Cloning and characterization of organic anion systems in the adrenal cortex and their role in steroid release
The adrenal steroid hormones have a central role in maintaining homeostasis, as they have influence on almost every physiological process. Their movement across the cell membrane is still poorly understood, although this is of great interest to basic biology and medicine. Previous studies have suggested transporter(s) may participate in this process. In this study the characteristic features of the previously demonstrated ROAT1-like exchange transport system in bovine adrenal cells were investigated with representative substrates. Corticotrophin (ACTH) stimulated 3H-PAH uptake into bovine adrenocortical cells, which could be inhibited by probenecid. Cortisol, glutarate and PAH in the incubation medium also cis-inhibited 3H-PAH uptake, and preincubation with PAH trans-stimulated 3H-PAH uptake. Preliminary studies on human adrenocortical cells also provided evidence for the existence of a probenecid inhibitable PAH-transporter. These results support the concept of an organic anion/dicarboxylate exchanger involved in cortisol release and PAH uptake into adrenocortical cells. Additionally, a sodium-dependent succinate uptake was also demonstrated in bovine adrenocortical cells. The uptake was inhibited by lithium, glutarate, fumarate, a-ketoglutarate and maleate, but not by 2,3-dimethylsuccinate or cis-aconitate. The lack of inhibition by citrate on succinate uptake at pH 7.4 is different from the data reported for the dicarboxylate transporters of all other organs investigated. These data are the first evidence for the existence of a Na+-dicarboxylate cotransporter in adrenocortical cells. The expression of two families of organic anion transporters in adrenal cells was also investigated. This study represents the first demonstration of the renal organic anion transporter (ROAT1) and the three members of the organic anion transporting polypeptide family (oatp1, oatp2, oatp3) by PCR from rat adrenal cDNA. The rat organic cation transporter 2 (OCT2) was also detected from the adrenal gland by in situ hybridization. In addition, in situ hybridization was performed to determine the localisation of the expression of the newly demonstrated transporters in the rat adrenal gland. In the case of ROAT1, intense signal was observed in the outer zona fasciculata, while oatp3 and OCT2 mRNAs were clearly shown to be expressed in the zona glomerulosa. The oatp1- and oatp2-specific probes produced signals in single cells or small groups of cells in the inner zona fasciculata and zona reticularis. For all transporters tested in the adrenal gland, only ROAT1 expression showed clear increase upon exposure to ACTH. The number of cells expressing ROAT1 mRNA was increased, especially in the inner zones of the adrenal cortex, while without ACTH, the signal was no longer detectable even in the inner zona fasciculata. In summary these experiments provide clear evidence for the existence of a variety of transport systems in the adrenal gland. The evidence presented here suggests that of these transporters, the adrenal ROAT1 seems to be involved in glucocorticoid release from adrenocortical cells.
570 Biowissenschaften, Biologie, Biologie, Adrenal cortex, bovine primary cells, human adrenocarcinoma cell line, cortisol, organic anion transport, dicarboxylate transport, in situ hybridization, PCR, 42.13, 42.15, 42.18, W.F., W.H.
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