SEARCH · PubMed Health

Results for “Necturus”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources

PubMed1983

Intracellular chloride activity and apical membrane chloride conductance in Necturus gallbladder.

Open-tip and Cl--selective microelectrodes were used to study the effect of external pH on apical membrane potential (Va) and intracellular chloride activity (aiCl) in epithelial cells of Necturus gallbladder. Increasing the pH from 7.2 to 8.2 in the mucosal, the serosal, or in both bathing solutions simultaneously, hyperpolarized Va (control value -60 +/- 5 mV) by about -6, -10 and -17 mV, respectively, but did not significantly change the transepithelial potential (VT = 0.3 +/- 0.5 mV). Identical hyperpolarizations were recorded with Cl--selective microelectrodes, even 40 min after changing external pH. Thus, aiCl (12 +/- 2 mM) remained essentially constant. The ratio fVa between the deflections in Va and VT produced by transepithelial current pulses, which is an approximate measure of the fractional resistance of the apical membrane, decreased when mucosal pH was increased, and increased when serosal pH was raised. The changes in Va and fVa are due, in part at least, to the known pH dependence of cell membrane K+ conductance (PK) in this tissue. The constancy of aiCl, despite significant increases in Va, indicates that cell membrane Cl- conductance (PCl) is virtually zero or decreases, with increased external pH, in a way that compensates for the increased driving force for Cl- exit. Experiments in which 90 mM gluconate or 90 mM methylsulfate were substituted for an equivalent amount of luminal Cl- did not provide any evidence for a significant contribution of Cl- ions, per se, to the emf or conductance of the apical membrane. They suggested, rather, a dependence of apical membrane cation permeability on luminal Cl- concentration. Since basolateral membrane PCl is known to be very low, the insensitivity of aiCl to Va is the consequence of a negligible electrodiffusive Cl- permeability at both cell membranes. Thus, overall, transcellular Cl- transport in Necturus gallbladder is, in large measure, effected by electroneutral processes.

Animals↗

PubMed1984

Feedback inhibition of NaCl entry in Necturus gallbladder epithelial cells.

When Necturus gallbladder epithelium is treated with ouabain the cells swell rapidly for 20-30 minutes then stabilize at a cell volume 30% greater than control. The cells then begin to shrink slowly to below control size. During the initial rapid swelling phase cell Na activity, measured with microelectrodes, rises rapidly. Calculations of the quantity of intracellular Na suggest that the volume increase is due to NaCl entry. Once the peak cell volume is achieved, the quantity of Na in the cell does not increase, suggesting that NaCl entry has been inhibited. We tested for inhibition of apical NaCl entry during ouabain treatment either by suddenly reducing the NaCl concentration in the mucosal bath or by adding bumetanide to the perfusate. Both maneuvers caused rapid cell shrinkage during the initial phase of the ouabain experiment, but had no effect on cell volume if performed during the slow shrinkage period. The lack of sensitivity to the composition of the mucosal bath during the shrinkage period occurred because of apparent feedback inhibition of NaCl entry. Another maneuver, reduction of the Na in the serosal bath to 10 mM, also resulted in inhibition of apical NaCl uptake. The slow shrinkage which occurred after one or more hours of ouabain treatment was sensitive to the transmembrane gradients for K and Cl across the basolateral membrane and could be inhibited by bumetanide. Thus during pump inhibition in Necturus gallbladder epithelium cell Na and volume first increase due to continuing NaCl entry and then cell volume slowly decreases due to inhibition of the apical NaCl entry and activation of basolateral KCl exit.

Animals↗

PubMed1991

Ca(2+)-dependent chloride conductance in Necturus taste cells.

This report describes the occurrence and localization of a Ca(2+)-dependent chloride conductance in taste cells of Necturus maculosus. Lingual epithelium from Necturus was removed with blunt dissection and mounted in a modified Ussing chamber which allowed individual taste cells to be impaled with intracellular micropipettes. Solutions in the mucosal and serosal chambers could be changed independently and the properties of apical and basolateral membranes tested separately. Action potentials in taste cells, elicited by brief depolarizing current pulses passed through the intracellular recording microelectrode, provided an accurate description of whether voltage-dependent conductances had been blocked or unmasked by the experimental conditions. We found that Ca2+ influx during the action potential triggers a prolonged depolarization due to Ca(2+)-dependent conductance changes, particularly in the presence of TEA to block repolarizing K+ currents. This afterdepolarization could last up to 7 sec and is due, in part, to a Ca(2+)-dependent Cl- conductance. Other Ca(2+)-dependent channels such as Ca(2+)-dependent K+ channels or nonselective cation channels may also contribute to the afterpotential. Calcium-dependent conductance channels were situated on apical and basolateral membranes of the taste cells. We speculate that Ca(2+)-dependent Cl- channels may play a role in discriminating chloride salts from salts of other anions and may help shape receptor cell responses elicited by taste stimuli.

