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.

Functional groups of the Na+ channel: role of carboxyl and histidyl groups

1983 ◽  
Vol 245 (6) ◽  
pp. F716-F725 ◽  
Author(s):  
C. S. Park ◽  
D. D. Fanestil
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Ringer Solution ◽  
Acid Dissociation ◽  
Na Channel ◽  
Toad Urinary Bladder ◽  
Carboxyl Group ◽  
Na Transport

Two titratable groups, with effect on Na+ transport and with apparent acid dissociation constants (pKaS) of 4.2 and 6.7, were found in the apical membrane of toad urinary bladder and are tentatively identified as a carboxyl and an imidazole. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a reagent selective for carboxyl residues, inhibits Na+ transport in the urinary bladder of toads. The underlying chemical reaction whereby EEDQ produces inhibition through potential modification of carboxyl residues was studied. The inhibitory action of EEDQ on Na+ transport was dependent on pH of reaction media and availability of nucleophile, indicating that formation of a covalent acyl-nucleophile bond is probably involved in the irreversible inhibition of Na+ transport. The kinetics of the inhibition showed a stoichiometry of formation of one acyl-nucleophile bond per closure of one Na+ transport site, presumably the Na+ channel. The nucleophile that appears to be involved in the formation of the acyl-nucleophile bond was tentatively identified as having an apparent pKa of 6.7. Amiloride and two analogues of amiloride added to the mucosal Ringer solution (but not serosal amiloride) protected against inhibition of Na+ transport by EEDQ--a finding consistent with the hypothesis that the EEDQ-activated carboxyl group undergoes reaction with a nucleophile at or near the site of specific binding of amiloride onto the apical membrane, most likely at the Na+ channel. Our findings led us to postulate that amiloride must interact with at least two sites on the Na+ channel in order to block the channel. One of the two sites appears to be an ionic interaction between the anionic carboxyl group at the Na+ channel and the cationic guanidinium group of amiloride.

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.

Tetracycline-induced inhibition of Na+ transport in the toad urinary bladder

1978 ◽  
Vol 235 (4) ◽  
pp. F359-F366 ◽  
Author(s):  
J. Guzzo ◽  
M. Cox ◽  
A. B. Kelley ◽  
I. Singer
Keyword(s):  
Urinary Bladder ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Lipid Solubility ◽  
Toad Urinary Bladder ◽  
Na Transport ◽  
Ussing Chambers ◽  
Incubation Periods ◽  
Active Conductance ◽  
Drug Protein Binding

The effects of three tetracyclines, demethylchlortetracycline (DMC), minocycline (MNC), and oxytetracycline (OTC), on Na+ transport (measured as short-circuit current) were examined in toad urinary bladders mounted in modified Ussing chambers. During a 1-h incubation period serosal DMC (but not MNC or OTC) inhibited basal Na+ transport, whereas MNC (but not DMC or OTC) inhibited ADH-stimulated Na+ transport. MNC also inhibited cyclic AMP-stimulated Na+ transport. During longer incubation periods all three drugs inhibited basal Na+ transport. The DMC-induced inhibition of basal Na+ transport and the MNC-induced inhibition of ADH-stimulated Na+ transport were paralleled by an inhibition of the active conductance of the bladders. Thus, although all three drugs inhibit basal Na+ transport, only MNC inhibits ADH-stimulated Na+ transport. This effect does not correlate with the known effects of the tetracyclines on ADH-stimulated water flow or with drug-protein binding, and may be related to the greater lipid solubility of MNC.

Insulin-mediated Na+ transport in the toad urinary bladder

1977 ◽  
Vol 232 (3) ◽  
pp. F270-F277
Author(s):  
M. Cox ◽  
I. Singer
Keyword(s):  
Urinary Bladder ◽  
Initial Rate ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Mechanisms Of Action ◽  
Maximal Response ◽  
Toad Urinary Bladder ◽  
Na Transport ◽  
Ussing Chambers ◽  
K Concentration

The characteristics of insulin-induced Na+ transport in the toad urinary bladder were determined and compared to those of aldosterone. Bladders were mounted in modified Ussing chambers, and standard short-circuit current techniques were employed to measure transepithelial Na+ transport. Insulin added to the serosal medium is much more effective than insulin added to the mucosal medium. Serosal insulin concentrations from 10(1) to 10(3) muU/ml increase both the initial rate and the final level of Na+ transport achieved, whereas concentrations from 10(3) to 10(5) muU/ml increase only the initial rate of Na+ transport. Insulin-induced Na+ transport probably does not require glucose. Both insulin- and aldosterone-induced Na+ transport are directly proportional to serosal (but not mucosal) K+ concentration over the physiologic range (2.0-7.0 meq/liter). However, cycloheximide abolishes aldosterone- but not insulin-induced Na+ transport. In addition, insulin stimulates Na+ transport after a maximal response to aldosterone, and aldosterone stimulates Na+ transport after a maximal response to insulin. Thus, although they have several similar characteristics, insulin and aldosterone have at least partially independent mechanisms of action on Na+ transport in the toad urinary bladder.

