α2-Adrenergic Receptors Stimulate Oligopeptide Transport in a Human Intestinal Cell Line1

  1. Françoise Berlioz1,3,
  2. Jean-José Maoret1,
  3. Hervé Paris2,
  4. Marc Laburthe1,
  5. Robert Farinotti3 and
  6. Claude Rozé1
  1. 1Institut National de la Santé et de la Recherche Médicale, Faculté X. Bichat, Paris (F.B., J.-J.M., M.L., C.R.); 2Institut National de la Santé et de la Recherche Médicale, Institut Louis Bugnard, Toulouse (H.P.); and3Unité Propre de Recherches de I′Enseignement Supérieure, Faculté de Pharmacie, Chatenay-Malabry, France (F.B., R.F.)
     
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    Abstract

    Di- and tripeptides, as well as peptidomimetic drugs such as cephalexin (CFX), are absorbed by enterocytes via the oligopeptide transporter PepT1. We recently showed that the α2-adrenergic agonist clonidine increases CFX absorption in anaesthetized rats. Herein, we investigated whether α2-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 α2A-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 α2-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 α2-adrenergic receptors) or in the HT29 19A clone (expressing α2-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 (α2-antagonists). Analysis of the kinetics of CFX transport in control and clonidine-treated Caco-2 3B cells showed that clonidine increased Vmaxof 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 α2-agonists to occur. In conclusion, clonidine can directly activate α2-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 di- and 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 β-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 β-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 α2-agonist clonidine induced a 2-fold increase of the intestinal absorption of CFX. In the small intestine, α2-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 α2-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 α2-adrenergic receptors. However, a cell model spontaneously expressing both proteins is not currently available. The HT29 colonic cells express α2-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 α2-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 α2C10 adrenergic receptor gene. This clone, referred to as Caco-2 3B (Schaak et al., 2000), expresses α2-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 α2-adrenergic receptors in the activation of CFX transport.

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    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 α2-adrenergic receptors was obtained by transfection of the parental cell-line with the bicistronic plasmid pα2C10ENeo containing the coding region of the human α2A-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. [14C]Mannitol (specific radioactivity, 57 Ci/mmol) and [3H]RX821002 (specific radioactivity, 53 Ci/mmol) were purchased from Amersham (Amersham, UK). [3H]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-cm2flasks at 37°C in a humidified 5% CO2incubator 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 × 104 cells/cm2) on Transwell Clear polyester membranes, 1 cm2 in surface and 0.4 μm 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 α2-adrenergic receptors in the different cell types was assessed by binding studies with [3H]RX821002 (α2-antagonist) and [3H]clonidine (α2-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 μl of membranes with the radioligand in a total volume of 400 μl 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 μM phentolamine. Final concentrations of radioligand ranged from 0.1 to 10 nM for [3H]RX821002 and from 0.05 to 8 nM for [3H]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 (RT-PCR).

    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 5′-TCCACCGCCATCTACCATAC-3′ and antisense 5′-GGACAAACACAATCAGGGCT-3′ primers allow amplification of a 479-base pair fragment corresponding to nucleotides 210 to 708 of the human PepT1 cDNA. Primers for GADPH were as follows: 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ (sense) and 5′CATGTGGGCCATGAGGTCCACCAC-3′ (antisense). Reaction mixtures were subjected to 35 cycles consisting of 30 s at 92°C, 30 s at 58°C, and 1 min at 72°C. The PCR products were electrophoresed on a 1% agarose gel and stained with ethidium bromide.

    Transport Studies.

