Deposition of BaSO4 in the tight junctions of amphibian epithelia causes their opening; apical Ca2+ reverses this effect

Selective deposition of BaSO₄ in the tight junctions (TJs) of frog skins led to profound and reversible functional alterations of these structures, as revealed by changes of tissue conductance (G), clamping current (I), and fluxes of extracellular markers (sulfate (J^SO ₄) and sucrose (J^SUC)). Experiments were performed with nominally Ca²⁺ -free simple salt solutions on the apical side (usually KCl) and Na₂SO₄-Ringer on the inner side of skins. The deposition of BaSO₄ in the TJs was obtained by diffusion and/or migration through the paracellular path of Ba²⁺ from the apical solution and SO ₄ ^2– from the inner solution. A brief presence (2 to 6 min) of apical Ba²⁺ (Ba²⁺ pulse) is followed (i.e., when Ba²⁺ is removed from the apical fluid) by a large increase of G, I, J^SO ₄ and J^SUC, above pre-Ba²⁺ levels. These attain a steady state within 15 to 30 min (overshoot phase), characterizing a conspicuous increase of the paracellular permeability. During the overshoot phase, a second Ba²⁺ pulse blocks the paracellular route while apical Ba²⁺ is present, leading to a new and larger overshoot when the Ba²⁺ pulse is terminated. Addition of apical Ca²⁺ triggers the resealing of the TJs, resulting in a full recovery of G, I, J^SO ₄ and J^SUC. This Ca²⁺ -induced recovery persists when apical Ca²⁺ is removed. The presence of a normal Ca²⁺ concentration in the inner bathing Ringer does not induce the recovery process. Tissues remain viable after being submitted to the Ba²⁺ treatment and the subsequent overshoot. Experiments performed in the urinary bladder of Rana catesbeiana and skins and urinary bladders of Bufo marinus indicate that Ba²⁺ effect can also be elicited in these tissues. The above results seem to report general properties of the TJs. Incidentally, they warn about the use of Ba²⁺ as an ion channel blocker in epithelial membranes in association with SO ₄ ^2– -containing solutions on the contralateral side.This project was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (91/0293-7 to F.L.V., and 90/1788-1 to A.S.), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (410068/90-0 and 303633-85/BF to F.L.V.). J.A.C. received a doctoral fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Fundação Universidade do Rio Grande. We thank Dr. Alice T. Ferreira for help in the measurements of free Ca²⁺ concentration.     

Comparison of label-free and GFP multiphoton imaging of hair follicle-associated pluripotent (HAP) stem cells in mouse whiskers

Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into many cell types, including neurons and heart muscle cells, and have been shown to repair peripheral nerves and the spinal cord in mice. HAP stem cells can be obtained from each individual patient for regenerative medicine which overcomes problems with immune rejection. Previously, we have demonstrated that genetically-encoded protein markers such as GFP in transgenic mice can be used to visualize HAP stem cells in vivo by multiphoton tomography. Detection and visualization of stem cells in vivo without exogenous labels such as GFP would be important for human application. In the present report, we demonstrate label-free visualization of hair follicle stem cells in mouse whiskers by multiphoton tomography due to the intrinsic fluorophores such as NAD(P)H/flavins. We compared multiphoton tomography of GFP-labeled HAP stem cells and unlabeled stem cells in isolated mouse whiskers. We show that observation of HAP stem cells by label-free multiphoton tomography is comparable to detection using GFP-labeled stem cells. The results described here have important implications for detection and isolation of human HAP stem cells for regenerative medicine.     

Insulin-releasing and cytotoxic properties of the frog skin peptide,tigerinin-1R: a structure–activity study

The frog skin host-defense peptide tigerinin-1R (RVCSAIPLPICH.NH₂) is insulinotropic both in vitro and in vivo. This study investigates the effects on insulin release and cytotoxicity of changes in cationicity and hydrophobicity produced by selected substitutions of amino acids by l-arginine, l-lysine and l-tryptophan. The [A5W], [L8W] and [I10W] analogs produced a significant (P < 0.01) increase in the rate of insulin release from BRIN-BD11 rat clonal β cells at concentration of 0.01 nM compared with 0.1 nM for tigerinin-1R. The increase in the rate of insulin release produced by a 3 μM concentration of the [S4R], [H12K], and [I10W] analogs from both BRIN-BD11 cells and mouse islets was significantly greater (P < 0.05) than that produced by tigerinin-1R. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM indicating that plasma membrane integrity had been preserved. [A5W] tigerinin-1R was the only analog tested that showed cytotoxic activity against human erythrocytes (LC₅₀ = 265 ± 16 μM) and inhibited growth of Escherichia coli (MIC = 500 μM) and Staphylococcus aureus (MIC = 250 μM). The circular dichroism spectra of tigerinin-1R and [A5W] tigerinin-1R indicate that the peptides adopt a mixture of β-sheet, random coil and reverse β-turn conformations in 50% trifluoroethanol/water and methanol/water. Administration of [S4R] tigerinin-1R (75 nmol/kg body weight) to high-fat fed mice with insulin resistance significantly (P < 0.05) enhanced insulin release and improved glucose tolerance over a 60 min period following an intraperitoneal glucose load. The study supports the claim that tigerinin-1R shows potential for development into novel therapeutic agents for treatment of type 2 diabetes mellitus.     

