bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell subpopulations within proliferative and differentiating compartments of epidermis
Authors: Pavlovitch J. H. , Rizk-Rabin M. , Gervaise M. , Metezeau P. , Grunwald D.
Published: December 2017

The heterogeneous population of newborn rat keratinocytes was separated into different subgroups according to their cell size. The relation between cell size, position in the cell cycle, RNA content, and proliferative potential in culture was examined. A reserve stem cell population of Go/G1 cells, low in RNA, giving rise to colonies of undifferentiated phenotype in cell culture, has been separated from more differentiated transit basal cells. In the fractions of the larger cells, several subgroups, probably corresponding to different stages of differentiation, were identified: G2M cells with low RNA content, large S-phase cells rich in RNA, and small Go/G1 cells low in RNA. The clonogenic cells from these fractions have limited growth potential and give rise to moderately or terminally differentiated colonies. The selective sorting of stem cell populations may be useful for elucidating the mechanism of carcinogenesis in epidermis and other proliferative tissues. Analysis of the relative proportions of cell subpopulations represents a novel approach leading to the refinement of the concepts of epidermal structure in physiological and pathological states. It also could, by extension, shed new light on the behavior of other proliferative tissues.
Role of chloride in potassium transport through a K-Cl cotransport system in human red blood cells
Authors: Brugnara C. , Van Ha T. , Tosteson D. C.
Published: December 2017

In this paper, we report experiments demonstrating the coupling of Cl and K movements in a volume-dependent K-Cl cotransport system in human red blood cells. We show that an outwardly directed Cl gradient can promote net K efflux against an inwardly directed K gradient at constant membrane potential. Red blood cell membrane potential was kept constant by using anions that are not transported through the K-Cl cotransport system but that are more permeable than Cl (NO3 and SCN). Under these conditions, when the activities of band 3 (capnophorin)-mediated anion exchange and of the carbonic anhydrase have been inhibited, it is possible to maintain a Cl gradient at constant membrane potential. Similar data were obtained in human red blood cells (least dense fraction from normal subjects and whole blood from patients with homozygous hemoglobin S disease), in rabbit red blood cells, and in low-K sheep red blood cells. These data confirm that the volume-dependent Cl-dependent K movement in these cells operates through coupled K-Cl cotransport.
Kinetics of volume-sensitive K transport in human erythrocytes: evidence for asymmetry
Authors: Kaji D. M.
Published: December 2017

The kinetic properties of volume-sensitive K fluxes in swollen human erythrocytes were investigated. Swelling-activated Cl-dependent K influx was a saturable function of external K concentration with a low affinity (apparent Km of 115-130 mM) and high capacity [maximal velocity (Vmax) = 20-30 mmol.l original cells-1.h-1 (mmol.loc-1.h-1)]. The Vmax and apparent Km for Cl-dependent K efflux were lower (Km = 47 mM; Vmax = 2.2 mmol.loc-1.h-1). The Hill coefficients for both K efflux and influx were close to unity, suggesting a single binding site for K. The increase of external K trans-stimulated K efflux, but the increase of intracellular K had no effect on Cl-dependent K influx in swollen cells. Under zero trans conditions, the Vmax (18 vs. 3 mmol.loc-1.h-1) and Km (138 vs. 32) were markedly different for influx and efflux, respectively. These results provide evidence for intrinsic functional asymmetry, such that the transporter is more prevalent and stable in the outward-facing conformation. The mean ratio of Km to Vmax for efflux (12.1) was 1.56 times larger than the same ratio for influx (7.8), but the difference between the means did not reach statistical significance. These kinetic observations are analyzed in terms of the simple carrier and the cotransport models.
Enhanced glucose transport in response to inhibition of respiration in Clone 9 cells
Authors: Mercado C. L. , Loeb J. N. , Ismail-Beigi F.
Published: December 2017

An acceleration of ATP synthesis by anaerobic glycolysis provides important compensation for interference with respiration in a variety of cells. Effective compensation for an inhibition of respiration, however, can occur in cells in which glucose entry is rate limiting only if sufficient glucose becomes available through an enhancement of transport. We present here a detailed study of the effects of inhibition of respiration in Clone 9 cells, a continuous cell line characterized by low internal glucose concentrations (less than 10% that of the external medium) and minimal stores of glycogen. Exposure of these cells to 5 mM cyanide results in a 90% fall in cell ATP and a twofold rise in cell Na+ within 20 min. By the end of 1 h, however, there is a 4.5- to 7-fold increase in cytochalasin B-inhibitable glucose transport that is accompanied by a parallel increase in the rate of lactate production, a partial recovery of cell ATP, and no further rise in cell Na+. The acute fall in ATP resulting from a submaximally effective concentration of cyanide (0.5 mM) is moreover followed by a time-dependent recovery of cell ATP to near-normal levels and subsequent resistance to challenge with even 5 mM cyanide. The stimulation of facilitative glucose transport resulting from exposure to cyanide is attributable to an increase in maximal velocity rather than to a change in Km and persists for more than 2 h after removal of the inhibitor. These results demonstrate that, in these cells characterized by low internal glucose concentrations, regulation of glucose entry is of central importance in ATP homeostasis and that a major component of the adaptive response to an inhibition of respiration is a time-dependent increase in glucose transport.
Regulation of Na-K-Cl cotransport in endothelial cells by atrial natriuretic factor
Authors: O'Donnell M. E.
Published: December 2017

