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Linoleic Acid Induces Calcium Signaling, Src Kinase Phosphorylation, and Neurotransmitter Release in Mouse CD36-positive Gustatory Cells

2008; Elsevier BV; Volume: 283; Issue: 19 Linguagem: Inglês

10.1074/jbc.m707478200

1083-351X

Abdelghani El-Yassimi, Aziz Hichami, Philippe Besnard, Naim Akhtar Khan,

Olfactory and Sensory Function Studies

We have recently demonstrated that the cells expressing CD36, localized apically on the taste buds of mouse lingual circumvallate papillae, act as gustatory cells. In the present study we isolated these CD36-positive cells from mouse circumvallate papillae and investigated intracellular signaling events, triggered by a long-chain polyunsaturated fatty acid, i.e. linoleic acid (LA). LA induced increases in free intracellular calcium concentrations, [Ca2+]i, by recruiting calcium from endoplasmic reticulum pool via inositol 1,4,5-triphosphate production followed by calcium influx via opening of store-operated calcium (SOC) channels. LA also induced phosphorylation of Src-protein-tyrosine kinases (Src-PTKs), particularly of Fyn59 and Yes62. LA-evoked phosphorylation of Fyn59 and Yes62 was implicated in the activation of SOC channels. Reverse transcription-quantitative PCR revealed that the CD36-positive gustatory cells possessed mRNA of enzymes like tryptophan hydroxylase-1, l-aromatic amino acid decarboxylase, tyrosine hydroxylase, and dopamine β-hydroxylase, involved in the synthesis of monoamine neurotransmitters. Interestingly, the addition of LA to these cells induced the release of 5-hydroxytryptamine and noradrenalin to the extracellular environment. The LA-induced release of these neurotransmitters was curtailed by SOC channel blockers and Src-PTK inhibitors. These results altogether demonstrate that LA binds to mouse CD36-positive gustatory cells, induces Src-PTKs phosphorylation, triggers calcium signaling, and evokes the release of 5-hydroxytryptamine and noradrenalin, which in turn may be implicated in the downstream signaling to the afferent nerve fibers, thus transmitting the output signal from taste buds to the central nervous system. We have recently demonstrated that the cells expressing CD36, localized apically on the taste buds of mouse lingual circumvallate papillae, act as gustatory cells. In the present study we isolated these CD36-positive cells from mouse circumvallate papillae and investigated intracellular signaling events, triggered by a long-chain polyunsaturated fatty acid, i.e. linoleic acid (LA). LA induced increases in free intracellular calcium concentrations, [Ca2+]i, by recruiting calcium from endoplasmic reticulum pool via inositol 1,4,5-triphosphate production followed by calcium influx via opening of store-operated calcium (SOC) channels. LA also induced phosphorylation of Src-protein-tyrosine kinases (Src-PTKs), particularly of Fyn59 and Yes62. LA-evoked phosphorylation of Fyn59 and Yes62 was implicated in the activation of SOC channels. Reverse transcription-quantitative PCR revealed that the CD36-positive gustatory cells possessed mRNA of enzymes like tryptophan hydroxylase-1, l-aromatic amino acid decarboxylase, tyrosine hydroxylase, and dopamine β-hydroxylase, involved in the synthesis of monoamine neurotransmitters. Interestingly, the addition of LA to these cells induced the release of 5-hydroxytryptamine and noradrenalin to the extracellular environment. The LA-induced release of these neurotransmitters was curtailed by SOC channel blockers and Src-PTK inhibitors. These results altogether demonstrate that LA binds to mouse CD36-positive gustatory cells, induces Src-PTKs phosphorylation, triggers calcium signaling, and evokes the release of 5-hydroxytryptamine and noradrenalin, which