Membrane Of Toad Urinary Bladder Research Articles

Cell swelling activates a poorly selective monovalent cation channel in the apical membrane of toad urinary bladder.

We measured the effects of conditions that increase cell volume on ion movements through the Ca(2+)-blockable poorly selective monovalent cation channel of the apical membrane of the toad urinary bladder. Three conditions were studied: dilution of the basolateral solution, basolateral perfusion with solutions prepared with solutes of low reflection coefficient and dilution of the apical solution in bladders treated with oxytocin. All three procedures markedly increased K+ movements and elevated the plateau of the Lorentzian component of the power spectrum by enhancing ion currents through the Ca(2+)-blockable pathway. Simultaneously there was a large increase in Ca(2+)-sensitive conductance. The magnitude of this increased conductance strongly suggests that the stimulation of ion flow is due to increased ion permeability and not solely to increases in driving force across the apical membrane. We were not able to detect an increase in the movements of alkali-earth ions induced by the conditions that increase cell volume. We speculate that activation of the Ca(2+)-blockable channel may play an important role in the regulation of cell volume and/or in K+ homeostasis.

Antidiuretic hormone moves membranes.

This review focuses on events at the apical plasma membrane of toad urinary bladder and mammalian collecting duct as their permeability to water changes in response to antidiuretic hormone (ADH) and to its withdrawal. The major marker of the permeability change is observed in freeze-fracture electron microscopy of the apical plasma membrane and consists of a dramatic increase in membrane particle aggregates and, in toad bladder but not in collecting duct, in fused vesicles (aggrephores) that contain particle aggregates in their limiting membranes. Withdrawal of ADH is accompanied by endocytosis at the apical membrane, reflecting retrieval of water-permeable, particle aggregate-containing membrane. Covalent labeling of the external surface of the apical membrane of toad bladder identifies specific proteins that are present in the apical membrane only during the response to ADH. Proteins of the same molecular weights are also present in the retrieved membrane when ADH is withdrawn. Several controversial areas are considered, including the extent of cell swelling as water flows across the epithelium from dilute apical solution to isotonic basal solution, whether only principal cells or principal cells and intercalated cells participate in the water permeability response of the collecting duct, the role of the cytoskeleton in the water permeability response, and the proposed second water permeability barrier that is affected by ADH, but not by adenosine 3',5'-cyclic monophosphate.

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

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.

Activation and blockage of a calcium‐sensitive cation‐selective pathway in the apical membrane of toad urinary bladder.

1. The properties of cation movements through a previously described Ca2+-sensitive oxytocin-stimulated pathway in the apical membrane of the toad urinary bladder were further investigated. 2. In the absence of Ca2+ and other polyvalent cations in the mucosal medium, oxytocin markedly stimulated the flow of current from mucosa to serosa when the major cation in the mucosal solution was any of the following ions: Na+, K+, Rb+, Cs+ or Li+. Analysis of the current noise showed a Lorentzian component associated with the movement of these cations. 3. Ca2+ and other divalent cations in the mucosal solution depressed both the current and the Lorentzian component of the fluctuation spectra. The Michaelis-Menten constants were 2.5, 10 and 58 mumol/l for Ca2+, Sr2+ and Mg2+ respectively. 4. The dihydropyridine Ca2+ channel blockers nitrendipine (10(-5) mol/l) and nicardipine (10(-6) mol/l) inhibited the Ca2+-sensitive current. 5. Alterations of the mucosal pH showed that the current and the plateau of the Lorentzian component increased by elevating the pH from 6 to 8. The Ca2+-sensitive current was further stimulated by increasing pH to 9. However, this manoeuvre resulted in the disappearance of the Lorentzian component in the noise spectrum. 6. Increasing either mucosal [Na+] or [K+] up to 115 mmol/l did not lead to saturation of the current passing through the Ca2+-sensitive channel. In contrast the amiloride-sensitive channel showed saturating behaviour when mucosal [Na+] was increased; half-maximum current was reached when mucosal [Na+] was about 15 mmol/l. 7. When a Na+-free mucosal solution, prepared with either choline or TEA as major cation, was rapidly replaced by a solution with Na+ (115 mmol/l), the current through the Ca2+-sensitive channel increased rapidly and then remained at a nearly constant level. This behaviour is in contrast with the response of the current through the amiloride-sensitive pathway. After suddenly increasing mucosal Na+ concentration ([Na+]m), the current through this channel first increased rapidly and then declined to values of nearly 50% of the peak about 10 min after the increase in [Na+]m.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein synthesis inhibitors attenuate water flow in vasopressin-stimulated toad urinary bladder

