Proc. Nail. Acad. Sci. USA Vol. 83, pp. 4942-4946, July 1986 Neurobiology Odorant-binding protein: Localization to nasal glands and secretions (olfaction/mucus/immunohistochemistry/pyrazines) JONATHAN PEVSNER, PAMELA B. SKLAR, AND SOLOMON H. SNYDER* Departments of Neuroscience, Pharmacology, and Experimental Therapeutics, Psychiatry, and Behavioral Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205 Contributed by Solomon H. Snyder, January 6, 1986

ABSTRACT An odorant-binding protein (OBP) was iso- formed at an antiserum dilution of 1:8100 in 0.1 M Tris HCl, lated from bovine olfactory and respiratory mucosa. We have pH 8.0/0.5% Triton X-100, in a final vol of50 A.l. Incubations produced polyclonal antisera to this protein and report its were carried out at 370C for 90 min with 35,000 cpm of immunohistochemical localization to mucus-secreting glands of '251-labeled OBP per tube. Immunoprecipitation was accom- the olfactory and respiratory mucosa. Although OBP was plished by using 25 1LI of 5% Staphylococcus aureus cells originally isolated as a pyrazine binding protein, both rat and (Calbiochem) in 0.1 M Tris HCl (pH 8.0) at 370C for 30 min. bovine OBP also bind the odorants [3H]methyldihydrojasmon- Bound 1251I-labeled OBP was separated from free OBP by ate and 3,7-dimethyl-octan-1-ol as well as 2-isobutyl-3-[3HI filtration over glass fiber filters (No. 32, Schleicher & methoxypyrazine. We detect substantial odorant-binding ac- Schuell) pretreated with 10% fetal bovine serum, using a tivity attributable to OBP in secreted rat nasal mucus and tears Brandel cell harvester (Brandel, Gaithersburg, MD). In but not in saliva, suggesting a role for OBP in transporting or typical experiments, maximal and nonspecific binding were concentrating odorants. 30% and 2% of added radioactivity, respectively. Immunoblots. Whole bovine nasal epithelia were homog- In an effort to clarify molecular mechanisms of olfaction, enized in buffer A (50 mM Tris HCl, pH 7.6/1 mM EDTA) several groups have examined the binding of radioactive and filtered over cheesecloth. Samples of purified OBP, odorants to (1-6). Recently, we purified to bovine serum albumin, and nasal homogenates prepared in homogeneity an odorant-binding protein based on its inter- buffer A were electrophoresed into 14% NaDodSO4/poly- actions with the potent odorant 2-isobutyl-3-[3H]methoxy- acrylamide gels. Transfer of proteins to nitrocellulose was pyrazine ([3H]IBMP) (7), a finding obtained independently by accomplished in 12 hr at 60 V (11). The nitrocellulose was Bignetti et al. (8). Odorant-binding protein (OBP) is a soluble incubated for 12 hr in buffer A supplemented with 0.1% dimeric protein with subunits of =19 kDa. OBP may have a gelatin and 0.1% Triton X-100 to decrease nonspecific staini- selective function in olfaction, since it occurs in nasal mucosa ing. Immunoblots were incubated with a 1:1000 dilution of and not in other tissues, and the relative potencies of a antisera for 2 hr as indicated in Fig. 2. Antisera were pre- homologous series of pyrazine derivatives in competing for adsorbed for 24 hr at 40C with either bovine serum albumin the binding sites parallels their potencies as odorants. or purified bovine OBP that had been further purified by We have produced antisera to bovine OBP and now report HPLC (see below). Immunoblots were developed by using the immunohistochemical localization ofOBP to bovine nasal the avidin/biotin/peroxidase technique (Vector Laborato- glands, which secrete mucus. Odorant-binding studies dem- ries, Burlingame, CA) with 4-chloro-1-naphthol and hydro- onstrate high levels of OBP in rat nasal mucus and tears, gen peroxide as substrates. suggesting a role for OBP in concentrating odorants from the Immunohistochemistry. Whole bovine nasal epithelia were air. obtained immediately after slaughter and were fixed in i% glutaraldehyde in 0.15 M sodium phosphate buffer (pH 7.4) MATERIALS AND METHODS for 2 hr. Tissue was embedded in a mixture of 50% Materials. [3H]IBMP (43.8 Ci/mmol; 1 Ci = 37 GBq), paste/50% Tissue-Tek (Miles Scientific, Naperville, IL) and 2-[3H]methoxypyrazine (63.6 Ci/mmol), and [pentyl-2,3- rapidly frozen. Cryostat tissue sections (8 ,um) were cut with 3H]methyldihydrojasmonate ([3H]MDHJ; 65.5 Ci/mmol) a microtome and immunohistochemically stained with the were prepared by New England Nuclear Dupont. 