DRUGS AND REAGENTS

[³H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2- imidazoline (RX821002) (59 Ci/mmol) and [³H]clonidine (66 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Courtaboeuf, France), [³²P]NAD⁺ (800 Ci/mmol) from New England Nuclear (Boston, Massachusetts, USA) and [α³²P]UTP (800 Ci/mmol) from ICN (Costa Mesa, California, USA). Phentolamine was donated by Ciba Geigy (Basel, Switzerland), and prazosin hydrochloride and 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK14304) tartrate by Pfizer (Sandwich, Kent, UK). Oxymetazoline, idazoxan, chlorpromazine, forskolin, pertussis toxin, GppNHp, phorbol 12-myristate 13-acetate (PMA), and all other chemicals were from Sigma (St Louis, Missouri, USA). Fetal calf serum (FCS) and G418 sulphate were purchased from Gibco BRL (Cergy Pontoise, France). 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) and genistein were obtained from Calbiochem (La Jolla, California, USA). Radioimmunoassay kits for cAMP determination were from Immunotech (Luminy, France). The human α_2A adrenoceptor gene (α2C10) was kindly provided by Dr R J Lefkowitz (Duke University, Durham, North Carolina, USA).

EXPRESSION VECTOR

The expression vector used to transfect Caco2 cells (pα2C10Eneo) was a bicistronic plasmid obtained as follows. The HPalpha2GEN construct,22 which contains the BamHI-BamHI fragment (5.5 kb) of the α2C10 gene, was digested with KpnI and HindIII restriction enzymes. The KpnI-HindIII fragment corresponding to nucleotides −1280/+1526 relative to the translation start was subcloned into pKS+ (pBluescriptII KS+, Stratagene, La Jolla, California, USA). This construct was then digested by NheI at position −201 of the α2C10 sequence and NotI in the pKS+ polylinker. The purified insert was cloned into the XbaI and NotI sites of the bicistronic vector, pEN.23 The resulting pα2C10Eneo vector (fig 1) contains an expression cassette comprising the human cytomegalovirus early promoter/enhancer (pCMV), the entire ORF encoding for α_2Aadrenoceptor (α2C10), an internal ribosome entry site derived from encephalomyocarditis virus (EMCV), the neomycin phosphotransferase gene, and a rabbit β globin genomic sequence containing an intron and a polyadenylation signal (IVS2-β).

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Figure 1

Schematic map of the bicistronic expression vector used for transfection of Caco2 cells. The pα2C10Eneo vector is 7.63 kb in size. It contains an expression cassette comprising the human cytomegalovirus early promoter/enhancer (pCMV), the gene encoding human α_2A adrenoceptor subtype (α2C10), the internal ribosomal entry site derived from the encephalomyocarditis virus (EMCV), the neomycin phosphotransferase gene (Neo), a rabbit β globin genomic fragment containing sequences for mRNA stabilisation and polyadenylation (IVS2-β).

CELL CULTURE AND TRANSFECTION

The human colon adenocarcinoma cell line, Caco2, was routinely subcultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and non-essential amino acids. Cells were transfected using the calcium-phosphate method. Two days after transfection, they were subcultured in the presence of G418 sulphate (1 mg/ml), and antibiotic resistant clones were collected individually using cloning cylinders.

ENTEROCYTIC DIFFERENTIATION AND HYDROLASE ACTIVITIES

Immunofluorescence studies were performed on post-confluent cells cultured on Costar Transwell filters (Dutscher, Brumath, France). Cell monolayers were fixed with 3.7% paraformaldehyde-30 mM sucrose in phosphate buffered saline (PBS) for 15 minutes and treated for 10 minutes with 50 mM NH₄Cl in PBS to reduce non-specific background. The cells were permeated with 0.05% saponin in PBS containing 1% skimmed milk and all subsequent steps were performed in the same buffer. The filters were cut out and incubated for one hour with phalloidin-FITC conjugate. After extensive washing, samples were mounted with Mowiol and viewed under a confocal laser microscope (Zeiss LSM). Brush border associated enzyme activities were measured on whole cell homogenates. Alkaline phosphatase and dipeptidyl peptidase IV were assayed usingp-nitrophenyl phosphate and glycyl-l-proline-4-nitroanilide, respectively, as substrates.24 ,25 Values are expressed as milliunits per mg of protein, one unit being the activity that hydrolyses 1 μmol of substrate per minute at 37°C.

