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At least 631 records · Page 35Linked to original sources
PubMed1984

Identification of membrane transport processes in renal cells, by means of liquid ion exchanger microelectrodes.

The development of liquid-ion-exchanger microelectrodes displaying tip diameters less than or equal to 1 micron has permitted direct measurement of the transmembrane chemical and electrochemical gradients of several permeant ion species in cells of small size. We have used Cl- and H+ resins to study the intracellular Cl- activity (alpha iCl) and cell pH (pHi) in the proximal tubule of Necturus kidney. These determinations were performed in association with perfusion of peritubular capillaries by several artificial solutions, in order to assess the dependence of alpha iCl and pHi on the composition of physiologic plasma constituents and selected inhibitors. The main findings are: Intracellular chloride activity, alpha iCl, is higher than the theoretical value predicted from electrochemical equilibrium. Peritubular application of SITS resulted in a decrease of alpha iCl and increase of pHi; these observations are taken to indicate that Cl- uptake is achieved across the basolateral membrane in exchange for HCO-3 by a mechanism sensitive to SITS. Na+ removal from peritubular fluid elicited a small reduction of alpha iCl, suggesting the presence of carrier-mediated Cl--Na+ cotransport from interstitium to cell, contributing to the rise of alpha iCl above equilibrium. In conclusion, two carrier-mediated processes (Cl-/HCO-3 exchange and Cl--Na+ symport) located at the basolateral membrane of the proximal tubule may account for the establishment of alpha iCl values above equilibrium, at steady state. The physiologic role of these carriers is discussed in relation to proximal electrolyte absorption.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1984

Effect of aspirin on epithelial cell membrane potentials of gastric fundic mucosa.

The effects of aspirin on epithelial cell membrane potentials of Necturus fundic mucosa were examined by in vitro experiments according to intracellular microelectrode techniques. Stable intracellular impalements were obtained with 15 to 50 M-ohm microelectrodes filled with 3M KCl. In neutral mucosal solutions (pH 7.0) aspirin (5.0 mM) resulted in a significant increase in apical cell membrane potential (Vmc) from -36.7 +/- 1.5 mV to -43.3 +/- 2.3 mV (p less than 0.001) and basolateral cell membrane potential (Vcs) from -42.7 +/- 1.8 mV to -50.6 +/- 2.4 (p less than 0.001). This hyperpolarization of the cell was associated with an increase in transmucosal potential from -5.8 +/- 0.7 to -7.4 +/- 0.9 (p less than 0.05) and an increase in the ratio of apical to basolateral membrane resistances from 5.1 +/- 1.2 to 8.8 +/- 1.9 (p less than 0.05). These changes were consistent with an increase in potassium conductance induced by the salicylate anion. In acidic mucosal solutions (pH 4.5) aspirin caused a reduction in Vmc and Vcs. This hypopolarization of the cell membrane is consistent with acidification of the epithelial cells. These observations support the proposed mechanisms of aspirin injury: (1) back diffusion of H+ into the cells and (2) influx of the salicylate anions into the cells, which may interfere with intracellular metabolism.

Animals↗
PubMed1982

Alternative methods for measurement of membrane potentials in epithelia.

Methods for the measurement of membrane potentials in cells not easily penetrated by microelectrodes were assessed for use in epithelia. The lipophilic cation triphenylmethylphosphonium appeared to distribute in a Nernstian fashion in the epithelial cells of Necturus gallbladder as judged by parallel microelectrode measurements. In the toad urinary bladder, the distribution of this cation gave a value for the membrane potential of epithelial cells under short-circuit conditions of -62 mV in normal Ringer's solution and -51 mV after 2 h treatment with ouabain. In our laboratory the dye 3,3'-dipropylthiadicarbocyanine iodide, when used with cell suspensions, yielded results comparable to those of other workers, but we were unable to record a redistributional signal from epithelial sheets or scraped cells. The dye appeared to enter cells and become irreversibly bound. The membrane-bound merocyanine dyes have not been used on epithelial cells. They appear to hold the greatest promise for dynamic experiments on epithelial membrane potentials.

