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PubMed1968

Ionic environment of neurones and glial cells in the brain of an amphibian.

1. Experiments were performed to determine the relative contribution of the blood plasma and of the cerebrospinal fluid (c.s.f.) to the ionic environment of neurones and glial cells within the brain of the amphibian Necturus maculosus.2. The concentrations in the blood plasma of untreated control animals were 99 +/- 2 mM for Na(+) and 2.0 +/- 0.1 mM for K(+). In the c.s.f. the corresponding values were 112 +/- 2 mM-Na(+) and 1.9 +/- 0.1 mM-K(+).3. By keeping animals in K(+)-rich water it was possible to raise chronically the concentrations of K(+) in the blood plasma up to almost 5 times the normal value, close to 9 mM, while the c.s.f. concentration of K(+) was only doubled, to about 4 mM. This behaviour of Necturus, tending to keep the K(+) in the c.s.f. low, resembles that of mammals.4. The membrane potential of glial cells in the optic nerve can be used as an accurate indicator for determining the K(+) concentration in the intercellular spaces. Such determinations were made in vivo, and it was shown that the glial cells adjust their membrane potential to the changes of K(+) concentrations in the c.s.f. and not to those of the blood plasma. In contrast, the membrane potential of skeletal muscle fibres changes according to the K(+) concentration in the blood plasma.5. It is concluded that the cells within the optic nerve are surrounded by an ionic environment which corresponds to that of the c.s.f. and not to that of the blood plasma. The intercellular spaces are open and ions diffuse freely into them from the c.s.f. A homeostatic mechanism operates, keeping the ion concentrations around neurones and glia within a narrow range and relatively independent of large changes in the blood plasma. This may provide relative stability for the signalling system. Similarities between the optic nerve and other parts of the central nervous system in respect to their relation to c.s.f. and blood are discussed. It seems likely that the mechanisms which control the electrolyte concentrations are similar in Necturus and in mammals.

Amphibians↗

PubMed1970

Responses to directional stimuli in retinal preganglionic units.

1. Extracellular recordings were made from directionally selective ganglion cell units in the isolated frog retina and decapitated Necturus preparation.2. Intracellular recordings were made from individual photoreceptor cells in the frog and Necturus retinae while stimuli which had evoked directionally selective responses at the ganglion cell level were presented. No evidence for inhibition of photoreceptors for any direction of movement of the light stimulus was found. This appeared to rule out a mechanism for directional selectivity involving inhibition of photoreceptor potentials.3. Intracellular recordings were made from the nuclear layer between photoreceptors and ganglion cells in Necturus. The responses were of two types: either transitory or sustained.4. The sustained type responses could be divided into two classes depending on their receptive field organization. One type of sustained potential had a large receptive field without any evidence for a centre-surround antagonism and corresponded to the luminosity type S-potential recorded in fish. The other type had a smaller receptive field and showed a difference in sign of response between centre and surround if the centre was flooded with a steady light. This is very similar to what has been described for a type of on-centre, off-surround ganglion cell.5. The transitory type of responses showed some centre-surround antagonistic organization. Some of these transitory units also appeared to show some discrimination in response as a function of the distribution of light on the retina.6. No specific directional selectivity was found from units at the inner nuclear layer. This further excluded any mechanism of directional sensitivity which involves selectivity at the photoreceptor level.7. It was concluded that although inner nuclear layer units may play a role in the mechanism of directional selectivity, no specific directionality was found at the first synaptic level of the retina.

Animals↗

PubMed1979

Dissociation of proximal tubular glucose and Na+ reabsorption by amphotericin B.