3,7-Di- avidin/biotin/peroxidase complex technique (Vector Labo- methyl[6,7(N)-3H]octan-1-ol ([3H]DMO; 57 Ci/mmol), ratories) using diaminobenzidine and hydrogen peroxide as [amyl-3H]isoamyl acetate (49 Ci/mmol), and [3H]isovaleric substrates (12-14). Tissue sections were incubated with a acid (4-[2,3(n)3H]methylbutanoic acid; 52 Ci/mmol) were 1:20,000 dilution of antisera against either bovine OBP, prepared by Amersham. Unlabeled odorants were from bovine serum albumin (Sigma), or normal rabbit serum for 48 International Flavors and Fragrances (Union Beach, NJ) or hr at 4°C. Additional sections were counterstained with Pyrazine Specialties (Atlanta, GA). Protein molecular size toluidine blue. markers were obtained from Bio-Rad. All other reagents Purification and Characterization of Bovine OBP and Rat were from commercial sources. Mucus OBP. Bovine OBP was purified from bovine olfactory Preparation of Antibodies to OBP and Radioimmunoassays. and respiratory epithelium as described (7) by sequential Bovine OBP was purified as described (7), and antibodies to centrifugation; ammonium sulfate fractionation; and DEAE- this protein were raised by standard techniques (9). Bovine cellulose, hydroxylapatite, and gel filtration chromatogra- OBP was iodinated with lodo-Beads (Pierce) according to the method of Markwell (10). Radioimmunoassays were per- Abbreviations: IBMP, 2-isobutyl-3-methoxypyrazine; MDHJ, methyldihydrojasmonate (3-oxo-2-pentyl-cyclopentane-acetic acid The publication costs of this article were defrayed in part by page charge methyl ester); DMO, 3,7-dimethyl-octan-1-ol; OBP, odorant-binding payment. This article must therefore be hereby marked "advertisement" protein. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 4942 Downloaded by guest on September 25, 2021 Neurobiology: Pevsner et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4943 phy. A single protein was purified from rat olfactory and respiratory epithelium by using the same purification proce- dure. Purified bovine OBP and proteins present in crude rat mucus were separated by HPLC on a Vydac C4 protein column (4.6 x 200 mm; 5-gim particle size) (15). The column was equilibrated with 0.1% (vol/vol) aqueous trifluoroacetic o 1: acid, and the samples were loaded in the same solvent. After x a 5-min wash, the proteins were eluted with a linear gradient E of 0-100% organic solvent over 40 min [0.1% (vol/vol) CL trifluoroacetic acid in acetonitrile/n-propanol (2:1)]. Proteins 0 were analyzed by NaDodSO4/PAGE (16) on 14% polyacryl- a E C- amide gels in the presence of 2-mercaptoethanol and were 0 ._-- .0 stained with Coomassie brilliant blue. Protein was deter- 0 mined according to the method of Bradford (17) using bovine 0- serum albumin as a standard. .0 Odorant Binding to Secretions from Rat. Male Sprague- Dawley rats (6-10 weeks old) were anesthetized with sodium Cu I pentobarbital (60 mg/kg, i.p.) and injected with isoproterenol ct (30 mg/kg, i.p.) to induce secretions (18-20). After 5 min, secreted nasal mucus, tears, and saliva were collected with 5-,41 or 100-gl glass micropipettes. Mucus was collected from the external nares and tears were collected from the puncta. Typically, 10 Al of nasal mucus, 20 Al of tears, and 200 A.l of saliva were obtained from a single rat in 1 hr. Fraction Binding assays were performed by filtration over polyeth- ylenimine-coated filters as described (7). Assay mixtures FIG. 1. Partial purification of OBP from bovine and rat nasal typically contain secretions (2-20 jig of protein), 3-25 nM epithelium by DEAE-cellulose chromatography. After ammonium radioactive odorant, and unlabeled odorants in a final vol of sulfate fractionation, bovine (A) or rat (B) protein was dialyzed and 100 ,ul, and they were