Neuropeptide Control of Ion and Fluid Transport Across Locust Hindgut

The anterior (ileum) and posterior (rectum) segments of locusthindgut constitute the reabsorptive part of the locust excretorysystem. They are functionally analogous to the proximal convolutedtubule and more distal parts, respectively, of the mammaliankidney tubule. Transport mechanisms are well understood in therectum, and an epithelial model has been proposed. Electrogenicactive absorption of Cl^– at the apical membrane drivesK⁺ transport (electrical coupling) and hence fluid transport(J_v). A partially purified neuropeptide (CTSH) from the CorpusCardiacum (CC) stimulates KC1 transport, and therefore presumablyfluid absorption via cAMP as second messenger. Another neuropeptide(purified and sequenced) from locust CC, neuroparsins, is reportedto stimulate rectal Jv via the inositol triphosphate (Ca²⁺)system, but actions on ion transport processes are unknown There is considerable similarity of transport processes in locustileum and rectum. A neuropeptide (ITP) acting on the ileum hasbeen purified and partially sequenced from locust CC (storagelobe). ITP has high sequence homology with a family of crustaceanneuropeptides. ITP, apparently acting via cAMP, stimulates ilealreabsorption of Cl^–, K⁺, Na⁺ and fluid by several-fold.It also inhibits active H⁺ secretion in the ileum, a processinvolved in hemolymph acid-base regulation. ITP has negligibleeffectson rectal transport processes. Thus separate neuropeptides apparentlycontrol transportevents in locust ileum and rectum.     

KCl transport across an insect epithelium: II. Electrochemical potentials and electrophysiology

Summary The cellular mechanism of K-stimulated Cl transport in locust hindgut was studied using double-barrelled ionsensitive microelectrodes and electrophysiological techniques. Steady-state net electrochemical potentials for Cl and K and the conductances of apical and basal membranes and paracellular pathway were determined under control conditions, during exposure to 1mm cAMP, and following ion substitutions. Under control open-circuit conditions, intracellular Cl activity (a _Cl ^c ) was 3.5 times that predicted for passive equilibrium across the apical membrane. The net electrochemical potential opposing Cl entry from the mucosal side increased by 50% during cAMP stimulation of transepithelial Cl absorption whereas the net electrochemical potential favoring Cl exit across the basal membrane was unchanged. No correlation was observed between and the net electrochemical potential across the apical membrane for Na. The net electrochemical potential favoring K entry across the apical membrane was negligible underI _sc conditions when Cl transport rate was approximately 10 eq cm^–2 hr^–1. Locust rectal cells showed electrical and dye coupling. The results also indicate that most transepithelial diffusion of ions is transcellular and that epithelial tightness effectively increases during exposure to cAMP becauseR _a andR _b both decrease, by 80% whileR _j is unchanged. The cAMP-induced R _b was abolished in Cl-free saline whereas R _a was insensitive to Cl removal, but was blocked by removing K from the saline. Based on these findings, our model for Cl absorption in locust hindgut features i) an active entry step for Cl at the apical membrane which is stimulated by cAMP and by low levels of K on the mucosal side, but is not energized by or a large cAMP-stimulated Cl conductance in the basal membrane and a similar cAMP-stimulated K conductance in the apical membrane. cAMP dose-response curves are similar for the stimulation of active Cl absorption and Cl-independent (i. e. K) conductance, indicating that cAMP exerts dual control over active Cl transport and counter-ion permeability.     

