Net Cl Research Articles

Salt Tolerance, Ion Relations and the Effect of Root Medium on the Response of Citrus to Salinity

This paper describes the ion relations of two citrus genotypes, Etrog citron (Citrus medica L.), a salt-sensitive genotype, and Rangpur lime (Citrus reticulata var. austera hybrid?), a salt-tolerant genotype, under conditions of high NaCl concentrations. Root morphology was modified by establishing seedlings for 6 months in solution or sand culture. When established in sand culture, both genotypes displayed a high efficiency, relative to plants established in solution culture, to exclude Na from leaves, but differed in their capacities to exclude Cl; this capacity was much lower in Etrog citron than Rangpur lime. The net Cl root to shoot transport rates over 7 weeks for Etrog citron and Rangpur lime, treated with 50 mol m-3 NaCl, were 0.25 and 0.07 μmol g-1 root FW h-1, respectively. The net transport rates of Cl in Etrog citron were 10 times those in Rangpur lime between weeks 4 and 7. In Rangpur lime Cl reached pseudo steady-state levels in root and leaf tissue by week 4, thereafter, the net Cl root to shoot transport rate of Rangpur lime decreased while the rates in Etrog citron increased. The accumulation of Na and Cl in leaves of Rangpur lime, in contrast to Etrog citron, was not affected by transpiration. The high salt tolerance of Rangpur lime appears to be associated with low rates of net uptake and transport of Cl, feedback control of net root to shoot transport by Cl levels in the shoot, a low interaction between water flow and Cl and Na fluxes, and a reduction in the leafiroot ratio in response to salinity. When plants were established in solution culture the pioneer roots became elongated and the normal development of the laterals into a fibrous root system was suppressed. Plants of both genotypes, previously established in solution culture and then treated with 50 mol m-3 NaCl, accumulated levels of Na and Cl in leaves up to 200-400 mol m-3. Under these conditions the rates of net Cl root to shoot transport over the 7 weeks were 0.53 and 0.56 μmol g-1 root FW h-1 for Etrog citron and Rangpur lime, respectively. These high rates of Cl uptake and transport were attributed to changes in root permeability and increases in passive ion fluxes. Keywords: citrus, root morphology, root medium, transpiration, Cl, Na, salt accumulation, salinity.

Use of MQAE for measurement of intracellular [Cl-] in cultured aortic smooth muscle cells.

A novel fluorescent indicator, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide (MQAE), was used to measure intracellular chloride concentration ([Cl-]i) in primary cultures of rat aortic smooth muscle cells (VSMC). The hydrolytic and fluorescent properties of the dye were characterized. The intracellular Stern-Volmer constant was calculated to be 25 M-1. Cl- efflux curves were characteristic of saturation-type kinetics, with an apparent Michaelis-Menten constant value of 11 +/- 4.8 (SD) mM, a maximum velocity of 0.038 +/- 0.021 mM/s, and a half time (t1/2) of 9.0 +/- 3.7 min. The average efflux rate in the first 10 min (0.023 +/- 0.004 mM/s) was reduced in the presence of either 130 microM 4,4'-diisothiocyanato-dihydrostilbene-2,2'-disulfonic acid (H2DIDS) (0.014 +/- 0.006, P = 0.02) or 40 microM furosemide (0.017 +/- 0.004, P = 0.04). Restoration of physiological extracellular chloride concentration ([Cl-]o) after zero Cl- resulted in net Cl- influx with a t1/2 of 3.6 +/- 1.0 min. The initial Cl- influx rate was reduced after exposure to furosemide, from 0.069 +/- 0.006 to 0.046 +/- 0.008 mM/s, P < 0.002, and was reduced after exposure to H2DIDS from 0.102 +/- 0.013 to 0.033 +/- 0.003 mM/s, P < 0.001. Furosemide reduced the steady-state [Cl-]i from 31.6 +/- 3.2 to 26.1 +/- 2.4 mM, P < 0.01, whereas H2DIDS had little effect on [Cl-]i. Our results demonstrate that MQAE can be used to measure [Cl-]i in primary cultures of VSMC.

The molecular basis of chloride transport in shark rectal gland.

