Ole Frederiksen

Sensitive osmometer function of juxtaglomerular cells in vitro.

The rate of renin release from viable juxtaglomerular cells was studied during prolonged superfusion of isolated rat renal glomeruli with Ringer solutions of differing osmolarities. 2. Reduction in osmolarity from 305 to 285 m‐osmole/l. by lowering sucrose concentration caused renin release rate to double. A rise in osmolarity of 30 m‐osmole/l. by raising sucrose concentration halved release rate. 3. The response to osmolarity was graded. During the first 30 min following a 20 m‐osmole/l. decrease in osmolarity, 1‐57 +/‐ 0‐22% (S.E. of mean) of cellular renin content was released; three times this amount was released with a decrease of 50 m‐osmole/l. The effect persisted at lower release rates for 60‐90 min. 4. The juxtaglomerular cells were four to five times more sensitive to changes in osmolarity through sucrose than sodium chloride concentration. Changes in potassium chloride concentration (7‐57 mM) had little effect. 5. Sodium chloride had no direct ionic effect on renin release outside its osmotic properties. 6. The findings support a previous proposal that the rate of renin release in vitro relates directly to the volume of the juxtaglomerular cell. The hypothesis is developed that a similar mechanism may underlie renin secretion in vivo.

Transcellular transport of isosmotic volumes by the rabbit gall‐bladder in vitro

1. Fluid transport rate and oxygen consumption (QO2) were studied in rabbit gall‐bladder preparations in vitro exposed on both sides to identical Ringer solutions with NaCl concentrations (and osmolarities) varying from 70 to 140 m‐equiv Na+/l.).

Ion transport in epithelial spheroids derived from human airway cells

In the present study, we describe a novel three-dimensional airway epithelial explant preparation and demonstrate its use for ion transport studies by electrophysiological technique. Suspension cultures of sheets of epithelial cells released by protease treatment from cystic fibrosis (CF) and non-CF nasal polyps developed free-floating, monolayered epithelial spheres, with the apical, ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. Microelectrode impalement of both non-CF and CF spheroids revealed lumen-positive transepithelial electrical potential differences (PDs) that were inhibited by amiloride, indicating that the spheroids were inflated due to amiloride-sensitive Na+ absorption followed by water. Transformation to a Cl- secretory state was achieved by addition of ATP to the bath, leading to the development of a diphenylamine-2-carboxylate-sensitive PD. A cAMP-induced increase in PD was seen in non-CF spheroids only. In response to hydrocortisone treatment, Na+ transport reflected by amiloride-sensitive PD increased and more so in CF than in non-CF spheres. We concluded that this preparation is a useful model for the airway surface epithelium and is suitable for studies of transport mechanisms and regulation.In the present study, we describe a novel three-dimensional airway epithelial explant preparation and demonstrate its use for ion transport studies by electrophysiological technique. Suspension cultures of sheets of epithelial cells released by protease treatment from cystic fibrosis (CF) and non-CF nasal polyps developed free-floating, monolayered epithelial spheres, with the apical, ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. Microelectrode impalement of both non-CF and CF spheroids revealed lumen-positive transepithelial electrical potential differences (PDs) that were inhibited by amiloride, indicating that the spheroids were inflated due to amiloride-sensitive Na+absorption followed by water. Transformation to a Cl- secretory state was achieved by addition of ATP to the bath, leading to the development of a diphenylamine-2-carboxylate-sensitive PD. A cAMP-induced increase in PD was seen in non-CF spheroids only. In response to hydrocortisone treatment, Na+ transport reflected by amiloride-sensitive PD increased and more so in CF than in non-CF spheres. We concluded that this preparation is a useful model for the airway surface epithelium and is suitable for studies of transport mechanisms and regulation.

Fluid absorption related to ion transport in human airway epithelial spheroids

Airway epithelium explants from cystic fibrosis (CF) patients and non-CF subjects formed monolayered spheres, with the apical ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. With the use of two microelectrodes, transepithelial potential difference and changes in potential difference in response to passage of current pulses were recorded, and epithelial resistance and the equivalent short-circuit current were calculated. Non-CF control potential difference and short-circuit current values were significantly lower than the CF values, and amiloride inhibited both values. Fluid transport rates were calculated from repeated measurements of spheroid diameters. The results showed that 1) non-CF and CF spheroids absorbed fluid at identical rates (4.4 μl ⋅ cm-2 ⋅ h-1), 2) amiloride inhibited fluid absorption to a lower residual level in non-CF than in CF spheroids, 3) Cl--channel inhibitors increased fluid absorption in amiloride-treated non-CF spheroids to a level equal to that of amiloride-treated CF spheroids, 4) hydrochlorothiazide reduced the amiloride-insensitive fluid absorption in both non-CF and CF spheroids, and 5) osmotic water permeabilities were equal in non-CF and CF spheroids (∼27 × 10-7cm ⋅ s-1 ⋅ atm-1).

