Cyclic nucleotide-gated channel activation is not required for activity-dependent labeling of zebrafish olfactory receptor neurons by amino acids

The olfactory epithelium of fish is heterogeneous both with respect to the types of receptor cells (ORNs) present and the families of odorant receptors expressed in these cells. As a consequence of this diversity, the transduction cascade(s) activated by odorants has yet to be unambiguously established. In the current study, electrophysiological and activity-dependent labeling techniques were used to assess the role of the cyclic nucleotide-gated channel in zebrafish olfactory transduction. Both amino acid and bile salt odorants elicited robust electrophysiological responses, however, activity-dependent labeling of ORNs could be stimulated only by the amino acid odorants. An adenylate cyclase (AC) activator (forskolin) and a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) also elicited robust electrophysiological responses; generally larger than the responses elicited by either the amino acid or bile salt odorants. However, neither forskolin alone or a mixture of forskolin and IBMX stimulated activity-dependent labeling. Bathing the olfactory epithelium with forskolin, which presumably increased the intracellular concentration of cAMP, reduced the responses to bile salt odorants to a significantly greater extent than amino acid odorants. Collectively, these findings suggest that the transduction of amino acid input does not rely primarily on cyclic nucleotide-gated (CNG) channel activation and that CNG channel activation may be required for the transduction of bile salt input. Copyright Copyright 1999 S. Karger AG, Basel     

Identification,release and olfactory detection of bile salts in the intestinal fluid of the Senegalese sole (<Emphasis Type="Italic">Solea senegalensis</Emphasis>)

Olfactory sensitivity to bile salts is wide-spread in teleosts; however, which bile salts are released in sufficient quantities to be detected is unclear. The current study identified bile salts in the intestinal and bile fluids of Solea senegalensis by mass spectrometry–liquid chromatography and assessed their olfactory potency by the electro-olfactogram. The main bile salts identified in the bile were taurocholic acid (342 mM) and taurolithocholic acid (271 mM) plus a third, unidentified, bile salt of 532.3 Da. These three were also present in the intestinal fluid (taurocholic acid, 4.13 mM; taurolithocholic acid, 0.4 mM). In sole-conditioned water, only taurocholic acid (0.31 μM) was released in sufficient quantities to be measured (release rate: 24 nmol kg⁻¹ min⁻¹). Sole had high olfactory sensitivity to taurocholic acid but not to taurolithocholic acid. Furthermore, olfactory sensitivity was higher in the upper (right) olfactory epithelium than the lower (left). These two bile acids contribute about 40% of the olfactory potency of intestinal fluid and account for the difference in potency at the two epithelia. Taurocholic acid (but not taurolithocholic acid), and possibly other types of bile acid not tested, could be used as chemical signals and the upper olfactory epithelium is specialised for their detection.     

Lake char (<Emphasis Type="Italic">Salvelinus namaycush</Emphasis>) olfactory neurons are highly sensitive and specific to bile acids

Bile acids have been implicated as chemical signals in spawning behaviour of lake char (Salvelinus namaycush). In this study, we investigated olfactory responses of lake char to bile acids by using the electro-olfactogram recording. Lake char detected 9 out of 38 bile acids tested at thresholds 0.02–0.5 nM. The most stimulatory included chenodeoxycholic acid, cholic acid, taurochenodeoxycholic acid, taurocholic acid, and taurolithocholic acid 3α-sulphate. Structure–activity analysis indicated that substituents in the side chain or hydroxyl sulphation were determinant elements for the recognition of individual bile acid receptors, while the position and orientation of hydroxyls or the type of amidation were important for effective stimulation. Three distinct types of concentration–response relationships were found, representing free, taurine- or glycine-amidated, and 3α-sulphated bile acids. Cross-adaptation and binary mixture experiments revealed the presence of multiple olfactory receptors for bile acids. Lake char were also capable of detecting petromyzonol sulphate at 1 nM, possibly via its own receptors. Our study further showed that the olfactory responses to bile acids were independent of those of known odorants including amino acids, prostaglandins and gonadal steroids. We conclude that lake char possess multiple olfactory receptors capable of discriminating bile acids produced and released by conspecifics.     

