The arginine taste receptor. Physiology, biochemistry, and immunohistochemistry.

The amino acid, L-arginine (L-Arg), is a potent taste stimulus for the channel catfish, Ictalurus punctatus. Receptor binding studies demonstrated a high-affinity binding of L-Arg to putative taste receptor sites. This binding could be inhibited by preincubation of the tissue in the lectins Phaseolus vulgaris agglutinin (PHA) and Ricinus communis agglutinin I (RCA I). Neurophysiological studies demonstrated that the L-Arg receptor is a stimulus-gated ion channel type receptor whose conductance was stimulated by L-Arg and inhibited by D-arginine (D-Arg). To purify the receptor we subjected CHAPS solubilized partial membrane preparation from barbel epithelium to RCA I lectin affinity chromatography. The bound proteins were eluted with D-galactose. When these proteins were reconstituted into lipid bilayers, L-Arg activated single channel currents with conductances between 45 and 85 pS. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted protein showed a distinct band at approximately 83 kDa. Polyclonal antibodies raised against this 83-kDa band in guinea pigs reacted with numerous small (approximately 1 micron) sites within the taste pore of every taste bud when applied to fixed nonpermeabilized barbels. This observation suggests that the antibodies recognize an externally-facing epitope of the putative Arg receptor. The antibodies also inhibited L-Arg-stimulated currents in reconstitution studies. Sephacryl S-300 HR chromatography of the eluant from the affinity column showed a high molecular weight peak (> 700 kDa) which was recognized by the antibodies. Reconstitution of the protein from this peak into a lipid bilayer resulted in L-Arg-stimulated channels that could be inhibited by D-Arg. This high molecular weight component may be aggregates of the arginine taste receptor.

G-protein(S), G alpha q/G alpha 11, in the olfactory neuroepithelium of the channel catfish (Ictalurus punctatus) is altered by the herbicide, dichlobenil.

The immunohistochemical localization of G alpha q/G alpha 11 was studied in the olfactory neuroepithelium of the channel catfish. Antigenicity in the rosette was found at the apical surfaces of cells, within the apical neck regions of some cells, and within the area of the basal nerve tracts. Specific labeling was eliminated by preincubation of the G alpha q/G alpha 11 antibodies with the cognate peptide. Catfish, exposed to a 20 ppm dose of the herbicide, dichlobenil, displayed a reduction in G alpha q/G alpha 11 antigenicity. Proteins were extracted from olfactory tissues and solubilized. Using SDS-PAGE and Western blotting, bands corresponding in apparent molecular weight to a 38 kD protein were found. These data demonstrate that the herbicide may be a potent nasal olfactory toxicant in aquatic situations.

G alpha 9/G alpha 11: immunolocalization in the olfactory epithelium of the rat (Rattus rattus) and the channel catfish (Ictalurus punctatus).

The immunohistochemical localization of G alpha 9/G alpha 11 was studied in the olfactory and respiratory epithelium of two representative vertebrates, the rat and the channel catfish. Localization in the rat was found at the apical surface of cells in the epithelium and within nerve tracts in the lamina propria. Immunostaining of neuronal cilia and supporting cell microvilli was confirmed by electron microscopy. Immunoreactivity on the ipsilateral neuroepithelium was abolished five weeks following unilateral bulbectomy. An emergence of patchy immunoreactivity was found, however, after fifteen weeks. In catfish, G alpha 9/G alpha 11 antigenicity was found at the apical surface of cells within the olfactory epithelium, at supranuclear regions within some cell bodies and in basal nerve tracts of the olfactory rosette. Immunoreactivity was removed with unilateral bulbectomy. Specific labelling in both rat and catfish was eliminated by preincubation of the G alpha 9/G alpha 11 antibodies with the cognate peptide. Proteins were extracted from olfactory tissues of both species and solubilized. Using western blotting, bands corresponding in apparent molecular weight to a 38,000 mol. wt protein were found. These data demonstrate the presence of G alpha 9/G alpha 11 in the olfactory tissues of these vertebrates and suggest a role in olfaction for this class of G-protein.

Differential localization of putative amino acid receptors in taste buds of the channel catfish, Ictalurus punctatus.