Action Potentials↗

PubMed1985

Cyclic AMP-induced changes in membrane conductance of Necturus gallbladder epithelial cells.

Enhanced cellular cAMP levels have been shown to increase apical membrane Cl- and HCO3- conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels in Necturus gallbladder. We used conventional open-tip and double-barreled Cl- -selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl- activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23 degrees C and pH 7.4. In HCO3- -free media, 0.1 mM IBMX added to the mucosal medium depolarized the apical membrane potential Va, decreased the fractional resistance FR, and significantly reduced intracellular Cl- activity (aCli). Under control conditions, aCli was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25 mM HCO3-, IBMX caused a small transient hyperpolarization of Va followed by a depolarization not significantly different from that observed in HCO3- -free Ringer's. Removal of mucosal Cl-, Na+ or Ca2+ did not affect the IBMX-induced depolarization in Va. The basolateral membrane of Necturus gallbladder is highly K+ permeable. Increasing serosal K+ from 2.5 to 80 mM, depolarized Va. Mucosal IBMX significantly reduced this depolarization. Addition of 10 mM Ba2+, a K+ channel blocker, to the serosal medium depolarized Va and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO3- conductance and decreases basolateral K+ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization of Va.

1-Methyl-3-isobutylxanthine↗

PubMed1983

Regulation of intracellular chloride activity during perfusion with hypertonic solutions in the Necturus proximal tubule.

In a previous study we presented evidence that chloride transport across the basolateral membrane in Necturus proximal tubule cells occurs predominantly via exchange for both Na+ and HCO3-. In this study the regulation of intracellular chloride was further examined in the doubly-perfused kidney preparation using conventional and chloride-sensitive microelectrodes. Application of hypertonic basolateral solutions containing 80 mM raffinose stimulated an efflux of chloride such that chloride activity remained unchanged at control levels. Membrane potential did not change in these experiments. Inhibition of Cl- exit across the basolateral cell membrane by removal of either HCO3- or Na+ from the perfusion solution resulted in a significant increase in intracellular chloride activity, aiCl, when basolateral osmolarity was raised. Hypertonic basolateral solutions also produced a significant rise in aiCl in the presence of SITS. This study provides further evidence that chloride is transported across the basolateral cell membrane in exchange for both Na+ and HCO3-. Since this exchange mechanism is activated in response to hypertonic solutions, these studies suggest a functional role for this exchanger in the regulation of aiCl in the Necturus proximal tubule cell during volume changes.

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

PubMed1981

Cyclic AMP and intracellular ionic activities in Necturus gallbladder.

Open-tip and liquid ion-exchanger microelectrodes were used to study the effects of cAMP (6mM, added to the serosal medium) on apical membrane potential (Em) and intracellular sodium, potassium, and chloride activities (alphaiNa, alphaiK, alphaiCl) in Necturus gallbladder under open-circuit conditions. Transepithelial potential difference (ETr) was also measured. In the presence of cAMP, alphaiCl fell from about 1.5 times its equilibrium value to a level that corresponded to electrochemical equilibrium across the apical and basolateral cell membranes. Under these conditions alphaiNa decreased and alphaiK increased, Em was unchanged and ETr increased from virtually zero to a small but significant serosal positive value. The cAMP-induced increase in alphaiK was abolished when Cl- -free incubation media were used. Addition of the Ca++-ionophore A23187 (0.5 microgram/ml) to the serosal medium had no effect on Em, ETr, or alphaiCl. When A23187 was added to the mucosal medium, Em and the basolateral membrane potential hyperpolarized by about 20 mV and an increase in the outwardly directed electrochemical driving force for Cl- was observed. These results indicate that cAMP inhibits coupled transapical Na-Cl entry into epithelial cells of Necturus gallbladder and suggest that this inhibition may not be mediated by an increase in intracellular Ca++ concentration.