RNA EXTRACTION AND RPA

Total cellular RNAs were isolated using the guanidine isothiocyanate/phenol chloroform extraction method.26Synthesis of the α2C10 antisense has been described previously.27 RNase protection assays (RPA) were performed as follows: 100 μg of RNA were added to 30 μl of hybridisation buffer (80% deionised formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM Pipes, pH 6.7) containing an excess of [³²P] labelled riboprobe. Samples were heated to 95°C for five minutes and then immediately kept at 55°C for 14 hours. Non-hybridised probe was eliminated by addition of 0.3 ml of RNase A (40 μg/ml) and RNase T1 (2 μg/ml) in 300 mM NaCl, 5 mM EDTA, and 10 mM Tris HCl (pH 7.5). After two hours at 37°C, digestion was stopped by addition of 5 μl of proteinase K (10 mg/ml) and the samples were further incubated for 15 minutes at 37°C. Carrier tRNA (10 μg) and 0.3 ml of solution D (4 M guanidine isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.1 M 2-mercaptoethanol and 0.5% sarkosyl) were added to each tube and protected hybrids were precipitated with isopropyl alcohol. RNA pellets were washed with 70% ethanol, air dried, taken up in sample buffer (97% deionised formamide, 0.1% sodium dodecyl sulphate (SDS), 10 mM Tris HCl, pH 7.0), and run on a 5% acrylamide gel containing 7 M urea. The gels were exposed for 48 hours at −80°C to xray film for autoradiography.

RECEPTOR QUANTIFICATION AND Gi PROTEIN IDENTIFICATION

Receptors were quantified on crude membrane preparations using the selective radioligands [³H]RX821002 (α₂antagonist) and [³H]clonidine (α₂agonist).28 Specific binding was defined as the difference between total and non-specific binding measured in the presence of 10 μM phentolamine. Saturation isotherms and inhibition curves were analysed using EBDA-LIGAND computer programs.29 ADP ribosylation was carried out essentially as described previously.30 Membranes (50–75 μg of protein) suspended in Tris HCl buffer containing 1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.05% lubrol (v/v) were incubated for 60 minutes at 30°C in the presence of 1 μCi [³²P]NAD⁺ and 100 ng of preactivated pertussis toxin in a final volume of 60 μl. The reaction mixture contained 70 mM Tris HCl (pH 8.0), 0.5 μM NAD⁺, 1 mM ATP, 10 mM thymidine, 1 mM EDTA, 0.1 mM MgCl₂, 1 mg/mll-myristyl phosphatidylcholine, 10 mM nicotinamide, and 25 mM DTT. The reaction was stopped by addition of 2 μg of bovine serum albumin (BSA) in 0.04% SDS and the proteins were precipitated with 70 μl of 10% trichloracetic acid. After centrifugation, pellets were washed twice with diethylether and finally dissolved in Laemmli sample buffer. The labelled proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and revealed by autoradiography.

cAMP MEASUREMENT

Cells were detached in PBS containing 0.6 mM EDTA and collected by gentle centrifugation (400 g, five minutes). The pellet was suspended in DMEM buffered with 25 mM Hepes (pH 7.4). Aliquots of the cell suspension were incubated in a 200 μl final volume of Hepes buffered DMEM containing 0.2 mM IBMX and the indicated concentration of the drug to be tested. After 15 minutes at 37°C, the reaction was stopped by adding 1.8 ml of methanol/formic acid (95/5, v/v). The alcoholic extract was centrifuged (3000g, 10 minutes, 4°C) and an aliquot of supernatant evaporated. The dry samples were taken up in acetate buffer containing 0.1% NaN₃, and cAMP content was determined by radioimmunoassay.

DETECTION OF PHOSPHORYLATED Erk1/Erk2 AND Shc

Cells were seeded at low density (10⁵cells/cm²) in 100 mm culture dishes. Three days later, they were rendered quiescent by incubation in FCS free DMEM for 48 hours. They were then treated for the indicated period of time with the compound to be tested, rapidly rinsed with ice cold PBS, and lysed with 1 ml of radio-immunoprecipitation (RIPA) buffer (10 mM Tris HCl, pH 7.4, 1% Triton X100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulphonyl fluoride, and 0.5 μM aprotinin). Cell lysates were sonicated, clarified by centrifugation for 15 minutes at 15 000 g, and stored at −80°C until analysis.

