Decrease In Isc Research Articles

Endothelin‐1 potently stimulates chloride secretion and inhibits Na(+)‐glucose absorption in human intestine in vitro.

1. Serosally added synthetic endothelin-1 (ET-1) increased short-circuit current (Isc) across isolated muscle-stripped human colonic mucosa in vitro. Bumetanide inhibited Isc responses, indicating that ET-1 stimulates electrogenic Cl- secretion. 2. In isolated human jejunal mucosa, ET-1 exhibited a concentration-dependent dual action. At low concentrations it induced rapid increases in Isc and these were inhibited by bumetanide. At a higher concentration (0.1 microM), ET-1 provoked a drastic and progressive decrease in Isc below the baseline value. 3. Pretreatment with phlorizin or omission of glucose from the Krebs-Ringer solution at the apical (luminal) side of the jejunal mucosa prevented the decreases in Isc evoked by ET-1, suggesting that the peptide inhibits the glucose-coupled electrogenic Na+ absorption. Indeed, flux experiments with D-[14C]glucose demonstrated that ET-1 decreases jejunal glucose absorption by approximately 80% within 30 min. 4. Electron microprobe analyses of cryosections of human jejunum showed that ET-1 (0.1 microM) evokes a significant decrease in intracellular Na+ concentrations of villus (not crypt) epithelial cells, suggesting that the peptide attenuates apical Na(+)-glucose entry by reducing the activity of the Na(+)-glucose cotransporter, SGLT1. 5. In the presence of tetrodotoxin (TTX), ET-1-induced Cl- secretion was significantly reduced, in both human jejunal and colonic mucosa. However, the inhibitory effect on jejunal Na(+)-glucose absorption was not affected by TTX. 6. ET-1 increases electrogenic Cl- secretion across human intestinal mucosa in vitro. This effect is mediated in part via the activation of enteric nerves. Responses of the human jejunal mucosa to high ET-1 concentrations exhibit a second component, namely the rapid inhibition of electrogenic Na(+)-glucose absorption, which might be mediated by an inhibition of the transport activity of SGLT1. This effect is independent from neuronal mediators. Our results suggest different cellular action sites for ET-1 in human small and large intestine.

Effects of a short-chain phospholipid on ion transport pathways in rabbit nasal airway epithelium.

We investigated the mechanism of interference of mucosal application of the short-chain phospholipid didecanoyl-L-alpha-phosphatidylcholine (DDPC; 0.1-0.5%) with ion transport pathways in isolated rabbit nasal airway epithelium (RNAE). Transports of Na+ and Cl- were evaluated from tracer ion fluxes, short-circuit current (Isc), and epithelial conductance (Gt) under short-circuit conditions in Ussing chambers. DDPC rapidly and reversibly abolished net Na+ absorption, reduced control Isc (approximately 110 microA/cm2) by approximately 80%, and induced a small Cl secretion. Intracellular Ca2+ concentration ([Ca2+]i) increased dose dependently and transiently (measured by fura 2 in cultured rabbit airway epithelium), but ionomycin failed to mimic the decrease in Isc. The rise in [Ca2+]i may explain a Ba(2+)-sensitive transient activation of a basolateral K+ conductance. Indomethacin-sensitive prostaglandin E2 production in RNAE increased severalfold, but cyclooxygenase and lipoxygenase inhibitors did not prevent DDPC-induced changes in Isc. DDPC initially decreased control Gt (approximately 13 mS/cm2) by approximately 25% due to inhibition of amiloride-sensitive Na+ channels, and then reversibly increased Gt to approximately 45% above control values. Passive Na+ fluxes increased more than Cl fluxes, suggesting that the increase in Gt is due to formation of a paracellular shunt conductance in parallel with unaffected, anion-selective tight junction channels. The results suggest that DDPC inhibits apical membrane Na+ channels and causes structural changes in tight junctions after incorporation in apical cell membranes.

Segmental heterogeneity of swelling-induced Cl- transport in rat small intestine.

