- Open Access
Interaction of arachidonic acid with electrogenic properties of mouse chemosensory neurons
© I. Holzapfel Publishers 2010
Published: 4 November 2010
Chemosensory neurons respond to stimulation induced by gasses, volatile and non-volatile compounds. Neuronal excitation mediated via second messengers involves typically: cGMP, cAMP, or IP3. Transduction pathways based on cyclic nucleotide have three-phosphate nucleotide as substrate, while IP3 has a membrane lipid substrate. These derivatives of cholesterol are signaling molecules with modulator-like effects on many proteins, including membrane ion channels. In the present study, spontaneous and induced activities were recorded in a whole-cell configuration, in current and voltage clamp modes, in isolated chemosensory neurons obtained from the mouse. Chemosensory neurons responded with an inward depolarizing current to application of arachidonic acid, which suggests a role for it in putative mechanisms of signal transduction.
An association between lung function and chemosensory activity has been demonstrated . Chemosensory neurons respond to stimulation induced by a wide range of chemical agents, ranging from gasses (CO2, NO), volatile (odorants, steroid derivates), and nonvolatile compounds (hormones, neurotransmitters). Neuronal excitation involve typically second messengers such as cGMP, cAMP and inositol-1,4,5-trisphosphate (IP3). Furthermore, important transduction pathways are based on the polyunsaturated fatty acids (PUFAs) components of the neuronal plasma membrane phospholipids. PUFAs behave as signaling molecules with modulator effects on many proteins, including membrane ion channels [2–4]. Membrane-bound PUFAs modulate membrane fluidity as well as the functional properties of membrane proteins [5, 6]. In particular, arachidonic acid (AA), a 20-carbon PUFA, is normally found esterified to cell membrane glycerophospholipids. In response to many a first messenger, including neurotransmitters, AA can be released from these cellular pools by phospholipases and can act as a precursor to several biologically active compounds [3, 7]. The three major enzymatic ways for AA oxidation are the cyclooxygenase, lipooxygenase, and cytochrome P450 monooxygenase pathways [8–10]. In the cyclooxygenase pathway, PGH synthases generate PGH2, which can be further metabolized to other PG, thromboxane, and prostacyclin. Lipooxygenases generate HPETEs, which are converted to leukotrienes and dienols (HETEs). Cytochrome P450 monooxygenases generate four regioisomeric epoxyeicosatrienoic acids (5,6-, 8,9-, 11,12- and 14,15-EET), several mid-chain cis, trans-conjugated HETEs, and alcohols of AA (19-OH-AA and 20-OH-AA) [8–10]. EETs are further metabolized by epoxide hydrolases to four regioisomeric dihydroxyeicosatrienoic acids (5,6-, 8,9-, 11,12- and 14,15-DHETs) . Both EETs and DHETs have been shown to influence a variety of biological processes, including control of vascular [12, 13] and airway  smooth muscle tone, regulation of pituitary/hypothalamic and pancreatic peptide hormone release [15–17], inhibition of platelet aggregation , and modulation of fluid and electrolyte transport . These compounds are dietary essential omega-3 fatty acid and possess both cardio- and neuroprotective properties [20, 21].
AA levels increase during inflammation and it behaves as an inflammatory mediator . Furthermore, proinflammatory mediators, such as bradykinin  and the cytokine tumor necrosis factor alpha (TNF-α) [24, 25] can increase AA synthesis by activating cytosolic PLA2. AA epoxygenase metabolites cause significant changes in rat airway electrical parameters and may be involved in the control of lung fluid and electrolyte transport . It has been shown that airway smooth muscle metabolizes AA through various enzymatic pathways, including cytochrome P450 hydroxylase, which leads to the production of HETE .
