Rescue of murine silica-induced lung injury and fibrosis by human embryonic stem cells.

Alveolar type II pneumocytes (ATII cells) are considered putative alveolar stem cells. Since no treatment is available to repair damaged epithelium and prevent lung fibrosis, novel approaches to induce regeneration of injured alveolar epithelium are desired. The objective of this study was to assess both the capacity of human embryonic stem cells (HUES-3) to differentiate in vitro into ATII cells and the ability of committed HUES-3 cells (HUES-3-ATII cells) to recover in vivo a pulmonary fibrosis model obtained by silica-induced damage. In vitro differentiated HUES-3-ATII cells displayed an alveolar phenotype characterised by multi-lamellar body and tight junction formation, by the expression of specific markers such as surfactant protein (SP)-B, SP-C and zonula occludens (ZO)-1 and the activity of cystic fibrosis transmembrane conductance regulator-mediated chloride ion transport. After transplantation of HUES-3-ATII cells into silica-damaged mice, histological and biomolecular analyses revealed a significant reduction of inflammation and fibrosis markers along with lung function improvement, weight recovery and increased survival. The persistence of human SP-C, human nuclear antigen and human DNA in the engrafted lungs indicates that differentiated cells remained engrafted up to 10 weeks. In conclusion, cell therapy using HUES-3 cells may be considered a promising approach to lung injury repair.

Association of pS2 (TFF1) release with breast tumour proliferative rate: in vitro and in vivo studies.

Although cytosolic expression of the protein pS2 (TFF1) is considered to be a marker of oestrogen receptor (OR) function, there exists some clinical data to suggest an inverse relationship of cytosolic pS2 to tumour proliferation. Although secreted from breast cancer cells, the relationship of pS2 secretion to tumour natural history has been little studied. The mechanisms and kinetics of pS2 release and its relation to tumour cell proliferation were studied in a human breast cancer cell line MCF-7 and verified in a preliminary clinical study. Stimulation by stripped serum or oestradiol resulted in parallel increases of proliferation and pS2 release in both time course and dose-response experiments. Direct pharmacological alterations of proliferation were followed by identical changes in pS2 release. The relationship between serum pS2 levels and tumour proliferative activity when analysed as a function of steroid status showed a slope of 0.56 in OR+ vs. 0.19 in OR- tumours. It is concluded that pS2 release from breast cancer cells is associated with their proliferation and measurement of serum pS2 levels might be a good predictor of tumour proliferative state and could permit noninvasive monitoring of this tumour parameter.

TGF-beta1 and IGF-1 expression are differently regulated by serum in metastatic and non-metastatic human breast cancer cells.

Transforming growth factor-beta (TGF-beta) exerts an inhibitory effect on epithelial cell proliferation while insulin-like growth factor-1 (IGF-1) is a positive regulator of proliferation and together they may participate in driving neoplastic progression. The regulation of TGF-beta1 and IGF-1 gene expression was analyzed in an in vitro model of an estrogen receptor positive (ER+), non-metastatic (MCF-7) and an (ER-), metastatic (MDA-MB-435) breast cancer cell line, respectively. Our results indicate a loss of the regulation of TGF-beta1 and the gain of the expression and upregulation of IGF-1 pathways during malignant progression. These data demonstrate that two factors, convergent on cell growth, can have divergent roles in the regulation of the expression of TGF-beta1.

NHE3 inhibits PKA-dependent functional expression of CFTR by NHERF2 PDZ interactions.

It has been shown that when CFTR and NHE3 are co-expressed on the apical membrane of the A6-NHE3 cell monolayers, the two transporters interact via a shared regulatory complex composed of NHERF2, ezrin, and PKA. We observe here that co-expression of NHE3 reduced both PKA-dependent apical CFTR expression and its activation once in place by approximately 50%. To analyze the role of NHERF2 in this process, we transfected NHE3 expressing and non-expressing A6 monolayers with NHERF2 cDNA in which its binding domains had been deleted. When only CFTR is expressed on the apical membrane, deletion of any of the NHERF2 binding domains inhibited both PKA-dependent apical CFTR expression and its activation, while when NHE3 was co-expressed with CFTR PDZ2 deletion was without effect on CFTR sorting and activity. This suggests that when the PDZ2 domain is "sequestered" by interacting with NHE3 it can no longer participate in CFTR functional expression.

Calcium role in base-line and ADH-stimulated sodium transport in frog skin.

