Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons.

Odorant signal transduction occurs in the specialized cilia of the olfactory sensory neurons. Considerable biochemical evidence now indicates that this process could be mediated by a G protein-coupled cascade using cyclic AMP as an intracellular second messenger. A stimulatory G protein alpha subunit is expressed at high levels in olfactory neurons and is specifically enriched in the cilia, as is a novel form of adenylyl cyclase. This implies that the olfactory transduction cascade might involve unique molecular components. Electrophysiological studies have identified a cyclic nucleotide-activated ion channel in olfactory cilia. These observations provide evidence for a model in which odorants increase intracellular cAMP concentration, which in turn activates this channel and depolarizes the sensory neuron. An analogous cascade regulating a cGMP-gated channel mediates visual transduction in photoreceptor cells. The formal similarities between olfactory and visual transduction suggest that the two systems might use homologous channels. Here we report the molecular cloning, functional expression and characterization of a channel that is likely to mediate olfactory transduction.

Olfactory neurons express a unique glycosylated form of the neural cell adhesion molecule (N-CAM).

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.

Expression of catfish amino acid taste receptors in Xenopus oocytes.

We demonstrate that poly (A+)RNA isolated from catfish barbels directs the expression of functional amino acid taste receptors in the Xenopus oocyte. The activity of these receptors is monitored in ovo by the two electrode voltage clamp technique. Specific conductance changes recorded in response to amino acid stimulation are analogous to those recorded electrophysiologically from intact catfish barbels. These responses exhibit specificity, reproducibility, rapid onset and termination, and desensitization to repetitive stimulation. A functional assay system that encompasses the full complement of transduction events from the ligand-receptor interaction to subsequent conductance changes is necessary to identify molecular components responsible for these events. Our results demonstrate that the Xenopus oocyte can be used to characterize and identify clones coding for amino acid taste receptors analogous to its use in studying receptor molecules for other neuroactive compounds.

Ligand binding characteristics of homologous rat and mouse urinary proteins and pyrazine-binding protein of calf.

1. The binding affinities of three classes of homologous proteins, alpha 2u protein from rat urine, major urinary protein of mouse and pyrazine-binding protein of calf nasal mucosa have been determined for a panel of ligands. 2. Best ligands were low threshold odorants for man, but chemically unrelated. 3. The binding spectra of the homologous proteins were different.

Does the olfactory system mediate water- and mineral-regulating mechanisms? Evidence of immunoreactive atrial natriuretic factor within olfactory mucosa.

The immunoreactivity of atrial natriuretic factor (ANF) was studied in the rat olfactory mucosa (OM). Endogenous immunoreactive ANF (IR-ANF) was purified from OM using Vycor glass beads for extraction and reverse-phase HPLC: two of three IR-ANF peaks, identified by retention time, were identical to both the circulating form of ANF (Ser99-Tyr126) and the ANF pro-hormone (Asn1-Tyr126). A radioreceptor assay, employing rat renal glomerular membranes, revealed that endogenous IR-ANF competed with radiolabelled ANF. IR-ANF was localized by immunocytochemistry in secretory cells of Bowman's gland and in some cells of the epithelial layer. The relatively low concentration of IR-ANF in the OM (2.5 ng/mg protein) suggests a local role of ANF in this tissue. This hypothesis is supported by the presence in OM of ANF-binding sites, characterized by a KD of 95 pM and a Bmax of 130 fmol/mg protein. We propose that ANF could be released from the OM and act throughout in a paracrine (if not autocrine) manner on some yet-unidentified targets containing ANF-binding sites.

[Ca2+-protease--an enzyme of the metabolism of cytoskeletal proteins in the olfactory membrane of vertebrates]. / Ca2+-proteaza--ferment metabolizma belkov tsitoskeleta oboniatel'noi vystilki pozvonochnykh.

Two forms of Ca(++)-activated protease (calpain I and calpain II) associated with an endogenous inhibitor (calpastatin) were detected in a cytosolic fraction of the olfactory tissue of vertebrates (pig, rat). Using ion exchange chromatography on DEAE-cellulose column, calpain I is divided into 2 peaks (eluting by 0.07-0.15 and 0.22-0.25 M NaCl), and calpain II is eluted by 0.35-0.40 M NaCl. The calpain activity was detected in fractions eluted by 0.1-0.17 M NaCl. The Ca(++)-activated protease was demonstrated also in a fraction of cytoskeleton of olfactory tissue insoluble in a 1% solution of Triton X-100. The activity can be detected by Ca(++)-dependent destruction of exogenous substrate (casein), and by Ca(++)-dependent degradation of cytoskeletal endogenous proteins (16, 18 and 20 kDa), of which one may be calmodulin.

Characterization of the total lipid and fatty acid composition of rat olfactory mucosa.