The effect of cell swelling induced by hypotonic media was studied in segments of rat small intestine. In the Ussing chamber, exposure to a hypotonic medium caused a decrease in short-circuit current (Isc) and potential difference (Vms) in the jejunum, whereas the ileum responded with an increase in Isc and Vms. The transition from one pattern to the other was located about in the middle of the small intestine. Tissue conductance decreased in both segments, probably due to a reduction of paracellular shunt conductance induced by the cell swelling. Voltage scanning experiments revealed that the observed decrease in total tissue conductance in the ileum was caused solely by a decrease in local conductance in the villus region while the crypt conductance did not change, suggesting that the decrease in paracellular conductance of the crypts is compensated by an increase in cellular conductance. The response in both segments was dependent on the presence of Cl- and was blocked by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). It was not affected by the neurotoxin tetrodotoxin. In the jejunum the swelling-induced decrease in Isc was reduced in the presence of the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, nordihydroguaiaretic acid. In the ileum the Cl- secretion induced by hypotonicity was blocked by the K+ channel blocker quinine and was reversed into a decrease in Isc when serosal Ca2+ was zero. We conclude that the observed volume regulatory changes are initiated in the jejunum by an eicosanoid-mediated opening of basolateral Cl- channels and in the ileum by a Ca2+-mediated opening of K+ channels which enhances apical Cl- efflux.

Mechanism for the anomalous degradation of Si solar cells induced by high fluence 1 MeV electron irradiation

An anomalous increase in the short-circuit current Isc of n-on-p Si space solar cells, followed by an abrupt decrease in Isc and cell failure has been observed under high fluence (≳1016 cm −2) 1 MeV electron irradiation. A model to explain these phenomena by expressing the change in carrier concentration p, of the base region as a function of the electron fluence is proposed in addition to the well-known model where Isc decreases by minority-carrier lifetime reduction under irradiation. The reduction in p due to majority-carrier trapping by radiation-induced defects has two effects: broadening of the depletion layer which increases the generated photocurrent, and also an increase in the resistivity of the base layer, resulting in the abrupt decrease of Isc and failure of the solar cells.

Mechanisms of acid injury in porcine gastroesophageal mucosa

Abstract Objective To identify the cause and mechanisms of injury in gastroesophageal ulcer disease in market weight swine. Design Comparison of mechanisms of injury caused by HCI with those caused by short chain fatty acids (SCFA) in gastric mucosa. Animals Pigs weighing 30 to 40 kg. Procedure Gastric tissues were studied in Ussing chambers; short-circuit current (Isc) and electrical resistance (R) were recorded in response to treatment, and tissues were examined histologically. Results 60 mM mucosal acetate abruptly (≤ 75 minutes) and irreversibly abolished Isc at pH ≤ 4.5, whereas R decreased more slowly. These data were associated with cell swelling and vesicle formation in mid-zonal layers, followed by sloughing of the outer barrier, erosion into deeper zones, and finally, ulceration. Mucosal HCI at pH > 1.5 was ineffective; however, at pH 1.5, HCI induced an abrupt decrease in R, followed by a slow decrease in Isc, an effect opposite to that caused by SCFA. Serosal addition of HCI rapidly abolished Isc suggesting a barrier to free H+ diffusion from the mucosal solution. Conclusions Undissociated SCFA rapidly penetrate the outer barrier and acidify underlying viable tissue. Cellular acidification inhibits Na pumping and osmoregulation, resulting in cell swelling and necrosis. In contrast, HCI induces an increase in outer barrier permeability before accessing the transporting cells, a much longer process (≥ 5 hours) requiring a lower pH. These studies suggest that microbial production of SCFA may be important in the pathogenesis of porcine gastric ulcers. (Am J Vet Res 1996;57:564-573)

Effect of Melittin on Electrophysiological Parameters in the Frog Cornea Epithelium

