Basolateral K+ conductances in surface epithelium of Necturus antrum: effects of Ca2+ and divalent cations

1992 ◽  
Vol 262 (4) ◽  
pp. G651-G659
Author(s):  
D. I. Soybel ◽  
S. W. Ashley ◽  
L. Y. Cheung
Keyword(s):  
Membrane Permeability ◽  
Divalent Cations ◽  
Basolateral Membrane ◽  
Membrane Resistance ◽  
Membrane Potentials ◽  
Time Dependent ◽  
Physiological Significance ◽  
Surface Epithelium ◽  
Antral Mucosa ◽  
Microelectrode Techniques

Intracellular microelectrode techniques were used to characterize basolateral membrane K+ conductances in isolated Necturus antral mucosa. Exposure of tissues to progressively higher levels of serosal K+ (4, 20, 40, or 60 mM) resulted in progressively greater depolarizations of basolateral membrane potentials and decreases in membrane resistance, consistent with the presence of a significant K+ conductance. Ba2+ (2 mM) partially blocks these conductances. Exposure of tissues to increased levels of serosal Ca2+ (from 1.8 to 6.8 mM) elicited significant hyperpolarization of basolateral potentials and decreases in basolateral resistance. These effects are also elicited by Sr2+ (5 mM), but not by Mg2+ (5 mM). Ba2+ (5 mM) elicits complex and time-dependent effects, but transiently elicits an effect similar to high Ca2+. Ion substitutions in the serosal perfusate suggest that the Ca(2+)-induced effects are due to enhancement of basolateral K+ conductances. Further work is necessary to identify the processes that mediate this increase in basolateral K+ conductance and to evaluate the physiological significance of this change in membrane permeability to K+.

Characteristics of basolateral Cl- transport by gastric surface epithelium in Necturus antral mucosa

1993 ◽  
Vol 264 (5) ◽  
pp. G910-G920 ◽  
Author(s):  
D. I. Soybel ◽  
M. B. Davis ◽  
L. Y. Cheung
Keyword(s):  
Nutrient Solution ◽  
Cell Membranes ◽  
Basolateral Membrane ◽  
Membrane Potentials ◽  
Gastric Antrum ◽  
Ion Exchanger ◽  
Surface Epithelium ◽  
Antral Mucosa ◽  
Liquid Ion Exchanger ◽  
Electrochemical Equilibrium

Conventional and ion-selective microelectrodes were used to characterize transport of Cl- across the basolateral cell membranes of gastric surface epithelium in isolated preparations of gastric antrum of Necturus. Conventional, voltage-sensing electrodes were used to evaluate changes in membrane potentials and resistances during removal of Cl- from the nutrient perfusate. Liquid ion exchanger Cl(-)-selective microelectrodes were constructed and validated to measure intracellular Cl- activity (aiCl). Our data indicate that 1) aiCl (range 12-25 mM) is close to that predicted if Cl- is distributed across the cell membranes by electrochemical equilibrium, 2) aiCl is not influenced by changes in luminal Cl- content but is susceptible to changes in nutrient Cl- content, 3) Cl- conductances cannot be detected in the basolateral membrane and changes in membrane potentials do not influence aiCl, and 4) Cl- accumulation across the basolateral membrane depends on Na+ and the level of [K+] in the nutrient solution. Inhibition of K(+)-dependent Cl- accumulation, in the absence of nutrient Na+ or in the presence of the inhibitor bumetanide, was demonstrated. These findings suggest that basolateral Na(+)-K(+)-Cl- cotransport is important in regulating cell Cl- levels in surface cells of the gastric antrum in Necturus.

Effects of luminal salt concentration on electrical pathways in Necturus antrum

1987 ◽  
Vol 252 (1) ◽  
pp. G19-G27
Author(s):  
D. I. Soybel ◽  
S. W. Ashley ◽  
R. A. Swarm ◽  
C. D. Moore ◽  
L. Y. Cheung
Keyword(s):  
Cell Membranes ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Membrane Resistance ◽  
Paracellular Pathway ◽  
Surface Epithelium ◽  
Reversible Inhibitor ◽  
Apical Cell Membrane ◽  
Paracellular Pathways ◽  
Microelectrode Techniques