incubated at 4°C for 1 hr. applied to a DEAE-cellulose column with a linear NaCl gradient In Vivo Binding of Individual male (0-400 mM NaCl; bovine OBP elutes at 330 mM NaCl; rat binding [3H]DMO. Sprague- protein elutes at 270 mM NaCl). All fractions were assayed for the Dawley rats were anesthetized and injected with isoproter- binding activities of [3H]DMO (8 nM), [3H]IBMP (10 nM), and enol as described above. Two minutes after the isoproterenol [3H]MDHJ (6 nM). All three odorants bind with a single major peak injection, a polypropylene test tube (1.5 ml capacity) con- of binding activity. Binding of [3H]amyl acetate and 2-[3H]methoxy- taining 50 ,Ci in 50 ,ul of [3H]DMO was positioned around the pyrazine also reveal single peaks of binding activity (not shown). animal's nose, eyes, or mouth for 3 min. Secretions were Data are from a single experiment. collected and assayed for protein content. Labeled odorant present in the mucus or saliva was measured by liquid DEAE-cellulose chromatography, column fractions were scintillation spectrometry. Radioactivity bound to protein assayed for the binding of four tritiated odorants. A single was measured by filtration (as described above) within 1 min major peak of activity was found (Fig. 1A). Using the same of collection of the secretions. techniques, we have purified an OBP from rat olfactory and respiratory epithelium. DEAE-cellulose chromatography re- RESULTS veals a single peak of binding activity (Fig. 1B) and the purified protein has an apparent subunit molecular size of 21 Odorant Binding to Purified Bovine OBP. OBP was first kDa by NaDodSO4/PAGE (data not shown). detected on the basis ofits pyrazine-binding properties (7, 8). We wondered if four chemically distinct 3H-labeled odor- Homogeneous bovine OBP also binds three nonpyrazine ants, which bind to a single protein, interact at the same or odorants-[3H]DMO, [3H]MDHJ, and [3H]amyl acetate (Ta- different sites on bovine OBP. In competitive binding studies, ble 1). Bovine OBP was purified as described (7). After each of the odorants displays similar potency in competing for the four different 3H-labeled odorants (Table 2). These Table 1. Binding constants for odorants to purified bovine OBP results suggest that all four odorants bind to the same site on and rat mucus and tears OBP. However, one cannot rule out the possibility that they Bovine OBP Rat mucus Rat tears bind to different sites that display allosteric interactions. Odorant Kd,,4M Bm, Kd,,LM Blx Kd, juM Bmx Table 2. Inhibition of 3H-labeled odorant binding to purified [3H]Amyl acetate 68 22 >1000 ND >1000 ND bovine OBP [3H]DMO 0.3 30 44 1.6 100 15.7 [3H]IBMP 3 27 20 2.3 11 1.7 IC50, JIM [3H]MDHJ 8 18 35 0.3 25 0.3 Amyl 2-[3H]Methoxy- Odorant acetate DMO IBMP MDHJ pyrazine >1000 ND >1000 ND >1000 ND [3H]Amyl acetate 0.5 18 30 Binding assays were performed as described (7) using bovine OBP [3H]DMO 30 13 23 (1-2 ,j.g ofprotein per tube), crude rat mucus (30-60 jig ofprotein per [3H]IBMP 70 0.5 - 13 tube), crude rat tears (30-60 Ag of protein per tube), and 5-15 nM of each tritiated odorant. B.. values are nmol bound per mg ofprotein. [3H]MDHJ 65 2 2 No specific binding was detected for the binding of 10 nM IC50 values (concentration of unlabeled odorant that inhibits [3H]isovaleric acid to bovine OBP or rat mucus. Less than 5 fmol per response by 50%o) were calculated from displacement curves by using mg of protein of [3H]IBMP or [3H]DMO binding to crude rat saliva bovine OBP (1-2 ,ug ofprotein per tube), 30-45 nM [3H]amyl acetate, was detected (n = 2). ND, not determined. Results are the mean of 3-10 nM [3H]DMO, 3-10 nM [3H]IBMP, and 3-10 nM [3H]MDHJ. 3-6 determinations. Results are the mean of at least two determinations. Downloaded by guest on September 25, 2021 4944 Neurobiology: Pevsner et al. Proc. Natl. Acad. Sci. USA 83 (1986)