in turn may be implicated in the downstream signaling to the afferent nerve fibers, thus transmitting the output signal from taste buds to the central nervous system. The tongue contains primarily four types of papillae. Filiforms are involved in the somesthesic perception of foods, whereas fungiforms, foliates, and circumvallates, which contain taste buds, are responsible for the chemosensory perception of basic taste modalities (sweet, bitter, salt, sour, umami). Recent evidences have strongly suggested the existence of an additive oro-sensory system devoted to the dietary fat perception in rodents. Gilbertson et al. (1Gilbertson T.A. Fontenot D.T. Liu L. Zhang H. Monroe W.T. Am. J. Physiol. 1997; 272: C1203-C1210Crossref PubMed Google Scholar) have reported that lipids may be sensed by isolated rat fungiform taste receptor cells (TRC) 2The abbreviations used are: TRC, taste receptor cells; CVP, circumvallate papillae; 5-HT, 5-hydroxytryptamine (serotonin); PLC, phospholipase C; PTK, protein-tyrosine kinase; SSO, sulfo-N-succinimidyl oleate; PBS, phosphate-buffered saline; IP3, inositol 1,4,5-triphosphate; LA, linoleic acid; PVDF, polyvinylidene difluoride; TBS, Tris-buffered saline; RT, reverse transcription; Ct, cycle threshold; HPLC, high performance liquid chromatography; ER, endoplasmic reticulum; NA, noradrenalin; SOC, store-operated calcium. via the inhibition of the delayed rectifying K+ channels by polyunsaturated fatty acids. Fukuwatari et al. (2Fukuwatari T. Kawada T. Tsuruta M. Hiraoka T. Iwanaga T. Sugimoto E. Fushiki T. FEBS Lett. 1997; 414: 461-464Crossref PubMed Scopus (208) Google Scholar) and Laugerette et al. (3Laugerette F. Passilly-Degrace P. Patris B. Niot I. Febbraio M. Montmayeur J.P. Besnard P. J. Clin. Investig. 2005; 115: 3177-3184Crossref PubMed Scopus (518) Google Scholar) documented the expression of the receptor-like lipid-binding protein CD36 in rat and mouse taste bud cells, respectively. Kawai and Fushiki (4Kawai T. Fushiki T. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2003; 285: 447-454Crossref PubMed Scopus (189) Google Scholar) demonstrated that the addition of a lipase inhibitor diminished the spontaneous preference for triglycerides. These investigators proposed that the lingual lipase, present in the rodent saliva, might release free fatty acids that would be detected by gustatory cells. The immunolocalization of CD36 in the apical side of few TRC in circumvallate papillae (3Laugerette F. Passilly-Degrace P. Patris B. Niot I. Febbraio M. Montmayeur J.P. Besnard P. J. Clin. Investig. 2005; 115: 3177-3184Crossref PubMed Scopus (518) Google Scholar) is especially adequate for this function since CD36 is known to exhibit a very high affinity for long-chain fatty acids (5Baillie A.G. Coburn C.T. Abumrad N.A. J. Membr. Biol. 1996; 153: 75-81Crossref PubMed Scopus (188) Google Scholar). Consistent with this assumption, we have recently provided the first evidence that CD36-positive gustatory cells play a significant role in dietary lipid perception in the mouse (3Laugerette F. Passilly-Degrace P. Patris B. Niot I. Febbraio M. Montmayeur J.P. Besnard P. J. Clin. Investig. 2005; 115: 3177-3184Crossref PubMed Scopus (518) Google Scholar). Indeed, the CD36 gene inactivation fully abolished the spontaneous preference for long-chain fatty acids observed in wild-type mice (3Laugerette F. Passilly-Degrace P. Patris B. Niot I. Febbraio M. Montmayeur J.P. Besnard P. J. Clin. Investig. 2005; 115: 3177-3184Crossref PubMed Scopus (518) Google Scholar). It is noteworthy that this effect on feeding behavior is lipid-specific since sweet preference and bitter aversion are not affected in these transgenic mice (3Laugerette F. Passilly-Degrace P. Patris B. Niot I. Febbraio M. Montmayeur J.P. Besnard P. J. Clin. Investig. 