The extent of MAPK phosphorylation was measured either on immunoprecipitated tyrosyl phosphorylated proteins using antibodies to extracellular regulated protein kinase 1 (Erk1) and 2 (Erk2) or directly on total cell protein extract using antibody to active MAPK. Tyrosyl phosphorylated proteins were immunoprecipitated (three hours at room temperature) by incubating 2 mg of total cell proteins with protein G-sepharose beads conjugated with an antiphosphotyrosine monoclonal antibody (PY20, Transduction Laboratories, Lexington, Kentucky, USA). The beads were washed four times with RIPA, dried, suspended in 50 μl of Laemmli buffer, boiled for five minutes, and centrifuged for 10 minutes at 15 000 g. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Blots were incubated for two hours at room temperature in TBST (10 mM Tris HCl buffer, pH 8.0, 150 mM NaCl, 0.2% Tween 20) containing 5% skimmed milk and then overnight at 4°C in the same buffer containing rabbit polyclonal antibodies to Erk1 and Erk2 (anti-Erk1, 1:250; anti-Erk2, 1:400; Santa Cruz Biotechnologies, Santa Cruz, California, USA). After extensive washing, blots were exposed for one hour to horseradish peroxidase conjugated donkey antirabbit IgG. Immunoreactive proteins were visualised by chemiluminescence (ECL Western blotting system, Amersham Pharmacia Biotech, Courtaboeuf, France). The intensity of the bands was analysed using ImageQuant software (Molecular Dynamics, Sunnyvale, California, USA). In some experiments, activated Erk1/Erk2 were detected with a rabbit polyclonal antibody recognising the phosphorylated sequence of the active forms of MAPK (Promega, Madison, Wisconsin, USA). In this case, total cell proteins (30 μg) were separated by SDS-PAGE and transferred onto a Polyscreen/polyvinylidene difluoride (PVDF) membrane (NEN Life Science, Belgium). Blots were treated for one hour in TBST containing 0.1% BSA and then incubated overnight at 4°C in the same buffer containing antiactive MAPK antibody (1:3000). After extensive washing, bound antibody was detected with the horseradish peroxidase conjugated secondary antibody and revealed by chemiluminescence. The membrane was then stripped of Ig and reprobed using anti-Erk2 antibody to assess equal loading. For determination of Shc phosphorylation, 500 μg of cell proteins were immunoprecipitated using 5 μg of rabbit polyclonal Shc antibody (Upstate Biotechnology, Lake Placid, New York, USA). After overnight incubation at 4°C, the immunocomplex was precipitated by addition of 50 μl of protein A-agarose beads (Transduction Laboratories, Lexington, Kentucky, USA). After several washes in RIPA, beads were dried, suspended in Laemmli sample buffer, boiled for five minutes, and centrifuged for 10 minutes at 15 000 g. The proteins were subjected to SDS-PAGE, electrotransferred to a PVDF membrane, and probed with an antiphosphotyrosine-horseradish peroxidase conjugated monoclonal antibody (ECL phosphorylation detection system, Amersham Pharmacia Biotech, Courtaboeuf, France). The membrane was stripped of Ig and reprobed using anti-Shc antibody to assess equal loading.

CELL PROLIFERATION ASSAY

Cells were seeded at a density of 10⁵cells/cm² in 24 well plates. Three days after seeding they were rendered quiescent by incubation in FCS free culture medium for 48 hours. Experiments were started when replacing the cells in DMEM supplemented with 0.5% FCS with or without 1 μM UK14304. The medium was changed every day. At the indicated time following reinitiation of cell proliferation, the effect of the α₂ agonist was estimated by measuring total cellular protein and DNA content. Protein concentration was determined using the Coomassie blue method.31 DNA was measured by a fluorometric method using DAPI.32

STATISTICAL ANALYSIS

Results are expressed as mean (SEM) for the number of observations indicated (n). Data were analysed using the Student'st test, and a value of p<0.05 was considered statistically significant.