Phospholipid accounted for 81% (by weight) of the total lipid of rat olfactory mucosa. Phosphatidylcholine (46% of total phospholipids) and phosphatidylethanolamine (26%) were the predominant phospholipids. Phosphatidylinositol (8%), sphingomyelin (6%), and phosphatidylserine (7%) were the next most abundant phospholipids, with cardiolipin (4%) and phosphatidic acid (1%) present in lesser amounts. Only trace amounts of the polyphosphoinositides, phosphatidylinositol monophosphate, and phosphatidylinositol bisphosphate were detected. Sterol was the major neutral lipid present (83% of the total neutral lipid mass) with lesser amounts of triacylglycerols (7%), steryl esters (6%), free fatty acids (4%), and diacylglycerols (1%). Monoacylglycerols were detected only in trace amounts. The sterol to phospholipid ratio was 0.39:1. Most of the phospholipids of the olfactory mucosa showed a high polyunsaturated fatty acid content, with the arachidonic acid (20:4) and docosahexaenoic acid (22:6) residues predominating. The fatty acids in sphingomyelin, however, were almost totally saturated and included the 24:0 and 24:1 residues, which were not detected in other phospholipids. Polyunsaturated fatty acids accounted for less than 25% of the total fatty acid of any individual neutral lipid and comprised largely linoleic and arachidonic acids. The results are discussed in relation to the putative role of lipids in olfactory signal transduction.

Golf: an olfactory neuron specific-G protein involved in odorant signal transduction.

Biochemical and electrophysiological studies suggest that odorants induce responses in olfactory sensory neurons via an adenylate cyclase cascade mediated by a G protein. An olfactory-specific guanosine triphosphate (GTP)-binding protein alpha subunit has now been characterized and evidence is presented suggesting that this G protein, termed Golf, mediates olfaction. Messenger RNA that encodes Golf alpha is expressed in olfactory neuroephithelium but not in six other tissues tested. Moreover, within the olfactory epithelium, Golf alpha appears to be expressed only by the sensory neurons. Specific antisera were used to localize Golf alpha protein to the sensory apparatus of the receptor neurons. Golf alpha shares extensive amino acid identity (88 percent) with the stimulatory G protein, Gs alpha. The expression of Golf alpha in S49 cyc- kin- cells, a line deficient in endogenous stimulatory G proteins, demonstrates its capacity to stimulate adenylate cyclase in a heterologous system.

Expression of intermediate filaments and desmoplakin in vertebrate olfactory mucosa.

The expression of intermediate filaments (IF) and desmoplakin was investigated in frog, bovine, and human (fetal) olfactory mucosa. IF are tissue-specific molecular cytoskeletal markers; desmoplakin is the major desmosomal protein. Positive immunoreactivity was observed in the epithelium and in the subepithelial Bowman's glands to keratin and to desmoplakin, indicating the epithelial nature of this tissue. Desmin, neurofilaments, and glial fibrillary acidic protein (GFAP) were not detected in the mucosa. The absence of neurofilaments and GFAP in the tissue containing sensory neurons and glia-like supporting cells is a unique feature and may be related to the fact that the chemosensory neurons are situated in a bonafide epithelium and are known to undergo continuous turnover. In view of the controversy regarding the expression of vimentin in the olfactory neurons, three independently derived antibodies to vimentin were used; weak or no labeling was found in the epithelium, whereas mesenchymal cells in the lamina propia were labeled with all three antibodies. Olfactory nerve fascicles in the lamina propia were heterogenously labeled: VIM 13.2 gave very weak labeling; aVimAS showed mild labeling and SBV-21 showed intensive labeling in the nerve fascicle. This heterogenous labeling pattern may suggest that olfactory vimentin is distinct in reacting only with some of the antivimentin antibodies.

Effects of antibodies against odorant binding proteins on electrophysiological responses to odorants.

Monoclonal antibodies against two olfactory mucosal proteins, one with affinity for anisole-like and the other for benzaldehyde-like compounds, were applied to mouse olfactory epithelium. Responses to three odorants (anisole, benzaldehyde and amyl acetate) were measured. Of 26 antibodies, three (12%) inhibited responses only to the odorant with affinity for the antigen, nine (35%) inhibited responses to all three odorants, and 14 (54%) were without effect. None reduced responses by as much as 50%. The data support the hypothesis that there is a class of related proteins in olfactory neuronal cell membranes that function as receptor molecules and that other mechanisms also mediate odorant stimulation.

Activation by odorants of a multistate cation channel from olfactory cilia.