Many vasoactive agents have been shown to bind to specific receptors on endothelial cells. Among these is atrial natriuretic factor (ANF). Binding of ANF to endothelial cells has been demonstrated to induce elevation of intracellular guanosine 3',5'-cyclic monophosphate (cGMP). Other vasoactive agents have been shown to cause elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP), Ca, and diacylglycerol. However, the endothelial cell response that occurs subsequent to elevation of cGMP or other second messengers is not well understood. Recently, endothelial cells have been shown to possess a Na-K-Cl cotransport system that is stimulated by vasopressin and bradykinin and inhibited by isoproterenol. Thus it is possible that modulation of Na-K-Cl cotransport may play a role in the endothelial cell response to second messengers that are elevated by ANF and other vasoactive agents. This possibility was examined in the present study by evaluating the effects of a variety of vasoactive agents and their second messengers on endothelial cell Na-K-Cl cotransport. Cotransport was assessed as bumetanide-sensitive K influx in cultured bovine aortic endothelial cells. A number of agents were found to reduce Na-K-Cl cotransport, including ANF, acetylcholine, histamine, and norepinephrine. Cotransport was found to be stimulated by angiotensin II, as well as vasopressin and bradykinin. Na-K-Cl cotransport was also inhibited by elevation of intracellular cGMP or cAMP or by treatment of the cells with phorbol ester to activate protein kinase C. However, A23187-induced elevation of intracellular Ca caused stimulation of Na-K-Cl cotransport.(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence for a Ca-activated inwardly rectifying K channel in human macrophages
Authors: Gallin E. K.
Published: December 2017

Cell-attached patch studies of cultured human macrophages demonstrate that exposure to ionomycin induces inward-rectifying single-channel currents that differ from the voltage-dependent 28 pS inward-rectifying K currents previously described in these cells (J. Membr. Biol. 103: 55-66, 1988). With 150 mM KCl in the electrode and NaCl Hanks' solution in the bath, the ionomycin-induced single-channel conductance for inward currents was 37 pS, and the reversal potential was 57 mV. Channel activity was often associated with a shift in the base-line current level indicating that the cell membrane potential hyperpolarized. The ability of ionomycin to induce channel activity depended on extracellular [Ca] supporting the view that the channels were gated by calcium. Ionomycin-induced channels were permeable to K, relatively impermeable to Cl or Na, exhibited bursting kinetics, and had no apparent voltage dependence. Barium (3 mM in the patch electrode) did not significantly block the ionomycin-induced channel at rest but blocked channel activity when the patch was hyperpolarized beyond the resting membrane potential. Exposure of macrophages to platelet-activating factor, which is known to increase intracellular [Ca] [( Ca]i) (J. Cell Biol. 103: 439-450, 1986), also transiently induced channel activity. In excised patches with 3 microM [Ca]i bursting inward-rectifying channels with a 41 pS conductance were noted that probably correspond to the ionomycin-induced channels present in cell-attached patches. Increasing [Ca]i from 10(-8) to 3 x 10(-6) M induced inward-rectifying channel activity in previously quiescent excised patches.(ABSTRACT TRUNCATED AT 250 WORDS)
Stimulation of Cl- self exchange by intracellular HCO3- in rabbit cortical collecting duct
Authors: Matsuzaki K. , Stokes J. B. , Schuster V. L.
Published: December 2017

In rabbit cortical collecting duct, Cl- self exchange accounts for most of the transepithelial Cl- tracer rate coefficient, KCl (nm/s); a small fraction is effected by Cl--HCO3- exchange and Cl- diffusion. We previously reported that changing from a CO2-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) bath to a 5% CO2-25 mM HCO3- bath stimulates Cl- self exchange. Here, we examine in further detail the individual components of the CO2-HCO3- system that stimulate KCl. Addition of 0.5% CO2 to a HEPES bath (final pH = 7.24) stimulated KCl by 70 +/- 19 nm/s, a delta KCl comparable to that induced by 1% CO2 (pH 7.12), 6% CO2 (pH 6.6), or 6% CO2-25 mM HCO3- (pH 7.4). The roles of intracellular pH (pHi) and HCO3- concentration were examined by clamping pHi using high K+ and nigericin. Increasing pHi from 6.9 to 7.6 in solutions without exogenous CO2 or HCO3- increased KCl by 71 +/- 17 nm/s. These results suggest that pHi might regulate anion exchange. However, during such a pHi-shift experiment, metabolically derived CO2 produces a concomitant change in intracellular HCO3- concentration [( HCO3-]i). To determine whether an increase in [HCO3-]i could stimulate Cl- self exchange, we replaced HEPES with 6% CO2-5 mM HCO3- isohydrically (pHi clamped at 6.9). With this increase in [HCO3-]i at constant pHi, KCl increased by 51 +/- 10 nm/s. These maneuvers had negligible effects on Cl- diffusion and Cl--HCO3- exchange. These experiments demonstrate that increases in cell [HCO3-] (or perhaps CO2) can stimulate transepithelial anion exchange.(ABSTRACT TRUNCATED AT 250 WORDS)
Synthesis of angiotensinogen by renin-containing neuroblastomas
Authors: Petrossian G. , Oliver J. A.
Published: December 2017