Animals↗

PubMed2001

Extracellular ATP activates a P2 receptor in necturus erythrocytes during hypotonic swelling.

We recently reported that ATP is released from Necturus erythrocytes via a conductive pathway during hypotonic swelling and that extracellular ATP potentiates regulatory volume decrease (RVD). This study was designed to determine whether extracellular ATP exerts its effect via a purinoceptor. This was accomplished using three different experimental approaches: 1) hemolysis studies to examine osmotic fragility, 2) a Coulter counter to assess RVD, and 3) the whole-cell patch-clamp technique to measure membrane currents. We found extracellular ATP and ATPgammaS, two P2 agonists, decreased osmotic fragility, enhanced cell volume recovery in response to hypotonic shock, and increased whole-cell currents. In addition, 2-methylthio-ATP potentiated RVD. In contrast, UTP, alpha,beta-methylene-ATP, and 2'-& 3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate and the P1 agonist adenosine had no effect regardless of experimental approach. Furthermore, the P2 antagonist suramin increased osmotic fragility, inhibited RVD, and reduced whole-cell conductance in swollen cells. Consistent with a previous study that indicated cell swelling activates a K+ conductance, suramin had no effect in the presence of gramicidin (a cationophore used to maintain a high K+ permeability). We also found the P2 antagonist pyridoxal-5-phosphate-6-azophenyl-2'4-disulfonic acid (PPADS) increased osmotic fragility; however, reactive blue 2 and the P1 antagonists caffeine and theophylline had no effect. Our results show that extracellular ATP activated a P2 receptor in Necturus erythrocytes during hypotonic swelling, which in turn potentiated RVD by stimulating K+ efflux. Pharmacological evidence suggested the presence of a P2X receptor subtype.

Adenosine↗

PubMed1983

Peculiarities of the Na+/D-glucose cotransport system in Necturus renal tubules.

The effects of D-glucose addition to a glucose-free luminal perfusate were investigated in the proximal tubule of Necturus kidney, by electrophysiological techniques. The main findings are: (1) In the presence of sodium, D-glucose produces 10.5 mV +/- 1.1 (S.E.) depolarization. (2) Phlorizin reduces the magnitude of this response to 2.1 +/- 0.1 mV. (3) The glucose-evoked depolarization, delta VG, does not alter the intracellular K+ activity nor is it affected by peritubular addition of ouabain. (4) Isosmotic reduction of Na+ concentration in luminal perfusate from 95 to 2 mmol/l (choline or Li+ substituting for Na+) does not change the magnitude of delta VG; complete removal of sodium from the lumen lowers the value of delta VG (3.2 +/- 0.2 mV) but the response is not abolished. This observation suggests that the D-glucose carrier of renal tubules in Necturus is poorly specific with regard to the cotransported cation species.

Animals↗

PubMed1990

pH dependence of protamine action on apical membrane permeability in Necturus gallbladder epithelium.

Protamine reversibly decreases cation permeability and alters the structure of Necturus gallbladder tight junctions. Conflicting results, however, have been published whether or not it also affects apical cell membrane permeability. We investigated this issue more systematically by measuring voltage (psi mc) and fractional resistance (fRa) of the apical membrane at varying concentrations of protamine, K+, and H+ in the bathing solution. At pH 7.6 and [K+] 2.5 mM, (Poler, M.S. and Reuss, L. (1987) Am. J. Physiol. 253, C662) 6 microM protamine caused psi mc to depolarize from -58 to -51 mV and fRa to decrease from 0.74 to 0.67. If we increased pH to 8.1 these effects were even more pronounced. At [K+] 2.5 mM, but not 4.5 mM, psi mc transiently hyperpolarized for about 5 min after adding protamine. Most importantly, if [K+] was 4.5 mM and pH was adjusted to 7.1 (Bentzel et al. (1987) J. Membr. Biol. 95, 9) no significant changes of psi mc and fRa occurred. In any case, at a supramaximal concentration of 200 microM, protamine did not further increase the paracellular response but produced decreasing psi mc and fRa. We conclude that 6 microM protamine decreases K+ conductance of the apical membrane, if it is already tuned high by high pH. At low control K+ conductance as observed at lower pH, protamine action is restricted to the paracellular pathway. Thus, conflicting results were due to different experimental conditions. At a solution pH of 7.1, 6 microM protamine fulfills criteria of a selective tool for reversibly altering structure and function of the tight junction in Necturus gallbladder.