The effect of amphotericin B on glucose and Na+ transport was studied in the Necturus proximal tubule and in microvillus membrane vesicles isolated from the rabbit renal cortex. In the Necturus experiments, the rate constants for disappearance of radiolabeled glucose (kG) and mannitol (kM) from the tubular lumen were determined by stop-flow microperfusion. Saturability and Na+-dependence of glucose reabsorption was confirmed, since kG was reduced by raising intratubular glucose from 1 to 5 mM or by replacing intratubular Na+ with choline. Neither maneuver affected kM. Intratubular amphotericin B (10 microgram/ml), previously shown to stimulate active Na+ reabsorption in the Necturus proximal tubule, inhibited kG with no effect on kM. In the membrane vesicle preparation, amphotericin inhibited the uphill glucose uptake which results from imposing a NaCl gradient from outside to inside, but had no effect on glucose uptake in either the absence of Na+ or in the presence of Na+ when there was no Na+ gradient. Amphotericin B stimulated the uptake of Na+ by the vesicles. The observed dissociation of glucose and Na+ transport by amphotericin B is consistent with the concept that proximal tubular glucose reabsorption is energized by the luminal membrane Na+ gradient and is not directly linked to active Na+ transport per se.

Amphotericin B↗

PubMed1979

Stimulation of alkaline secretion in amphibian-isolated gastric mucosa by 16,16-dimethyl PGE2 and PGF2 alpha. A proposed explanation for some of the cytoprotective actions of prostaglandins.

The mechanism of the gastric cytoprotective action of prostaglandins is unknown but seems to be unrelated to inhibition of acid secretion. In the present study, effects of the prostaglandins, 16,16-dimethyl E2 and F2 alpha on H+ and HCO-3 secretion and electrical properties in amphibian-isolated gastric mucosa were studied. Spontaneous net secretion in fundic mucosa from Rana temporaria and Necturus was acid, whereas Necturus antrum secreted only HCO-3. The histamine H2-receptor antagonist, metiamide (10(-3) M), was used to inhibit acid secretion for studies on fundic alkalinization. Nutrient side administration of 16,16-dimethyl E2 (10(-6) M) for 60 min inhibited H+ secretion and stimulated HCO-3 secretion in Rana temporaria fundus. The drug (10(-5) M) also stimulated antral alkalinization. There was a dose-related increase in HCO-3 secretion in Necturus fundus after administration of F2 alpha (10(-5)-10(-4) M), but this drug had no significant effect on H+ secretion. Inhibition of acid secretion by 16,16-dimethyl E2 was associated with an increase in potential difference (PD), but there was no change in electrical resistance. Neither of the prostaglandins affected PD or resistance in alkaline-secreting tissues. Previous work has suggested that gastric HCO-3 secretion has a physiologic role in protecting the mucosal surface. The ability of prostaglandins to stimulate alkaline secretion may contribute to the cytoprotective action of these drugs in the stomach.

Animals↗

PubMed1980

Hydrostatic pressure changes related to paracellular shunt ultrastructure in proximal tubule.

We examined the effets of changes in hydrostatic pressures on the ultrastructural geometry of the lateral intercellular space and tight junctions in proximal tubules of contrtol (C) and volume-expanded (VE) Necturus kidney. The following groups of tubules were studied: (1) C, free-flow pressure, (2) C, stopped-flow, high-luminal pressure, (3) C, stopped-flow, low-luminal pressure, (4) VE, free-flow pressure, and (5) VE, stopped-flow, high-luminal pressure. Intratubular and peritubular capillary pressures were monitored before and during standardized perfusion-fixation for electron microscopy, and complete cross-sections of all sampled tubules were subjected to morphometric analysis. Average lateral intercellular space widths decreased significantly in C and VE stopped-flow tubules with high-luminal pressures but widened greatly in C stopped-flow tubules with low-luminal pressures. The length or width of the tight junctions did not change between the five experimental conditions. The ultrastructural changes correlate with the applied transepithelial pressure gradients rather than with transepithelial volume fluxes. The narrowing of lateral intercellular spaces in high pressure tubules correlate with the previously described increase in electrical resistance expressed per unit length tubule indicating that in these conditions part of the paracellular resistance is located in the free interspaces. The geometry of the lateral intercellular space in the proximal tubule of Necturus favors models of near-isotonic transport that do not depend on long and narrow interspaces.