The taste system of catfish, having distinct taste receptor sites for L-alanine and L-arginine, is highly sensitive to amino acids. A previously described monoclonal antibody (G-10), which inhibits L-alanine binding to a partial membrane fraction (P2) derived from catfish (Ictalurus punctatus) taste epithelium, was found in Western blots to recognize a single band, at apparent MW of 113,000 D. This MW differs from the apparent MW for the presumed arginine receptor identified previously by PHA-E lectin affinity. In order to test whether PHA-E lectin actually reacts with the arginine-receptor, reconstituted membrane proteins partially purified by PHA-E affinity were used in artificial lipid bilayers. These reconstituted channels exhibited L-arginine-activated activity similar to that found in taste cell membranes. Accordingly, we utilized the PHA-E lectin and G-10 antibody as probes to differentially localize the L-alanine and L-arginine binding sites on the apical surface of catfish taste buds. Each probe labels numerous, small (0.5-1.0 micron) patches within the taste pore of each taste bud. This observation suggests that each bud is not tuned to a single taste substance, but contains putative receptor sites for both L-arginine and L-alanine. Further, analysis of double-labeled tissue reveals that the PHA-E and G-10 sites tend to be separate within each taste pore. These findings imply that in catfish, individual taste cells preferentially express receptors to either L-arginine or L-alanine. In addition, PHA-E binds to the apices of solitary chemoreceptor cells in the epithelium, indicating that this independent chemoreceptor system may utilize some receptor sites similar to those in taste buds.

NADPH diaphorase staining suggests localization of nitric oxide synthase within mature vertebrate olfactory neurons.

Nitric oxide, a simple gas which serves as a neurotransmitter in the CNS, has been proposed to serve as an interneuronal second messenger in olfactory transduction. However, the role of nitric oxide in olfaction has been questioned by experiments in which nitric oxide synthase, the enzyme that generates nitric oxide, could not be localized to the olfactory epithelium. We have localized nitric oxide synthase to the olfactory neurons in adult rat and catfish olfactory epithelia using a modified nicotinamide adenine dinucleotide phosphate diaphorase technique. In the rat, staining was also found in cells with morphology reminiscent of microvillar olfactory cells. In contrast, the respiratory epithelium and the sustentacular cells in the olfactory epithelium displayed no staining. The nicotinamide adenine dinucleotide phosphate diaphorase reaction, which has been shown to co-localize with immunohistochemical staining for nitric oxide synthase in the brain, was stimulated by addition of the nitric oxide synthase substrate L-arginine, and was inhibited by the nitric oxide synthase inhibitor L-NG-nitro arginine, indicating that staining was specific for nitric oxide synthase. Unilateral bulbectomy, which causes degeneration of mature olfactory neurons on the bulbectomized size, markedly reduced nicotinamide adenine dinucleotide phosphate diaphorase staining. These observations were substantiated by biochemical assays for nitric oxide synthase by monitoring the production of [3H]-L-citrulline from [3H]-L-arginine. This is the first demonstration of specific NADPH diaphorase staining of mature olfactory neurons in rat and catfish olfactory epithelial suggesting the presence of nitric oxide synthase in these cells. Our histological and biochemical findings, in conjunction with data from other research, are supportive of a role for nitric oxide synthase in olfactory function.

Identification of synaptophysin-like immunoreactivity in the sea anemone Condylactis gigantea (Cnidaria: Anthozoa).

Synaptophysin is a membrane protein of synaptic vesicles that serves as an antigenic marker for nervous and endocrine systems in mammals. Monoclonal antisera generated against synaptophysin were used for immunocytochemical staining in tissues of the tentacles of the sea anemone Condylactis gigantea (Cnidaria: Anthozoa). Specific staining, visible at the light and electron microscope levels, was found in the tentacle. Proteins were extracted from the tissues and solubilized. Using SDS-polyacrylamide gel electrophoresis and Western blotting, we identified proteins with apparent molecular weights of 38,000, 78,000, and 114,000. The data suggest the tissues of this anthozoan contain synaptophysin-like proteins with molecular properties similar to those of mammalian neurons.

Neurofilament-like immunoreactivity in the sea anemone Condylactis gigantea (Cnidaria: Anthozoa).