Because lung function is related to chemosensory activity , AA could affect several pathophysiological reactions due to pulmonary diseases, such as COPD. The AA cascade is possibly one of the most intricate signaling systems, since it generates multiple messenger molecules that may act both outside and inside of the neuron. In the present study we sought, therefore, to determine whether chemosensory neurons can respond to AA. We addressed the issue using patch-clamp methods to study electrophysiological properties of the isolated mouse chemosensory neurons in response to AA application.
Materials and methods
All experiments conformed to the international guidelines on the ethical use of animals (86/609/EEC). C57BL isolated mouse chemosensory neurons were used for the study. The neurons were isolated according to standard enzymatic-mechanical dissociation protocols . Dissociated cells were plated onto Petri dishes and stored for stabilization for 1 h. Isolated neurons were then used for experiments up to two hours and constantly perfused with normal Ringer solution (mM: 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPESNa0.5, 1 sodium pyruvate, 10 D-(+)glucose, pH 7.4, and osmolality 300-310 mOsm). The intracellular pipette solution contained in mM: 145 KCl, 4 MgCl2, 10 HEPESNa0.5, 0.5 EGTA, 1 ATP, 0.1 GTP, pH 7.3, and 310-315 mOsm. Borosilicate pipette have resistance ranging from 2 to 10 MΩ. All chemicals used in this study were obtained from Sigma-Aldrich (St. Louis, MO). 50 μM AA or 50 mM KCl was applied for 2 s using a fast perfusion stepper system . To prevent oxidation, AA was dissolved in DMSO under nitrogen and stored at -80°C. To avoid micelle formation, solutions were briefly sonicated and then vortexed. Electrophysiological recordings were made using an Axopatch system (Axon Instruments, CA) in a whole-cell configuration in both current and voltage-clamp modes . Currents data were filtered at 5 kHz and digitized at 12 kHz. Analysis was performed using Clampfit 9 (Axon Instruments, CA) and Origin (OriginLab, Northampton, MA).
The purpose of the present study was to investigate whether the chemosensory neurons' electrical activity could be affected by AA. To verify this purpose we performed patch-clamp experiments on isolated mouse chemosensory neurons found to have inherent spontaneous firing activity and to be responsive in a regular incremental way to current injections. The major finding of the study was that AA provoked a depolarization of chemosensory neurons. A regular pattern of neuronal responses, repeatability, and a complete recoverability after the electrical stimulus had ceased acting demonstrate the ability of AA to interact with electrogenic properties of the mouse chemosensory neurons. AA elicits an intricate signaling system as it generates multiple messenger molecules that may act both intra- and extracellularly. The present finding of electrophysiological effects exerted by AA in chemosensory neurons strongly implicates its potential role in signal transduction mechanisms of the cells. The implication is strengthened by other reports showing the effects of AA exerted on sensory neurons [31, 32].
AA is involved in a number of physiological pathways depending on the enzyme that acts on it. Interestingly, AA has been found in abundance in nasal mucosa , and the airway smooth muscles metabolize it as well . AA also may be involved in the control of fluid and electrolyte transport in the lung . AA has a short lifespan, during which it may interact with the activity of ion channels and protein kinases within the cell. Alternatively, it may be transformed into a family of metabolites, the eicosanoids, which may also produce separate effects on intracellular targets. In either case, the AA cascade affects neuronal excitability. Furthermore, these compounds can cross the cell membrane, diffuse through the extracellular space, and interact with high-affinity receptors located on neighboring neurons. Therefore, the AA cascade may give rise to both intracellular second messengers and to local mediators, bridging the gap between transmembrane and transcellular communication. This two-pronged role may be important in integrating the responses of postsynaptic neurons with the activity of presynaptic terminals and of other contacting cells.