EXPRESSION AND FUNCTIONAL ACTIVITY OF HUMAN α_2A ADRENOCEPTOR IN Caco2 CELL TRANSFECTANTS

The Caco2 human colonic adenocarcinoma cell line undergoes enterocytic differentiation under standard culture conditions. This process is growth related. In the latter stages of the culture, cells are organised as a polarised monolayer with tight junctions and an apical brush border,33 and form domes that are indicative of transepithelial transport properties.34 RPA and RT-PCR experiments have previously demonstrated that Caco2 cells do not spontaneously contain α₂adrenoceptors.27 ,35 In order for this model to express the same receptor subtype as normal human mucosa, the Caco2 cells were transfected with the bicistronic construct pα2C10Eneo (fig 1). Of the 20, G418 resistant clones which were isolated and subcultured, five were submitted to a first round of screening using RPA with a specific riboprobe derived from the α2C10-AR gene. As shown in the autoradiogram in fig 2, one of the five clones (clone 1A) was negative whereas the others contained substantial amounts of transcript. Binding experiments using [³H]RX821002 (α₂antagonist) were thus performed to check the level of receptor expression. As expected, no specific radioligand binding was detected in the parental cell line or in clone 1A. All other clones displayed radioligand binding sites but at densities ranging from 28 to 300 fmol/mg of membrane protein. The clone 3B (Caco2-3B), which expresses the receptor at a level similar to that in epithelial cells from normal human intestinal mucosa,9 was selected for further studies. Data from a typical saturation binding experiment on Caco2-3B membranes is presented in fig 3. The number of [³H]RX821002 binding sites was not dependent on the stage of culture (230 (15) and 195 (23) fmol/mg of protein in pre-confluent and post-confluent cells, respectively) and remained stable over at least 30 successive cell passages. From phase contrast and confocal microscopy, Caco2-3B was seen to exhibit the same pattern of enterocytic differentiation as the parental cell line. In addition, measurement of enzymatic activities in cell extracts showed that brush border associated hydrolases were expressed (alkaline phosphatase 85 (12) mU/mg protein; dipeptidyl peptidase IV, 59 (7) mU/mg protein, n=3). Inhibition of [³H]RX821002 binding by various adrenergic compounds indicated that the Ki values for yohimbine (2.9 (1.3) nM), idazoxan (3.7 (1.2) nM), phentolamine (9.2 (3.1) nM), chlorpromazine (2630 (350) nM), and prazosin (2200 (420) nM) matched the pharmacological properties expected for an α_2Aadrenoceptor subtype.

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Figure 2

Screening of positive clones by RPA. RNA from HT29 (used as a positive control), Caco2, and G418 resistant clones were hybridised with [³²P]labelled α2C10 probe. Samples were digested with a mixture of RNases A and T1. The resistant hybrids were analysed by electrophoresis. A representative autoradiogram is shown. Lanes P and (−) correspond to the undigested probe and negative control, respectively, carried out on 100 μg of tRNA.

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Figure 3

Saturation isotherm of [³H]RX821002 binding to Caco2 and Caco2-3B cell membranes. Membranes prepared from Caco2 and Caco2-3B were incubated in the presence of various concentrations of radioligand. The amount of specifically bound [³H]RX821002 was determined using 10 μM phentolamine to estimate non-specific binding. The data presented are from a typical experiment. The inset shows the corresponding Scatchard plot. Computer assisted analysis of the results from this specific experiment indicated that the Bmax and Kd values of [³H]RX821002 were, respectively, 198 (12) fmol/mg of protein and 1.05 (0.11) nM in membranes from Caco2-3B. No specific binding was detected on membranes from Caco2 cells.

The degree of Gi protein coupling was analysed by examining the ability of the receptor to exist under a high affinity state for agonists (fig4). In contrast with the situation with antagonists, the UK14304 competition curve was shallow and was better fitted to a two site inhibition model. According to computer assisted analysis of the data from three independent experiments, the Ki values for UK14304 for the high and low affinity state receptor were, respectively, 1.05 (0.23) and 108 (30) nM. The proportion with the high affinity conformation for agonist represented 62 (6)% of the whole receptor population. This fraction was abolished in the presence of Gpp(NH)p/Na⁺ or in membranes prepared from cells treated for 16 hours with pertussis toxin (250 ng/ml). Furthermore, [³²P]ADP ribosylation demonstrated that Caco2-3B expressed the same pertussis toxin sensitive G proteins as the parental cell line (fig 4, inset). Two peptides with apparent molecular masses of 40 and 41 kDa were labelled. Immunoblotting with specific antibodies indicated that they corresponded to αi2 and αi3 subunits (not shown). Further evidence for receptor coupling to Gi proteins was provided by the direct study of [³H]clonidine binding. Indeed, this [³H]agonist labelled 122 (18) fmol of sites/mg of protein with high affinity (Kd 2.5 (0.9) nM). As expected, no significant [³H]clonidine binding was detected in membranes from Caco2-3B cells treated with pertussis toxin. In cells isolated from human and rat colonic crypts,11 ,12 or in the human adenocarcinoma cell line HT29,13 α₂ adrenoceptors were negatively coupled to adenylyl cyclase. In the next series of experiments we asked whether this pathway of signal transduction is also efficient in Caco2-3B. Measurement of intracellular cAMP levels indicated that Caco2 and Caco2-3B cells were similarly responsive to forskolin exposure (table 1). UK14304, clonidine, or (−)adrenaline were ineffective in the parental cell line but significantly inhibited forskolin induced cAMP accumulation in Caco2-3B. It is worth mentioning that neither UK14304 nor any of the other tested agonists lowered basal cAMP levels. Further experiments demonstrated that the inhibitory effect of UK14304 on forskolin induced cAMP production was dose dependent (EC₅₀ 12 (5) nM) and abolished by addition of α₂ antagonist (not shown).