By use of microelectrode techniques the electrical resistances of the cell membranes and paracellular pathways of surface epithelium in Necturus antrum were determined under control conditions (Ringer solutions containing 106.6 mM Na+, 91.4 mM Cl-) and during exposure to mucosal solutions containing high Na+ and Cl- concentrations. Resistances were determined by briefly exposing tissues to mucosal solutions containing 10(-4) M amiloride, a reversible inhibitor of Na+ conductances. Under control conditions in eight tissues, measurements obtained by exposure to amiloride were not significantly different from those obtained by an independent method, intraepithelial cable analysis, thus indicating the validity of the measurements obtained by the amiloride method. In 10 tissues, high luminal NaCl concentrations (Ringer salts + 125 mM NaCl) increased the apical cell membrane resistance from 5,778 +/- 267 to 7,714 +/- 422 omega X cm2 (P less than 0.01) and the basolateral membrane resistance from 2,973 +/- 186 to 3,869 +/- 335 omega X cm2 (P less than 0.01). The resistance of the paracellular pathway decreased from 625 +/- 13 to 505 +/- 13 omega X cm2 (P less than 0.001). Similar alterations in these resistances were observed when Na+ or Cl- were increased individually, when added as salts of isethionate- and N-methyl-D-glucamine+, respectively. These effects were not attributable to increases in luminal osmolarity, since mucosal solutions made equally hyperosmotic with 250 mM sucrose elicited increases in paracellular pathway resistance and decreases in resistances of the cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Identification and functional assay of an extracellular calcium-sensing receptor in Necturusgastric mucosa

1997 ◽  
Vol 273 (5) ◽  
pp. G1051-G1060 ◽  
Author(s):  
Robert R. Cima ◽  
Ivan Cheng ◽  
Mary E. Klingensmith ◽  
Naibedya Chattopadhyay ◽  
Olga Kifor ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Basal Membrane ◽  
Membrane Resistance ◽  
Immunohistochemical Localization ◽  
Functional Assay ◽  
Antral Mucosa ◽  
Electrical Potentials ◽  
Necturus Maculosus

In mammals and amphibians, increases in extracellular Ca2+ can activate bicarbonate secretion and other protective functions of gastric mucosa. We hypothesized that the recently cloned extracellular Ca2+-sensing receptor (CaR) is functioning in the gastric mucosa. In Necturus maculosus gastric mucosa, reverse transcription-polymerase chain reaction using primers based on previously cloned CaR sequences amplified a 326-bp DNA fragment that had 84% nucleotide sequence identity with the rat kidney CaR. Immunohistochemical localization of the CaR using specific anti-CaR antiserum revealed its presence on the basal aspect of gastric epithelial cells. In microelectrode studies of Necturus antral mucosa, exposure to elevated Ca2+ (4.8 mM) and the CaR agonists NPS-467 and neomycin sulfate resulted in significant hyperpolarizations of basal membrane electrical potentials and increases in apical-to-basal membrane resistance ratios. Circuit analysis revealed that these changes reflected specific decreases in basolateral membrane resistance. Inhibition of prostaglandin synthesis using indomethacin significantly attenuated these effects. We conclude that the CaR is present and functioning in Necturus gastric antrum.

Electrophysiological effects of cholinergic agonists in surface epithelium of Necturus gastric antrum

1996 ◽  
Vol 270 (3) ◽  
pp. G449-G462
Author(s):  
A. E. Gadacz ◽  
M. E. Klingensmith ◽  
D. I. Soybel
Keyword(s):  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Early Spring ◽  
Transport Processes ◽  
Surface Epithelium ◽  
Antral Mucosa ◽  
Cholinergic Stimulation ◽  
Cholinergic Agonists ◽  
Ion Substitution

Intracellular microelectrode techniques were used to characterize voltage and conductance properties of the basolateral membrane of surface epithelial cells in in vitro Necturus antral mucosa. Flux studies confirmed that this tissue secretes HCO3- under resting conditions and during response to cholinergic stimulation. In studies using intracellular microelectrodes, exposure to cholinergic agonists such as acetylcholine, bethanechol, or carbachol elicited an initial hyperpolarization followed by depolarization of the basolateral cell membranes associated with up to fourfold increases in basolateral membrane conductance. Effects of acetylcholine were dose dependent (10(-6) - 10(-4) M) and prevented by pretreatment of tissues with the nonselective muscarinic receptor blocker atropine. Some variation in this response to cholinergic stimulation was observed and appeared to be related to the season (fall/winter/early spring vs. late spring/summer). Despite such variability, circuit analysis and ion substitution studies indicated that the carbachol-induced increases in basolateral conductance were due to increases in conductance to K+ and Cl- . These increases in basolateral transport processes may serve to stabilize cell ion composition and membrane electrical properties during cholinergic stimulation of mucus and HCO3- secretions.

