Demonstration of a Probenecid-Inhibitable Anion Exchanger Involved in the Release of Cortisol and cAMP and in the Uptake of <i>p</i>-Aminohippurate in Bovine Adrenocortical Cells

Abstract

Recently we provided evidence for the involvement of a probenecid-inhibitable anion exchanger in cortisol release from primary cultures of bovine adrenocortical cells. In the present study, we further characterized this exchange transporter. Adrenocorticotropic hormone stimulated ³H-p-aminohippurate (³H-PAH) uptake into as well as cortisol release from the cells about two- and tenfold, respectively. Probenecid inhibited both ³H-PAH uptake and cortisol release by about 55 and 63%. Preincubation of the cells with 1 mM PAH trans-stimulated ³H-PAH uptake by 30%, whereas cortisol release was inhibited by 30%. ³H-PAH uptake was cis-inhibited by 1 mM glutarate or by 1 mM cortisol in the medium, while cortisol release was trans-stimulated by glutarate. PAH in the incubation medium showed saturable cis-inhibition of ³H-PAH uptake. The release of cyclic adenosine monophosphate, a substrate of the renal PAH exchanger, was also inhibited by probenecid and trans-stimulated by glutarate. In summary, the trans-stimulation and cis-inhibition experiments support the concept of an anion exchanger involved in cortisol and cyclic adenosine monophosphate release from and PAH uptake into adrenocortical cells.

The rapid release of corticosterone from the adrenal induced by ACTH is mediated by nitric oxide acting by prostaglandin E 2

“…The last few years have witnessed the description of a steroid transport mechanism involving organic anion transport. Because ACTH-stimulated corticosterone release was partially reduced by an organic anion transporter inhibitor, this mechanism could also be involved in steroid secretion from the AG other than that which is proposed here (31). The mechanism of organic acid change is of utmost importance to the transporter of corticosterone in facilitating the mobilization and fusion of the vesicles with the cell membrane and extrusion of corticosteroids in the adrenal.…”

Regulation of steroid hormone biosynthesis enzymes and organic anion transporters by forskolin and DHEA-S treatment in adrenocortical cells

“…Indeed, earlier experiments on bovine adrenal cells in primary culture provided evidence for the involvement of transporters for organic anions in the release of cortisol. The inhibitor of OATs, probenecid decreased cortisol release, and extracellular PAH and glutarate increased it by trans-stimulation (17,18). Bovine adrenal cells exhibited probenecid-sensitive PAH uptake that was inhibited by cortisol (18), indicating a transporter common for PAH, glutarate, and cortisol.…”

“…The inhibitor of OATs, probenecid decreased cortisol release, and extracellular PAH and glutarate increased it by trans-stimulation (17,18). Bovine adrenal cells exhibited probenecid-sensitive PAH uptake that was inhibited by cortisol (18), indicating a transporter common for PAH, glutarate, and cortisol. In rat adrenal glands, OAT1 was localized by immunocytochemistry to the zona fasciculata cells and was shown to increase in amount after stimulation by ACTH (2).…”

“…The concentration of cortisol in the culture supernatants was determined by radioimmunoassay (RIA) as described before (18). Briefly, the antiserum used was raised against corticosterone but showed 100% cross-reactivity with cortisol.…”

“…Further investigations on bovine adrenocortical cells demonstrated an uptake of radioactively labeled PAH, which was inhibited by probenecid and unlabeled PAH. The uptake of organic anions (e.g., PAH) into the cells, as well as the cortisol release from the cells, was stimulated by ACTH (18). Investigations applying RT-PCR on rat adrenals revealed the expression of organic anion transporter 1 (OAT1).…”