K+ transport and capacitance of the basolateral membrane of the larval frog skin

Skin from larval bullfrogs was mounted in an Ussing-type chamberin which the apical surface was bathed with a Ringer solution containing 115 mM K⁺ and thebasolateral surface was bathed with a Ringer solution containing 115 mMNa⁺. Ion transport was measured asthe short-circuit current(I_sc) with alow-noise voltage clamp, and skin resistance(R_m) wasmeasured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine wavesto the command stage of the voltage clamp. From the ratio of theFourier-transformed voltage and current signals, it was possible tocalculate the resistance and capacitance of the apical and basolateralmembranes of the epithelium(R_a andR_b,C_a and C_b,respectively). With as the anion,R_m decreasedrapidly within 5 min following the addition of 150 U/ml nystatin to theapical solution, whereasI_sc increasedfrom 0.66 to 52.03 µA/cm² over a60-min period. These results indicate that nystatin becomes rapidlyincorporated into the apical membrane and that the increase inbasolateral K⁺ permeabilityrequires a more prolonged time course. Intermediate levels ofI_sc were obtainedby adding 50, 100, and 150 U/ml nystatin to the apical solution. Thisproduced a progressive decrease in R_a andR_b whileC_a andC_b remainedconstant. With Cl as theanion, I_sc valuesincreased from 2.03 to 89.57 µA/cm² following treatment with150 U/ml nystatin, whereas with gluconate as the anionI_sc was onlyincreased from 0.63 to 11.64 µA/cm². This suggests that theincrease in basolateral K⁺permeability produced by nystatin treatment, in the presence of morepermeable anions, is due to swelling of the epithelial cells of thetissue rather than the gradient for apicalK⁺ entry. Finally,C_b was notdifferent among skins exposed toCl,, or gluconate, despite the largedifferences inI_sc, nor didinhibition of I_scby treatment with hyperosmotic dextrose cause significant changes inC_b. These resultssupport the hypothesis that increases in cell volume activateK⁺ channels that are alreadypresent in the basolateral membrane of epithelial cells.

     

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers onfilters to analyze the apical membrane mechanisms that help mediate ionand fluid transport across the epithelium. RT-PCR showed the presenceof cystic fibrosis transmembrane conductance regulator (CFTR) andepithelial Na⁺ channel (ENaC) message, and immunomicroscopyshowed apical membrane staining for both proteins. CFTR was alsolocalized to the apical membrane of native human mammary ductepithelium. In control conditions, mean values of transepithelialpotential (apical-side negative) and resistance(R_T) are 5.9 mV and 829  · cm², respectively. The apical membranepotential (V_A) is 40.7 mV, and the mean ratioof apical to basolateral membrane resistance (R_A/R_B) is 2.8. Apicalamiloride hyperpolarized V_A by 19.7 mV andtripled R_A/R_B. AcAMP-elevating cocktail depolarized V_A by 17.6 mV, decreased R_A/R_B by60%, increased short-circuit current by 6 µA/cm²,decreased R_T by 155  · cm², and largely eliminated responses toamiloride. Whole cell patch-clamp measurements demonstratedamiloride-inhibited Na⁺ currents [linear current-voltage(I-V) relation] and forskolin-stimulated Clcurrents (linear I-V relation). A capacitance probe methodshowed that in the control state, MEC monolayers either absorbed orsecreted fluid (2-4µl · cm² · h¹). Fluidsecretion was stimulated either by activating CFTR (cAMP) or blockingENaC (amiloride). These data plus equivalent circuit analysis showedthat 1) fluid absorption across MEC is mediated byNa⁺ transport via apical membrane ENaC, and fluid secretionis mediated, in part, by Cl transport via apicalCFTR; 2) in both cases, appropriate counterions move throughtight junctions to maintain electroneutrality; and 3)interactions among CFTR, ENaC, and tight junctions allow MEC to eitherabsorb or secrete fluid and, in situ, may help control luminal[Na⁺] and [Cl].

     

Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium

In vitro mammary epithelial cell models typically fail to form a consistently tight barrier that can effectively separate blood from milk. Our hypothesis was that mammary epithelial barrier function would be affected by changes in luminal ion concentration and inflammatory cytokines. Bovine mammary epithelial (BME-UV cell line) cells were grown to confluence on permeable supports with a standard basolateral medium and either high-electrolyte (H-elec) or low-electrolyte (L-elec) apical medium for 14 days. Apical media were changed to/from H-elec medium at predetermined times prior to assay. Transepithelial electrical resistance (R_te) was highest in monolayers continuously exposed to apical L-elec. A time-dependent decline in R_te began within 24 h of H-elec medium exposure. Change from H-elec medium to L-elec medium time-dependently increased R_te. Permeation by FITC-conjugated dextran was elevated across monolayers exposed to H-elec, suggesting compromise of a paracellular pathway. Significant alteration in occludin distribution was evident, concomitant with the changes in R_te, although total occludin was unchanged. Neither substitution of Na⁺ with N-methyl-D-glucosamine (NMDG⁺) nor pharmacological inhibition of transcellular Na⁺ transport pathways abrogated the effects of apical H-elec medium on R_te. Tumor necrosis factor alpha, but not interleukin-1 nor interleukin-6, in the apical compartment caused a significant decrease in R_te within 8 h. These results indicate that mammary epithelium is a dynamic barrier whose cell-cell contacts are acutely modulated by cytokines and luminal electrolyte environment. Results not only demonstrate that BME-UV cells are a model system representative of mammary epithelium but also provide critical information that can be applied to other mammary model systems to improve their physiological relevance. transepithelial electrical resistance; apical cation concentration; paracellular permeability; mastitis; inflammatory cytokines; occludin     

Overexpression of protein kinase C-delta increases tight junction permeability in LLC-PK1 epithelia

The Ca²⁺-independent-isoform of protein kinase C (PKC-) was overexpressed inLLC-PK₁ epithelia and placed undercontrol of a tetracycline-responsive expression system. In the absenceof tetracycline, the exogenous PKC- is expressed. Westernimmunoblots show that the overexpressed PKC- is found in thecytosolic, membrane-associated, and Triton-insoluble fractions.Overexpression of PKC- produced subconfluent and confluentepithelial morphologies similar to that observed on exposure ofwild-type cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Transepithelialelectrical resistance(R_T) in cellsheets overexpressing PKC- was only 20% of that in cell sheetsincubated in the presence of tetracycline, in which the amount ofPKC- and R_Twere similar to those in LLC-PK₁ parental cell sheets. Overexpression of PKC- also elicited a significant increase in transepithelial flux ofD-[¹⁴C]mannitoland a radiolabeled 2 × 10⁶-molecular-weight dextran,suggesting with theR_T decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC- overexpression results in amultilayered cell sheet, the tight junctions of which are almost uniformly permeable to ruthenium red. Freeze-fracture electron microscopy indicates that overexpression of PKC- results in a moredisorganized arrangement of tight junctional strands. As withLLC-PK₁ cell sheets treated with12-O-tetradecanoylphorbol-13-acetate, the reducedR_T, increasedD-mannitol flux, and tightjunctional leakiness to ruthenium red that are seen with PKC-overexpression suggest the involvement of PKC- in regulation oftight junctional permeability.

     

Electrotonic Coupling between Adjacent Internodal Cells of Chara braunii: Transmission of Action Potentials beyond the Node

Many nodal cells are interposed between two internodal cellsof Chara braunii. When an action potential conducted in an internodereached the node, no electrical activation in the nodal cellscould be found, although an area of the membrane bordering thenodal cells in this internode was partially activated (end-membraneaction potential). When the action potential approached thenode along the stimulated internode, an electrotonic potentialchange (depolarization) was produced in the other internode.This depolarization was greatly depressed by the end-membraneaction potential of the stimulated internode, so that hardlyany transmission took place. The ratio of the potential changein the surface membrane of the adjoining ("postsynaptic") internode(cell b) to that of the stimulated one (cell a), the couplingratio, e_b/e_a, can be estimated from a simple equivalent circuitof the nodal region composed of two surface-membrane resistances(R_a, R_b) and intercellular resistance (R_n). If R_n remains thesame, a higher ratio should be produced with a larger R_b, butthe ratio does not depend on any change in R_a, which could beproved experimentally. Transmission of the action potentialbeyond the node was frequent when the coupling ratio was increasedand when the threshold that elicits the action potential waslowered by immersing the node in a K or Na salt solution. ¹ Present address: Department of Physiology, Tohoku UniversitySchool of Dentistry, Sendai 980, Japan. (Received December 1, 1980; Accepted January 23, 1981)     

Chloride secretion by canine tracheal epithelium: II. The cellular electrical potential profile

