scholarly journals Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder.

1983 ◽  
Vol 81 (6) ◽  
pp. 785-803 ◽  
Author(s):  
H Garty ◽  
I S Edelman
Keyword(s):  
Hormonal Regulation ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Toad Urinary Bladder ◽  
Apical Surface ◽  
Na Channels ◽  
Na Transport ◽  
Na Permeability ◽  
Trypsin Inhibition

Incubation of the mucosal surface of the toad urinary bladder with trypsin (1 mg/ml) irreversibly decreased the short-circuit current to 50% of the initial value. This decrease was accompanied by a proportionate decrease in apical Na permeability, estimated from the change in amiloride-sensitive resistance in depolarized preparations. In contrast, the paracellular resistance was unaffected by trypsinization. Amiloride, a specific blocker of the apical Na channels, prevented inactivation by trypsin. Inhibition of Na transport by substitution of mucosal Na, however, had no effect on the response to trypsin. Trypsinization of the apical membrane was also used to study regulation of Na transport by anti-diuretic hormone (ADH) and aldosterone. Prior exposure of the apical surface to trypsin did not reduce the response to ADH, which indicates that the ADH-induced Na channels were inaccessible to trypsin before addition of the hormone. On the other hand, stimulation of short-circuit current by aldosterone or pyruvate (added to substrate-depleted, aldosterone-repleted bladders) was substantially reduced by prior trypsinization of the apical surface. Thus, the increase in apical Na permeability elicited by aldosterone or substrate involves activation of Na channels that are continuously present in the apical membrane in nonconductive but trypsin-sensitive forms.

scholarly journals Acute ENaC Stimulation by cAMP in a Kidney Cell Line is Mediated by Exocytic Insertion from a Recycling Channel Pool

2004 ◽  
Vol 125 (1) ◽  
pp. 81-101 ◽  
Author(s):  
Michael B. Butterworth ◽  
Robert S. Edinger ◽  
John P. Johnson ◽  
Raymond A. Frizzell
Keyword(s):  
Hormonal Regulation ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Membrane Capacitance ◽  
Apical Surface ◽  
Cortical Collecting Duct ◽  
Ussing Chambers ◽  
Camp Stimulation

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na+ transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, MPKCCD14) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (ISC), membrane capacitance (CT), and conductance (GT) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 ± 6% (n = 31) increase in ISC and a 15.3 ± 1.5% (n = 15) increase in CT. Selective membrane permeabilization demonstrated that the CT increase was due to an increase in apical membrane capacitance. ISC and CT declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (∼1 h each cycle) resulted in response fatigue; ΔISC decreased ∼10% per stimulation–recovery cycle. When channel production was blocked by cycloheximide, ΔISC decreased ∼15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated ISC, and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the ISC responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.

Complex response of epithelial cells to inhibition of Na+ transport by amiloride

1988 ◽  
Vol 254 (2) ◽  
pp. C297-C303 ◽  
Author(s):  
R. S. Fisher ◽  
J. W. Lockard
Keyword(s):  
Urinary Bladder ◽  
Frog Skin ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Second Phase ◽  
Toad Urinary Bladder ◽  
Apical Surface ◽  
Na Transport ◽  
Transport Inhibition

When toad urinary bladder or frog skin epithelia are treated with amiloride, short-circuit current (Isc), which represents the net active transepithelial Na+ transport rate from the apical to basolateral surface, decreases rapidly (2-5 s) to approximately 15-20% of control values and then slowly, over several minutes, continues falling toward zero. The contribution of this second phase of the decline is dependent on the transporting condition of the tissue before administration of amiloride. Attenuation of the second phase was observed if tissues were subjected to a period of transport inhibition. Tissues preincubated in 0 Na+ Ringer solution on the apical surface were returned to control Na+ Ringer, which caused an approximately 25% increase of Isc above control values. Immediate reapplication of amiloride caused Isc to decrease more rapidly than the previous exposure to values near zero, substantially reducing or eliminating the secondary slow decline. After long-term reincubation of tissues in control, 100 mM Na+ solution, another treatment with amiloride indicated that the magnitude of the secondary decline increased in frog skin but not in urinary bladder epithelia. We conclude that the effect of amiloride is complex and may cause additional effects besides simply blocking entry of Na+ into the apical membrane channel, and we suggest that regulatory mechanisms may be invoked in response to transport inhibition.

