a 2-Adrenergic Receptors Stimulate Oligopeptide Transport in a Human Intestinal Cell Line 1

Diand tripeptides, as well as peptidomimetic drugs such as cephalexin (CFX), are absorbed by enterocytes via the oligopeptide transporter PepT1. We recently showed that the a2-adrenergic agonist clonidine increases CFX absorption in anaesthetized rats. Herein, we investigated whether a2-adrenergic receptors can directly affect PepT1 activity in a clone of the differentiated human intestinal cell line Caco-2 (Caco-2 3B) engineered to stably express a2A-adrenergic receptors at a density similar to that found in normal mucosa. Measurement of CFX fluxes across cell monolayers cultured on transwell filters demonstrated that the a2-agonists clonidine and UK14304 caused a 2-fold increase of CFX transport in Caco-2 3B cells, but not in Caco-2 (expressing PepT1 but not a2-adrenergic receptors) or in the HT29 19A clone (expressing a2-adrenergic receptors but not PepT1). The stimulatory effect of clonidine was abolished by glycyl-sarcosine (a competitor for the transporter) and blocked by yohimbine or RX821002 (a2-antagonists). Analysis of the kinetics of CFX transport in control and clonidine-treated Caco-2 3B cells showed that clonidine increased Vmax of CFX transport without changing Km. Clonidine action was abolished by colchicine but not altered by amiloride, demonstrating that microtubule integrity but not Na/H exchanger activity is necessary for the effect of a2-agonists to occur. In conclusion, clonidine can directly activate a2-adrenergic receptors located on epithelial cells. The precise molecular mechanisms whereby these receptors modulate PepT1 activity remain to be elucidated but an increased translocation to the apical membrane of preformed cytoplasmic transporter molecules is likely to be involved. The H/oligopeptide cotransporter PepT1 is a 12 transmembrane domain protein located in the brush-border membrane of enterocytes that is specific for diand tripeptides arising from digestion of dietary proteins (for review, see Leibach and Ganapathy, 1996). Besides its key role in nutrient absorption, PepT1 is also pharmacologically relevant because its activity is responsible for absorption of peptidomimetic drugs such as b-lactam antibiotics. Although several studies have been carried out on the functional and molecular characteristics of PepT1 (Liang et al., 1995; Mackenzie et al., 1996), little information is available on the regulation of its activity (Brandsch et al., 1994; Muller et al., 1996; Fujita et al., 1997, 1999; Thamotharan et al., 1999b). A recent study demonstrated transcriptional enhancement of PepT1 expression in rats fed with protein-rich diet. However, in a previous work with a single-pass jejunal perfusion technique in anaesthetized rats, we showed that intestinal absorption of the b-lactam antibiotic cephalexin (CFX), which is carried by PepT1, was influenced by the nervous system (Berlioz et al., 1999). In our experiments, stimulation of PepT1 occurred very rapidly, excluding a transcriptional control, and depended on the activity of intramural and/or extramural neuron networks, including nicotinic synapses, intestinal sensory neurons, and sympathetic noradrenergic fibers. Among the agents acting on neurotransmitter receptors that were tested, administration of the a2agonist clonidine induced a 2-fold increase of the intestinal absorption of CFX. In the small intestine, a2-adrenergic receptors are present on enteric neurons, on extrinsic sympathetic postganglionic neurons (Cooke and Reddix, 1994), and also on intestinal epithelial cells (Laburthe et al., 1982; Nakaki et al., 1983). Thus, the precise location of the receptor responsible for the stimulatory effect of clonidine in vivo is unclear. The purpose of this study was to investigate whether Received for publication February 10, 2000. 1 This study was funded in part by Institut de Recherches sur les Maladies de l9Appareil Digestif and by Association Charles Debray. F.B. was the recipient of a grant from the Fondation pour la Recherche Médicale. ABBREVIATIONS: PepT1, H/peptide cotransporter; CFX, cephalexin; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; UK14304, 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline; RX821002, 2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; RT-PCR, reverse transcription-polymerase chain reaction; TEER, transepithelial electrical resistance. 