Animals↗

PubMed1990

Norepinephrine stimulation of sodium transport in Necturus urinary bladder.

Norepinephrine alters the transepithelial electrical properties of an open-circuited urinary bladder from the mud puppy, Necturus maculosus. When 10(-5) M norepinephrine is superfused over the serosa of the epithelium, the transepithelial voltage (Vt) and short-circuit current (Isc) increase as the resistance (Rt) decreases. The norepinephrine-mediated changes are reversed by the addition of amiloride (5.10(-5) M) to the mucosal Ringer's solution. The serosal adrenoceptors mediating the Na+ transport are more sensitive to norepinephrine (EC50 = 1.2.10(-6) M) than to epinephrine or isoproterenol. Since the Isc is blocked selectively by the antagonist, phenoxybenzamine, stimulation of active transepithelial Na(+)-flux by catecholamines is mediated by an alpha-adrenoceptor. The apical cell membrane voltage (Va) and fractional resistance (fRa) were recorded using conventional KCl-filled microelectrodes. Untreated tissues have Va close to 0 mV while the basolateral membrane voltage (Vb) is between -85 and -95 mV. About 90% of Rt is apical cell membrane resistance (fRa). When amiloride inhibits sodium transport, Va becomes negative, Vb hyperpolarizes slightly and fRa increases to 97%. On the other hand, if the bladders are treated with norepinephrine, fRa decreases to 79% as Va becomes positive and Vb depolarizes. When Rt changes, the resistance of the paracellular pathway (Rp) is unaltered. Changes in the electrical properties of the tissue appear to be mediated primarily by alterations in Ra. Since the Necturus bladder does not respond to antidiuretic hormone, this study implies that biogenic amines regulate Na+ transport in the epithelium.

Animals↗

PubMed1981

Differential distribution of different classes of Necturus retinal ganglion cells.

The responses of Necturus retinal ganglion cells were recorded extracellularly. Each cell was characterized by its response type and the distance between its receptive field center and the center of the optic disc. There was a statistically reliable difference between the locations of sustained-ON cell and the locations of sustained-OFF and ON-OFF cells. Thus, the different classes of Necturus retinal ganglion cells are differentially distributed across the surface of the retina.

Animals↗

PubMed1983

The differentiation of Leydig cells, steroidogenesis, and the spermatogenetic wave in the testis of Necturus maculosus.

The study of seminiferous tubule--Leydig cell interactions in relation to specific germ cell stages during the cycle of the seminiferous epithelium is extremely difficult in most mammalian species due to the continual presence of different spermatogenetic stages in the testis from the onset of puberty. The problem is also compounded by the uniform distribution of both seminiferous tubules and interstitial tissue throughout the entire testis. This difficulty can be circumvented, however, by studying certain species where there is a topographical distribution of germ cell stages within the testis. The urodele amphibian Necturus maculosus exhibits a breeding cycle during which a longitudinal wave of spermatogenesis occurs along the length of the testis, resulting in a spatial and temporal segregation of differentiating germ cells. Moreover, this topographical pattern of spermatogenesis is also reflected in the degree of development of adjacent Leydig cells. This anatomical arrangement allows distinct testicular regions to be obtained using a dissecting microscope. The isolated zones, containing germ cells and Leydig cells in various stages of development, were analyzed for 17 alpha-hydroxylase, C-17,20-lyase, and aromatase activities (key enzymes for the synthesis of androgens and estrogen), estrogen binding, and cytochrome P-450 content. Functional parameters were then correlated with the morphology of Leydig cells in the various zones observed by both light and electron microscopy. It was found that there existed a distinct correlation between the state of differentiation of the leydig cells, their steroidogenic potential, and the distribution of estrogen receptors. These results in Necturus indicate indicate in this species, at least, the steroidal microenvironment of different germ cell associations may be quite specific.

Androgens↗

PubMed1983

Paracellular shunt ultrastructure and changes in fluid transport in Necturus proximal tubule.