Transepithelial Cl- secretion in vertebrates is accomplished by a secondary active transport process brought about by the coordinated activity of apical and basolateral transport proteins. The principal basolateral components are the Na+/K(+)-ATPase pump, the Na+/K+/2Cl- cotransporter (symporter) and a K+ channel. The rate-limiting apical component is a cyclic-AMP-stimulated Cl- channel. As postulated nearly two decades ago, the net Cl- movement from the blood to the lumen involves entry into the epithelial cells with Na+ and K+, followed by active Na+ extrusion via the pump and passive K+ exit via a channel. Intracellular [Cl-] is raised above electrochemical equilibrium and exits into the lumen when the apical Cl- channel opens. Cl- secretion is accompanied by a passive paracellular flow of Na+. The tubules of the rectal glands of elasmobranchs are highly specialized for secreting concentrated NaCl by this mechanism and hence have served as an excellent experimental model in which to characterize the individual steps by electrophysiological and ion flux measurements. The recent molecular cloning and heterologous expression of the apical Cl- channel and basolateral cotransporter have enabled more detailed analyses of the mechanisms and their regulation. Not surprisingly, since hormones acting through kinases control secretion, both the Cl- channel, which is the shark counterpart of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), and the cotransporter are regulated by phosphorylation and dephosphorylation. The primary stimulation of secretion by hormones employing cyclic AMP as second messenger activates CFTR via the direct action of protein kinase A (PKA), which phosphorylates multiple sites on the R domain. In contrast, phosphorylation of the cotransporter by as yet unidentified kinases is apparently secondary to the decrease in intracellular chloride concentration caused by anion exit through CFTR.

Vasopressin activates a chloride conductance in cultured cortical collecting duct cells

In rabbit cortical collecting duct (CCD) cells, arginine vasopressin (AVP) causes a transient increase followed by a sustained depression of transepithelial potential difference (PDte). Mechanisms underlying the decrease in PDte are not well understood. In this study, we used primary cultures of rabbit CCD cells to study effects of AVP. Basolateral addition of AVP caused a dose-dependent increase in transepithelial conductance (Gte) and a corresponding decrease in PDte. A significant effect was observed at 1 pM AVP, and half-maximal response occurred at 30 pM AVP; 1 nM AVP increased Gte and decreased PDte. Replacement of apical Na+ with N-methyl-D-glucamine did not prevent the effect of AVP on Gte, suggesting that it is not mediated by an increase in apical Na+ conductance. Similarly, apical Ba2+ (1 mM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.1 mM) failed to prevent the effect of AVP. On the other hand, 5-nitro-2(3-phenylpropylamino)benzoic acid (0.1 mM) caused partial inhibition, whereas substitution of apical Cl- with gluconate or cyclamate almost completely prevented the AVP-induced increase in Gte. In unidirectional ion-flux studies, 1 nM AVP caused only a modest increase in apical-to-basolateral (A-->BL) flux of 22Na and had no effect on transepithelial flux of 86Rb in either direction. On the other hand, AVP caused a pronounced increase in A-->BL flux and BL-->A flux of 36Cl, resulting in an increased net Cl- absorption. The effect of AVP on Gte could be mimicked by 8-bromo-adenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) and isoproterenol, and effects of AVP and isoproterenol were not additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin.

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS-insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.

Regulatory volume decrease in HL-60 cells: importance of rapid changes in permeability of Cl- and organic solutes.