Single-channel records were obtained after fusion of ciliary membranes from the olfactory epithelium of Rana catesbeiana to planar lipid bilayers, and odorant-activated cation-selective channels were identified. In addition, a 190-pS potassium-selective channel and a 40-pS cation-selective channel were found in a 0.2 M salt-containing buffer. Odorant-sensitive channels were directly and reversibly activated by nanomolar concentrations of the bell pepper odorant 3-isobutyl-2-methoxypyrazine and the citrus odorant 3,7,-dimethyl-2,6-octadienenitrile. These channels display burst kinetics, multiple conductance levels between 35 and 420 pS, and open times in the millisecond range. With increasing concentrations of odorant, the probability of populating the higher conductance levels increases. These results show that direct activation of channels by odorants may mediate excitation of the olfactory receptor cell.

Properties of odour-binding glycoproteins from rat olfactory epithelium.

The specific membrane glycoproteins with high affinity for camphor and decanal were isolated from rat olfactory epithelium. Antibodies to these glycoproteins inhibited both the electroolfactogram and the binding of odorants. The enzyme immunoassay has shown these glycoproteins to be present in the olfactory epithelium of rat, mouse, guinea-pig and hamster but not in that of frog and carp. The molecular mass of the odour-binding glycoproteins from rat olfactory epithelium solubilized by Triton X-100 was approx. 140 kDa. They consisted of two subunits (88 and 55 kDa). The 88 kDa subunit was capable of binding odorants. The data obtained suggest that the glycoproteins isolated have some properties that make them plausible candidates for olfactory receptor molecules.

Spectrophotometric determination of cation concentrations in olfactory mucus.

Spectrophotometric techniques were used to determine the concentrations of Na+, K+ and Ca2+ in the olfactory mucus of frogs. The mean concentrations in mEq/l were: [Na+], 52.7 +/- 4.1; [K+], 10.6 +/- 1.9 and [Ca2+], 10.7 +/- 1.7. Topical application of the odorant cineole was associated with statistically significant increases in [Na+] and [Ca2+]; the secretagogues methacholine and isoproterenol induced transient increases in [Na+]. Cineole and methacholine caused sustained increases in [Na+]/[K+] from the control value of 5:1, while isoproterenol caused a transient increase followed by a decline. The results indicate that the cation concentrations in olfactory mucus samples are more similar to those derived from secretory tissue than to those found in the extracellular fluids surrounding typical neural tissue.

High substance P-like immunoreactivity (SPLI) in the olfactory and electrosensory cells of gymnotid fish.

Substance P has been proposed as a candidate neurotransmitter or neuromodulator in the nociceptive system. Using a light microscopial immunohistochemical peroxidase-anti-peroxidase technique we have detected high substance P-like immunoreactivity (SPLI) in several types of sensory organs of 4 species of gymnotiform teleost fish: olfactory epithelium, vestibular, lateral line and electrosensory organs. The olfactory nerve and its endings within the olfactory bulb and the telencephalon were also strongly labelled. At these sites no SPLI was revealed in other teleosts (Carassius auratus, Gnathonemus petersii). The findings suggest that substance P may be involved in neurotransmission or neuromodulation in these specific sensory systems of these species.

The subunits of specific odor-binding glycoproteins from rat olfactory epithelium.

The specific odor-binding glycoproteins have been isolated from rat olfactory epithelium. They consist of two subunits, gp88 and gp55. Subunit gp88 is capable of odorant binding.

Isolation of an olfactory cDNA: similarity to retinol-binding protein suggests a role in olfaction.

Molecular cloning techniques were used to isolate and characterize a protein possibly involved in the signal transducing system in olfactory tissue of the frog Rana pipiens. A complementary DNA library was constructed with messenger RNA obtained from frog olfactory neuroepithelium. A 700-base pair complementary DNA clone encoding a protein with a molecular weight of 20,300 was identified by differential hybridization analysis with polyadenylated RNA from olfactory epithelium and nonsensory respiratory epithelium. The messenger RNA corresponding to this clone was abundant in the cells of Bowman's glands in olfactory tissue but not in respiratory epithelium nor in several other tissues. The predicted sequence of this protein is homologous to members of a family of proteins that bind and transport small molecules in serum, suggesting that this protein may also bind and transport odorants in the mucus secreted by Bowman's glands.

Immunohistochemical method for the diagnosis of olfactory disturbance.

To confirm the diagnosis and determine the cause of the olfactory disturbance, we used an immunohistochemical method to examine biopsy specimens of the olfactory mucosa from a patient who complained of anosmia after head injury. Neuron-specific enolase immunoreactivity was found in the olfactory vesicles and dendrites of the receptor cells in the olfactory epithelium. S-100 protein immunoreactivity was found in the ductal cells of Bowman's gland in the olfactory epithelium and in the acinar cells of Bowman's gland in the lamina propria. This suggests that the olfactory receptor cells and Bowman's gland were normal. The olfactory disturbance in this patient was not caused by nerve transection due to the head injury, but by already existing chronic sinusitis. Immunohistochemical methods are useful for diagnosing olfactory disturbance when used in combination with biopsy of olfactory mucosa.