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Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP.

The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toad Bufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to th serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.

Animals↗

Cellular mechanism of HCO-3 and Cl- transport in insect salt gland.

Active HCO-3 secretion in the anterior rectal salt gland of the mosquito larva, Aedes dorsalis, is mediated by a 1:1 Cl-/HCO-3 exchanger. The cellular mechanisms of HCO-3 and Cl- transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO2 and HCO-3 from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (Va) and had little effect on basolateral potential (Vbl). Changes in luminal Cl- concentration altered Va in a rapid, linear manner with a slope of 42.2 mV/decalog a1Cl-. Intracellular Cl- activity was 23.5 mM and was approximately 10 mM lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl- concentration had no effect on Vbl, indicating an electrically silent basolateral Cl- exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated acHCO-3 was 14.4 mM. These results show that under control conditions HCO-3 enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl- enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl- -selective channel, and HCO-3 exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl- exit and HCO-3 entry, and the effects of serosal addition of the Cl-/HCO-3 exchange inhibitor, DIDS, on JCO2net and transepithelial potential (Vte) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl- and HCO-3 movements.

Aedes↗

Modulation of apical Na permeability of the toad urinary bladder by intracellular Na, Ca, and H.

The Na conductance of the apical membrane of the toad urinary bladder was measured at different concentrations of Na both in the external medium and in the cell. Bladders were bathed in high K-sucrose medium to reduce basal-lateral resistance and voltage, and the transepithelial currents measured under voltage-clamp conditions. Amiloride was used as a specific blocker of the apical Na channel. At constant external Na, the internal Na concentration was increased by blocking the basal-lateral Na pump with ouabain. With high Na activity in the mucosal medium (86 mM), increases in intracellular Na activity from 10 to over 40 mM increased the amiloride-sensitive slope conductance at zero voltage while apical Na permeability, estimated from current-voltage plots using the constant field equation, decreased by less than 20%. Lowering the serosal Ca concentration from 1 to 0.1 mM had no effect on the change in PNa with increasing Nac, but increasing serosal Ca to 5 mM enhanced the reduction in PNa with increasing Nac, presumably by increasing Ca influx into the cell. PNa was also reduced by serosal vanadate (0.5 mM), a putative blocker of ATP-dependent Ca extrusion from the cell, and by acute exposure to CO2, which presumably acidifies the cytoplasm. Current-voltage relationships of the amiloride-sensitive transport pathway were also measured in the absence of a Na gradient across the apical membrane. These plots show that outward current passes through the channels somewhat less easily than does inward current. The shape of the I-V relationships was not significantly altered by changes in cellular Na, Ca or H, indicating that the effects of these ions on PNa are voltage independent.

Amiloride↗

Current-voltage analysis of apical sodium transport in toad urinary bladder: effects of inhibitors of transport and metabolism.

The basal-lateral surface of the epithelium of the urinary bladder of the toad (Bufo marinus) was depolarized by exposure of the serosal surface to 85 mM KCL and 50 mM sucrose. The extent of depolarization appeared to be virtually complete, as evaluated by the invariance in the transepithelial electrical potential difference and conductance on addition of nystatin (a monovalent cation ionophore) to the serosal medium. The Na-specific current (INa) was defined as the current sensitive to the removal of Na from the mucosal medium or inhibitable by addition of amiloride to this medium. In the presence of the high K-sucrose serosal medium, rapid, serial, stepwise clamping of the transepithelial voltage (V) yielded a curvilinear dependence of INa on V; which is taken to represent the I--V curve of the apical Na channels. The constant field equation (Goldman, D.E. 1943; J. Gen. Physiol. 27:37) fits the I--V data points closely, allowing estimates to be made of the permeability to Na of the apical membrane (PNa) and of the intracellular Na activity (Nac). Exposure of the apical surface to amiloride (5 X 10(-7) M) decreased PNa in proportion to the decrease in INa (i.e., approximately 70%) but decreased Nac only 25%. In contrast, an equivalent reduction in INa elicited by exposure of the basal-lateral surface to ouabain was accompanied by only a 20% decrease in PNa and a sixfold increase in Nac. The effects of amiloride on PNa and ouabain on Nac are consistent with the primary pharmacological actions of these drugs. In addition, PNa appears to be under metabolic control, in that 2-deoxyglucose, a specific inhibitor of glycolysis, decreased INa and PNa proportionately, and lowered Nac marginally, effects indistinguishable from those obtained with amiloride.