The effect of melittin on electrophysiological parameters of the bullfrog cornea was studied using an in vitro preparation. Epithelial cells of corneas were impaled with microelectrodes. Experiments were done under short-circuit current (Isc) conditions. Melittin was added in concentrations of 10(-5)M to the stromal or 10(-6)M to the tear solution. The effects of melittin were as follows: (i) stromal side, a decrease in Isc; an increase in the apical membrane fractional resistance, fRo; no change in the transepithelial conductance, gt; and a depolarization of the intracellular potential, Vo; (ii) tear side, an initial (first 10 min) increase and then a decrease in Isc; a decrease in fRo; an initial (first 10 min) increase with subsequent small decrease in gt; and a depolarization of Vo. Changes in tear Na+, but not in tear K+, with melittin present in tear solution, induced changes in some electrical parameters. The effects on the tear side may be explained by opening of nonspecific channels in the apical membrane with some specificity for Na+ channel. The subsequent effects on the tear and the effects on the stromal side may be explained by an inhibition of the primary transport system, that is, of the Na+/K+ -ATPase pump located in the basolateral membrane. In these experiments, there was no evidence of opening of channels in the basolateral membrane.

Chronic regulation of transepithelial Na+ transport by the rate of apical Na+ entry.

In several settings in vivo, prolonged inhibition of apical Na+ entry reduces and prolonged stimulation of apical entry enhances the ability of renal epithelial cells to reabsorb Na+, an important feature of the load-dependent regulation of renal tubular Na+ transport. To model this load dependency, apical Na+ entry was inhibited or stimulated for 18 h in A6 cells and vectorial transport was measured as short-circuit current (Isc) across monolayers on filter-bottom structures. Basal amiloride-sensitive Isc represents the activity of apical Na+ channels, whereas Isc after permeabilization of the apical membrane to cations with nystatin represents maximal activity of the basolateral Na(+)-K(+)-ATPase. Chronic inhibition of apical Na+ entry by 18-h apical exposure to amiloride or replacement of apical Na+ with tetramethylammonium (TMA+), followed by washing and restoration of normal apical medium, revealed a persistent decrease in Isc that remained despite exposure to nystatin. Both basal and nystatin-stimulated Isc recovered progressively after restoration of normal apical medium. In contrast, chronic stimulation of apical Na+ entry by short circuiting the epithelium increased Isc in the absence and presence of nystatin, indicating upregulation of both apical Na+ channels and basolateral Na(+)-K(+)-ATPase. Basolateral equilibrium [3H]ouabain binding was reduced to 67 +/- 5% in TMA+ vs. control cells, whereas values in 18-h short-circuited cells increased by 42 +/- 19%. The results demonstrate that load dependency of tubular Na+ transport can be modeled in vitro and indicate that the regulation of Na(+)-K(+)-ATPase observed in these studies occurs in part by changes in the density of functional transporter proteins within the basolateral membrane.

Inhibitory effect of erythromycin on ion transport by stria vascularis and vestibular dark cells.

A previous study showed that systemic administration of erythromycin caused a reversible decline in the endocochlear and cochlear microphonic potentials. Those data were thought to suggest that erythromycin caused hearing loss by interference with ion transport processes in the stria vascularis. The present study was undertaken to test this hypothesis by measuring the effects of erythromycin perfused on either the apical or basolateral side on the transepithelial short circuit current (Isc), a measure of the K+ secretion rate. Isc was measured from preparations of strial marginal cells and the homologous vestibular dark cells in vitro with a micro-Ussing chamber. Erythromycin was found to have no effect when perfused on the apical side but to cause a reversible decrease in Isc when perfused on the basolateral side for both epithelia. These data are consistent with the notion that at least one ototoxic effect of erythromycin is the inhibition of K+ secretion in the inner ear.

Effects of Ca2+ on rabbit translens short-circuit current: evidence for a Ca2+ inhibitable K+ conductance.