    The day of the experiment, the transepithelial resistance of each cell layer was measured with a Millicel-ERS ohmmeter (Millipore, St. Quentin en Yvelines, France). Monolayers exhibiting a transepithelial resistance above 150 Ω/cm2 were considered as satisfactory and were further used for the transport experiments. Before transport studies, the culture medium was removed and the apical and basolateral compartments were washed three times with Krebs' solutions [137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, 0.3 mM NaHPO4, 0.3 mM KH2PO4) buffered with either 10 mM HEPES/Tris, pH 7.4 (basolateral compartment) or 10 mM Mes/Tris, pH 6.0 (apical compartment)]. Cell monolayers were incubated for 15 min at 37°C under continuous circular shaking in Krebs' modified buffer (pH 6.0 in the apical compartment and 7.4 in the basolateral compartment). Unless otherwise specified, at zero time of the experiment, CFX (final concentration 1 mM) was added to the apical compartment. The rate of CFX transport was estimated over a 30 min-period by measuring CFX concentration (see below) in 50-μl aliquots taken from the basolateral compartment every 5 min. For competition experiments, glycyl-sarcosine (50 mM) was added to the apical compartment before the addition of CFX. The α2-adrenergic drugs (clonidine, UK14304, yohimbine, or RX821002) were added to the basolateral compartment 15 min after CFX. The effects of these pharmacological agents were evaluated on the same cell monolayer by comparing basal CFX flux (calculated from the three samples taken during the 0- to 15-min incubation period) with the flux measured after their addition (calculated from the three samples taken during the 15- to 30-min incubation period). In some experiments, cells were pretreated with colchicine (20 μM) or amiloride (10 μM); these inhibitors were added to the apical compartment 25 and 10 min, respectively, before the addition of CFX.

    The formation of tight junctions was estimated by measuring passive diffusion of mannitol across cell monolayer. To do so, [14C]mannitol (1 μCi) was added to the apical compartment of cultures at day 3 to 24 postseeding. After 30 min at 37°C, the amount of radioactivity in the basolateral compartment was determined by liquid spectrometry.

    Determination of CFX Concentration.

    CFX concentration was measured by HPLC on a Supelcosil LC18 column (250 × 4.6 mm, 5-μm particle size; Supelco, Touzart et Matignon, France). The HPLC apparatus comprised a WISP 712 automatic sampler (Waters, Paris, France), an SPD-6AV pump, and an SPD-6AV UV detector (Shimadzu, Paris, France) set at 260 nm to monitor the CFX peak that came off at 14 min. The mobile phase was a mixture of sodium acetate buffer (0.01 M, pH 5.2) and acetonitrile (94.5:4.5, v/v). The flow rate was 1.5 ml/min.

    Data Analysis.

    The experiments were performed in triplicate and repeated twice. The data are presented as mean ± S.E.M. The means were compared by paired or unpaired Student's t test, as appropriate; P < .05 was considered significant. The kinetic constants of CFX transport were determined by applying a nonlinear regression method to fit the Michaelis-Menten kinetic equation with the GRAFIT program. Vmaxis the maximal CFX flux and Km is the concentration of CFX that yielded one-half ofVmax. To determine the number of systems involved in CFX transport, the CFX fluxes were transformed according to the Eadie-Hofstee method, by plotting V againstV/S were V is the CFX flux in nanograms per square centimeter per minute and S the apical CFX concentration in millimolar.

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    Results

    Junctional Integrity and Expression of hPepT1 in Caco-2 3B Cell Line.

    Caco-2 is a human colon cancer cell line that has retained the remarkable property to spontaneously differentiate and to behave as a functional epithelium. Thus, this cell line has been extensively used as a model to study transepithelial transport of a large panel of molecules, including dipeptides. Emergence of the differentiated phenotype is growth-related and it can be monitored by measuring changes in transepithelial electrical resistance (TEER) and in paracellular diffusion, which both reflect appearance of tight junctions. The Caco-2 3B clone was recently generated by stable transfection and this model was never used before for transport study. Preliminary experiments were therefore designed to assess its epithelial properties. TEER and [14C]mannitol flux were measured in Caco-2 3B monolayers from day 3 to day 24 postseeding. During the time interval between days 3 and 15, TEER increased 4-fold from 45 ± 6 to 180 ± 5 Ω/cm2. Conversely, the percentage of passive diffusion of mannitol decreased 17-fold from 0.50 ± 0.02 to 0.030 ± 0.002%/cm2/min. The two parameters remained stable between days 15 and 24. All further experiments were carried out on monolayers at days 16 to 17 postseeding.