Animals↗
PubMed1982

Effects of ouabain on the electrophysiological properties of proximal tubular cells.

The properties of the Na-K pump were studied by electrophysiological techniques in the proximal tubule of Necturus kidney. Peritubular capillaries were perfused for a short time with different artificial solutions, then ouabain (50 microM-1 mM) was added to each perfusate and its effects on cell membrane potential were assessed. Addition of ouabain, 1 mM, in a physiologic Ringer's solution (K concentration = 3 mM) failed to produce immediate depolarization. The introduction of the cardiac glycoside (50 microM) in solutions in which external K+ concentration was lowered to 0.75 or 0.3 mM resulted in depolarization by 4.1 +/- 0.8 and 9.5 +/- 1.4 mV, respectively. Furthermore, ouabain brought about an increase of membrane input resistance when added in a solution of lowered K+ concentration, but not in the presence of physiologic K+ concentrations. Readmission of K+ into a K-free perfusate flowing through the renal capillaries for more than 15 min resulted in hyperpolarization, 7.2 + 2.0 mV. These and other observations suggest that the Na-K pump operates in electrogenic fashion when external K+ concentration is lowered. Yet, part of the depolarizing response to ouabain under these conditions is mediated via ouabain-elicited changes of cell membrane permeabilities. The stoichiometry of the Na-K pump at physiologic external K concentrations cannot be assessed from the present experiments.

Animals↗
PubMed1981

Intracellular sodium activity and transcellular sodium transport in gallbladder.

As stated in the introduction, the purpose of this report has been to illustrate how the measurement of steady-state intracellular ionic activities, a technique that has proved to be of great importance in studying the energetics of transmembrane ionic transfer processes, can, with appropriate assumptions, be used to obtain information concerning the kinetics of these processes. Specifically, our analysis has focussed on transcellular Na+ transport in Necturus gallbladder and has shown that, given the steady-state values of Em, a1Na, and apical Na+ conductance for a particular set of conditions, it is possible to obtain estimates of net baso-lateral Na+ efflux (Na+ pump rate), net (and/or unidirectional) diffusive apical Na+ influx, and net coupled NaCl influx. It should be emphasized that the analysis outlined above is a preliminary essay in this direction. We present it here in the hope that, wit appropriate refinements, it may prove useful in unraveling the mechanisms by which drugs, hormones and other specific agents affect membrane function in epithelial and other systems.

Animals↗
PubMed1982

Heterogeneity in the distal nephron of the salamander (Ambystoma tigrinum): a correlated structure function study of isolated tubule segments.

Studies on isolated perfused tubules of the tiger salamander (Ambystoma tigrinum) have shown that the distal nephron is heterogeneous with respect to function (Stoner, 1977). In this study, the initial portion of the distal tubule (diluting segment) exhibited a voltage, positive in the lumen, and a net absorption of chloride. Since the chloride was transported against an electro-chemical gradient, its transport was active. More distad, the junctional segment exhibited a lumen-negative voltage and sodium, rather than chloride, was transported actively. More recently Delaney and Stoner (1981) have demonstrated in vitro that the collecting duct of this species also has a lumen-negative voltage which is probably associated with active sodium reabsorption. The primary objective of the present paper was to correlate the morphology of the diluting and junctional segments of the Ambystoma distal tubules with the physiologic data from the same isolated perfused tubules. The results indicate that the morphological heterogeneity previously demonstrated in distal tubules of Necturus exists with respect to both structure and function in Ambystoma. The cell types found in the amphibian distal nephron appear to be homologous to those seen in the mammalian nephron.

Animals↗
PubMed1992

Histochemical study of the heart of the axolotl (Ambystoma mexicanum).

We have investigated the presence of cells containing monoamines, substance P, and neuron-specific enolase (NSE) in the heart and in the pericardial wall of a urodele amphibian, the axolotl. Fibers containing substance P-like immunoreactivity were present in the heart but not in the pericardial wall. Also present in the heart were small branched cells, which stained metachromatically with toluidine blue. Similar cells were found in the peritoneum and were tentatively identified as mast cells. NSE-immunoreactive fibers were found both in the heart and in the pericardial wall. Small intensely fluorescent (SIF) cells of the pericardial wall contained a high concentration of norepinephrine but no other monoamines, substance P, or NSE. Comparison with data available for the mudpuppy, Necturus maculosus, a closely related amphibian species, suggests that the innervation of the heart in the axolotl is substantially different.

