cells that mediate hormonal regulation of glomerular filtration. To gain better understanding of ionic events that accompany contraction/relaxation of these cells, flux pathways of K+ and their regulation by vasoactive agents were investigated in cultured rat mesangial cells using 86Rb+ as a tracer. Of total 86Rb+ influx (16.6 +/- 0.6 nmol x mg protein -1 x min-1), 46% was inhibited by 2 mM ouabain. Loop diuretics inhibited 43% of ouabain-insensitive 86Rb+ influx (3.9 +/- 0.2 nmol x mg protein-1 x min-1). Half-maximum inhibition was observed at 2 and 0.4 microM for furosemide and bumetanide, respectively. Loop diuretic-sensitive 86Rb+ influx was dependent on extracellular Na+ and Cl-; a hyperbolic dependency on extracellular Na+ was noted with apparent Michaels constant of 39 mM while a sigmoidal dependency on extracellular Cl- was present, which, assuming 1:1:2 stoichiometry for Na(+)-K(+)-Cl-, produced an apparent mean affinity constant of 64 mM. Moreover, a fraction of amiloride-insensitive 22Na+ influx was found to be sensitive to furosemide and dependent on extracellular K+ and Cl-, further indicating the presence of Na(+)-K(+)-Cl- cotransport. Efflux of 86Rb+ followed first-order kinetics, of which 60% was inhibitable by furosemide. Manipulations of extracellular osmolarity revealed that these furosemide-sensitive 86Rb+ flux pathways were coordinately regulated in response to osmotic stress. Concentration-dependent stimulation of Na(+)-K(+)-Cl- cotransport-mediated 86Rb+ influx was induced by two vasoconstrictors, angiotensin II (ANG II) and arginine vasopressin (AVP), and by a vasodilator, atrial natriuretic peptide (ANP).(ABSTRACT TRUNCATED AT 250 WORDS)
Na(+)-K(+)-ATPase gene expression during in vitro development of rat fetal forebrain
Authors: Corthesy-Theulaz I. , Merillat A. M. , Honegger P. , Rossier B. C.
Published: December 2017

The existence of at least three isoforms of Na(+)-K(+)-ATPase in adult brain tissues [alpha 1, kidney type; alpha 2 [or alpha(+)]; alpha 3] suggests that these genes might be regulated in a cell-specific and time-dependent manner during development. We have studied this question in serum-free aggregating cell cultures of mechanically dissociated rat fetal telencephalon. At the protein level, the relative rate of synthesis of the pool of alpha 1-, alpha 2-, and alpha 3-subunits increased approximately twofold over 15 days of culture, leading to a marked increase in the immunochemical pool of alpha-subunits as measured by a panspecific polyclonal antibody. Concomitantly, Na(+)-K(+)-ATPase enzyme-specific activity increased three- (lower forebrain) to sixfold (upper forebrain). The transcripts of all three alpha-isoforms and beta-subunit were detected in vitro in similar proportion to the level observed in vivo. alpha 3-mRNA (3.7 kb) was more abundant than alpha 1 (3.7 kb) or alpha 2 (5.3 and 3.4 kb). Cytosine arabinoside (0.4 microM) and cholera toxin (0.1 microM) were used to selectively eliminate glial cells or neurons, respectively. It was found that alpha 2-mRNA is predominantly transcribed in glial cell cultures, whereas alpha 3- and beta 1-mRNA (2.7, 2.3, and 1.8 kb) are predominant in neuronal cultures.
Characterization of a muscarinic receptor controlling Cl- secretion in hen trachea
Authors: Winding B. , Bindslev N.
Published: December 2017