Summary We used intracellular microelectrode techniques to study the mechanisms responsible for Cl secretion by canine tracheal epithelium. Tissues were treated with indomethacin (10^–6 m, added to the mucosal solution) to reduce the baseline rate of Cl secretion and then stimulated by addition of epinephrine (10^–6 m) or prostaglandin E₁ (10^–6 m) to the submucosal solution.Three conclusions emerged from our findings: First, secretagogues enhance the rate of transepithelial Cl transport primarily by increasing apical membrane Cl permeability, since: (i) stimulation of secretion produced parallel decreases in transepithelial resistance (R _t) and the membrane resistance ratioR _a/R_b, whereR _a andR _b refer to the resistances of the apical and basolateral membranes; (ii) there was an inverse relation between the short-circuit current andR _a/R_b; (iii) secretagogues depolarized the electrical potential difference across the apical membrane (_a) and produced an equivalent hyperpolarization of the transepithelial electrical potential difference (₁) so that, in the steady-state, the basolateral membrane potential (_b) was unchanged; and (iv) substitution of sulfate or gluconate for Cl in the bathing solutions prevented secretagogue-induced changes inR _t, R_a/R_b, (_a) and (₁).Second, Cl entry into the cell across the basolateral membrane appears to be electrically-neutral since omission of Cl from the submucosal solution had no effect on (_b) and did not decreaseR _a/R_b as would be expected if Cl entered the cell by a conductive process.Third, secretagogues decreaseR _b. Approximately 20 sec after the onset of the secretory responseR _a/R_b underwent a secondary increase whileR _t continued to fall. The decrease inR _b may reflect an increase in basolateral membrane K permeability.     

Effect of HCO(3)(-) on TPA- and IBMX-induced anion conductances in Necturus gallbladder epithelial cells

Effects of HCO₃ on protein kinase C (PKC)-and protein kinase A (PKA)-induced anion conductances were investigatedin Necturus gallbladder epithelial cells. InHCO₃-free media, activation of PKC via12-O-tetradecanoylphorbol 13-acetate (TPA) depolarizedapical membrane potential (V_a) and decreased fractional apical voltage ratio (F_R). These effects wereblocked by mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid(NPPB), a Cl channel blocker. In HCO₃media, TPA induced significantly greater changes inV_a and F_R. These effects wereblocked only when NPPB was present in both mucosal and basolateralcompartments. The data suggest that TPA activates NPPB-sensitive apicalCl conductance (g_Cl^a) in theabsence of HCO₃; in its presence, TPA stimulated bothNPPB-sensitive g_Cl^a and basolateralCl conductance (g_Cl^b).Activation of PKA via 3-isobutyl-1-methylxanthine (IBMX) also decreased V_a and F_R; however, thesechanges were not affected by external HCO₃. Weconclude that HCO₃ modulates the effects of PKC ong_Cl^b. In HCO₃ medium, TPAand IBMX also induced an initial transient hyperpolarization andincrease in intracellular pH. Because these changes were independent ofmucosal Na⁺ and Cl, it is suggested that TPAand IBMX induce a transient increase in apical HCO₃ conductance.

     

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia

It has been reported thatsecretory mammary epithelial cells (MEC) release ATP, UTP, and UDP uponmechanical stimulation. Here we examined the physiological changescaused by ATP/UTP in nontransformed, clonal mouse mammary epithelia(31EG4 cells). In control conditions, transepithelial potential (apicalside negative) and resistance were 4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4  · cm², respectively. The apicalmembrane potential was 43.9 ± 1.7 mV, and the ratio of apicalto basolateral membrane resistance (R_A/R_B) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranescaused large voltage and resistance changes with an EC₅₀ of~24 µM (apical) and ~30 µM (basal). Apical ATP/UTP (100 µM)depolarized apical membrane potential by 17.6 ± 0.8 mV (n = 7) and decreasedR_A/R_B by a factor of3. The addition of adenosine to either side (100 µM) hadno effect on any of these parameters. The ATP/UTP responses werepartially inhibited by DIDS and suramin and mediated by a transientincrease in free intracellular Ca²⁺ concentration (427 ± 206 nM; 15-25 µM ATP, apical; n = 6). This Ca²⁺ increase was blocked by cyclopiazonic acid, by BAPTA,or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbedfluid in the resting state, and ATP or UTP increased fluid secretion by5.6 ± 3 µl · cm² · h¹(n = 10). Pharmacology experiments indicate that 31EG4epithelia contain P2Y₂ purinoceptors on the apical andbasolateral membranes, which upon activation stimulate apicalCa²⁺-dependent Cl channels and cause fluid secretion acrossthe monolayer. This suggests that extracellular nucleotides could playa fundamental role in mammary gland paracrine signaling and theregulation of milk composition in vivo.