2005; 115: 3177-3184Crossref PubMed Scopus (518) Google Scholar). To further explore whether a sixth taste modality devoted to the "fat" functionally exists in rodents, the downstream signaling events triggered by the free fatty acid/CD36 interaction in gustatory cells must be studied. We have for the first time purified the CD36-positive gustatory cells from mouse CVP and demonstrated that linoleic acid induced increases in [Ca2+]i in these cells via CD36 (6Gaillard, D., Laugerette, F., Darcel, N., El-Yassimi, A., Degrace-Passily, P., Hichami, A., Khan, N. A., Montmayeur, J. P., and Besnard, P. (2007) FASEB J., in pressGoogle Scholar). In the present study we have extended these investigations to characterize the mechanisms of action of linoleic acid on calcium signaling/protein phosphorylation, leading to the release of neurotransmitters, which might be implicated in the activation of afferent nerve fibers. Materials—C57BL/6J mice were obtained from Janvier Elevage (Le Genest-st-isle, France). Sulfo-N-succinimidyloleate (SSO) was a generous gift from JF Glatz (Maastricht, The Netherlands). The culture medium RPMI 1640 and l-glutamine were purchased from Lonza Verviers SPRL (Verviers, Belgium). Fura-2/AM was procured from Invitrogen. All other chemicals including linoleic acid (18:2 n-6), collagenase type-I, and trypsin inhibitor were obtained from Sigma. SKF96365, econazole, and SU6656 were purchased from Calbiochem. Elastase and dispase were purchased from Serlabo (Bonneuil/Marne, France) and Roche Diagnostics, respectively. Anti-CD36 antibody coupled to phycoerythrin and anti-α-gustducin antibody were procured from Santa Cruz Biotechnology, Inc. Anti-p-Fyn59, anti-p-Lyn53/56, and anti-p-Yes62 antibodies were bought from Tebu-Bio (Paris, France), and anti-phosphotyrosine kinase (p-PTK) antibody was obtained from Chemicon (Dundee, UK). Anti-CD36 antibody was procured from Cell Sciences®. All the experiments on animals were conducted as per declaration of Helsinki and European ethical guidelines, and the protocols were approved by the Regional Ethical Committee. Isolation of CD36-positive Cells from Mouse Circumvallate Papillae (CVP)—Mouse lingual CVP were isolated according to previously published procedure (3Laugerette F. Passilly-Degrace P. Patris B. Niot I. Febbraio M. Montmayeur J.P. Besnard P. J. Clin. Investig. 2005; 115: 3177-3184Crossref PubMed Scopus (518) Google Scholar). Lingual epithelium was separated from connective tissues by enzymatic dissociation (elastase and dispase mixture, 2 mg/ml each, in Tyrode buffer: 120 mm NaCl, 5 mm KCl, 10 mm HEPES, 1 mm CaCl2, 1 mm MgCl2, 10 mm glucose, 10 mm sodium pyruvate, pH 7.4), and papillae were dissected under a microscope. The CD36-positive cells from mouse CVP were purified as described elsewhere (6Gaillard, D., Laugerette, F., Darcel, N., El-Yassimi, A., Degrace-Passily, P., Hichami, A., Khan, N. A., Montmayeur, J. P., and Besnard, P. (2007) FASEB J., in pressGoogle Scholar). In brief, cells were isolated by incubation in RPMI 1640 medium containing 2 mm EDTA, 1.2 mg/ml elastase, 0.6 mg/ml collagenase (type I), and 0.6 mg/ml trypsin inhibitor at 37 °C for 20 min followed by a centrifugation (600 g × 10 min). The mixture of different cell populations was incubated with anti-CD36 antibody coupled to phycoerythrin for 2 h followed by a wash with PBS (600 g × 10 min) and resuspended in a solution containing microbeads coupled to anti-phycoerythrin IgG. The CD36-positive cells were isolated by passing through the MACS columns of the Miltenyi magnet system. The CD36-negative cells passed through the column, whereas CD36-positive cells were retained therein. Both the cells populations, after separation, were suspended in a fresh