We have attempted to characterize a muscarinic receptor subtype involved in Cl- secretion in isolated epithelium of hen trachea, taking advantage of drugs developed in the last 15 yr. Hen trachea can be stimulated to secrete Cl- equal to approximately 80-90 microA/cm2 by application of acetylcholine (ACh) to the serosal side. The process has an apparent dissociation constant (Kd) for ACh of 740 nM. The Cl- secretion is completely inhibited by 20 microM bumetanide at the serosal side. Of five selective antagonists for muscarinic receptors, pirenzepine, hexahydrosiladifenidol, dicyclomine, 11-([2-[(diethylamino)-methyl]-1-piperidinyl]acetyl)-5,11- dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, and 4 diphenyl acetoxy-N-methylpiperidine methobromide, only the latter had a high affinity for the functional receptor with an apparent Kd of 3 nM. The receptor may be classified as an M4-muscarinic receptor subtype and probably belongs to a group of muscarinic receptors on exocrine glands and mucosal cells involved in ion transport. All the functional responses caused by muscarinic agonists and antagonists tested exhibited exponents (apparent Hill coefficients) in the range from 1.3 to 2.4, indicating a gain in the stimulus secretion coupling mechanism, an aspect of muscarinic receptor function that is not revealed in radioligand binding studies.
Effects of low extracellular Ca2+ on cytosolic free Ca2+, Na+, and pH of MDCK cells
Authors: Borle A. B. , Borle C. J. , Dobransky P. , Gorecka-Tisera A. M. , Bender C. , Swain K.
Published: December 2017

The effects of lowering the Ca2+ electrochemical potential (delta mu Ca2+) by removing extracellular calcium (Ca2+o) was studied in Madin-Darby canine kidney (MDCK) cells. We measured cytosolic free calcium (Ca2+i), intracellular Na+ (Na+i), and intracellular pH (pHi) as well as Ca2+ and Na+ influx and efflux and H+ secretion. As soon as Ca2+o was lowered to zero, Ca2+ efflux increased 2-fold, and Na+ influx increased 4.3-fold. As a result, Ca2+i fell 71% from 120 to 34.7 nM, and Na+i rose 56% from 16.7 to 26.1 mM. At the same time, H+ secretion was depressed 38%, and pHi fell from 7.32 to 7.22. When Ca2+o was restored to normal (1.3 mM), there was a sharp rise in Ca2+i from 34.7 to 243 nM (+600%) caused by an immediate increase in Ca2+ influx (+112%). At the same time, Na+ efflux increased 20%. Ten to fifteen minutes after restoring Ca2+o to 1.3 mM, Ca2+i, Na+i, and pHi were back to control levels. These results suggest that reducing delta mu Ca2+ activates the forward mode of Na(+)-Ca2+ exchange which leads to a fall in Ca2+i and a decreased delta mu Na+ by increasing Na+i. The fall in delta mu Na+ secondarily depresses Na(+)-H+ exchange which results in a reduced H+ secretion and a fall in pHi. When Ca2+o is restored to 1.3 mM, the reverse mode of Na(+)-Ca2+ exchange is activated, since delta mu Ca2+ is larger than normal because of the low Ca2+i, and delta mu Na+ is reduced because of the high Na+i.(ABSTRACT TRUNCATED AT 250 WORDS)
Decrease in Na(+)-K(+)-ATPase associated with maturation of sheep reticulocytes
Authors: Blostein R. , Grafova E.
Published: December 2017

