Determination of basolateral Na(+)/H(+) exchange activity in MDCK cells using a multiwell-multilabel reader

Area of Science:

• Physiology
• Molecular Biology
• Cell Biology

Background:

• Sodium-hydrogen exchangers (NHE) are crucial membrane proteins regulating cellular pH and volume.
• NHE-1 specifically maintains pH and volume homeostasis and is located in the basolateral membrane of epithelial cells.

Purpose of the Study:

• To evaluate the utility of multiwell-multilabel readers for investigating basolateral NHE-1.
• To compare the performance of multiwell-multilabel readers with an imaging system for NHE-1 studies.

Main Methods:

• Confluent Madin-Darby canine kidney (MDCK) cells were used to assess basolateral NHE-1.
• Experiments were conducted using both a multiwell-multilabel reader and an imaging system with filter perfusion.
• Cellular pH recovery after acid load was measured under various conditions, including the presence of Na+ and the NHE-1 inhibitor HOE642.

Main Results:

• The multiwell-multilabel reader yielded comparable results to the imaging system for steady-state pH and NHE-1 activity.
• Na+-dependent pH recovery was observed primarily after basolateral Na+ addition with both methods.
• NHE-1 inhibition by HOE642 reduced Na+-dependent pH recovery by over 80%.
• Protein kinase C stimulation via phorbol myristate acetate induced similar increases in NHE-1 activity with both techniques.

Conclusions:

• Multiwell-multilabel readers are suitable for studying the physiology and regulation of basolateral NHE.
• These readers offer a valid and convenient alternative to imaging systems for investigating basolateral transporters.