The neuronal cytoskeleton contains neurofilament proteins that serve as markers for nervous tissue in many species across phyla. Antiserum generated to mammalian neurofilaments was used for immunocytochemical staining of tissues in the sea anemone Condylactis gigantea (Cnidaria: Anthozoa). Specific staining, visible at the light and electron microscope levels, was found in the tissues of the tentacle. Proteins were extracted from the tissues and solubilized. SDS-polyacrylamide gel electrophoresis and Western blotting revealed two bands of MWr 156 kD and 74 kD that reacted with antiserum generated to neurofilaments. The protein bands also bound a monoclonal antibody shown to react with a highly conserved epitope in many classes of intermediate filaments. These data suggest that the neurons of this anthozoan contain neurofilament-like proteins with molecular properties similar to those of mammalian neurons.

The taste system of the channel catfish: from biophysics to behavior.

Catfish, described as 'swimming tongues', are unique experimental models for studies of taste reception because of the extensive distribution of taste buds over their external body surface and within their oropharyngeal cavity. Both the extraordinary numbers of taste buds and their high sensitivity to amino acids have made it possible to perform in the same species: biochemical and biophysical studies of stimulus recognition and signal transduction; electrophysiological recordings of taste activity from receptor cells, afferent nerve fibers and CNS relays; and behavioral studies of taste-controlled food search, biting and mastication. The close correspondence of results obtained with these diverse experimental approaches has provided critical information concerning vertebrate gustation.

Evidence for an InsP3-gated channel protein in isolated rat olfactory cilia.

Stimulation of rat olfactory cilia (ROC) with odorants leads to a transient elevation in the levels of either cAMP or inositol trisphosphate (InsP3). We have characterized the binding of [3H]InsP3 to isolated ROC. Unlabeled InsP3 displaced [3H]InsP3 binding in a dose-dependent manner (dissociation constant = 3.9 +/- 0.65 microM). Binding was stereospecific and dependent on the number of phosphates in the inositol ring. A ciliary protein of 120 kDa molecular mass was labeled specifically upon exposure of cilia membranes to ultraviolet light in the presence of the 125I-labeled InsP3 analogue 1-O-[N-(4-azidosaliciloxy)-3-aminopropyl-1-phospho]-myo-inositol 4,5-bisphosphate. Labeling of this protein displayed the same stereospecificity as binding of [3H]InsP3 to ROC. In addition, ROC membranes incorporated into a phospholipid bilayer at the tip of a patch pipette displayed an increase in conductance upon exposure to micromolar D-myoinositol 1,4,5-trisphosphate in 45% of the trials (n = 88). The InsP3-gated conductance is relatively nonspecific for cations and is distinct from the cAMP-gated conductance. The conductance displayed stereospecificity consistent with the InsP3 binding experiments. The results suggest that the site of action for odorant-stimulated elevations in InsP3 concentration in rat olfactory cilia is at a ciliary InsP3-gated channel.

Characterization of a novel inositol 1,4,5-trisphosphate receptor in isolated olfactory cilia.

Inositol 1,4,5-trisphosphate (InsP3), a product of G-protein-mediated receptor activation of phosphoinositide turnover, plays the role of a second messenger when olfactory neurons are stimulated with certain olfactory stimuli. In this paper we examine the specific binding of [3H]InsP3 to isolated olfactory cilia, microsomes and brain membranes from the channel catfish (Ictalurus punctatus) and, by photoaffinity labelling with an InsP3 analogue (125I-labelled 1-[3-(4-azidosalicyloxy)-aminopropyl]inositol 1,4,5-trisphosphate (125I-ASA-InsP3)], we tentatively identify the major InsP3-binding protein in catfish olfactory cilia. InsP3 binding to ciliary membranes is specific and saturable, with a Kd of 1.10 +/- 0.31 microM and a maximum number of binding sites (Bmax) of 17.6 +/- 5.8 pmol/mg. The rank order for potency of inhibition of [3H]InsP3 binding is Ins(1,4)P2 less than Ins(1,3,4)P3 less than Ins(1,3,4,5)P4 = Ins(1,4,5)P3 less than Ins(2,4,5)P3. Exposure of cilia membranes to u.v. light in the presence of 125I-ASA-InsP3 results in the labelling of a protein with apparent Mr 107,000. Labelling is specifically prevented by Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(1,3,4,5)P4, but not by Ins(1,4)P2 or Ins(1,3,4)P3. Both specific [3H]InsP3 binding and photoaffinity labelling of the Mr-107,000 protein were displaced by heparin. The Kd and the inhibition of [3H]InsP3 binding and of photoaffinity labelling by inositol phosphates and heparin are consistent with the ability of micromolar concentrations of Ins(1,4,5)P3 [but not Ins(1,3,4)P3] to activate the InsP3-gated currents in patch-clamp experiments with olfactory neurons. These results suggest that InsP3 binding to a Mr-107,000 cilia membrane protein may represent binding to the olfactory InsP3-gated cation channel.