Angiotensinogen in plasma is of hepatic origin, but many organs possess the ability to synthesize this protein because messenger RNA for angiotensinogen is widely distributed in the body. The cell types responsible for the extrahepatic synthesis of angiotensinogen remain to be identified. To examine whether renin-containing cells synthesize angiotensinogen, we have utilized a polyclonal antibody to angiotensinogen and immunoprecipitated metabolically labeled cells of two neuroblastomas known to contain renin. The results indicate that the cell line Neuro 2a synthesizes and releases a protein with a molecular mass of 57 kDa that is specifically recognized by the angiotensinogen antibody, indicating that Neuro 2a synthesizes angiotensinogen. Similarly, the cell line NB41A3 was also found to synthesize a protein specifically recognized by the antibody to angiotensinogen.
Effects of systemic inhibition of prostaglandin production on protein metabolism in tumor-bearing rats
Authors: Strelkov A. B. , Fields A. L. , Baracos V. E.
Published: December 2017

Two rat tumors, Morris hepatoma 7777 (MH) and Yoshida ascites hepatoma AH130 (YAH) were compared, and the influence of systemic inhibition of prostaglandin (PG) synthesis on muscle protein metabolism was evaluated. Tumor-bearing rats were compared with ad libitum- and pair-fed controls. Rats were also treated with naproxen, an inhibitor of PG synthesis. Tumors caused progressive anorexia and weight loss and resulted in decreased weight and/or protein content of the soleus, extensor digitorum longus, and epitrochlearis muscles. The extent of this wasting varied with muscle and tumor type. Muscle wasting induced by the tumors appeared to result from increased protein degradation and/or decreased protein synthesis, as determined in isolated epitrochlearis muscle. In YAH, reduced feed intake did not appear to be responsible for muscle wasting; however, in MH, it accounted for a significant proportion of the muscle loss. YAH produced large amounts of PGE2. Treatment of rats with naproxen inhibited tumor PGE2 production and muscle protein loss in rats bearing YAH. Naproxen had no effect on muscle weight or protein degradation in rats bearing MH. These results would appear to implicate PGE2 in the development of cachexia in the laboratory rat.
Fast Na+ and slow Ca2+ channels in single uterine muscle cells from pregnant rats
Authors: Ohya Y. , Sperelakis N.
Published: December 2017

Whole cell voltage-clamp method was applied to single smooth muscle cells freshly isolated from the longitudinal layer of 18-day pregnant rat uterus. Inward currents were isolated after outward currents were minimized by use of high Cs+ in the pipette solution and 4-aminopyridine (3 mM) in the bath solution. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed and disappeared within 30 ms and depended on extracellular Na+ concentration. This fast current was inhibited by tetrodotoxin (TTX) dose dependently (KD = 27 nM). These results suggest that the fast inward current was a TTX-sensitive Na+ channel current. In contrast, the slow inward current decayed slowly, dependent on extracellular Ca2+ (or Ba2+) concentration, and was inhibited by the Ca2+ channel blocker, nifedipine, dose dependently (10 nM-10 microM). These results suggest that the slow inward current was a Ca2+ channel current. A fast-inactivating Ca2+ channel current was not evident when Ba2+ was the charge carrier. We conclude that the major ion channels in the cell membrane of pregnant rat uterus, which generate inward currents, are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels (L-type, high-threshold type).
Cyanide toxicity in hepatocytes under aerobic and anaerobic conditions
Authors: Aw T. Y. , Jones D. P.
Published: December 2017

The effect of cyanide on cell viability and mitochondrial function was studied in hepatocytes exposed to air or argon. Cells were more susceptible to cyanide toxicity under air than under argon. Analysis of the disposition of cyanide showed that the difference in susceptibility to KCN was not due to O2-dependent differences in cyanide metabolism or elimination. Studies of mitochondrial function revealed that cyanide under aerobic conditions resulted in substantial swelling of the mitochondria, which corresponded to a matrix loading of phosphate. In addition, cyanide caused a loss of the mitochondrial protonmotive force. This was in contrast to the results for cells exposed to 30 min of anoxia alone in which there was no loss of mitochondrial delta pH, no detectable change in mitochondrial volume, and little matrix loading of phosphate. These results show that at least some of the protective mechanisms elicited by anoxia (B. S. Andersson, T. Y. Aw, and D. P. Jones. Am. J. Physiol. 252 (Cell Physiol. 21): C349-C355, 1987) are not elicited by cyanide alone. Thus cyanide under aerobic conditions does not provide a completely valid model for simple anoxia. Moreover, the results suggest that the molecular sensor necessary to signal suppression of metabolic and transport functions during neahypoxia is dependent on O2 and is neither stimulated nor antagonized by KCN.
Characterization of macroscopic outward currents of canine colonic myocytes
Authors: Cole W. C. , Sanders K. M.
Published: December 2017

