Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits.

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha- or beta-subunits of the epithelial Na(+) channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominantly subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na(+) transport. The basal level of Na(+) transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na(+) transport was 64% greater than control in both the alpha- and beta-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na(+) transport.

Assessment of the antitumour activity of targeted immunospecific albumin microspheres loaded with cisplatin and 5-fluorouracil: toxicity against a rodent ovarian carcinoma in vitro.

The 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay is used successfully to estimate the number of viable cells in drug screening trials. We used the MTT assay to assess the viability of a rodent ovarian carcinoma cell line (DMBA-OC-1R) after exposure to combinations of cisplatin and 5-fluorouracil as free drug and in encapsulated (conjugated and unconjugated) forms. After 48 h of exposure to free drugs, a significant trend towards cell cytotoxicity could be observed and this was well established by 120 h. Cells treated with drug-containing immuno-microspheres showed a similar initial decrease in cell viability after 96 h, and this was maintained for 128 h. These results suggest that immuno-microspheres loaded with chemotherapeutic drugs have the potential to be successfully used in the treatment of ovarian cancer.

cAMP-sensitive endocytic trafficking in A6 epithelia.

Blocker-induced noise analysis and laser scanning confocal microscopy were used to test the idea that cAMP-mediated vesicle exocytosis/endocytosis may be a mechanism for regulation of functional epithelial Na+ channels (ENaCs) at apical membranes of A6 epithelia. After forskolin stimulation of Na+ transport and labeling apical membranes with the fluorescent dye N-(3-triethylammoniumpropyl)4-(6-4 diethylaminophenyl) hexatrienyl pyridinium dibromide (FM 4-64), ENaC densities (N(T)) decreased exponentially (time constant approximately 20 min) from mean values of 320 to 98 channels/cell within 55 min during washout of forskolin. Two populations of apical membrane-labeled vesicles appeared in the cytosol within 55 min, reaching mean values near 18 vesicles/cell, compared with five vesicles per cell in control, unstimulated tissues. The majority of cAMP-dependent endocytosed vesicles remained within a few micrometers of the apical membranes for the duration of the experiments. A minority of vesicles migrated to >5 microm below the apical membrane. Because steady states require identical rates of endocytosis and exocytosis, and because forskolin increased endocytic rates by fivefold or more, cAMP/protein kinase A acts kinetically not only to increase rates of cycling of vesicles at the apical membranes, but also principally to increase exocytic rates. These observations are consistent with and support, but do not prove, that vesicle trafficking is a mechanism for cAMP-mediated regulation of apical membrane channel densities in A6 epithelia.

Differential effects of phorbol ester (PMA) on blocker-sensitive ENaCs of frog skin and A6 epithelia.

Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) caused complex transient perturbations of amiloride-sensitive short-circuit Na+ currents (INa) in A6 epithelia and frog skins that were tissue and concentration dependent. A noninvasive channel blocker pulse method of noise analysis (18) was used to investigate how PMA caused time-dependent changes of apical membrane epithelial Na+ channel (ENaC) single-channel currents, channel open probabilities (Po), and channel densities (NT). In A6 epithelia, 5 and 50 nM PMA caused within 7 min concentration-dependent sustained decreases of Po (approximately 55% below control, 50 nM) and rapid compensatory transient increases of NT within 7 min ( approximately 220% above control, 50 nM), resulting in either small transient increases of INa at 5 nM PMA or small biphasic decreases of INa at 50 nM PMA. In contrast to A6 epithelia, 50 and 500 nM PMA in frog skin caused after a delay of at least 10 min transient increases of NT to approximately 60-70% above control at 30-60 min. Unlike A6 epithelia, Po was increased approximately 15% above control within 7 min and remained within +/-10-15% of control for the duration of the 2-h experiments. Despite differences in the time courses of secondary inhibition of transport in A6 epithelia and frog skin, the delayed downregulation of transport was due to time-dependent decreases of NT from their preelevated levels in both tissues. Whereas Po is decreased within minutes in A6 epithelia as measured by noise analysis or by patch clamp (8), the discrepancy in regulation of NT in A6 epithelia as measured by noise analysis and patch clamp is most likely explained by the inability of on-cell patches formed before treatment of tissues with PMA to respond to regulation of their channel densities.

Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia.

To study and define the early time-dependent response (< or = 6 h) of blocker-sensitive epithelial Na+ channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current (iNa) channel open probability (Po), and channel density (NT) under transient conditions of transport as measured by macroscopic short-circuit currents (Isc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 microA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. NT varied linearly over a 70-fold range of transport (0.5-35 microA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of Po occurred during control (10-20% over 2 h) and aldosterone experimental periods (10-30% over 6 h). When the Po of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, Po was observed to vary inversely with Isc, falling from approximately 0.5 to approximately 0.15 at the highest rates of Na+ transport or approximately 25% per 3-fold increase of transport. Because decreases of Po from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane NT.

Cytochemical localization of adenylate cyclase in cultured renal epithelial (A6) cells.

To characterize the vasopressin-adenylate cyclase (AC) signaling pathway in control of Na+ reabsorption in cultured renal (A6) cells, we determined the distribution of AC with a cytochemical technique using 5'-adenylylimidodiphosphate as substrate and cerium chloride as capturing agent. The addition of forskolin to the medium to stimulate AC activity increased the production of reaction deposits at the enzyme sites. To ensure that the cells were close to their physiological states, cytochemical reactions were performed on unfixed tissues. Subsequent postfixation adequately preserved the morphological features of the cells. AC was mainly restricted to the lateral folds of the cells while the apical membranes were devoid of any deposits. This result provided evidence that the V2-AC pathway is not present in the apical membrane and, hence, any vasopressin action on apical Na+ channels from the luminal side of the cell must involve other signaling pathways. The cytochemical results provided further morphological evidence of the functional coupling between the basolateral and apical membranes of renal cells. We examined the idea that highly variable basal rates of Na+ transport in young differentiating cell cultures may be related to the degree of AC activity. Cytochemical results apparently revealed highly variable amounts of deposits in these cells, but by quantitative analysis of AC activity we could find no significant differences between cells of 6, 14, and 21 days.

Steroid hormone-dependent expression of blocker-sensitive ENaCs in apical membranes of A6 epithelia.

Weak channel blocker-induced noise analysis was used to determine the way in which the steroids aldosterone and corticosterone stimulated apical membrane Na+ entry into the cells of tissue-cultured A6 epithelia. Among groups of tissues grown on a variety of substrates, in a variety of growth media, and with cells at passages 73-112, the steroids stimulated both amiloride-sensitive and amiloride-insensitive Na+ transport as measured by short-circuit currents in chambers perfused with either growth medium or a Ringer solution. From baseline rates of blocker-sensitive short-circuit current between 2 and 7 microA/cm2, transport was stimulated about threefold in all groups of experiments. Single channel currents averaged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and were decreased 6-20% from controls by steroid due to the expected decreases of fractional transcellular resistance. Irrespective of baseline transport rates, the steroids in all groups of tissues stimulated transport by increase of the density of blocker-sensitive epithelial Na+ channels (ENaCs). Channel open probability was the same in control and stimulated tissues, averaging approximately 0.3 in all groups of tissues. Accordingly, steroid-mediated increases of open channel density responsible for stimulation of Na+ transport are due to increases of the apical membrane pool of functional channels and not their open probability.

Effects of disassembly of actin microfilaments on the AVP-induced regulation of sodium channel densities in frog skin epithelium.

