Zanthoxylum piperitum, an Asian spice, inhibits food intake in rats.

UNLABELLED: We investigated the effects of a total extract from Zanthoxylum piperitum fruit, of the volatile components of extract, and of a non-volatile fraction containing the major alkylamides of Zanthoxylum (NVA) on food intake in rats. In Experiment 1, three groups (A, B, C) of mildly food deprived rats were given one hour long feeding trials during which they were offered choices between an oat-bran wafer paired with vegetable oil and a wafer paired with a Zanthoxylum preparation in vegetable oil. Trials were divided into three blocks of two tests each. During Blocks 1 and 3, two wafers coated with vegetable oil were offered to establish baseline consumption. During Block 2, Group A was given choices between wafers coated with oil and wafers coated with total extract. Group B was offered oil coated wafers and wafers coated with the NVA fraction. Group C received two oil coated wafers. One was served on top of a screened dish containing a sample of total extract. In this manner, the rats were exposed to volatile compounds emanating from the extract but could not contact the extract. The second wafer was served on top of a screened sample of oil. RESULTS: In Blocks 1 and 3 trials, the rats fed indiscriminately from both wafers. During Block 2, total extract, the NVA fraction and the volatile compounds from extract all significantly reduced food consumption. In Experiment 2, habituation to the NVA fraction and to the volatile constituents of extract was examined in two additional groups (D, E), using the methods described above. Baseline consumption was tested in Blocks 1 and 3 by offering two oil coated wafers. During Block 2, group D was given 10 trials with oil coated and NVA-fraction coated wafers. Group E was given 10 trials with oil coated wafers paired with the volatile constituents of total extract and with those of oil. RESULTS: Both groups failed to habituate to the Zanthoxylum stimuli. Wafers treated with the NVA fraction and wafers paired with the volatile constituents of extract were avoided throughout the 10 test days.

Reduction in carbonic anhydrase activity in the tongue epithelium and submandibular gland in zinc-deficient rats.

We investigated the effects of zinc deficiency on carbonic anhydrase (CA) activity in the tongue epithelium and submandibular gland in rats. Male 4-week-old SD rats were given free access to a diet containing 2.2 (zinc-deficient), 4.1 (low-zinc), or 33.7 (zinc-sufficient) mg zinc/kg diet for 6 weeks. Rats in the fourth group (receiving 33.7 mg zinc/kg) were pair-fed against the zinc-deficient rats. Biochemical analysis at the end of the experimental period indicated that zinc deficiency significantly reduced CA activity in the tongue epithelium and submandibular gland, and the CA activity levels in these tissues seemed to parallel the dietary zinc levels. By enzyme histochemistry, an intensely positive reaction for CA was observed in the middle and basal regions of the taste buds in the circumvallate papilla in the zinc-sufficient and pair-fed (control) rats. The cells in von Ebner's glands also showed a strong positive reaction in control rats, although only a weak reaction product was found in zinc-deficient rats. These results suggest that CA activity is affected by the dietary content of zinc, which is considered to be an indispensable factor for the maintenance of normal taste sensation.

Alkylamides that produce tingling paresthesia activate tactile and thermal trigeminal neurons.

Alkylamides isolated from the fruit of Xanthoxylum, Szechuan pepper, produce a strong tingling sensation in the mouth. In order to determine the peripheral basis of this sensation, extracellular nerve recordings were obtained from the lingual nerve of rats. The primary pungent compound, hydroxy-alpha-sanshool (HO-alpha-S), altered the levels of spontaneous activity in cool-sensitive fibers as well as inducing activity in tactile fibers, cold nociceptors and silent fibers that were insensitive to innocuous thermal or tactile stimuli. Moreover, tactile or thermal sensitivity was induced in fibers that were initially insensitive to touch or cooling. The neuronal distribution of sensitivities to capsaicin and to HO-alpha-S indicate that this compound affects neurons mediating innocuous sensations. HO-alpha-S may be useful as a model stimulus for studies of paresthesia.

