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D J Benos

Publications and source records attributed to D J Benos.

At least 163 records · Page 9Linked to original sources
PubMed1992

cAMP-stimulated ion currents in Xenopus oocytes expressing CFTR cRNA.

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in stage V/VI Xenopus oocytes by injection of cRNA transcribed in vitro from a pBluescript vector containing 6.2-kb wild-type cDNA. This clone was also used for the preparation of antisense RNA. Double-electrode voltage clamp was employed to measure transmembrane currents. In sense RNA-injected oocytes, cAMP depolarized the membrane potential (Vm) from -52 to -31 mV and increased membrane conductance (Gm) 10-fold. However, cAMP had no effect on Vm or Gm in uninjected oocytes or in oocytes injected with antisense RNA. The endogenous Ca-activated Cl currents of control oocytes were abolished by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 50 microM) or bath Cl replacement. In contrast, the cAMP-stimulated currents of CFTR-expressing oocytes were DIDS insensitive and were inhibited only approximately 50% when bath Cl was replaced by gluconate or glutamate. In addition, the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB; 50 microM) and diphenylamine-2-carboxylic acid (DPC; 3 mM) reduced the cAMP-evoked currents by only approximately 10%. The stimulated currents of CFTR-expressing oocytes were reduced approximately 30% by 10 mM Ba, suggesting that the Cl-independent current component is due to an increase in K conductance. Our results indicate that expression of CFTR in Xenopus oocytes produces a cAMP-activated Cl current. The Cl-independent current may represent a regulatory action of CFTR on K conductance pathways or a secondary response of the oocyte membrane to the high Cl conductance induced by CFTR expression.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Immunocytochemical and functional characterization of Na+ conductance in adult alveolar pneumocytes.

The purpose of this study was to document the existence, assess the spatial localization, and characterize some of the transport properties of proteins antigenically related to epithelial Na+ channels in freshly isolated rabbit and rat alveolar type II (ATII) cells. ATII cells, isolated by elastase digestion of lung tissue and purified by density-gradient centrifugation, were incubated with polyclonal antibodies raised against Na+ channel protein purified from beef kidney papilla (NaAb), followed by a secondary antibody (goat antirabbit immunoglobulin G conjugated to fluorescein isothiocyanate). Rat ATII cells exhibited specific staining with NaAb at the level of the plasma membrane, which, in most cells, colocalized with that of the lectin Maclura pomiferra agglutinin, an apical surface marker. In Western blots, NaAb specifically recognized a 135 +/- 10-kDa protein in rat ATII membrane vesicles. When patch clamped in the whole cell mode using symmetrical solutions (150 mM Na+ glutamate), ATII cells exhibited outwardly rectified Na+ currents that were diminished by amiloride (10-100 microM) instilled into the bath solution. Ion substitution studies showed that the conductive pathways were three times more permeable to Na+ than K+. Amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride were equally effective in diminishing 22Na+ flux into rabbit and rat ATII cells (45% inhibition at 100 microM, with IC50 of approximately 1 microM for all inhibitors). Tetraethylammonium chloride (10 mM) or BaCl2 (2 mM), well-known K+ channel blockers, had no effect on 22Na+ uptake. These results indicate that ATII cells express an amiloride-sensitive Na+ conductance, probably a channel, with a lower affinity for amiloride and its structural analogues than the well-established amiloride-sensitive Na+ channels found in bovine renal papila and cultured amphibian A6 kidney cells.

Amiloride↗
PubMed1992

CFTR!