To characterize the effects of medium Ca2+ levels on rabbit lens electrical properties. Early studies with wholly submerged lenses had shown that Ca2+ removal from the bath resulted in an increased Rb+ efflux, a consequence of an increased Na+ Permeability and lens depolarization. Lenses were bathed with Ussing-type chambers under short-circuited conditions, an arrangement in which the translens short-circuit current (Isc) is carried out across the posterior lens surface mainly by an influx of Na+, and across the anterior face largely by a K+ efflux. Under the present conditions in which the effects of Ca2+ were characterized unilaterally, the above established effects could only be ascribed to the posterior surface. When Ca2+ removal was limited to the anterior face, the Isc increased from 11.87 +/- 1.17 to 17.04 +/- 1.52 microA/cm2 (means +/- SE's, n = 18; an accompanying translens resistance (Rt) decrease of 0.23 +/- 0.049 K omega.cm2 was also recorded). Conversely, increasing the control, anterior-bath [Ca2+] from 1.8 to 3.6 mM reduced the K+ efflux-dependent Isc from 10.54 +/- 1.09 to 8.93 +/- 1.02 (n = 10, with an Rt increase of 0.11 +/- 0.013). These changes were reversible Na(+)-independent, and fully inhibited by the presence of K+ channel blockers (quinidine or Ba2+). Inhibitions of the Ca2+ effects were also obtained with strontium, a Ca2+ surrogate. The Isc was less responsive to changes in the Ca2+ content of the posterior bath. Removal of the cation caused a gradual 1.65 +/- 0.72 microA/cm2 increase (n = 9, with an Rt decrease of 0.090 +/- 0.021 K omega.cm2). In the absence of posterior Na+, Ca2+ withdrawal resulted in highly variable responses, with some specimens exhibiting salient current increases, suggesting that an outwardly directed, posterior efflux of an anion could also have been affected. During the course of this study it was consistently observed that the removal of Na+ from the anterior bath led to an Isc decrease of 2.62 +/- 0.22 microA/cm2 (n = 32, with an Rt increase of 0.35 +/- 0.029 k omega.cm2). This change occurred in both the presence of ouabain and the absence of Ca2+, suggesting that it did not result from an inhibition of the Na(+)-K+ pump current nor from a reversal in putative Na+/Ca2+ exchange activity. Small Isc increases upon anterior Na+ withdrawal (1.68 +/- 0.17, n = 7), consistent with Na+ efflux from the lens, could only be observed with K+ channels inhibited with Ba2+. Also congruent with the observations of a relatively limited anterior Na+ permeability, was the finding that the induction of nonspecific cation channels with amphotericin B reduced the Isc by following Na+ from the anterior bath to enter the lens. Thus, changes in lens Isc can differentiate changes in K+ permeability across the native anterior epithelium from changes in Na+ permeability. Overall, these results suggest that lens Ca2(+)-mobilizing agents (e.g. acetylcholine) could trigger the inhibition of epithelial K+ conductance(s) by the direct action of Ca2+ on K+ channels.

The effect of neuropeptide Y on sodium, chloride and potassium transport across the rat distal colon.

1. Neuropeptide Y (NPY; 10(-10)-10(-7) mol l-1) reduced basal short-circuit current (Isc) in a concentration-dependent manner in the rat distal colon but was ineffective in the proximal colon. 2. The action of NPY was dependent upon the presence of Cl- and HCO3- anions and was blocked by prior treatment of the tissue with a Cl- channel blocker. The decrease in Isc was associated with an increase in mucosa-to-serosa fluxes of Na+, Rb+ (K+) and Cl-, whereas the serosa-to-mucosa flux of Cl- was decreased. 3. The size of the inhibitory NPY effect was linearly correlated with the height of the basal Isc, i.e. it inhibited 55% of basal secretory Isc. 4. The action of NPY was unaffected by indomethacin and tetrodotoxin, when given alone, but was abolished, when the basal Isc was decreased to values near zero by a combination of both inhibitors. This inhibition could be overcome by restoring basal Isc with prostaglandin E2, indicating that the effect of NPY is not mediated by nerves or prostaglandins, but that NPY is only effective, when anion secretion is stimulated by the spontaneous release of neurotransmitters and prostaglandins. 5. NPY inhibited the increase in Isc induced by veratridine and prostaglandin E2, but it had no effect on the Isc induced by direct stimulation of the adenylate cyclase with forskolin, or on Isc induced by stimulation of the Ca(2+)-pathway with carbachol. Inhibition of the response to veratridine or prostaglandin E2 by NPY showed the same dependence on the height of the ISC just prior to addition of NPY as seen in control conditions, i.e. NPY inhibited 55% of cyclic AMP-mediated secretion.6. These results suggest that the effect of NPY is mediated by an inhibition of cyclic AMP-stimulated secretion, which is stimulated in the rat distal colon by a continuous release of prostaglandins and neurotransmitters.