Outward currents of colonic smooth muscle cells were characterized by the whole cell voltage-clamp method. Four components of outward current were identified: a time-independent and three time-dependent components. The time-dependent current showed strong outward rectification positive to -25 mV and was blocked by tetraethylammonium. The time-dependent components were separated on the basis of their time courses, voltage dependence, and pharmacological sensitivities. They are as follows. 1) A Ca2+-activated K current sensitive to external Ca2+ and Ca2+ influx was blocked by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (0.1 X 10(-3) M) and nifedipine (1 X 10(-6) and was increased by elevated Ca2+ (8 X 10(-6) M) and BAY K 8644 (1 X 10(-6) M). 2) A "delayed rectifier" current was observed that decayed slowly with time and showed no voltage-dependent inactivation. 3) Spontaneous transient outward currents that were blocked by ryanodine (2 X 10(-6) M) were also recorded. The possible contributions of these currents to the electrical activity of colonic muscle cells in situ are discussed. Ca2+-activated K current may contribute a significant conductance to the repolarizing phase of electrical slow waves.
Utility of AICAr for metabolic studies is diminished by systemic effects in situ
Authors: Foley J. M. , Adams G. R. , Meyer R. A.
Published: December 2017

It has been reported that intraperitoneal infusion of 5-amino-4-imidazolecarboxamide riboside (AICAr) inhibits the purine nucleotide cycle, resulting in rapid fatigue of isometric twitch force during stimulation of rat muscle. In this study, peak isometric twitch force was recorded from gastrocnemius muscles of pentobarbital-anesthetized rats stimulated in situ at 0.75 Hz after intraperitoneal infusion of 9ml/100 g body wt of 250 mM AICAr or isotonic saline. Excluding the two AICAr-infused rats that died during infusion or stimulation, there was no difference in twitch force between groups, although mean arterial pressure in the AICAr group declined to values 20 Torr below those of the control group during drug infusion. High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy of extracts verified the presence of AICAr and AICA ribotide (AICAR) in muscles of experimental animals. In a second study in which rats were mounted head down in an NMR spectrometer, AICAr infusion resulted in decreased mean arterial pressure and impairment of phosphocreatine recovery after stimulation but no significant difference in twitch force during stimulation. These results demonstrate that AICAr infusion has systemic effects and that the previously reported decline in muscle force with AICAr infusion may not be attributable to purine nucleotide cycle inhibition.
Abnormal expression of glycogen phosphorylase genes in regenerated muscle
Authors: Gorin F. , Ignacio P. , Gelinas R. , Carlsen R.
Published: December 2017

Physiological and molecular biological properties of free, orthotopic grafts of rat extensor digitorum longus (EDL) muscle were determined at 28-, 42-, and 76-days postgraft. cDNA probes for the rat fetal (B), liver (L), and muscle (M) isozymes of glycogen phosphorylase were used to assay isozyme mRNA levels. Regenerating muscle grafts did not express nonmuscle phosphorylase isozymes in vivo in contrast to primary rat skeletal muscle explants in vitro. Low levels of M-phosphorylase mRNA were present at all stages of regeneration in the grafts. However, M-phosphorylase mRNA levels and activity increased markedly and nonuniformly in a subset of functionally and morphologically stabilized regenerated muscle fibers between 42- and 76-days postgraft. Biochemical, physiological, and histochemical characterization of the stabilized grafts demonstrated that all fibers present were innervated and indicated that innervation might be a necessary, but not sufficient, condition for the increase in M-phosphorylase expression. The nonuniform appearance of phosphorylase activity suggests that a differential activity profile imposed on muscle fibers by their motoneuron may govern M-phosphorylase gene expression.
Dexamethasone effects on microvascular endothelial cell lipid composition
Authors: Medow M. S. , Intrieri L. , Moatter T. , Gerritsen M. E.
Published: December 2017

Previous studies have shown that treatment of cultured rabbit coronary microvessel endothelial (RCME) cells with dexamethasone results in a dose-, time-, and glucocorticoid-dependent inhibition of prostaglandin release. In the present study, the effects of dexamethasone on RCME membrane lipid composition and release of arachidonic acid were examined. This study demonstrated that dexamethasone treatment did not significantly alter the relative distribution of membrane phospholipids but did result in changes of fatty acid composition. There was an increase in saturated and monounsaturated fatty acids and a decrease in polyunsaturated fatty acids. Dexamethasone treatment did not reduce A23187-stimulated arachidonic acid release, despite inhibiting prostaglandin release by 50%. Studies with radiolabeled arachidonic acid suggest that dexamethasone may exert some actions on membrane remodeling, an effect that will require further investigation. Our data strongly suggest that the inhibitory actions of glucocorticoids on prostaglandin release in cultured RCME cells are not the result of a generalized inhibition of arachidonic acid release, and alternate mechanisms must therefore be considered.
Cl-Cl exchange in promyelocytic HL-60 cells follows simultaneous rather than ping-pong kinetics
Authors: Restrepo D. , Kozody D. J. , Spinelli L. J. , Knauf P. A.
Published: December 2017

The intra- and extracellular chloride concentration dependencies of the rate of Cl-Cl exchange in human promyelocytic leukemic HL-60 cells were studied by means of radioactive isotope (36Cl) efflux measurements. Efflux of isotope from cells follows an exponential time course. The Cl-Cl exchange flux follows Michaelis-Menten kinetics as a function of both intra- and extracellular chloride concentrations. The ratio of the maximum exchange velocity to the apparent Michaelis constant for both extracellular and intracellular substrate increases as a function of trans Cl concentration, indicating that Cl-Cl exchange in the HL-60 cell does not follow ping-pong kinetics. A kinetic scheme in which extracellular and intracellular chloride ions bind in random order to the transporter and are then translocated simultaneously can adequately model the experimental data.
Properties and physiological function of the polyamine transport system
Authors: Byers T. L. , Pegg A. E.
Published: December 2017