Na(+)-K(+)-ATPase of immature and mature sheep red blood cells of both the high-K+ and low-K+ genotype and of immature cells matured in vitro was detected using polyclonal antiserum to purified sheep kidney Na(+)-K(+)-ATPase. This antiserum detects both alpha (alpha 1)- and alpha + (alpha 2 and/or alpha 3)-isoforms of the catalytic subunit as well as the beta-subunit of brain and kidney Na(+)-K(+)-ATPase. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, a single major immunologically reactive component corresponding to the alpha-subunit was detected in membranes of immature and mature cells from sheep of both genotypes. Cells separated according to increasing density showed a corresponding decrease in ouabain binding sites on the cells and Na(+)-activated ATP hydrolysis of membranes isolated from the cells. A progressive decrease in immunologically reactive alpha-subunit was also observed. After in vitro culture of reticulocytes, reduction in ouabain binding to the cells was also associated with loss in alpha-subunit. As well, appearance of immunologically reactive alpha-subunit was detected in membranous material shed into the incubation medium, accounting for a fraction (less than or equal to 30%) of the material lost from the cells. Proteolytic sensitivity of the alpha-subunit indicates that, in this material, the cytoplasmic surface of the enzyme is exposed to the medium. The shed material was largely devoid of function as evidenced in little, if any, Na(+)-dependent phosphorylation of Na(+)-K(+)-ATPase. The existence in reticulocytes of an intracellular pool of ouabain binding sites was indicated by the transient appearance on the cell surface of ouabain binding sites after rapid ATP depletion and also after addition of chloroquine to cells during culture. Taken together, these findings indicate that the maturation-associated loss of sodium pump protein involves, at least partly, energy-dependent endocytosis and, presumably, processing whereby inactivation of function occurs as well as release of pump protein into the extracellular milieu.
Increased secretion of erythropoietin in human renal carcinoma cells in response to atrial natriuretic factor
Authors: Ueno M. , Rondon I. , Beckman B. , Brookins J. , Nakashima J. , Cole F. E. , Fisher J. W.
Published: December 2017

The present studies were undertaken to assess the effects of atrial natriuretic factor (ANF) on erythropoietin (Ep) secretion in Ep-producing renal carcinoma (RC) cells using a sensitive radioimmunoassay for Ep. Human ANF produced a significant dose-related increase in Ep secretion at concentrations of 10(-7) and 10(-6) M when compared with vehicle controls. ANF (greater than or equal to 10(-9) M) also significantly increased the intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration after 5-min incubation with the RC cells. Scatchard analysis of the human 125I-labeled ANF binding data indicated that the RC cells contain a single class of binding sites with a dissociation constant (Kd) of 93 +/- 1 pM and a binding capacity of 2,190 +/- 750 sites/cell. Incubation of the RC cells with 8-bromo-cGMP in concentrations of 10(-7)-10(-5) M also produced a significant dose-related enhancement of Ep secretion. These findings suggest that the increase in Ep secretion in response to ANF can be attributed, at least in part, to activation of guanylate cyclase, which is coupled to specific ANF receptors on the RC cell.
Duramycin enhances chloride secretion in airway epithelium
Authors: Cloutier M. M. , Guernsey L. , Mattes P. , Koeppen B.
Published: December 2017

The effect of duramycin, a polypeptide antibiotic, on Cl- transport in canine tracheal epithelium mounted in Ussing chambers was studied. Over a narrow concentration range, duramycin increased short-circuit current (Isc) and net Cl- secretion and had no effect on mannitol flux when added to the mucosal bathing solution. The maximum increase in Isc was observed at a duramycin concentration of 2 X 10(-6) M and was associated with an increase in both unidirectional Cl- fluxes. Higher duramycin concentrations produced a decrease in Isc. Submucosal addition of duramycin had no effect on Isc except at high concentrations. Pretreatment of tissues with mucosal amiloride (10(-4) M) to reduce basal Na+ transport had no effect on the subsequent response to duamycin. In other tissues pretreated with 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), duramycin produced a further increase in Isc and net Cl- secretion similar to its effect in nonpretreated tissues. In all instances the increase in Isc was entirely accounted for by an increase in net Cl- secretion. We conclude that duramycin increases Isc and Cl- secretion in airway epithelium. Although the mechanism of activation is not known, these data demonstrate that duramycin increases Cl- secretion by a pathway other than cAMP. An understanding of the mechanism of action of duramycin may further our understanding of Cl- secretion regulation in airway epithelium.
Phorbol ester-stimulated human neutrophil membrane depolarization is dependent on Ca2(+)-regulated Cl- efflux
Authors: Myers J. B. , Cantiello H. F. , Schwartz J. H. , Tauber A. I.
Published: December 2017

