John H. Teeter

Inhibition by amiloride of chorda tympani responses evoked by monovalent salts.

The diuretic, amiloride, is a potent yet reversible inhibitor of passive sodium transport in many epithelia. It has been shown to inhibit sodium transport in dorsal lingual epithelia and to inhibit both psychophysical and neural measures of salt taste. The present results demonstrate that amilorides action as an inhibitor of integrated whole chorda tympani nerve recordings in the rat is specific for Li and Na salts, displaying little inhibition of neural responses evoked by KCl and RbCl. Amiloride reduces both the phasic and tonic portion of the nerve recording equally. When amiloride inactivates the tonic portion of the nerve response to 250 mM NaCl, only a portion of the response is affected. Complete inactivation does not occur even at high amiloride concentrations. With amiloride flowing constantly over the tongue at 1 microM, 10 microM, or 50 microM a reciprocal plot of stimulus NaCl concentration versus response is non-linear. This result suggests that the inhibition of the NaCl-induced response has both competitive and non-competitive properties. These results support the hypothesis that salt taste is mediated in part by amiloride sensitive Na-channels located in taste receptor cell plasma membranes.

Measurement of membrane potential and [Ca2+]i in cell ensembles: application to the study of glutamate taste in mice

We have studied the spectral properties of the voltage-sensitive dye, 1-(3-sulfonatopropyl)-4-[beta [2-(di-n-octylamino)-6-naphtyl]vinyl] pyridinium betaine (di-8-ANEPPS), and the Ca(2+)-sensitive dye, fura-2, in azolectin liposomes and in isolated taste buds from mouse. We find that the fluorescence excitation spectra of di-8-ANEPPS and fura-2 are largely nonoverlapping, allowing alternate ratio measurements of membrane potential and intracellular calcium ([Ca2+]i). There is a small spillover of di-8-ANEPPS fluorescence at the excitation wavelengths used for fura-2 (340 and 360 nm). However, voltage-induced changes in the fluorescence of di-8-ANEPPS, excited at the fura-2 wavelengths, are small. In addition, di-8-ANEPPS fluorescence is localized to the membrane, whereas fura-2 fluorescence is distributed throughout the cytoplasm. Because of this, the effect of spillover of di-8-ANEPPS fluorescence in the [Ca2+]i estimate is < 1%, under the appropriate conditions. We have applied this method to study of the responses of multiple taste cells within isolated taste buds. We show that membrane potential and [Ca2+]i can be measured alternately in isolated taste buds from mouse. Stimulation with glutamate and glutamate analogs indicates that taste cells express both metabotropic and ionotropic receptors. The data suggest that the receptors responding to 2-amino-4-phosphonobutyrate (L-AP4), presumably metabotropic L-glutamate receptors, do not mediate excitatory glutamate taste responses.

Cluster Organization of Ion Channels Formed by the Antibiotic Syringomycin E in Bilayer Lipid Membranes

The cyclic lipodepsipeptide, syringomycin E, when incorporated into planar lipid bilayer membranes, forms two types of channels (small and large) that are different in conductance by a factor of sixfold. To discriminate between a cluster organization-type channel structure and other possible different structures for the two channel types, their ionic selectivity and pore size were determined. Pore size was assessed using water-soluble polymers. Ion selectivity was found to be essentially the same for both the small and large channels. Their reversal (zero current) potentials with the sign corresponding to anionic selectivity did not differ by more than 3 mV at a twofold electrolyte gradient across the bilayer. Reduction in the single-channel conductance induced by poly(ethylene glycol)s of different molecular weights demonstrated that the aqueous pore sizes of the small and large channels did not differ by more than 2% and were close to 1 nm. Based on their virtually identical selectivity and size, we conclude that large syringomycin E channels are clusters of small ones exhibiting synchronous opening and closing.

Properties of Voltage-gated Ion Channels Formed by Syringomycin E in Planar Lipid Bilayers

Abstract. Using the planar lipid bilayer technique we demonstrate that the lipodepsipeptide antibiotic, syringomycin E, forms voltage-sensitive ion channels of weak anion selectivity. The formation of channels in bilayers made from dioleoylglycerophosphatidylserine doped with syringomycin E at one side (1–40 μg/ml) was greatly affected by cis-positive voltage. A change of voltage from a positive to a negative value resulted in (i) an abrupt increase in the single channel conductance (the rate of increase was voltage dependent) simultaneous with (ii) a closing of these channels and an exponential decrease in macroscopic conductance over time. The strong voltage dependence of multichannel steady state conductance, the single channel conductance, the rate of opening of channels at positive voltages and closing them at negative voltages, as well as the observed abrupt increase of single channel conductance after voltage sign reversal suggest that the change of the transmembrane field induces a significant rearrangement of syringomycin E channels, including a change in the spacing of charged groups that function as voltage sensors. The conductance induced by syringomycin E increased with the sixth power of syringomycin E concentration suggesting that at least six monomers are required for channel formation.

