Elena Fabbri, 1 Maria Enrica Ferretti, 1 Marco Buzzi, 1 Roberta Cavallaro, 1 Giancarlo Vesce, 2 and Carla BiondP z. (Accepted February 2, 1995). The enzyme ...
Neurochemical Research, Vol. 20, No. 6, 1995, pp. 719-725
Olfactory Transduction Mechanisms in Sheep E l e n a F a b b r i , 1 M a r i a E n r i c a Ferretti, 1 M a r c o Buzzi, 1 R o b e r t a C a v a l l a r o , 1 G i a n c a r l o V e s c e , 2 and Carla BiondP z
(Accepted February 2, 1995)
The enzyme adenylyl cyclase from sheep olfactory epithelium is dually regulated by GTP and is highly sensitive to the nucleotide analogues GTP~/S and GppNHp, as well as to fluoride ions and forskolin. Many, but not all, odorants tested are able to stimulate adenylyl cyclase in a dosedependent manner and with different potencies. Such an effect is detectable only in the presence of GTP. The odorants belonging to the putrid class are the least effective in stimulating adenylyl cyclase activity, and only furfuryl mercaptan significantly increases cAMP biosynthesis. Mixtures of two odorants, chosen among those able to activate adenylyl cyclase, induce additive or supraadditive effects, suggesting the presence of many different receptor types. The presence of an alternative olfactory signal transduction process, i.e. the inositol phospholipid second messenger system, has been evaluated. Triethylamine, a putrid odorant completely ineffective on cAMP levels, is able to significantly increase inositol phosphate accumulation, indicating the coexistence of both cAMP- and InsP3-mediated signalling pathways in sheep olfactory epithelium. KEY WORDS: Olfaction; sheep; odorants; adenylyl cyclase; phospholipase C.
INTRODUCTION
activity, and Bakalyar and Reed (3) have recently cloned a Ca2+-dependent adenylyl cyclase of type III, selectively concentrated in such compartment. Olfactory adenylyl cyclase of different animal species is activated by many odorants (4,5), and its coupling to a novel G protein, called Golf, has already been demonstrated (6). Golf is a stimulatory protein that, when activated by odorant-receptor interaction, positively affects the adenylyl cyclase catalytic moiety in heterologous systems (7). This renders the olfactory adenylyl cyclase similar to hormoneand neurotransmitter-stimulated enzymes in other tissues (8,9). The functional role of cAMP in mediating the olfactory response has been emphasized by the demonstration that the nucleotide directly gates cation channels in amphibian ciliary plasma membrane (10). Although greater interest has been addressed to the role of the adenylyl cyclase system, the involvement of .phosphoinositide turnover in the olfactory transduction mechanism has also been reported (11,5,12), raising the hypothesis of a cross-talk between these two second
The odorous sensation is produced by a chemoelectrical signalling cascade which, upon specific interaction of chemical compounds with olfactory receptor neurons, leads to membrane depolarization and, ultimately, to a burst of action potentials. The mechanism by which odors regulate membrane conductance is not yet completely known, but a body of evidence points to cyclic AMP (cAMP) as an intracellular messenger in this phenomenon. It has been reported that olfactory cilia, the dendritic membrane extensions of chemosensory neurons (1,2), are highly enriched in adenylyl cyclase Dipartimento di Biologia, sezione di Fisiologia Generale, Universith degli Studi di Ferrara, Ferrara, Italia 2 Istituto di Clinica Chirurgica, Facolt~ di Medicina Veterinaria, Universith degli Studi di Napoli, Napoli, Italia 3 Address reprint requests to: Prof. Carla Biondi, Dipartimento di Biologia, Sezione di Fisiologia Generale, Universit~t di Ferrara, 44100Ferrara- ITALIA.
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m e s s e n g e r systems w h i c h m a y m o d u l a t e the olfactory response. In v i e w o f the lack o f information available about the olfactory transduction m e c h a n i s m in non-rodent m a m m a l s , the present research was undertaken as part o f a larger project to r e v e a l the p r e s e n c e o f a d e n y l y l cyclase activity in sheep olfactory epithelium, as w e l l as to characterize its r e s p o n s i v e n e s s to odorant m o l e c u l e s and to p h y s i o l o g i c a l and n o n - p h y s i o l o g i c a l ligands. W i t h i n the same context, w e h a v e recently characterized adenylyl cyclase activity in goat olfactory bulb, epithelium and cilia (13). Our data will be c o m p a r e d later with field observations on sheep f o o d preference or aversion in the course o f a study pointed to assess the role o f the olfactory system on small r u m i n a n t grazing behaviour.