To define the structure-function relationships of the lateral intercellular space (LIS), we examined the ultrastructure of Necturus proximal tubule following experimental variation of fluid absorption with or without alteration of active sodium transport. Comparisons were made between (1) control (blood-perfused) kidneys and kidneys doubly perfused using either (2) Ringer solution with organic substrate, (3) Ringer solution with substrate in capillaries but steady-state solution in tubules, (4) Ringer without substrate, or (5) Ringer with low sodium. Intratubular and peritubular capillary pressures were monitored before and during standardized perfusion-fixation for electron microscopy and complete cross-sections of sampled proximal tubules were analyzed by morphometry. All morphometric parameters were the same for proximal tubules in kidneys perfused with blood and kidneys perfused with substrate Ringer. Morphometric parameters of cells and lateral intercellular spaces were the same for tubules with normal volume reabsorption rate (Jv) and with Jv = 0 in substrate-perfused kidneys, and for tubules in kidneys perfused without substrate. However, in kidneys perfused with low sodium, cell height, cell volume, LIS-volume, and average maximum width of LIS were significantly decreased while tight junction length and peritubular length of LIS were unchanged. The results suggest that the measured dimensions of the lateral intercellular space in Necturus proximal tubule are independent of the magnitude of transepithelial salt and fluid movement per se but influenced by hydrostatic pressure gradients and active sodium transport.

Animals↗

PubMed1989

Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.

Basolateral membrane vesicles from Necturus enterocytes, highly (greater than 20-fold) enriched in Na+,K+-ATPase, were reconstituted into planar lipid bilayers. The principal channel activity observed is selective for K+ over Na+ and Cl-. This K+ channel is blocked by Ba2+ and Leiurus quinquestriatus venom but is not affected by Ca2+ over the range of 10(-3) to less than 10(-7) M and is not inhibited by charybdotoxin. L. quinquestriatus venom also markedly reduces the conductance of the basolateral membrane of intact villus cells of Necturus small intestine. The open-time probability (Po) of the channel displays a voltage-dependence characteristic of an "inward rectifier"; i.e., the channel inactivates when the basolateral membrane is depolarized and Po increases with increasing hyperpolarization of that barrier. Assuming that similar properties prevail under physiological conditions, this characteristic could provide, in part, an explanation for the parallelism between Na+-pump and K+-leak activities of the basolateral membrane observed in this epithelium. Thus, an increase in rheogenic Na+-pump activity at the basolateral membrane would hyperpolarize that barrier and, in turn, increase the open time of this K+ channel.

Animals↗

PubMed1992

A peptide from the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membranes of Necturus enterocytes.

A synthetic peptide composed of the first 22 amino acid residues of the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membrane vesicles from Necturus enterocytes reconstituted in planar phospholipid bilayers when added to the solution bathing the inner surface of this channel but not when added to the solution bathing its outer surface. A modified peptide in which the leucine in the 7 position is replaced with phenylalanine is also an effective inhibitor, but replacement of the leucine-7 with lysine or glutamate, or digestion with trypsin, renders the peptide ineffective; replacement of the leucine-7 with glycine markedly reduces but does not abolish the effectiveness of the peptide as an inhibitor. These results are analogous to those reported for the Shaker K+ channel +ADHoshi, T., Zagotta, W.N. & Aldrich, R.W. (1990) Science 250, 533-538; and Zagotta, W.N., Hoshi, T. & Aldrich, R.W. (1990) Science 250, 568-571.+BD and suggest that the molecular anatomy of the receptor at the inner face of the Necturus K+ channel with which the peptide interacts to bring about inhibition of that channel may be similar to that of the Shaker K+ channel.

Amino Acid Sequence↗

PubMed1999

Volume regulatory responses of basolateral membrane vesicles from Necturus enterocytes: role of the cytoskeleton.

Previous studies from this laboratory have demonstrated that basolateral membrane vesicles isolated from Necturus maculosus small intestinal epithelial cells possess a K(+) channel that is inhibited by ATP. In the present studies, we demonstrate that these vesicles, which are essentially devoid of soluble cytoplasmic contaminants, exhibit volume regulatory responses that parallel those of intact epithelial cells. Thus, suspension of these vesicles in a solution that is hypotonic to the intravesicular solution increases channel activity whereas suspension in a solution that is hypertonic to the intravesicular solution decreases, and may abolish, channel activity. These volume regulatory responses appear to be mediated by the same K(ATP) channel and depend on an intact actin cytoskeletal network. The responses to both hypotonic and hypertonic challenge are abolished by cytochalasin D or by incubating the vesicles under conditions that are known to depolymerize actin. Phalloidin, which is known to stabilize actin filaments, partially prevents the action of cytochalasin D. Thus, the present results indicate that the K(ATP) channel activity of basolateral membrane vesicles from Necturus basolateral membranes respond to hypo- and hypertonic challenge monotonically around an isotonic "set point" and that these responses depend on an intact actin cytoskeleton.

