A Transporter of Ibuprofen is Upregulated in MDCK I Cells under Hyperosmotic Culture Conditions

Abstract

Ibuprofen is a widely used drug. It has been identified as an inhibitor of several transporters, but it is not clear if ibuprofen is a substrate of any transporter itself. In the present work, we have characterized a transporter of ibuprofen, which is upregulated by hyperosmotic culture conditions in Madin-Darby canine kidney I (MDCK I) renal cells. [(3)H]-Ibuprofen uptake rate was measured in MDCK I cell cultured under normal (300 mOsm) and hyperosmotic (500 mOsm) conditions. Hyperosmotic conditions were obtained by supplementing urea, NaCl, mannitol, or raffinose to culture medium. The effect of increased osmolarity was investigated for different incubation times. [(3)H]-Ibuprofen uptake in MDCK I cells was upregulated by hyperosmotic culture condition, and was saturable with a Km value of 0.37 ± 0.08 μM and a Vmax of 233.1 ± 17.2 pmol· cm(-2)· min(-1). Racemic [(3)H]-ibuprofen uptake could be inhibited by (R)-(-)- and (S)-(+)-ibuprofen with IC50 values of 19 μM (Log IC50 1.39 ± 0.34) and 0.47 μM (Log IC50 -0.36 ± 0.41), respectively. Furthermore, the [(3)H]-ibuprofen uptake rate was increased by decreased extracellular pH but not dependent on Na(+) or Cl(-) ions. The mRNA of Mct1, -2, -4, and -6 as well as Oat1 and -3 were not upregulated by hyperosmolarity. Our findings present strong evidence for the presence of a yet unknown ibuprofen transporter in MDCK I cells. The transporter was upregulated under hyperosmotic culture conditions, and the present study is therefore a starting point for identification of the molecular correlate and potential impact on ibuprofen disposition.

Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis

“…Figure 4 F). These concentrations overlap with the ∼70–240 μM therapeutic range of the drug that occurs in human blood plasma/serum associated with its conventional use (relating to a low apparent volume of distribution of ∼0.1–0.2 L/kg) [ 55 , 56 ]. As such, appreciable stimulation of thermogenesis might be possible if a similar UCP1 activation could be achieved in brown adipose tissue.…”

“…Though in both cases, the metabolism would have to be rapid. Alternatively, the drug may not be taken up by these cells in contrast to kidney cell lines where uptake has been demonstrated to be mediated by an unknown transporter [ 56 ]. Ibuprofen transport across the blood brain barrier also relies on an unknown solute carrier, rather than passive diffusion [ 58 ].…”

Transcriptome analysis identifies activated signaling pathways and regulated ABC transporters and solute carriers after hyperosmotic stress in renal MDCK I cells

“…Further functional studies need to be conducted to confirm these findings. In two recent studies, Nielsen and co-workers showed that a transporter with the anionic drug substance ibuprofen as a substrate was up-regulated in MDCK I cells under condition similar to the ones employed here [37,38]. However, there was only seen a functional effect, and the responsible gene(s) is not yet identified.…”

Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis

“…by triglyceride incorporation, as can occur in white adipocytes (48)) that, if rapid enough, could prevent it from activating UCP1. Alternatively, the drug may not be taken up by these cells in contrast to kidney cell lines where uptake has been demonstrated to be mediated by an unknown transporter (49). Compound variants circumventing such barriers, therefore, are an attractive avenue for further investigation.…”