Stimulation by cGMP of apical Na channels and cation channels in toad urinary bladder

1991 ◽  
Vol 260 (2) ◽  
pp. C234-C241 ◽  
Author(s):  
S. Das ◽  
M. Garepapaghi ◽  
L. G. Palmer
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Toad Urinary Bladder ◽  
Na Channels ◽  
Serosal Side ◽  
Cyclic Monophosphate ◽  
Stimulation Of

The effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) on apical membrane cation conductances in the toad urinary bladder were investigated. 8-BrcGMP (1 mM) added to the serosal solution increased the amiloride-sensitive short-circuit current (INa) after a delay of 5 min to a steady-state value 1.8 times that of controls achieved after 30 min. Similar effects were seen when the bladders were bathed on the serosal side with a normal NaCl Ringer solution and with a high-K sucrose solution to depolarize the basolateral membrane. Under the latter conditions, the amiloride-sensitive transepithelial conductance increased in parallel with the short-circuit current, indicating stimulation of apical membrane Na channels. The threshold concentration for observing the stimulation of INa was 100 microM, 10-100 times larger than the concentration of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) required to elicit an increase in INa. Currents through an outwardly rectifying Ca-sensitive cation conductance (Iout) were also increased by 1.8-fold relative to controls. This stimulatory effect occurred after a delay of 15 min and reached maximal levels 90-120 min after addition of the nucleotide. The effects of cGMP on INa were not additive with those of 8-BrcAMP or with antidiuretic hormone, an agent known to act by increasing cAMP within the cell. Addition of 1 mM 3-isobutyl-1-methylxanthine to the serosal side of the bladders stimulated INa by 1.3-fold and Iout by 2.4-fold. In both cases, subsequent addition of cGMP produced no further activation of either conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of carboxyl group in Na+-entry step at apical membrane of toad urinary bladder

1983 ◽  
Vol 245 (6) ◽  
pp. F707-F715 ◽  
Author(s):  
C. S. Park ◽  
J. Kipnowski ◽  
D. D. Fanestil
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Complete Recovery ◽  
Na Channel ◽  
Toad Urinary Bladder ◽  
Carboxyl Group ◽  
Na Transport ◽  
Tight Epithelia

Mucosal addition of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and some lipid-soluble carbodiimides, agents which are selective for carboxyl groups, irreversibly inhibited Na+ transport as measured by short-circuit current (SCC) in the urinary bladder of the toad. The inhibition of Na+ transport by EEDQ had the following characteristics: 1) the inhibition was accompanied by a significant increase in the transepithelial electrical resistance; 2) the decrease in SCC was accounted for by a comparable decrease in 22Na+ influx without effect on Na+ efflux; 3) amphotericin B produced complete recovery of SCC inhibited with EEDQ but not with antimycin A or ouabain; 4) mucosal EEDQ decreased the amiloride-sensitive reversal of Na+ current that is induced by serosal nystatin in the absence of mucosal Na+; 5) vasopressin and acid mucosal pH caused an increase in SCC in proportion to the SCC remaining after EEDQ inhibition; and 6) Vmax of the SCC was decreased without alteration in the apparent Km for Na+. Based on these characteristics of EEDQ inhibition of Na+ transport, we infer that a carboxyl group of the Na+ channel is involved in the Na+-entry step across the apical membrane of “tight” epithelia. The inhibition of Na+ transport with EEDQ most likely involves closing the Na+ channel through a chemical reaction involving a carboxyl group of the channel.