Leukotrienes in inflammatory bowel disease with special reference to their influence on mucosal circulation, ion transport and bowel movement

Leukotrienes in inflammatory bowel disease with special reference to their influence on mucosal circulation, ion transport and bowel movement

Tannin inhibition of protein kinase C in airway epithelium.

Tannin, a polydisperse polyphenol extracted from cotton bracts (CBE), has been implicated in the pathogenesis of byssinosis, a lung disease of mill workers. CBE tannin inhibits chloride secretion in airway epithelial cells by means of an unknown mechanism(s). Activation of protein kinase C (PKC) by PMA (phorbol 12-myristate 13-acetate) in airway cells increases chloride secretion. The effect of tannin on this PKC pathway was examined, using canine tracheal epithelium mounted in Ussing chambers. PMA addition (10 nM) to the mucosal bath resulted in a 0.36 +/- 0.07 microEq/cm2.h (mean +/- SEM, n = 20) increase in short-circuit current (Isc) and a 0.38 +/- 0.17 microEq/cm2.h increase in net chloride secretion (Jnet). The inactive 4 alpha-phorbol had no effect. Tannin addition to the mucosal bath produced a dose-dependent decrease in Isc and Jnet. In tissues pretreated with 2-50 micrograms/ml tannin, and subsequently stimulated with PMA, tannin inhibited PMA stimulation of chloride secretion beginning at a tannin concentration of 10 micrograms/ml (0.09 +/- 0.05 microEq/cm2.h [n = 10] increase in Isc and 0.08 +/- 0.03 microEq/cm2.h increase in Jnet with PMA after tannin pretreatment). At 50 micrograms/ml tannin, the stimulatory effect of PMA was completely abolished. The known PKC inhibitor, H-7 (20 microM), inhibited PMA stimulation, while chelerythrine (2 microM) had not effect on PMA-stimulated Isc and Jnet, and calphostin C was toxic to the airway epithelium. In membrane fragments, 2.5 micrograms/ml tannin inhibited the rate of histone III phosphorylation by PMA from 32.1 +/- 4.4 nmol/mg protein per min to 20.1 +/- 2.7 nmol/mg protein per min (n = 7). In bovine airway cells, tannin pretreatment (2.5 micrograms/ml) decreased the cytosolic activity of PKC but had no effect on PKC translocation to the membrane. We conclude that tannin inhibits chloride secretion in airway epithelial cells in part by inhibiting PKC.

Electron spin resonance in PPV-photodiodes: detection via photoinduced current

Optically (ODMR) and electrically (EDMR) detected magnetic resonance experiments were performed on poly-(paraphenylene-vinylene) (PPV) films and photodiodes based on PPV. The photoinduced short circuit current ISC was found to be dependent on the carriers spin polarization in the space charge region of the A1/PPV/ITO-photodiode. A large decrease in ISC of up to ten percent was observed in the temperature range between 1.5 and 293 K, when ESR conditions were fulfilled. The effect is at least two orders of magnitude stronger than the enhancement of photoluminescence (PL) induced by ESR, where the recombination of thermalized non-geminate excess charge carriers takes place. The ESR signal shows a strong dependence on the applied voltage, the behaviour of which is similar to a diode current-voltage characteristic in a range between −10 and +10 V. In this voltage range the dark current is a singly injected current, so that only the photoinduced current and not the dark current is subjected by ESR. The ESR-induced change of ISC is discussed in terms of the recombination of non-thermalized, non-geminate polaronic pairs. The inequilibrium steady-state magnetization in the spin system is believed to be on account of the difference in the generation and emission rates of particular spin states in a polaronic pair, from which the large effect of ESR on polaronic recombination by means of the intersystem crossing resulted.

