SUMMARY1. The validity of the macroscopic laws of ion diffusion was critically examined within the microenvironment of the extracellular space in the rat cerebellum using ion-selective micropipettes and ionophoretic point sources.2. The concepts of volume averaging, volume fraction (a) and tortuosity (A) were defined and shown to be theoretically appropriate for quantifying diffusion in a complex medium such as the brain.3. Diffusion studies were made with the cations tetramethylammonium and tetraethylammonium and the anions a-naphthalene sulphonate and hexafluoroarsenate, all of which remained essentially extracellular during the measurements. Diffusion parameters were measured for a period of 50 s and over distances of the order of 0-1 mm. 4. Measurements ofthe diffusion coefficients of the ions in agar gel gave values that were very close to those derivable from the literature, thus confirming the validity of the method.5. Measurements in the cerebellum did not reveal any systematic influences of ionophoretic current strength, electrode separation, anisotropy, inhomogeneity, charge discrimination or uptake, within the limits tested. 6. The pooled data from measurements with all the ions gave a = 0-21 +002 (mean+s.E. of mean) and A = 1-55+0-05 (mean+S.E. of mean).7. These results show that the extracellular space occupies about 20 % of the rat cerebellum and that the diffusion coefficient for small monovalent extracellular ions is reduced by a factor of 2-4 (i.e. A2) without regard to charge sign. The over-all effect of this is to increase the apparent strength of any ionic source in the cerebellum by a factor of A2/a, about 12-fold in the present case, and to modify the time course of diffusion.8. These conclusions confirm that the laws of macroscopic diffusion are closely obeyed in the cerebellum for small ions in the extracellular space, provided that volume fraction and tortuosity are explicitly taken into account. It is likely that these conclusions are generally applicable to other brain regions and other diffusing substances. PHY 321C. NICHOLSON AND J. M. PHILLIPS

Cigarette smoke is composed of approximately 5% particulate phase and 95% vapour phase by weight. However, routine in vitro toxicological testing of smoke normally only measures the activity of the particulate phase. This study describes a new system for exposing cells at an air–liquid interface to serial dilutions of gaseous smoke. Confluent monolayers of NCI-H292 human lung epithelial cells on semi-permeable membranes were placed in a purpose-designed Perspex chamber at an air–liquid interface. The cells were exposed to dilute whole mainstream cigarette smoke for 30 minutes, followed by a 20-hour recovery period. Firstly, high and low delivery cigarettes were compared, and cytotoxicity was determined by using the neutral red uptake assay. Clear differential cytotoxic responses were observed with the two cigarette types, which correlated positively with the concentrations of components in smoke, and particularly compounds in the vapour phase, such as aldehydes. Secondly, low doses of smoke were found to up-regulate mRNA levels of the secreted mucin, MUC5AC, and to stimulate the production of interleukin (IL)-6, IL-8 and matrix-metalloprotease-1, but had no effect on growth-related oncogene alpha. This system will facilitate further investigations into the toxicological mechanisms of cigarette smoke components, and may be useful for studying other gaseous mixtures or aerosols.

Cigarette smoke is a complex mixture of more than 4,000 constituents. Its effects on cell biology are poorly understood, partly because whole smoke exposure in vitro is technically challenging. To investigate the effects of smoke on cell signaling and function, a three-dimensional air-liquid interface model of tracheobronchial epithelium, grown from primary human lung epithelial cells, was exposed to air or whole mainstream cigarette smoke for 1 h in a purpose-designed chamber. Gene expression profiles were then determined at 1, 6, and 24 h postexposure using Affymetrix HGU133-2 Plus microarrays. Cells from three different donors were used in the study, and the experiment was performed in triplicate for each donor. Genes significantly regulated by smoke, compared with the air control, in all experiments were determined. Genes exhibiting differential expression were assigned to functional categories and mapped to signaling pathways. Effects were observed on many cellular processes including xenobiotic metabolism, oxidant/antioxidant balance, and DNA damage and repair. Notably, there was marked downregulation of the transforming growth factor-beta pathway, which has not been previously reported. This study provides important data on the acute effects of whole cigarette smoke on mucociliary epithelium and may be used to gain a greater understanding of smoke toxicity.

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