Animals↗

PubMed1988

Influence of lateral intercellular spaces on current propagation in tubular epithelia as estimated by a multi-cable model.

A multiple cable model has been developed for tubular epithelia which allows current flow along the tubular lumen, along the cell layer and inside the lateral intercellular space (LIS) to be quantitatively assessed. In this model tubular lumen and cell layer are represented by two concentric cylinders and the LIS by n concentric interconnected fluid layers which are interposed between the cells, contact the lateral cell membranes and extend all along the tubular length. The innermost LIS layer connects to the tight junctions and the outermost layer to the peritubular space. Modelling each element by a cable-like structure the mathematical solution leads to n + 2 linear combinations of n + 2 exponential functions. Based on morphometric data and resistance measurements on Necturus proximal tubule [4,10] model calculations have been performed of the voltage attenuation along tubular lumen, cell layer and LIS for n = 3 or n = 6 assuming different LIS widths (0.02, 0.2, and 2.0 micron). The results show that the influence of LIS is insignificant in Necturus proximal tubule under control conditions, but may become significant in other functional states or other tubules. Collapsing the LIS increases predominantly the shunt resistance and the effective resistance of the lateral cell membrane but longitudinal current propagation along the LIS remains negligible at all space widths. In addition, model calculations are presented which allow errors in determining tight junction resistance and cell membrane resistances from a simple cable model to be quantified as function of LIS width.

Animals↗

PubMed1981

Chloride movement across the basolateral membrane of proximal tubule cells.

Electrophysiologic and tracer experiments have shown that Cl- enters Necturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na+, and that Cl- entry is electrically silent. The mechanism of Cl- exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl- ions across the basolateral membrane, the relative conductance of Cl- to K+ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl- and K+ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl- conductance was about 10% of K+ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl- activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl- activity was virtually unaffected by the PD changes. We conclude that Cl- leaves Necturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na+ or K+.

Animals↗

PubMed1997

A cyclic nucleotide-dependent chloride conductance in olfactory receptor neurons.

Whole-cell membrane currents were recorded from olfactory receptor neurons from the neotenic salamander Necturus maculosus. Cyclic nucleotides, released intracellularly by flash photolysis of NPE-caged cAMP or NPE-caged cGMP, activated a transient chloride current. The chloride current could be elicited at constant voltage in the absence of extracellular Ca2+ as well as in the presence of 3 mM intracellular Ca2+, suggesting that the current did not require either voltage or Ca2+ transients for activation. The current could be elicited in the presence of the protein kinase inhibitors H-7 and H-89, and in the absence of intracellular ATP, indicating that activation was independent of protein kinase A activity. These results suggest that Necturus olfactory receptor neurons contain a novel chloride ion channel that may be directly gated by cyclic nucleotides.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1982

Gallbladder epithelial cell hydraulic water permeability and volume regulation.

The hydraulic water permeability (Lp) of the cell membranes of Necturus gallbladder epithelial cells was estimated from the rate of change of cell volume after a change in the osmolality of the bathing solution. Cell volume was calculated from computer reconstruction of light microscopic images of epithelial cells obtained by the "optical slice" technique. The tissue was mounted in a miniature Ussing chamber designed to achieve optimal optical properties, rapid bath exchange, and negligible unstirred layer thickness. The control solution contained only 80% of the normal NaCl concentration, the remainder of the osmolality was made up by mannitol, a condition that did not significantly decrease the fluid absorption rate in gallbladder sac preparations. The osmotic gradient ranged from 11.5 to 41 mosmol and was achieved by the addition or removal of mannitol from the perfusion solutions. The Lp of the apical membrane of the cell was 1.0 X 10(-3) cm/s . osmol (Posm = 0.055 cm/s) and that of the basolateral membrane was 2.2 X 10(-3) cm/s . osmol (Posm = 0.12 cm/s). These values were sufficiently high so that normal fluid absorption by Necturus gallbladder could be accomplished by a 2.4-mosmol solute gradient across the apical membrane and a 1.1-mosmol gradient across the basolateral membrane. After the initial cell shrinkage or swelling resulting from the anisotonic mucosal or serosal medium, cell volume returned rapidly toward the control value despite the fact that one bathing solution remained anisotonic. This volume regulatory response was not influenced by serosal ouabain or reduction of bath NaCl concentration to 10 mM. Complete removal of mucosal perfusate NaCl abolished volume regulation after cell shrinkage. Estimates were also made of the reflection coefficient for NaCl and urea at the apical cell membrane and of the velocity of water flow across the cytoplasm.