The ionic basis of phorbol 12-myristate 13-acetate (PMA)-stimulated membrane depolarization in the human neutrophil has not previously been established. Alterations in cation permeability are probably not directly responsible for the depolarization response, since the rate or Rb+ influx or efflux is unchanged upon PMA stimulation, and although Na+ fluxes are increased, depolarization is not changed by either the addition of ouabain or reduction of extracellular Na+ from 140 to 0 meq. Furthermore, the enhanced Na+ influx in stimulated cells is blocked by amiloride at 10(-3) M, but not by 10(-5) M, suggesting Na+ influx proceeds through the electroneutral Na(+)-H+ antiporter and is therefore not responsible for depolarization. Upon stimulation, Cl- content of PMA-stimulated neutrophils decreases without change in Na+ or K+ content, as determined by electron probe analysis. In addition, acute reduction in extracellular Cl- enhances the rate and extent of depolarization induced by PMA. This change in intracellular Cl- and effect of reduction in extracellular Cl- concentration on depolarization can best be accounted for by an enhanced efflux via an electrogenic mechanism. Thus enhanced conductive Cl- efflux can account for the observed depolarization. That Ca2+ regulates depolarization is evidenced by the dependence of depolarization on external Ca2+ (Cao2+). Depolarization is absent in Ca2(+)-depleted cells [internal Ca2+ (Cai2+) less than 15 nM] and is restored with titration of extracellular Ca2+, exhibiting a 50% effective dose (ED50) of 100 mM. Thus PMA-initiated depolarization is regulated by Ca2+, either from intra- or extracellular sources, but the Ca2(+)-dependent activity responsible for control of Cl- efflux is as yet uncharacterized.
Nonmetabolizable glucose analogues and ornithine decarboxylase expression in LLC-PK1 cells
Authors: Lundgren D. W. , Vacca C. V.
Published: December 2017

This report examines the effect of nonmetabolizable glucose analogues on ornithine decarboxylase (ODC) activity in LLC-PK1 cells. The addition of Na(+)-dependent cotransported glucose analogues, 1-O-methyl-alpha-D-glucopyranoside (alpha-MDG) and 1-O-methyl-beta-D-glucopyranoside, to Earle's balanced salt solution minus glucose (EBSS-G) increased ODC activity five- to sevenfold above basal levels. The passive carrier-mediated transported glucose analogue 3-O-methyl-D-glucopyranose had very little effect on enzyme activity. alpha-MDG increased ODC activity in quiescent but not growing cells. ODC activity increased as a function of both the incubation time in EBSS-G + alpha-MDG and the concentration of alpha-MDG in EBSS-G. Phlorizin significantly reduced the level of enzyme activity induced by alpha-MDG. ODC expression by alpha-MDG was reduced in cells incubated in hypertonic EBSS-G + alpha-MDG. Enzyme activity, in the absence of extracellular organic substrates, was markedly elevated in cells incubated in hypotonic media. It is suggested that an influx of Na+ and/or an increase in cell volume elevates one or more signal transducers that regulate ODC expression.
Anion selectivity and block of the small-conductance chloride channel on pancreatic duct cells
Authors: Gray M. A. , Pollard C. E. , Harris A. , Coleman L. , Greenwell J. R. , Argent B. E.
Published: December 2017

Rat and human pancreatic duct cells have small-conductance Cl- channels in their apical plasma membranes. These channels are regulated by secretin and adenosine 3',5'-cyclic monophosphate and may function in parallel with Cl(-)-HCO3- exchangers to allow HCO3- secretion from the duct cell. Using the patch-clamp technique, we have now determined the anion permeability sequence of the channel as NO3- greater than Br- approximately I- approximately Cl- much greater than HCO3- much greater than gluconate. From this we conclude 1) that anion permeation involves a weak interaction with charged sites inside the channel pore, 2) that because of the low HCO3-/Cl- permeability ratio it is unlikely that significant amounts of HCO3- could be secreted directly via the channel, and 3) that channel permeability may determine the anion selectivity of secretion. We also show that 5-nitro-2-(3-phenylpropylamino)benzoic acid blocks the small-conductance Cl- channel, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid has no effect.
Basal and stretch-augmented natriuretic peptide secretion by quiescent rat atria
Authors: Page E. , Goings G. E. , Power B. , Upshaw-Earley J.
Published: December 2017

