Selective enhancement and suppression of frog gustatory responses to amino acids

Properties of the receptor sites for L-amino acids in taste cells of the bullfrog (Rana catesbeiana) were examined by measuring the neural activities of the glossopharyngeal nerve under various conditions. (a) The frogs responded to 12 amino acids, but the responses to the amino acids varied with individual frogs under natural conditions. The frog tongues, however, exhibited similar responses after an alkaline treatment that removes Ca2+ from the tissue. The variation in the responses under natural conditions was apparently due to the variation in the amount of Ca2+ bound to the receptor membrane. (b) The responses to hydrophilic L-amino acids (glycine, L-alanine, L-serine, L- threonine, L-cysteine, and L-proline) were of a tonic type, but those to hydrophobic L-amino acids (L-valine, L-leucine, L-isoleucine, L- methionine, L-phenylalanine, and L-tyrptophan) were usually composed of both phasic and tonic components. (c) The properties of the tonic component were quite different from those of the phasic component: the tonic component was largely enhanced by the alkaline treatment and suppressed by the acidic treatment that increases binding of Ca2+ to the tissue. Also, the tonic component was suppressed by the presence of low concentrations of salts, or the action of pronase E, whereas the phasic component was unchanged under these conditions. These properties of the phasic component were quite similar to those of the response to hydrophobic substances such as quinine. These results suggest that the hydrophilic L-amino acids stimulate receptor protein(s) and that the hydrophobic L-amino acids stimulate both the receptor protein and a receptor site similar to that for quinine. (d) On the basis of the suppression of the responses to amino acids by salts, the mechanism of generation of the receptor potential is discussed.     

Contribution of electrostatic and hydrophobic interactions of bitter substances with taste receptor membranes to generation of receptor potentials

The effects of changed ionic environments on the frog taste nerve responses to the bitter substances were examined. The responses to quinine and strychnine carrying a positive charge were suppressed by an increase in ionic strength of stimulating solutions. It was concluded that electrostatic interaction of these positive bitter substances with the receptor membranes greatly contributes to the adsorption of the substances on the membranes and that this interaction was suppressed by an increase in ionic strength. The responses to neutral bitter substances (caffeine and theophylline) were unchanged by an increase in salt concentration. The zeta potential of the mouse neuroblastoma (N-18 clone), which was depolarized by various bitter substances similarly to a taste cell, was measured in the presence of the bitter substances. The zeta potential was a little changed by quinine and practically unchanged by strychnine, caffeine and theophylline. The membrane fluidity of the N-18 cell monitored with 2-(9-anthroyloxy)stearic acid was changed in response to the bitter substances, while the fluidity monitored with 12-(9-anthroyloxy)stearic acid or 1,6-diphenyl-1,3,5-hexatriene was unchanged. This suggested that the bitter substances are adsorbed on the hydrophobic region near the surface and induce a conformational change at the region. The depolarization by the bitter substances seems to stem from changes in the “boundary potential” at the region near the surface within the membrane interior.     

A comparison of the actions of 4-aminopyridine, caffeine and quinine on the toad isolated rectus abdominis muscle

1. 4-Aminopyridine (4-AP)-induced contractures have been compared with those evoked by caffeine and quinine on the toad rectus abdominis muscle. 2. All three compounds produced slowly-developing sustained contractures. The time to half maximal contracture and relaxation was significantly longer for 4-AP than for caffeine or quinine. 3. Verapamil and manganese inhibited 4-AP, caffeine and quinine-induced contractures. 4. Ca2+-free-EGTA Ringer and procaine severely inhibited caffeine and quinine responses, but 4-AP contractures were relatively unaffected. 5. In depolarizing (100 mM K+) Ringer solution, caffeine and quinine responses were reduced to 6-9% of their controls. 4-AP responses were reduced by about 25%. 6. It is concluded that in the toad rectus muscle, 4-AP-induced contractures differ from those produced by caffeine and quinine, and appear to rely mainly on the release of intracellular located Ca2+, while caffeine and quinine are considered to act predominantly on plasma membrane sites.     

Electrical responses of supporting cells in the frog taste organ to chemical stimuli

1. The mean resting potential of supporting cells in the frog taste organ was -19.1 mV. The supporting cells responded to the four basic taste stimuli with a depolarization but responded to water with a depolarization or a hyperpolarization. 2. The membrane resistances of supporting cells decreased during stimulation with sucrose, NaCl and acetic acid, but increased during stimulation with Q-HCl and water. 3. Reversal potential of the depolarizing response for 0.5 M NaCl in supporting cells was +7.6 mV. The depolarizing responses for Q-HCl and acetic acid were independent of the membrane potential level. 4. These results suggest that the characteristics of taste responses in supporting cells are similar to those in taste cells.     

Vasopressin increases frog gustatory neural responses elicited by NaCl and HCl.