RPMI 1640 medium containing 10% fetal calf serum, 200 units/ml penicillin, and 0.2 mg/ml streptomycin, seeded onto a Biocoat poly-d-lysine-coated dishes, and cultured for 24 h. At the end of this period cells were used for the experiments or stained with trypan blue to assess their viability. To check the purity of CD36-positive cells, we performed an immunocytochemical detection of CD36 and α-gustducin after selection. Hence, cytospin-prepared slides were fixed in 95% ethanol and rehydrated in 0.1 m PBS, pH 7.4. Slides were blocked in PBS containing 5% fetal calf serum and 0.2% Triton X-100 for 30 min at room temperature before overnight incubation at 4 °C with a 1:100 dilution of anti-rat CD36 antibody (UA009) or a 1:150 dilution of polyclonal anti-mouse α-gustducin antibody. It is noteworthy that the clone UA009 also recognizes mouse CD36 (3Laugerette F. Passilly-Degrace P. Patris B. Niot I. Febbraio M. Montmayeur J.P. Besnard P. J. Clin. Investig. 2005; 115: 3177-3184Crossref PubMed Scopus (518) Google Scholar). After washing, slides were incubated for 2 h at room temperature with Cy3-conjugated anti-mouse IgG (1:600 dilutions) for CD36 expression or with fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:600) for α-gustducin detection. Staining specificity was assessed by treating slides in the absence of primary antibodies. After three washings with PBS, a drop of Aqua Poly-Mount mounting medium was added on the slide for the analysis under fluorescent microscope (Zeiss Axioskop). Measurement of Ca2+ Signaling—The CD36-negative and CD36-positive gustatory cells, cultured for 24 h, were washed with PBS, pH 7.4. The composition of PBS was as follows: 3.5 mm KH2PO4, 17.02 mm Na2HPO4, 136 mm NaCl. The cells were then incubated with Fura-2/AM (1 μm) for 60 min at 37 °C in loading buffer which contained 110 mm NaCl, 5.4 mm KCl, 25 mm NaHCO3, 0.8 mm MgCl2, 0.4 mm KH2PO4, 20 mm Hepes-Na, 0.33 mm Na2HPO4, 1.2 mm CaCl2, and the pH was adjusted to 7.4. After loading, the cells (2 × 106/ml) were washed 3 times (600 g × 10 min) and remained suspended in the identical buffer. For experiments in Ca2+-free (0% Ca2+) medium, CaCl2 was replaced by EGTA (2 mm). [Ca2+]i was measured according to Grynkiewicz et al. (7Grynkiewicz G.M. Ponie M. Tsein R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). The fluorescence intensities were measured in the ratio mode in PTI spectrofluorometer at 340 and 380 nm (excitation filters) and 510 nm (emission filter). The cells were continuously stirred throughout the experimentation. The test molecules were added into the cuvettes in small volumes with no interruptions in recordings. The intracellular concentrations of free Ca2+, [Ca2+]i, were calculated by using the equation [Ca2+]i = Kd × (R – Rmin)/(Fmax – F) × (Sf2/Sb2), where Kd is the dissociation constant for Fura-2/calcium complex, R is the ratio emission with excitation at 340 nm divided by excitation at 380 nm, Rmin is the ratio in the presence of no Ca2+, Fmax is the ratio of saturating [Ca2+]i, and Sf2/Sb2 is the ratio of 380 nm excitation fluorescence at zero and saturating [Ca2+]i. A value of 224 nm for Kd was added into the calculations. Rmax and Rmin values were obtained by the addition of ionomycin (5 μm) and MnCl2 (2 mm), respectively. The SOC channel blockers (SKF96365 and econazole), CD36 inhibitor (SSO), or protein-tyrosine kinase (PTK) inhibitors (PP2, genistein, and SU6656) were added to the cells for 20 min before the addition of linoleic acid. Measurement of Inositol 1,4,5-Triphosphate (IP3) Production— The CD36-negative and CD36-positive gustatory cells were treated with LA in the presence or absence of SSO for 20 min. The vehicle control received 0.1% ethanol (v/v) for 20 min. After stimulation, cells were washed