    Caco-2 cells were previously demonstrated to express the H+/oligopeptide cotransporter PepT1 (Liang et al., 1995). To investigate whether the Caco-2 3B clone has kept this feature, the presence of hPepT1 mRNA was first searched by RT-PCR. RNAs from Caco-2 and HT29 19A cells were used as positive and negative controls in these experiments. As shown in Fig.1A, hPepT1 transcripts also are present in Caco-2 3B clone. The functionality of hPepT1 was then evaluated by measuring CFX transport at pH 6.0 in the apical compartment (Fig. 1B), the optimal pH for CFX absorption in Caco-2 cells (Gochoco et al., 1994). Under these conditions, the basal rate of CFX absorption in Caco-2 3B appears similar to that in Caco-2. Furthermore, addition of an excess of glycyl-sarcosine inhibited CFX fluxes to the same extent (61% diminution in Caco-2 and 57% diminution in Caco-2 3B), indicating that both cell lines displayed PepT1 activity. As already demonstrated by others, the residual CFX flux observed in presence of glycyl-sarcosine is due to passive diffusion (Dantzig and Bergin, 1990;Gochoco et al., 1994). Finally, the expression of α2-adrenergic receptors was estimated by radioligand binding (Fig. 1C). Analysis of [3H]RX821002 saturation isotherms confirmed that Caco-2 cells do not express this receptor. It also indicated that receptor density was 119 ± 14 fmol/mg of protein in Caco-2 3B (n = 12) and 158 ± 11 fmol/mg of protein in HT29 clone 19A (n = 3). Moreover, according to [3H]clonidine binding, 60 to 70% of the receptor population was under high-affinity state for agonists, indicative of efficient coupling to G-protein.

    Figure 1
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    Figure 1

    A, detection by PCR amplification of hPepT1 mRNA in Caco-2 3B, Caco-2, and HT29 clone 19A cell lines. Ten micrograms of total RNA from the indicated cell line was reverse transcribed and first-strand cDNA synthesized was amplified with a set of specific primers described in Experimental Procedures. The PCR products were separated by electrophoresis through a 1% agarose gel and stained with ethidium bromide. GADPH was taken as control for a housekeeping protein. B, effect of glycyl-sarcosine on CFX transport in Caco-2 and Caco-2 3B cell lines. Transport of CFX (1 mM) was measured in confluent monolayers of Caco-2 and Caco-2 3B cells for 30 min at 37°C under continuous circular shaking. pH was 6.0 in the apical compartment and 7.4 in the basolateral compartment. After 15 min of equilibration, CFX ± glycyl-sarcosine (GS; 50 mM) was added to the apical compartment. CFX alone fluxes were measured between 0 and 30 min (CFX alone) and compared with CFX + GS 50 mM fluxes between 0 and 30 min (CFX + GS). Data are mean ± S.E.M. of triplicate measurements repeated twice. *P < .05 versus corresponding CFX alone (unpaired t test). C, α2-adrenergic receptor quantification in Caco-2 3B, Caco-2, and HT29 cl 19A cell lines with Scatchard plots. The expression of α2-adrenergic receptors was assessed by binding studies with [3H]RX821002 (α2-antagonist) as specific radioligand. Binding experiments were performed on crude membranes prepared from frozen cells as described previously (Paris et al., 1990). Specific binding was defined as the difference between total and nonspecific binding measured as described above but in the presence of 10 μM phentolamine.

    Effect of α2-Adrenergic Receptor Stimulation on CFX Absorption.

