Evidence for the Involvement of an Anion Exchanger in Cortisol Release from Bovine Adrenocortical Cells

Abstract

In this study the influence of probenecid as inhibitor of the renal excretory system for organic anions was tested on the release of cortisol from primary cultures of bovine adrenocortical cells. ACTH-stimulated cortisol release (30 min to 24 h) was inhibited by probenecid (Ki = 0.093 mM). Analysis of steroid synthesis with ³H-pregnenolone as precursor indicated that part of the inhibition resulted from an attenuation of steroid synthesis by probenecid. The increased intracellular cortisol levels in the presence of probenecid however indicate an inhibitory effect on an efflux mechanism. Cortisol efflux was trans-stimulated by p-aminohippurate (PAH) and tetrafluorosuccinate, substrates of the renal PAH/anion exchanger. Further evidence for the PAH exchanger provided the expression of a probenecid-sensitive PAH transporter after injection of mRNA from adrenal cells into Xenopus laevis oocytes.

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.…”

“…Using the stop-flow peritubular capillary microperfusion method, Ullrich et al (19) described cortisol transport across the basolateral membrane of the proximal tubule cell of the rat kidney. Recent studies showed an inhibition of cortisol release from primary cultures of bovine adrenocortical cells by probenecid and trans-stimulation of cortisol release by p-aminohippurate (PAH) and glutarate in the incubation medium (17). Further investigations on bovine adrenocortical cells demonstrated an uptake of radioactively labeled PAH, which was inhibited by probenecid and unlabeled PAH.…”

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

“…In our former study [7], we have shown that 1 mM PAH in the incubation medium trans-stimulated cortisol release out of the cells approximately 1.6-fold. If cortisol and PAH share a common transporter, unlabeled PAH or cortisol at the outside, i.e., in the incubation medium, should cis-inhibit 3 H-PAH uptake.…”

“…Recently we demonstrated that adrenocorticotropic hormone (ACTH) stimulated cortisol release from bovine adrenocortical cells was inhibited by probenecid and trans-stimulated by PAH [7]. In that paper, we also described the expression of probenecid-inhibitable PAH transport after injection of mRNA from adrenocortical cells in Xenopus oocytes which suggested the existence of mRNA encoding a PAH transporter in the adrenocortical cells.…”

“…Bovine adrenal glands were obtained from freshly slaughtered 8-to 15-month-old steers, and adrenocortical cells were isolated as previously described [7,9] by digestion with 2.5% trypsin. The isolated cells were dispersed in culture medium [Ham's F12/Dulbecco's modified Eagle's medium containing in addition 100 U penicillin/ml, 100 Ìg streptomycin/ml and 10% (w/v) bovine calf serum], filtered through gauze, and seeded onto six-well plates at a density of approximately 6 !…”