twice with ice-cold PBS. Cell suspension was then lysed with 20% ice-cold perchloric acid (0.2:1; v/v). After centrifugation, the supernatant was assayed for IP3 production according to the manufacturer's instructions, furnished with [3H] Biotrak Assay System (GE Healthcare). Briefly, the assay is based on competition between [3H]IP3 (tracer) and unlabeled (cellular) IP3 for binding to bovine adrenal d-myo-inositol 1,4,5-triphosphate-binding protein. The bound IP3 is separated from the free IP3 by centrifugation, which brings the binding protein to the bottom of the tube. The free IP3 in the supernatant is then discarded by decantation, leaving the bound fractions. The radioactivity is measured with a β-scintillation counter. Immunoprecipitation and Immunodetection of PTKs—First, we detected the presence of CD36 and Src-related kinases (Src total, Fyn, Yes, and Lyn) in CD36-positive cells by employing the specific antibodies (Fig. 3A). Second, we detected LA-induced phosphorylation at tyrosine resides by employing anti-phosphorylated PTK antibodies. Hence, the cells were incubated with or without SSO (50 μm) for 20 min then stimulated or not with LA (20 μm) as described in the legends of Fig. 3, B and C. After stimulation, cells were lysed for 1 h at 4 °C with 800 μl of ice-cold lysis buffer A (20 mm HEPES, pH 7.4, 2 mm EDTA, 125 mm NaCl, 1 mm dithiothreitol, 1 mm sodium orthovanadate, 0.5 mg/ml benzamidine, 1% Nonidet P-40) in the presence of protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 2.5 μg/ml pepstatin, 5 μg/ml leupeptin). The lysates were centrifuged (13,500 g × 15 min at 4 °C), and supernatants were subjected to Western blotting. To ensure equal loading of proteins, immunodetection of β-actin was performed in the same assays. To detect the phosphorylation of Src-related kinases, we first immunoprecipitated the treated-CD36-positive or CD36-negative gustatory cells with anti-phospho-PTK and then probed them with antibodies against Fyn, Yes, and Lyn (Fig. 3D). Briefly, 1 mg of protein was immunoprecipitated with 1 μg of anti-PTK antibody and 25 μl of A/G-Sepharose beads. Phosphotyrosine protein-containing immunoprecipitates were washed three times with buffer A and diluted with Laemmli sample buffer. For Western blotting, denatured proteins (20 μg) were separated on SDS-polyacrylamide/bisacrylamide (8%) gel and transferred onto the PVDF membrane. After saturation for 1 h in the TBS/Tween 20 (10 mm Tris base, 0.15 m NaCl, 0.05% Tween 20) supplemented with delipidated milk 1% (v/v), the PVDF membrane was incubated with primary antibodies for 2 h at room temperature. After washing 4 times in the TBS/Tween 20, the PVDF membrane was incubated for 1 h with horseradish peroxidase-conjugated secondary anti-mouse antibody (1:1000) in TBS/Tween 20 supplemented with 1% delipidated milk (v/v). The PVDF membrane was washed 3 times in 0.05% TBS/Tween 20 and once in TBS. The phosphorylated bands were revealed with the horseradish peroxidase substrate (Super Signal Substrate, Western blotting kit from Pierce). To ensure the specificity of the detection of phosphorylated Src-related kinases, immunodetection of unphosphorylated Src, Fyn, Yes, and Lyn was performed in each sample (Fig. 3D). Detection of mRNA of Enzymes Involved in the Synthesis of Neurotransmitters by RT-quantitative PCR and Semiquantitative RT-PCR—Total RNA was extracted from CD36-negative and CD36-positive gustatory cells by using Trisol (Invitrogen) and subjected to DNase treatment using the RNase-free DNase Set (Qiagen). One μg of total RNA was reverse-transcribed with Super script II RNase H-reverse transcriptase using oligo(dT) according to the manufacturer's instructions (Invitrogen). Real time PCR was performed on the iCycler iQ real time