0022-3565/00/2942-0466$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 294, No. 2 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 294:466–472, 2000 /2582/834825 466 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from a2-adrenergic receptors can directly affect the transport of peptidomimetic drugs in cultured epithelial cells devoid of innervation. To fulfill this objective, we needed to use a polarized intestinal cell line possessing PepT1 and a2-adrenergic receptors. However, a cell model spontaneously expressing both proteins is not currently available. The HT29 colonic cells express a2-adrenergic receptors but not PepT1 (Langin et al., 1989, 1995). Conversely, Caco-2 cells, frequently used to study intestinal drug absorption (Zweibaum et al., 1991) show enterocytic differentiation and express the PepT1 transporter (Liang et al., 1995) but not the a2-adrenergic receptor (Devedjian et al., 1991). The absence of suitable model was recently circumvented by the generation of a clone of Caco-2 cells stably transfected with the a2C10 adrenergic receptor gene. This clone, referred to as Caco-2 3B (Schaak et al., 2000), expresses a2-adrenergic receptors at a density similar to that found on enterocytes and colonocytes of different species, including humans (Paris et al., 1990; Senard et al., 1990; Valet et al., 1993). As in normal intestinal cells, the receptor is coupled to Gi2 and Gi3 and its stimulation inhibits forskolin-stimulated cAMP production. We thus decided to use Caco-2 3B cells to study a possible direct involvement of a2-adrenergic receptors in the activation of CFX transport. Experimental Procedures Materials. Caco-2 cells were purchased from the American Type Culture Collection (Rockville, MD). HT-29 clone 19A cells were a generous gift from C. Laboisse (CJF 9404, Nantes, France). The clone of Caco-2 cells (Caco-2 3B) expressing a2-adrenergic receptors was obtained by transfection of the parental cell-line with the bicistronic plasmid pa2C10ENeo containing the coding region of the human a2A-adrenergic receptor subtype (Schaak et al., 1999). Dulbecco’s modified Eagle’s medium (DMEM), trypsin solution, and fetal calf serum (FCS) were purchased from Gibco-BRL (Cergy Pontoise, France). Cephalexin, clonidine, yohimbine, glycyl-sarcosine, amiloride, and colchicine were obtained from Sigma (St. Louis, MO). UK14304 and RX821002 were donated by Pfizer (Sandwich, UK) and Reckitt and Colman Laboratories (Kingston-upon-Hull, UK), respectively. [C]Mannitol (specific radioactivity, 57 Ci/mmol) and [H]RX821002 (specific radioactivity, 53 Ci/mmol) were purchased from Amersham (Amersham, UK). [H]Clonidine (specific radioactivity, 66 mCi/mmol) was from New England Nuclear (Boston, MA). Cell Culture. Caco-2 3B (passages 18–27) and Caco-2 (passages 35–37) cells were propagated in 25-cm flasks at 37°C in a humidified 5% CO2 incubator in DMEM supplemented with 20% FCS and 1% nonessential amino acids (Zweibaum et al., 1991). When reaching confluency, cells were trypsinized and plated (starting density, 5 3 10 cells/cm) on Transwell Clear polyester membranes, 1 cm in surface and 0.4 mm in pore size (Costar, Dutscher, France). Culture medium was changed every day and, except where noted, monolayers at day 16 to 17 postseeding were used for transport experiments. HT-29 clone 19A cells (passage 154) were subcultured and plated as Caco-2, except they were grown in DMEM supplemented with 10% FCS and 1% nonessential amino acids. Adrenergic Receptor Quantification. The expression of a2adrenergic receptors in the different cell types was assessed by binding studies with [H]RX821002 (a2-antagonist) and [H]clonidine (a2-agonist) as specific radioligands. Binding experiments were performed on crude membranes prepared from frozen cells as described previously (Paris et al., 1990). Briefly, total binding was measured by incubating 100 ml of membranes with the radioligand in a total volume of 400 ml of binding buffer (50 mM Tris-HCl, 0.5 mM MgCl2, pH 7.5). After a 45-min incubation at 25°C, bound radioactivity was separated from free by filtration through GF/C Whatman filters with a Millipore manifold sampling unit. Filters were rapidly washed with ice-cold buffer and bound radioactivity was determined by liquid spectrometry. Specific binding was defined as the difference between total and nonspecific binding measured as described above but in the presence of 10 mM phentolamine. Final concentrations of radioligand ranged from 0.1 to 10 nM for [H]RX821002 and from 0.05 to 8 nM for [H]clonidine. Saturation isotherms were analyzed with the EBDA-LIGAND computer programs (McPherson, 1985) and protein concentration was determined with the Coomassie blue method (Bradford, 1976). Reverse Transcription-Polymerase Chain Reaction (RTPCR). Total RNAs were extracted from Caco-2, Caco-2 3B, and HT-29 19A cells with RNAXEL (Eurobio, Les Ulis, France) according to the manufacturer’s instructions. Ten micrograms of total RNA was reverse transcribed with Moloney murine leukemia virus RNase at 37°C for 45 min and then heated at 80°C for 5 min. The synthesized cDNA was used for subsequent PCR with two sets of primers allowing us to amplify either PepT1 or GAPDH, taken as a control for housekeeping gene. The primers for PepT1 were identical with those used in Liang et al. (1995). The sense 59-TCCACCGCCATCTACCATAC-39 and antisense 59-GGACAAACACAATCAGGGCT-39 primers allow amplification of a 479-base pair fragment corresponding to nucleotides 210 to 708 of the human PepT1 cDNA. Primers for GADPH