Results obtained through the use of inhibitors and isotope flux and equilibration techniques indicate that the regulatory volume decrease (RVD) response of human promyelocytic leukemic HL-60 cells occurs largely through the efflux of K+ and Cl- through separate conductive membrane pathways. These "channels" differ pharmacologically and in their modes of activation from those described in lymphocytes and Ehrlich ascites tumor cells. With use of measured 86Rb+ and 36Cl- fluxes, together with a diffusion kinetic model, the membrane potential (Em) and apparent K+ and Cl- permeabilities (PK and PCl) were estimated under various isotonic and hypotonic conditions. Under isotonic (300 mosM) conditions, Em is close to the Nernst potential for K+ and PCl is < 0.1 PK. Rapid and steeply graded increases in the measured Cl- efflux rate and calculated PCl occur with decreasing tonicity, with the largest increases at tonicities < 80% of isotonic. K+ efflux and the apparent PK increase only modestly with decreasing tonicity. At 50% tonicity, PCl rises to nearly 10 times PK, which should cause substantial membrane depolarization, with Em approaching the Nernst potential for Cl-. Gramicidin treatment markedly accelerates the rate of RVD and net 36Cl- efflux in hypotonic Na(+)-and Cl(-)-free media, providing further evidence that PK is rate limiting during RVD. K+ loss exceeds Cl- loss during RVD, and the total loss of K+ and Cl- is insufficient to account for the observed degree of volume recovery in 50% tonicity media, indicating that other (organic) osmolytes must take part in the HL-60 cell RVD response.

Control of intracellular pH during regulatory volume decrease in HL-60 cells.

Intracellular pH (pHi) homeostasis was investigated in human promyelocytic leukemic HL-60 cells as they undergo regulatory volume decrease (RVD) in hypotonic media to determine how well pHi is regulated and which transport systems are involved. Cells suspended in hypotonic (50-60% of isotonic) media undergo a small (< 0.2 pH units), but significant (P < 0.05), intracellular acidification within 5 min. However, after 30 min of RVD, pHi is not significantly different from the initial pHi in 20 mM HCO3- medium and is significantly higher in HCO3(-)-free medium. Experiments performed in media with or without 150 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and HCO3- demonstrate that the anion exchanger (AE) mediates a net Cl- influx, with compensating HCO3- efflux, during RVD. To determine which transport systems are involved in counteracting this tendency toward acidification, we measured transport rates and examined the effect of transport system inhibitors on pHi. We found that inhibition of Na+/H+ exchange (NHE) with 12.5 microM ethylisoproplamiloride (EIPA) causes pHi to fall significantly by the end of 30 min of RVD. As assessed by EIPA-sensitive 22Na+ uptake measurements, NHE, largely dormant under resting isotonic conditions, becomes significantly activated by the end of 30 min of RVD, despite recovery of pHi and cell volume to near-normal levels. Thus a shift in the normal pHi dependence and/or volume dependence of NHE activity must occur during RVD under hypotonic conditions. In contrast, H(+)-monocarboxylate cotransport appears to play only a supportive role in pH regulation during RVD, as indicated by lack of stimulation of [14C]lactate efflux during RVD.

Effects of Ion Exchange on Stream Solute Fluxes in a Basin Receiving Highway Deicing Salts.

At Fever Brook, a 1260-ha forested basin in central Massachusetts, highway deicing salt application increased the solute flux in streamflow by 120% above background flux (equivalent basis) during a 2-yr period. Attempts to isolate the nonsalt component of stream solute fluxes have commonly subtracted salt contributions based on the net Cl flux (Cl output in streamflow minus Cl input in precipitation). In these studies, any net Na flux in excess of the amount needed to balance the net Cl flux has been attributed to weathering. At Fever Brook, however, the net output of Na was less than the net output of Cl, suggesting a loss of Na within the basin. The Na sink was inferred to be cation exchange of Na for Ca and Mg in the soil. A method was developed to quantify the exchange based on a Na budget, which included an independent estimate of the Na flux from weathering. The amount of exchange was apportioned to Ca and Mg based on their relative concentrations in the stream. The background fluxes of Ca and Mg (i.e., those that would occur in the absence of deicing salts) were calculated by subtracting the amounts from ion exchange plus the much smaller direct contributions in deicing salts from the observed fluxes. Ion exchange and direct salt contributions increased the net output fluxes of Ca and Mg, each by 44% above background. In basins that receive deicing salts, failure to account for cation exchange thus may result in an underestimate of the flux of Na from weathering and overestimates of the fluxes of Ca and Mg from weathering.