Absolute rates of immunoreactive atrial natriuretic peptide (ANP) secretion were measured in vitro at 37 degrees C in noncontracting preparations of combined right and left rat atria at constant distending pressures of 0 or 5.1 mmHg in presence of 0.2 mM extracellular Ca2+ concentration [( Ca2+]o), 10 microM ryanodine, and either 1 microM saxitoxin or 10 microM tetrodotoxin. By systematic deletion of external Na+, K+, Mg2+, Cl-, or HCO3-, and reduction of [Ca2+]o, and by selective ion transport inhibitors, neither net transplasmalemmal fluxes of Na+, Cl-, HCO3-, and Mg2+ nor ryanodine-sensitive Ca2+ release from sarcoplasmic reticulum (SR) was found necessary for stretch augmentation of secretory rate (Ra). Ra 1) was near zero within 20 min when extracellular Na+ concentration = 0 and [Ca2+]o = 20 microM and within 5 min or less after preincubation with caffeine, 8-chlorophenylthioadenosine 3',5'-cyclic monophosphate, or at 18 degrees C; 2) was significantly decreased by Cd2+, Ni2+, the isoquinoline H-7, and trifluoperazine but not 100 microM ryanodine; 3) was increased by neomycin; and 4) had an apparent activation energy of 18.5 +/- 4.1 x 10(3) cal/mol between 23 and 42 degrees C. These experiments strongly implicated transplasmalemmal Ca2+ influx and cAMP but not SR Ca2+ release in control of Ra under the experimental conditions studied.
Calcium oscillations and morphological transformations in single cultured gastric parietal cells
Authors: Ljungstrom M. , Chew C. S.
Published: December 2017

Calcium is an important regulator of cellular activities including HCl secretion by parietal cells. With cholinergic agonists, a role for calcium is established; however, with histamine, at least two signaling pathways may be involved including calcium and adenosine 3',5'-cyclic monophosphate (cAMP). Because chelation of medium and/or cellular calcium has pronounced inhibitory effects on cholinergic but lesser effects on histamine-stimulated acid secretory responses in cell populations, the calcium pathway may not be of central importance for HCl secretion regulated by histamine. We have used digitized video imaging of fura-2 fluorescence ratios and cellular morphology to determine more precisely the relationship between cellular calcium signaling mechanisms and acid secretion in single cultured rabbit parietal cells. Calcium signaling patterns were found to exhibit striking differences with histamine as compared with the cholinergic agonist carbachol. Maximal doses of histamine initiated repetitive oscillations in intracellular calcium ([Ca2+]i) in approximately 50% of cells, whereas the maximal carbachol response was characterized by a typical initial spike followed by a sustained elevation in [Ca2+]i. Oscillations in response to carbachol were detected only at doses below the half-maximal concentration for initiation of acid secretion. Correlation of gradual expansion of acidic vacuoles with increases in [Ca2+]i in the same cells indicated that approximately 20% of cells increased acid secretory-related activities in response to histamine with no detectable rise in [Ca2+]i. These data suggest two possibilities: 1) a rise in [Ca2+]i is not necessary for histamine-stimulated HCl secretion, or 2) heterogeneous receptor-coupling mechanisms exist in parietal cell populations with either calcium or cAMP mechanisms predominating in different subpopulations. The ability to assess simultaneously acid secretory-related responses and calcium signaling patterns allows, for the first time, correlation of "physiological" and biochemical responses in single parietal cells. This methodology is expected to provide new insight into second messenger control mechanisms that are not possible either in cell populations or acutely isolated parietal cells that do not exhibit morphological transformations detectable at the light microscope level.
Uncoupling of cardiac cells by fatty acids: structure-activity relationships
Authors: Burt J. M. , Massey K. D. , Minnich B. N.
Published: December 2017