Vasopressin (AVP) stimulates the absorption of Na+ across frog skin epithelium by increasing the number of open apical channels (N(o)) mainly through a large expansion of the total number of channels (NT) at the membrane. This study investigates with blocker-induced noise analysis the potential role of actin in the regulation of AVP-induced changes in channel densities. Particularly we examined the idea that actin regulates the insertion of channels into apical cell membranes, consistent with the model for its hydrosmotic action. Treating cells with cytochalasin B (CB) for 2 h to disassemble the prominent subapical actin network did not significantly alter the stimulation of the Isc by AVP. Importantly, the treatment had no significant effect on the AVP-induced activation of Na+ channels, nor on the increases in NT indicating that an intact actin network is not required for the natriferic actions on the channels. This result is disparate from well known effects of CB on the AVP-induced hydrosmotic response and we assume that these distinct responses are produced by different mechanisms. Other mechanisms need to be considered to explain the mechanism whereby new Na+ channels are recruited to the apical membrane. In particular, mechanisms of intracellular trafficking involved in the redistribution of epithelial Na+ channels remain unresolved and need to be refined.

Dual role of prostaglandins (PGE2) in regulation of channel density and open probability of epithelial Na+ channels in frog skin (R. pipiens).

Prostaglandins are important in signaling pathways involved in modulating the rates of Na+ transport in a diverse group of tissues possessing apical membrane epithelial channels. PGE2 is known to cause either stimulation, inhibition or transient stimulatory changes of Na+ transport. We have continued our studies of frog skins that are known to respond to forskolin and PGE2 with large steady-state increases of transport and have used noninvasive methods of blocker-induced noise analysis of Na+ channels to determine their channel densities (NT) and open probabilities (Po). In the absence of exogenous hormones, baseline rates of Na+ transport are especially high in scraped skins (R. pipiens pipiens) studied in the fall of the year. Na+ transport was inhibited by indomethacin and by removal of the unstirred layers of the corium (isolated epithelia) alone suggesting that PGE2 is responsible for the sustained and elevated rates of transport in scraped skins. Changes of transport caused by indomethacin, forskolin or PGE2 were unquestionably mediated by considerably larger changes of NT than compensatory changes of Po. Since cAMP caused no change of Po in tissues pretreated with indomethacin, PGE2 appears in this tissue to serve a dual role, increasing the steady state NT by way of cAMP and decreasing Po by unknown mechanisms. Despite appreciable PGE2-related decreases of Po, the net stimulation of transport occurs by a considerably greater cAMP-mediated increase of NT.

Distribution of actin microfilaments in frog skin epithelial granular cells.

Microfilaments were localised by immunofluorescence and immunogold cytochemistry to examine their distribution in granular cells of the isolated frog skin epithelium. Strongly fluorescent bundles of actin were observed beneath the plasma membrane with little evidence for actin in the central regions. Higher resolution offered by cytochemistry revealed that bundles of actin filaments comprised a substantial portion of the cortical cytoskeleton. Quantitative analysis of the frequency of gold label revealed an extremely rich array of filaments beneath the apical membrane of granular cells, with markedly less label along the basolateral membrane and in the central cytoplasm. Treating cells with cytochalasin B or arginine vasopressin caused an apparent disruption of the apical actin fibres, concurrent with a decrease in gold label density. Assumably these signs are indicative of depolymerization of the filaments. Although the significance of this distribution is unknown, the apical polarisation of actin is consistent with a role in regulating the Na+ permeability of the apical membrane. The data are discussed in relation to possible roles of the cytoskeleton in the regulation of transepithelial sodium transport by vasopressin.

Cytochemical localization of adenylate cyclase in the sodium-transporting epithelium isolated from frog skin.