A distinctive electrophysiological signature from the Peyer's patches of rabbit intestine.

1. Rabbit small intestinal segments containing Peyer's patches (PP) were examined in Ussing chambers using short-circuit current (Isc) recording. By comparison with control small intestinal mucosal segments, rabbit PP-containing epithelia exhibited decreased basal Isc, increased transepithelial resistance (TER) and unchanged potential difference (PD). 2. Carbachol caused a decrease in Isc in rabbit PP epithelia. Forskolin, dibutyryl cyclic GMP, histamine and the calcium ionophore, A23187, were without effect. In contrast, control epithelial segments of rabbit intestine responded to carbachol and forskolin with an increased Isc, indicative of electrogenic chloride secretion. The EC50 for carbachol was approximately 2 microM in both types of epithelia. Methacholine also caused an outward current in rabbit PP epithelia which had similar properties to that of carbachol. The effect of the cholinomimetics on rabbit PP was basolateral-sided, reversible, and sensitive to low concentrations of the general muscarinic cholinoceptor blockers, atropine, scopolamine and also to the M1 cholinoceptor blocker, pirenzepine. 3. The Isc response to cholinomimetics in rabbit PP was insensitive to bumetanide, amiloride, TEA, barium, acetazolamide, piroxicam and omeprazole, but was attenuated in the presence of ouabain. Using bilaterally-substituted solutions, the carbachol effect on rabbit PP Isc was abolished in chloride/bicarbonate-free, but not in chloride-free solutions, suggestive of stimulation of electrogenic bicarbonate absorption by the agent. Substitution for sodium abolished both the basal current and the Isc response to carbachol. Part of the effect of carbachol on PP Isc appeared to be mediated by submucosal neurones because addition of tetrodotoxin reduced the effect by 60%. 4 As microfold (M) epithelial cells predominate in the PP of the rabbit, the unusual phenotype of cholinomimetic-induced outward current may be used as an electrophysiological marker for these potential sites of oral vaccine delivery, and in particular it may also be of use as a marker for rabbit M cells.

Effects of dinitrophenol on active-transport processes and cell membranes in the Malpighian tubule of Formica

In Formica Malpighian tubules KCl secretion is driven by a V-type H+ ATPase in the luminal membrane in parallel with a H+/K+ antiporter. The effect of the protonophore dinitrophenol (DNP) was investigated on the isolated, symmetrically perfused tubule. DNP was applied in two different concentrations: 0.2 mmol/l and 1 mmol/l. The effects were fast and rapidly reversible. The equivalent short-circuit current (Isc) was reduced significantly to respectively 25 +/- 3% Cn = 4) and -3 +/- 7% (n = 11) of the control value when 0.2 mmol/l or 1 mmol/l was added to the bath. When 1 mmol/l DNP was applied the transepithelial resistance (Rte) decreased significantly to 74 +/- 11% of the control value (n = 11), and the luminal over basolateral voltage divider ratio (VDR), providing an estimate of luminal over basolateral membrane resistance, decreased to 37 +/- 12% of the control (n = 6). A concentration of 1 mmol/l DNP was also applied from the lumen. The decrease in Isc was significant, but much less pronounced (74 +/- 5% of control; n = 6) and no significant changes in Rte and VDR were observed. It is argued that, when the concentration in the bath is high enough, DNP may cross the cell and have a protonophoric effect not only on the mitochondria but also across the luminal cell membrane explaining the drop in transepithelial and in relative luminal membrane resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of volume‐sensitive CI channels in the stimulation of chloride absorption by short‐chain fatty acids in the rat colon