The permeability and conductance of gap junctions between pairs of neonatal rat heart cells were rapidly and reversibly decreased by oleic acid in a dose- and time-dependent manner. Other unsaturated fatty acids (C-18: cis 6, 9, or 11, and C-18, 16, and 14, cis 9), saturated fatty acids (C-10, 12, and 14), and saturated fatty alcohols (C-8, 10, and 12) also caused uncoupling. The most effective compounds of the unsaturated and saturated fatty acid and saturated fatty alcohol series caused essentially complete uncoupling at comparable aqueous concentrations. However, oleic acid uncoupled cells at membrane concentrations as low as 1 mol%, whereas decanoic acid required upwards of 35 mol%. The channels that support the action potential remained functional at these same membrane concentrations. The data are discussed in terms of the possible mechanism by which these compounds cause uncoupling and the possible role of uncoupling by nonesterified free fatty acids in the initiation of arrhythmias during and after ischemic insults.
Unusual [Ca2+] dependence of vascular smooth muscle cell shortening velocity
Authors: Dougherty T. J. , Driska S. P.
Published: December 2017

The relationship between unloaded shortening velocity and Ca2+ concentration was determined for hog carotid arterial smooth muscle cells, freshly isolated by digestion with papain. Cells were exposed to various [Ca2+] for 60 s at 37 degrees C and then stimulated with 10 microM histamine. Cell length was measured by a video analysis system. Shortening velocity was expressed as an exponential rate constant by fitting the cell lengths to the following equation: length = Lmin + (Lmax-Lmin)exp[-v (time-latency)], where Lmax is length before contraction, Lmin is shortest length reached, time is time elapsed after addition of agonist, latency is time from addition of agonist until contraction starts, and v is the exponential rate constant (s-1). Cells shortened substantially, usually reaching one-fourth to one-third of their initial length within 1 min. Shortening velocities of the cells were much faster than published values of maximum shortening velocity in muscle strips from this same tissue. At 1.6 mM Ca2+, v was 0.173 +/- 0.015 s-1. When Ca2+ was increased to 5 or 10 mM, v was not significantly different. However, when Ca2+ was decreased to 0.5 and 0.16 mM, v increased to 0.288 and 0.258 s-1, respectively. The difference between 0.5 and 1.6 mM was significant. The unexpected increase in shortening velocity at low Ca2+ was also seen when 10 mM caffeine was used as a stimulus: v at 1.6 mM Ca2+ was 0.156 s-1, whereas v at 0.16 mM Ca2+ was 0.272 s-1. The high shortening velocities we measured suggest that measurements made on multicellular tissues seriously underestimate the potential shortening velocity of isolated individual cells.
Role of cytosolic Ca in renal tubule damage induced by anoxia
Authors: Jacobs W. R. , Sgambati M. , Gomez G. , Vilaro P. , Higdon M. , Bell P. D. , Mandel L. J.
Published: December 2017