A modified cytochemical technique with 5'-adenylylimidodiphosphate as substrate, was used to examine the distribution of adenylate cyclase in cells comprising the transepithelial Na+ transport pathway in isolated frog skin epithelium. Particular attention was paid to the effects of fixation on the activity and localization of adenylate cyclase. Fixation in glutaraldehyde alone or in combination with paraformaldehyde reduced the amount of reaction product, while better results were obtained using unfixed tissues. Optimum results were obtained following stimulation of adenylate cyclase with forskolin and in the presence of specific metabolic inhibitors. Adenylate cyclase was localized in the basolateral membranes of the principal cells which constitute a functional syncytium for Na+ transport and was absent from the apical membranes of the outermost granulosum cells. This distribution is consistent with the transepithelial Na+ transport model and defines the functional morphology of the cells involved in Na+ transport across frog skin. The results are compatible with the process of Na+ re-absorption across other epithelial cells, verifying that frog skin is a convenient model-tissue to study Na+ transport mechanisms. Adenylate cyclase was also found in membranes of the mitochondria-rich cells, a minor and parallel Na+ transporting pathway.

Sodium-dependent regulation of epithelial sodium channel densities in frog skin; a role for the cytoskeleton.

1. A weak electroneutral sodium channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide was used to perform noise analysis on isolated epithelium from Rana fuscigula to determine the cellular mechanism underlying autoregulation of Na+ channel densities in response to a reduction in the mucosal Na+ concentration. 2. The inherent transport rates of these tissues were generally lower than in other frog skins. The macroscopic sodium current, INa, averaged 10.71 microA/cm2 and was mainly determined by the number of open channels (N(o)) which averaged 21.6 million/cm2. The calculated mean channel open probability (beta') was 0.38, and corresponded very closely to values previously determined by patch clamp. 3. Reducing the mucosal Na+ from 110 to 10 mM caused large increases in the open channel density, which stabilized the Na+ transport rate. N(o) increased from a mean value of 26.6 to 64.3 million/cm2 within 2 min. 4. Autoregulatory changes were induced primarily by increasing beta' by about 60% and to a lesser extent by an increase in NT, the total number of open and closed channels. 5. We also examined the role of the cytoskeleton in the regulation of Na+ channel densities. Colchicine treatment, which disrupted microtubules, had no apparent effect on the ability of the tissues to autoregulate their Na+ channel densities. 6. The integrity of the microfilaments were essential for autoregulatory changes in N(o). After we had disrupted the microfilaments with cytochalasin B, we observed a marked reduction in the ability of the tissues to increase N(o). 7. The mean N(o) did not increase in response to a drop in mucosal Na+ despite the fact that beta' increased by 69%. We, therefore, assumed that cytochalasin B did not affect Na+ channels already present in the membrane but interfered with recruitment of new channels. Significantly, we did not observe any increase in NT. 8. In kidney and other tight epithelia, microfilaments are responsible for regulating the delivery of newly synthesized membrane proteins. We believe that our results with cytochalasin-treated tissues support the theory that autoregulatory changes in N(o) are also regulated by the recruitment of channels from a cytoplasmic pool.

Activation of epithelial Na channels by hormonal and autoregulatory mechanisms of action.

Methods of blocker-induced noise analysis were used to investigate the way in which forskolin and vasopressin stimulate Na transport at apical membranes of short-circuited frog skin transporting Na at spontaneous rates of transport. Experiments were done under conditions where the apical Ringer solution contained either 100 mM Na or a reduced Na concentration of 5 or 10 mM Na and buffered with either HCO3 or HEPES. Reduction of apical solution Na concentration caused a large autoregulatory increase of Na channel density (NT) similar in magnitude to that observed previously in response to blocker (amiloride) inhibition of apical membrane Na entry. Forskolin at 2.5 microM caused maximal and reversible large increases of NT, which were larger than could be elicited by 30 mU/ml vasopressin. In both the absence and presence of the autoregulatory increase of NT (caused by reduction of apical Na concentration), forskolin caused large increases of NT. Although the fractional increases of NT in response to forskolin were roughly similar, the absolute increases of NT were considerably larger in those tissues studied at reduced Na concentration and where baseline values of NT were markedly elevated by reduction of apical Na concentration. Because the effects on NT were additive, it is likely that the cAMP-dependent and autoregulatory mechanism that lead to changes of NT are distinct. We speculate that autoregulation of NT may involve change of the size of a cytosolic pool of Na-containing vesicles that are in dynamic balance with the apical membranes. cAMP-dependent regulation of NT may involve change of the dynamic balance between vesicles and the apical membranes of these epithelial cells. Alternative hypotheses cannot at present be ruled out, but will require incorporation of the idea that regulation of NT can occur both by hormonal and nonhormonal (autoregulatory) mechanisms of action.