The effect of some lissamine greens on gall bladder fluid absorption and frog skin sodium transportin vitro

Summary1.The effects of three well-defined, but chemically different, Lissamine Greens (LGs) on sodium transport across isolated skins of Rana temporaria and on transcellular fluid transport across rabbit gall bladder sac preparationsin vitro were investigated.2.In the frog skin either of the three LGs (LG 240 SF, LG 162 B, and LG 280 V) reduced short circuit current (SCC) and electrical potential difference (PD) about 80% when applied to the epithelial side at a concentration of 400 mg-%. The effect was time dependent, and in the case of LG 162 B and LG 280 V it was partly reversible. When applied to the corium side at the same concentration the effect was less pronounced (LG 240 SF: about 70%; LG 280 V: about 30%) but more completely reversible; in the case of LG 280 V SCC even increased above control values in the recovery period.3.Gall bladders were exposed to LGs bilaterally at a concentration of 400 mg-% on each side. All three types of LGs inhibited net fluid transfer when the spontaneous (control) transport rate was above a certain threshold level; this level apparently differed for each of the LGs. Above the threshold levels the degree of inhibition increased with increasing control transport rate. Thus, the inhibitory action seemed to be dependent on the type of LG used and the functional state of the epithelium. The effect was time dependent and partly reversible.4.It is concluded that the effects of Lissamine Greens on transport depend essentially on 1. the chemical composition of the dyes and 2. the type of epithelium investigated.

Comparative Aspects of Actions of a Short-Chain Phospholipid on Epithelial Na+ Channels and Tight Junction Conductance

Ion transport in both the frog skin (a high-resistance epithelium) and the rabbit nasal airway epithelium (a low-resistance epithelium) are dominated by electrogenic Na+ absorption via apical membrane amiloride-sensitive Na+ channels, and short-circuit current (ISC) is essentially a measure of Na+ absorption in both epithelia. In both epithelia, mucosal application of the short-chain phospholipid didecanoyl-L-alpha-phosphatidylcholine (DDPC) dose-dependently inhibited the amiloride-sensitive ISC and caused an initial decrease in epithelial conductance (Gt) followed by an increase in Gt to steady-state values above control level. The effects were reversible. It is concluded that DDPC (a) inhibits epithelial amiloride-sensitive Na+ channels and (b) induces an increase in paracellular tight junction conductance. These effects may involve changes in non-specific lipid-protein interactions at the cell membrane level.

Water permeability in human airway epithelium

Osmotic water permeability (Pf) was studied in spheroid-shaped human airway epithelia explants derived from nasal polyps by the use of a new improved tissue collection and isolation procedure. The fluid-filled spheroids were lined with a single cell layer with the ciliated apical cell membrane facing the outside. They were capable of surviving hours of experiment involving continuous superfusion of the bathing medium and changes of osmolarity. A new image analysis technique was developed for measuring the spheroid diameters, giving high time and measurement resolutions. The transepithelial Pf, determined by the changes of the apical solution osmolarity, was not influenced by the presence of glucose, Na+, or Na+/glucose-cotransport inhibitors in the bath, but was sensitive to the aquaporin (AQP) inhibitor HgCl2. The measured Pf levels and the values of activation energy were in the range of those seen in AQP-associated water transport. Together, these results indicate the presence of an AQP in the apical membrane of the spheroids. Notably, identical values for Pf were found in CF and non-CF airway preparations, as was the case also for the calculated spontaneous fluid absorption rates.

INTERFERENCE OF A SHORT-CHAIN PHOSPHOLIPID WITH ION TRANSPORT PATHWAYS IN FROG SKIN

Abstract The effects of mucosal application of the short-chain phospholipid didecanoyl-L-α-phosphatidylcholine (DDPC; with two saturated 10-carbon acyl chains) on active Na+ transport and transepithelial conductance (G) in the frog skin (Rana temporaria) were investigated. Active Na+ transport was measured as the amiloride-sensitive short-circuit current (ISC) and G was determined from transepithelial voltage-clamp pulses under short-circuit conditions. DDPC dose-dependently inhibited ISC with an ID50 of about 0.05% (w/v) and a maximal effect (≈55%) at ≥ 1% DDPC. G increased to steady-state values above control level. Simultaneously, equal increases in unidirectional sucrose permeabilities (PSu; measured from [14C]sucrose fluxes) were observed, and a positive correlation was demonstrated between DDPC-induced changes in PSu and G. Since amiloride did not prevent the increase in G by DDPC, these results suggest that the DDPC-induced increase in G represents an increase in the paracellular shunt conductance. The effects of mucosal DDPC were almost fully reversible within 8 h. The results indicate that DDPC inhibits amiloride-sensitive Na+ channels in the apical membrane of the frog skin epithelium and opens a paracellular tight junction pathway. Both effects may be caused by incorporation of DDPC in the apical cell membrane.