Polyamine transport was examined in Chinese hamster ovary (CHO) cells because of the unique potential these cells hold for utilizing genetic approaches to study the mechanisms of polyamine transport, its regulation, and its function. Parental (control) CHO cells were shown to contain a polyamine transport system with characteristics consistent with polyamine-uptake properties described in other cell types. Polyamines appear to cross the plasma membrane via an energy-requiring transport system specific for putrescine, spermidine, spermine, and their analogues. A mutant line, CHOMG, selected for resistance to the toxicity of methylglyoxal bis(guanylhydrazone), was shown to lack a functional polyamine transport system. CHOMG cells provided the negative controls necessary to examine the role of polyamine transport in maintenance of intracellular polyamine levels and in the regulation of the polyamine metabolic enzymes. It was found that the repression of ornithine decarboxylase activity by polyamines and the induction of spermidine/spermine-N1 acetyltransferase by polyamine analogues including bis(ethyl)spermine derivatives required the presence of a functional polyamine transport system. The CHO-CHOMG model was also shown to provide a means for establishing the importance of the polyamine transport system in the toxicity of polyamine analogues. The inability of alpha-difluoromethylornithine-treated CHOMG cells to utilize extracellular polyamines to replenish depleted intracellular polyamine levels suggested a means by which polyamine transport-positive cells may be identified. Such a selection procedure will permit the use of CHOMG cells in the isolation of genes encoding proteins involved in polyamine transport.
Mechanism of angiotensin II stimulation of Na-K-Cl cotransport of vascular smooth muscle cells
Authors: Owen N. E. , Ridge K. M.
Published: December 2017

Previous studies from this laboratory have demonstrated that Na-K-Cl cotransport of vascular smooth muscle cells is inhibited by hormones that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels (e.g., catecholamines) and is stimulated by hormones that increase intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels (e.g., atrial natriuretic peptides). Others have suggested that calcium may also modulate Na-K-Cl cotransport of vascular smooth muscle cells. The goal of the present study was to characterize the mechanism of angiotensin II stimulation of Na-K-Cl cotransport of early passage cultured vascular smooth muscle cells. We found that when vascular smooth muscle cells were treated with angiotensin II or a calcium ionophore, Na-K-Cl cotransport was markedly enhanced above basal levels. We found that when calcium influx was blocked with the calcium chelator EDTA or with three different chemical types of calcium-channel blockers, the stimulatory effects of angiotensin II on Na-K-Cl cotransport were markedly inhibited. Furthermore, when intracellular calcium mobilization was blocked with high concentrations of the calcium chelator quin2 or with the intracellular calcium antagonist 8-(diethyl-amino)octyl 3,4,5-trimethoxybenzoate (TMB-8), the stimulatory effects of angiotensin II on Na-K-Cl cotransport were also substantially inhibited. These results suggest that both calcium influx via receptor-operated calcium channels and intracellular calcium mobilization may play a role in stimulation of Na-K-Cl cotransport of vascular smooth muscle cells in response to angiotensin II.
Cardiac antisecretory peptide inhibits intestinal chloride secretion
Authors: O'Grady S. M. , Wolters P. J.
Published: December 2017

A heat-stable low-molecular-weight peptide isolated from porcine heart inhibited vasoactive intestinal polypeptide and ionomycin (a Ca ionophore) -stimulated Cl secretion across the rat ileum. The antisecretory effects of this peptide were resistant to the neuronal conduction blocker tetrodotoxin, suggesting that submucosal nerves were not involved in mediating its effects on Cl transport. Activity was found in extracts of the right and left atria and in the ventricles of the heart. The antisecretory effect was not mimicked by atrial natriuretic factor, brain natriuretic peptide, or by putative antisecretory factors (neuropeptide Y, somatostatin, enkephalin, and norepinephrine), suggesting that cardiac antisecretory peptide is a unique regulatory factor that may regulate Cl transport in the intestinal mucosa and other epithelia.
Expression of amiloride-blockable sodium channels in Xenopus oocytes
Authors: Hinton C. F. , Eaton D. C.
Published: December 2017

This report describes the expression of a sodium-selective, amiloride-blockable conductance in Xenopus oocytes that have been injected with RNA prepared from a distal nephron cell line (A6). After injecting the RNA into mature oocytes (stage V or VI) and incubating the oocytes for 2-4 days, the oocytes were examined for amiloride-blockable current. The RNA induced a substantial amiloride-blockable current. Uninjected or water-injected oocytes had no measurable amiloride-blockable current. RNA prepared from aldosterone-treated A6 cells was much more effective in inducing amiloride-blockable sodium current than RNA prepared from aldosterone-depleted A6 cells. Oocytes injected with RNA prepared from mineralocorticoid-depleted cells appeared very similar to water-injected oocytes. The amiloride-blockable current in oocytes has a reversal potential of approximately +50 - +60 mV, which varies 61 mV/decade change in external sodium concentration, suggesting that the current is highly selective for sodium over other ions. In addition, the concentration of amiloride that produces half block of the current is 48 +/- 8 nM. Thus the current expressed in oocytes appears very similar to sodium-selective currents observed from the apical membranes of various tight epithelial tissues.
Lowering Po2 induces epithelium-dependent relaxation in isolated canine bronchi
Authors: Gao Y. S. , Vanhoutte P. M.
Published: December 2017

