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-CI3 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-CI3 cells shown a resting pHi, in the absence of CO2/HCO3-, of 6.94 +/- 0.1; after an acid load PC-CI3 cells recovered toward resting pHi value, using a Na-dependent H+ extrusion mechanisms which was amiloride sensitive (K-i = 23 muM). 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-CI3 cells express a functional Na/H exchange activity and different isciforms (NHE1, NHE2 and NHE3) are expressed in the plasma membrane.
Characterization of Na+/H+ antiporter activity in PC-Cl3 thyroid cells
VILELLA, Sebastiano;MARSIGLIANTE, Santo;STORELLI, Carlo
2003-01-01
Abstract
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-CI3 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-CI3 cells shown a resting pHi, in the absence of CO2/HCO3-, of 6.94 +/- 0.1; after an acid load PC-CI3 cells recovered toward resting pHi value, using a Na-dependent H+ extrusion mechanisms which was amiloride sensitive (K-i = 23 muM). 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-CI3 cells express a functional Na/H exchange activity and different isciforms (NHE1, NHE2 and NHE3) are expressed in the plasma membrane.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
