Culture of sweat gland epithelial cells from normal individuals and patients with cystic fibrosis

Collie, Gail; Buchwald, Manuel; Harper, Patricia A.; Riordan, John R.

doi:10.1007/bf02620892

Cited by 27 publications

(11 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Individual segments of coiled reabsorptive duct (1-2 mm) were dissected freehand from skin samples that were treated with collagenase (1 mg/ml) for 18-24 hr (13). Each ductal segment was placed on a no.…”

Section: Methodsmentioning

confidence: 99%

Cl- permeability of human sweat duct cells monitored with fluorescence-digital imaging microscopy: evidence for reduced plasma membrane Cl- permeability in cystic fibrosis.

Ram

Kirk

1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Salt reabsorption by the human sweat duct is markedly reduced in cystic fibrosis (CF). We used fluorescence-digital imaging microscopy in combination with a halidespecific fluorescent dye [6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ)] to determine if this defective salt reabsorption is referable to a reduced plasma membrane Cl-permeability of the epithelial cells that line the sweat duct. Sweat duct cells were cultured from explants of normal and CF reabsorptive duct and loaded with SPQ, the fluorescence of which is specifically quenched by halide ions (Br-> Cl) and provides a relative index of intracellular halide concentration. Two lines of evidence indicate that normal sweat duct cells exhibit a substantial permeability to Cl-and Br-. First, the replacement of extracellular Cl-with an impermeant anion (i.e., gluconate) resulted in a rapid and reversible increase in the intracellular fluorescence, as expected if the cells rapidly lost Cl-to the extracellular media. Second, the replacement of extracellular Cl-with Br-resulted in a rapid and reversible quenching of the intracellular fluorescence, as expected if the cells accumulated Br-(a more effective quencher of SPQ fluorescence) in exchange for CI. The rate of fluorescence change that was induced by either maneuver was inhibited by the Cl-channel blocker, diphenylamine-2-carboxylate (10 ,AM). Moreover, CF cells exhibited markedly reduced rates of fluorescence change in response to either maneuver. Our results document the utility of this imaging strategy for assessing the Cl-permeabilities of individual epithelial cells that are affected in cystic fibrosis and indicate that the defective salt reabsorption by the CF sweat duct is referable, at least in part, to a reduced plasma membrane Cl-permeability of sweat duct cells. Cystic fibrosis (CF) is a common genetic disorder amongCaucasians that is clinically expressed as a triad of symptoms (1): (i) pancreatic insufficiency; (ii) pulmonary congestion and infection, and (iii) elevated salt concentrations in sweat. Underlying this symptomology is a generally reduced Clpermeability of CF-affected epithelia, such as airway epithelium (2-4) and reabsorptive duct of sweat gland (5, 6). The mechanistic basis for the reduced Cl-permeability has been rigorously established for only one class of CF-affected tissue, the airway epithelium (3,4,7,8). The permeability defect expressed by this tissue is attributable to the inability of Cl--secreting cells to activate plasma membrane Clchannels in response to secretagogues that generate cyclic AMP (3,4,7,8).At present there is insufficient information concerning the nature of the reduced Cl-permeabilities of other CF-affected tissues to determine if an altered regulation of plasma membrane Cl-channels is a "universal" functional defect in CF. In this regard it is important to note that the transepithelial Clpermeability of the normal sweat duct is tonically elevated in vitro in the absence of extrinsic humoral or neural factors (5, 6, 9)-a finding that implies that the...

show abstract

Section: Methodsmentioning

confidence: 99%

Cl- permeability of human sweat duct cells monitored with fluorescence-digital imaging microscopy: evidence for reduced plasma membrane Cl- permeability in cystic fibrosis.

Ram

Kirk

1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…T84 cells were cultured in a 50 : 50 mixture of DMEM and F-12 media supplemented with 5% FBS, penicillin (100 U/ml) and streptomycin (100 ~g/ml). Sweat ducts were isolated from skin biopsies from young adult donors and cultured as described previously [14]. Briefly, skin samples were digested for 18 hr with 1 mg/ml Type I collagenase dissolved in alpha MEM medium containing 1% fetal bovine serum.…”

Section: Cellsmentioning

confidence: 99%

Bicarbonate permeability of the outwardly rectifying anion channel

et al. 1989

View full text Add to dashboard Cite

Single anion-selective channels have been studied in cultured human epithelial cells using the patch-clamp technique. Three cell types were used as models for different anion transport systems: (i) PANC-1, a cell line derived from the pancreatic duct, (ii) T84, a Cl-secreting colonic cell line, and (iii) primary cultures of sweat duct epithelium. Outwardly rectifying anion-selective channels were observed in all three preparations and were indistinguishable with respect to conductance, selectivity and gating. Striking similarities between HCO3- and Cl-secreting epithelia, and the high density of outward rectifiers in pancreatic cells prompted us to study HCO3 permeation through this channel. HCO3 permeability was significant when channels were bathed in symmetrical 150 mM HCO3 solutions, Cl-HCO3 mixtures, and under bi-ionic conditions with outwardly and inwardly directed HCO3 gradients. Permeability ratios (PHCO3/PCl) estimated from bi-ionic reversal potentials ranged from 0.50 to 0.64, although conductance ratios greater than 1.2 were observed with high extracellular pH. Chloride did not inhibit HCO3 permeation noticeably but rather had a small stimulatory effect when present on the opposite side of the membrane. The prevalence of outward rectifiers in PANC-1 and their permeability to bicarbonate suggests the channel may have a dual role in HCO3 secretion; to allow Cl recycling at the apical membrane and to mediate some of the HCO3 flux. Defective modulation of this channel in cystic fibrosis might provide a common basis for dysfunction in epithelia having very different anion transport properties (e.g., HCO3 secretion, Cl secretion and Cl absorption.

