As long as the question of which channels are responsible for cAMP-mediated epithelial Cl- secretion remains unsolved, it is still important to search for specific inhibitors that might help to relate macroscopic to microscopic events. Following the report by Sheppard and Welsh (J Gen Physiol 100: 573, 1992) that glibenclamide inhibits whole-cell Cl- currents in genetically manipulated fibroblasts expressing the cystic fibrosis transmembrane conductance regulator (CFTR), we have studied the effect of glibenclamide on different types of Cl- channels of HT29 and T84 cells at the single-channel level. Our results confirm that micromolar concentrations of glibenclamide inhibit the linear, low-conductance Cl-channel, which appears to represent CFTR and show that the inhibition results from a typical flicker block. However, the same concentrations of glibenclamide inhibit also the outwardly rectifying intermediate conductance Cl- channel which, potentially, may contribute to transepithelial Cl- secretion.

Myelination of axons by oligodendrocytes in the central nervous system is crucial for fast, saltatory conduction of action potentials. As myelination is central for brain development and plasticity, and deficits are implicated in several neural disorders such as multiple sclerosis, major depressive disorder, bipolar disorder and schizophrenia, it is important to elucidate the underlying mechanisms regulating myelination. Numerous mechanisms have been proposed by which the communication between oligodendrocytes and active axons may regulate the onset and maintenance of activity‐dependent myelination. We compared two models of ‘silencing' layer V and/or VI cortical projection neurons from early stages by either decreasing their excitability through Kir2.1 expression, an inward rectifying potassium channel, introduced through in utero electroporation at embryonic day (E)13.5, or inhibiting regulated vesicular release through Cre‐dependent knock‐out of synaptosomal associated protein 25 kDA (SNAP25). SNAP25 is a component of the soluble N‐ethylmaleimide fusion protein attachment protein receptor (SNARE) complex, which, among others, is needed for calcium‐dependent regulated vesicle release from synapses. In layer VI cortical projection neurons in the Ntsr1‐Cre;Ai14;Snap25 fl/fl mouse, we found that inhibiting regulated vesicular release significantly decreased the amount of myelin basic protein (MBP, used as marker for myelination) and the amount of myelinated projections at postnatal day (P)14 without affecting the initial timing of onset of myelination in the brain (at P7/P8). Additionally, overall oligodendrocyte maturation appears to be affected. A strong trend towards reduced node of Ranvier (NoR) length was also observed in Ntsr1‐Cre;Ai14;Snap25 fl/fl corpus callosum. An equally strong trend towards reduced NoR length was observed in Rbp4‐Cre;Ai14;Snap25 fl/fl corpus callosum at P14, and the g‐ratio in the spinal cord dorsal column was reduced at P18. However, no measurable differences in levels of MBP were detected in the striatum when comparing Rbp4‐Cre;Ai14;Snap25 fl/fl and control brains. Conversely, Kir2.1 in utero electroporation at E13.5 did not significantly affect the amount of MBP or number of myelinated callosal axons at P14 but did significantly decrease the NoR length measured in the corpus callosum. It therefore seems likely that the excitability of the neuron can potentially perform a modulating function of myelin characteristics, whereas regulated vesicular release has the potential to have a more pronounced effect on overall myelination, but in a cell‐type specific manner.

It has been known for several years that the outwardly rectifying 30-pS chloride channel, the regulation of which has been reported to be defective in cystic fibrosis, can be activated by excision of a membrane patch from a cell. This suggested that the cytosol contains an inhibitory factor, which diffuses away after excision, thereby releasing the channel block. To test for such an inhibitor we have isolated cytosol from two epithelial cell lines, and in larger quantities from pig kidney cortex. Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl- conductance in the brush-border membrane, which is activated during isolation of membrane vesicles. The inhibitory effect of the cytosol preparations was assessed by: (a) measuring conductive Cl- fluxes on renal proximal tubular brush-border membrane vesicles preloaded with or without cytosol, and (b) recording single Cl- channel currents from excised membrane patches of nasal polyp epithelia and CFPAC-1 cells in the presence and absence of cytosol. All cytosol preparations tested were found to inhibit both conductive Cl- flux in membrane vesicles and single Cl- channels in patch-clamp experiments. In the latter case a type of flicker block was observed with a reduction of channel open probability. Stepwise dilution of the cytosol consistently reduced the inhibitory potency. Since the inhibition was preserved after boiling the cytosol for 10 min, we conclude that the inhibitor is a heat-stable substance. Whether it is identical with the postulated intracellular regulator that couples the defective function of the cystic fibrosis gene product to Cl- channel inhibition cannot be decided at present.

Cytosol prepared from human placenta inhibits outwardly rectifying Cl^- channels excised from HT₂₉ cells. Size exclusion chromatography of cytosol on Superose 12 revealed two inhibitory fractions suggesting Cl^- channel inhibitors of different size or molecular weight (MW). The ‘high MW’ inhibitor co-migrated with the proteins, whereas the ‘low MW inhibitor eluted together with tryptophan, xanthine, hypoxanthine, and nicotinamide. Although these substances did not inhibit Cl^– channels we tentatively assume that the ‘low MW cytosolic inhibitor may be a heterocyclic aromatic compound. Alternatively, the ‘low MW inhibitor may be a steroid, because aldosterone has a similar retention time on Superose 12. We can as yet exclude a number of putative cytosolic Cl^– channel inhibitors including glutathione, cAMP, cGMP, ATP, GTP, NADH, NADPH, and arachidonic acid.