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PubMed1985

Cyclic AMP inhibits Na+/H+ exchange at the apical membrane of Necturus gallbladder epithelium.

The effects of elevating intracellular cAMP levels on Na+ transport across the apical membrane of Necturus gallbladder epithelium were studied by intracellular and extracellular microelectrode techniques. Intracellular cAMP was raised by serosal addition of the phosphodiesterase inhibitor theophylline (3 mM) or mucosal addition of either 8-Br-cAMP (1 mM) or the adenylate cyclase activator forskolin (10 microM). During elevation of intracellular cAMP, intracellular Na+ activity (alpha Nai) and intracellular pH (pHi) decreased significantly. In addition, acidification of the mucosal solution, which contained either 100 or 10 mM Na+, was inhibited by approximately 50%. The inhibition was independent of the presence of Cl- in the bathing media. The rates of change of alpha Nai upon rapid alterations of mucosal [Na+] from 100 to 10 mM and from 10 to 100 mM were both decreased, and the rate of pHi recovery upon acid loading was also reduced by elevated cAMP levels. Inhibition was approximately 50% for all of these processes. These results indicate that cAMP inhibits apical membrane Na+/H+ exchange. The results of measurements of pHi recovery at 10 and 100 mM mucosal [Na+] and a kinetic analysis of recovery as a function of pHi suggest that the main or sole mechanism of the inhibitory effect of cAMP is a reduction in the maximal rate of acid extrusion. In conjunction with the increase in apical membrane electrodiffusional Cl- permeability, produced by cAMP, which causes a decrease in net Cl- entry (Petersen, K.-U., and L. Reuss, 1983, J. Gen. Physiol., 81:705), inhibition of Na+/H+ exchange contributes to the reduction of fluid absorption elicited by this agent. Similar mechanisms may account for the effects of cAMP in other epithelia with similar transport properties. It is also possible that inhibition of Na+/H+ exchange by cAMP plays a role in the regulation of pHi in other cell types.

Animals↗
PubMed1987

Characterization of the basolateral membrane conductance of Necturus urinary bladder.

Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that permitted rapid changes in the ion composition of the serosal solution. The transepithelial electrical properties exhibited a marked seasonal variation that could be attributed to variations in the conductance of the shunt pathway, apical membrane selectivity, and basolateral Na+ transport. In contrast, the passive electrical properties of the basolateral membrane remained constant throughout the year. The apparent transference numbers (Ti) of the basolateral membrane for K+ and Cl- were determined from the effect on the basolateral membrane equivalent electromotive force of a sudden increase in the serosal K+ concentration from 2.5 to 50 mM/liter or a decrease in the Cl- concentration from 101 to 10 mM/liter. TK and TCl were 0.71 +/- 0.05 and 0.04 +/- 0.01, respectively. The basolateral K+ conductance could be blocked by Ba2+ (0.5 mM), Cs+ (10 mM), or Rb+ (10 mM), but was unaffected by 3,4-diaminopyridine (100 microM), decamethonium (100 microM), or tetraethylammonium (10 mM). We conclude that a highly selective K+ conductance dominates the electrical properties of the basolateral membrane and that this conductance is different from those found in nerve and muscle membranes.

4-Aminopyridine↗
PubMed1987

Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder.

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.

Amiloride↗
PubMed1987

Cyclic AMP inhibits Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder epithelium.