Electrophysiological demonstration of independent olfactory receptor types and associated neuronal responses in the trout olfactory bulb

The present study attempts to highlight the principles by which peripheral olfactory information of across- and within-class odorant signals is transformed into bulbar neuron responses. For this purpose, we performed electro-olfactogram cross-adaptation and mixture experiments as well as single unit recording of olfactory bulb neurons using amino acid, bile acid and F-prostaglandin stimulants in brown and rainbow trout. The results show that amino acids, a bile acid and a F-prostaglandin activate independent receptor types. However, within the class of amino acids, different receptor types are only partially independent. Neurons responsive to bile acid and amino acids were segregated to the mid-dorsal and latero-posterior olfactory bulb, respectively. Of the 43 responsive olfactory bulb neurons studied in brown trout, 41 showed specificity for one odorant class. Olfactory bulb neurons gained responsiveness to new amino acids with increasing stimulant concentration. We conclude that different odorant classes activate specific neurons located in different regions of the trout olfactory bulb, and that information distinguishing related amino acids can be represented in a limited number of bulbar neurons with distinct response profiles under the conditions investigated.     

Partitioning of amino acids in the aqueous biphasic system containing the water‐miscible ionic liquid 1‐butyl‐3‐methylimidazolium bromide and the water‐structuring salt potassium citrate

In biotechnology, extraction by means of aqueous biphasic systems (ABS) is known as a promising tool for the recovery and purification of bio‐molecules. Over the past decade, the increasing emphasis on cleaner and environmentally benign extraction procedures has led to enhanced interest in the ABS containing ionic liquids (ILs)—a new class of non‐volatile alternative solvents. ABS composed of the hydrophilic IL {1‐butyl‐3‐methylimidazolium bromide ([C₄mim]Br)} and potassium citrate—which is easily degraded—represents a clean media to green separation of bio‐molecules. In this regard, here, the extraction capability of this ABS was evaluated through its application to the extraction of some amino acids. To gain an insight into the driving forces of amino acid partitioning in the studied IL ‐based ABS, the distribution of five model amino acids (L ‐tryptophan, L ‐phenylalanine, L ‐tyrosine, L ‐leucine, and L ‐valine) at different aqueous medium pH values and different phase compositions was investigated. The studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting‐out effects were also important for the transfer of the amino acids. Moreover, based on the statistical analysis of the driving forces of amino acid partitioning in the studied IL ‐based ABS, a model was established to describe the partition coefficient of three model amino acids, L ‐tryptophan, L ‐phenylalanine, and L ‐valine, and employed to predict the partition coefficient of two other model amino acids, L ‐tyrosine and L ‐leucine. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Evidence for functional asymmetry in the olfactory system of the Senegalese sole (Solea senegalensis)

The two olfactory epithelia of flatfish of the family Soleidae are essentially in contact with two distinct environments; the upper (right) side samples open water while the lower (left) side samples interstitial water. This study assessed whether there are differences in the responsiveness of the two epithelia by use of the electro-olfactogram in the Senegalese sole (Solea senegalensis). The upper epithelium was significantly more responsive to the basic amino acids (L-lysine and L-arginine), glycine, and L-threonine than the lower epithelium. The lower epithelium was significantly more responsive to aromatic amino acids (L-tryptophan, L-tyrosine, L-DOPA, and L-phenylalanine), L-leucine, and L-asparagine than the upper. Both epithelia had similar responsiveness to the sulphur-containing amino acids (L-cysteine and L-methionine), L-alanine, L-serine, and L-glutamine. Neither side was responsive to the acidic amino acids (L-aspartate and L-glutamate) or the D-isomers of any amino acid tested. The upper olfactory organ was much more responsive to conspecific-derived stimuli (bile and intestinal fluid) than the lower organ. We suggest that these differences in responsiveness may be related to different functional roles of the upper and lower epithelia in feeding and chemical communication.     

Electrophysiological evidence for acidic, basic, and neutral amino acid olfactory receptor sites in the catfish

Electrophysiological experiments indicate that olfactory receptors of the channel catfish, Ictalurus punctatus, contain different receptor sites for the acidic (A), basic (B), and neutral amino acids; further, at least two partially interacting neutral sites exist, one for the hydrophilic neutral amino acids containing short side chains (SCN), and the second for the hydrophobic amino acids containing long side chains (LCN). The extent of cross-adaptation was determined by comparing the electro-olfactogram (EOG) responses to 20 "test" amino acids during continuous bathing of the olfactory mucosa with water only (control) to those during each of the eight "adapting" amino acid regimes. Both the adapting and test amino acids were adjusted in concentrations to provide approximately equal response magnitudes in the unadapted state. Under all eight adapting regimes, the test EOG responses were reduced from those obtained in the unadapted state, but substantial quantitative differences resulted, depending upon the molecular structure of the adapting stimulus. Analyses of the patterns of EOG responses to the test stimuli identified and characterized the respective "transduction processes," a term used to describe membrane events initiated by a particular subset of amino acid stimuli that are intricately linked to the origin of the olfactory receptor potential. Only when the stimulus compounds interact with different transduction processes are the stimuli assumed to bind to different membrane "sites." Four relatively independent L-alpha-amino acid transduction processes (and thus at least four binding sites) identified in this report include: (a) the A process for aspartic and glutamic acids; (b) the B process for arginine and lysine; (c) the SCN process for glycine, alanine, serine, glutamine, and possibly cysteine; (d) the LCN process for methionine, ethionine, valine, norvaline, leucine, norleucine, glutamic acid-gamma-methyl ester, histidine, phenylalanine, and also possibly cysteine. The specificities of these olfactory transduction processes in the catfish are similar to those for the biochemically determined receptor sites for amino acids in other species of fishes and to amino acid transport specificities in tissues of a variety of organisms.     