Microelectrode studies of Necturus antral mucosa. II. Equivalent circuit analysis

1985 ◽  
Vol 248 (5) ◽  
pp. G574-G579
Author(s):  
S. W. Ashley ◽  
D. I. Soybel ◽  
L. De ◽  
L. Y. Cheung
Keyword(s):  
Equivalent Circuit ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Ringer Solution ◽  
Antral Mucosa ◽  
Electrical Parameters ◽  
Equivalent Circuit Analysis ◽  
Equivalent Circuit Parameters ◽  
The Difference ◽  
Microelectrode Techniques

Intracellular microelectrode techniques were employed to determine the equivalent circuit parameters in Necturus antral mucosa. Stable intracellular impalements were obtained using 15- to 50-M omega microelectrodes. Measured transepithelial and cellular potentials and voltage deflections produced by transepithelial current pulses were used to calculate the electrical resistances of the cell membranes and the equivalent electromotive forces (EMF) at both cell borders. The measured potentials were -4.1 +/- 0.8 mV for the entire epithelia, -41.8 +/- 5.1 mV for the apical membrane, and -45.9 +/- 5.0 mV for the basolateral membrane. Values for the resistances were 7,300 +/- 1,900 omega X cm2 for the apical, 3,990 +/- 1,170 omega X cm2 for the basolateral, and 710 +/- 40 omega X cm2 for the shunt. Assuming that the shunt EMF is zero with control Ringer solution on both sides of the tissue, the effect of this relatively low-resistance shunt on electrical parameters can be determined. The cell membrane EMFs are both oriented with the interior negative and are -1.2 +/- 9.7 mV (apical) and -69.7 +/- 11.3 mV (basolateral). The difference between these values and the measured potentials is the result of a flow of current through the shunt from serosa to mucosa, hyperpolarizing the apical and depolarizing the basolateral membranes.

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

1992 ◽  
Vol 99 (2) ◽  
pp. 241-262 ◽  
Author(s):  
G A Altenberg ◽  
J S Stoddard ◽  
L Reuss
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Resistance ◽  
Membrane Voltage ◽  
Gallbladder Epithelium ◽  
K Channels ◽  
Necturus Gallbladder ◽  
Electrophysiological Effects ◽  
Paracellular Diffusion ◽  
Cl Conductance

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.

Bumetanide blocks CFTRG Cl in the native sweat duct

1999 ◽  
Vol 276 (1) ◽  
pp. C231-C237 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Basolateral Membrane ◽  
Electrical Conductance ◽  
Anion Channel ◽  
Membrane Resistance ◽  
Activation Process ◽  
Transmembrane Conductance Regulator ◽  
Sweat Duct ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Human Sweat Duct

Bumetanide is well known for its ability to inhibit the nonconductive Na+-K+-2Cl−cotransporter. We were surprised in preliminary studies to find that bumetanide in the contraluminal bath also inhibited NaCl absorption in the human sweat duct, which is apparently poor in cotransporter activity. Inhibition was accompanied by a marked decrease in the transepithelial electrical conductance. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is richly expressed in the sweat duct, we asked whether bumetanide acts by blocking this anion channel. We found that bumetanide 1) significantly increased whole cell input impedance, 2) hyperpolarized transepithelial and basolateral membrane potentials, 3) depolarized apical membrane potential, 4) increased the ratio of apical-to-basolateral membrane resistance, and 5) decreased transepithelial Cl− conductance ( G Cl). These results indicate that bumetanide inhibits CFTR G Clin both cell membranes of this epithelium. We excluded bumetanide interference with the protein kinase A phosphorylation activation process by “irreversibly” phosphorylating CFTR [by using adenosine 5′- O-(3-thiotriphosphate) in the presence of a phosphatase inhibition cocktail] before bumetanide application. We then activated CFTR G Clby adding 5 mM ATP. Bumetanide in the cytoplasmic bath (10−3 M) inhibited ∼71% of this ATP-activated CFTR G Cl, indicating possible direct inhibition of CFTR G Cl. We conclude that bumetanide inhibits CFTR G Clin apical and basolateral membranes independent of phosphorylation. The results also suggest that >10−5 M bumetanide cannot be used to specifically block the Na+-K+-2Cl−cotransporter.

Low doses of ethanol have Ca2+ ionophore-like effects on apical membrane potential of in vitro Necturus antrum

1991 ◽  
Vol 261 (1) ◽  
pp. G92-G103
Author(s):  
M. J. Rutten ◽  
C. D. Moore
Keyword(s):  
Membrane Potential ◽  
Apical Membrane ◽  
Periodic Acid ◽  
Ethanol Exposure ◽  
Membrane Potentials ◽  
Antral Mucosa ◽  
Low Doses ◽  
Periodic Acid Schiff ◽  
Induced Changes