The short-chain fatty acids acetate, propionate and butyrate induced a concentration-dependent decrease in short-circuit current (Isc) of the rat colon in vitro. The decrease in Isc, being more pronounced in the distal than in the proximal colon, was dependent on the presence of Cl- ions and partly on the presence of HCO3-. In the distal colon, the fall in Isc could be inhibited by amiloride, indicating that the activity of the Na+/H+ exchanger is necessary for the induction of this response. The decrease in Isc was diminished by the Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, and the lipoxygenase inhibitor, nordihydroguaiaretic acid. In contrast, inhibitors of the leukotriene pathway or a Cl- channel blocker did not affect the Isc response in the proximal colon. Measurements of unidirectional fluxes revealed that butyrate caused a stimulation of the mucosa to serosa fluxes (Fms) of Na+ and Cl- in the distal, but only of FNams in the proximal colon. Unidirectional Rb+ fluxes were not altered. The stimulation of Fclms correlated with the degree of metabolism of the short-chain fatty acid. The increase in FClms was most pronounced for butyrate, smaller for acetate and not observed with the poorly metabolizable short-chain fatty acid, isobutyrate. Consequently, two factors seem to be responsible for the stimulation of Cl- absorption by short-chain fatty acids in the distal colon: (1) the intracellular production of HCO3- during the oxidation of short-chain fatty acids as substrate for the apical Cl-/HCO3- exchanger, and (2) the activation of volume-sensitive basolateral Cl- channels.

Potassium secretion by vestibular dark cell epithelium demonstrated by vibrating probe

Detection of motion and position by the vestibular labyrinth depends on the accumulation of potassium within a central compartment of the inner ear as a source of energy to drive the transduction process. Much circumstantial evidence points to the vestibular dark cell (VDC) epithelium as being responsible for concentrating K+ within the lumen. We have used the vibrating probe technique to directly observe voltage and ion gradients produced by this tissue to put this assumption on a solid experimental footing. Relative current density (Isc,probe) over the apical membrane of VDC epithelium was measured with the vibrating voltage-sensitive probe, and this technique was validated by performing maneuvers known to either stimulate or inhibit the transepithelial equivalent short circuit current. Basolateral bumetanide (5 x 10(-5) M) and ouabain (1 x 10(-3) M) caused a decrease in Isc,probe by 55 +/- 6% and 39 +/- 3%, respectively while raising the basolateral K+ concentration from 4 to 25 mM caused an increase by 35 +/- 8%. A K+ gradient directed toward the apical membrane was detected with the vibrating K(+)-selective electrode, demonstrating that, indeed, the VDC epithelium secretes K+ under control conditions. This secretion was inhibited by bumetanide (by 94 +/- 7%) and ouabain (by 52 +/- 8%). The results substantiate the supposition that dark cells produce a K+ flux and qualitatively support the correlation between this flux and the transepithelial current.

Endothelial-derived nitric oxide inhibits sodium transport by affecting apical membrane channels in cultured collecting duct cells.

Previously, it has been shown that the addition of bradykinin (Bk) to M-1 cortical collecting duct cells in the presence of endothelial cells decreased short-circuit current (Isc), a measure of net active transport. This effect is presumably due to the release of endothelium-derived nitric oxide (EDNO), because the decrease in Isc could be blocked with Nw-nitro-L-arginine. To show that the inhibition of Isc was due to EDNO rather than prostaglandins, the ability of a cyclooxygenase inhibitor to block the inhibition was examined. When Bk was added to cocultures in the presence of meclofenamate (10(-5) M), Isc decreased from 62 +/- 12 to 44.5 +/- 7 muA/cm2, not significantly different from that in the absence of meclofenamate. To determine if the effect was due to an alteration of sodium absorption, Bk (10(-9) M) was added to cocultures, resulting in a decrease in Na flux from 28 +/- 3.1 to 20 +/- 2.2 nEq/min (P < 0.05), with Isc decreasing from 25 +/- 2.4 to 20 +/- 3.6 nEq/min (P < 0.05). To examine if the inhibition was due to blockade at the apical membrane sodium channel or the basolateral Na+/K+ ATPase, the cation-selective ionophore nystatin was used. Nystatin reversed the effect of EDNO on Isc. The effects of EDNO on Na+/K+ ATPase were also measured directly. Under maximum rate conditions, the Na+/K+ ATPase activity of control and Bk-treated cocultures was 5.2 +/- 0.3 and 6.8 +/- 1.0 nmol/min per square centimeter, respectively (not significantly different).(ABSTRACT TRUNCATED AT 250 WORDS)