Renewing olfactory receptor neurons in goldfish do not require contact with the olfactory bulb to develop normal chemical responsiveness

This study investigated whether contact with the olfactory bulb was necessary for developing and renewing olfactory receptor neurons (ORNs) to attain normal odorant responsiveness, and whether the anatomical and functional recoveries of the olfactory epithelium were similar in both bulbectomized (BE) and bilaterally axotomized (AX) preparations. In vivo electrophysiological recordings were obtained in response to amino acids, a bile acid [taurolithocholic acid sulfate(TLCS)] and a pheromonal odorant [17α, 20β,-dihydroxy-4-pregnen-3-one (17,20P)] from sexually immature goldfish. Both transmission and scanning electron microscopy indicated that the olfactory epithelium degenerated in BE and AX goldfish. Within 1–2 weeks subsequent to the respective surgeries, responses to high concentrations (>0.1 mmol · l⁻¹) of the more stimulatory amino acids remained, whereas responses were no longer obtainable to TLCS and 17,20P. At 4 weeks, responses to amino acid stimuli recovered to control levels, while responses to TLCS and 17,20P were minimal. By 7 weeks post bilateral axotomy, the olfactory epithelium recovered to a condition similar to control sensory epithelium; however, the rate of degeneration and proliferation of receptor neurons in BE preparations appeared to remain in balance, thus blocking further recovery of the olfactory epithelium. At 7 weeks post surgery, odorant responses of AX and BE goldfish to TLCS and 17,20P were still recovering. Accepted: 14 June 1997     

Signal transduction for taurocholic acid in the olfactory system of Atlantic salmon

Conjugated bile acids such as taurocholic acid (TChA) are potentolfactory stimuli for Atlantic salmon (Salmo solar). A plasmamembrane rich fraction was derived from salmon olfactory rosettesand used to investigate TChA signal transduction and receptorbinding. In the presence of GTP     

Metabotropic glutamate receptor expression in olfactory receptor neurons from the channel catfish,Ictalurus punctatus

Metabotropic glutamate receptors (mGluRs) were identified in olfactory receptor neurons of the channel catfish, Ictalurus punctatus, by polymerase chain reaction. DNA sequence analysis confirmed the presence of two subtypes, mGluR1 and mGluR3, that were coexpressed with each other and with the putative odorant receptors within single olfactory receptor neurons. Immunocytochemical data showed that both mGluR subtypes were expressed in the apical dendrites and some cilia of olfactory neurons. Pharmacological analysis showed that antagonists to each mGluR subtype significantly decreased the electrophysiological response to odorant amino acids. α-Methyl-L -CCG1/(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a known antagonist to mGluR3, and (S)-4-carboxyphenylglycine (S-4CPG), a specific antagonist to mGluR1, each significantly reduced olfactory receptor responses to L -glutamate. S-4CPG and MCCG reduced the glutamate response to 54% and 56% of control, respectively, which was significantly greater than their effect on a neutral amino acid odorant, methionine. These significant reductions of odorant response by the antagonists, taken with the expression of these receptors throughout the dendritic and ciliated portions of some olfactory receptor neurons, suggest that these mGluRs may be involved in olfactory reception and signal transduction. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 94–104, 1998     

Functional identification of a goldfish odorant receptor.

The vertebrate olfactory system utilizes odorant receptors to receive and discriminate thousands of different chemical stimuli. An understanding of how these receptors encode information about an odorant's molecular structure requires a characterization of their ligand specificities. We employed an expression cloning strategy to identify a goldfish odorant receptor that is activated by amino acids-potent odorants for fish. Structure-activity analysis indicates that the receptor is preferentially tuned to recognize basic amino acids. The receptor is a member of a multigene family of G protein-coupled receptors, sharing sequence similarities with the calcium sensing, metabotropic glutamate, and V2R class of vomeronasal receptors. The ligand tuning properties of the goldfish amino acid odorant receptor provide information for unraveling the molecular mechanisms underlying olfactory coding.     