Ambystoma mexicanum↗
PubMed1976

Functional role of efferents to the avian retina. II. Effects of reversible cooling of the isthmo-optic nucleus.

Efferents to the retina in the bird arise in the isthmo-optic nucleus of the caudal midbrain, and terminate on amacrine cells in the retina. The functional role of these efferents was studied by determining the receptive field properties of 107 optic tract fibers in the lightly anesthetized adult pigeon, and quantitating their responses to specific moving stimuli. While the recording from these fibers continued, the isthmo-optic nucleus was cooled by a thermoelectric cooling probe, and the response properties of the cells redetermined. Recording was maintained in half of the units long enough to observe recovery from cooling, and in several units the entire procedure was repeated. In 77 of the 107 units, responsiveness to all stimuli was decreased by removing efferent influences, whereas specific receptive field properties such as motion sensitivity or directionaltiy were not altered. All of the major receptive field types were affected in a similar fashion, irrespective of their position in the visual field. Responses to stimuli that did not involve the antagonistic surround were similarly affected by removal of the efferents, as were units were both weak and strong antagonistic surrounds. Efferents exert their influence on retinal ganglion cells by way of the amacrine cells on which they terminate. Data available on amacrines in Necturus indicates that they are inhibitory to ganglion cells. If amacrines have a similar role in the pigeon, then it may be stated that decreased activity in the centrifugal fibers leads to enhanced inhibition throughout the receptive fields of ganglion cells, and increased activity in the efferents produces disinhibition.

Animals↗
PubMed1988

Putative neurotransmitters in the retinae of three urodele species (Triturus alpestris, Salamandra salamandra, Pleurodeles waltli).

The immunocytochemical localization of several substances with putative neurotransmitter or modulator properties was investigated in the retinae of three urodele species. Gamma-aminobutyric acid-like immunoreactive labelling appeared in different types of amacrine and horizontal cells. In addition, labelled fibres in the optic nerve were detected. It was not possible to determine whether these fibres were ganglion-cell axons or part of an efferent projection. Endogenous serotonin was found in several populations of amacrine cells including stratified and diffuse types. Glucagon-like immunoreactivity appeared in one bistratified amacrine cell type, and neurotensin-like immunoreactivity was detected in a single monostratified amacrine cell type. Metenkephalin-like-immunoreactive labelling was type. Metenkephalin-like-immunoreactive labelling was rare but found in several sublaminae of the inner plexiform layer. Thus each peptide-like-immunoreactive cell type makes up a distinct and unique population of cells and probably has a special functional role in retinal processing. There are striking similarities in the peptide-like immunoreactive patterns of Triturus alpestris and Necturus maculosus whereas in Ambystomatidae the peptide-like-immunoreactive systems appear to be differently organized. This supports the hypothesis that Salamandridae and Proteidae are more closely related to each other than to the Ambystomatidae.

Animals↗
PubMed1976

DNA reassociation kinetics in relation to genome size in four amphibian species.

DNA reassociation kinetics were studied, by means of the hydroxyapatite chromatography method, for four species of Amphibians with different nuclear DNA content: Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) of the Anura subclass and Triturus cristatus (23 pg) and Necturus maculosus (52 pg) of the Urodela subclass. Within each subclass the two species studied were found to have about the same absolute amount of unique DNA. The differences of total nuclear DNA can be accounted for by quantitative variations of the repetitive sequence classes, at least in part due to changes in the number of copies of the various sequences. On the contrary the great difference in nuclear DNA between the two subclasses, Anura and Urodela, involves all sequence classes in parallel; the slowly reassociating fraction appears to be unique in spite of a tenfold difference in absolute amount. The dependence of reassociation kinetics on DNA fragment length for the four species indicates for all of them an interspersed organization of the various sequence classes.

Amphibians↗
PubMed1982

Transcription of complementary repeat sequences in amphibian oocytes.