Treatment of ventral frog skin with serosal A23187 calcium ionophore caused an initially transient increase in transepithelial sodium transport. After 60 min of treatment with A23187, a steady-state transport value was reached which was significantly lower than the initial one. Furthermore, it was found that ionophore treatment greatly inhibited the natriferic response to ADH and to 8br-cAMP. A further analysis on the possible ionophore action mechanism was carried out through pretreatment of the skin with indomethacin, very powerful prostaglandin synthesis inhibitor. In the experimental conditions reported, A23187 seems no longer capable of inducing a transient increase in sodium transport, although it does inhibit the natriferic response to ADH.

Evidence for the role of calcium in the hydrosmotic response to antidiuretic hormone in frog skin.

Treatment with the calcium ionophore A23187 on either the serosal or mucosal sides of frog skin, strongly inhibits the hydrosmotic response to vasopressin. On the contrary, the hydrosmotic response to 8-br-cAMP is not affected by treatment with the A23187. Trifluoperazine, a drug which inhibits the Ca2+-calmodulin complex, selectively inhibits vasopressin-induced water transport. Collectively, our results suggest that an increase in the intracellular concentration of Ca2+, obtained by treatment with the ionophore A23187, interferes with a pre-cAMP step of the hydrosmotic response to the antidiuretic hormone. Calcium ions could regulate adenyl-cyclase activity and consequently intracellular levels of cAMP. This effect may probably involve calmodulin.

Protective effect of hydrocortisone on vasopressin response in frog skin.

The effect of microtubular-poisons, such as colchicine and vincristine, on frog skin permeability has been investigated. Three-hour treatment with the drugs has no effect on nonelectrolyte basal transepithelial permeability, but completely suppresses the effect of ADH. Colchicine and vincristine, in addition, affect both basal sodium transport and the rise in short circuit current induced by vasopressin. The inhibition produced by microtubular-poisons disappears, however, when hydrocortisone, a glucocorticoid known to preserve junctional communications is used. Together with the results previously obtained with isolated epithelial cells (Svelto et al. 1979), these findings provide further support for our hypothesis that the microtubular-microfilament-system, is involved in cell-to-cell exchange.

Hydrosmotic response to vasopressin in frog skin. Control by endogenous factors.

The A23187 calcium ionophore strongly inhibits the hydrosmotic response to vasopressin. On the contrary neither exogenous cAMP nor theophylline-induced hydrosmotic response are affected by Ca++-ionophore. The effects obtained with A23187 suggest that calcium ions modulate vasopressin-induced osmotic water flow by interfering with a pre-cAMP step. The increase in intracellular calcium concentration induced by A23187, in fact, may inhibit the rate of cAMP formation by interfering with the vasopressin-stimulated adenylate cyclase system. This inhibition seems to be restricted to a locus proximal to the catalytic subunit of adenylate cyclase, whereas the hydrosmotic response to forskolin, a non-hormonal activator, is not affected by calcium ionophore addition. In order to clarify whether calcium ions act directly on the adenylate cyclase system or activate a more complex pathway, we studied the interaction between calcium and prostaglandins in modulating the hydrosmotic response to vasopressin. Direct measurements by radioimmunoassay of PGE2 release in the serosal medium show that A23187 notably increases PGE2 release. Furthermore, the inhibition of prostaglandin biosynthesis by hydrocortisone (a phospholipase inhibitor) or by indomethacin and naproxen (agents that inhibit arachidonic acid oxygenase) results in augmented vasopressin-stimulated water flow and prevents the inhibitory effect of the ionophore. Collectively these results strongly suggest that the effects obtained with A23187 on hydrosmotic response to ADH, are closely linked to calcium-stimulated PGE2 biosynthesis.

Phorbol ester effect on the hydrosmotic response to vasopressin in frog skin.

Serosal preincubation of frog skin with tetradecanoyl phorbol acetate, TPA, an activator of protein kinase C, inhibits the hydrosmotic response elicited by vasopressin (AVP) but not that induced by 8br-cAMP. This proves that serosal TPA primarily influences a pre-cAMP step. The TPA-induced inhibition of AVP response appears to be related to TPA-induced prostaglandin synthesis. The pretreatment with naproxen, in fact, prevents the inhibition induced by serosal TPA on the AVP response. On the contrary, mucosal TPA produces a more marked inhibition of the response to AVP and significantly diminishes the water flow induced by 8br-cAMP; this suggests that mucosal TPA interferes mainly with a post-cAMP step. Furthermore, naproxen is unable to completely prevent the inhibition induced by mucosal TPA on AVP response thus indicating that mucosal TPA may also activate a prostaglandin-independent mechanism able to inhibit one of the last steps of the hydrosmotic response to AVP.

cAMP levels and alpha and beta receptors stimulation by noradrenaline in frog skin.