    To investigate whether activation of α2-adrenergic receptors had any effect on CFX transport, clonidine was added to the basolateral compartment, 15 min after adding CFX to the apical compartment. As depicted in the Fig.2, addition of the α2-agonist caused a rapid increase of the flux of CFX through the Caco-2 3B monolayer. Calculation of the slope of the absolute values of CFX fluxes against time under basal and stimulated conditions (Fig. 2) revealed a doubling of the mean rate of CFX transport from 5.9 ± 0.5 to 12.0 ± 0.6 ng/cm2 · min. The stimulatory effect of clonidine was not observed in the presence of glycyl-sarcosine, indicating that it reflects an increase of PepT1 activity (Fig.3). The effect of clonidine was mimicked by UK14304 (Fig. 3). However, the effects of the two α2-agonists were totally blocked by two specific α2-antagonists with different chemical structures, yohimbine and RX821002, strongly suggesting the involvement of α2-adrenergic receptors in the observed effect (Fig. 3).

    Figure 2
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    Figure 2

    Mean concentrations of CFX versus time in the basolateral compartment and effect of clonidine (clo) 10−5M. Columns represent CFX fluxes measured between 0 and 15 min (CFX alone) compared with fluxes between 15 and 30 min (CFX + clo 10−5 M). Transport of CFX (1 mM) was measured in confluent monolayers of Caco-2 3B cells for 30 min at 37°C under continuous circular shaking. pH was 6.0 in the apical compartment and 7.4 in the basolateral compartment. After 15 min of equilibration, CFX was added to the apical compartment. clo (10−5 M) was added to the basolateral compartment 15 min after CFX. Data are mean ± S.E.M. of triplicate measurements repeated twice. *P < .05 versus corresponding CFX alone (paired t test).

    Figure 3
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    Figure 3

    Effect of α2-adrenergic receptor agonists clonidine (clo), UK14304 (UK) alone, or associated with α2-adrenergic receptor antagonists yohimbine (yo), RX821002 (RX), or with glycyl-sarcosine (GS) on CFX transport. Transport of CFX (1 mM) was measured in confluent monolayers of Caco-2 3B cells for 30 min at 37°C under continuous circular shaking. pH was 6.0 in the apical compartment and 7.4 in the basolateral compartment. After 15 min of equilibration, CFX ± GS (50 mM) was added to the apical compartment. GS (50 mM) was added to the apical compartment just before CFX. clo (10−5 and 10−6 M) and UK (10−5 M) were added to the basolateral compartment 15 min after CFX. The α2-adrenergic receptor antagonists yo (10−5 M) and RX (10−5 M) were added to the basolateral compartment 15 min after CFX, just before adding clo or UK. Results are expressed as the percentage of CFX fluxes measured between 15 and 30 min in the presence of the various agents compared with CFX fluxes between 0 and 15 min (CFX alone). The effect of the α2-adrenergic receptor agonists was suppressed by the antagonists. Data are mean ± S.E.M. of triplicate measurements repeated twice. *P < .05 versus corresponding CFX alone (paired t test).

    The conclusion that clonidine increases CFX transport via stimulation of α2-adrenergic receptor and subsequent activation of PepT1 was further confirmed by the study of the effect of this α2-agonist on Caco-2 and HT29 19A monolayers. Indeed, clonidine failed to activate CFX transport in these two models, which lack either α2-adrenergic receptor or PepT1 (Caco-2, 7.8 ± 0.6 versus 8.4 ± 0.6 ng/cm2 · min; HT29 19A, 0.30 ± 0.01 versus 0.32 ± 0.01 μg/cm2/min), respectively.

    Mechanism of PepT1 Stimulation.

