Extracellular nucleotides modify ion transport in cortical and medullary collecting duct-derived cell lines from mouse kidney. The inner medullary collecting duct cell line mIMCD-K2 expresses both P2Y and P2X receptors (McCoy et al. 1999). In M1 cells activation of P2Y₂ receptors inhibits sodium transport (Cuffe et al. 2000); however, P2X receptor expression and function in this cell line has not been defined. Here we present our preliminary results aimed at determining P2X expression in M1 cells.

M1 cells (P25-P27) were cultured to confluence on plastic T25 flasks in a defined, serum-free medium (PC-1: BioWhittaker) at 37 °C in a humidified, 95 % air: 5 % CO₂ atmosphere. Total RNA was extracted by standard methods (Trizol^TM) from 3-5 flasks of M1 cells and 3-5 whole kidneys of adult mice that were killed humanely. Reverse transcription polymerase chain reaction (RT-PCR) was performed initially using degenerate primers designed against rat P2X₁-P2X₃ sequences and subsequently with specific primers designed against rat P2X₃ and P2X₅. Amplification of β-actin and GAPDH were used to verify efficacies of RNA extraction and reverse transcription. PCR products were separated by electrophoresis on 3 % agarose gels and visualised by ethidium bromide staining. Each determination was performed with and without reverse transcriptase and all RT-PCR reactions were performed at least three times.

P2X₅ protein expression was investigated by fluorescence immunostaining of confluent M1 cell monolayers fixed in Zamboni’s fixative (15 % Picric acid, 2 % formaldehyde in PBS) for 20 min at room temperature. Fixed monolayers were blocked in normal goat serum for 30 min and then incubated with a polyclonal antibody against P2X₅ overnight at 4 °C. Staining was observed by FITC fluorescence.

RT-PCR products of 500 bp and 265 bp were observed (n = 3-5) corresponding to the predicted product sizes for P2X₃ and P2X₅, respectively, in both M1 cells and in mouse whole kidney. Fluorescence immunostaining showed strong expression of P2X₅. Staining was abolished by appropriate controls, which included addition of cognate peptide or omission of primary antiserum (n = 3).

We conclude that M1 cells express both P2X₃ and P2X₅ mRNA and P2X₅ protein. Possible function of these receptors in M1 cells remains to be determined.

Cuffe, J.E., Bielfeld-Ackermann, A., Thomas, J., Leipziger, J. & Korbmacher, C. (2000). J. Physiol. 524, 77-90. abstract

McCoy, D.E., Taylor, A.L., Kudlow, B.A., Karlson, K., Slattery, M.J., Schwiebert, L.M., Schwiebert, E.M. & Stanton, B.A. (1999). Am. J. Physiol. 277, F552-559.