Sex Pheromone Communication in the Sea Lamprey: Implications for Integrated Management

Studies in the past two decades have demonstrated that sexually mature sea lampreys (Petromyzon marinus) release sex pheromones that attract conspecific individuals of the opposite sex. Recently, electrophysiological, behavioral, and biochemical studies have shown that male sea lampreys, during spermiation (spermatogenesis), release 7α,12α,24-trihydroxy-3-one-5α-cholan-24-sulfate, or 3 keto-petromyzonol sulfate, which induces searching behavior in ovulatory females. This behavior appears to lead the females to nests occupied by males. The biosynthesis and release of this pheromone are likely to be regulated by the endocrine system and by environmental factors such as water temperature. Synthetic copies of this pheromone, if available, could be used for attraction and annihilation of mature female lampreys, or for disruption of mating. It may also be possible to develop procedures that promote pheromone biosynthesis in sterile males, which when stocked into spawning grounds, may make them highly effective in competing for mates with resident males. To develop protocols for implementing these potential strategies in integrated sea lamprey management, it is critical to fully understand pheromone biosynthesis and release in male sea lampreys, and to determine the role of sex pheromones in male reproductive fitness.

Ampullary electroreceptors in the sturgeonScaphirhynchus platorynchus (rafinesque)

Summary1.Ampullary receptors in the skin of the shovelnose sturgeon,Scaphirhynchus platorynchus, were examined morphologically and physiologically. As in the chondrostean paddlefish,Polyodon (Jørgensen etal., 1972), and in the ampullae of Lorenzini of elasmobranchs (Waltman, 1966; Szabo, 1972; Szamier and Bennett, 1980) each receptor cell has a cilium on its apical (lumenal) surface (Figs. 2, 3). Several synaptic projections from the basal part of the receptor cell fit into invaginations of the innervating nerve terminals (Fig. 3). A dense ribbon extends into each projection and the ribbons are covered with vesicles above the projections. Each ampulla is innervated by a single nerve fiber.2.The primary afferent fibers innervating these receptors are spontaneously active (20–60 impulses/s) and this activity is modulated by electrical stimuli of less than 1 mV applied at the receptor opening. Cathodal stimuli, which make the outside of the skin negative with respect to the inside, accelerate the resting nerve discharge (Fig. 4). As a cathodal stimulus is increased beyond the level at which the maximum nerve discharge is evoked, the nerve response decreases in frequency until it is completely blocked (Figs. 5, 7). Anodal stimuli of increasing strength decelerate and eventually block the resting nerve discharge (Fig. 4). Increasing the strength of an anodal stimulus beyond the level at which the resting discharge is blocked results in a progressive return of nerve impulses (Figs. 5, 7).3.Application of 10 mM CoCl2 or MgCl2 to the receptor openings usually produces a rapid and reversible block of both the resting and evoked nerve discharge (Fig. 8). Recovery from this suppression is facilitated by application of 10 mM CaCl2.4.These results are similar to those obtained with elasmobranch ampullae of Lorenzini and suggest that a similar mode of operation is present (Clusin and Bennett, 1979a). We conclude that the ampullary organs of chondrostean fishes are electroreceptors. Their morphological and functional similarities to the ampullary receptors in elasmobranchs suggest that they should be classified as ampullae of Lorenzini.