Fasting alters basal and stimulated ion transport in piglet jejunum

Three-week-old piglets were used to study the effects of short-term fasting on jejunal ion transport. A 48-h fast significantly reduced mucosal weight, villus height, and crypt depth. Fasting increased basal short-circuit current (Isc), which reflects active ion transport, and total tissue conductance (Gt) of muscle-stripped jejunal sheets mounted in Ussing chambers. Increases in Isc evoked by carbachol, serotonin, histamine, prostaglandin E2, or Escherichia coli heat-stable enterotoxin were significantly greater in the fasted piglets. Isc responses to mucosal D-glucose were also enhanced by the fast. Under basal conditions, unidirectional and net fluxes of Na+ and Cl-, as well as serosal-to-mucosal inulin fluxes, were significantly increased in fasted piglets. In fed piglets, carbachol increased net Cl- secretion by stimulating serosal-to-mucosal Cl- flux; Gt was not affected. In fasted piglets, carbachol increased net Cl- secretion by inhibiting mucosal-to-serosal fluxes with no effect on serosal-to-mucosal fluxes. In addition, carbachol significantly inhibited mucosal-to-serosal Na+ fluxes and reduced Gt in this group. Thus a 48-h fast increased unidirectional and net ion fluxes in piglet jejunum and enhanced ion transport responses to secretory agonists. The mechanism by which carbachol stimulated net Cl- secretion was also altered by the fast. These results suggest that the absence of luminal nutrition changes the ion transport characteristics of the jejunal epithelium.

Potassium-induced chloride secretion across the frog retinal pigment epithelium

In the intact eye, a transition from light to dark increases K concentration ([K]o) from approximately 2 to 5 mM in the extracellular (subretinal) space between the photoreceptors and the retinal pigment epithelium (RPE) apical membrane. In control (HCO3/CO2) Ringer solution, 36Cl was actively absorbed across isolated bullfrog RPE (retina to choroid) at a rate of 0.31 +/- 0.02 (SE) mu eq.cm-2.h-1 (n = 15). Elevating apical [K]o from 2 to 5 mM reversed active 36Cl transport to secretion (choroid to retina), with a rate of 0.76 +/- 0.17 mu eq.cm-2.h-1. This reversal was completely inhibited by 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in either the apical or basal bath. In open circuit, elevating [K]o induced a similar reversal of net 36Cl flux and inhibited fluid absorption by approximately 25%. Apical Ba2+ (1 mM), decreased CO2 (5 to 1%), or increased apical bath HCO3 concentration ([HCO3]o) also caused a DIDS-inhibitable reversal of active 36Cl flux. A 10-fold reduction of apical bath Na or [HCO3]o significantly inhibited [K]o, Ba2+, and low CO2-induced Cl secretion. All of these results can be understood in terms of an intracellular pH-dependent stimulation of the basolateral membrane Cl-HCO3 exchanger.

Acidification stimulates chloride and fluid absorption across frog retinal pigment epithelium.

Radioactive tracers and a modified capacitance-probe technique were used to characterize the mechanisms that mediate Cl and fluid absorption across the bullfrog retinal pigment epithelium (RPE)-choroid. In control (HCO3/CO2) Ringer solution, 36Cl was actively absorbed (retina to choroid) at a mean rate of 0.34 mu eq.cm-2.h-1 (n = 34) and accounted for approximately 25% of the short-circuit current. Apical bumetanide (100 microM) or basal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 1 mM) inhibited active Cl transport by 70 and 62%, respectively. Active Cl absorption was doubled, either by removing HCO3 from the bathing media or by elevating CO2 from 5 to 13%, and the increased flux was inhibited by apical bumetanide or basal DIDS. Open-circuit measurements of fluid absorption rate (Jv) and the net fluxes of 36Cl, 22Na, and 86Rb (K substitute) indicated that CO2-induced acidification stimulated NaCl and fluid absorption across the RPE. During acidification, bumetanide produced a twofold larger inhibition of Jv compared with control. Stimulation of net Cl absorption was most likely caused by inhibition of the the basolateral membrane intracellular pH-dependent Cl-HCO3 exchanger.