kDa 1 2 3 4 5 6 7 8 9 Based on the binding of [3H]IBMP, the levels of OBP in rat 66.2:- nasal mucosa are about the same as in bovine nasal mucosa (data not shown). However, radioimmunoassay for OBP in rat olfactory mucosal homogenates detects levels only 0.1% 45 iw- of those found in bovine , indicating very FIG. 2. Immunoblot analysis little antiserum cross-reactivity between the species. of antisera to bovine OBP. Puri- To ensure that the antiserum is selective for OBP, we fied OBP (20 ,g; lanes 1-3), crude performed immunoblot analysis of bovine olfactory and 31 nasal homog- respiratory mucosa (Fig. 2). Coomassie blue staining of enates (40 /Lg; lanes 4-6), crude purified OBP detects the major band of protein at 19 kDa and nasalrespiratoryepitheliumhomog- at 66 kDa (see Fig. 4 Inset, lane 2). In enates (40 ,ug; lanes 7-9). Anti-OBP a minor contaminant antiserum at a dilution of 1:1000 immunoblots of purified bovine OBP, antisera against OBP (lanes 1, 4, and 7); anti-OBP antise- recognize a band of 19 kDa and a minor contaminant at 63 21 .5 I .nrum (1:1000), preadsorbed for 24 hr kDa (Fig. 2, lane 1). The 66-kDa contaminant observed with with HPLC-purified bovine OBP Coomassie blue staining is not recognized by the antiserum 144 (50 (lanes 2, 5, and 8); nor- to OBP. In crude olfactory and respiratory epithelium 14.4 3W- malpHg/ml)rabbit serum (1:1000) (lanes 3, homogenates, only the 19-kDa protein is recognized, con- 6, and 9). firming that the immunohistochemical stain images only authentic 19-kDa protein (Fig. 2, lanes 4 and 7). Preadsorp- Characterization of the Antiserum to Bovine OBP. The titer tion of antiserum with HPLC-purified bovine OBP abolishes of the polyclonal antiserum and its affinity for OBP were staining ofthe 19-kDa protein (lanes 2, 5, and 8). The staining evaluated by radioimmunoassay. Half-maximal binding of and adsorption patterns are the same for both olfactory and OBP occurs at an antibody dilution of 1:8100. To determine respiratory mucosa, although the olfactory mucosa has the affinity ofthe antibody for OBP, we examined the binding somewhat less OBP, confirming earlier results based on of 115I-labeled OBP to the antiserum in the presence of [3H]IBMP binding (7). increasing concentrations ofunlabeled OBP. Fifty percent of Immunohistochemical Loclizaon of Bovine OBP. Immu- maximal binding is apparent at 0.6 nM OBP. In the radio- nohistochemical staining of bovine nasal epithelium reveals immunoassay, the lowest detectable level of OBP is 30 pM. OBP-like immunoreactivity in the glands of the lamina

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FIG. 3. Immunohistochemical localization of bovine aD,'~~~~~~J OBP. Bovine olfactory (A, C, and E) and respiratory (B and D) epithelium are shown. Normal histology was demonstrat- KI ; - A 'a' ed by toluidine blue staining (A and B). The antiserum !WIF-~ produced against purified bovine OBP as described in Ma- terials and Methods was used at a dilution of 1:20,000 to immunohistochemically stain bovine olfactory (C) and res- piratory (D) epithelium. (E) Staining of bovine olfactory epithelium by normal rabbit serum (1:20,000). OE, olfactory epithelium; RE, respiratory epithelium; RBC, erythrocyte; G, glands; LP, lamina propria; A, arteriole. Downloaded by guest on September 25, 2021 ws*s - -FS;;_*8X**8S.:q Neurobiology: Pevsner et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4945

(Table 1). [3H]DMO, [3H]IBMP, and [3H]MDHJ bind spe- 120 A cifically and saturably to secreted rat mucus and tears, but not to saliva. We also evaluated [3H]amyl acetate, 2-[3H]me- thoxypyrazine and [3H]isovaleric acid, all of which have distinctive odors. Only very low levels of [3H]amyl acetate and 2-[3H]methoxypyrazine binding are detectable, and bind- C., 60 ing constants cannot be obtained by filtration binding assays. 0 We have failed to detect any binding of [3H]isovaleric acid at x neutral pH to nasal mucus or OBP. (n Using a two-step procedure consisting of DEAE-cellulose C and HPLC chromatographic steps, we have purified OBP to 0) apparent homogeneity from rat nasal mucus. A single peak of CQ 20 B binding activity is observed after DEAE-cellulose chroma- tography (data not shown). This peak (40 8% of the total 0 kDa 1 2 3 4 ,ug; 92 protein) is further purified by reverse-phase HPLC (Fig. 4B). 