Histological features and histochemistry of the mucous glands in ventral skin of the frog (Rana fuscigula).

The glycoconjugate components of secretory granules were analyzed in cells of mucous glands in ventral skin from Rana fuscigula. The analysis was done with standard histochemical methods on semithin glycol methacrylate-embedded tissues. The staining patterns in semithin sections were comparable to those using paraffin-embedded tissue while the cytological detail was better preserved. The mucous glands contained at least two different types of secretory cells lining the lower two-thirds of the mature gland: a principal cell type filled with dense staining secretory granules and a solitary type containing paler staining, globular secretory granules. The principal type of cell contained variable amounts of acid glycoconjugates; predominantly carboxylated but also variably carboxylated and weakly sulfated glycoproteins. Other secretory cells contained mainly neutral glycoproteins. The results indicated that the mucus is a heterogeneous substance and that one cell type may produce different secretory products. We suggested that the variability in histochemical staining might be related to the sequence of biosynthesis of the secretory granule.

Micro-electrode studies on the effects of exogenous cyclic adenosine monophosphate on active sodium transport in frog skin.

1. The electrical parameters of the sodium-transporting cells in frog skin of Rana angolensis were determined under control conditions by using the micro-electrode technique. The data were analysed in terms of an electrical model (Helman, 1979). 2. The control intracellular voltages averaged -84.7 mV while the electromotive force of the inner barrier, E'1, averaged 103.9 mV. The major portion (82%) of the transcellular resistance was situated at the outer, apical, barrier. 3. Exogenous cyclic AMP stimulated active sodium transport and the short-circuit current (Isc) increased by an average 88%. The change in Isc was mediated primarily by decreasing the resistance of the apical barrier (Ro) with little effect on the electromotive force or resistance (Ri) of the inner membranes. 4. Isoprenaline increased the Isc by an average of 165%. The major effect of isoprenaline was to decrease the apical resistance by an average 77%. 5. Forskolin (2.5 microM) stimulated the Isc by an average of 138%. Amiloride would not completely reduce the Isc, but with the low concentration of 0.2 microM-forskolin, the Isc was typically inhibited to values close to zero. The major effect of forskolin was also to reduce the resistance of the apical barrier, although it concurrently also caused the E'1 to decrease by about 13%. 6. Theophylline increased the Isc by reducing the resistance of the apical barrier by an average 61%, with little or no effect on the other parameters. Theophylline augmented the effect of cyclic AMP. 8. Our results are consistent with the theory that cyclic AMP is a second messenger in hormonal control of active sodium transport in frog skin.

Vasopressin, theophylline, PGE2, and indomethacin on active Na transport in frog skin: studies with microelectrodes.

Active transepithelial Na transport in frog skin is influenced by vasopressin, theophylline, indomethacin, and PGE2. During stimulation or inhibition of the short-circuit current, the transapical membrane voltage of short-circuited skins was recorded using an intracellular microelectrode. The microelectrode also permitted determination of the fractional resistance of the apical barrier of the cells (fRo) and the E'1 (transepithelial voltage at which the apical membrane voltage is zero). Analysis of the data according to an electrical model proposed previously indicated that changes of ISC were mediated primarily via changes of the slope resistance Rfo (Vo negative) of the apical barrier of the cells with little or no effect on the Thévenin emf or resistance of the basolateral membranes. These data are in accordance with previous observations that ADH had no effect on the ENa and are discussed in relation to the origin of the ENa at the basolateral membranes of the epithelial cells.