Related Experiment Videos
Characterization of Na(+)/H(+) antiporter activity in PC-Cl3 thyroid cells
Sebastiano Vilella1, Roberta Schiavone, Loredana Zilli
1Laboratory of Comparative Physiology, Department of Biological and Environmental Sciences and Technology, University of Lecce. sebastiano.viella@unile.it
Background/Aims:
In thyroid cells, the intracellular pH plays a key role in the control of the iodide uptake, since iodide accumulation is associated with an intracellular acidification. In the present paper we studied the kinetic proprieties of the Na(+)/H(+) antiporter (NHE) and the molecular expression of different NHE isoforms in rat thyroid PC-Cl3 cells. In addition the intracellular buffer capacity was also evaluated.
Methods:
pHi was measured using the pH sensitive fluorescent dye BCECF-AM. Amiloride, 5-(N,N-Dimethyl) hydrochloride was used to inhibit NHE activity. RT-PCR and western blot analyses were used to study the expression of NHE mRNA and protein isoforms.
Results:
PC-Cl3 cells shown a resting pHi, in the absence of CO(2)/HCO(3)(-), of 6.94 +/- 0.1; after an acid load PC-Cl3 cells recovered toward resting pHi value, using a Na-dependent H(+) extrusion mechanisms which was amiloride sensitive (K(i) = 23 microM). The kinetic parameters were K((Na)app) = 10 +/- 2 mM and V(max) = 0.23 +/- 0.02 DeltapH/min x 10(5) cells. NHE1, NHE2 and NHE3 were expressed at the mRNA level as well as at the protein level.
Conclusion:
PC-Cl3 cells express a functional Na/H exchange activity and different isoforms (NHE1, NHE2 and NHE3) are expressed in the plasma membrane.