Cystic fibrosis (CF) is a fatal genetic disease primarily affecting Caucasians, although cases have been reported from other ethnic groups. CF has a complex etiology, but it is chiefly a disease of electrolyte transport and is characterized by defects in fluid secretion by several epithelia, including the sweat duct, exocrine pancreas, and the pulmonary airways. The link between CF and a defect in cAMP-mediated Cl- transport in secretory epithelia was established in the early 1980s. Since then, numerous electrophysiological studies have focused on the characterization and regulation of individual Cl- channels underlying the macroscopic Cl- currents of secretory epithelia in the airways, sweat ducts, and gut. In this review the results of these studies in the light of current knowledge of the function of the CF gene product, the CF transmembrane conductance regulator (CFTR) protein, will be analyzed. The CFTR protein is a member of a family of ATP-binding proteins that act as unidirectional solute pumps. These proteins are membrane spanning, are found in both prokaryotic and eukaryotic cells, and have two ATP-binding domains. The family includes the p-glycoproteins that are involved with the expression of multidrug resistance in certain tumor cells. The majority of CF chromosomes (70%) have a single codon deletion that translates to a missing phenylalanine residue at position 508 (delta F508) of the protein. Unique for this family of proteins, the CFTR protein possesses an additional highly charged domain (the R domain) containing several consensus polypeptide sequences for kinase phosphorylation. Although CFTR bears structural resemblance to this family of ATP-dependent pumps, overexpression of the protein in a variety of different cell types is associated with the appearence of a cAMP-sensitive Cl- channel. We critically examine current information concerning the structure-function relationships of the CFTR protein obtained from both electrophysiological and biochemical approaches. We also summarize recent evidence suggesting that the CFTR protein may act as a pump and a channel, a hypothesis in keeping with the multifaceted nature of the disease.

Adenosine Triphosphate↗
PubMed1991

Interferon-gamma-induced astrocyte class II major histocompatibility complex gene expression is associated with both protein kinase C activation and Na+ entry.

Astrocytes can be induced by interferon-gamma (IFN-gamma) to express class II major histocompatibility complex (MHC) antigens. This study was undertaken to elucidate the intracellular signaling pathways involved in IFN-gamma induction of class II MHC. We examined the effects of Na+/H+ antiporter and protein kinase C (PKC) inhibitors on class II expression and Na+ influx in astrocytes. We found that amiloride and ethyl isopropylamiloride, inhibitors of Na+/H+ exchange, blocked IFN-gamma-induced class II gene expression. IFN-gamma stimulated Na+ influx, and this increased influx was inhibited by amiloride. Treatment of astrocytes with the PKC inhibitor H7 also blocked the increase in Na+ uptake induced by IFN-gamma, indicating that IFN-gamma-induced PKC activation is required for subsequent Na+ influx. IFN-gamma treatment produced an increase of total PKC activity, which was associated with a rapid translocation of PKC activity from cytosolic to particulate fraction. H7 and another PKC inhibitor, staurosporine, inhibited IFN-gamma-induced class II gene expression. However, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, a potent PKC activator, did not affect class II expression. Taken together, our data indicate that both IFN-gamma-induced PKC activation and Na+ influx are required for class II MHC expression in astrocytes but that activation of PKC alone is not sufficient for ultimate expression of this gene.

Amiloride↗
PubMed1991

Amiloride-sensitive sodium channel is linked to the cytoskeleton in renal epithelial cells.

Amiloride-sensitive sodium channels are localized to the microvillar domain of apical membranes in sodium-transporting renal epithelial cells. To elucidate the elements that maintain sodium channel distribution at the apical membrane, we searched for specific proteins associating with the channel. Triton X-100 extraction of A6 epithelial cells reveals that sodium channels are associated with detergent-insoluble and assembled cytoskeleton. Indirect immunofluorescence and confocal microscopy show that sodium channels are segregated to the apical microvillar membrane and colocalize with ankyrin, fodrin, and actin. We document by immunoblot analysis that ankyrin and fodrin remain associated with sodium channels after isolation and purification from bovine renal papillae. 125I-labeled ankyrine can be precipitated by anti-sodium-channel antibodies only in the presence of purified bovine sodium-channel complex. Direct binding of 125I-labeled ankyrin shows ankyrin binds to the 150-kDa subunit of the channel. Fluorescence photobleach lateral-diffusion measurements indicate sodium channels are severely restricted in their lateral mobility. We conclude that ankyrin links the amiloride-sensitive sodium channel to the underlying cytoskeleton and this association may sequester sodium channels at apical microvilli and maintain their polarized distribution in renal epithelial cells.