Intracellular microelectrode techniques were employed to study the effect of cyclic AMP on apical membrane Cl-/HCO3- exchange and electrodiffusive HCO3- transport in Necturus gallbladder epithelium. Intracellular cAMP levels were raised by addition of either the phosphodiesterase inhibitor theophylline (3 X 10(-3) M) or the adenylate cyclase activator forskolin (10(-5) M) to the serosal bathing solution. Measurements of pH in a poorly buffered control mucosal solution upon stopping superfusion show acidification, owing to secretion of both H+ and HCO3-. When the same experiment is performed after addition of amiloride or removal of Na+ from the mucosal bathing medium, alkalinization is observed since H+ transport is either inhibited or reversed, whereas HCO3- secretion persists. The changes in pH in both amiloride or Na-free medium were significantly decreased in theophylline-treated tissues. Theophylline had no effect on the initial rates of fall of intracellular Cl- activity (aCli) upon reducing mucosal solution [Cl-] to either 10 or 0 mM, although membrane voltage and resistance measurements were consistent with stimulation of apical membrane electrodiffusive Cl- permeability. Estimates of the conductive flux, obtained by either reducing simultaneously mucosal [Cl-] and [HCO3-] or lowering [Cl-] alone in the presence of a blocker of anion exchange (diphenylamine-2-carboxylate), indicate that elevation of intracellular cAMP inhibited the anion exchanger by approximately 50%. Measurements of net Cl- uptake upon increasing mucosal Cl- from nominally zero to levels ranging from 2.5 to 100 mM suggest that the mechanism of inhibition is a decrease in Vmax. Consistent with these results, the rate of intracellular alkalinization upon reducing external Cl- was also inhibited significantly by theophylline. Reducing mucosal solution [HCO3-] from 10 to 1 mM under control conditions caused intracellular acidification and an increase in aCli. Theophylline inhibited both changes, by 62 and 32%, respectively. These data indicate that elevation of intracellular cAMP inhibits apical membrane anion (Cl-/HCO3-) exchange. Studies of the effects of rapid changes in mucosal [HCO3-] on membrane voltages and the apparent ratio of membrane resistances, both in the presence and in the absence of theophylline, with or without Cl- in the mucosal solution, do not support the hypothesis that cAMP produces a sizable increase in apical membrane electrodiffusive HCO3- permeability.

Animals↗
PubMed1990

Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium.

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.

Animals↗
PubMed1991

Effects of changes in mucosal solution Cl- or K+ concentration on cell water volume of Necturus gallbladder epithelium.

An electrophysiologic technique was used to measure changes in cell water volume in response to isosmotic luminal solution ion replacement. Intracellular Cl- activity (aCl-i) was measured and net flux determined from the changes in volume and activity. Reduction of luminal solution [Cl-] from 98 to 10 mM (Cl- replaced with cyclamate) resulted in a large fall in aCl-i with no significant change in cell water volume. Elevation of luminal solution [K+] from 2.5 to 83.5 mM (K+ replaced Na+) caused a small increase in aCl-i with no change in cell water volume. Exposure of the Necturus gallbladder epithelium to agents that increase intracellular cAMP levels (forskolin and/or theophylline) induces an apical membrane electrodiffusive Cl- permeability accompanied by a fall in aCl-i and cell shrinkage. In stimulated tissues, reduction of luminal solution [Cl-] resulted in a large fall in aCl-i and rapid cell shrinkage, whereas elevation of luminal solution [K+] caused a large, rapid cell swelling with no significant change in aCl-i. The changes in cell water volume of stimulated tissues elicited by lowering luminal solution [Cl-] or by elevating luminal solution [K+] were reduced by 60 and 70%, respectively, by addition of tetraethylammonium (TEA+) to the luminal bathing solution. From these results, we conclude that: (a) In control tissues, the fall in aCl-i upon reducing luminal solution [Cl-], without concomitant cell shrinkage, indicates that the Cl- entry mechanism is electroneutral (Cl-/HCO3-) exchange. (b) Also in control tissues, the small increase in aCl-i upon elevating luminal solution [K+] is consistent with the recent demonstration of a basolateral Cl- conductance. (c) The cell shrinkage elicited by elevation of intracellular cAMP levels results from conductive loss of Cl- (and probably K+). (d) Elevation of cAMP inhibits apical membrane Cl-/HCO-3-exchange activity by 70%. (e) The cell shrinkage in response to the reduction of mucosal solution [Cl-] in stimulated tissues results from net K+ and Cl- efflux via parallel electrodiffusive pathways. (f) A major fraction of the K+ flux is via a TEA(+)-sensitive apical membrane K+ channel.

Animals↗
PubMed1991

Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium.

The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-.

Adenosine Triphosphate↗
PubMed1992

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium.

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.

