[HTML][HTML] In Vivo Readout of CFTR Function: Ratiometric Measurement of CFTR-Dependent Secretion by Individual, Identifiable Human Sweat Glands

JJ Wine, JE Char, J Chen, H Cho, C Dunn, E Frisbee… - PLoS …, 2013 - journals.plos.org
JJ Wine, JE Char, J Chen, H Cho, C Dunn, E Frisbee, NS Joo, C Milla, SE Modlin, IH Park…
PLoS One, 2013journals.plos.org
To assess CFTR function in vivo, we developed a bioassay that monitors and compares
CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (∼ 50)
individual, identified glands in each subject. Sweating was stimulated by intradermally
injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-
layer that contained dispersed, water soluble dye particles that partitioned into the sweat
bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated …
To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (∼50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ∼0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with ‘CFTR-related’ conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.
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