     

Estrogen modulates paracellular permeability of human endothelial cells by eNOS- and iNOS-related mechanisms

Estradiol had abiphasic effect on permeability across cultures of human umbilical veinendothelial cells (HUVEC): at nanomolar concentrations it decreased theHUVEC culture permeability, but at micromolar concentrations itincreased the permeability. The objective of the present study was totest the hypothesis that the changes in permeability were mediated bynitric oxide (NO)-related mechanisms. The results revealed dualmodulation of endothelial paracellular permeability by estrogen.1) An endothelial NO synthase (eNOS)-, NO-, and cGMP-related,Ca²⁺-dependent decrease inpermeability was activated by nanomolar concentrations of estradiol,resulting in enhanced Clinflux, increased cell size, and increases in the resistance of thelateral intercellular space(R_LIS) and inthe resistance of the tight junctions(R_TJ); theseeffects appeared to be limited by the ability of cells to generate cGMPin response to NO. 2) An inducibleNO synthase (iNOS)- and NO-related,Ca²⁺-independent increase inpermeability was activated by micromolar concentrations of estradiol,resulting in enhanced Clefflux, decreased cell size, and decreasedR_LIS andR_TJ. We conclude that the net effect on transendothelial permeability across HUVEC depends on the relative contributions of each of these two systems tothe total paracellular resistance.     

Luminal L-alanine stimulates exocytosis at the K+-conductive apical membrane of Aplysia enterocytes

In Aplysia intestine,stimulation of Na⁺ absorption withluminal alanine increases apical membraneK⁺ conductance(G_K,a), whichpresumably regulates enterocyte volume during stimulatedNa⁺ absorption. However, themechanism responsible for the sustained increase in plasma membraneK⁺ conductance is not known forany nutrient-absorbing epithelium. In the present study, we have begunto test the hypothesis that the alanine-induced increase inG_K,a inAplysia enterocytes results fromexocytic insertion of K⁺ channelsinto the apical membrane. We used the fluid-phase marker horseradishperoxidase to assess the effect of alanine on apical membraneexocytosis and conventional microelectrode techniques to assess theeffect of alanine on fractional capacitance of the apical membrane(fC_a). Luminalalanine significantly increased apical membrane exocytosis from 1.04 ± 0.30 to 1.39 ± 0.38 ng · min¹ · cm².To measure fC_a,we modeled the Aplysia enterocyte as adouble resistance-capacitance (RC) electric circuit arranged in series. Several criteria were tested to confirm application of the model to theenterocytes, and all satisfied the model. When added to the luminalsurface, alanine significantly increasedfC_a from 0.27 ± 0.02 to 0.33 ± 0.04 (n = 10)after 4 min. There are two possible explanations for our findings:1) the increase in exocytosis, whichadds membrane to the apical plasma membrane, prevents plasma membranefracture, and 2) the increase inexocytosis delivers K⁺ channels tothe apical membrane by exocytic insertion. After the alanine-induceddepolarization of apical membrane potential (V_a), there isa strong correlation (r = 0.96)between repolarization ofV_a, whichreflects the increase inG_K,a, andincrease in fC_a. This correlation supports the exocytic insertion hypothesis for activation ofG_K,a.