Cl− channels in basolateral renal medullary vesicles VIII. Partial purification and functional reconstitution of basolateral mTAL Cl− channels

Cl- channels fused from basolateral mTAL membranes into planar bilayers have distinctive functional characteristics which, when taken together, are unique among Cl- channels. The properties of these 50 to 60 pS channels can account for the characteristics of basolateral Cl- conductances in microperfused mTAL segments and thus may mediate net basolateral Cl- absorption in the intact mTAL. In the present studies, we solubilized basolateral membranes from rabbit mTAL. Since basolateral mTAL Cl- channels contain arginine- and lysine-rich domains, we exposed these solubilized membranes to sequential cation- and anion-exchange chromatography. The bound and unbound eluates from cation- and anion-exchange chromatography were reconstituted into proteoliposomes which, when fused into bilayers, yielded Cl- channels whose properties were virtually identical to those described above for native basolateral mTAL channels fused into bilayers. As judged by valinomycin-sensitive conductive 36Cl- uptake, proteoliposomes reconstituted from the unbound eluates after anion-exchange chromatography were enriched at least 30-fold in Cl- channel activity and had about 30% of the total Cl- channel activity solubilized in native vesicles.

Different Mechanisms of Acid-Base Regulation in Rainbow Trout (Oncorhynchus mykiss) and American Eel (Anguilla rostrata) during NaHCO3Infusion

Rainbow trout (Oncorhynchus mykiss) and American eels (Anguilla rostrata) were infused intra-arterially with NaHCO₃ (∼800 μmol/kg/h) to assess their acid-base regulatory mechanisms during metabolic alkalosis. In both species, plasma pH and [HCO₃ ⁻] increased during infusion, although the changes were more pronounced in the eel. In trout, pH and [HCO₃⁻] were returned to preinfusion values by 12 h after infusion was stopped, while in the eel there was only incomplete clearance of accumulated [HCO₃⁻] during the 24-h postinfusion period. In each case, approximately 20 mmol/L of HCO₃⁻ was removed from the plasma during postinfusion. The predominant mechanism employed by both trout and eel to regulate blood acid-base status during alkalosis was manipulation of branchial net Cl⁻ flux $(J_{net}^{Cl-})$, although this was accomplished with markedly different strategies. Trout relied primarily on adjustments of branchial Cl⁻ uptake $(J_{in}^{Cl-})$ to control $J_{net}^{Cl-}$. In the eel, however, owing to the absen...

Delivery of marine chloride in precipitation and removal by rivers in the Murray-Darling Basin, Australia

Rising groundwater tables and increasing river salinities are major problems in the semi-arid Murray-Darling (M-D) Drainage Basin of SE Australia. Chemical data from precipitation, rivers and groundwaters are integrated here to estimate chloride fluxes in the context of dramatic alteration of the hydrologic balance in the basin initiated during European settlement approximately 150 years ago. Our analysis indicates that about half of Cl deposition from the atmosphere in the western Murray Basin is derived from resuspended regional continental dust, resulting in high Ca/Na ratios in bulk precipitation samples. Estimates of recent marine deposition of Cl to the M-D Basin were made by excluding rainfall data from sites having high Ca/Na ratios. Basin-wide budgets for Cl delivery from the atmosphere and removal by rivers indicate that the Darling Basin retains about 90% of current annual input of marine Cl whereas the River Murray exports two to three times current annual input to the Murray Basin (exclusive of the Darling). Some implications from this study are as follows: 1. (1) Re-evaluation of net marine Cl input by rain implies that recent model calculations of 36 Cl Cl inputs from the atmosphere to the Murray Basin may differ significantly from actual values. In particular, net 36 Cl Cl input ratios from the atmosphere to the western Murray Basin may be a factor of two higher than those derived assuming all Cl in rain is of recent marine origin. 2. (2) The very large inventories of salt within the two catchments, especially the saline groundwaters of the Murray Basin, accumulated over very long periods, probably of the order of a few million years. The current rearrangements of stored salinity as a result, at least in part, of human perturbations of the hydrologic balance are likely to continue for an extended period because of the massive quantities of salts involved, and thus present major limitations on options for long-term management of the M-D Basin.