Capsaicin modifies responses of rat chorda tympani nerve fibers to NaCl.

Single-fiber preparations of the rat chorda tympani (CT) nerve were used to study the mechanism of action of capsaicin on salt-taste transduction. Capsaicin selectively suppressed the responses to NaCl of the CT nerve fibers (N-fibers) that are sodium-specific (insensitive or poorly sensitive to potassium). Among the more broadly responsive, cation-sensitive fibers (E-fibers) there are two subtypes, both of which responded to capsaicin but in different ways ('enhanced' type and 'suppressed' type). In both N- and E-fibers, 5% ethanol (the vehicle for capsaicin) slightly reduced the response to 100 mM NaCl. The suppressive effect of capsaicin on the response of the N-type fibers to 100 mM NaCl was significantly stronger than the effect of 5% ethanol. The suppression lasted for at least 20 s after the simultaneous application of 100 p.p.m. capsaicin-100 nM NaCl. These results indicate that 100 p.p.m. capsaicin can modify the response of CT fibers to NaCl. The observed effect of capsaicin on gustatory fibers could be the net result of opposite suppressive and enhancing processes in the taste buds cells and excited intra- or extragemmal trigeminal nerve endings.

Effectiveness of thirteen vertebrate repellents as rodent trigeminal stimulants.

Repellent chemicals are presumed to activate trigeminal neurons, including polymodal nociceptors, but few data are available that bear on this notion. In the present experiment, we assessed multi-unit and single-unit responses of neurons in the rat lingual trigeminal nerve to 13 candidate repellents and a thermal stimulus. All of the chemicals evoked trigeminal responses, and neural activity was predictable from available behavioral data. These results are consistent with the view that repellents are irritants. The results also suggest that electrophysiological methods may represent a useful method for screening candidate repellent compounds.

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.

Capsaicin and its analogs induce ion channels in planar lipid bilayers.

The irritating, pungent compound, capsaicin (10-20 microM), induces the formation of non-selective ion channels with a wide variety of conductances in protein-free lipid bilayers form from a mixture of zwitterionic phospholipids. The channel-forming activity of capsaicin and four of its analogs followed the sequence: resiniferatoxin > capsaicin = pelargonic acid vanillylamide > methylcapsaicin >> veratrylamine. The ability to form channels correlated with the biological activity of these compounds observed in other studies that measured 45Ca uptake into rat dorsal root ganglion cells. The correlation obtained suggests that an interaction with the lipid bilayer may be an important component of the biological activity of capsaicin.

Computational analysis of binding affinity and neural response at the L-alanine receptor.

A model of analogue-receptor binding is developed for the L-alanine receptor in the channel catfish using the AM1-SM2 and ab initio SCRF computational methods. Besides interactions involving the zwitterionic moiety of the amino acid analogue and complementary subsites on the receptor, the model suggests the presence of a hydrophobic pocket with dispersion interactions between the receptor and the residue on the amino acid analogue. Conformational analysis suggests not only a small compact active site on the receptor, but also that the analogues with the highest affinity occupy nearly identical regions of space. Although the binding interaction is dominated by the ionic terms, AM1-SM2 calculations indicate that free energy terms associated with cavity formation, solvent reorganization, and dispersion interactions can be correlated to activation and neural response. From a consideration of this model, molecular features of the analogues that are important for binding and neural response were deduced and other analogues or ligands were developed and tested.

Factors affecting the sensitivity of the lingual trigeminal nerve to acids.