    The stimulation of CFX uptake caused by the α2-agonist may be the consequence of an increase in the affinity of the oligopeptide transporter for its substrate, and/or of an augmentation of the number of transporter molecules functionally available at the apical membrane of Caco-2 3B cells. The effect of clonidine on the kinetics of CFX transport was thus investigated to clarify this point. The transformation of the kinetic data yielded linear Eadie-Hofstee plots (Fig.4), indicating the presence of a single class of transporters in both control and clonidine-treated cells. Furthermore, the treatment with the α2-agonist resulted in a significant (P < .05) increase of theVmax value from 54.4 ± 9.1 to 92.8 ± 13.4 ng/cm2 · min without any modification of the Km value (2.64 ± 0.79 versus 2.78 ± 0.69 mM). These data indicate that another transporter system is not recruited after α2-agonist exposure. They also eliminate the possibility that a change in the intrinsic properties of the oligopeptide transporter occurred.

    Figure 4
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    Figure 4

    Effect of the α2-adrenergic receptor agonist clonidine (clo) on kinetic parameters of CFX flux. Transport conditions as described in Fig. 3. After 15 min of equilibration, CFX was added to the apical compartment. clo (10−5 M) was added to the basolateral compartment 15 min after CFX. Fluxes were measured between 0 and 15 min (CFX alone, ○) and between 15 and 30 min (CFX + clo, ●). A, CFX flux versus CFX concentration. B, CFX flux versus CFX flux/CFX concentration (Eadie-Hofstee plot). Data are mean ± S.E.M. of triplicate measurements repeated twice.

    Previous studies carried out on epithelial cells isolated from intestinal villi demonstrated that α2-adrenergic receptor stimulation by clonidine provokes a raise in the intracellular pH of enterocytes by increasing the Na+/H+ exchanger activity (Sundaram, 1995). Because CFX flux is highly dependent on the H+ gradient between the luminal and the intracellular compartment (Gochoco et al., 1994), the possibility exists that activation of the Na+/H+ exchanger may account for the effect of clonidine on CFX absorption. Transport experiments were therefore conducted in the presence of 10 μM amiloride to check this possibility. As shown in Fig.5, preincubation with the inhibitor of Na+/H+ exchanger modified neither the basal transport of CFX nor the extent of its stimulation by clonidine.

    Figure 5
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    Figure 5

    Effect of amiloride and of colchicine on CFX transport. Transport conditions as described in Fig. 3. Caco-2 3B cells were pretreated with amiloride (10 μM) in the apical compartment, 10 min before CFX, or with colchicine (20 μM) in the apical and basolateral compartments, 25 min before CFX. CFX was then added to the apical compartment. Clonidine (clo; 10−5 M) was added to the basolateral compartment 15 min after CFX. Fluxes were measured between 0 and 15 min (CFX alone) and between 15 and 30 min (CFX alone in a group and CFX + clo at 10−5 M in another). Amiloride did not affect neither basal transport of CFX nor clo stimulation of CFX transport. Colchicine did not affect basal transport of CFX but suppressed clo stimulation of CFX transport. Data are mean ± S.E.M. of triplicate measurements repeated twice. *P < .05 versus CFX alone.

    Because clonidine affected theVmax only, a possible mechanism for α2-agonist effect is an increase of the membranous population of hPepT1 by translocation from a preformed cytoplasmic pool. Such a mechanism was already demonstrated for the effect of insulin on hPepT1 in Caco-2 cells (Thamotharan et al., 1999b). To investigate this possibility we determined whether clonidine still stimulated CFX absorption after treatment of the cells with colchicine. As shown in the Fig. 5, addition of 20 μM colchicine 25 min before CFX load had no effect on the basal flux of CFX, but it completely abolished the stimulatory effect of clonidine.

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    Discussion

    Previous experiments conducted in anaesthetized rats have demonstrated that administration of clonidine resulted in a rapid enhancement of intestinal absorption of CFX through increased activity of the oligopeptide transporter PepT1. This effect might either be the consequence of the stimulation of α2-adrenergic receptors located on neurons from the central nervous system, sympathetic system, or intramural network or be due to the direct activation of α2-adrenergic receptors located on enterocytes. A clone of Caco-2 stably expressing α2-adrenergic receptors (Caco-2 3B; Schaak et al., 2000) was therefore used as a model to determine whether α2-agonists can directly modulate the activity of the dipeptide carrier PepT1 in the absence of nerve endings.