30 I ~~~~~~~~~~~66.2- co A single peak is observed, closely corresponding to the 6 45 - elution volume of bovine OBP (Fig. 4A). This HPLC peak 20 I- ~~~~~31-- contains 21 pmol of [3H]DMO bound per mg of protein ([3H]DMO concentration in the assay, 41 nM). NaDodSO4/ 21.5- .w_ PAGE of the peak reveals a single band of 21 kDa (Fig. 4 10 _ 14.4 Inset) corresponding to that observed for purified rat OBP derived from olfactory epithelium (data not shown). Concentration of [3H]DMO by OBP in Rat Mucus. A I 0 _ function of OBP might be to interact with certain odorants in 30 10 20 vivo. To examine this possibility, rats were anesthetized and Time, min injected with isoproterenol, and then either the nose, eyes, or mouth was exposed to [3H]DMO in ambient air as described FIG. 4. Reverse-phase HPLC analysis of bovine OBP and rat in Materials and Methods. mucus. HPLC analysis was performed after fractionation on a Total [3H]DMO levels in the nasal mucus are -'60 nM, DEAE-cellulose column as described in Materials and Methods. (A) nM Bovine OBP (50 jig; retention time, 20.58 min); (B) partially purified while filter-bound radioactivity is "14 (Table 3). Slightly rat mucus (40 ,g; retention time, 20.88 min). (Inset) NaDodSO4/ lower concentrations of tritiated odorant accumulate in PAGE analysis of bovine and putative rat OBP was performed as secreted tears. No bound radioactivity is detected in saliva described. Lanes: 1, molecular size markers; 2, bovine OBP (15 jg) exposed to [3H]DMO in vivo, and saliva contains only 1% of purified as described (7); 3, the major HPLC peak ofabsorbance from the concentration of [3H]DMO measured in nasal mucus. A; 4, the major HPLC peak of absorbance from B. DISCUSSION propria and along the surface of the epithelium (Fig. 3 C and D). Anatomically, mammalian nasal glands are differentiated The major findings of the present study are that OBP, into Bowman's glands in the olfactory mucosa and respira- originally isolated as a pyrazine-binding protein, binds sev- tory glands, which underlie the respiratory mucosa (21). eral structurally unrelated odorants, that OBP is localized in Specific staining for OBP occurs in both types of glands. bovine nasal glands, and that OBP is secreted into the nasal Preadsorption of the antiserum with HPLC-purified OBP mucus of rats, where it comprises 2% of total protein. eliminates glandular staining (data not shown). Preadsorption The binding of several structurally unrelated odorants to ofthe antiserum with bovine serum albumin does not alter the OBP and the lack ofbinding ofnon-odorants (7) indicates that staining pattern. A band ofimmunoreactivity along the ciliary OBP is involved in olfaction. Of numerous nasal proteins (outer) surface of the olfactory and respiratory epithelium is (22-27), only OBP binds odorants selectively. Moreover, the also seen in the normal rabbit serum control and thus is not micromolar Kd of OBP for certain odorants is in the range of specific for OBP. Reaction product is deposited in erythro- concentrations relevant to olfactory transduction both cytes when either OBP antiserum or normal rabbit serum is electrophysiologically (28) and biochemically (29). Our evi- used and presumably reflects endogeneous peroxidase activ- dence that OBP interacts with odorants in the ambient air ity. suggests a specific role in olfaction. Odorants subjectively Demonstration of Odorant Binding in Rat Nasal Mucus and detected when present at apparent nanomolar concentrations Tears. A major function ofthe nasal glands is the secretion of in the air are then concentrated to the micromolar levels in mucus into the (22). Based on the localization of mucus required to influence olfactory neuronal firing (28) and OBP to bovine mucus-secreting glands, we investigated the adenylate cyclase (29). The parallel between sensory poten- binding of odorants to secreted mucus, tears, and saliva in cies of pyrazine odorants and their affinities for OBP sup- rats whose secretions were stimulated with isoproterenol ports such a role.

