title: In vivo readout of CFTR function: ratiometric measurement of CFTR-dependent secretion by individual, identifiable human sweat glands.
abstract: 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.
authors.affiliation: Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California, United States of America ; Department of Pediatrics, Stanford University School of Medicine, Stanford, California, United States of America ; Department of Psychology, Stanford University, Stanford, California, United States of America.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Wine
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Char
authors.forename: Jonathan
authors.initials: J
authors.lastname: Chen
authors.forename: Hyung-Ju
authors.initials: HJ
authors.lastname: Cho
authors.forename: Colleen
authors.initials: C
authors.lastname: Dunn
authors.forename: Eric
authors.initials: E
authors.lastname: Frisbee
authors.forename: Nam Soo
authors.initials: NS
authors.lastname: Joo
authors.forename: Carlos
authors.initials: C
authors.lastname: Milla
authors.forename: Sara E
authors.initials: SE
authors.lastname: Modlin
authors.forename: Il-Ho
authors.initials: IH
authors.lastname: Park
authors.forename: Ewart A C
authors.initials: EA
authors.lastname: Thomas
authors.forename: Kim V
authors.initials: KV
authors.lastname: Tran
authors.forename: Rohan
authors.initials: R
authors.lastname: Verma
authors.forename: Marlene H
authors.initials: MH
authors.lastname: Wolfe
mesh_terms: D000318:Adrenergic beta-Agonists / Q000008:administration & dosage; D000328:Adult; D000628:Aminophylline / Q000008:administration & dosage; D001285:Atropine / Q000008:administration & dosage; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D006579:Heterozygote; D006801:Humans; D007271:Injections, Intradermal; D007545:Isoproterenol / Q000008:administration & dosage; D008297:Male; D016210:Methacholine Chloride / Q000008:administration & dosage; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists / Q000008:administration & dosage; D009154:Mutation; D058915:Purinergic P1 Receptor Antagonists / Q000008:administration & dosage; D013542:Sweat / Q000187:drug effects / Q000378:metabolism*; D013545:Sweat Glands / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000318:Adrenergic beta-Agonists; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D058915:Purinergic P1 Receptor Antagonists; D016210:Methacholine Chloride; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000628:Aminophylline; D001285:Atropine; D007545:Isoproterenol