Amiloride↗

Membrane electrical parameters in turtle bladder measured using impedance-analysis techniques.

Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; inhibition of proton secretion using acetazolamide in CO2 and HCO3- -free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; stimulation of proton transport with CO2 and HCO3- -containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.

Animals↗

Intracellular gradients of electrical potential in the epithelial cells of the Necturus gallbladder.

When single-barrelled electrodes (5-60 Momega) were advanced under manual control from the mucosal side of the epithelium the mucosal membrane was on average indented by about 40 micron before the microelectrode pentrated the cell. Since this dimpling was comparable with the total depth of the cell, which recovered its original shape within 0.5 sec, the steady intracellular potential was recorded only about 14 micronm from the basal (serosal) membrane. Fast recording of the associated change in potential revealed an abrupt drop to -26 mV at a mean rate of 84 V/sec, followed by a further slow drop to a steady value of about -50 mV at a mean rate of 0.28 V/sec. The initial level of -26 mV may be regarded as the potential difference across the mucosal membrane. This conclusion was confirmed by mounting the microelectrode on a piezoelectric probe, which delivered 3 micron jabs in less than 0.5 msec. With this device in operation to prevent dimpling, the mean potential difference across the mucosal membrane was recorded as -29 mV. In all cases the potential across the basal membrane was recorded as -52 mV. Manual advance of the microelectrode tip within the cytoplasm yielded an intracellular potential gradient of 0.6 mV/micron. The same potential profile and membrane potentials were demonstrated on penetrating the epithelium from the serosal side, and measurements with multibarrelled electrodes whose tips were staggered in depth gave roughly the same internal potential gradient. The resistivity of the cytoplasm was determined by a triple-barrelled microelectrode, and varied from 10 times that of Necturus saline at the mucosal end of the cell to 4 times in the middle and 6 times at the serosal end.

Animals↗

Electron microprobe analysis of the different epithelial cells of toad urinary bladder. Electrolyte concentrations at different functional states of transepithelial sodium transport.

The electrolyte composition of toad urinary bladder epithelial cells has been measured using the technique of electron microprobe analysis. Portions of hemibladders, which had been mounted in chambers and bathed with a variety of media, were layered with albumin solution on their mucosal surfaces and immediately shock-frozen in liquid propane at -180 degrees C. From the frozen material 1--2 micrometer thick cryosections were cut and promptly freeze-dried for 12 hr at-80 degrees C and 10(-6) Torr. Electron microprobe analysis using a scanning electron microscope, an energy dispersive X-ray detector, and a computer programme, to distinguish between characteristic and uncharacteristic radiations, allowed quantification of cellular ionic concentrations per kg tissue wet wt by comparison of the intensities of the emitted radiations from the cells and from the albumin layer. Granular, mitochondrial-rich, and basal cells, and the basal portions of goblet cells, showed a similar composition, being high in K (about 110 mM/kg wet wt) and low in Na (about 13 mM/kg wet wt). The apical portions of goblet cells were higher in Ca and S and lower in P and K, presumably reflecting the composition of the mucus within them. With Na-Ringer's as the mucosal medium, cells gained Na and lost K, when their serosal surfaces were exposed to ouabain, 10(-2) M. Replacement of mucosal Na by choline virtually prevented these ouabain-induced changes. Cellular ion contents were unchanged when Na in the serosal medium was replaced by choline. No differences in Na and K concentrations were detected between nuclei and cytoplasm. These results provide independent support for the hypothesis the the cellular Na transport pool in toad bladder epithelial cells derives exclusively from the mucosal medium and that no important recycling of Na occurs from the serosal medium to the cells.

Animals↗

Ionic and metabolic requirements for the hydroosmotic response to antidiuretic hormone in toad urinary bladder.