     

Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO₃ exchange as the mechanism for ³⁶Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of ³⁶Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO₃ exchange. In the absence of voltage clamping, both outward OH gradient (pH_out/pH_in: 7.5/5.5) and outward HCO₃ gradient stimulated transient ³⁶Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven ³⁶Cl uptake, whereas HCO₃ gradient-driven ³⁶Cl uptake was only partially inhibited (38%). Both electroneutral HCO₃ and OH gradient-driven ³⁶Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant (K_i) of 26.9 and 30.6 µM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a K_m for Cl of 12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pH_i) was also determined with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pH_i both in HCO₃-buffered and in the nominally HCO₃-free buffered solutions (0.28 ± 0.02 and 0.11 ± 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO₃ exchange is present in basolateral membranes and that, in the absence of HCO₃, Cl-HCO₃ exchange can function as a Cl-OH exchange and regulate pH_i across basolateral membranes of rat distal colon. crypt glands; superfusion; intracellular pH; membrane vesicles; ³⁶Cl uptake; Cl-anion exchange     

The Movement of Ions to the Xylem Exudate of Maize Roots: II. A COMPARISON OF THE ELECTRICAL POTENTIAL AND ELECTROCHEMICAL POTENTIALS OF IONS IN THE EXUDATE AND IN THE ROOT CELLS

The electrical potential differences (E_ex) and the electrochemicalpotential differences for potassium and chloride in the xylemexudate of excised maize roots were determined and the valuesobtained indicated active transport of both ions to the xylemexudate. The potential differences were depolarized by increasesin the external KCl concentration ([KCl]_o), and E_ex was a linearfuction of log[KCl]_o. The corresponding data for the cell vacuolesof the same roots were also determined and trends similar tothose found for the exudate were obtained. The electrochemicalpotential differences between the cell vacuoles and the externalsolution for potassium (µ_rac.k) and chloride (µ_vac.Cl)indicated that both ions were actively transported to the vacuoles.Both µ_vac.k and µ_vac.cl decreased in a linear manneras [KCl]_o was increased. However, in spite of the similar trendsin both sets of data, the values of the electrical potentialand chloride electrochemical potential differences were alwaysmarkedly larger for the vacuoles than for the exudate. For potassium,there were no significant differences. It is concluded thatthe discrepancy between the data for the vacuoles and the wholeroots does not support the idea that the exuding root systembehaves like a single cell, as has been suggested by some workers.     

Cl- secretory effects of EBIO in the rabbit conjunctival epithelium

Experiments were conducted to determine whether the Cl^– secretagogue, 1-ethyl-2-benzimidazolinone (EBIO), stimulates Cl^– transport in the rabbit conjunctival epithelium. For this study, epithelia were isolated in an Ussing-type chamber under short-circuit conditions. The effects of EBIO on the short-circuit current (I_sc) and transepithelial resistance (R_t) were measured under physiological conditions, as well as in experiments with altered electrolyte concentrations. Addition of 0.5 mM EBIO to the apical bath stimulated the control I_sc by 64% and reduced R_t by 21% (P < 0.05; paired data). Under Cl^–-free conditions, I_sc stimulation using EBIO was markedly attenuated. In the presence of an apical-to-basolateral K⁺ gradient and permeabilization of the apical membrane, the majority of the I_sc reflected the transcellular movement of K⁺ via basolateral K⁺ channels. Under these conditions, EBIO in combination with A23187 elicited nearly instantaneous 60–90% increases in I_sc that were sensitive to the calmodulin antagonist calmidazolium and the K⁺ channel blocker tetraethyl ammonium. In the presence of an apical-to-basolateral Cl^– gradient and nystatin permeabilization of the basolateral aspect, EBIO increased the Cl^–-dependent I_sc, an effect prevented by the channel blocker glibenclamide (0.3 mM). The latter compound also was used to determine the proportion of EBIO-evoked unidirectional ³⁶Cl^– fluxes in the presence of the Cl^– gradient that traversed the epithelium transcellularly. Overall, EBIO activated apical Cl^– channels and basolateral K⁺ channels (presumably those that are Ca²⁺ dependent), thereby suggesting that this compound, or related derivatives, may be suitable as topical agents to stimulate fluid transport across the tissue in individuals with lacrimal gland deficiencies. Ussing chamber; short-circuit current; electrolyte transport; chloride secretagogue; potassium conductance; 1-ethyl-2-benzimidazolinone; 1,10-phenanthroline     