The effect of noradrenaline on intracellular levels of cAMP in epithelial cells isolated from frog skin, has been studied both in the presence and in the absence of alpha (phentolamine) or beta (propranolol) adrenergic blocking agents. Such levels are increased by hormonal treatment and are linked to the stimulation of beta-adrenergic receptors, while alpha-stimulation has an inhibitory effect. Higher levels of cAMP are reached in the presence of both Ca2+ and Mg2+, confirming the existence of a Ca2+ and Mg2+-dependent adenyl-cyclase, stimulated by noradrenaline. The experimental conditions necessary to induce the highest intracellular cAMP levels, are those in which the permeabilizing effect reaches its maximum.

Effect of cycloheximide on urea facilitated transport through toad gallbladder epithelium.

Transepithelial urea outfluxes across toad gallbladder were determined before and after the addition of cycloheximide. The drug inhibits the movement of urea but has no effect on thiourea and antipyrine outfluxes. The inhibition of amide transport is time dependent as also shown in counterflow experiments. These results are consistent with the hypothesis that cycloheximide inhibits the synthesis of membrane proteic sites involved in urea mediated transport.

Facilitated transport of urea across the gall-bladder luminal membrane.

Counterflow experiments demonstrate the existence of urea counter-transport on the epithelium luminal surface. This phenomenon disappears when 10(-4) M phloretin is added to the perfusion fluid. Moreover counterflow experiments made using thiourea as elicitor, demonstrate that the phenomenon is specific for the urea.

Permeability pathways for non-electrolytes through Bufo bufo gall-bladder.

Amphotericin B treatment increases the thiourea, D-xylose and mannitol fluxes and lowers those of urea, N-methyl-urea, acetamide, formamide, and N-N'-dimethyl-thiourea. The degree of flux inhibition is related to the cellular permeability of these compounds. Most probably Amphotericin B increases the permeability of all those molecules across the luminal plasma membrane, but simultaneously elicits a cellular swelling, which reduces the diffusion across the lateral plasma membranes. This effect masks the polyene effect especially for molecules showing a mainly cellular permeation pathway such as amides and lipid soluble molecules.

Isolation of frog urinary bladder plasma membranes with polycation coated beads.

It is now generally accepted that the increase in water permeability induced by antidiuretic hormone (ADH) in responsive epithelia is accompanied by the insertion of specific structures in the apical membrane of epithelial cells. There are strong indications that these particles, probably proteic in nature, represent water channels. In order to evaluate the nature and role of such proteins, plasma membranes were isolated by the affinity chromatography technique. The method is based on the firm attachment of the external face of the membrane to polycations covalently bound to the surface of polyacrylamide beads, followed by shearing of the rest of the cells. Maximal binding of epithelial cells to beads was achieved in a medium of low ionic strength and pH 5.2 (i.e. sucrose-MES buffer). By this procedure plasma membranes were obtained from both cAMP-stimulated cells and control cells. Membranes isolated on beads were enriched in the activity of typical membrane marker enzymes (LAP; H+ ATPase; Na+, K+ ATPase) with respect to a whole cell homogenate, whereas contamination of plasma membrane fraction by endoplasmic reticulum, lysosomes, and mitochondria was relatively low. Analysis by SDS polyacrylamide gel electrophoresis showed an interesting difference between cAMP-treated and control samples.

Fluorescence labeling of proteins related to ADH-induced change in frog bladder luminal membrane.

Sulfhydryl (SH) reactive reagents, such as eosin derivatives, have been found to be useful in labeling water pathways in red cells. In the present study we used an impermeable SH-reagent, a fluorescent maleimide analogue EMA (eosin-5'-maleimide), in order to identify proteins involved in water permeability response to antidiuretic hormone (ADH). We observed that: 1) EMA (1 mM) mucosal pretreatment did not modify either the basal water flux or the subsequent ADH-induced hydrosmotic response; 2) EMA added to the mucosal bath at the maximum response to ADH, significantly decreased net water flux by about 40%; similar results were obtained when 10(-5) M forskolin was used as a hydrosmotic agent. These results suggest that the inhibitory effect of EMA occurs at a post cAMP step, possibly at the level of the sulfhydryl groups of the water channels themselves. Fluorescence distribution in SDS-PAGE of Triton X-100 extracted proteins from bladder labeled with EMA in both control conditions and under ADH stimulation allowed us to identify apical membrane proteins, labeled during ADH stimulation and not labeled in water impermeable controls. Of particular importance are four proteins of 52, 32-35, 26, 17, kDa. These polypeptides are probably involved in ADH-stimulated water transport and may be components of the water channels.