A study has been conducted to determine the ionic and metabolic requirements for full expression of the hydroosmotic response to antidiuretic hormone in the toad urinary bladder. By appropriate manipulation of incubation conditions it can be shown that there is a pool of serosal sodium necessary for a full hormone response. This serosal sodium pool is not related to the transepithelial sodium transport pool A full hydroosmotic response also requires serosal potassium; however, no specific anion requirement was demonstrated. Additionally, anaerobic or aerobic metabolism support a full hydroosmotic response equally well.

Animals↗

Metabolic regulation of apical sodium permeability in toad urinary bladder in the presence and absence of aldosterone.

In the present study, further evidence was adduced for energy-dependent regulation of passive apical transport of Na in toad bladder epithelium. In potassium-depolarized preparations studied by current-voltage analysis, additions of pyruvate or glucose to the media of substrate-depleted bladders evoked proportionate increases in the transepithelial Na current and in apical Na permeability. These responses were large in aldosterone pretreated hemibladders and almost absent in the aldosterone-depleted preparations or when hormonal action was blocked by spironolactone or cycloheximide. The substrate-induced increases in apical Na permeability were fully reversed by appropriate metabolic inhibitors, i.e. 2-deoxyglucose and oxythiamine. Moreover, the inhibitory effect of 2-deoxyglucose was bypassed by the addition of pyruvate to the serosal medium. Thus apical Na permeability is clearly sensitive to the supply of cellular energy. The possibility that changes in intracellular free Na activity may mediate metabolic regulation of apical Na permeability was evaluated by prolonged exposure to Na-free mucosal and serosal media, with and without inhibition of the Na/K-pump by ouabain. The stimulatory and inhibitory effects of pyruvate, 2-deoxyglucose and oxythiamine on Na currents and Na conductances were preserved under these circumstances. Furthermore, reduction of serosal Ca to a minimal level of 3 microM, was without effect on the response to metabolic inhibition. These experiments demonstrate the existence of Na-independent metabolic regulation of apical Na transport and imply that neither basal-lateral nor mitochondrial Na/Ca exchange is required for this regulatory process under the imposed conditions. The possibility that a Na-independent, Ca transport mechanism in mitochondria or endoplasmic reticulum may be involved in metabolic regulation of apical Na transport, however, remains to be evaluated.

Aldosterone↗

The sodium transport pool in toad urinary bladder epithelial cells.

The sodium which equilibrates with 24-Na in epithelial cells of toad urinary bladders has been determined. With sodium Ringer's bathing both mucosal and serosal surfaces, 24-Na in the mucosal medium equilibrated with about 35 mmoles cellular sodium/kg cellular dry weight, representing about 20% of the total cellular sodium determined flame photometrically; 24-Na in the serosal medium equilibrated with 120 mmoles cellular sodium/kg cellular dry weight, about 80% of the total cellular sodium. With 24-Na in both media all cellular sodium was labeled within 30 min. In the absence of serosal sodium, total cellular sodium and that sodium which equilibrated with mucosal 24-Na in sodium Ringer's were both similar to the cellular sodium of mucosal origin which had been determined in epithelial cells exposed on both surfaces to sodium Ringer's. Sodium-free mucosal medium, and sodium Ringer's containing amiloride 10-4 or 10-3 M in the mucosal medium, both virtually completely inhibited transepithelial sodium transport. But, whereas the cellular sodium of mucosal origin fell to only 2 mmoles/kg cellular dry weight with sodium-free mucosal medium, an appreciable labeling of cellular sodium was found whether amiloride was present before, or only after, exposure of tissue to mucosal 24-Na. Rapid washing of the mucosal surface of hemibladders just before removal of epithelial cells for analysis removed most of this sodium labeled in the presence of amiloride, suggesting that the cellular sodium of mucosal origin consists of at least two fractions with only about two-thirds truly intracellular. The sodium transport pool measured directly in these experiments is appreciably smaller than any previous estimates of pool size all of which have been obtained by indirect techniques involving use of whole hemibladders rather than epithelial cells alone.

Amiloride↗

Aldosterone control of the density of sodium channels in the toad urinary bladder.

