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

Abstract

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