¹Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA

Correspondence: Dr D Merlin, PhD, Emory University, Division of Digestive Diseases, Department of Medicine, 615 Michael Street, Atlanta, GA 30322, USA. E-mail: dmerlin@emory.edu

Received 4 January 2006; Revised 15 February 2006; Accepted 16 February 2006; Published online 27 March 2006.

Abstract

Here, we examined hPepT1 expression in the monocytic cell line, KG-1. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that hPepT1 is expressed in KG-1 cells, while cDNA cloning and direct sequencing confirmed the sequence of KG-1 hPepT1 (accession number, AY634368). Immunoblotting of cell lysates from KG-1 cells or macrophages isolated from human peripheral blood revealed a 100 kDa immunoreactive band mainly present in the membrane fraction. Uptake experiments showed that the transport of 20 M radiolabeled Gly-Sarcosine ([¹⁴C]Gly-Sar) in KG-1 cells was Na⁺, Cl^- dependent and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS)-sensitive. In addition, hPepT1 activity was likely to be coupled to a Na⁺/H⁺ exchanger, as evidenced by the fact that [¹⁴C]Gly-Sar uptake was not affected by the absence of Na⁺ when cells were incubated at low pH (5.2). Interestingly, hPepT1-mediated transport was reduced in KG-1 cells incubated at low pH as it was also observed in nonpolarized Caco2-BBE cells. This pattern of pH-dependence is due to a disruption of the driving force of hPepT1-mediated transport events. This was supported by our finding that nonpolarized cells, Caco2-BBE cells and KG-1 cells, have an increased permeability to H⁺ when compared to polarized Caco2-BBE cells. Finally, we showed that hPepT1 is responsible for transporting fMLP into undifferentiated and differentiated (macrophage-like) KG-1 cells. Together, these results show that hPepT1 is expressed in nonpolarized immune cells, such as macrophages, where the transporter functions best at the physiological pH 7.2. Furthermore, we provide evidence for hPepT1-mediated fMLP transport, which might constitute a novel immune cell activation pathway during intestinal inflammation.