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.
Cnidarians are the simplest metazoans to exhibit satiety after feeding. When hydra are fed to repletion, they close their mouths and cease to capture prey. As feeding stops, contractions of the tentacles and body column increase. Our earlier experiments showed that a gel chromatographic fraction of prey substances inhibits prey capture. We now present evidence that the same fraction reduces the duration of mouth opening induced by reduced glutathione (GSH) and inhibits the binding of GSH to its putative receptor. The fraction also induces column contractions which are similar to those normally seen in sated animals. Prey substances, of unfractionated homogenate, also induce post-feeding tentacle contractions similar to those seen in sated animals. Gut distention does not appear to induce behavior associated with satiety. Therefore, these experiments suggest that chemoreception of prey substances induce satiety in hydra.
Background: The channel catfish, Ictalurus punctatus, is invested with a high density of cutaneous taste receptors, particularly on the barbel appendages. Many of these receptors are sensitive to selected amino acids, one of these being a receptor for L-arginine (L-Arg). Previous neurophysiological and biophysical studies suggested that this taste receptor is coupled directly to a cation channel and behaves as a ligand-gated ion channel receptor (LGICR). Earlier studies demonstrated that two lectins, Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and that PHA-E inhibited the L-Arg-stimulated ion conductance of barbel membranes reconstituted into lipid bilayers.
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.
Inhibition of desmoneme and stenotele nematocyst discharge occurs when Hydra attenuata are fed to repletion. Inhibition can be induced by the application of prey homogenates in the external medium. The onset of inhibition is relatively rapid (<30 s) while the release from inhibition is much slower (>20 min). Inhibition is concentration-depedent. Gel chromatography separation of homogenate shows that the inhibitory substance(s) have a molecular weight greater than 5000. These substances cause the strongest stenotele inhibition and are least effective in activating the feeding reflex (mouth opening and tentacle concerts) which is caused by smaller molecular weight substances. The receptor sites for the inhibitory substance(s) are located on the external surface of the hydra tentacle. Accumulation of prey substances may be the mechanism by which stenotele discharge is inhibited when hydra are fed to repletion.
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