Amiloride↗
PubMed1991

Isolation and functional reconstitution of a 38-kDa chloride channel protein from bovine tracheal membranes.

Secretion of chloride ions via apically located anion-selective channels in epithelia regulates fluid formation and cytosolic Cl- homeostasis. In order to understand the biochemical basis of Cl- channel function, we attempted to isolate this transporter from bovine tracheal apical membranes. Initially, peripheral polypeptides were removed from apically enriched vesicles by washing with alkaline buffer (pH 10.8) containing 2 mM CHAPS. The resulting pellet contained 50-60% of the original protein and displayed 2-fold enhanced Cl- channel activity compared to untreated vesicles. The pellet was treated with Triton X-100, and the solubilized proteins were separated on the cationic exchanger CM-cellufine. Washing the resin with a pH 8.0-8.3 buffer eluted a fraction with enriched Cl- channel activity. This fraction contained less than 5% of the total solubilized protein. A subsequent separation was performed using the anionic exchanger AM-cellufine. The highest activity was found in the fractions eluted by 80-120 mM KCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed a major 38,000-Da protein band. This band was electroeluted from the gel under nondenaturing and nonreducing conditions and reconstituted into phosphatidylcholine liposomes. KCl-loaded vesicles containing the purified 38-kDa protein transported up to 5 nmol of 125I-/mg of protein/5 min. This value was 15-fold higher than the uptake measured in vesicles reconstituted with total solubilized membrane proteins and 4-fold higher compared to the CM-cellufine-enriched fraction. The observed 125I- uptake was 90% inhibited by 100 microM 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate or 10 microM valinomycin. In summary, we have developed a biochemical protocol for the isolation of a 38 kDa protein mediating potential-dependent and 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate-sensitive Cl- channel activity.

Animals↗
PubMed1991

Epithelial sodium conductance in rabbit preimplantation trophectodermal cells.

We examined the development of epithelial Na+ conductance in 6- and 7-day post coitus (p.c.) preimplantation rabbit embryos using the whole-cell patch-clamp technique on dissociated rabbit trophectodermal cells and by immunocytochemical localization using a polyclonal antibody directed against subunits of an apical epithelial Na+ channel on the intact blastocyst. In Day 6 and 7 p.c. trophectodermal cells, we observed an outwardly rectified whole-cell Na+ current. The current-voltage characteristics did not differ between the 6- and the 7-day p.c. cells. Replacement of Na+ with the impermeant cation N-methyl-D-glucamine in the pipette or bath reduced outward currents and inward currents, respectively, indicating that the current was Na(+)-dependent. Treatment of 7-day p.c. cells with 100 microM amiloride, benzamil, or ethylisopropyl amiloride (EIPA) blocked the whole-cell currents within 5 min. However, the current of the Day 6 p.c. embryo was not blocked by amiloride. The amiloride block at Day 7 p.c. was only partially reversible after 15 min of continuous perfusion of the bath with an amiloride-free solution. The apparent dissociation constant (Ki) for amiloride, benzamil, and EIPA was 12, 50, and 16 microM, respectively, when measured 5 min after drug addition. Immunolocalization studies of blastocysts with a polyclonal antibody raised against a high amiloride affinity Na+ channel isolated from bovine kidney revealed no specific binding to the trophectodermal cells at Day 6 p.c.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1991

Amiloride-inhibitable Na+ conductive pathways in alveolar type II pneumocytes.