Animals↗

PubMed1980

Cytoplasmic regulation of tight-junction permeability: effect of plant cytokinins.

The significance of the "leaky" tight junction might be understood better if cells of the epithelial monolayer possessed mechanisms to regulate molecular flow through the junction. To test this possibility, Necturus gallbladder, a representative leaky epithelium, was studied before, during, and after mucosal exposure to plant cytokinins and two other microfilament-active drugs, cytochalasin B and phalloidin. Concomitant with morphological changes in microfilaments, cytokinins induced rapid reversible increases in transepithelial resistance and potential difference (PD) and decreases in NaCl dilution potentials, with no change in the ratio of relative cell membrane resistances. Cytochalasin B (0.2-1.2 microM) and phalloidin (0.6-12.7 microM) caused similar changes in transepithelial resistance and PD. When the intramembranous structure of tight junctions was studied by freeze fracture, peak cytokinin-induced increments in transepithelial resistance were associated with more disorder in the strand meshwork resulting in a small increase in tight junction depth, but there was no evidence of de novo strand assembly. These studies suggest that permeability of the tight junction of Necturus gallbladder is subject to rapid reversible modulation, possibly under cytoskeletal control.

Animals↗

PubMed1985

NBD-taurine fluorescence as a probe of anion exchange in gallbladder epithelium.

Previous work has demonstrated that after osmotic shrinkage of Necturus gallbladder epithelial cells, their volumes are restored to control levels despite the continued presence of the hyperosmotic medium. It has been proposed that activation of parallel neutral Na+-H+ and Cl--HCO-3 exchangers in the apical membrane is necessary for regulatory volume increase. As an independent technique to determine whether and for how long ion flux through the anion exchanger is actually enhanced by exposure to hypertonicity, fluorescence measurements of N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine), a substrate of the anion exchanger in red blood cells, have been made in intact Necturus gallbladder. The cells were loaded with the dye by incubation. The tissue was perfused in a miniature chamber placed on the stage of a microscope and viewed with high-magnification optics combined with video. Fluorescence was monitored at frequent intervals with a photomultiplier tube, and transmittance of the tissue to the laser excitation light was monitored with a photodiode. The epithelium was simultaneously observed with transmitted light to control for changes in focus or lateral movement. Exposure of the tissue to a mucosal medium made hypertonic by the addition of mannitol transiently enhanced the efflux of NBD-taurine from the cells in approximately 70% of the tissues examined. In the presence of the anion-exchange inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 100 microM), hypertonicity enhanced NBD-taurine efflux in only 14% of the preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

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

PubMed1986

Segregation of gastric Na and Cl transport: a vibrating probe and microelectrode study.