Estrogen increases the permeability of the cultured human cervical epithelium by modulating cell deformability

Estrogens increase secretion of cervical mucusin females. The objective of this research was to study the mechanismsof estrogen action. The experimental models were human CaSki(endocervical) and hECE (ectocervical) epithelial cells cultured onfilters. Incubation in steroid-free medium increased transepithelialelectrical resistance(R_TE) anddecreased epithelial permeability to the cell-impermeant acid pyranine.Estrogen treatment reversed the effects, indicating estrogen decreasesepithelial paracellular resistance. The estrogen effect was time anddose related (EC₅₀ ~1 nM) andspecific (estradiol = diethylstilbestrol > estrone, estriol; noeffect by progesterone, testosterone, or cortisol) and was blocked byprogesterone, tamoxifen, and ICI-182780 (an estrogen receptorantagonist). Estrogen treatment did not modulate dilution potential orchanges in R_TE inresponse to diC8 or to low extracellularCa²⁺ (modulators of tightjunctional resistance). In contrast, estrogen augmented decreases inR_TE in responseto hydrostatic and hypertonic gradients [modulators of resistanceof lateral intercellular space (R_LIS)],suggesting estrogen decreasesR_LIS. Estrogendecreased cervical cell size, shortened response time relative tochanges in cell size after hypertonic challenge, and augmented thedecrease in cell size in response to hypertonic and hydrostaticgradients. Lowering luminal NaCl had no significant effect onR_TE, and the Cl channel blockerdiphenylamine-2-carboxylate attenuated the hypertonicity-induced decrease in cell size to the same degree in control andestrogen-treated cells, suggesting estrogen effects on permeability andcell size are not mediated by modulatingNa⁺ orCl transport. In contrast,estrogen increased cellular G-actin levels, suggesting estrogens shiftactin steady-state toward G-actin and the cervical cell cytoskeletontoward a more flexible structure. We suggest that the mechanism bywhich estrogens decreaseR_LIS and increasepermeability is by fragmenting the cytoskeleton and facilitatingdeformability and decreases in cervical cell size.

     

Potential Dry Matter and Water Import Rates in the Tomato Fruit in Relationship to Fruit Size

The tomato fruit was compared to a sphere with a radius R. Radialgrowth rates in the fruit (F_IW and F_ID) due to water importor to dry matter import, respectively, which are also the waterimport rate or dry matter import rate per unit surface areaof fruit, were calculated from two sets of published results.This data referred to fruits which swelled in such a way thatthe availability of assimilates had little effect on growth.Two varieties differentiated the two series of results and inone series, three trials were differentiated by the salinityof the nutrient solution. In all trials, it was found that F_IW and F_ID decreased whenR increased. Two phases were observed for F_IW: after a firstphase, F_IW decreased more quickly and almost linearly when Rincreased. F_ID was constant or decreased with respect to R.Except at the beginning of growth at the greatest salinity,there were clearly linear regressions between F_IW and F_ID suchas F_ID = aF_IW-b; where b was lower with higher salinity. Thechanges of the concentration of imported dry matter (F_ID/F_IWwere examined in terms of R and F_IW/R. The mechanisms controllingthe changes in F_IW and F_ID were discussed. The results suggestedfruit radius was an important parameter of these mechanisms.Thus, water import rate and dry matter import rate could eachbe considered to be the product of two factors: fruit surfacearea, which is directly dependent on fruit radius, and waterimport rate or dry matter import rate per unit of fruit surfacearea.Copyright 1993, 1999 Academic Press Dry matter, fruit growth, logistic model, sink size, tomato, water transfer     

Regulation of water permeability in rabbit conjunctival epithelium by anisotonic conditions