AA modulates directly or indirectly by its metabolites, a wide variety of ion channels . One proposed mechanism of this modulation is through a binding to the ion channel, since the channel modulation depends on the membrane potential [35, 36] and mutations of specific residues in the pore region . AA potentiates acid-sensing ion channels (ASICs) in sensory neurons by a direct action . The ASICs family underlies the inward current induced by acidity , such as vanilloid receptor activation by AA or its metabolites . Stimulation of cells leads to an increase of [Ca2+]i due to Ca2+ release from intracellular organelles and Ca2+ entry across the plasma membrane. This process is initiated upon the binding of a stimulus-released neurotransmitter to its receptors and activation of phospholipase C (PLC). PLC, in turn, converts phosphatidylinositol-4,5-bisphosphate (PIP2) to IP3 and diacylglycerol (DAG). Plasma membrane channels, e.g., TRP channels, are activated and extracellular cations, including Ca2+, flow into the cell. The activation of plasma membrane channels might involve a reduction of PIP2 concentration or an increase in DAG or its metabolites, such as polyunsaturated fatty acids .
The finding that chemosensory neurons responded to AA administration with an inward depolarizing current raises the question of a physiological role and the exact determinants of the possible transduction pathway underlain by AA or its metabolites. We could speculate that such a pathway could be specific for a subtype of neurons involved in transduction of chemical stimuli, control of electrolyte transport, or inflammatory conditions. Alternative study designs are required to verify these mechanisms and to attribute a role for AA in signaling pathways.
Conflicts of interest
The authors declare that they have no competing interests.
Prof. M. Pokorski was supported by grants from Convenzione tra l'Universita degli Studi "G.d'Annunzio" di Chieti e Pescara and Al Ministero Affari Esteri in Rome.
- Di Giulio C, Data PG, Lahiri S: Chronic cobalt causes hypertrophy of glomus cells in the rat carotid body. Am J Physiol 1991, 261: 102–5.Google Scholar
- Piomelli D, Greengard P: Lipoxygenase metabolites of arachidonic acid in neuronal signalling. Trends Pharmacol Sci 1990, 11: 367–73. 10.1016/0165-6147(90)90182-8View ArticlePubMedGoogle Scholar
- Axelrod J: Receptor-mediated activation of phospholipase A 2 and arachidonic acid release in signal transduction. Biochem Soc Trans 1990, 18: 503–7.View ArticlePubMedGoogle Scholar
- Liu Y, Liu D, Heath L, Meyers DM, Krafte DS, Wagoner PK, Silvia CP, Yu W, Curran ME: Direct activation of an inwardly rectifying potassium channel by arachidonic acid. Mol Pharmacol 2001, 59: 1061–8.PubMedGoogle Scholar
- Stubbs CD, Smith AD: The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biophys Acta 1984, 779: 89–137. 10.1016/0304-4157(84)90005-4View ArticlePubMedGoogle Scholar
- Piomelli D: Eicosanoids in synaptic transmission. Crit Rev Neurobiol 1994, 8: 65–83.PubMedGoogle Scholar
- Baker RR, Chang HY: The hydrolysis of natural phosphatidylethanolamines by phospholipase A 2 from rat serum: a degree of selectivity is shown for docosahexaenoate release. Biochim Biophys Acta 1992, 1125: 56–61. 10.1016/0005-2760(92)90155-OView ArticlePubMedGoogle Scholar
- Capdevila JR, Falck JR, Estabrook W: Cytochrome P450 and arachidonate cascade. FASEB J 1992, 6: 731–6.PubMedGoogle Scholar