detection system, and amplification was undertaken by using SYBR Green I detection. Oligonucleotide primers were based on the sequences of mice gene in GenBank™ data base (see Table 1). The amplification was carried out in a total volume of 25 μl that contained 12.5 μl of SYBR® Green Supermix buffer (50 mm KCl, 20 mm Tris-HCl, pH 8.4, 3 mm MgCl2, 0.2 mm each dNTPs, 0.63 units of iTaq DNA polymerase, and SYBR®Green 1.0 nm fluorescein) and 12.5 μl (0.3 μm) of each primer and diluted cDNA.TABLE 1Sequences of the primers used to study the expression of mRNA of different genesGenePrimer sequencesβ-ActinForward, 5′-AGAGGGAAATCGTGCGTGAC-3′Reverse, 5′-CAATAGTGATGACCTGGCCGT-3′TPH-1Sense, 5′-GAGTTGCTCCACGCTTTGC-3′Antisense, 5′-ACACTCAGTCTACATCCATCCC-3′AADCForward, 5′-TCCTGCTGTTCTTTTGACAATCTC-3′Reverse, 5′-GCGTAAGCAGCGTCAATGTG-3′THForward, 5′-AAGCTGATTGCAGAGATTGCC-3′Reverse, 5′-TTCCTCCTTTGTGTATTCCACGT-3′DBHForward, 5′-GAGAGCCCCTTCCCCTACC-3′Reverse, 5′-TGGAGCTGGAAGTGGATGATCT-3′VMAT-2Forward, 5′-GGTTGCCAAGCCTTATCGGA-3′Reverse, 5′-ACCTGCTCCACTGCCTTGCT-3′ Open table in a new tab The amplification conditions consisted of an initial denaturation step at 95 °C for 5 min as a "hot start" followed by 40 cycles at 95 °C for 30s and 60 °C for 30s with a single fluorescence detection point at the end of the relevant annealing or extension segment. At the end of the PCR, the temperature was increased from 60 to 95 °C at a rate of 2 °C/min, and the fluorescence was measured every 15 s to construct the melting curve. The standard curves were generated for each gene using serial dilutions of positive control template to establish PCR efficiencies. All determinations were performed at least in duplicate using two dilutions of each assay to achieve reproducibility. Results were evaluated by iCycler iQ software including standard curves, amplification efficiency (E), and cycle threshold (Ct). Relative quantification of mRNA in different groups was determined as ΔΔCt =ΔCt of gene of interest – ΔCt of β-actin. ΔCt = Ct of CD36-positive or CD36-negative cells – Ct of total cells. Relative quantity (RQ) was calculated as RQ = (1 + E)(–ΔΔCt). For semiquantitative determinations, amplification was performed in the same conditions as described above for 30 cycles followed by a final extension period of 72 °C for 10 min. Reaction products were electrophoresed on a 1.5% agarose gel impregnated with ethidium bromide. The RNA pattern was visualized by UV transillumination. Determination of Monoamine Neurotransmitters by High Performance Liquid Chromatography (HPLC)—HPLC separation of monoamine neurotransmitters was performed as described elsewhere (8Moret F. Guilland J.C. Coudouel S. Rochette L. Vernier P. J. Comp. Neurol. 2004; 468: 135-150Crossref PubMed Scopus (58) Google Scholar). Cultured CD36-negative and CD36-positive gustatory cells were incubated with LA, as mentioned in the legends, and at the end of incubation supernatant was removed and supplemented with equal volume of solution A (0.2 m perchloric acid, 2.7 mm EDTA, 5.26 mm sodium metabisulfite), then 100 pg of an internal standard (3,4-dihydroxy-benzylamine hydrobromide) was added to each sample. Samples were centrifuged for 6 min at 4 °C at 13,000 × g. The supernatants were analyzed by HPLC with a reverse phase column (60 RP Select B, 250 × 4 mm, 5 μm, Merck) with electrochemical detection (Waters, Milford, MA) before or after passing through alumina column, as described previously (9Riggin R.M. Kissinger P.T. Anal. Chem. 1977; 49: 2109-2111Crossref PubMed Scopus (234) Google Scholar). The potential of the electrochemical detector was set at +0.85 V. The mobile phase consisted of 0.08 m sodium phosphate, 0.27 mm EDTA, 3.7 mm octan-1-sulfonic acid, pH 3.5. The biogenic amines were identified