Effects of barium Chloride on electrolyte transport across isolated colons from normal and Aldosterone-treated lizards (Gallotia Galloti)

Addition of BaCl2 to the solution bathing colons of normal lizards did not alter the absorptive Na+ flux, but did reverse the net absorption of Cl- to become net secretion. Cl- secretion resulted from an increase in its serosal-to-mucosal movement and was positively correlated to an increase in short-circuit current. Ba2+-induced short-circuit current was dependent on the presence of both Na+ and Cl- in the serosal medium. Ba2+-induced Cl- secretion could be reversed by serosal amiloride (10(&shy;4) mmol l-1). Colons from acutely or chronically aldosterone-treated lizards exhibited a considerably higher short-circuit current, potential difference and net Na+ absorption than did untreated colons. Net Cl- transport was unaltered by acute treatment, but was totally abolished after chronic treatment. BaCl2 rapidly decreased the potential difference, short-circuit current and tissue conductance across colons from aldosterone-treated lizards. Net Na+ transport was markedly inhibited by Ba2+ in both acutely and chronically treated tissues, but barium did not change unidirectional or net Cl- fluxes in these. The present results support the following hypotheses: (1) that BaCl2 inhibits electrogenic Na+ absorption induced by acute or chronic aldosterone treatment and (2) that Ba2+ induces an electrogenic Cl- secretion by stimulating a basolateral Na+-dependent Cl- intake in normal but not in aldosterone-treated colons. This also suggests that aldosterone could exert an antisecretory influence in colonic epithelia.

Nacl flux in the flounder ( Pseudopleuronectes americanus) intestine: Effects of ph and transport inhibitors

1. 1. The effects of extracellular pH on Na + and Cl − absorption were studied in vitro in the small intestine of the winter flounder, Pseudopleuronectes americanus. 2. 2. Reductions in bathing solution pH inhibited J ms na (mucosal-to-serosal flux) and J net na (net flux) ( r = 0.90) and J net Cl ( r = 0.92) [due to an increase in J sm Cl, (serosal-to-mucosal)] and decreased short circuit current (Isc). 3. 3. Luminal bumetanide (0.1 mM) and amiloride (1 mM) inhibited Na + and Cl − absorption by reducing J ms. 4. 4. Luminal barium (5mM) and luminal copper (100 μM) decreased J ms Cl and increased J sm Cl. 5. 5. We conclude that reductions in extracellular pH inhibit a luminal membrane NaCl absorptive process (Na +-K +-2Cl −) and stimulate an electrogenic Cl − secretory process.

Intestinal l-methionine transport in the cultured gilthead bream ( Sparus aurata)

1. 1. The influx and transepithelial movements of l-methionine and its effects on the electrophysiology and Na-Cl-transport in upper and lower intestine of the cultured fish, Spanis aurata, were measured. 2. 2. The K m and V max of l-methionine influx into the tissues were higher in lower intestine than in upper intestine. A prominent diffusion-like transport component was also measured in both segments during influx experiments. 3. 3. Net transepithelial fluxes of l-methionine (1 mM) were observed in both upper and lower intestine, this transport being Na +-dependent. 4. 4. The two intestinal segments exhibited an electrical potential difference (PD) and a short circuit current ( I s c ) serosa negative or near zero. Tissue conductance ( G t ) was higher in posterior than in lower intestine. 5. 5. Addition of l-methionine to the mucosal side of lower or upper intestine did not induce changes in PD in either part. 6. 6. Isotopic fluxes of Cl − or Na + measurements under short circuit conditions showed that there were no net Cl − or Na + transport in either part. 7. 7. l-Methionine additions to the mucosa did not induce changes in unidirectional fluxes of Cl − or Na + or in the ( I sc ) in either the anterior or posterior intestine.

Na(+)-K(+)-2Cl- cotransport and Cl- secretion evoked by heat-stable enterotoxin is microfilament dependent in T84 cells.