The spatial distribution of ciliated and microvillous olfactory receptor neurons in the channel catfish is not matched by a differential specificity to amino acid and bile salt stimuli

The distribution of two receptor cell types, stellate microvillousand ciliated, was quantitatively examined in the sensory regionsof the olfactory lamellae of the channel catfish, Ictaluruspunctatus. Using scanning electron microscopy (SEM), these twocell types were seen to exhibit an incomplete segregation acrossthe sensory epithelium. The density of stellate microvillouscells was greatest in the dorso-medial portion of the sensoryregion of the lamella and markedly decreased ventrally and laterally.The ciliated cells were more numerous in the ventro-lateralportion of the sensory region and decreased in number dorso-medially.Multi-unit olfactory neural responses to amino acids and bilesalts were recorded from the dorso-medial and ventro-lateralportions of the sensory region. In both portions of the sensoryregion, L-cysteine, L-alanine and L-norleucine were highly stimulatory,while the sodium salts of cholic acid, taurocholic acid andtaurolithocholic acid (i.e., bile salts) were the least stimulatorycompounds tested. With the exception of L-norleucine, no significantdifferences were found in the responses from the dorso-medialand ventro-lateral portions of the sensory region for the compoundstested. These results indicate that in I. punctatus the responsespecificities of the microvillous and ciliated olfactory receptorcells are similar for amino acids and bile salts.     

Correlation between signal input and output in PctA and PctB amino acid chemoreceptor of Pseudomonas aeruginosa

The PctA and PctB chemoreceptors of Pseudomonas aeruginosa mediate chemotaxis toward amino acids. A general feature of signal transduction processes is that a signal input is converted into an output. We have generated chimeras combining the Tar signaling domain with either the PctA or PctB ligand binding domain (LBD). Escherichia coli harboring either PctA‐Tar or PctB‐Tar mediated chemotaxis toward amino acids. The responses of both chimeras were determined using fluorescence resonance energy transfer, and the derived EC₅₀ values are a measure of output. PctA‐Tar and PctB‐Tar responded to 19 and 11 L‐amino acids respectively. The EC₅₀ values of PctA‐Tar responses differed by more than three orders of magnitude, whereas PctB‐Tar responded preferentially to L‐Gln. The comparison of amino acid binding constants and the corresponding EC₅₀ values for both receptors revealed statistically significant correlations between inputs and outputs. PctA and PctB possess a double PDC (PhoQ‐DcuS‐CitA) LBD – a family of binding domain found in various other amino acid chemoreceptors. Similarly, various chemoreceptors share the preferential response to certain amino acids (e.g. L‐Cys, L‐Ser and L‐Thr) that we observed for PctA. Defining the specific inputs and outputs of these chemoreceptors is an important step toward better understanding of their physiological role.     

Evidence for multiple calcium response mechanisms in mammalian olfactory receptor neurons

Olfactory receptor neurons employ a diversity of signaling mechanisms for transducing and encoding odorant information. The simultaneous activation of subsets of receptor neurons provides a complex pattern of activation in the olfactory bulb that allows for the rapid discrimination of odorant mixtures. While some transduction elements are conserved among many species, some species-specificity occurs in certain features that may relate to their particular physiology and ecological niche. However, studies of olfactory transduction have been limited to a relatively small number of vertebrate and invertebrate species. To better understand the diversity and evolution of olfactory transduction mechanisms, we studied stimulus-elicited calcium fluxes in olfactory neurons from a previously unstudied mammalian species, the domestic cat. Isolated cells from cat olfactory epithelium were stimulated with odorant mixtures and biochemical agents, and cell responses were measured with calcium imaging techniques. Odorants elicited either increases or decreases in intracellular calcium; odorant-induced calcium increases were mediated either by calcium fluxes through the cell membrane or by mobilization of intracellular stores. Individual cells could employ multiple signaling mechanisms to mediate responses to different odorants. The physiological features of these olfactory neurons suggest greater complexity than previously recognized in the role of peripheral neurons in encoding complex odor stimuli. The investigation of novel and unstudied species is important for understanding the mechanisms of odorant signaling that apply to the olfactory system in general and suggests both broadly conserved and species-specific evolutionary adaptations.     