The short-circuit current (Isc) of resting Necturus gastric mucosa (approximately 20 microA/cm2) can be attributed to the algebraic sum of the net Cl- secretion and amiloride-inhibitable net Na+ absorption. We have attempted to identify the cell types [surface epithelial cells (SCs) or oxyntic cells (OCs)] responsible for the transport of these ions in Necturus gastric mucosa using microelectrodes (ME) and a vibrating probe (VP). Mucosae were mounted horizontally in an open-topped Plexiglas chamber either serosal side up for basolateral ME impalements of OCs or mucosal side up for apical impalements of SCs and VP measurements. Cell impalements were made under open-circuit conditions, and VP measurements were performed under short-circuit conditions. Impalements of OCs indicate that neither the ratio of their apical to basolateral cell membrane resistances (Ra/Rb = 1.3 +/- 0.2) nor their cell membrane potentials were affected by 10(-6) M mucosal amiloride. In contrast, impalements of SCs indicate that amiloride increased their Ra/Rb from 3.5 +/- 0.2 to 15.6 +/- 1.8 and hyperpolarized both cell membrane potentials by greater than 20 mV. VP measurements showed that the amiloride-induced change in the current from SCs (5.6 microA/cm2) accounted for the amiloride-induced change in the Isc (5.5 microA/cm2). A non-zero current (4.4 +/- 1.0 microA/cm2) measured over SCs in the presence of amiloride was due to contamination from current arising from the gastric crypts that contain the OCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗

PubMed1999

Isolated epithelial cells from amphibian urinary bladder express functional gap junctional hemichannels.

Exposure of the urinary bladder epithelium of Necturus maculosus (NUB) to protease and collagenase yields approximately 50% isolated polarized cells. These cells express a membrane current slowly activated by depolarization or by removal of external divalent cations. The biophysical and pharmacological properties of the current are largely consistent with those of gap junctional hemichannels. After removal of divalent cations, the cells can also be loaded with 5(6)-carboxyfluorescein, a hydrophilic fluorescent anionic dye, and exposure to dye reduces the current in a manner dependent on membrane voltage and side of application. In contrast, Necturus gallbladder (NGB) cells exhibit no membrane conductance attributable to gap junctional hemichannels, although previous studies reveal the persistence of gap junction plaques on the plasma membrane. We conclude that functional gap junctional hemichannels can be expressed on the surface of certain isolated epithelial cells and that this is not a necessary consequence of the isolation procedure. These structures may contribute to cell damage under pathological conditions involving cell detachment.

Animals↗

PubMed1991

Biochemical and functional characterization of H(+)-K(+)-ATPase in distal amphibian nephron.

Because proton secretion and K+ reabsorption in the late distal tubule of amphibians are active, we evaluated whether these processes could be mediated by an H(+)-K(+)-ATPase similar to the gastric H(+)-K+ pump and to the K(+)-ATPase previously described in the terminal segments of the mammalian nephron. K(+)-stimulated ATPase activity was detected in microdissected segments of frog and Necturus nephron: its activity was high in the late distal and collecting tubules, whereas it was undetectable in the proximal convoluted tubule and early distal tubule. In frog collecting tubule, K(+)-ATPase had a high affinity for K+ (Km approximately 0.30 mM), was inhibited by vanadate, omeprazole, and the imidazopyridine Sch 28080, and was insensitive to ouabain. Furthermore, in vivo administration of Sch 28080 to anesthetized Necturus induced a significant rise of the steadystate intratubular pH in the late distal tubule, demonstrating that this drug inhibited tubular fluid acidification. It is suggested that K(+)-ATPase present in the terminal segments of amphibian nephron is similar to the gastric H(+)-K+ pump and is involved in urinary acidification.

Adenosine Triphosphatases↗

PubMed1986

Potassium channels along the nephron.

The K+ channels that are present in three different nephron segments, the Necturus proximal, Amphiuma early distal (diluting segment), and rabbit collecting tubule have been examined. Ca2+-sensitive K+ channels were present in the apical membranes of the cells lining all these segments. The channels were all voltage-sensitive and their open probability increased with membrane depolarization. Because of the ubiquitous distribution, it is suggested that this channel is responsible for K+ secretion by the nephron and that the same intracellular regulators act throughout the various segments. Basolateral K+ channels have been examined only in Necturus proximal tubules. This channel is apparently insensitive to Ca2+; the voltage dependence is exactly opposite to that of the apical K+ channels; that is, hyperpolarizing potentials caused an increase in open probability. These differences in regulatory factors permit the independent regulation of apical and basolateral membrane K+ permeabilities that must occur in renal cells.