This study was designed to investigate whether the respiratory epithelium can modulate the tone of the underlying smooth muscle in response to decreases in partial pressure of O2 (PO2). Canine bronchial segments with or without epithelium (diameter, 4-6 mm; length, 50-60 mm) were mounted in organ chambers and perfused intraluminally with modified Krebs-Ringer bicarbonate solution [temperature, 37 degrees C; PO2 varying from 600 (control) to 40 mmHg; PCO2, 36 mmHg]. Isometric tension was recorded by means of stirrups passed through the wall of the central part of the bronchial segment. During contractions to carbachol, the tissues with epithelium showed epithelium-dependent relaxations when the PO2 was decreased. The level of relaxation was dependent on the PO2. The epithelium-dependent relaxation could not be blocked by the following agents: indomethacin, methylene blue, propranolol, or tetrodotoxin (antagonists or blockers of cyclooxygenase, guanylate cyclase, beta-adrenoceptors, and sodium channels, respectively). The epithelium-dependent relaxation was not accompanied by the release of an assayable relaxing factor in the bronchial lumen. The experiments suggest that 1) lowering the PO2 induces the epithelium to release a relaxing factor(s), which is neither a product of cyclooxygenase nor endothelium-derived relaxing factor; 2) a local reflex mechanism is not involved in the phenomenon; and 3) the relaxing factor(s) either is not released into the bronchial lumen or, if it is, is catalyzed rapidly in the lumen on release.
Role of Mg in the activation of Na-H exchange in dog red cells
Authors: Parker J. C. , Gitelman H. J. , McManus T. J.
Published: December 2017

Depleting dog red cells of Mg prevents activation of Na-H exchange by cell shrinkage but has no effect on activation by acidification of the cytosol. Replacing cell Mg restores the activation of Na-H exchange by cell shrinking. We conclude that Mg is required, not for the transport process per se, but for the transduction of the volume stimulus.
Mechanisms and regulation of water permeability in renal epithelia
Authors: Verkman A. S.
Published: December 2017

Water transport occurs in all biological membranes. A few selected membranes in the kidney, amphibian urinary bladder, and erythrocyte have very high water permeability and are thought to contain specialized water transporting units termed "water channels." The known biophysical properties of membranes containing water channels are a high osmotic water permeability coefficient (Pf), an osmotic-to-diffusional water permeability coefficient ratio (Pf/Pd) greater than unity, a low activation energy (Ea), and inhibition by mercurial compounds. The biochemical and molecular characteristics of water channel pathways are not known at present. Established and new methods to measure Pf and Pd in kidney tubules and in isolated membrane vesicles from kidney cells are reviewed and evaluated. In the mammalian proximal tubule, a high Pf results from transcellular movement of water across highly permeable apical and basolateral membranes containing water channels. It has been assumed that proximal tubule Pf is unregulated; however, recent results indicate that apical water channels are retrieved by endocytosis and that Pf is decreased fivefold with increasing transepithelial osmotic gradients. In the thin and thick ascending limbs, Pf is nearly the lowest of all biological membranes and is not subject to regulation. In contrast, collecting tubule Pf is subject to hormonal regulation by vasopressin. Vasopressin binding to receptors located at the basal membrane of principal cells initiates adenosine 3',5'-cyclic monophosphate production, which is thought ultimately to activate the exocytic insertion of intracellular vesicles containing water channels into the cell apical membrane. Vasopressin-induced endosomes from kidney collecting tubule and toad urinary bladder contain functional water channels but no proton pumps or urea transporters, supporting a membrane shuttle hypothesis that is selective for water channels. Future directions for the isolation and molecular cloning of kidney water channels are evaluated.
Suppressive action of cobalt on exocytosis and respiratory burst in neutrophils
Authors: Elferink J. G. , Deierkauf M.
Published: December 2017

Activation of exocytosis and respiratory burst in rabbit neutrophils by the chemotactic peptide fMet-Leu-Phe is inhibited by Co2+. Inhibition is antagonized by extracellular Ca2+ and is dependent on the time of preincubation of cells with Co2+ before addition of activator. Co2+ inhibits the enhancement of 45Ca association that occurs during activation with fMet-Leu-Phe. Interference with Ca2+ translocation across the plasma membrane by Co2+ is probably not the cause of inhibition of neutrophil activation, because activation in the absence of extracellular Ca2+ is inhibited by Co2+. Activation of neutrophils by phorbol myristate acetate is inhibited at higher Co2+ concentrations than activation by fMet-Leu-Phe. Inhibition of the superoxide production by Co2+ occurs both in the presence or in the absence of cytochalasin B. Fluorescence of neutrophils loaded with quin2 is diminished by Co2+, indicating that Co2+ had entered into the cytoplasm. The results are compatible with the view that Co2+ inhibits exocytosis and respiratory burst in neutrophils by an interaction with a Ca2+-dependent intracellular target.
Role of endothelium in endotoxin blockade of voltage-sensitive Ca2+ channels in smooth muscle cells
Authors: Goligorsky M. S.
Published: December 2017