Time-dependent aldosterone metabolism in toad urinary bladder

1988 ◽  
Vol 254 (4) ◽  
pp. F547-F553 ◽  
Author(s):  
A. S. Brem ◽  
M. Pacholski ◽  
D. J. Morris
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Time Dependent ◽  
Toad Urinary Bladder ◽  
Matched Pairs ◽  
Na Transport ◽  
Reduced Products ◽  
Metabolic Transformation

Aldosterone (Aldo) metabolism was examined in the toad bladder. Bladders were incubated with [3H]aldosterone (10(-7) M) for 5 h, 1 h, or 10 min. Tissues were analyzed for metabolites using high-pressure liquid chromatography (HPLC). In separate experiments, Na+ transport was assessed by the short-circuit current (SCC) technique. Following a 5-h tissue incubation, about 25% of the [3H]-aldosterone was converted into metabolites including a polar monosulfate metabolite, 20 beta-dihydroaldo (20 beta-DHAldo), small quantities of 5 beta-reduced products, and a variety of 5 alpha-reduced Aldo products including 5 alpha-DHAldo, 3 alpha,5 alpha-tetrahydroaldo (3 alpha,5 alpha-THAldo), and 3 beta,5 alpha-THAldo. Tissues metabolized approximately 10% of the labeled hormone into the same compounds by 1 h. Measurable quantities of these metabolites were also synthesized by bladders exposed to Aldo for only 10 min and then incubated in buffer for an additional 50 min without Aldo. Bladders pretreated with the spironolactone, K+-canrenoate (3.5 X 10(-4) M), and stimulated with Aldo (10(-7) M) generated a peak SCC 44 +/- 6% of that observed in matched pairs stimulated with Aldo (P less than 0.001; n = 6). K+-canrenoate also markedly diminished [3H]aldosterone metabolism at both 5 and 1 h. Thus, metabolic transformation of Aldo begins prior to hormone-induced increases in Na+ transport. Both the generation of certain metabolites (e.g., 5 alpha-reductase pathway products) and the increase in Na+ transport can be selectively inhibited by K+-canrenoate.

Transport properties of toad kidney epithelia in culture

1981 ◽  
Vol 241 (3) ◽  
pp. C154-C159 ◽  
Author(s):  
F. M. Perkins ◽  
J. S. Handler
Keyword(s):  
Electrical Resistance ◽  
Hormonal Regulation ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transepithelial Electrical Resistance ◽  
Apical Surface ◽  
Osmotic Water ◽  
Sodium Flux ◽  
Regulation Of Transport ◽  
Osmotic Water Flow

The characteristics of a continuous line of toad kidney epithelial cells (A6) are described. These cells form a monolayer epithelium of high transepithelial electrical resistance (about 5,000 omega . cm2). The cells generate a transepithelial potential difference (apical surface negative) of about 9 mV. The short-circuit current is equivalent to net sodium flux. Net sodium flux is stimulated by aldosterone and by analogues of cAMP. The stimulation is readily reversible. Neither urea permeability nor osmotic water flow is altered by analogues of cAMP. Amiloride eliminates 90% of the short-circuit current. Thus A6 cells form an epithelium with several differentiated properties including hormonal regulation of transport.

Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

1986 ◽  
Vol 251 (2) ◽  
pp. C186-C190 ◽  
Author(s):  
J. P. Johnson ◽  
D. Jones ◽  
W. P. Wiesmann
Keyword(s):  
Enzyme Activity ◽  
Epithelial Cells ◽  
Atpase Activity ◽  
Hormonal Regulation ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Toad Urinary Bladder ◽  
A6 Cells ◽  
Cultured Epithelial Cells ◽  
Stimulation Of

Aldosterone and insulin stimulate Na+ transport through mechanisms involving protein synthesis. Na+-K+-ATPase has been implicated in the action of both hormones. We examined the effect of aldosterone and insulin on Na+-K+-ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (ISC) in TB6C cells. Aldosterone increases Na+-K+-ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in ISC, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase ISC in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na+-K+-ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na+ entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na+-K+-ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on ISC.