and quantified in comparison with commercial standards using the KromaSystem 2000 software (BIO-TEK Instruments, Milano, Italy). Measurement of Membrane Potential, Vm—The probe bisoxonol was used to measure membrane potential in isolated CD36-positive gustatory cells. This probe is chemically unrelated to the cyanines and is not toxic to the cells (10Rink T.J. Motecucco C. Hesketh T.R. Tsein R.Y. Biochim. Biophys. Acta. 1980; 595: 15-30Crossref PubMed Scopus (253) Google Scholar). For these experiments the cells were prepared as described for [Ca2+]i determinations. After washing, the cells (106/1.5 ml) were transferred to the fluorometer cuvettes, and 150 nm of bisoxonol was added. The stock solution of bisoxonol was prepared in DMSO. The cells were allowed to equilibrate with the dye, and after 10 min different test molecules were added. The fluorescent intensities were determined at 540 nm (excitation filter) and 580 nm (emission filter). Upward deflections represent depolarizations. Statistical Analysis—Statistical analysis of data was carried out using Statistica (Version 4.1, Statsoft, Paris, France). The significance of the differences between mean values was determined by analysis of variance one way followed by a least significant difference test. For all the tests the significance level chosen was p < 0.05. Linoleic Acid Induces Increases in [Ca2+]i in CD36-positive Gustatory Cells—To explore whether LA triggers a calcium response via CD36 receptor, we purified the CD36-positive gustatory cells which expressed CD36 (Fig. 1A) and α-gustducin, a G-protein considered as a specific marker of TRC (Fig. 1B). We observed that LA induced a rapid increase in [Ca2+]i which was followed by a stationary phase both in calcium-free (0% Ca2+) and calcium-enriched (100% Ca2+) buffers (Fig. 1C). LA-induced increases in [Ca2+]i were more important in 100% Ca2+ buffer than those in 0% calcium buffer. We also measured the production of IP3, known to recruit calcium from endoplasmic reticulum (ER) pool. Fig. 1E shows that LA induced 4-fold increases in IP3 production in comparison to unstimulated cells. To shed light on whether LA induces the production of IP3 via binding to CD36, we employed sulfo-N-succinimidyl derivative of oleate (SSO) which is known to inhibit the binding of fatty acids to CD36 (11Harmon C.M. Abumrad N.A. J. Membr. Biol. 1993; 133: 43-49Crossref PubMed Scopus (168) Google Scholar). We observed that SSO curtailed LA-induced increases in IP3 in these cells. As far as the CD36-negative cells are concerned, we observed that these cells, in response to LA, exhibited a weak increase in [Ca2+]i in 100% Ca2+ buffer without influencing the same in 0% Ca2+ buffer (Fig. 1D). Furthermore, in CD36-negative cells, LA failed to induce IP3 production, and SSO exerted no effect on it (Fig. 1E). It is important to mention that SSO treatment exerted no cytotoxic effect on either of the cell populations as assessed by trypan bleu exclusion assay (not shown). LA Stimulates Capacitative Calcium Influx via a PTK-dependent Mechanism in CD36-positive Gustatory Cells—Because LA-induced increases in [Ca2+]i are more important in 100% calcium buffer than those in 0% Ca2+ buffer, it is possible that LA activates a capacitative calcium entry due to calcium recruitment from internal stores in CD36-positive gustatory cells. To support this hypothesis we conducted experiments in 100% calcium buffer in the presence of SKF96365 and econazole, the inhibitors of store-operated calcium (SOC) channels. Fig. 2A shows that the addition of either SKF96365 or econazole significantly diminished the LA-induced increases in [Ca2+]i in CD36-positive gustatory cells. These two SOC channel blockers exerted no effect on CD36-negative gustatory cells (Fig. 