Vasopressin stimulates the introduction of aggregated particles, which may represent pathways for water flow, into the luminal membrane of toad urinary bladder. It is not known whether water transport pathways are degraded on removal from membrane or whether they are recycled. We examined the effect of the protein synthesis inhibitors cycloheximide and puromycin using repeated 30-min cycles of vasopressin followed by washout of vasopressin, all in the presence of an osmotic gradient, a protocol that maximizes aggregate turnover. "High dose" cycloheximide (200 micrograms/ml) inhibited flow immediately. "Low dose" cycloheximide (1 microgram/ml) did not affect initial flow; however, flow was inhibited by the fourth restimulation. On further rechallenge, inhibition persisted but did not increase. In the absence of vasopressin, inhibition did not develop. Despite the inhibition of flow in vasopressin-treated tissues, the cAMP-dependent protein kinase ratio (-cAMP/+cAMP), an index of in vivo cAMP effect, was elevated in cycloheximide-treated tissues, suggesting modulation at a distal site in the stimulatory cascade. Cycloheximide inhibited flow when 10 microM forskolin or 0.2 mM 8-BrcAMP was substituted for vasopressin in the fourth period; however, MIX (4 mM)-stimulated flow was enhanced by 1 microgram/ml cycloheximide but inhibited by 200 micrograms/ml cycloheximide. [14C]urea permeability was not inhibited by cycloheximide. Puromycin (0.5 mM) also inhibited water flow by the fourth challenge with vasopressin. The data suggest that protein synthesis inhibitors attenuate flow at a site that is distal to cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

An amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder.

Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 omega cm2. The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5 mM Na+, and serosal Na+ is substituted by K+, Cs+, or N-methyl-D-glucamine, the basolateral membrane expresses what appears to be a large Na+ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca2+. In Cl-free SO2-(4) Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60 mM K+. This pathway can be blocked by serosal amiloride (Ki = 13.1 microM) or serosal Na+ ions (Ki approximately 10 to 20 mM). It also conducts Li+ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec-1 at 0 mV.

Oxytocin and cAMP stimulate monovalent cation movements through a Ca2+-sensitive, amiloride-insensitive channel in the apical membrane of toad urinary bladder.

The effects of oxytocin and cAMP on ion transport were investigated in toad urinary bladders incubated with Ca2+-free solutions on the apical side. Under these conditions both oxytocin and cAMP markedly stimulated the movements of Na+, K+, Rb+, Cs+, Li+, and NH4+ through a pathway that is insensitive to amiloride. The amiloride-insensitive currents were inhibited by the addition of Ca2+, Sr2+, or Mg2+ to the apical solution. The movement of the monovalent cations was associated with a spontaneous Lorentzian component in the power spectrum of the fluctuation in short-circuit current. The plateau of the Lorentzian component was enhanced by oxytocin and cAMP and was depressed by divalent cations. Methohexital inhibited the stimulation of monovalent cation movements caused by oxytocin. These findings suggest that oxytocin and cAMP activate at least two kinds of ionic channels in the apical membrane of toad urinary bladder: the well-known amiloride-sensitive channel and an amiloride-insensitive channel that allows the movement of several monovalent cations and is blocked by Ca2+ and other divalent cations.