Amiloride↗
PubMed1992

Pseudo-streaming potentials in Necturus gallbladder epithelium. I. Paracellular origin of the transepithelial voltage changes.

Apparent streaming potentials were elicited across Necturus gallbladder epithelium by addition or removal of sucrose from the apical bathing solution. In NaCl Ringer's solution, the transepithelial voltage (Vms) change (reference, basolateral solution) was positive with sucrose addition and negative with sucrose removal. Bilateral Cl- removal (cyclamate replacement) had no effect on the polarity or magnitude of the Vms change elicited by addition of 100 mM sucrose. In contrast, bilateral Na+ removal (tetramethylammonium [TMA+] replacement) inverted the Vms change (from 2.7 +/- 0.3 to -3.2 +/- 0.2 mV). Replacement of Na+ and Cl- with TMA+ and cyclamate, respectively, abolished the change in Vms. Measurements of cell membrane voltages and relative resistances during osmotic challenges indicate that changes in cell membrane parameters do not explain the transepithelial voltage changes. The initial changes in Vms were slower than expected from concomitant estimates of the time course of sucrose concentration (and hence osmolality) at the membrane surface. Paired recordings of the time courses of paracellular bi-ionic potentials (partial substitution of apical Na+ with tetrabutylammonium [TBA+]) revealed much faster time courses than those produced by sucrose addition, although the diffusion coefficients of sucrose and TBACl are similar. Hyperosmotic and hypoosmotic challenges yielded initial Vms changes at the same rate; thereafter, the voltage increased with hypoosmotic solution and decreased with hyperosmotic solution. These late voltage changes appear to result from changes in width of the lateral intercellular spaces. The early time courses of the Vms changes produced by osmotic challenge are inconsistent with the expectations for water-ion flux coupling in the junctions. We propose that they are pseudo-streaming potentials, i.e., junctional diffusion potentials caused by salt concentration changes in the lateral intercellular spaces secondary to osmotic water flow.

Action Potentials↗
PubMed1992

Pseudo-streaming potentials in Necturus gallbladder epithelium. II. The mechanism is a junctional diffusion potential.

The mechanisms of apparent streaming potentials elicited across Necturus gallbladder epithelium by addition or removal of sucrose from the apical bathing solution were studied by assessing the time courses of: (a) the change in transepithelial voltage (Vms). (b) the change in osmolality at the cell surface (estimated with a tetrabutylammonium [TBA+]-selective microelectrode, using TBA+ as a tracer for sucrose), and (c) the change in cell impermeant solute concentration ([TMA+]i, measured with an intracellular double-barrel TMA(+)-selective microelectrode after loading the cells with TMA+ by transient permeabilization with nystatin). For both sucrose addition and removal, the time courses of Vms were the same as the time courses of the voltage signals produced by [TMA+]i, while the time courses of the voltage signals produced by [TBA+]o were much faster. These results suggest that the apparent streaming potentials are caused by changes of [NaCl] in the lateral intercellular spaces, whose time course reflects the changes in cell water volume (and osmolality) elicited by the alterations in apical solution osmolality. Changes in cell osmolality are slow relative to those of the apical solution osmolality, whereas lateral space osmolality follows cell osmolality rapidly, due to the large surface area of lateral membranes and the small volume of the spaces. Analysis of a simple mathematical model of the epithelium yields an apical membrane Lp in good agreement with previous measurements and suggests that elevations of the apical solution osmolality elicit rapid reductions in junctional ionic selectivity, also in good agreement with experimental determinations. Elevations in apical solution [NaCl] cause biphasic transepithelial voltage changes: a rapid negative Vms change of similar time course to that of a Na+/TBA+ bi-ionic potential and a slow positive Vms change of similar time course to that of the sucrose-induced apparent streaming potential. We conclude that the Vms changes elicited by addition of impermeant solute to the apical bathing solution are pseudo-streaming potentials, i.e., junctional diffusion potentials caused by salt concentration changes in the lateral intercellular spaces secondary to osmotic water flow from the cells to the apical bathing solution and from the lateral intercellular spaces to the cells. Our results do not support the notion of junctional solute-solvent coupling during transepithelial osmotic water flow.