The purpose of these studies was to document the existence of electrogenic Na+ uptake by membrane vesicles of rabbit alveolar type II (ATII) cells and the extent to which this process was inhibited by amiloride. ATII cells (greater than 85% pure) were obtained by elastase digestion of lung tissue followed by Percoll centrifugation, and an enriched plasma membrane vesicle fraction was obtained by differential centrifugation. 22Na+ uptake into these vesicles was measured in the presence of a negative inside membrane potential, produced by the addition of the K+ ionophore valinomycin (10 microM) after all external K+ was removed. Electrogenic (valinomycin-sensitive) Na+ uptake (ELNa) was defined as the difference in uptake in the presence and absence of valinomycin. ELNa, normalized per milligram protein, was twice as high across ATII cells than alveolar macrophage membrane vesicles, was inhibited by amiloride (50% inhibitory concentration = 10 microM), and was decreased in the presence of an outwardly directed proton gradient (pHin 6.8; pHout 7.8), suggesting that it was not mediated by Na(+)-H+ antiport. Furthermore, ELNa was equally inhibited by increasing concentrations of amiloride and benzamil but was more sensitive to 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride in concentrations of 10-1,000 microM. These findings indicate that a fraction of Na+ transport across ATII membrane vesicles occurs through a conductive pathway, probably a channel, that has different sensitivity to amiloride and its analogues than the previously described epithelial high amiloride-affinity Na+ channel.

Alkaline Phosphatase↗
PubMed1991

Glucose metabolism in the trophectoderm and inner cell mass of the rabbit embryo.

The metabolism of glucose in the intact Day-6 and -7 post coitum (p.c.) rabbit blastocyst and in the separated trophectoderm and inner cell mass (ICM) of the Day-7 p.c. embryo was investigated. At Day-6 p.c., glucose traversed the trophectoderm with a half-time of 39 +/- 9.3 min, and was metabolized to CO2 at a rate of 25.5 +/- 1.6 nmol.cm-2.h-1. Neither the Na+ ionophore, amphotericin B, nor cyclic AMP had an effect on glucose metabolism to CO2. Lactate production by the Day-6 blastocyst was largely independent of glucose. At Day-7 p.c. in the intact embryo, CO2 production from glucose significantly decreased to 7.76 +/- 2.8 nmol.cm-2.h-1. Per unit surface area, the metabolism of glucose to CO2 was similar in the separated Day-7 p.c. trophectoderm and ICM. We conclude that the rabbit blastocyst is not highly dependent on glucose, and that the ICM does not utilize glucose as a metabolite to a greater extent than does the trophectoderm, at least in the Day-7 p.c. embryo.

Animals↗
PubMed1991

Localisation of putative mechanoelectrical transducer channels in cochlear hair cells by immunoelectron microscopy.

Displacement of the apical stereociliary bundle of cochlear hair cells mechanically gates transducer channels. Knowing the position of the channels with regard to the apical structures of the hair cell could indicate how this mechanism operates. At present, there is conflicting evidence regarding their precise location; the channels have been suggested to be located either towards the base of the stereocilia or at the tips where they could be operated by extracellular links running from the top of shorter stereocilia to the sides of adjacent taller ones. The channels have been shown to be reversibly blocked by amiloride. This has prompted us to use a polyclonal antibody raised against another amiloride-sensitive channel to search for them using immunolabelling. The location of the primary antibody has been revealed using pre-embedding labelling with a colloidal gold-conjugated secondary antibody followed by scanning transmission electron microscopy of semi-thin sections. In this way, more complete information on the relationship of the labelling to the three-dimensional organisation of the stereociliary bundle has been obtained in comparison with previous immunofluorescence and transmission electron microscopic results. Labelling occurs in discrete areas towards the tips of the stereocilia, one of the possible sites for the transducer channels, predominantly between the membranes of shorter and taller stereocilia.

Amiloride↗
PubMed1990

A non-selective cation channel in the apical membrane of cultured A6 kidney cells.