show abstract

“…With the introduction of sweat duct cell culture (Pedersen, 1984;Collie, Buchwald, Harper & Riordan, 1985; Lee, Carpenter, Coaker & Kealey, 1986), the mechanism and regulation of electrolyte reabsorption by the duct was facilitated by: (1) supply of sufficient material for extended biochemical analysis (Dearborn, Waller & Yike, 1988;Riorden, Vokarty, Jensen, Tsui & Buchwald, 1988;Doughney, Pedersen, McPherson & Dormer, 1989;Fogh, Pedersen, Herlin & Kragballe, 1989); (2) cultureinduced formation of a flat-type epithelium instead of a poorly accessible microtubule, which facilitates intracellular microelectrode studies (Jones, Bell & Quinton, 1988; Reddy, Riordan & Quinton, 1988), microfluorescence studies (Bijman, Scholte, Jonge, Hoogeveen, Kansen, Sinaasappel & Van der Kamp, 1988;Dearborn et al 1988), and transepithelial electrical and isotope flux measurements (Pedersen & Larsen, 1986;Pedersen, 1987;Pedersen, Larsen & Brandt, 1987 a;Pedersen, Larsen, Hainau & Brandt, 1987 b;Brayden, Cuthbert & Lee, 1988;Pedersen, 1989); (3) access to the mucosal side of the epithelium for patch-clamp studies of this membrane (Bijman et al 1988;Joris, Krouse, Hagiwara, Bell & Wine, 1989). …”

Section: Introductionmentioning

confidence: 99%

Chloride permeability regulation via a cyclic AMP pathway in cultured human sweat duct cells.

Pedersen

1990

The Journal of Physiology

View full text Add to dashboard Cite

SUMMARY1. Isolated coiled reabsorptive sweat ducts from normal subjects and patients with cystic fibrosis (CF) were cultured in vitro. Cells were harvested and plated onto permeable supports to form confluent cell sheets. The Ussing chamber technique was used to study pharmacological regulation of the transepithelial ion transport in these membranes.2. Addition of a stable cyclic AMP analogue, 8-Br-cyclic AMP, to normal cell cultures resulted in a decrease of the transepithelial potential difference (PD).3. Forskolin exposure resulted in a similar PD decrease, which was augmented by the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX).4. Exposure to isoprenaline, prostaglandin E2 (PGE2), and phenylephrine resulted in a response mimicking the forskolin-induced response, that was also amplified by IBMX.5. Pre-incubation with cholera toxin abolished the isoprenaline response and reduced the control resistance.6. Propranolol abolished the responses induced by isoprenaline and phenylephrine, whereas phentolamine had no effect. PGE2-induced responses were inert to both types of blockers.7. Indomethazine addition to an unstimulated membrane resulted in a weak PD increase, i.e. a response opposite to that induced by isoprenaline.8. IBMX addition to an unstimulated membrane resulted in a weak isoprenalinelike response. When the cells were pre-treated with indomethazine this IBMX response was absent.9. Unidirectional Cl-isotope flux studies demonstrated a large increase of net Clreabsorption in response to isoprenaline and PGE2.10. Mannitol isotope flux studies revealed that the paracellular permeability was unaffected by isoprenaline exposure. 11. Membranes derived from CF patients did not respond similarly to any of these agents. However, a weak spike, occasionally followed by a gradual increase of the short-circuit current VS,,), was observed in both normal subjects and CF patients.12. It is concluded that the primary effect on ion transport of factors increasing the cyclic AMP in normal cultured sweat duct cells is an activation of a transcellular Cl-permeability. This effect was missing in cells derived from CF patients.MS 7617 P. S. PEDERSEN

show abstract

Culture of sweat gland epithelial cells from normal individuals and patients with cystic fibrosis

Cited by 27 publications

References 19 publications

Cl- permeability of human sweat duct cells monitored with fluorescence-digital imaging microscopy: evidence for reduced plasma membrane Cl- permeability in cystic fibrosis.

Cl- permeability of human sweat duct cells monitored with fluorescence-digital imaging microscopy: evidence for reduced plasma membrane Cl- permeability in cystic fibrosis.

Bicarbonate permeability of the outwardly rectifying anion channel

Chloride permeability regulation via a cyclic AMP pathway in cultured human sweat duct cells.

Contact Info

Product

Resources

About