Specific L-arginine taste receptor sites in the catfish, Ictalurus punctatus: biochemical and neurophysiological characterization.

We report here the characterization of the arginine binding site(s) and corroborative neurophysiological studies. Binding of L-[3H]arginine to Fraction P2 from taste epithelium was measured by a modification of the method of Krueger and Cagan. Parameters for measuring maximal binding activity were established for both duration of incubation and pH of medium. At pH 7.8, the apparent single rate constant for association (kobs) at 4 degrees C was 4.72 x 10(+5).M-1.min-1. Dissociation was more complex, yielding two rate constants of 1.77.min-1 and 8.34 x 10(-3).min-1. These data suggest the presence of two affinity states for L-arginine. The KD values as calculated from the ratio k-1/k+1 were 1.3 x 10(-6) M and 1.8 x 10(-8) M. Homologous inhibition studies of L-arginine binding were not fit by a simple mass action relationship (Hill Coefficient 0.79), but were best fit by a two-site model with IC50 values of 1.6 x 10(-6) M for the high affinity state and 9 x 10(-4) M for the low affinity state. Multiunit neural recordings examined the stimulatory effectiveness of a number of guanidinium-containing compounds. Compared with L-arginine, only L-arginine methyl ester and L-alpha-amino-beta-guanidino propionic acid (L-AGPA) were effective stimuli. Cross-adaptation experiments demonstrated that at 10(-4) M L-arginine methyl ester, L-AGPA and, to a lesser extent, D-arginine were effective cross-adapting stimuli to 10(-6) M L-arginine. In competition binding studies L-arginine methyl ester, L-AGPA and D-arginine also inhibited binding of L-[3H]arginine (10(-6) M), but each recognized only one affinity state. Inhibition by the poorly cross-adapting stimuli L-glutamate, glycine and L-alanine occurred only above 10(-3) M, indicating that the binding sites for L-arginine are selective. These studies suggest that there are at least two affinity states of L-arginine binding, that the binding sites are specific, and that effective agonists of L-arginine receptors must contain a guanidinium group and an unblocked L-alpha-amino group.

Differential interaction of lectins with chemosensory receptors.

L-Alanine and L-arginine bind with similar affinity (Kd 10(-7)-10(-6) M) to receptors in both a sedimentable fraction (P2) from taste epithelium and isolated olfactory cilia from the channel catfish, Ictalurus punctatus. Lectins of differing carbohydrate specificity were used to determine the glycoprotein nature of the chemosensory plasma membranes and to differentially affect receptors for L-alanine and L-arginine. The peroxidase-conjugated lectins concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) were used to identify the glycoprotein components of the chemosensory plasma membranes after polyacrylamide gel electrophoresis. In both chemosensory membranes, numerous protein components were labelled by Con A and WGA. In contrast, a single predominant component was labeled by PNA in olfactory cilia, whereas several proteins in taste membranes were labeled by this lectin. When unconjugated lectins were preincubated with olfactory cilia, 60-70% of binding to L-alanine and L-arginine receptors was inhibited by Con A and WGA. PNA inhibited L-alanine but not L-arginine binding to olfactory receptors. Inhibition of olfactory receptor binding by lectins was time- and dose-dependent. By contrast, no inhibition of either L-alanine or L-arginine receptor binding in taste membranes was observed with any of the lectins. The differential labeling of the chemosensory membranes and the differential inhibition of receptor binding by lectins suggest that, despite ligand similarity, the chemosensory receptors in these membranes are not identical molecular species.

Biochemical studies of taste sensation. XIII. Enantiomeric specificity of alanine taste receptor sites in catfish, Ictalurus punctatus.