The patch-voltage clamp technique was used to investigate the characteristics of a non-selective cation channel (NSCC) identified in the apical membrane of cultured A6 toad kidney cells. The NSCC was present in cell-attached and inside-out membrane patches. The characteristics of this NSCC are as follows: (a) linear current-voltage relationship with a channel conductance of 21 +/- 2 pS; (b) a low selectivity between Na+ and K+ (1.5:1); (c) a high selectivity of Na+ to Cl- (greater than 45:1); (d) this channel has a single open state and two closed states; (e) the open-time constant and the second closed-time constant of this channel are voltage dependent; and (f) this NSCC is insensitive to amiloride (10(-7) M). We conclude that the NSCC resembles previously described non-selective cation channels. The NSCC of the apical membrane of A6 cells may aid in the movement of Na+ and K+ in response to varying ionic concentrations across the apical membrane.

Amiloride↗
PubMed1990

Glycolytic component of rat spermatid energy and acid-base metabolism.

The impact of glycolysis on rat spermatid energy metabolism is made apparent by the simultaneous occurrence of the following three events upon glucose addition to the extracellular medium of a rat spermatid cell suspension: decrease in ATP content, exit of acid equivalents, and increased lactate production and efflux. In this work, we have studied the interrelations between these three phenomena. By measuring ATP content, net acid transport, lactate exit, oxygen consumption, intracellular pH, CO2 production, and glycolytic intermediates in the presence of glucose and glucose analogues, we conclude that 1) lactate production, decrease in ATP content, and acid equivalent exit are dependent on the metabolism of glucose up to different stages in glycolysis. 2) The decrease in ATP content is not directly related to the exit of acid equivalents from rat spermatids. 3) Glucose metabolism is a net ATP-consuming process at high intracellular ATP content but is a net ATP-producing process at low intracellular ATP concentration in rat spermatids. 4) Acid equivalent production arises from the metabolism of glucose beyond glyceraldehyde 3-phosphate dehydrogenase. 5) Lactic acid diffusion and/or lactate transport and CO2 production and exit could account for the glucose-dependent acid equivalent efflux in rat spermatids.

Acid-Base Equilibrium↗
PubMed1990

Hexose transport in preimplantation rabbit blastocysts.

Transtrophectodermal 3-0-methyl glucose (3-0MG) transport in the rabbit blastocyst at Days 6 and 7 post coitum was investigated to understand better how the trophectoderm can regulate inner cell mass growth by controlling substrate availability. 3-0MG rapidly traversed the trophectoderm and displayed saturation kinetics (Km = 4.3 +/- 0.5 mM, Vmax = 79 +/- 3.8 nmol.cm-2). The flux of 3-0MG was inhibited nearly 95% by 10(-4) M-phloretin, and only 15% by 10(-4) M-phlorizin. Furthermore, 3-0MG influx was inhibited by cytochalasin B (5 microM) and was unaffected by removal of sodium. The transport system had a high specificity for 2-deoxy-D-glucose and glucose, and a very low specificity for fructose and 4-alpha-methyl glucoside. Western blots probed with a polyclonal antibody to the human erythrocyte glucose transport protein and also with a polyclonal antibody to the C-terminus of the glucose transport protein of the rat brain revealed a broad band with a molecular weight of 55,000. Using immuno-gold labelling techniques, Na(+)-independent glucose transporters were localized to both the apical and basolateral borders of the trophectodermal cell. These results suggest that the mechanism in the trophectoderm responsible for transport of glucose is similar to other sodium-independent glucose transport systems. In addition, 3-0MG influx was unaffected by short-term incubation with progesterone, the progesterone antagonist mifepristone (RU-486), PGF-2 alpha, PGE-2, insulin, or cAMP. Day-7 p.c. embryos also transported hexoses by a similar system because the influx rate and the phlorizin/phloretin sensitivity were the same as in the Day-6 p.c. embryo.

3-O-Methylglucose↗
PubMed1989

The biology of amiloride-sensitive sodium channels.