    Preliminary experiments were designed to verify that the transfection and the selection of the Caco-2 3B clone had no incidence on epithelial properties and PepT1 expression. The measurement of TEER and the estimation of mannitol diffusion demonstrated that tightness of Caco-2 3B monolayers was identical with that of the parental cell line. The expression of PepT1 was assessed by RT-PCR and the functionality of the oligopeptide transporter was evidenced by the large inhibition of CFX transport by glycyl-sarcosine. Finally, theKm of PepT1 for CFX in Caco-2 3B (2.64 ± 0.79 mM) was fairly similar to that previously reported in Caco-2 cells (Gochoco et al., 1994).

    Exposure of Caco-2 3B cells to clonidine resulted in a doubling of CFX transport. The involvement of α2-adrenergic receptors in this response is obvious because the effect of clonidine was mimicked by UK14304 (another α2-agonist), blocked by RX821002 or yohimbine (α2-antagonists), and absent in Caco-2 cells, which express PepT1 but not the receptor. However, the implication of PepT1 is proved because the clonidine-induced increase of CFX transport was abolished in the presence of an excess of glycyl-sarcosine and was not observed in HT29 clone 19A, which expresses the α2-receptor but not PepT1.

    Changes in PepT1 activity have been reported in the rat intestine as a consequence of starvation (Ogihara et al., 1999; Thamotharan et al., 1999a), dietary protein content (Erickson et al., 1995; Shiraga et al., 1999), or epithelium injury by 5-fluorouracil (Tanaka et al., 1998). In Caco-2, an augmentation in the amount of PepT1 also was found after complementation of the culture medium with glycyl-glutamine (Walker et al., 1998) and after cell exposure to pentazocine, a selective ligand of ς-receptor (Fujita et al., 1999). In all these cases, up-regulation of PepT1 correlated with increased level of its mRNA. Although mRNA levels were not examined in this study, a modification of PepT1 gene expression is unlikely because the increase of CFX transport occurred within minutes after exposure to the α2-agonist.

    Clonidine also was previously shown as able to raise the intracellular pH of intestinal epithelial cells by increasing the activity of the Na+/H+ exchanger (Sundaram, 1995). An alkalinization of the intracellular compartment might indirectly affect CFX flux because oligopeptide transport is electrogenic and PepT1 activity is highly dependent on the H+-gradient (Fei et al., 1994; Mackenzie et al., 1996). In our experiments, amiloride, however, did not alter the extent of clonidine effect, suggesting that the Na+/H+ exchanger is not involved.

    The determination of PepT1 kinetic parameters showed that the stimulation of CFX flux by clonidine solely resulted from an increase in the population of functionally active PepT1 (increase inVmax but no change inKm). Furthermore, this effect was abolished after microtubule disruption by colchicine. It is thus possible that α2-agonists act by increasing the insertion of transporter molecules, recruited from a preformed cytoplasmic pool, into the apical membrane of Caco-2 3B. A similar mechanism of translocation was demonstrated to account for the effect of insulin on PepT1 activity in Caco-2 cells (Thamotharan et al., 1999b). Further study with anti-PepT1 antibody on brush-border membrane purified from Caco-2 3B is necessary to verify this hypothesis.