Separate regulatory control of apical and basolateral Na+/H+ exchange in renal epithelial cells.

Epithelial layers of LLC-PK1/PKE20 cells, a renal epithelial cell line which expresses Na+/H+ exchange activities in the apical as well as basolateral membrane domains, are examined in the single cell mode by microspectrofluorometry. We provide evidence that basolateral Na+/H+ exchange is more sensitive to amiloride inhibition than is apical Na+/H+ exchange. Furthermore, we demonstrate that the two exchange activities differ in their regulatory control: kinase A activation (forskolin, 8-Br-cAMP) leads to inhibition of both exchange activities, whereas kinase C activation (phorbol ester) stimulates basolateral and inhibits apical Na+/H+ exchange. Thus, renal epithelial cells may contain two Na+/H+ exchange activities: an apical ("epithelial") and basolateral ("housekeeping") which may serve different cellular functions and are under separate regulatory controls.

Evidence for a two-site model of forskolin action in frog urinary bladder.

In the present study several aspects of the osmotic water-flow-regulation mechanism in frog urinary bladder were examined, utilizing forskolin either as a direct hydrosmotic agent or in association with vasopressin. It was found that forskolin induces a hydrosmotic effect similar to the one induced by vasopressin. This effect is rapid, reversible and dose-dependent. The half-maximally effective concentration (Ec50FSK) is 1.37 microM forskolin. No additional effect on the osmotic water flow was observed when maximal concentrations of forskolin and vasopressin were given simultaneously. Moreover, forskolin can also markedly potentiate vasopressin-induced hydrosmotic response. This potentiation was maximal with submaximal doses of vasopressin, whereas it disappeared when the hormonal concentration was increased to very high levels. Therefore, forskolin increases vasopressin potency without affecting vasopressin efficacy. The Ec50FSK for the forskolin-induced increase in vasopressin potency was 11 nmol, about two orders of magnitude lower than the Ec50FSK for the direct effect of forskolin on the osmotic water transport. On the whole, our results are compatible with a two-site model of forskolin action in frog urinary bladder: a low affinity site (Ec50FSK = 1.37 microM) that mediates the direct action of forskolin on the osmotic water flow and a high affinity site (Ec50FSK = 11 nmol), which mediates the synergic effect of forskolin with the antidiuretic hormone.

[Effect of ionophore A23187 on the response to ADH in the ventral skin of Rana esculenta]. / Azione dello ionoforo A23187 sulla risposta all'ADH in pelle ventrale di Rana esculenta.

The addition of the Ca++ ionophore A23187 (10 microM) to the inside solution of the frog skin induced a transient increase in the active Na+ transport in frog skin (Rana esculenta) which decayed to the control values 60 minutes after the addition. At the same time the skin resistance failed significantly; antidiuretic hormone addition resulted in no-more increase of the Na+ active transport; the skin resistance remained unchanged. To further investigate the role of intracellular calcium on the skin transepithelial permeability, the effect of A23187 ionophore on thiourea permeability has been tested. Increase in intracellular Ca++ concentration brought about by calcium ionophores have been shown to modify both basal and ADH-stimulated thiourea transport.

Phloretin sensitive active urea absorption in frog skin.

This report presents evidence for urea active absorption by isolated skin of Rana esculenta. One of the supporting factors of such evidence is that at a low concentration the urea influx is five times greater than the outflux, in the absence of a chemical gradient. The transport shows a saturation kinetics with an apparent Km = 1.33 mM and is inhibited by un uncoupling agent (FCCP). 5 x 10(-4) M Phloretin, added to the external side, markedly inhibits inward urea transport, whereas it is ineffective when added to the serosal fluid. This provides evidence for a phloretin-sensitive mechanism located at the external side of the epithelium. Phloretin stimulates the sodium active transport; the possible coupling of urea and sodium movement is analysed.

Facilitated transport of urea across the toad gallbladder.

The toad gallbladder epithelium is much more selective than that of the rabbit especially as to the permeability of two molecules like urea and thiourea. These observations can probably be attributed to different permeation mechanisms of the 2 molecules. Neither active transport nor solvent drag can explain these phenomena. 10(-4) M phloretin strongly inhibits urea movement, but does not alter either thiourea fluxes or isotonic net water transport: these results suggest that a specific mechanism is involved in urea movement. The urea transport shows saturation kinetic which is consistent with the presence of a facilitated mechanism.