1. The effect of arginine vasopressin (AVP) on frog gustatory responses was investigated by recording integrated responses of the whole glossopharyngeal nerve by stimulation of the tongue with tastants. 2. After AVP (100 mUnits/ml) was perfused to the basolateral side of taste cells through the lingual artery, gustatory neural responses for NaCl and hydrochloric acid (HCl) stimuli were greatly enhanced, but the responses for CaCl2, quinine hydrochloride (Q-HCl) and galactose were not affected. 3. Three hours after the onset of AVP perfusion, the responses for NaCl and HCl increased to 260% and 270% of the respective controls. 4. The NaCl response which was insensitive to amiloride during normal saline perfusion became sensitive to amiloride during AVP perfusion. 5. When membrane-permeable 8-bromo-cyclic AMP (8-Br-cAMP, 0.1 mM) was perfused to the basolateral side of taste cells, the responses for NaCl and HCl decreased to 41 and 63% of the respective controls. 6. These results suggest that AVP may regulate the gustatory responses for monovalent salts and acids by a mechanism which is not necessary to activate adenylate cyclase.     

Taste responses to amino acids in the southern leopard frog, Rana sphenocephala

Integrated taste recordings of the glossopharyngeal (IX) nerve innervating the tongue of the southern leopard frog were studied in response to various amino acids and quinine hydrochloride. Amino acids and quinine hydrochloride elicited primarily phasic taste responses. Acidic (L-aspartic and L-glutamic) and basic (L-lysine and L-arginine) amino acids, adjusted to pH8, were effective taste stimuli. All glossopharyngeal nerve twigs that responded to amino acid stimuli also responded to quinine; however, not all quinine-sensitive IX nerve bundles were responsive to amino acids. Electrophysiological thresholds for amino acids were estimated to be 2.5-10 mM, whereas threshold for quinine hydrochloride averaged approximately 10 microM.     

Interaction Between Gustatory Depolarizing Receptor Potential and Efferent-Induced Slow Depolarizing Synaptic Potential in Frog Taste Cell

Electrical stimulation of parasympathetic nerve (PSN) efferent fibers in the glossopharyngeal nerve induced a slow depolarizing synaptic potential (DSP) in frog taste cells under hypoxia. The objective of this study is to examine the interaction between a gustatory depolarizing receptor potential (GDRP) and a slow DSP. The amplitude of slow DSP added to a tastant-induced GDRP of 10 mV was suppressed to 60% of control slow DSPs for NaCl and acetic acid stimulations, but to 20–30% for quinine–HCl (Q-HCl) and sucrose stimulations. On the other hand, when a GDRP was induced during a prolonged slow DSP, the amplitude of GDRPs induced by 1 M NaCl and 1 M sucrose was suppressed to 50% of controls, but that by 1 mM acetic acid and 10 mM Q-HCl unchanged. It is concluded that the interaction between GDRPs and efferent-induced slow DSPs in frog taste cells under hypoxia derives from the crosstalk between a gustatory receptor current across the receptive membrane and a slow depolarizing synaptic current across the proximal subsynaptic membrane of taste cells.     

Topographical difference in taste organ density and its sensitivity of frog tongue

Distribution density of the taste disks of the fungiform papillae in the frog tongue was larger at the proximal portion than at the apical and middle portions. The number of myelinated afferent nerve fibres and taste cells per cm2 area of the tongue increased in the order of proximal greater than middle greater than apical portion. The amplitudes of gustatory neural responses for 0.5 M NaCl, 0.5 M KCl, 0.5 M NH4Cl, 0.05 M CaCl2, 1 mM acetic acid and 1 mM quinine-HCl (Q-HCl) were significantly larger with lingual stimulation of the proximal region than with the stimulation of the apical region. With these stimuli the mean ratio of the apical response to the proximal response was 1.00:1.54. On the other hand, this ration with deionized water was 1.00:5.00. The mean magnitudes of receptor potentials in taste cells for 1 mM acetic acid and 10 mM Q-HCl were the same among the apical, middle and proximal portions of the tongue. The mean magnitudes of receptor potentials for 0.5 M NaCl were significantly larger at the apical portion than at the other portions, whereas those for deionized water tended to be the largest at the proximal portion. It is concluded that the larger magnitude of the gustatory neural responses at the proximal portion of the tongue is due to morphological and physiological properties of the taste organ.     

Electrical properties and gustatory responses of various taste disk cells of frog fungiform papillae

We compared the electrical properties and gustatory response profiles of types Ia cell (mucus cell), Ib cell (wing cell), and II/III cell (receptor cell) in the taste disks of the frog fungiform papillae. The large depolarizing responses of all types of cell induced by 1 M NaCl were accompanied by a large decrease in the membrane resistance and had the same reversal potential of approximately +5 mV. The large depolarizing responses of all cell types for 1 mM acetic acid were accompanied by a small decrease in the membrane resistance. The small depolarizing responses of all cell types for 10 mM quinine-HCl (Q-HCl) were accompanied by an increase in the membrane resistance, but those for 1 M sucrose were accompanied by a decrease in the membrane resistance. The reversal potential of sucrose responses in all cell types were approximately +12 mV. Taken together, depolarizing responses of Ia, Ib, and II/III cells for each taste stimulus are likely to be generated by the same mechanisms. Gustatory depolarizing response profiles indicated that 1) each of Ia, Ib, and II/III cells responded 100% to 1 M NaCl and 1 mM acetic acid with depolarizing responses, 2) approximately 50% of each cell type responded to 10 mM Q-HCl with depolarizations, and 3) each approximately 40% of Ia and Ib cells and approximately 90% of II/III cells responded to 1 M sucrose with depolarizations. These results suggest that the receptor molecules for NaCl, acid, and Q-HCl stimuli are equivalently distributed on all cell types, but the receptor molecules for sugar stimuli are richer on II/III cells than on Ia and Ib cells. Type III cells having afferent synapses may play a main role in gustatory transduction and transmission.     