Transduction mechanisms for the taste of amino acids

Amino acids are important taste stimuli for a variety of animals. One animal model, the channel catfish, I. punctatus, possesses sensitive taste receptor systems for several amino acids. Neurophysiological and biochemical receptor binding studies suggest the presence of at least three receptor pathways: one is a relatively nonspecific site(s) responsive to short-chain neutral amino acids such as L-alanine (L-ALA); another is responsive to the basic amino acid L-arginine (L-ARG); still another is a low affinity site for L-proline (L-PRO). Several possible transduction pathways are available in the taste system of this animal model for these amino acids. One of these, formation of inositol trisphosphate (IP3) and cyclic AMP (cAMP), is mediated by GTP-binding regulatory proteins, while another involves ion channels directly activated by stimuli. L-ALA is a potent stimulus to cAMP and IP3 accumulation, while L-ARG at low concentrations is without effect. On the other hand, L-ARG and L-PRO, but not L-ALA, are able to activate stimulus-specific and cation-selective channels in taste epithelial membranes reconstituted in phospholipid bilayers at the tips of patch pipettes. Preliminary studies using mouse taste tissue demonstrate that monosodium-L-glutamate (MSG) did not enhance production of IP3 or cAMP. However, in reconstitution experiments using taste epithelium of mouse, conductance changes due to MSG are observed. The specificity of this channel(s) and its uniqueness have yet to be determined.

Human olfactory neurons respond to odor stimuli with an increase in cytoplasmic Ca2

The sense of smell allows terrestrial animals to collect information about the chemical nature of their environment through the detection of airborne molecules. In humans smell is believed to play an important role in protecting the organism from environmental hazards such as fire, gas leaks and spoiled food, in determining the flavor of foods, and perhaps in infant-parent bonding. In addition, the study of human olfaction is relevant to a number of medical problems that result in olfactory dysfunction, which can affect nutritional state, and to the study of the etiology of neurodegenerative diseases which manifest themselves in the olfactory epithelium. Although much is known about behavioral aspects of human olfaction, little is understood about the underlying cellular mechanisms in humans. Here we report that viable human olfactory neurons (HON) can be isolated from olfactory tissue biopsies, and we find that HON respond to odorants with an increase in intracellular calcium concentration ([Cai]).

Spatial gradient detection of chemical cues by catfish

Summary1.Unrestrained brown bullhead catfish (Ictalurus nebulosus) were trained to turn toward the higher concentration when two concentrations of a mixture of amino acids were simultaneously flowed over the cutaneous taste epithelium on the right and left maxillary barbels.2.The minimum detectable Weber fraction was dependent upon the stimulus solution concentration with smaller Weber fractions being detected at weaker concentrations. Brown bullheads were capable of detecting a concentration difference of 0.3 Weber fractions at the highest stimulus solution concentration and 0.1 Weber fractions at the lowest.3.Variations in the time-of-arrival of the stimuli at the two barbels, greater than those occurring during normal testing, did not alter the values of the minimum discernable Weber fraction. This indicates that spatial rather than temporal differences in concentration were the basis of these responses.4.Anosmic bullheads were as sensitive to concentration differences as intact fish, indicating that discrimination was made using the taste receptors on the barbels.5.These experiments show that bullheads can compare the intensity of a taste stimulus at two distinct receptor fields and thus define a gustatory chemical space. This ability is a requirement for tropotactic orientation to chemical gradients and suggests that such a mechanism may be important in nature.

A stimulus-activated conductance in isolated taste epithelial membranes.

Membrane vesicles isolated from the cutaneous taste epithelium of the catfish were incorporated into phospholipid bilayers on the tips of patch pipettes. Voltage-dependent conductances were observed in approximately 50% of the bilayers and single-channel currents having conductances from 8 to greater than 250 pS were recorded. In 40% of the bilayers displaying no voltage-dependent conductances, micromolar concentrations of L-arginine, a potent stimulus for one class of catfish amino acid taste receptors, activated a nonselective cation conductance. The L-arginine-gated conductance was concentration-dependent, showing half-maximal activation in response to approximately 15 microM L-arginine. L-Arginine-activated channels had unitary conductances of 40-50 pS and reversed between -6 and +18 mV with pseudointracellular solution in the pipette and Ringer in the bath. L-Alanine, a potent stimulus for the other major class of catfish amino acid taste receptors, did not alter bilayer conductance. D-Arginine, which is a relatively ineffective taste stimulus for catfish but a good cross-adapter of the L-arginine-induced neural response, had no effect on bilayer conductance at concentrations below 200 microM. However, increasing concentrations of D-arginine from 1 to 100 microM progressively suppressed the L-arginine-activated conductance, suggesting that D-arginine competed for the L-arginine receptor, but did not activate the associated cation channel. This interpretation is consonant with recent biochemical binding studies in this system. These results suggest that L-arginine taste receptor proteins in the catfish are part of or closely coupled to cation-selective channels which are opened by L-arginine binding.