Actins↗

PubMed1963

THE VISUAL CELLS AND VISUAL PIGMENT OF THE MUDPUPPY, NECTURUS.

Electron microscopy of the visual cells of the mudpuppy Necturus have revealed several new or hitherto neglected features of organization: (a) A system of deeply staining micelles in virtually crystalline array, probably located in the lamellae of the rod outer segments. These particles may contain the visual pigment, porphyropsin. Counts of the micelles, and microspectrophotometric measurements of porphyropsin in the retina and single rods yield the estimate that each lamellar micelle may contain about 50 molecules of porphyropsin. (b) Systems of about 30 cytoplasmic filaments (here called dendrites), continuous with the cytoplasm of the inner segment, and standing like a palisade about the outer segments of the rods and cones. In the rods, one such filament stands in the mouth of each of the approximately 30 deep fissures that carve the outer segment into a radial array of lobules. (c) A system of deeply staining particles in the membranes of the dendrites, and another in the membranes of the pigment epithelial processes. It is suggested that these may have a part in interchanges of material with the outer segments. The ciliary process is found to penetrate more deeply than is commonly supposed into the outer segments of the rods and cones. The edge of each double-membrane disc in rods forms a differentiated rim structure, both around the disc circumference and bordering the fissures. These anatomical arrangements are summarized in Figs. 13 and 14, and the relevant measurements in Table I. The dilution of visual pigment in Necturus rods and cones and a general consideration of their microstructures make it seem unlikely that such typically solid state processes as exciton migration or photoconduction can transport the effects of light far from the site of absorption. Excitation must, therefore, be conveyed to the receptor as a whole by some axial structure. Among axial structures, the plasma membrane is most likely to be the site of nervous excitation. The ciliary process probably plays its main role in the embryogenesis and regeneration of outer segments; and the dendrites and pigment epithelial processes in exchanges of material with the outer segments and perhaps with one another.

Animals↗

PubMed1984

Na+-H+ exchange and Na+ entry across the apical membrane of Necturus gallbladder.

The role of Na+-H+ exchange in Na+ transport across the apical membrane was evaluated in Necturus gallbladder epithelium by means of intracellular Na+ activity (aNai) and 22Na+ uptake measurements. Under control conditions, complete replacement of Na+ in the mucosal solution with tetramethylammonium reduced aNai from 14.0 to 6.9 mM in 2 min (P less than 0.001). Mucosal addition of the Na+-H+ exchange inhibitor amiloride (10(-3) M) reduced aNai from 15.0 to 13.3 mM (P less than 0.001), whereas bumetanide (10(-5) and 10(-4) M) had no effect. Na+ influx across the apical membrane was studied by treating the tissues with ouabain, bathing them in Na-free solutions, and suddenly replacing the mucosal solution with an Na-containing solution. When the mucosal solution was replaced with Na-Ringer's, aNai increased at approximately 11 mM/min. This increase was inhibited by 54% by amiloride (10(-3) M, P less than 0.001) and was unaffected by bumetanide (10(-5) M). Amiloride-inhibitable Na+ fluxes across the apical membrane were also induced by the imposition of pH gradients. Na+ influx was also examined in tissues that had not been treated with ouabain. Under control conditions, 22Na+ influx from the mucosal solution into the epithelium was linear over the first 60 s and was inhibited by 40% by amiloride (10(-3) M, P less than 0.001) and by 19% by bumetanide (10(-5) M, P less than 0.025). We conclude that Na+-H+ exchange is a major pathway for Na+ entry in Necturus gallbladder, which accounts for at least half of apical Na+ influx both under transporting conditions and during exposure to ouabain. Bumetanide-inhibitable Na+ entry mechanisms may account for only a smaller fraction of Na+ influx under transporting conditions, and cannot explain influx in ouabain-treated tissues. These results support the hypothesis that NaCl entry results primarily from the operation of parallel Na+-H+ and Cl--HCO-3 exchangers, and not from a bumetanide-inhibitable NaCl cotransporter.

Amiloride↗