2A). Because PTKs have been shown to modulate the activation of SOC channels (12Vazquez G. de Boland A.R. Boland R.L. J. Biol. Chem. 1998; 273: 33954-33960Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), we investigated the involvement of PTKs in the LA-induced SOC influx. We used the inhibitors of PTKs: PP2 (known to inhibit the PTK activity of Src in a non-competitive manner against ATP), genistein (a soybean isoflavone, known to selectively inhibit PTK), and SU6656 (a selective inhibitor of Fyn, Yes, and Lyn). We observed that in CD36-positive gustatory cells, these inhibitors significantly abolished the increases in [Ca2+]i, induced by LA; however, these agents exerted no effects in CD36-negative cells (Fig. 2B). LA Induces the Phosphorylation of Src, Fyn, and Yes in CD36-positive Gustatory Cells—We observed that mouse lingual CD36-positive gustatory cells expressed abundantly CD36, Src, and Src-PTK-like Fyn and Yes. The CD36-negative cells also expressed these Src-PTK but to a lesser extent than CD36-positive cells. The two cell populations do not express the Lyn isoform of Src-PTK (Fig. 3A). Fig. 3B shows that LA induced the phosphorylation of PTKs, which was well apparent at 5 min and started declining at 15 min of incubation. Moreover, the LA-induced phosphorylation of PTKs was completely abolished by SSO in these cells (Fig. 3C). In CD36-negative cells, LA failed to induce PTK phosphorylation, and SSO exerted no effect on the same (Fig. 3C). Furthermore, we immunoprecipitated the cell lysates with total anti-phospho-PTKs and detected different isoforms of Src-PTKs. We noticed that LA induced the phosphorylation of both Fyn59 and Yes62, but not of Lyn56/53, at 5 and 15 min of incubation (Fig. 3D). mRNA Expression of the Enzymes Involved in the Synthesis of Monoamine Neurotransmitters in CD36-positive Gustatory Cells—By employing RT-PCR, we have assessed the expression of mRNA of the enzymes involved in the synthesis of 5-HT and catecholamines. We have measured tryptophan hydroxylase-1 mRNA as the tph1 gene that encodes for the synthesis of tryptophan hydroxylase in peripheral tissues (13Izikki M. Hanoun N. Marcos E. Savale L. Barlier-Mur A.M. Saurini F. Eddahibi S. Hamon M. Adnot S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2007; 293: 1045-1052Crossref PubMed Scopus (52) Google Scholar). Tryptophan hydroxylase-1 converts l-tryptophan to l-5-hydroxytryptophan. We have also detected l-aromatic amino acid decarboxylase that converts l-5 hydroxytryptophan to 5-HT. l-Aromatic amino acid decarboxylase has been detected both in neuronal and non-neuronal cells (14Nagatsu T. Neurosci. Res. 1991; 12: 315-345Crossref PubMed Scopus (105) Google Scholar). We have also determined the mRNA of the enzymes involved in the biosynthesis of catecholamines, like tyrosine hydroxylase that converts tyrosine to l-3,4-dihydroxyphenylalanine and dopamine β-hydroxylase that converts dopamine to NA. We evaluated the expression of vesicular monoamine transporter-2, implicated in the vesicular storage and subsequent release of neurotransmitters. We observed that CD36-positive gustatory cells expressed a high level of tryptophan hydroxylase-1 and l-aromatic amino acid decarboxylase (AADC) mRNA (Fig. 4, A and B). However, tyrosine hydroxylase mRNAs are less abundantly expressed in purified CD36-positive gustatory cells (Fig. 4, A and B). Dopamine β-hydroxylase mRNAs were identically present in unpurified (total), CD36-positive, and CD36-negative cells (Fig. 4B). Vesicular monoamine transporter-2 mRNA is highly expressed in CD36-positive gustatory cells (Fig. 4, A and B). It is also noteworthy that the unpurified total cells also express in relatively small quantities the aforementioned mRNAs (Fig. 4B). Production and Releas