Isolated rat foetal distal lung epithelial (FDLE) cells were plated on Transwell clear supports and placed into plastic petri-dishes containing a 1 x 0.1cm (dia. x depth) glass-bottomed centre well. As cultures were confluent and presumably resistive, compounds applied to the interior of the Transwell support act effectively at the apical surface, whereas those applied to the exterior (ie. the petri-dish side) act at the basolateral surface. For experiments where the concentration of Cl- was altered over a range of concentrations, cells were maintained in supplemented 100mM Na+-phosphate buffer. Osmolality was maintained at 297mmol.kg-1 using mannitol and divalent cation concentrations to avoid PO4 precipitation. As experiments were carried out on an inverted Zeiss microscope the angle of electrode oscillation was in the vertical (Z)-plane; although the field of view was slightly distorted, we found that we could easily position the electrode to within a micron of the apical surface o f the monolayer, and could clearly see whether or not there was any contact with the cell. Notethat these results are confidential as they will be submitted for publication. Na+ flux experiments: Although an amiloride-inhibitable Na+ -current has been detected from monolayers maintained in Ussing chambers we were unable to detect a meaningful, regulatedNa+ fluxwith the Na+ ionophore that we used. In the interestof time, we did not pursue this further; the problem likely stemmed from the long response time of the electrode (minutes), prohibitively high background concentrations of Na+ and, possibly, poor electrode design. Cl- flux experiments: These experiments were highly encouraging. Clearly, part of the strength of the Self-referencing (SrE) system is the rapidity with which data can be obtained, albeit within the operational limitations of the electrode used. From the body of this work we were able to establish a foundation for using ion-selective and (particularly Cl-) SrE with FDLE monolayers by demonstrating that 1) we could measure a directional Cl- flux, 2) which could be manipulated with blockers of Cl- transport (NPPB and bumetanide data not shown), 3) which in the unstimulated state demonstrated a net Cl- efflux over a physiologically relevant range of external Cl- (70-140mM), similar to that of the foetal lung in situ and 4) which could be predictably manipulated with ?2-adrenoreceptors and P2Y2 receptor agonists.Future work will build on this using an SrE system now installed in our Department at Ninewells Hospital and Medical School to monitor the effects of transfected knockout oligonucleotides targeted to various parts of the G-protein signalling pat hway. This represents a novel and particularly appropriate use of the system where the number of positively transfected cells falls below the resolving capability of other techniques (eg Isc measures the ion-transport characteristics of the monolayer (cf patch clamp).
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