Ion-transporting activity in the murine colonic epithelium of normal animals and animals with cystic fibrosis.

Electrogenic ion transport in the isolated colonic epithelium from normal and transgenic mice with cystic fibrosis (CF mice) has been investigated under short-circuit current (Isc) conditions. Normal tissues showed chloride secretion in response to carbachol or forskolin, which was sensitive to the Na-K-2Cl cotransport inhibitor, frusemide. Responses to both agents were maintained for at least 12 h in vitro, but the responses to carbachol changed in format throughout this period. By contrast CF colons failed to show the normal secretory responses to carbachol and forskolin, most preparations showing a decrease in Isc that was immediately reversed by frusemide. In CF colons addition of Ba2+ ions or tetraethylammonium (TEA+) to the apical bathing solution antagonised the reduction in Isc caused by the secretagogues. It is concluded that the reduction in Isc in CF colons is due to electrogenic K+ secretion and this was confirmed by flux studies using rubidium-86. In normal colons exposed to TEA+ the responses to forskolin were greater, but not significantly so, presumably because the minor K(+)-secretory responses are dominated by major chloride-secretory responses. Again rubidium-86 fluxes showed an increase of K+ secretion in normal colons receiving forskolin. Since the amiloride-sensitive current was not different in CF and normal colons there was no evidence that the CF mice were stressed in a way that increased mineralocorticoid levels and hence K+ secretion. Knowledge of the phenotype of the colonic epithelium of the CF mouse sets the baseline from which attempts at gene therapy for the gut must be judged.

Regulation of K secretion across the porcine gallbladder epithelium

Porcine gallbladder epithelium from the neck and the fundus of the organ was stripped of serosal muscle and mounted in Ussing chambers to investigate the mechanisms of K secretion. The sensitivity to K channel blockers and regulation by norepinephrine (NE), adenosine 3',5'-cyclic monophosphate (cAMP), and increases in intracellular Ca concentration ([Ca]) were studied. The porcine gallbladder secretes K (approximately 0.8 mu eq/cm2.h) under basal conditions. Mucosal tetraethylammonium (TEA) produced a concentration-dependent increase in short-circuit current (Isc) and inhibited the unidirectional serosal-to-mucosal 86Rb flux JsmRb, resulting in a > 60% reduction in net Rb secretion. In contrast, serosal Ba produced a concentration-dependent decrease in Isc and stimulated JsmRb, resulting in a > 200% increase in net Rb secretion. NE inhibited JsmRb and net Rb secretion in both regions. In the fundic region the mucosal-to-serosal Rb flux (JmsRb) was also significantly increased, suggesting that active K absorption was activated. Exogenous cAMP increased JsmRb and net Rb secretion by > 85% in both regions. This increase in net Rb secretion was blocked by mucosal TEA but unaffected by NE. The Ca ionophore ionomycin also increased JsmRb and net Rb secretion and reduced the Isc by approximately 50%. Neither mucosal TEA nor Ba blocked changes in steady-state Rb secretion induced by ionomycin. Although both serosal Ba and ionomycin produced significant reductions in Isc, the effects of Ba were blocked by ionomycin pretreatment. These findings indicate that basal K secretion occurs through TEA-sensitive apical K channels and is regulated by intracellular cAMP. NE likely reduces K secretion by decreasing intracellular concentration of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)