Cytosolic free Ca (Caf) was measured in three different preparations of freshly prepared proximal tubules from the rabbit kidney during energy deprivation using fura-2. Isolated perfused tubules, tubules immobilized on glass cover slips, and tubules in suspension were subjected to inhibitors of oxidative phosphorylation (“chemical hypoxia”); the latter two preparations were also subjected to 40 min of anoxia. During normoxia, Caf ranged from 100 to 180 nM in all three preparations, and chemical hypoxia caused either no change or a small (30-100%) increase in Caf values. Subsequent addition of Ca ionophores increased Caf to 300-500 nM in the first 2 min and to greater than 1 microM after 15 min. In individual experiments, anoxia produced similar responses to those of chemical hypoxia, eliciting no average significant change in Caf, despite clear evidence for impaired respiration and plasma membrane damage after 40 min of anoxia. This lack of change in Caf was unrelated to “Ca buffering” by fura-2 or inactivation of the dye, since Caf increased to 666 +/- 59 nM upon addition of Ca ionophore during anoxia. These data suggest that increased Caf is not a prerequisite for cellular damage during anoxia in proximal renal tubules. Furthermore, no apparent alteration in plasma membrane permeability to Ca occurs before membrane disruption. Decreased ATP seems to initiate a series of Caf-independent events that cause irreversible injury.
Several cation transporters and volume regulation in high-K dog red blood cells
Authors: Fujise H. , Yamada I. , Masuda M. , Miyazawa Y. , Ogawa E. , Takahashi R.
Published: December 2017

Normal dog red blood cells lack the Na-K pump, and their cation composition is low K and high Na (LK). Recently, a dog was found with red blood cells containing high K and low Na concentrations (HK) due to the existence of the Na-K pump. In the present study, cation transport and volume regulation in HK cells were compared with those of LK cells. HK cells showed not only Rb influx through a Na-K pump, but also Rb influx through a Cl-dependent K transporter. The Rb influx rate through the Na-K pump was 0.65-1.44 mmol.l cells-1.h-1 in Cl and 1.75-2.24 mmol.l cells-1.h-1 in NO3, in HK cells, but only trace activities are found in LK cells. In HK cells, the Rb influx rate through Cl-dependent K transport was 0.36-0.96 mmol.l cells-1.h-1, and it was enhanced in swollen cells but vanished in shrunken cells. In LK cells, the transport was evident only in swollen cells. The original volume of swollen HK cells was restored by water extrusion promoted by Cl-dependent transport. The Na-Ca exchange transporter, which works as a volume regulator in LK cells, functioned in HK cells only when they were loaded with Na. Hence, the exchange transporter is latent in HK cells under physiological conditions. Moreover, the exchange transporter could restore the cell volume in swollen and Na-loaded HK cells. However, the volume in HK cells was still larger than that in LK cells, while the Na-Ca exchange transporter was working.(ABSTRACT TRUNCATED AT 250 WORDS)
Cholinergic stimulation produces oscillations of cytosolic Ca2+ in a secretory epithelial cell line, T84
Authors: Devor D. C. , Ahmed Z. , Duffey M. E.
Published: December 2017

The effects of carbamylcholine (carbachol) on intracellular Ca2+ concentration ([Ca2+]c) of T84 cells were examined using the fluorescent Ca2+ indicator fura-2 and microfluorometric techniques. In single isolated cells, carbachol (100 microM) caused a rapid increase in [Ca2+]c of 184 +/- 15 nM (SE, n = 44) from a resting value of 56 +/- 7 nM. This initial transient was followed by a series of oscillations in 68% of the cells. Atropine (10 microM) blocked this response. Removal of bath Ca2+ did not inhibit the rise in [Ca2+]c or oscillations, but the response duration was shortened in 47% of the cells. The amplitude and latency of the initial Ca2+ rise, frequency of oscillations, and number of responding cells varied with the agonist concentration. We have previously shown that carbachol induces an oscillating K+ conductance in T84 cells [D. Devor, S. Simasko, and M. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990]. Simultaneous measurement of membrane K+ current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca2+]c and increase in K+ current. These results show that a rise in [Ca2+]c and oscillations is likely to underlie the membrane K+ current responses to carbachol in T84 cells. Responses from a single cell within a subconfluent monolayer were different from those of isolated cells. In cells of a monolayer the initial [Ca2+]c rise (111 +/- 8 nM; n = 41) was followed by a decline to a stable plateau, and oscillations were not seen. Removal of bath Ca2+ both reduced the initial transient and eliminated the plateau phase of the response. These results suggest that cell-to-cell contact or differentiation during monolayer formation influences the Ca2+ handling mechanisms of T84 cells.
K(+)-H+ exchange, a fundamental cell acidifier in corneal epithelium
Authors: Bonanno J. A.
Published: December 2017