EXPERIMENTAL
PROCEDURE
Materials. 3H-cAMP (Sp.Act. 28 Ci/mmol) was purchased from the Radiochemical Centre, Amersham, UK, and 3H-PIP2 (Sp.Act. 5.45 Ci/mmol) from NEN, Florence, Italy. PIP2 was from Boheringer Mannheim, Milan, Italy. ATP, GTP, cAMP, GTP3,S, GppNHp, triethylamine, pyrrolidine, isobutyric and isovaleric acids were purchased from Sigma Chem. Co., St. Louis, MO, USA; forskolin was from Calbiochem Bhering Corp. La Jolla, CA, USA. Eugenol, isoeugenol, ethylvanillin, D-carvone, L-carvone, furfuryl mercaptan, citralva and cresol were purchased from Aldrich, Milan, Italy, while menthol, menrhone, [3-ionone, geraniol, a-damascone, [3-damascone, pyridine and 2-hexylpyridine were kindly provided by Prof. G. P. Pollini, Dept. of Pharmaceutical Chemistry, University of Ferrara, Italy. The nomenclature for most of the odorants is listed in the Merck Index (Ninth edition, 1976, Merck and Co., NJ). As for compounds not listed in the
Merck Index, nomenclature is as follows: citralva: 3,7,dimethyl-2,6 octadienenitrile; c~-damascone: 2-buten-l-one-l-(2,6,6-trimethyl-l-cyclohexen-l-yl); [3-damascone: 2-buten-l-one-l-(2,6,6-trimethyl-2-cyclohexen-l-yl). All other chemicals were the highest reagent grade commercially available. Tissue Preparation. Adult sheep of both sexes were taken from the local slaughter-house. Nasal cavities were dissected open, then sheets of the olfactory epithelium were removed and collected in icecold Ringer's solution. The tissue was homogenized in 10 mM TrisHC1, pH 8.0, and centrifuged at 500 g for 10 rain. Supematant aliquots were utilized for adenylyl cyclase and phospholipase C activity determination. Adenylyl CyclaseActivity. Adenylyl cyclase activity was evaluated according to the method of Clement-Cormier et al. (14), with slight modifications. Briefly, 50 ~tl of tissue preparation (about 200/.tg of proteins) were preincubated for 20 min at 0~ in a final volume of 400 ~tl containing: (raM) 80.2 Tris-maleate buffer pH 7.4, 2 MgSO4, 0.6 EGTA, 10 I.tM GTP, 10 anainophylline, and test substances at the opportune concentration. Odorants were dissolved in dimethylsulphoxide, per se ineffective at the tested concentrations. 0.5 mM ATP was added to start the reaction, and incubation was carried out in a shaking bath for 5 rain at 30~ The enzyme activity was stopped by immersing the tubes in boiling water for 2 rain; the samples were then frozen at -20~ After melting, the samples were centrifuged at 2,000 g for 10 rain, and 100 ~l-aliquots of clear supernatant were transferred into separate tubes for cAMP determination, following the radiochemical method described by Brown et al. (15). Phospholipase C Activity. Basal and odorant-stimulated phospholipase C activity was determined as reported by Mazzoni et al. (16), with slight modifications. Briefly, the assay was carried out in glass tubes at 37~ for different times, according to the experimental protocol. 20 l.tl of tissue preparation (200 ~tg of proteins) were added to a final volume of 120 [tl containing: 3 n_M phosphatidyl-D-myo-inositol-4,5-bisphosphate (P1P2), 3H-PIP2 (about 30,000 dpm), 2% Nataurodeoxycholate, 0.9% NaC1, 1 M Tris-HC1 pH 7.0, 1 mM CaCI2 and opportune concentrations of test substances. The hydrolysis of PIPz was blocked by the addition of 2 ml CHCI~/ CH~OH/ HC1 mixture (2:1:0.75 by vol.). After vortexing, 500 ~tl of 0.6 M HC1 were added. Samples were again vigorously vortexed and centrifuged for 10 rain at 4,000 g. 200 I.tl of the upper phase - containing the watersoluble products of PIPz hydrolysis - were then transferred into plastic vials and 4 ml of scintillation cocktail were added. Radioactivity was determined by a Beckman 1801 liquid spectrometer. Protein Determination. Protein content of the samples was evaluated according to the colorimetric method reported by Lowry et al. (17), using bovine serum albumin as standard. StatisticalAnalysis. Statistical significance was assessed by Student's t test using a p value of < 0.05.
RESULTS
Characterization of Adenylyl Cyclase Activity in Sheep Olfactory Epithelium. A s a first step, we tested the d e p e n d e n c e o f a d e n y l y l cyclase activity on two essential parameters: protein concentration and incubation time. A s s h o w n in Fig. I, c A M P biosynthesis is proportional to the protein content up to 400 g g per assay. Fig. 2 shows the t i m e - d e p e n d e n c e o f basal as w e l l as odorant-activated adenylyl cyclase. In both cases, en-
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Fig. 2. Time course of the basal (e) and citralva-stimulated (o) adenylyl cyclase activity of sheep olfactory epithelium. Citralva is 100 gM. Data are the mean of three separate experiments run in duplicate. For each value SEM is within 10%. *p