Effects of unilateral exposure to anisotonic conditions on diffusional water permeability of the isolated rabbit conjunctiva were determined. A segment of the bulbar-palpebral conjunctiva was mounted between Ussing-type hemichambers under short-circuit conditions. Unidirectional water fluxes (J_dw) were measured in either direction by adding ³H₂O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously. J_dw were determined under control isosmotic conditions and after introduction of either hyper- or hypotonic solutions against the tear or stromal sides of the preparations. In each of these four separate conditions, the anisotonic medium produced an 20–30% reduction in J_dw across the tissue, with the exception that to obtain such reduction with increased tonicity from the stromal side (medium osmolality increased by adding sucrose), conditions presumptively inhibiting regulatory volume increase mechanisms (e.g., pretreatment with amiloride and bumetanide) were also required. All reductions in J_dw elicited by the various anisotonic conditions were reversible on restoration of control tonicity. In experiments in which preparations were pretreated with the protein cross-linking agent glutaraldehyde, anisotonicity-elicited reductions in J_dw were not observed. Such reductions were also not observed in the presence of HgCl₂, implying the involvement of aquaporins. However, it is possible that the mercurial may be toxic to the epithelium, preventing the tonicity response. Nevertheless, from concomitant changes in transepithelial electrical resistance, as well as [¹⁴C]mannitol fluxes, [¹⁴C]butanol fluxes, and Arrhenius plots, arguments are presented that the above effects are best explained as a cell-regulated reduction in membrane water permeability that occurs at the level of water-transporting channels. Presumably both apical and basolateral membranes can downregulate their water permeabilities as part of a protective mechanism to help maintain cell volume. unidirectional water fluxes; net water fluxes; Ussing chamber; short-circuit current; electrolyte transport; cell volume regulation; paracellular mannitol permeability     

Membrane cholesterol extraction decreases Na+ transport in A6 renal epithelia

In this study, we have investigated the dependence of Na⁺ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current (I_sc), transepithelial conductance (G_T), and transepithelial capacitance (C_T) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl--cyclodextrin (mCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mCD treatment. However, apical mCD application hampered the responses of I_sc and G_T to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in I_sc demonstrated that apical mCD treatment decreased the epithelial Na⁺ channel (ENaC) open probability (P_o) in the steady state as well as after activation of Na⁺ transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by C_T measurements. Na⁺ transport activation by hypotonicity was abolished during basolateral mCD treatment as a result of reduced Na⁺/K⁺ pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na⁺/K⁺ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na⁺ transport by reducing ENaC P_o. epithelial Na⁺ channel; Na⁺-K⁺-ATPase activity; short-circuit current; methyl--cyclodextrin; channel open probability     

Regulation of anion exchanger Slc26a6 by protein kinase C

SLC26A6 (CFEX, PAT1) is an anion exchanger expressed in several tissues including renal proximal tubule, pancreatic duct, small intestine, liver, stomach, and heart. It has recently been reported that PKC activation inhibits A6-mediated Cl/HCO₃ exchange by disrupting binding of carbonic anhydrase to A6. However, A6 can operate in HCO₃-independent exchange modes of physiological importance, as A6-mediated Cl/oxalate exchange plays important roles in proximal tubule NaCl reabsorption and intestinal oxalate secretion. We therefore examined whether PKC activation affects HCO₃-independent exchange modes of Slc26a6 functionally expressed in Xenopus oocytes. We found that PKC activation inhibited Cl/formate exchange mediated by Slc26a6 but failed to inhibit the related anion exchanger pendrin (SLC26A4) under identical conditions. PKC activation inhibited Slc26a6-mediated Cl/formate exchange, Cl/oxalate exchange, and Cl/Cl exchange to a similar extent. The inhibitor sensitivity profile and the finding that PMA-induced inhibition was calcium independent suggested a potential role for PKC-. Indeed, the PKC--selective inhibitor rottlerin significantly blocked PMA-induced inhibition of Slc26a6 activity. Localization of Slc26a6 by immunofluorescence microscopy demonstrated that exposure to PKC activation led to redistribution of Slc26a6 from the oocyte plasma membrane to the intracellular compartment immediately below it. We also observed that PMA decreased the pool of Slc26a6 available to surface biotinylation but had no effect on total Slc26a6 expression. The physiological significance of these findings was supported by the observation that PKC activation inhibited mouse duodenal oxalate secretion, an effect blocked by rottlerin. We conclude that multiple modes of anion exchange mediated by Slc26a6 are negatively regulated by PKC- activation. oxalate; formate; chloride; duodenum