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)

OSMOTIC INHIBITION OF THE NATRIFERIC RESPONSE OF THE TOAD URINARY BLADDER TO VASOPRESSIN

1975 ◽  
Vol 67 (1) ◽  
pp. 119-125
Author(s):  
P. J. BENTLEY
Keyword(s):  
Urinary Bladder ◽  
Water Movement ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Toad Bladder ◽  
Toad Urinary Bladder ◽  
Electrical Potential Difference ◽  
Osmotic Gradient

SUMMARY The electrical potential difference and short-circuit current (scc, reflecting active transmural sodium transport) across the toad urinary bladder in vitro was unaffected by the presence of hypo-osmotic solutions bathing the mucosal (urinary) surface, providing that the transmural flow of water was small. Vasopressin increased the scc across the toad bladder (the natriferic response), but this stimulation was considerably reduced in the presence of a hypo-osmotic solution on the mucosal side, conditions under which water transfer across the membrane was also increased. This inhibition of the natriferic response did not depend on the direction of the water movement, for if the osmotic gradient was the opposite way to that which normally occurs, the response to vasopressin was still reduced. The natriferic response to cyclic AMP was also inhibited in the presence of an osmotic gradient. Aldosterone increased the scc and Na+ transport across the toad bladder but this response was not changed when an osmotic gradient was present. The physiological implications of these observations and the possible mechanisms involved are discussed.

Apical nonspecific cation conductances in rabbit cecum

1994 ◽  
Vol 266 (3) ◽  
pp. G475-G484 ◽  
Author(s):  
J. H. Sellin ◽  
W. P. Dubinsky
Keyword(s):  
Epithelial Transport ◽  
Apical Membrane ◽  
Divalent Cations ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Na Channel ◽  
Similar Response ◽  
Electrogenic Transport

Rabbit cecum exhibits electrogenic Na absorption in vitro. However, because this transport process is not inhibited by amiloride nor does it demonstrate saturation kinetics typical of the amiloride-inhibitable Na channel, we considered whether the cecal transporter represented one of a recently described family of nonselective cation conductances or channels (NSCC). Both transepithelial and vesicle studies demonstrated that K, Cs, and Rb were transported via an apical conductance. Electrogenic transport was inhibited by divalent cations including Ca, Mg, and Ba but was unaffected by either lanthanum or gadolinium. Parallel studies in distal colon did not exhibit a similar response to either K substitution or Ba inhibition. Phenamil, verapamil, and nicardipine significantly inhibited the short-circuit current (Isc). stimulated by nominal Ca- and Mg-free conditions. Flux studies demonstrated a correlation between changes in Isc and Na transport. Microelectrode impalement studies suggested that there may be both NSCC and K conductance in the apical membrane. Planar bilayer studies identified a 190-pS cation channel that may correlate with the macroscopic transport properties of this epithelium. These studies are consistent with a model of cecal Na absorption mediated by a NSCC in the apical membrane; this may be the mechanism underlying the distinct epithelial transport characteristics of this intestinal segment.

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.

Hormone effects on transport in cultured epithelia with high electrical resistance

1981 ◽  
Vol 240 (3) ◽  
pp. C103-C105 ◽  
Author(s):  
J. S. Handler ◽  
F. M. Perkins ◽  
J. P. Johnson
Keyword(s):  
Urinary Bladder ◽  
Cell Lines ◽  
Sodium Transport ◽  
Time Course ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transepithelial Resistance ◽  
Striking Difference ◽  
Toad Urinary Bladder ◽  
Hormone Effects

Three continuous lines of amphibian epithelial cells form epithelia with a high transepithelial resistance (greater than 4,000 omega . cm2) in culture. The cell lines are TB-M and TB-6c, derived from the urinary bladder of Bufo marinus, and A6, derived from the kidney of Xenopus laevis. Short-circuit current is equivalent to net mucosa-to-serosa sodium transport in two cell lines and slightly exceeds sodium transport in epithelia formed by TB-6c cells. None of the cell lines has an adenylate cyclase response or a transport or permeability response to vasopressin. Water permeability is low in all three cell lines and is not affected by adenosine 3',5–-cyclic monophosphate (cAMP). In the three lines of cells, cAMP and aldosterone each increases short-circuit current with a time course similar to that seen in naturally occurring epithelia. In contrast to the toad urinary bladder and epithelia of line TB-M in which the aldosterone stimulation of short-circuit current is associated with a fall in transepithelial resistance, there is no change in resistance across epithelia of lines TB-6c and A6. There is also a striking difference in the sensitivity of the three lines to inhibition of short-circuit current by amiloride.

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