Coculture of endothelial and smooth muscle cells was used to study effects of endotoxin on depolarization-induced Ca2+ transients in fura-2-loaded individual smooth muscle cells. Although endotoxin did not modify the response of cultured smooth muscle cells to depolarization, endotoxin resulted in an attenuation of cytosolic Ca2+ (Cai2+) transients in response to K+ depolarization and failure of KCl-induced contractions in smooth muscle cells when they were cocultured with endothelial cells. The observed endothelial modulation of smooth muscle responses was not accomplished via gap junctions. The possible role of free radical species secreted by endothelial cells in conditioning of smooth muscle responses to depolarization was supported by the results of three sets of experiments: 1) endothelial cells did respond to endotoxin with oxidative burst, 2) pretreatment of cocultured cells with catalase prevented endotoxin-induced downregulation of Ca2+ transients, and 3) in isolated smooth muscle cells, the addition of hydrogen peroxide virtually abolished depolarization-induced Ca2+ transients. Hence vascular endothelium stimulated by endotoxin generates reduced oxygen intermediates, which in turn downregulate depolarization-induced Cai2+ transients in smooth muscle cells. This phenomenon may contribute to the development of hypotension in septicemia.
Augmentation of bleomycin-induced DNA damage in intact cells
Authors: Moseley P. L.
Published: December 2017

Bleomycin, an important cause of pulmonary fibrosis, is known to produce DNA damage. The mechanism for this damage in vitro is related to free radical production by a bleomycin and iron complex. To determine whether bleomycin causes damage to DNA in vivo by a similar mechanism, we used a viral minichromosome that is replicated in cultured cells. Bleomycin causes dose-dependent damage to intracellular DNA, and this damage is augmented by Fe2+ but not Fe3+. The augmentation of the bleomycin-induced DNA damage caused by Fe2+ is also dose dependent in that increasing DNA damage occurs with increasing amounts of Fe2+. These studies demonstrate that bleomycin causes damage to DNA in vivo and suggest that bleomycin must rely on Fe2+ to donate an electron for oxygen radical-induced DNA strand scission.
Repolarization of Na+-K+ pumps during establishment of epithelial monolayers
Authors: Contreras R. G. , Avila G. , Gutierrez C. , Bolivar J. J. , Gonzalez-Mariscal L. , Darzon A. , Beaty G. , Rodriguez-Boulan E. , Cereijido M.
Published: December 2017

Madin-Darby canine kidney (MDCK) cells plated at confluence and incubated for 20 h in low (5 microM) Ca2+ have no tight junctions (TJs), and their Na+-K+-ATPase is randomly distributed over the surface. On transfer to normal Ca2+ levels (1.8 mM) ("Ca2+ switch"), TJs and transepithelial resistance develop quickly, trapping a considerable fraction (35%) of the surface Na+-K+-ATPase on the apical (incorrect) side. This misplaced enzyme is subsequently removed from this region or inactivated, demonstrating that polarization proceeds despite TJs. Simultaneously, the amount of Na+-K+-ATPase on the basolateral side increases in a higher proportion (125%), than could be accounted for by relocation of the misplaced apical enzyme. This incorporation is prevented by cycloheximide, ammonium chloride, primaquine, or chloroquine, suggesting that Na+-K+-ATPase originates in an intracellular pool and that its surface insertion requires synthesis of new enzyme or of a protein factor, since it is carried to the surface membrane through a mechanism of exocytosis. In summary, asymmetric distribution of ion pumps depends 1) on polarized insertion of Na+-K+-ATPase as well as 2) on removal or inactivation of misplaced enzyme.
Glucocorticoids inhibit acetylcholine-induced current in chromaffin cells
Authors: Inoue M. , Kuriyama H.
Published: December 2017

The effects of dexamethasone, a synthetic glucocorticoid, on membrane excitability and on the acetylcholine (ACh)-induced inward current (IACh) were studied in dispersed guinea pig adrenal medullary cells by using the whole cell version of the patch-clamp technique. Bath application of 10 microM dexamethasone had no effect on the resting membrane conductance or voltage-gated Na+, Ca2+, or K+ channels, whereas it reversibly inhibited the IACh at a holding potential of -70 mV. Intracellular application of dexamethasone through the patch electrode did not modify the IACh. Application of 10 microM dexamethasone neither shifted the dose-response curve of the peak IACh to the right [dissociation constant (Kd) = 50 microM] nor affected its Hill coefficient (1.2) but inhibited the current amplitudes by approximately 41% in the cells sufficiently pretreated with dexamethasone. Furthermore, fractional inhibition of the IACh at the end of approximately 50-s application was the same for any concentration of ACh (3-100 microM). The dose-response curve of the inhibition by dexamethasone showed a good fit to the theoretical line assuming an inhibition constant (Ki) of 10 microM and a Hill coefficient of 1. Hydrocortisone 21-hemisuccinate sodium salt (25 microM) and prednisolone 21-hemisuccinate sodium salt (25 microM) inhibited the IACh to a lesser extent than 25 microM dexamethasone. These results suggest that dexamethasone binds to the specific site on the outer cell membrane, probably on the ACh receptor-coupled channel, and inhibits the IACh in a noncompetitive manner, thus controlling the immediate catecholamine release from the adrenal medulla.
Protein metabolism during nutrient deprivation and refeeding of neonatal heart cells
Authors: Schroedl N. A. , Bacon C. R. , Huang Y. C. , Hartzell C. R.
Published: December 2017