Near-instantaneous current-voltage relationships and shot-noise analysis of amiloride-induced current fluctuations were used to estimate apical membrane permeability to Na (PNa), intraepithelial Na activity (Nac), single-channel Na currents (i) and the number of open (conducting) apical Na channels (N0), in the urinary bladder of the toad (Bufo marinus). To facilitate voltage-clamping of the apical membrane, the serosal plasma membranes were depolarized by substitution of a high KCl (85 mM) sucrose (50 mM) medium for the conventional Na-Ringer's solution on the serosal side. Aldosterone (5 X 10(-7) M, serosal side only) elicited proportionate increases in the Na-specific current (INa and in PNa, with no significant change in the dependence of PNa on mucosal Na (Nao). PNa and the control of PNa by aldosterone were substrate-dependent: In substrate-depleted bladders, pretreatment with aldosterone markedly augmented the response to pyruvate (7.5 X 10(-3) M) which evoked coordinate and equivalent increases in INa and PNa. The aldosterone-dependent increase in PNa was a result of an equivalent increase in the area density of conducting apical Na channels. The computed single-channel current did not change. We propose that, following aldosterone-induced protein synthesis, there is a reversible metabolically-dependent recruitment of preexisting Na channels from a reservoir of electrically undetectable channels. The results do not exclude the possibility of a complementary induction of Na-channel synthesis.

Aldosterone↗

Unstirred layer effects in osmotic water flow across gallbladder epithelium.

The standard one-dimensional model of the unstirred layer is applied in a re-examination of the experimental results of Wright, Smulders and Tormey (Wright, E.M., Smulders, A.P., Tormey, J. McD., 1972, J. Membrane Biol. 7:198) who reported large transients in the osmotic flux of water from the serosal to the mucosal side of rabbit gallbladder epithelium. They initiated osmosis by the addition of sucrose to the mucosal bathing solution (initially, approximately 300 nOsm NaCl) and observed that the initial flux was more than ten times its eventual steady-state value; they interpreted this as a consequence of the piling-up of NaCl in the unstirred tissue layer on the serosal side of the epithelium. The present analysis (both steady-state and unsteady) shows that if measured values of layer thickness delta are used, together with reasonable values of the reduced diffusivity of NaCl in the tissue and of the fraction of tissue available for water flow, then one would predict a discrepancy of only about 10%, not tenfold, between the initial and final values of the flux. Thus the standard model is inconsistent with the observations. Furthermore, Wright et al's results cannot be used to infer that the osmotic permeability of epithelial cell membranes is much larger than steady-state measurements on whole epithelia would indicate. Mucosal-to-serosal flow is also analyzed, and in this case a considerably greater osmotic permeability is predicted; this result is consistent with the observed changes in structure of the lateral intercellular spaces when the direction of flow is reversed.

Animals↗

Anti-IgE-induced histamine release from rat tissues passively sensitized in vivo with myeloma IgE differs with strain and mast cell source.

Four different strains of rats, BDII, E3, LE, and OM/N, each with a low basal serum level of IgE, were examined with respect to anti-IgE- and ConA-induced release of histamine from serosal mast cells and chopped lung tissue. Tests were performed before and three days after i.p. injection of a graded dose of myeloma IgE (IR 162)-containing ascitic fluid. There was a clearcut strain difference in response capacity with respect to ConA- and anti-IgE-induced release of histamine from serosal mast cells before myeloma IgE-injection. Response capacity increased in some but not all strains after myeloma IgE injection; increase in response capacity of serosal mast cells did not correlate to that of chopped lung tissue. Analogous findings were observed when two of the strains, LE and E3, were passively sensitized by i.p. injection with serum containing another myeloma IgE (IR2). These results indicate that differences exist between mast cells of different rat strains, and within strain between mast cells of various tissues in capacity to become sensitized to myeloma IgE.

Animals↗

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↗

Effects of potassium-free media and ouabain on epithelial cell composition in toad urinary bladder studied with X-ray microanalysis.