The effects of low doses of luminal ethanol on the amiloride-sensitive apical membrane potential of Necturus antral mucosa were studied using conventional microelectrode techniques. Luminal ethanol (0.250-4.0% vol/vol) caused a dose-dependent hyperpolarization of the apical membrane potential (Vmc), an increase in transepithelial resistance (Rt) and resistance ratio (Ra/Rb), and a decrease in transepithelial potential (Vms). Luminal amiloride (100 microM) to 4% ethanol-treated antra did not cause any additional hyperpolarization of Vmc. Compared with luminal 2% ethanol-Ringer, an equivalent osmotic mannitol solution depolarized Vmc and basolateral potential (Vcs), decreased Rt and Ra/Rb, and increased Vms. A single dose of 0.50% ethanol attenuated the effects of a second 2% ethanol exposure on Vmc. No change in periodic acid-Schiff (PAS)-positive mucous granule content could be found between control and 2% ethanol-treated antra. The Ca2+ ionophores A23187 or ionomycin (0.25-5.0 microM) dose dependently hyperpolarized the Vmc and Vcs, increased Rt and Ra/Rb, and decreased Vms. Luminal Ca(2+)-free Ringer had no effect on luminal 2.00% ethanol-induced changes in membrane potentials or resistances. Pretreatment with BAPTA blocked by approximately 70 and 55% the Vmc hyperpolarization of 2 and 4% ethanol, respectively. Pretreatment with ruthenium red (10-50 microM) also dose dependently reduced the 2% ethanol-induced changes in Vmc. The data indicate that 1) low doses of luminal ethanol and Ca2+ ionophores have similar effects on Necturus gastric antral membrane potentials and resistances, 2) ethanol-induced hyperpolarizations of the Vmc are partially mediated through an alteration in intracellular Ca2+, and 3) low doses of luminal ethanol do not cause the release of antral epithelial mucous granules at the time when significant changes are occurring in the Vmc.

Electrophysiological analysis of beta-receptor stimulation in salamander proximal tubules

1987 ◽  
Vol 253 (4) ◽  
pp. F636-F641
Author(s):  
N. S. Morgunov
Keyword(s):  
Basolateral Membrane ◽  
Membrane Resistance ◽  
Proximal Tubules ◽  
Sodium Reabsorption ◽  
Organic Substrates ◽  
Receptor Stimulation ◽  
Electrophysiological Response ◽  
Control Value ◽  
Beta Receptor ◽  
Absolute Requirement

Electrophysiological response to isoproterenol stimulation was studied in isolated perfused salamander proximal tubules. The addition of 10(-5) M isoproterenol to the bath superfusate depolarized both the cell membrane, and transepithelial potentials by 2.2 +/- 0.2 and 0.31 +/- 0.04 mV, respectively (P less than 0.01, n = 35) and significantly reduced the apical-to-basolateral membrane resistance ratio by 30% (P less than 0.01, n = 7) from a control value of 3.7 +/- 0.6. These responses were blocked by 10(-6) M propranolol but not mimicked by 10(-4) M adenosine 3',5'-cyclic monophosphate. Qualitatively similar effects were observed with 10(-3) and 10(-7) M isoproterenol. Further characterization of the 10(-5) M isoproterenol response revealed 1) a 50% reduction in the response following the removal of organic substrates from the luminal perfusate, 2) an absolute requirement for sodium, and 3) an absolute requirement for a functioning basolateral Na+-K+-ATPase. The data suggest that beta-receptor stimulation may increase sodium reabsorption by activating sodium cotransport systems.

Na+-K+ pump stoichiometry and basolateral membrane permeability of frog corneal epithelium

1986 ◽  
Vol 250 (5) ◽  
pp. F850-F859 ◽  
Author(s):  
O. A. Candia ◽  
P. Cook
Keyword(s):  
Membrane Permeability ◽  
Amphotericin B ◽  
Corneal Epithelium ◽  
Flow Condition ◽  
Basolateral Membrane ◽  
Flux Ratio ◽  
Na Currents ◽  
Static Head ◽  
K Permeability ◽  
Variable Stoichiometry

The Na+-K+ pump flux ratio and the Na+ and K+ permeability of the basolateral membrane of the isolated frog corneal epithelium were studied with the aid of microelectrodes by analyzing the effects of ouabain, Ba2+, and amphotericin B. The experiments were done in Cl(-)-free solutions, a situation that approximates that of static head. Ouabain produced a quick depolarization of the potential difference across the basolateral membrane (PDb) from -72 to -62 mV without a change in resistance. Ba2+ (3 mM) rapidly lowered PDb from -74 to -57 mV and decreased the apical-to-basolateral resistance ratio. The effects of ouabain and Ba2+ were additive. The Na+-K+ flux ratio at the pump was calculated to be 1.78, substantially less than when the tissue is in a level flow condition, suggesting a variable stoichiometry. The K+ and Na+ resistances of the basolateral membrane were 15.7 and 5.5 k omega X cm2, respectively, allowing K+ and Na+ currents that approximately matched those produced by the Na+-K+ pump. The resistance of the basolateral membrane (4.0 k omega X cm2) was double that reported in Cl(-)-rich solutions, suggesting that Cl- contributes to the conductance of this membrane.

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