Oral sensitivity to acids mediates a range of irritation, from desirable sensations of pungency in various foods and beverages to the pain associated with harmful levels of acidity and inflammation. To characterize the role of the epithelial and vascular processes in neural sensitivity to acid applied to the lingual mucosa, we measured lingual trigeminal nerve responses in rats to acidic stimuli of varying physicochemical properties. Stimulatory efficacy was strongly correlated with lipophilicity (octanol-H2O partition coefficient) for the series of straight-chain fatty acids from one to six carbons. Neither acid dissociation constant nor the pH of stimulus solutions correlated with stimulatory efficacy for the fatty acids and six other organic acids. The hypothesis that weakly acidic stimuli gain access to trigeminal nerve endings primarily through the lipid phase of the epithelium was supported by these findings. Further support for this hypothesis is the result that LaCl3, an inhibitor of epithelial tight junctions, had no effect on the neural response to n-pentanoic acid. The potential relevance of buffering-clearance in modulating responses to acid was indicated by the finding that the application of 1 mM capsaicin to the tongue reversibly inhibited the neural response to acid, as well as causing plasma extravasation in lingual epithelium.

Enhancement of gustatory nerve fibers to NaCl and formation of ion channels by commercial novobiocin.

Single fibers of the rat chorda tympani nerve were used to study the mechanism of action of the antibiotic novobiocin on salt taste transduction. In the rat, novobiocin selectively enhanced the responses of sodium-specific and amiloride-sensitive chorda tympani nerve fibers (N type) without affecting more broadly responsive cation-sensitive and amiloride-insensitive fibers (E type). In the presence of amiloride, novobiocin was ineffective at enhancing the response of N-type fibers toward sodium chloride. Novobiocin also increased the conductance of bilayers formed from neutral lipids by forming nonrectifying ion channels with low conductance (approximately 7 pS in 110 mM NaCl), long open times (several seconds and longer), and high cation selectivity. Amiloride did not alter either the conductance or kinetics of these novobiocin channels. These observations suggest that even though novobiocin is able to form cation channels in lipid bilayers, and possibly in cell membranes as well, its action on the salt-taste response is through modulation of existing amiloride-sensitive sodium channels.

Acetazolamide specifically inhibits lingual trigeminal nerve responses to carbon dioxide.

The goal of this study was to examine the role of the enzyme, carbonic anhydrase, in oral trigeminal chemoreception with particular regard to the reception of CO2. Using both single and multiunit recordings of trigeminal neurons in the lingual nerve of rat, we measured responses to cool (24 degrees C), noxiously hot (55 degrees C) and cold (8 degrees C) H2O, NH4Cl and supersaturated solutions of CO2 (24 degrees C and 33 degrees C). The importance of peripheral carbonic anhydrase was tested by inhibiting enzyme activity with acetazolamide (15 mg/kg b.w.). Single unit responses to CO2 and HCl suggest that neural sensitivity to CO2 is not simply a function of extraepithelial pH. Responses to CO2 were significantly inhibited by acetazolamide while the responses to thermal stimuli and NH4Cl were not. The results support a role for carbonic anhydrase in trigeminal responses to CO2. Furthermore, the results suggest that intraepithelial acidification mediated by carbonic anhydrase may be the basis for sensitivity to CO2.

Molecular structural requirements for binding and activation of L-alanine taste receptors.

L-Alanine binds to and activates specific taste receptors ofIctalurus punctatus, the channel catfish. In order to determine the structural requirements for receptor binding and activation in this model system, a number of analogues of L-alanine were tested using a neurophysiological assay and a competitive ligand binding assay. These assays measured the ability of analogues to activate taste receptors and to displace L-[(3)H]alanine from L-alanine binding sites. Of those derivatives with modifications of the sidechain, L-serine, glycine,ß-chloro-L-alanine and 1-amino-cyclopropane-1-carboxylic acid were the most potent analogues with IC50s similar to and neural responses slightly decremented from that of L-alanine. Derivatives containing branched sidechains or sidechains of otherwise increased volume were considerably less active. All modifications of theα-carboxylic acid and theα-amine, including amides, esters and various isosteres, led to substantial reduction in the analogues' ability to displace L-[(3)H]alanine and, in most cases, very weak stimulatory capability. However, L-lactic acid was a reasonably strong stimulus, but a poor competitor, suggesting that it acts at a different receptor site. Overall, these results indicate the importance of the charged amine and carboxylic acid groups for binding to and activation of the receptor for L-alanine. Moreover, modifications around the chiral center of L-alanine support the hypothesis that receptor binding and activation are separate processes in this model taste system.