The technique of X-ray microanalysis was used to study the composition of toad urinary bladder epithelial cells incubated in Na Ringer's and K-free medium, with and without ouabain. Following incubation under short-circuit conditions, portions of tissue were coated with an external albumin standard and plunge-frozen. Cryosections were freeze-dried and analyzed. In Na Ringer's, granular and basal cells, and also the basal portion of the goblet cells, had similar water and ion compositions. In contrast, mitochondria-rich cells contained less Cl and Na. On average, the granular cells and a subpopulation of the basal cells lost K and gained Na after ouabain and in K-free medium alone. However, there was considerable variation from cell to cell in the responses, indicating differences between cells in the availabilities of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control. In contrast, the compositions of both the low Cl, mitochondria-rich cells and a sub-population of the basal cells were little affected by the different incubation conditions. This is consistent with a comparatively low Na permeability of these cells. The results also indicate that (i) much, if not all, of the K in the dominant cell type, the granular cells, is potentially exchangeable with serosal medium Na, and (ii) Na is accumulated from the serosal medium under K-free conditions. They also provide information about the role of the (Na-K)-ATPase in the maintenance of cellular K in K-free medium, being consistent with other evidence that removal of serosal medium K inhibits transepithelial Na transport by decreasing Na entry to the cells from the mucosal medium, rather than solely by inhibiting the basolateral membrane (Na-K)-ATPase.

Animals↗

Ion activities in the lateral intercellular spaces of gallbladder epithelium transporting at low external osmolarities.

The ion activities in the lateral spaces of the unilateral preparation of the gallbladder of Rana catesbiana were measured by double-barrelled ion-selective microelectrodes. The bladders were bathed in a saline solution with a low osmolarity (62 mOsm) containing, in mM: 27 Na+, 27 Cl-, 2 K+, 1 Ca++, 4 HCO3-. Working at reduced osmolarities had the advantage of an increased volume transport and of widened intercellular spaces. The reference barrel recorded an electrical potential of +2.7 mV in the spaces; they contained a solution similar to the external solution. The electrodes recorded a Na+ concentration of 27 mM, a K+ concentration of 1.7 mM, a Ca++ concentration of 0.69 mM and a Cl- concentration of 28.5 mM. In the spaces there was a lower resistance between the tip of the electrode and the serosal bath than that recorded with the tip in the lumen, and injection of fluorescent dye (11 A diameter) via the electrodes did not stain the cells. The concentrations in the secretion were similar to those in the spaces. The intracellular compartment had an apparent K+ concentration of 95 mM, and the concentrations of Na+ and Cl- were both about 5 mM. These data indicate that when the gallbladder is bathed with hypotonic solutions and is transporting fluid at approximately three or four times the normal rate, there are no significant osmotic gradients between the lumen and the lateral spaces. It is suggested that transcellular transport of water is implemented by a combination of high osmotic permeabilities across both mucosal and serosal cell membranes and low reflection coefficients (for K+ salts) at the serosal cell membranes.

Animals↗

Calcium, histamine, and pentagastrin: speculations about the regulation of gastric acid secretion at cellular level.

The acid secretory response to acetylcholine, histamine, pentagastrin and db-cAMP was studied in dependence of serosal calcium and magnesium level in the isolated whole stomach of the mouse. Histamine- or db-cAMP-induced acid secretion from isolated stomach was not dependent on calcium. Secretory responses to pentagastrin and acetylcholine were strongly diminished in the absence of serosal calcium. Reduction of serosal magnesium concentration influenced neither histamine- nor pentagastrin-induced secretion. The calcium-antagonist verapamil reduced only acid response to pentagastrin. A working hypothesis has been discussed to understand the possible role of calcium in the regulation gastric acid secretion.

Acetylcholine↗

Epithelial cell electrolytes in relation to transepithelial sodium transport across toad urinary bladder.

Aspects of the relationships between cellular composition and transepithelial sodium transport across toad urinary bladder are reviewed. Changes in cellular sodium produced by amiloride, vasopressin, aldosterone, hypoxia, ouabain, and sodium-free media are consistent with a cellular sodium transport pool. Metabolic studies suggest that this pool gains its sodium from the mucosal medium and that there is little recycling of sodium between cell and serosal medium. One-third of the cellular potassium equilibrates readily with serosal potassium. The rate of exchange of potassium is much less than the rate of sodium transport supporting the contention that sodium transport in this tissue is electrogenic. Studies with 36Cl suggest that chloride does not cross the apical cellular membranes, but exchanges with serosal chloride. Possible relationships between transepithelial sodium transport and cellular volume regulation are discussed.

Aldosterone↗