    The intracellular messenger responsible for the redistribution of PepT1 molecules was not determined in this study. cAMP and protein kinase C may appear as possible candidates. Indeed, a previous study with Caco-2 has shown that elevation of cAMP levels induced by cholera toxin or heat-labile enterotoxin inhibits PepT1 activity (Muller et al., 1996). Because PepT1 is devoid of site for phosphorylation by protein kinase A but does possess two putative sites for phoshorylation by protein kinase C, and because phorbol esters inhibit the transporter activity (Brandsch et al., 1994), it is thought that the effect of cAMP may be indirectly mediated by protein kinase C (Muller et al., 1996). With the α2-adrenergic receptor being negatively coupled to adenylate cyclase (Remaury et al., 1993), one could expect that decreased intracellular level of cAMP may conversely result in enhanced PepT1 activity. However, two arguments make such a mechanism of action unlikely. First, regardless the cellular system examined, α2-agonists were never found to inhibit protein kinase C. Second, if clonidine and other α2-agonists are able to inhibit forskolin-induced cAMP production in Caco-2 3B cells (Schaak et al., 2000), they are unable to lower the level of this intracellular messenger in basal conditions such as those under which the effects on PepT1 activity are observed. In contrast, α2-agonists per se were found to activate mitogen-activated protein kinase in Caco-2 3B via a cascade of events comprising recruitment of Gi-proteins, Gβγ-subunit-mediated formation of Shc-Grb2-SOS complex, and subsequent activation of mitogen-activated protein kinase kinase 1. Furthermore, stimulation of α2-adrenergic receptors was proved to evoke focal adhesion kinase phosphorylation and a rapid rearrangement of actin cytoskeleton in smooth muscle cells (Richman and Regan, 1998) and preadipocytes (Betuing et al., 1996). In this latter case, the effect of α2-agonists was correlated with stimulation of RhoA via a Gβγ-subunit-independent mechanism. Activation of the RhoA/focal adhesion kinase pathway was not demonstrated yet in Caco-2 3B, but the possibility exists that the effect of α2-agonists on PepT1 is triggered by one of these two cAMP-independent mechanisms. In support to this view, it is worth mentioning that substantial amounts of Gi-proteins were found intracellularly in Caco-2 (Lacombe et al., 1996) and that these proteins were demonstrated to regulate trafficking of a number of transporters, including cystic fibrosis transmembrane conductance regulator (Schwiebert et al., 1994) and aquaporin (Valenti et al., 1998).

    Finally, another point that needs to be addressed is whether our results obtained on a cell line can be extrapolated to the in vivo situation. Indeed, previous studies have shown that α2-adrenergic receptors are abundant in crypt cells, whereas they are scarce in villus cells (Paris et al., 1990; Valet et al., 1993). Conversely, PepT1 amount is high in villus cells but low in crypt cells (Ogihara et al., 1996). It is therefore questionable whether the density of receptors in villus cells is sufficient to affect PepT1 function. The measurement of CFX transport on cells isolated from the villi is presently impossible; but experiments on other clones of Caco-2 expressing a lower amount of receptor may provide an answer to this issue. It is however noteworthy that the density of α2-adrenergic receptors in the villi is high enough to stimulate activity of the Na+/H+ exchanger (Sundaram, 1995). One could hypothesize that the same is true for PepT1.

    In conclusion, our data demonstrate that activation of an epithelial Gi-protein-coupled neurotransmitter receptor can increase the intestinal absorption of peptides and peptidomimetic drugs. Although the mechanisms involved in this effect remain to be elucidated and the relative participation of epithelial and neural receptors remains to be precisely delineated in vivo, the amplitude of the α2-agonist effect is sufficient to be of interest to peptidomimetic drugs poorly absorbed by the intestine and to potentially represent a new field for therapeutic application of α2-agonists.

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    Footnotes

    • Send reprint requests to: Dr. C. Rozé, Institut National de la Santé et de la Recherche Médicale U410, Faculté de Médecine X. Bichat, 16 rue H. Huchard, 75018 Paris, France. E-mail: roze{at}bichat.inserm.fr

    • ↵1 This study was funded in part by Institut de Recherches sur les Maladies de l′Appareil 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
      • Received February 10, 2000.
      • Accepted April 7, 2000.
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