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Figure 4

Analysis of receptor coupling and identification of Gi proteins. Caco2-3B cells were or were not treated with 250 ng/ml of pertussis toxin for 16 hours. Cell membrane preparations were incubated in the presence of 3 nM [³H]RX821002 and increasing concentrations of UK14304. The experiments were carried out in the absence or presence of 10 μM GppNHp with 100 mM NaCl. The curves presented are from a specific experiment. Inset: membranes from rat brain, Caco2, and Caco2-3B were ADP ribosylated with pertussis toxin in the presence of [³²P]NAD⁺. The labelled proteins were resolved on SDS-PAGE and autoradiographed as described in materials and methods.

EFFECT OF α₂ADRENOCEPTOR STIMULATION ON MAPK ACTIVATION AND CELL PROLIFERATION

The effect of receptor stimulation on MAPK was investigated by immunoprecipitation of tyrosine phosphorylated proteins followed by immunodetection with anti-Erk1/Erk2 antibodies (fig 5). In Caco2 and Caco2-3B, these antibodies recognised two proteins with apparent molecular masses of 44 and 42 kDa. Treatment of Caco2-3B with UK14304 resulted in a rapid increase in the extent of phosphorylation of the two forms of MAPK. Phosphorylation was observed as early as two minutes after treatment, was maximal at five minutes, and persisted for at least 15 minutes. On the basis of the analysis of seven independent experiments, the maximal effect observed after five minutes of exposure represents a twofold increase in phosphorylation. No significant change in phosphorylation of MAPK was detected in Caco2 cells, demonstrating that the effect of UK14304 was primarily due to stimulation of α₂ adrenoceptors. The mechanisms whereby stimulation of α_2A adrenoceptor increases MAPK phosphorylation were then investigated directly using antiactive MAPK antibody on total cellular extracts. Unlike anti-Erk antibodies on immunoprecipitated phosphoproteins (as in fig 5), the antiactive MAPK antibody revealed a major band corresponding to phosphorylated Erk2 (see figs 6, 7). The western blots presented in fig 6 clearly show that the increase in Erk2 phosphorylation following exposure to UK14304 was totally abolished by pretreatment of the cells with pertussis toxin (250 ng/ml, 16 hours). Thus the integrity of Gi proteins is necessary for the effect of the α₂ agonist to occur. In the same manner, addition of the inhibitor of protein tyrosine kinases, genistein (25 μM), or addition of the inhibitor of the MEK1 form of MAPK kinases, PD98059 (50 μM), prevented the effect of UK14304. As shown in fig 7, acute stimulation of protein kinase C (PKC) by addition of 2.5 μM PMA for five minutes also caused marked activation of MAPK. Long term treatment of cells with PMA totally abolished any further response to the phorbol ester but did not affect the response to UK14304, suggesting that α₂ adrenoceptors trigger their effects through a PKC independent pathway. As activation of MAPK by several G protein coupled receptors was shown to correlate with Shc phosphorylation in Rat-1 fibroblasts,36 we examined if such a phosphorylation also occurs on α_2A adrenoceptor stimulation in Caco2-3B cells. The use of anti-Shc antibody on extracts prepared from A431 and Caco2-3B cells (fig 8) indicated that our model predominantly expressed the 46 and 52 kDa isoforms of Shc. Furthermore, treatment of the cells with 1 μM UK14304 caused increased phosphorylation of both isoforms of Shc. Activation was notable after two minutes of treatment with the α₂ agonist and was transient, a decrease in phosphorylation being observed after 10 minutes. The time course of Shc phosphorylation was thus compatible with activation of Shc preceding that of MAPK.