Table 3. Concentration of [3H]DMO in rat secretions after odorant exposure in vivo Total [3H]DMO Bound [3H]DMO Secretion cpm/,ul Concentration, nM cpm/Al Concentration, nM % total

Nasal mucus 2280 572 (8) 60 15 539 ± 109 (8) 14 ± 3 24 ± 6 Tears 1942 821(6) 51 ±22 304 ± 95 (7) 8 ± 3 16 ± 8 Saliva 16± 14 (6) 0.4 0.4 0 ± 4 (6) 0 ± 0.1 0 ± 1 Individual rats were anesthetized and injected with isoproterenol, and the nose, eyes, or mouth were exposed to odorant (17.5 AM [3H]DMO; 7.3 x 101 cpm/Al) as described in Materials and Methods. Three to 10 A.l of mucus or tears (25-30 mg ofprotein per ml) and 50-100 A.l of saliva (20-30 mg of protein per ml) were collected and assayed as described. Data are expressed as cpm/pl ± SEM of (n) determinations. Scintillation counter background (12 cpm) was subtracted from each data point. Downloaded by guest on September 25, 2021 4946 Neurobiology: Pevsner et al. Proc. Natl. Acad. Sci. USA 83 (1986) Immunohistochemical localization of OBP to bovine nasal USA 77, 4412-4416. glands indicates that this protein is not a neuronal odorant 7. Pevsner, J., Trifiletti, R. R., Strittmatter, S. M. & Snyder, receptor. However, the olfactory epithelium is bathed in a S. H. (1985) Proc. Natl. Acad. Sci. USA 82, 3050-3054. 8. Bignetti, E., Cavaggioni, A., Pelosi, P., Persaud, K. C., Sorbi, layer of mucus secreted by the underlying glands, which R. T. & Tirindelli, R. (1985) Eur. J. Biochem. 149, 227-231. odorants must traverse prior to their interaction with 9. Eipper, B. A. & Mains, R. E. (1978) J. Supramol. Struct. 8, neuronal receptors (21). OBP is secreted into the nasal mucus 247-262. after isoproterenol stimulation. This finding is consistent with 10. Markwell, M. A. K. (1982) Anal. Biochem. 125, 427-432. the reduction in the secretory granule content of acinar cells 11. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. of the salamander olfactory glands after stimulation with Acad. Sci. USA 76, 4350-4354. isoproterenol (20) or with micromolar concentrations of 12. Childs, G. & Unabia, G. (1982) J. Histochem. Cytochem. 30, IBMP (30). OBP is presumably stored in the granules of the 713-716. olfactory glands and released upon appropriate stimulation. 13. Hsu, S. M., Raine, L. & Fauger, H. (1981) J. Histochem. Cytochem. 29, 577-580. The binding ofvarious odorants to OBP may be relevant to 14. Braas, K. M., Newby, A. G., Wilson, V. S. & Snyder, S. H. models proposed for transporting odorants (31, 32) prior to J. Neurosci., in press. their interaction with olfactory receptors or eliminating them 15. Tarr, G. E. & Crabb, J. W. (1983) Anal. Biochem. 131, 99-107. after signal transduction (21). The time required for an 16. Laemmli, U. K. (1970) Nature (London) 227, 680-685. odorant to traverse the olfactory mucosa from the surface to 17. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. receptor sites is in part a function of the diffusion coefficient 18. Burton, L. E., Wilson, W. H. & Shooter, E. M. (1978) J. Biol. of the molecule and the viscosity of the medium (33, 34). In Chem. 21, 7807-7812. addition to concentrating odorants in the mucosa relative to 19. Wallace, L. J. & Partlow, L. M. (1976) Proc. Natl. Acad. Sci. the air, OBP could decrease the diffusion delay of odorants USA 73, 4210-4214. 