Activation of K+ conductance in basolateral membrane of toad urinary bladder by oxytocin and cAMP

1988 ◽  
Vol 254 (6) ◽  
pp. C816-C821 ◽  
Author(s):  
W. Van Driessche ◽  
D. Erlij
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Potassium Conductance ◽  
Short Circuit Current ◽  
Membrane Properties ◽  
Toad Urinary Bladder ◽  
Electrical Behavior ◽  
Apical Side ◽  
Camp Analogue

We incubated toad urinary bladders with Na+-free, isotonic K+ solutions on the apical side and increased the cationic conductance of the apical membrane with nystatin (150 U/ml). Under these conditions, the short-circuit current is mostly carried by K+ flowing from mucosa to serosa. Impedance measurements showed that in nystatin-treated preparations, the electrical behavior of the tissue is dominated by the basolateral membrane properties. Oxytocin (0.1 U/ml) produced an increase of the current and the conductance of the basolateral membrane. Both the resting and the oxytocin-stimulated current were rapidly and reversibly blocked by serosal Ba2+. Addition of the adenosine 3',5'-cyclic monophosphate (cAMP) analogue [8-(4-chloropheylthio)-cAMP] to the basolateral solution mimicked the effects of oxytocin. These results show that oxytocin and cAMP stimulate a potassium conductance in the basolateral membrane and that the stimulation is not related to an increase in sodium entry through the apical membrane. Addition of ouabain (10(-3) M) to the serosal solution did not modify the stimulation by oxytocin, indicating that the activated pathway is not linked to the rate of turnover of the Na+ pump.

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

1987 ◽  
Vol 89 (4) ◽  
pp. 563-580 ◽  
Author(s):  
J R Demarest ◽  
A L Finn
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
K Channel ◽  
Channel Blockers ◽  
Na Transport ◽  
Transepithelial Na Transport ◽  
Pump Activity ◽  
Relationship Of

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.

scholarly journals Molecular mechanisms for intestinal HCO3- secretion and its regulation by guanylin in seawater-acclimated eels

2019 ◽  
Author(s):  
Yoshio Takei ◽  
Marty K.S. Wong ◽  
Masaaki Ando
Keyword(s):  
Hormonal Regulation ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Disulfonic Acid ◽  
Intestinal Epithelia ◽  
Mucosal Side

AbstractThe intestine of marine teleosts secretes HCO3- into the lumen and precipitates Ca2+ and Mg2+ in the imbibed seawater as carbonates to decrease luminal fluid osmolality and facilitate water absorption. However, reports on studies on the hormonal regulation of HCO3- secretion are just emerging. Here, we showed that guanylin (GN) applied to the mucosal side of intestinal epithelia increased HCO3- secretion in seawater-acclimated eels. The effect of GN on HCO3- secretion was slower than that on the short-circuit current, and the time-course of the GN effect was similar to that of bumetanide. Mucosal bumetanide and serosal 4,4’-dinitrostilbene-2,2’-disulfonic acid (DNDS) inhibited the GN effect, suggesting an involvement of apical Na+-K+-2Cl- cotransporter (NKCC2) and basolateral Cl-/HCO3- exchanger (AE)/Na+-HCO3- cotransporter (NBC) in the GN effect. However, mucosal DNDS and diphenylamine-2-carboxylic acid (DPC) failed to inhibit the GN effect, showing that apical AE and Cl- channel are not involved. To identify molecular species of possible transporters involved in the GN effect, we performed RNA-seq analyses followed by quantitative real-time PCR after transfer of eels to seawater. Among the genes upregulated after seawater transfer, those of Slc26a3a, b (DRAa, b) and Slc26a6a, c (Pat-1a, c) on the apical membrane of the intestinal epithelial cells, and those of Sls4a4a (NBCe1a), Slc4a7 (NBCn1), Slc4a10a (NBCn2a) and Slc26a1 (Sat-1) on the basolateral membrane were candidate transporters involved in HCO3- secretion. Judging from the slow effect of GN, we suggest that GN inhibits NKCC2b on the apical membrane and decreases cytosolic Cl- and Na+, which then activates apical DNDS-insensitive DRAa, b and basolateral DNDS-sensitive NBCela, n1, n2a to enhance transcellular HCO3- flux across the intestinal epithelia of seawater-acclimated eels.

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