Repeat sequences are transcribed in the germinal vesicles of amphibian oocytes. In the hnRNA population both complements of the repeats are found and can be readily detected because they form intermolecular duplex structures. The structure and formation of duplex regions have been studied in the hnRNA of Xenopus laevis, Triturus cristatus, Amphiuma means and Necturus maculosus, a series of amphibians of increasing genome size (C-value). In T. cristatus, the duplex structures are mostly 600-1200 bp in length, whereas in X. laevis they are shorter and in N. maculosus they tend to be longer. Although the proportion of RNA sequence capable of rapidly forming duplex structures is different in different organisms, this property bears no relationship to C-value. However the sequence complexity of complementary repeats, as estimated from the rate of duplex formation, does show an increasing trend with C-value. The complementary repeats found in oocyte hnRNA are transcribed from families of DNA sequence that are each represented in the genome by thousands of copies. The extent of cross-species hybridization is low, indicating that the repeat sequences transcribed in different amphibian genera are not the same. In situ hybridization experiments indicate that the repeat sequences are spread throughout the genome. The evolution and possible function of complementary repeats are considered.

Amphibians↗
PubMed1985

Pathways for volume flow and volume regulation in leaky epithelia.

Continuous pathways must pierce the cell membrane to be used by water during osmotic equilibration between proximal straight tubular cells and the external medium, because a) the water osmotic permeability coefficient of the basolateral plasma membrane, Poscb, is high; b) its activation energy, Ea, is as that of free water movement and c) pCMBS inhibits markedly (but reversibly) Poscb and increases Ea to values similar to those observed in lipid bilayers without pores. d) Preliminary measurements of Pd the water diffusive permeability coefficient using NMR indicate that Poscb/Pd is near 4 - 5. The following two observations indicate that a significant paracellular water flow must exist in leaky epithelia. Namely, a) large extracellular solutes are dragged by water in four leaky epithelia: gall bladder, Necturus proximal tubule, rat proximal tubule and Rhodnius malpighian tubule. b) The transcellular water osmotic permeability coefficient is smaller than the transepithelial values available in the rabbit proximal straight tubule. This requires a significant paracellular permeability.

Animals↗
PubMed1980

Versatile piezoelectric driver for cell puncture.

A simple and versatile tool facilitating micropuncture of small cells is described which utilizes a commercial piezoelectric element made from a stacked column of monomorph ceramic discs. The device is able to advance complete input stage-electrode-assemblies with high speed and can be used in combination with conventional micromanipulators. Advancing characteristics as recorded optically at high magnification demonstrated less axial vibration, although faster action, than two other modern micropositioners driven by step motors. In biological experiments on selected tissues (Necturus gallbladder epithelium, Amphiuma renal distal tubule cells, rabbit and human corneal endothelium) the combined use of micromanipulator and piezo-stepper was, in all cases, superior to the use of a micromanipulator alone: the percentage of successful cell penetrations increased, cell potentials were stable for a longer time, and the durability of electrode-tips improved.

Animals↗
PubMed1975

[The role of cyclic adenosine 3':5'-monophosphate as "second messenger" in gastric acids secretion (author's transl)].

The criteria to determine whether cyclic adenosine 3':5'-monophosphate (cyclic AMP) is or is not involved in a particular hormone response require to look for positive qualitive, quantitative and temporal correlations between the effects of the hormone on cyclic AMP levels and the physiological response. These requirements have been shown to be fulfilled in histamine and pentagastrin-stimulated gastric acid secretion of the frog (Necturus maculosus) and the rat. In dog and man, however, the available evidence does not support a role for cyclic AMP in the gastric secretory process; it remains a challenge for future research whether in those species cyclic guanosine 3':5'-monophosphate acts as an intracellular substitute for cyclic AMP.

Adenylyl Cyclases↗
PubMed1996

Modulation of the junctional integrity by low or high concentrations of cytochalasin B and dihydrocytochalasin B is associated with distinct changes in F-actin and ZO-1.

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2-1.2 microM) and higher concentrations (1.5 microM and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain 1 (MDCK-1) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 microgram/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.