20. Getchell, M. L. & Getchell, T. V. (1984) J. Comp. Physiol. traversing the mucosa by acting as a selective carrier. 155, 435-443. [3H]DMO in ambient air accumulates in the mucus but not 21. Getchell, T. V., Margolis, F. L. & Getchell, M. L. (1984) in tears or saliva. However, the observed levels ofodorant in Prog. Neurobiol. 23, 317-345. these secretions may not be directly comparable because 22. Widdicombe, J. G. & Wells, U. M. (1982) in The Nose: Upper tritiated odorant is actively inhaled into the nose, but it Airway Physiology and the Atmospheric Environment, eds. diffuses passively into the tears and saliva. The binding of Proctor, D. F. & Andersen, I. B. (Elsevier, New York), pp. inhaled [3H]DMO suggests that OBP can interact with odor- 215-244. ants in vivo. 23. Rosen, R. D., Alford, R. H., Butler, W. J. & Vannier, W. E. It is unclear whether odorant binding in rat tears involves (1966) J. Immunol. 97, 369-378. 24. Josephson, A. S. & Weiner, R. S. (1968) J. Immunol. 100, secretions from the lacrimal glands or from the nose with 1080-1092. subsequent transport to the tears via the nasolacrimal duct 25. Creeth, J. M. (1978) Brit. Med. Bull. 34, 17-24. (35). 26. Boat, T. F. & Cheng, P. W. (1980) Fed. Proc. Fed. Am. Soc. Exp. Biol. 39, 3067-3074. We thank Drs. Karen Braas and Randall Reed for valuable advice, 27. Gower, D. B., Hancock, M. R. & Bannister, L. H. (1981) in and Jeffrey Nye for helpful discussions. This work was supported by Biochemistry of Taste and Olfaction, eds. Cagan, R. H. & a grant from International Flavors and Fragrances, Incorporated. Kare, M. R. (Academic, New York), pp. 7-31. J.P., P.B.S., and S.H.S. are respective recipients of National 28. Kashiwayanagi, M. & Kurihara, K. (1984) Brain Res. 359, Institutes of Health Training Grant GM-07626, Training Grant 97-103. MH-15330, and RSA Award DA-00074. 29. Pace, U., Hanski, E., Salomon, Y. & Lancet, D. (1985) Nature (London) 316, 255-258. 1. Pelosi, P., Pisanelli, A. M., Baldaccini, N. E. & Gagliardo, A. 30. Getchell, T. V., Zielinski, B. & Getchell, M. L. (1985) Chem. (1981) Chem. Senses 6, 77-85. Senses 10, 398-399. 2. Pelosi, P., Baldaccini, N. E. & Pisanelli, A. M. (1982) 31. Mozell, M. M. (1970) J. Gen. Physiol. 56, 46-63. Biochem. J. 201, 245-248. 32. Hornung, D. E. & Mozell, M. M. (1981) in Biochemistry of 3. Topazzini, A., Pelosi, P., Pasqualetto, P. L. & Baldaccini, Taste and Olfaction, eds. Cagan, R. H. & Kare, M. R. (Aca- N. E. (1985) Chem. Senses 10, 45-49. demic, New York), pp. 33-45. 4. Gennings, J. N., Gower, D. B. & Bannister, L. H. (1977) 33. Getchell, T. V., Heck, G. L., DeSimone, J. A. & Price, S. Biochim. Biophys. Acta 496, 547-556. (1980) Biophys. J. 29, 397-411. 5. Fesenko, E. E., Novoselov, V. I. & Krapivinskaya, L. D. 34. Getchell, T. V. & Getchell, M. L. (1977) Chem. Senses Flavor (1979) Biochim. Biophys. Acta 587, 424-433. 2, 313-326. 6. Rhein, L. D. & Cagan, R. H. (1980) Proc. Natl. Acad. Sci. 35. Bojsen-Moller, F. (1964) Anat. Rec. 150, 11-24. Downloaded by guest on September 25, 2021