Specific binding of amino acid taste stimuli is known to occur to a sedimentable fraction (P2) from catfish (Ictalurus punctatus) taste epithelium or to purified plasma membranes from that fraction. L-Alanine, a potent taste stimulus for the catfish, binds in a reversible and saturable manner to these preparations. The extent to which the enantiomeric stimuli, L- and D-alanine, interact with the same or different receptor/transduction processes is investigated here both electrophysiologically and biochemically. With an electrophysiological assay, L-alanine was the more potent stimulus across a concentration range of 10(-9)-10(-3) M, yet both enantiomers displayed approximately the same threshold. The concentration-electrophysiological response functions for each enantiomer were different. That of L-alanine was approximately linear across the (log) concentration range while that of D-alanine was non-linear, with small but definitely observable responses being noted from 10(-9)-10(-5) M D-alanine, and larger incremental responses thereafter. With most of the nerve bundle preparations studies, L- and D-alanine cross-adapted one another, but this cross-adaptation was not always complete. Experiments in which both L- and D-alanine were present in a 1:1 mixture of equally stimulatory concentrations suggested the existence of receptor or transduction processes unique to each enantiomer. Biochemically binding studies demonstrated high affinity binding sites for both enantiomers with values of Kd-app for L-alanine of 1.5 microM and for D-alanine of 25 microM. For both enantiomers, additional lower-affinity binding sites were observable. The capacity of the lower-affinity sites was particularly great for D-alanine. The enantiomers competed one with the other for binding, with L-alanine showing greater competitive ability than D-alanine at low concentrations. For the high affinity sites, double-reciprocal plots of the data suggested a competitive mechanism. The lower affinity sites for D-alanine were less accessible to L-alanine compared with the high affinity sites of D-alanine. Both the biochemical and electrophysiological results indicate that while a portion of the responses to L- and D-alanine occurs through a common receptor/transduction process, there exist independent receptor/transduction processes for the enantiomers, L- and D-alanine.

Interaction of GTP-binding regulatory proteins with chemosensory receptors.

GTP-binding regulatory proteins (G-proteins) were identified in chemosensory membranes from the channel catfish, Ictalurus punctatus. The common G-protein beta-subunit was identified by immunoblotting in both isolated olfactory cilia and purified taste plasma membranes. A cholera toxin substrate (Mr 45,000), corresponding to the G-protein that stimulates adenylate cyclase, was identified in both membranes. Both membranes also contained a single pertussis toxin substrate. In taste membranes, this component co-migrated with the alpha-subunit of the G-protein that inhibits adenylate cyclase. In olfactory cilia, the Mr 40,000 pertussis toxin substrate cross-reacted with antiserum to the common amino acid sequence of G-protein alpha-subunits, but did not cross-react with antiserum to the alpha-subunit of the G-protein from brain of unknown function. The interaction of G-proteins with chemosensory receptors was determined by monitoring receptor binding affinity in the presence of exogenous guanine nucleotides. L-Alanine and L-arginine bind with similar affinity to separate receptors in both olfactory and gustatory membranes from the catfish. GTP and a nonhydrolyzable analogue decreased the affinity of olfactory L-alanine and L-arginine receptors by about 1 order of magnitude. In contrast, the binding affinities of the corresponding taste receptors were unaffected. These results suggest that olfactory receptors are functionally coupled to G-proteins in a manner similar to some hormone and neurotransmitter receptors.

The resolution of agonist alpha 2-adrenergic receptor complexes from unoccupied receptors or antagonist-alpha 2 receptor complexes using DEAE chromatography.

Alpha 2-adrenergic receptors linked to inhibition of adenylate cyclase activity in human platelet membranes can be isolated using DEAE chromatography following solubilization into digitonin-containing buffers. This procedure permits resolution of agonist-occupied receptors from antagonist-occupied receptors. Antagonist-occupied receptors co-elute with unoccupied receptors. Adenylate cyclase activity elutes independently of all alpha 2-receptor activities. A greater resolution of agonist-receptor complexes from antagonist-receptor complexes is obtained using DEAE chromatography than reported earlier using approaches that rely entirely on agonist-stabilized increases in apparent receptor size. Consequently, DEAE chromatography may be of considerable value in isolating these agonist-receptor complexes to permit identification of the membrane component(s) which are more stably associated with the receptor subsequent to agonist occupancy and thus might be involved in receptor-cyclase coupling.