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Figure 5

Effect of UK14304 on mitogen activated protein kinase (MAPK) phosphorylation. Caco2 and Caco2-3B cells were seeded at low density (10⁵ cells/cm²) and grown for three days in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. Pre-confluent cell layers were then maintained for 48 hours in serum free medium and treated for the indicated time with 1 μM UK14304. Left: tyrosine phosphorylated proteins were immunoprecipitated, separated by SDS-PAGE, and MAPK revealed by immunoblotting with anti-Erk1 and anti-Erk2 antibodies as described in materials and methods. Right: the extent of MAPK phosphorylation is expressed as a per cent of that in control cells (untreated). Results are mean (SEM) (n=3 for Caco2, n=7 for Caco2-3B). Significantly different from untreated cells: *p<0.05, **p<0.02.

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Figure 6

Effect of pertussis toxin (PTX), genistein, and PD98059 on mitogen activated protein kinase (MAPK) phosphorylation. Caco2-3B cells were grown and rendered quiescent as indicated in the legend of fig 5. The cells were treated for 16 hours with pertussis toxin (250 ng/ml) or for 30 minutes with either genistein (25 μM) or PD98059 (50 μM). They were then exposed (+) or not (−) to 1 μM UK14304 for five minutes. Top: MAPK phosphorylation was studied using the anti-active MAPK antibody. Bottom: the membrane was stripped of Ig and reprobed with anti-Erk2 antibody to assess equal protein loading.

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Figure 7

Effect of phorbol 12-myristate 13-acetate (PMA) on UK14304 induced mitogen activated protein kinase (MAPK) phosphorylation. The Caco2-3B cells were grown and rendered quiescent, as indicated in the legend of fig 5, and treated for 16 hours with 2.5 μM PMA (treated) or vehicle (control). They were then exposed for five minutes to vehicle (−), 2.5 μM PMA (PMA), or 1 μM UK14304 (+). Top: MAPK phosphorylation was studied using the anti-active MAPK antibody. Bottom: the membrane was stripped of Ig and reprobed with anti-Erk2 antibody to assess equal protein loading.

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Figure 8

Identification of Shc isoforms and effect of UK14304 on phosphorylation. Left: identification of the Shc isoforms expressed in Caco2-3B cells. Protein extracts from Caco2-3B and A431 cells (used here as positive control) were subjected to SDS-PAGE and Shc isoforms were revealed by western blot using anti-Shc antibody. Right: UK14304 induced phosphorylation of Shc. The Caco2-3B cells were grown, rendered quiescent as indicated in the legend of fig 5 and then treated for the indicated time with 1 μM UK14304. Shc proteins were immunoprecipitated with anti-Shc antibody and the extent of their phosphorylation estimated by western blot with an antiphosphotyrosine antibody (right upper panel). The membrane was stripped of Ig and reprobed using anti-Shc antibody to assess equal protein loading (right lower panel).

Activation of MAPK being generally associated with cellular proliferation, we finally examined the effect of α₂adrenoceptor stimulation on the growth rate of Caco2-3B. No effect was detected when UK14304 was tested in serum free or in 10% FCS medium. However, an accelerated proliferation was repeatedly observed when assays were carried out in culture medium containing 0.5% FCS. The proliferative effect of the α₂ agonist was modest but significant. As estimated by measurement of total cellular protein and DNA content, it represented a 20% increase in cell proliferation after 16 days of culture (fig 9). Furthermore, this effect was solely due to α₂ adrenoceptor activation because it was not observed in the parental cell line Caco2 (not shown).

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Figure 9

Effect of UK14304 on cell growth. Caco2-3B cells were seeded at low density (10⁵ cells/cm²). Three days after seeding, they were rendered quiescent in serum free medium for 48 hours. Cellular growth was then reinitiated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.5% fetal calf serum with or without 1 μM UK14304. Protein content (left) and DNA content (right) per well were determined as described in materials and methods. Data presented are from a typical experiment and the results are mean (SEM) (n=3). Significantly different from untreated cells: *p<0.05, **p<0.01.