Voltage dependence of Na channel blockage by amiloride: Relaxation effects in admittance spectra

Amiloride, present in the mucosal solution, causes the appearance of a distinct additional dispersion in the admittance spectrum of the apical membrane of toad urinary bladder. The parameters of this dispersion (characteristic frequency, amplitude) change with amiloride concentration and with membrane voltage. They allow the calculation of the overall rate constants for Na channel blockage by the positively charged form of amiloride, and the voltage dependence of these rate constants. The on-rate of blockage increases and the off-rate decreases when the membrane surface to which cationic amiloride has access, is made more positive. This result is suggestive of a blocking model where the cationic amidino group of amiloride, depending on its charge, senses 10 to 13% of the membrane voltage while invading the channel entrance by a single-step process, and rests at an electrical distance corresponding to 24 to 30% of membrane voltage while occupying the blocking position.

The water permeability of toad urinary bladder. I. Permeability of barriers in series with the luminal membrane.

Antidiuretic hormone (ADH) induces a large increase in the water permeability of the luminal membrane of toad urinary bladder. Measured values of the diffusional water permeability coefficient, Pd(w), are spuriously low, however, because of barriers within the tissue, in series with the luminal membrane, that impede diffusion. We have now determined the water permeability coefficient of these series barriers in fully stretched bladders and find it to be approximately 6.3 X 10(-4) cm/s. This is equivalent to an unstirred aqueous layer of approximately 400 microns. On the other hand, the permeability coefficient of the bladder to a lipophilic molecule, hexanol, is approximately 9.0 X 10(-4) cm/s. This is equivalent to an unstirred aqueous layer of only 100 microns. The much smaller hindrance to hexanol diffusion than to water diffusion by the series barriers implies a lipophilic component to the barriers. We suggest that membrane-enclosed organelles may be so tightly packed within the cytoplasm of granular epithelial cells that they offer a substantial impediment to diffusion of water through the cell. Alternatively, the lipophilic component of the barrier could be the plasma membranes of the basal cells, which cover most of the basement membrane and thereby may restrict water transport to the narrow spaces between basal and granular cells.

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

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.

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.

Anion-sensitive sodium conductance in the apical membrane of toad urinary bladder.

Membrane potentials and the electrical resistance of the cell membranes and the shunt pathway of toad urinary bladder epithelium were measured using microelectrode techniques. These measurements were used to compute the equivalent electromotive forces (EMF) at both cell borders before and after reductions in mucosal Cl- concentration ([Cl]m). The effects of reduction in [Cl]m depended on the anionic substitute. Gluconate or sulfate substitutions increased transepithelial resistance, depolarized membrane potentials and EMF at both cell borders, and decreased cell conductance. Iodide substitutions had opposite effects. Gluconate or sulfate substitutions decreased apical Na conductance, where iodide replacements increased it. When gluconate or sulfate substitutions were brought about the presence of amiloride in the mucosal solution, apical membrane potential and EMF hyperpolarized with no significant changes in basolateral membrane potential or EMF. It is concluded that: (a) apical Na conductance depends, in part, on the anionic composition of the mucosal solution, (b) there is a Cl- conductance in the apical membrane, and (c) the electrical communication between apical and basolateral membranes previously described is mediated by changes in the size of the cell Na pool, most likely by a change in sodium activity.

Anion-sensitive sodium conductance in the apical membrane of toad urinary bladder

Anion-sensitive sodium conductance in the apical membrane of toad urinary bladder

Functions of apical membrane of toad urinary bladder: effects of membrane impermeant reagents

ARTICLESFunctions of apical membrane of toad urinary bladder: effects of membrane impermeant reagentsV. Harms, and D. D. FanestilV. Harms, and D. D. FanestilPublished Online:01 Dec 1977https://doi.org/10.1152/ajprenal.1977.233.6.F607MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation More from this issue > Volume 233Issue 6December 1977Pages F607-F614 Copyright & PermissionsCopyright © 1977 the American Physiological Societyhttps://doi.org/10.1152/ajprenal.1977.233.6.F607PubMed202172History Published online 1 December 1977 Published in print 1 December 1977 Metrics