- Fitzpatrick FA, Murphy DA: Cytochrome P450 metabolism of arachidonic acid: formation and biological actions of 'epoxygenase'-derived eicosanoids. Pharmacol Rev 1989, 40: 229–41.Google Scholar
- McGiff JC: Cytochrome P-450 metabolism of arachidonic acid. Annu Rev Pharmacol Toxicol 1991, 31: 339–69. 10.1146/annurev.pa.31.040191.002011View ArticlePubMedGoogle Scholar
- Zeldin DC, Kobayashi J, Falck JR, Winder BS, Hammock BD, Snapper JR, Capdevila JH: Regio- and enantiofacial selectivity of epoxyeicosatrienoic acid hydration by cytosolic epoxide hydrolase. J Biol Chem 1993, 268: 6402–7.PubMedGoogle Scholar
- Carrol MA, Schwartzman M, Capdevila J, Falck JR, McGiff JC: Vasoactivity of arachidonic acid epoxides. Eur J Pharmacol 1987, 138: 281–3. 10.1016/0014-2999(87)90445-6View ArticleGoogle Scholar
- Gebremedhin D, Ma Y, Falck JR, Roman RJ, VanRollins M, Harder DR: Mechanism of action of cerebral epoxyeicosatrienoic acids on cerebral arterial smooth muscle. Am J Physiol 1992, 263: 519–29.Google Scholar
- Zeldin DC, Plitman JD, Kobayashi J, Miller RF, Snapper JR, Falck JR, Szarek JL, Philpot RM, Capdevila JH: The rabbit pulmonary cytochrome P450 arachidonic acid metabolic pathway: characterization and significance. J Clin Invest 1995, 95: 2150–60. 10.1172/JCI117904PubMed CentralView ArticlePubMedGoogle Scholar
- Cashman JR, Hanks D, Weiner RI: Epoxy derivatives of arachidonic acid are potent stimulators of prolactin secretion. Neuroendocrinol 1987, 46: 246–51. 10.1159/000124827View ArticleGoogle Scholar
- Falck JR, Manna S, Moltz J, Chacos N, Capdevila J: Epoxyeicosatrienoic acids stimulate glucagon and insulin release from isolated rat pancreatic islets. Biochem Biophys Res Commun 1983, 114: 743–9. 10.1016/0006-291X(83)90843-4View ArticlePubMedGoogle Scholar
- Snyder GD, Capdevila J, Chacos N, Manna S, Falck JR: Action of luteinizing hormone-releasing hormone: Involvement of novel arachidonic acid metabolites. Proc Natl Acad Sci USA 1983, 80: 3504–7. 10.1073/pnas.80.11.3504PubMed CentralView ArticlePubMedGoogle Scholar
- Malcolm KC, Fitzpatrick FA: Epoxyeicosatrienoic acids inhibit Ca 2+ entry into platelets stimulated by thapsigargin and thrombin. J Biol Chem 1992, 267: 19854–8.PubMedGoogle Scholar
- Hu S, Kim HS: Activation of K + channel in vascular smooth muscles by cytochrome P450 metabolites of arachidonic acid. Eur J Pharmacol 1993, 230: 215–21. 10.1016/0014-2999(93)90805-RView ArticlePubMedGoogle Scholar
- Maruyama K, Yoneya S, Miyauchi O, Adachi-Usami E, Nishikawa M: Fish oil (polyunsaturated fatty acid) prevents ischemic-induced injury in the mammalian retina. Exp Eye Res 2002, 74: 671–6. 10.1006/exer.2002.1151View ArticleGoogle Scholar
- McLennan P, Howe P, Abeywardena MY, Muggli R, Raederstorff D, Mano M, Rayner T, Head R: The cardiovascular protective role of docosahexaenoic acid. Eur J Pharmacol 1996, 300: 83–9. 10.1016/0014-2999(95)00861-6View ArticlePubMedGoogle Scholar
- Brash AR: Arachidonic acid as a bioactive molecule. J Clin Invest 2001, 107: 1339–45. 10.1172/JCI13210PubMed CentralView ArticlePubMedGoogle Scholar
- Dray A, Perkins M: Bradykinin and inflammatory pain. Trends Neurosci 1993, 16: 99–104. 10.1016/0166-2236(93)90133-7View ArticlePubMedGoogle Scholar
