Two-path impedance spectroscopy for measuring paracellular and transcellular epithelial resistance

Area of Science:

• Epithelial physiology
• Biophysical techniques

Background:

• Solute transport across epithelial cell layers occurs via transcellular and paracellular pathways.
• Tight junction proteins regulate paracellular channels, influencing barrier function in health and disease.
• Conventional transepithelial resistance (TER) measurements cannot differentiate between these pathways.

Purpose of the Study:

• To develop and validate an impedance spectroscopic approach for separately determining paracellular (R(para)) and transcellular (R(trans)) resistance.
• To provide a method for better understanding epithelial barrier function and solute transport regulation.

Main Methods:

• Utilized impedance spectroscopy in specialized Ussing chambers.
• Employed Ca(2+)-dependent modulation of R(para) using EGTA.
• Incorporated flux measurements of fluorescein as a nonradioactive paracellular marker.

Main Results:

• The developed method successfully differentiates between R(para) and R(trans).
• Verified prerequisites for routine application, including a marker varying with R(para) and experimental conditions altering R(para) without affecting R(trans).
• Validated the method on diverse cell lines (HT-29/B6, MDCK C7, C11).

Conclusions:

• The impedance spectroscopic approach offers a reliable method to distinguish paracellular and transcellular resistance.
• This technique is applicable for routine analysis of epithelial barrier function.
• Improved characterization of epithelial transport pathways has implications for understanding various physiological and pathological states.