Structure/activity relationships in the L-alanine taste receptor system of the channel catfish, Ictalurus punctatus.

In order to understand the molecular determinants of amino acid taste receptor binding and activation, structure/activity studies were performed using analogs of L-alanine in a competitive ligand binding assay and a taste neurophysiological preparation. The presence of both the amine and carboxylic acid in a charged and unhindered form is a primary requisite for a ligand to both bind and activate L-alanine receptors. Although a number of carboxylic acid derivatives are moderately good stimuli, their neural activity derives from action at receptors different from L-alanine receptors. Of the molecular parameters examined, chirality and molecular volume of the side chain are the most important factors in determining the binding and stimulatory efficacy of L-amino acid taste stimuli. Electronegativity of the side chain did not correlate with receptor site binding. Heterologous ligand-induced enhancement of the binding of L-[3H]alanine by a purified taste membrane preparation is described. Neurophysiological experiments support the hypothesis that this phenomenon may be a basis of peripheral sensory interactions.

Odor stimuli trigger influx of calcium into olfactory neurons of the channel catfish.

Olfactory transduction is thought to be mediated by a G protein-coupled increase in intracellular adenosine 3',5'-monophosphate (cAMP) that triggers the opening of cAMP-gated cation channels and results in depolarization of the plasma membrane of olfactory neurons. In olfactory neurons isolated from the channel catfish, Ictalurus punctatus, stimulation with olfactory stimuli (amino acids) elicits an influx of calcium that leads to a rapid increase in intracellular calcium. In addition, in a reconstitution assay a plasma membrane calcium channel has been identified that is gated by inositol-1,4,5-trisphosphate (IP3), which could mediate this calcium influx. Together with previous studies indicating that stimulation with olfactory stimuli leads to stimulation of phosphoinositide turnover in olfactory cilia, these data suggest that an influx of calcium triggered by odor stimulation of phosphoinositide turnover may be an alternate or additional mechanism of olfactory transduction.

Specific inhibition of the binding of the taste stimulus, L-alanine, by sulphydryl reagents, in Ictalurus punctatus.

1. Taste receptors for L-alanine and L-arginine in the channel catfish, Ictalurus punctatus, are differentially reactive to N-ethylmaleimide (NEM) and p-chloromercuribenzenesulphonic acid (pCMBS). 2. The binding of L-[3H]alanine by a sedimentable membrane fraction (Fraction P2) isolated from taste epithelium was inhibited by both NEM and pCMBS while the binding of L[3H]arginine was unaffected. 3. Inhibition of the binding of L-[3H]alanine by pCMBS was reversible with dithiothreitol (DTT). 4. NEM (10(-3) M) inhibited multi-unit neural responses to both 10(-4) M L-alanine and 10(-4) M L-arginine, while pCMBS had little effect on neural responses. 5. Pretreatment of intact taste epithelium before the preparation of Fraction P2 with NEM caused strong inhibition of L-[3H]alanine binding, while pretreatment with pCMBS caused weak inhibition. 6. The presence of L-alanine during the reaction of pCMBS or NEM with taste plasma membranes did not substantially protect against the inhibition of L-[3H]alanine binding.

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.

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.

Diet manipulation affects social behavior of catfish : Importance of body odor.

Diet manipulation, an habituation test, and chemical analysis of urinary free amino acids were used to demonstrate that bullhead catfish (Ictalurus nebulosus) naturally detect the body odors of conspecifics and respond to them in a predictable fashion. These signals are used in dominance and territorial relationships and lead to increased aggression toward chemical "strangers." The results support the general notion that nonspecific metabolites, as well as specific pheromones, are important in chemical mediation of social behavior.