We previously reported that adenosine 3',5'-cyclic monophosphate-mediated stimulation of Cl- secretion in the human intestinal epithelial cell line T84 is accompanied by significant remodeling of F-actin and that both the secretory and cytoskeletal responses may be inhibited by phalloidin derivatives, agents that polymerize actin and prevent dynamic reorganization of microfilaments. In contrast, the carbachol-elicited Cl- secretory response (Ca2+ mediated) was not attenuated by phalloidin (J. Clin. Invest. 87: 1903-1909, 1991). In the present study, we examine the effect of phalloidin on the Cl- secretory response elicited by the heat-stable enterotoxin of Escherichia coli (STa), which induces elevations in intracellular guanosine 3',5'-cyclic monophosphate. We find that apical administration of 1 microM STa results in a regionally restricted redistribution of F-actin confined to the basal pole of the cells. In monolayers pretreated with phalloidin, the Cl- secretory response to STa was inhibited by > 60%. Sequential treatment of phalloidin-loaded monolayers with STa followed by carbachol resulted in a synergistic secretory response that was not different from control (unloaded) monolayers. Examination of efflux/uptake through specific membrane transport pathways involved in STa-stimulated Cl- secretion indicated normal activation of apical Cl- and basolateral K+ channels under phalloidin-loaded conditions. The ability of STa-treated monolayers to pump Na+ in an absorptive direction was also unaffected by phalloidin. der phalloidin-loaded conditions, STa-stimulated Na(+)-K(+)-2Cl- cotransporter activity was reduced by approximately 60%, sufficient to account for the observed inhibition of net Cl- secretory response.(ABSTRACT TRUNCATED AT 250 WORDS)

DPC blockade of transepithelial chloride absorption and single anion channels in teleost urinary bladder

The columnar cell epithelium of the euryhaline goby (Gillichthys mirabilis) urinary bladder actively absorbs NaCl from the lumen, thereby driving water transport and reducing water loss to the hypertonic external environment. Transcellular transport of Cl- involves apical membrane entry via Na(+)-coupled cotransport driven by the Na+ electrochemical gradient and subsequent basolateral membrane exit. An anion channel in the basolateral cell membrane of columnar epithelial cells was identified using patch-clamp technique. This channel may be one avenue for basolateral Cl- exit from the urinary bladder columnar cell. Single-channel conductance (Gc) of channels in excised, inside-out membrane patches was approximately 75 pS in symmetrical solutions containing 140 mM Cl-. The channel was selective to Cl- over other anions [Cl- > 2-(N-morpholino)ethanesulfonic acid (MES) > F- approximately Br- approximately I- > NO3- approximately SO4(2-)). Channel activity, expressed as the open probability (Po), was voltage dependent in the physiological range of membrane potential, with membrane depolarization increasing Po. Decreasing the pH of the solution bathing the cytoplasmic face of the membrane patch over the range 8.4-6.0 reduced Po. There was no effect of pH on either Gc or ionic selectivity. Radiochloride flux technique was also applied to intact columnar epithelial cell sheets to relate anion channel activity to macroscopic transcellular transport. Serosal exposure to the anion channel blocker diphenylamine-2-carboxylic acid (DPC, 30 microM to 3 mM) reduced and abolished transcellular radiochloride fluxes and net Cl- absorption across short-circuited tissues in a dose-dependent fashion. DPC addition (10 microM to 1 mM) to the solution bathing the cytoplasmic face of excised, inside-out membrane patches reduced Po in a dose-dependent manner and had no effect on Gc. These parallel findings of DPC blockade on intact epithelia and on single anion channels support the notion that this anion channel is a basolateral membrane component of the pathway for Cl- movement in transcellular Cl- absorption.

The effect of colitis on large-intestinal electrolyte transport in early childhood.

The large intestine plays an important role in the salvage of electrolytes and water, although little is known of its function in early childhood in health or in disease. Using an in vitro voltage clamp technique, we have measured bidirectional sodium (Na) and chloride (Cl) fluxes (J), electrical potential difference (PD), short circuit current (Isc), and tissue resistance (R) in 10 pairs of healthy human infant left colon and four pairs of inflamed colon. In healthy tissue there was net Na and Cl absorption with JNa net approximating Isc. In contrast, inflamed colon exhibited significant reductions in PD, Isc, and R and increases in net serosa to mucosa flux of Na and Cl (JNasm and JClsm) resulting in net Na and Cl secretion. These results suggest that Na and Cl absorption in left colon is highly efficient in young children and that mucosal inflammation may seriously impair the colon's ability to conserve salt and water.