Dissimilar effects of L and D amino acids on the growth of Tetrahymena

Of the L and D configurations of four amino acids (phenylalanine, valine, tryptophan, tyrosine) tested for influence on the growth rate of Tetrahymena, only L-tyrosine was able to induce imprinting in Tetrahymena pyriformis Zeuthen. D-valine stimulated the division of T.pyriformis NT-1, but failed to induce imprinting. The experiments have substantiated the selectivity of the amino acid receptors of Y.pyriformis, and the extraordinary imprinting potential of tyrosine as well, as judged by its influence on the growth rate.     

Whole-cell response characteristics of ciliated and microvillous olfactory receptor neurons to amino acids, pheromone candidates and urine in rainbow trout.

Olfactory lamellae of teleosts contain two morphologically different types of olfactory receptor neurons (ORNs): ciliated ORNs (cORNs) and microvillous ORNs (mORNs). However, little is known about the functional difference between these two types of ORNs in fish olfaction. We isolated cORNs and mORNs using a Ca(2+)-free solution method from olfactory organs of the rainbow trout and examined their response characteristics to various odorants including fish pheromone candidates by whole-cell voltage-clamp techniques. Quadruple mixture of amino acids, single amino acids, steroids (analogues of DHP; 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one and ECG; etiocholan-3 alpha-ol-17-one glucuronide), prostaglandins (PGFs) and urine samples collected from immature and mature female fish were applied focally to olfactory cilia or microvilli using a multi-barreled stimulation pipette with a pressure ejection system. Inward current responses to odorants were recorded from both cORNs and mORNs at a holding potential of -60 mV. cORNs responded to the amino acid mixture, single amino acids, urine samples and ECG, whereas mORNs responded specifically either to the amino acid mixture or single amino acids. The response profiles of both cORNs and mORNs to various odorants varied widely. None of cORNs and mORNs responded to fish pheromone candidates, PGFs and DHPs. Androgen treatment of immature fish did not influence olfactory sensitivity of both cORNs and mORNs to the amino acid mixture and both urine samples. Amino acid and bile acid analyses by HPLC showed that both urine samples contained 35 amino acids (1-40 mM) and trace amounts of taurocholic acid and glycoursodeoxycholic acid. Our results suggest that cORNs are 'generalists' that respond to a wide variety of odorants, including pheromones, whereas mORNs are 'specialists', specific to amino acids, and also suggest that PGFs and DHPs are not pheromones for the rainbow trout.     

Discrimination of bile acids by the rainbow trout olfactory system: evidence as potential pheromone

Electro-olfactogram recording was used to determine whether the olfactory epithelium of adult rainbow trout is specifically sensitive to bile acids, some of which have been hypothesized to function as pheromones. Of 38 bile acids that had been pre-screened for olfactory activity, 6 were selected. The rainbow trout-specific bile acids, taurocholic acid (TCA), and taurolithocholic acid 3-sulfate (TLS) were the most potent compounds tested. TLS had a distinctive dose-response curve. Cross-adaptation experiments demonstrated that sensitivity to bile acids is attributable to at least 3 independent classes of olfactory receptor sites. Our data suggest that bile acids are discriminated by olfaction in rainbow trout, supporting the possibility that these compounds function as pheromones.     

Responses of Xenopus laevis water nose to water-soluble and volatile odorants.

Using the whole-cell mode of the patch-clamp technique, we recorded action potentials, voltage-activated cationic currents, and inward currents in response to water-soluble and volatile odorants from receptor neurons in the lateral diverticulum (water nose) of the olfactory sensory epithelium of Xenopus laevis. The resting membrane potential was -46.5 +/- 1.2 mV (mean +/- SEM, n = 68), and a current injection of 1-3 pA induced overshooting action potentials. Under voltage-clamp conditions, a voltage-dependent Na+ inward current, a sustained outward K+ current, and a Ca2+-activated K+ current were identified. Application of an amino acid cocktail induced inward currents in 32 of 238 olfactory neurons in the lateral diverticulum under voltage-clamp conditions. Application of volatile odorant cocktails also induced current responses in 23 of 238 olfactory neurons. These results suggest that the olfactory neurons respond to both water-soluble and volatile odorants. The application of alanine or arginine induced inward currents in a dose-dependent manner. More than 50% of the single olfactory neurons responded to multiple types of amino acids, including acidic, neutral, and basic amino acids applied at 100 microM or 1 mM. These results suggest that olfactory neurons in the lateral diverticulum have receptors for amino acids and volatile odorants.     

Influence of dietary bile acids on formation of bile acids in rat

Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.