Studies of the molecular nature of epithelial sodium channels are in their infancy and have largely involved experiments in which the interaction between amiloride and this transport process has been examined. Because of the inherent geometric complexity of epithelial tissues, these studies have in large measure been macroscopic in nature, with the molecular details of transport being deduced. In the past five years, however, the molecular biology of these critical ion channels has been studied directly. The development of radioactive high-affinity probes, the application of patch-clamp and reconstitution techniques, the generation of specific antibodies, and the formulation of epithelial cDNA expression libraries have propelled the field of epithelial ion channels into a new era. Now, for the first time, we can rigorously address questions concerning the molecular nature of the amiloride block, the channel's selectivity to alkali metal cations, and the modulation of ion transport through this channel by other ions (such as calcium), hormones (such as vasopressin, aldosterone, and atrial natriuretic factor), or intracellular second messengers (such as cAMP or cGMP). The complexity of the epithelial sodium channel's structure may reflect the constitutive and regulatory role this protein plays in sodium homeostasis. The epithelial sodium channel is continually operating, constantly changing its activity on a second-to-second basis. Hence, its tonic functions are probably modulated by a myriad of factors, most of which are unknown. With the application of molecular techniques, a much clearer understanding of the nature and regulation of epithelial sodium channel processes in health and disease will emerge in the years to come.

Aldosterone↗
PubMed1989

In vitro differentiation of rabbit blastocyst cells.

Six- and seven-day post-coitus (p.c.) rabbit embryos have been cultured in an attempt to establish a trophectodermal cell line. Results indicate that cells with epithelial characteristics (i.e. positive staining for cytokeratin) will survive in culture until Passage 3. At that time a fibroblastlike cell becomes predominant. In addition, we have found that the presence of the inner cell mass is required for trophectodermal cells from 6-d p.c. embryos to attach to the collagen substrate. Culture of 7-d p.c. embryo explants often results in the development of cells that spontaneously contract. These cells stain positively for myosin, which indicates that they may be precardiac cells. Maximum diastolic potential was -59 +/- 1.2 mV and the threshold potential was -53 +/- 2.3 mV. Spontaneously contracting cells did not respond to atropine, acetylcholine, epinephrine, isoproterenol, or propranolol. Action potential seems to be a result of an inward calcium current, because the beating rate is decreased in a dose-related manner with the calcium channel blocker verapamil, whereas the voltage-sensitive sodium channel blocker tetrodotoxin was without effect.

Animals↗
PubMed1989

Macromolecular transport in rabbit blastocysts: evidence for a specific uteroglobin transport system.

Though uterine proteins are found within the blastocoel of the rabbit blastocyst, the mechanisms involved in protein entry into the blastocoel have not been studied. To investigate potential avenues of protein entry into the blastocoel, we have monitored uptake of a fluorescent, fluid-phase marker (lucifer yellow) into the rabbit blastocyst trophectodermal cell. In addition, we have measured the transtrophectodermal permeabilities of several uterine proteins (uteroglobin (UTG), rabbit serum albumin, rabbit IgG), and radiolabeled fluid-phase markers (sucrose, polyethylene glycol, dextran) in the 6- and 7-day post-coitus rabbit blastocyst. Extrablastocoelic lucifer yellow (LY) was rapidly endocytosed by the trophectodermal cell and was subsequently trapped in a perinuclear compartment for at least 30 min after removal of the extracellular dye. The slow rate of LY turnover within the endocytic compartments implies that the rate of fluid-phase transcytosis was negligible. Permeability coefficients of the fluid-phase markers and proteins, with the exception of UTG, decreased with increasing molecular weight of each compound tested. These data are consistent with the conclusion that these compounds traverse the trophectoderm via a 'leak' pathway, as opposed to a transcytotic pathway. In contrast, permeability of 125I-UTG when compared to the other compounds, was 10-fold greater than would be predicted based on its molecular weight. Furthermore, the flux of radioiodinated UTG displayed saturation kinetics with a Km of 19.5 micrograms/ml and a Vmax of 132 ng.cm-2.h-1. Sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated that the radioiodinated protein recovered from the blastocoel was of a lower molecular weight than native UTG and was not immunoreactive with goat anti-UTG antibody. Our data are consistent with the idea that UTG is transported across the trophectoderm by a receptor-mediated system, and UTG is modified intracellularly during transport to a protein of a lower molecular weight.

Animals↗