Pathological conditions or nutrient deprivation in the heart cause an imbalance between rates of protein synthesis and degradation, often resulting in a severe depletion of cardiac protein. We used cultured neonatal rat heart cells, a model system exhibiting positive nitrogen balance, to examine the effects of 10 h of starvation on myocardial glucose and protein metabolism. Cellular capacity for glucose utilization was depressed after starvation, as evidenced by lower hexokinase and other glycolytic enzyme activities and a 21% decrease in glucose usage. A 21.0% decrease in protein synthetic rate and an increase in protein degradation rate combined to yield a 29.5% decrease in total cellular protein during starvation. Degradation rates increased 29.0, 46.7, and 59.6% in 2-, 24-, and 96-h prelabeled cells, respectively, indicating that lability increased with half-life of proteins. During refeeding of starved, cultured cells, at least three proteins were synthesized at a lower rate. At the same time, proteins with approximate molecular masses of 45, 84, 92, and 174 kDa exhibited increased synthesis.
Modification of leucine transport across bovine pigment epithelium by metabolic stress
Authors: Pautler E. L. , Tengerdy C. , Beyer J. , Beezley D.
Published: December 2017

The transport of leucine in the apical-to-basal (retina to choroid) direction across the isolated bovine retinal pigment epithelium is mediated predominantly by the L amino transport system at low carrier (10 microns) concentrations. There is no evidence of an active or facilitated transport system operating in the opposite direction. The identification of the L system is based on the lack of sodium dependence, specific inhibition of leucine transport by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), and the demonstration of trans-stimulation. Lysine, glutamate, and 2-methylaminoisobutyric acid (MeAIB) did not provide any competitive inhibition. Ouabain and iodoacetate were also ineffective in modifying leucine transport. The transport mediated by the L system was markedly temperature sensitive, whereas no temperature dependence was apparent in the transport of leucine in the basal-to-apical direction (choroid to retina). When treated with dinitrophenol (DNP), the transport of leucine in the apical-to-basal direction was greatly enhanced, but no effect was observed on the leucine movement in the opposite direction. Azide and rotenone had an effect similar to DNP, as did reducing the partial pressure of O2 to less than 40 Torr. The enhancement of transport appeared to be mediated by the activation of an ancillary system, since it was susceptible to different classes of metabolic and competitive inhibitors as well as the observed ionic dependency. After DNP treatment, the transport of leucine was inhibited by lysine and BCH, revealed a sodium dependence, and could be inhibited by iodoacetate. The characteristics of the enhanced transport appear to be similar to those of the recently described G system(s) of amino acid transport.
Voltage-activated cation transport in human erythrocytes
Authors: Halperin J. A. , Brugnara C. , Tosteson M. T. , Van Ha T. , Tosteson D. C.
Published: December 2017

We report here the effects of membrane potential on the permeability of the human erythrocyte to Na, K, and Ca. Membrane potential was changed either by varying the K concentration gradient in the presence of valinomycin or by varying the concentration gradient of the permeant anion nitrate in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. When the membrane potential was changed from inside negative (-10 mV) to inside positive (greater than 40 mV), influx, efflux, and net flux of Na and K increased. Marked net cation loss and cell shrinkage occurred in the absence of a chemical gradient for Na and K. This voltage-dependent increase in Na and K conductance is partially inhibited by 10 microM ruthenium red and persists when the membrane potential is returned to -10 mV after transient exposure to inside-positive potentials. A similar voltage-dependent behavior was found for Ca influx. The voltage-activated Ca influx is almost completely inhibited by 10 microM ruthenium red.
Effects of cysteine protease inhibitors on rabbit cathepsin D maturation
Authors: Samarel A. M. , Ferguson A. G. , Decker R. S. , Lesch M.
Published: December 2017

To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed (24 h) to media containing leupeptin (0-10 mM), E 64 (0-10 mM), or chloroquine (0-50 microM). Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D (Mr 53,000) within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D (Mr 48,000). Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate (not detectable in control fibroblasts). Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation.
p-aminohippurate transport in the airways: role of Na+ and HCO3-
Authors: Cloutier M. M.
Published: December 2017

The role of Na+ and HCO3- in the transport of p-aminohippurate (PAH) across the canine tracheal epithelium was investigated using Ussing chamber techniques and radiolabeled PAH. Under control conditions, net PAH absorption or a tendency toward net PAH absorption was observed. Neither amiloride (10(-4) M), furosemide (10(-3) M), ouabain (2 x 10(-4) M), nor Na+ substitution of the Ringer solution with choline had any effect on unidirectional PAH fluxes. When the Ringer solution was replaced with a HCO3(-)-free solution, net PAH absorption was consistently observed. In HCO3(-)-free experiments, unidirectional PAH absorptive fluxes were inhibited by mucosal addition of either of the stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 10(-4) M) or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 10(-4) M). DIDS was more effective than SITS and was also effective in inhibiting PAH absorption in tissues bathed in Ringer solution. Submucosal DIDS or SITS had no effect on PAH fluxes either in HCO3(-)-free or Ringer experiments. We conclude that PAH transport in canine tracheal epithelium occurs by a HCO3(-)-PAH exchange process located on the luminal membrane. PAH transport is not Na+ dependent but is inhibited by both DIDS and SITS.