Rabbit corneal epithelial cells, loaded with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxy-ethyl)-5(6)-carboxyfluorescein, show a profound acidification (pH 7.33 to 6.75) in HCO3(-)-free Ringer solution when exposed to the Na(+)-H+ exchange inhibitor amiloride. This indicates that the cells are under a constant acid load that is being countered by Na(+)-H+ exchange. Amiloride-induced acidification was affected neither by incubation in Cl(-)-free Ringer solution nor by hypoxia, indicating that the potential acid loaders, Cl(-)-HCO3- (or OH-) exchange or glycolytic metabolism, are not contributing to the acidification. The possibility of a H+ influx dependent on the outward K+ gradient was tested. Perfusion with a high-K+ Ringer solution (77 mM) caused Na(+)- and Cl(-)-independent alkalinizations. Membrane depolarization by gramicidin, Ba2+, Cl(-)-free Ringer solution, or ouabain all produced small (less than 0.1 pH units) acidifications, inconsistent with contribution by a membrane potential driven passive H+ influx. In Na(+)-free Ringer solution, intracellular pH (pHi) of 6.4-6.6, addition of nigericin (a 1K(+)-1H+ ionophore) produced no significant change in pHi, indicating that [K+]i/[K+]o = [H+]i/[H+]o. Both amiloride-induced acidifications and high-K(+)-induced alkalinizations were significantly stimulated by the presence of 1 mM ZnSO4 and unaffected by H2-DIDS (0.5 mM, an anion transport blocker) or 100 microM SCH28080 (K(+)-H(+)-ATPase blocker). In the absence of a demonstrable H+ conductance, it is concluded that amiloride-induced acidification and K(+)-induced pHi changes are via a carrier-mediated K(+)-H+ exchanger. In addition to pHi regulation, K(+)-H+ exchange may play a role in cell volume control.(ABSTRACT TRUNCATED AT 250 WORDS)
Increased Na(+)-H+ antiporter activity in apical membrane vesicles from mutant LLC-PK1 cells
Authors: Reilly R. F. , Haggerty J. G. , Aronson P. S. , Adelberg E. A. , Slayman C. W.
Published: December 2017

In whole cell experiments, the PKE20 mutant of the renal epithelial cell line LLC-PK1 displays a severalfold elevation of Na(+)-H+ antiporter activity at the apical surface (J.G. Haggerty, N. Agarwal, R.F. Reilly, E. A. Adelberg, and C.W. Slayman. Proc. Natl. Acad. Sci. USA 85: 6797-6801, 1988). The present study was undertaken to explore the properties of the mutant at the membrane level. Apical membrane vesicles were prepared by the magnesium-aggregation technique, with a similar enrichment (ca. 10-fold) of the marker enzyme gamma-glutamyltranspeptidase in vesicles from parent and mutant cell lines. In both cases, 22Na influx was stimulated by an inside-acid pH gradient, inhibited by ethylisopropylamiloride (EIPA), and unaffected by valinomycin, indicating that it was mediated by Na(+)-H+ antiport. Quantitatively, PKE20 vesicles showed a 4.2-fold increase in the maximal velocity of Na(+)-H+ antiporter activity compared with the parent, with only minor increases in the activity of two other Na(+)-dependent transporters (14-56% for alpha-methylglucoside and L-glutamate). Dose-response curves for EIPA indicated that the increased Na(+)-H+ antiport activity in PKE20 vesicles was due to an increased activity of the relatively amiloride-resistant form of the Na(+)-H+ antiporter with little or no change in the amiloride-sensitive form. No differences in polypeptide composition of the two vesicle preparations could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Taken together, the results indicate that the mutation in PKE20 is expressed at the membrane level and is specific for the relatively amiloride-resistant Na(+)-H+ antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)