Animals↗

PubMed1989

Acetylcholinesterase activity in the adrenal chromaffin vesicles of Urodela.

The presence of acetylcholinesterase (AChE) activity in the adrenal chromaffin cells of Necturus maculosus and Ambystoma maculatum (Amphibia, Urodela) has been demonstrated by cytochemical method at the electron microscope level. The enzymatic activity is localized in RER and perinuclear cisternae, on the plasma membrane and within the chromaffin vesicles, both in adrenaline (A) and noradrenaline (N) cells. Moreover N cells appear to be more reactive than A cells and Necturus more reactive than Ambystoma. The possible function of the AChE activity inside the vesicles is discussed as a mechanism of protons donor or as peptidasic activity acting on various peptides present in the vesicle.

Acetylcholinesterase↗

PubMed1985

Determinants of epithelial cell volume.

Epithelial cell volume is determined by the concentration of intracellular, osmotically active solutes. The high water permeability of the cell membrane of most epithelia prevents the establishment of large osmotic gradients between the cell and the bathing solutions. Steady-state cell volume is determined by the relative rates of solute entry and exit across the cell membranes. Inhibition of solute exit leads to cell swelling because solute entry continues; inhibition of solute entry leads to cell shrinkage because solute exit continues. Cell volume is then a measure of the rate and direction of net solute movements. Epithelial cells are also capable of regulation of the rate of solute entry and exit to maintain intracellular composition. Feedback control of NaCl entry into Necturus gallbladder epithelial cells is demonstrable after inhibition of the Na,K-ATPase or reduction in the NaCl concentration of the serosal bath. Necturus gallbladder cells respond to a change in the osmolality of the perfusion solution by rapidly regulating their volume to control values. This regulatory behavior depends on the transient activation of quiescent transport systems. These transport systems are responsible for the rapid readjustments of cell volume that follow osmotic perturbation. These powerful transporters may also play a role in steady-state volume regulation as well as in the control of cell pH.

Animals↗

PubMed1985

Effect of 16,16-dimethyl prostaglandin E2 on gastric epithelial cell membrane potentials and resistances.

Intracellular microelectrode techniques were employed to examine the effects of 16,16-dimethyl prostaglandin E2 (dmPGE2) on Necturus antral mucosa epithelial cell membrane potentials and resistances. Necturus antral mucosa was mounted in a modified Ussing chamber and stable intracellular impalements were obtained. Addition of 0.01 microgram/ml dmPGE2 to the mucosal solution produced an increase of the apical cell membrane potential (Vmc) from -44.3 +/- 3.1 to -53.5 +/- 3.8 mV (p less than 0.001) and of the basolateral cell membrane potential (Vcs) from -48.8 +/- 2.8 to -57.7 +/- 3.2 mV (p less than 0.001). This reversible, dose-dependent hyperpolarization of both cell membranes was accompanied by a decrease in the electrical resistance of the apical membranes (Ra) from 2550 +/- 250 omega/cm2 to 1870 +/- 210 omega/cm2 (p less than 0.05) and a decrease in the resistance of the basolateral membrane (Rb) from 1020 +/- 250 omega/cm2 to 630 +/- 80 omega/cm2 (p less than 0.05). In addition, there was an increase in the resistance of the shunt (intercellular junction, Rs), the major route of transepithelial ion flow, from 710 +/- 60 omega/cm2 to 750 +/- 80 omega/cm2 (p less than 0.05). Thus dmPGE2 increased the cell membrane potentials and reduced the ionic permeability of the intercellular junction.

16,16-Dimethylprostaglandin E2↗