Action Potentials↗
PubMed1992

Chemosensory responses in isolated olfactory receptor neurons from Necturus maculosus.

Olfactory receptor neurons were isolated without enzymes from the mudpuppy, Necturus maculosus, and tested for chemosensitivity. The cells responded to odorants with changes in firing frequency and alterations in excitability that were detected with tight-seal patch electrodes using on-cell and whole-cell recording conditions. Chemosensitive cells exhibited two primary response characteristics: excitation and inhibition. Both types of primary response were observed in different cells stimulated by mixtures of amino acids as well as by the single compound L-alanine, suggesting that there may be more than one transduction pathway for some odorants. Using the normal whole-cell recording method, the chemosensitivity of competent cells washed out rapidly; a resistive whole-cell method was used to record odorant responses under current-clamp conditions. In response to chemical stimulation, excitability appeared to be modulated in several different ways in different cells: odorants induced hyperpolarizing or depolarizing receptor potentials, elicited or inhibited transient, rhythmic generator potentials, and altered excitability without changing the membrane potential or input resistance. These effects suggest that olfactory transduction is mediated through at least three different pathways with effects on four or more components of the membrane conductance. Polychotomous pathways such as these may be important for odor discrimination and for sharpening the "odor image" generated in the olfactory epithelium.

Action Potentials↗
PubMed1992

Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli.

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.

Adenosine Triphosphate↗
PubMedDate not supplied

Carbonic anhydrase and red blood cell anion exchange in the neotenic aquatic salamander, Necturus maculosus.

Carbonic anhydrase (CA) activity in blood and other tissues and red blood cell (rbc) anion exchange were measured in the mud puppy, Necturus maculosus, in order to gain insight into the strategy for CO2 transport used by these neotenic salamanders and to further explore evolutionary relationships between rbc CA activity and anion exchange in nonmammalian vertebrates. CA activity was detectable in all of the tissues examined, but CA activity in blood was much lower than that in most vertebrates. There was no indication, however, that additional CA had been incorporated into the membrane fraction of other tissues to compensate for this low blood CA activity. In further contrast to most other animals, low levels of CA activity were also detectable in mud puppy plasma. Preliminary characterization of the rbc CA indicated that the Type II, fast-turnover enzyme was indeed present, but that there are a very low number of active sites in mud puppy rbc's. Further experiments showed that the rbc's were highly permeable to anions and that the relative rate of anion flux could be inhibited by 4, 4-diisothiocyanostilbene-2,2-disulphonic acid. Thus, the process of CO2 transport in the blood of mud puppies probably involves components of the Jacobs-Stewart cycle, as in most other vertebrates.

Adaptation, Physiological↗
PubMed2002

Two second messengers mediate amino acid responses in olfactory sensory neurons of the salamander, Necturus maculosus.

Odor transduction mediated by the adenylyl cyclase/cAMP pathway has been well studied, but it is still uncertain whether this pathway mediates the transduction of all odors in vertebrates. We isolated olfactory sensory neurons from the salamander Necturus maculosus and used calcium imaging with the indicator dye fura-2 to examine olfactory responses elicited by amino acids. The properties of approximately two-thirds of the odor responses suggested they were mediated by the adenylyl cyclase/cAMP pathway, but one-third of the responses were not mimicked by cAMP analogs nor blocked by inhibition of adenylyl cyclase, suggesting that these odor responses were mediated differently. Responses that were unaffected by inhibition of adenylyl cyclase were blocked by neomycin, an inhibitor of phospholipase C, implying that they were transduced by activation of phospholipase C. Some cells which responded to more than one amino acid appeared to employ both pathways, but each was used to transduce different odors. In addition, many responses that were mediated by the adenylyl cyclase/cAMP pathway were enhanced following inhibition of phospholipase C, suggesting that the phospholipase C pathway has a role not only in odor transduction, but also in the modulation of olfactory responses.

Action Potentials↗
PubMed1982

Fluid reabsorption by Necturus proximal tubule perfused with solutions of normal and reduced osmolarity.

