Organic anion transport cell culture

There have been several studies that underscore the importance of unbound concentration in cell-based studies of receptor function. In a model study of the effect of plasma protein binding on the renal transport of organic anions using the expression of various organic anion transporters (OATPs) in Xenopus oocytes, the transport of ochratoxin A, methotrexate, and estrone sulfate was found to be strongly inhibited by the addition of human serum albumin to the culture medium [16]. Similarly, the addition of oq-acid glycoprotein was found to reverse the blockade of sodium-ion current by cocaine in a preparation of cardiac myocytes [17]. 

OTA is thought to be transported into the proximal tubular epithelium via organic anion transporters. In primary rabbit proximal tubule cells cultured on microporus supports OTA was taken up from the basolateral compartment and secreted into the apical compartment. This process was sensitive to probenecid [198]. Mouse proximal tubular cells transfected with hOATs (1, 3 and 4) exhibited an enhanced uptake of OTA which was also inhibited by probenecid [199, 200]. However, it is also known that OTA has a high affinity for albumin and it has been demonstrated experimentally that human serum albumin dose depend-ently decreases OTA uptake via hOATl [201]. Whether albumin bound OTA can be transported via megalin mediated endocytosis has not been investigated. 

Ulvestad M, Darnell M, Molden E et al (2012) Evaluation of organic anion-transporting polypeptide 1B1 and CYP3A4 activities in primary human hepatocytes and HepaRG cells cultured in a dynamic three-dimensional bioreactor system. J Pharmacol Exp Ther 343(1 ) 145-156... 

Kouzuki et al. [157, 158] have proposed a method using reference compounds to determine the contribution of rat Oatplal and Ntcp to the hepatic uptake of bile adds and organic anions. This concept is originally established in the field of metabolic enzymes by Crespi et al. [159] and they named it relative adivity fador (RAF) method. In this method, they checked the transport activity of both test compounds and the reference compounds, which should be specific substrates for single transporters, in short-term cultured rat hepatocytes and transporter-expressing COS-7 cells. Then, they estimated the contribution from the following equations ... 

The Calu-3 human submucosal gland cell line forms polarized cell monolayers with tight junctions, produces mucus, and develops apical cilia when grown at an air interface. Transport studies can be performed after 10-14 days in culture, and it has been shown that Calu-3 cells express P-gp and actively transport amino acids, nucleosides, and dipeptide analogs organic anions, organic cations, polyamines, and efflux pump substrates are not actively transported.43,51,53-56 Because Calu-3 cells are not subject to the influence of multiple in vivo cell types, the expression of carrier proteins and enzymes may not reflect in vivo levels. Nevertheless, values obtained in Calu-3 permeability studies correlate well with those obtained from primary cultured rabbit tracheal epithelial cells and in vivo rat lung absorption studies.54 Mannitol permeation in Calu-3 cells is about 10 times less than that in vivo, but this is the same ratio difference between Caco-2 cells and in vivo intestinal epithelium.51... 

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