Actins↗
PubMed1985

Intracellular calcium activity in split frog skin epithelium: effect of cAMP.

Measurement of intracellular calcium activity (acCa) by ion-selective microelectrodes has previously been technically limited to relatively large cells (greater than or equal to 20 micron). We now report results obtained with this technique in the small epithelial cells (less than or equal to 10 micron) of split frog skin using microelectrodes having an outer tip diameter of less than 0.2 micron. The basolateral membrane potential was measured with Ca2+-selective microelectrodes (EscCa) and with reference micropipettes (psi sc) either sequentially or simultaneously in 15 successful experiments. Under baseline conditions, acCa was measured to be 215 +/- 39 nM (mean +/- SE), in close agreement with the mean values estimated from published data obtained with Necturus proximal tubule. Stimulation of Na+ transport across six skins with 1 mM serosal 8 p-chlorophenylthio-3,5' cyclic AMP (CPTcAMP) increased acCa by a factor of 2.6 +/- 0.6. The increase in acCa preceded the CPTcAMP-induced increase in Isc. The results of the present study indicate that electrometric determination of intracellular calcium activity is now feasible in a much wider range of cell systems than heretofore possible. CPT cAMP elevates intracellular Ca2+ activity; this phenomenon is an early event, preceding the natriferic effect of CPTcAMP.

Animals↗
PubMed1979

Intracellular ionic activities and transmembrane electrochemical potential differences in gallbladder epithelium.

Intracellular ion activities in Necturus gallbladder epithelium were measured with liquid ion-exchanger microelectrodes. Mean values for K, Cl and Na activities were 87, 35 and 22 mM, respectively. The intracellular activities of both K and Cl are above their respective equilibrium values, whereas the Na activity is far below. This indicates that K and Cl are transported uphill toward the cell interior, whereas Na is extruded against its electrochemical gradient. The epithelium transports NaCl from mucosa to serosa. From the data presented and the known Na and Cl conductances of the cell membranes, we conclude that neutral transport driven by the Na electrochemical potential difference can account for NaCl entry at the apical membrane. At the basolateral membrane, Na is actively transported. Because of the low Cl conductance of the membrane, only a small fraction of Cl transport can be explained by diffusion. These data suggest that Cl transport across the basolateral membrane is a coupled process which involves a neutral NaCl pump, downhill KCl transport, or a Cl-anion exchange system.

Animals↗
PubMed1979

Effects of external sodium and cell membrane potential on intracellular chloride activity in gallbladder epithelium.

Conventional and Cl-selective liquid ion-exchanger intracellular microelectrodes were employed to study the effects of extracellular ionic substitutions on intracellular Cl activity (aCli) in Necturus gallbladder epithelium. As shown previously (Reuss, L., Weinman, S.A., 1979; J. Membrane Biol. 49:345), when the tissue was exposed to NaCl-Ringer on both sides aCli was about 30 mM, i.e., much higher than the activity predicted from equilibrium distribution (aCleq) across either membrane (5--9 mM). Removal of Cl from the apical side caused a reversible decrease of aCli towards the equilibrium value across the basolateral membrane. A new steady-state aCli was reached in about 10 min. Removal of Na from the mucosal medium or from both media also caused reversible decreases of aCli when Li, choline, tetramethylammonium or N-methyl-D-glucamine (NMDG) were employed to replace Na. During bilateral Na substitutions with choline the cells depolarized significantly. However, no change of cell potential was observed when NMDG was employed as Na substitute. Na replacements with choline or NMDG on the serosal side only did not change aCli. When K substituted for mucosal Na, the cells depolarized and aCli rose significantly. Combinations of K for Na and Cl for SO4 substitutions showed that net Cl entry during cell depolarization can take place across either membrane. The increase of aCli in depolarized cells exposed to K2SO4-Ringer on the mucosal side indicates that the basolateral membrane Cl permeability (PCl) increased. These results support the hypothesis that NaCl entry at the apical membrane occurs by an electroneutral mechanism, driven by the Na electrochemical gradient. In addition, we suggest that Cl entry during cell depolarization is downhill and involves an increase of basolateral membrane PCl.

Animals↗