Fluid absorption in Necturus proximal tubule was studied when the kidneys were perfused with solutions of different osmolarities. The rate of fluid absorption was inversely proportional to the perfusion fluid osmolarity, while Na uptake remained constant. No difference was detected between the collected and injected luminal fluid, i.e. reabsorption was isotonic at normal and reduced osmolarities. The transtubular osmotic permeability remained fairly constant under the different perfusion osmolarities. Using our experimental results to test various models based on osmotic equilibration across the tubule wall we show that none of these provides an adequate mechanism for fluid absorption in this epithelium.

Animals↗
PubMed1995

Milk and egg phospholipids act as protective surfactants against luminal acid in Necturus gastric mucosa.

BACKGROUND: Our previous studies indicate that milk phospholipids have anti-ulcer properties in rats and humans, possibly by forming a hydrophobic surfactant layer at the epithelial surface. In the present study we measured intracellular pH and parameters of membrane resistances in gastric epithelium exposed to luminal acid using a microelectrode technique. METHODS: Chambered isolated Necturus maculosus antral mucosa was exposed to pH 2.3, with or without 20-25 min pre-treatment with milk or egg phospholipids. The pH in surface epithelial cells was measured with double-barrelled liquid sensor pH/PD-microelectrodes. RESULTS: Pre-treatment with phospholipids (2500-5000 micrograms P/mL) significantly (P < 0.01, n = 14) opposed intracellular acidification. Phospholipids significantly (P < 0.05, n = 14) increased the ratio of apical and basal membrane resistances, suggesting that they primarily affect the apical cell membrane. In contrast, there was no significant change in transmucosal resistance suggesting lack of effect on paracellular shunts in the 'leaky' epithelium. CONCLUSIONS: Exogenous phospholipids of dietary origin may be used to form a protective layer in the gastric mucosa against irritants.

Animals↗
PubMed1983

Influence of Ca2+ on the mechanosensitivity of the hair cells in the lateral line organs of Necturus maculosus.

The degree of synchronization (DOS) between the afferent spike activity from one stitch of the lateral line of Necturus maculosus (in vivo) and the mechanical stimulation of one neuromast of the same stitch was measured under different circumstances. The DOS was found to be independent of changes in the concentration of monovalent cations (Na+, K+ and choline+) in the bulk solution at high Ca concentration (1 mM). DOS was also independent of the Ca concentration in the range 1 mM-1 microM in Tris-HCl buffer, but was markedly reduced at Ca = 10 microM in MOPS-KOH buffer. The reduced DOS, however, could be restored by addition of 10-20 mM KCl. 5 mM of 4-aminopyridine did not influence the DOS at high Ca concentration, but completely reduced DOS at Ca = 10 microM. D600 (a methoxy derivative of verapamil) decreased DOS both at high and low Ca concentration.

4-Aminopyridine↗
PubMed1989

Effect of bumetanide on tubuloglomerular feedback in Necturus maculosus.

A non-invasive technique was developed to measure single-nephron glomerular blood flow (SNGBF) in Necturus maculosus. Erythrocytes labelled with rhodamine, a fluorescent dye, were injected systemically and the frequency at which labelled cells entered an arteriole was measured. Frequency was converted to flow by measuring the concentration of labelled erythrocytes in whole blood. Dependence of SNGBF on flow rate in early distal tubules was used to assess tubuloglomerular feedback (TGF). SNGBF decreased with increasing flow in the early distal tubule in a pattern typical of TGF; SNGBF decreased 25% at the highest flow rates. SNGBF increased when bumetanide was added to the perfusate, but the TGF response to flow rate persisted. IC50 (concentration that produces half-maximal inhibition) was 2.4 x 10(-10), 9.8 x 10(-10) and 1.2 x 10(-9) M bumetanide at distal perfusion rates of 5, 10 and 20 nl min-1 respectively. These results are consistent with modulation of SNGBF according to the rate of luminal entry of NaCl into early distal tubule cells. This transport rate depends on the luminal concentration of NaCl, which is tubular flow rate-dependent; NaCl and bumetanide compete.

Animals↗