Roles of external and cellular Cl− ions on the activation of an apical electrodiffusional Cl− pathway in toad skin

Summary This study is concerned with the short-circuit current,I _sc, responses of the Cl^–-transporting cells of toad skin submitted to sudden changes of the external Cl^– concentration. [Cl]₀. Sudden changes of [Cl]₀, carried out under apical membrane depolarization, allowed comparison of the roles of [Cl]₀ and [Cl]_cell on the activation of the apical Cl^– pathways. Equilibration of shortcircuited skins symmetrically in K-Ringer's solutions of different Cl^– concentrations permitted adjustment of [Cl]_cell to different levels. For a given Cl^– concentration (in the range of 11.7 to 117mm) on both sides of a depolarized apical membrane, this structure exhibits a high Cl^– permeability,P _(Cl)apical. On the other hand, for the same range of [Cl]_cell but with [Cl]₀=0,P _(Cl)apical is reduced to negligible values. These observations indicate that when the apical membrane is depolarizedP _(Cl)apical is modulated by [Cl]₀; in the absence of external Cl^– ions, intracellular Cl^– is not sufficient to activateP _(Cl)apical. Computer simulation shows that the fast Cl^– currents induced across the apical membrane by sudden shifts of [Cl]₀ from a control equilibrium value strictly follow the laws of electrodiffusion. For each experimental group, the computer-generatedI _sc versus ([Cl]_cell–[Cl]₀) curve which best fits the experimental data can only be obtained by a unique pair ofP _(Cl)apical andR _b (resistance of the basolateral membrane), thus allowing the calculation of these parameters. The electrodiffusional behavior of the net Cl^– flux across the apical membrane supports the channel nature of the apical Cl^– pathways in the Cl^–-transporting cell. Cl^– ions contribute significantly to the overall conductance of the basolateral membrane even in the presence of a high K concentration in the internal solution.     

Trypanosoma cruzi: in vitro interactions between cultured amastigotes and human skin-muscle cells.

Established cultures of human skin-muscle cells were used for determining the parasite—host cell relationship of Trypanosoma cruzi amastigotes (12–16 passages) cultured in a cell-free medium (F-69) at 37 C. The medium used for this experiment was tissue culture fluid M-199 enriched with 10% fetal bovine serum and relatively high concentrations of ATP, ADP and AMP. Amastigotes entered skin-muscle cells incubated at 32 or 35 C, multiplied and completed their intracellular life cycle in about 7 days. At 35 C, 23.6% of cells became infected in 7 days and at 32 C, 43.6% were infected in 5 days. The higher infection rate of cultured cells at 32 C was probably due to more frequent and prolonged cell-parasite contact, as amastigotes multiplied in the tissue culture medium and remained viable for a longer period at the lower temperature. As a control, epimastigotes were used to infect skinmuscle cells. Epimastigotes transformed into metacyclic trypomastigotes before entering host cells, multiplied, and completed the intracellular life cycle. We conclude that the amastigotes cultured in F-69 at 37 C are biologically similar to intracellular amastigotes from the vertebrate host, in that both can multiply and complete the life cycle intracellulary.     

A tracer method to study unidirectional fluxes of lithium: Application to frog skin

We describe a new tracer method to measure unidirectional fluxes of Li⁺, despite the lack of any utilizable radioisotope of lithium. This method uses the purified stable isotopes, ⁶Li and ⁷Li, detected with an ion-probe microanalyser. The accuracy is comparable to that obtained for other ions (e.g., Na⁺) with radiotracers.The method has been applied to frog skin with both faces bathed in a 20% lithium/80% sodium medium. Sodium and lithium unidirectional fluxes have been measured simultaneously. The results are consistent with lithium being actively pumped, the outflux of lithium being, however, much larger than that of sodium.     

Time course of pump inhibition by ouabain in amphibian epithelia

Inhibition by ouabain of rheogenic Na⁺ transport across the basolateral membranes of frog skin is found to be manifest within 3–4 min. This rate of pump inhibition is not different from the rate of diffusion through extracellular tissue layers between the serosal bath and the actual site of action, i.e., the epithelial cell layers. It is concluded that the well-known slow time course of decrease in transepithelial current flow is due ionic redistribution and conductance changes of the epithelial membranes secondary to pump inhibition.     

Contact sensitization: A new approach to risk assessment

The murine local lymph node assay is a novel predictive test for the identification of skin sensitizing chemicals. The method measures sensitization potential of a chemical in mice as a function of proliferative activity induced in lymph nodes draining the site of topical exposure to that test chemical. Here we describe the use of the local lymph node assay for evaluation of the relative potency of skin sensitizing chemicals via derivation of the concentration required to produce a threshold positive reaction. Subsequently, the development of risk assessments based on comparisons with index contact allergens is outlined.     

Single-file diffusion through K+ channels in frog skin epithelium

Summary The ratio between the unidirectional fluxes of K⁺ across the frog skin with K-permeable outer membranes was determined in the absence of Na⁺ in the apical solutions. The experiments were performed under presteady-state conditions to be able to separate the flux ratio for K⁺ through the cells from contributions to the fluxes through extracellular leaks. The cellular flux ratio deviated strongly from the value calculated from the flux ratio for electrodiffusion. The experiments can be explained if the passive K transport through the epithelial cells proceeds through specific channels by single-file diffusion with a flux ratio exponent of about 2.5.