- Hoeck WG, Ramesha CS, Chang DJ, Fan N, Heller RA: Cytoplasmic phospholipase A 2 activity and gene expression are stimulated by tumor necrosis factor: Dexamethasone blocks the induced synthesis. Proc Natl Acad Sci USA 1993, 90: 4475–9. 10.1073/pnas.90.10.4475PubMed CentralView ArticlePubMedGoogle Scholar
- Jupp OJ, Vandenabeele P, MacEwan DJ: Distinct regulation of cytosolic phospholipase A 2 phosphorylation, translocation, proteolysis and activation by tumor necrosis factor-receptor subtypes. Biochem J 2003, 374: 453 61.View ArticlePubMedGoogle Scholar
- Pascual JMS, McKenzie A, Yankaskas JR, Falck JR, Zeldin DC: Epoxygenase metabolites of arachidonic acid affect electrophysiologic properties of rat tracheal epithelial cells. J Pharm Exp Ther 1998, 286: 772–9.Google Scholar
- Morin C, Sirois M, Echave V, Gomes MM, Rousseau E: Functional effects of 20-HETE on human bronchi: hyperpolarization and relaxation due to BKCa channel activation. Am J Physiol Lung Cell Mol Physiol 2007, 293: L1037–44. 10.1152/ajplung.00145.2007View ArticlePubMedGoogle Scholar
- Maue RA, Dionne VE: Preparation of isolated mouse olfactory receptor neurons. Pflügers Arch 1987, 409: 244–50.PubMedGoogle Scholar
- Zhang P, Yang C, Delay RJ: Urine stimulation activates BK channels in couse vomeronasal neurons. J Neurophysiol 2008, 100: 1824–34. 10.1152/jn.90555.2008PubMed CentralView ArticlePubMedGoogle Scholar
- Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ: Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 1981, 391: 85–100. 10.1007/BF00656997View ArticlePubMedGoogle Scholar
- Eto K, Arimura Y, Mizuguchi H, Nishikawa M, Noda M, Ishibashi H: Modulation of ATP-induced inward currents by docosahexaenoic acid and other fatty acids in rat nodose ganglion neurons. J Pharmacol Sci 2006, 102: 343–46. 10.1254/jphs.SC0060053View ArticlePubMedGoogle Scholar
- Smith ES, Cadiou H, McNaughton PA: Arachidonic acid potentiates acid-sensing ion channels in rat sensory neurons by a direct action. J Neurosci 2007, 145: 686–98. 10.1016/j.neuroscience.2006.12.024View ArticleGoogle Scholar
- Russell Y, Evans P, Dodd GH: Characterization of the total lipid and fatty acid composition of rat olfactory mucosa. J Lipid Res 1989, 30: 877–84.PubMedGoogle Scholar
- Meves H: Modulation of ion channels by arachidonic acid. Prog Neurobiol 1994, 43: 175–86. 10.1016/0301-0082(94)90012-4View ArticlePubMedGoogle Scholar
- Barrett CF, Liu L, Rittenhouse AR: Arachidonic acid reversibly enhances N-type calcium current at an extracellular site. Am J Physiol Cell Physiol 2001, 280: 1306–18.Google Scholar
- Vellani V, Reynolds AM, McNaughton PA: Modulation of the synaptic Ca 2+ current in salamander photoreceptors by polyunsaturated fatty acids and retinoids. J Physiol (Lond) 2000, 529: 333–44. 10.1111/j.1469-7793.2000.00333.xView ArticleGoogle Scholar
- Hamilton KL, Syme CA, Devor DC: Molecular localization of the inhibitory arachidonic acid binding site to the pore of hIK1. J Biol Chem 2003, 278: 16690–7. 10.1074/jbc.M212959200View ArticlePubMedGoogle Scholar
- Krishtal O: The ASICs: signaling molecules? Modulators? Trends Neurosci 2003, 26: 477–83. 10.1016/S0166-2236(03)00210-8View ArticlePubMedGoogle Scholar
- Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D: The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997, 389: 816–24. 10.1038/39807View ArticlePubMedGoogle Scholar
- Hardie RC: Regulation of TRP channels via lipid second messengers. Annu Rev Physiol 2003, 65: 735–59. 10.1146/annurev.physiol.65.092101.142505View ArticlePubMedGoogle Scholar