Depression of gustatory receptor potential in frog taste cell by parasympathetic nerve-induced slow hyperpolarizing potential

Parasympathetic nerve (PSN) innervates taste cells of the frog taste disk, and electrical stimulation of PSN elicited a slow hyperpolarizing potential (HP) in taste cells. Here we report that gustatory receptor potentials in frog taste cells are depressed by PSN-induced slow HPs. When PSN was stimulated at 30 Hz during generation of taste cell responses, the large amplitude of depolarizing receptor potential for 1 M NaCl and 1 mM acetic acid was depressed by approximately 40% by slow HPs, but the small amplitude of the depolarizing receptor potential for 10 mM quinine-HCl (Q-HCl) and 1 M sucrose was completely depressed by slow HPs and furthermore changed to the hyperpolarizing direction. The duration of the depolarizing receptor potentials depressed by slow HPs prolonged with increasing period of PSN stimulation. As tastant-induced depolarizing receptor potentials were increased, the amplitude of PSN-induced slow HPs inhibiting the receptor potentials gradually decreased. The mean reversal potentials of the slow HPs were approximately -1 mV under NaCl and acetic acid stimulations, but approximately -14 mV under Q-HCl and sucrose stimulations. This implies that when a slow HP was evoked on the same amplitude of depolarizing receptor potentials, the depression of the NaCl and acetic acid responses in taste cells was larger than that of Q-HCl and sucrose responses. It is concluded that slow HP-induced depression of gustatory depolarizing receptor potentials derives from the interaction between gustatory receptor current and slow hyperpolarizing current in frog taste cells and that the interaction is stronger for NaCl and acetic acid stimulations than for Q-HCl and sucrose stimulations.     

Inhibitory effect of gurmarin on palatal taste responses to amino acids in the rat

Gurmarin (10 microg/ml), a protein extracted from Gymnema sylvestre, depressed significantly (40-50%) the phasic taste responses to sugars (sucrose, fructose, lactose, and maltose) and saccharin sodium recorded from the greater superficial petrosal nerve (GSP) innervating palatal taste buds in the rat. However, no significant effect of gurmarin was observed for taste responses to NaCl, HCl, and quinine hydrochloride. Phasic responses to D-amino acids that taste sweet to humans (His, Asn, Phe, Gln) were also depressed, but gurmarin treatment was without significant effect on taste responses to D-Trp and D-Ala, six L-amino acids (His, Asn, Phe, Gln, Trp, and Ala), and two basic amino acid HCl salts (Arg and Lys). With the exception of D-Trp, these inhibitory effects of gurmarin on GSP taste responses were related to the rat's preference for these substances.     

Neural responses and aversion to bitter stimuli in rats

Gustatory responses of rats to quinine, nicotine, caffeine,MgCl₂ and bitter peptides such as glycyl-L-phenylalanine (Gly-Phe)and glycyl-L-isoleucine (Gly-lle), were studied by recordingintegrated responses to bitter stimuli from the chorda tympaniand IXth nerve as well as by measuring fluid intakes with atwo-bottle choice method. In addition, the effect of L-glutamyl-L-glutamicacid (Glu-Glu), which has an ability of masking bitterness inhumans, on the IXth nerve response was examined. Quinine, nicotine and caffeine elicited aversive behavior atconcentrations similar to those eliciting the IXth nerve responses,while MgCl₂ induced aversion above 0.1 M although it producedneural responses at much lower concentrations in both nerves.No aversion was produced by the bitter peptides at 30 mM, althougha small response was elicited by Gly-Phe in the chorda tympaniand by Gly-Ile in the IXth nerve. Responses in the IXth nerveto mixtures of Glu-Glu with bitter substances was smaller thanthe sum of those for single solution alone, indicating depressionof nerve responses to bitter stimuli by Glu-Glu.     

Phasic contractions of the rat portal vein depend on intracellular Ca2+ release stimulated by depolarization

The phasic contraction to phenylephrine of the rat isolated portal vein was investigated using functional studies. Phasic contractions to phenylephrine and caffeine could be produced after several minutes in Ca(2+)-free Krebs solution, which were inhibited by cyclopiazonic acid or ryanodine. The phenylephrine and caffeine contractions were abolished, however, within 10 min in Ca(2+)-free Krebs solution and by nifedipine. This indicated the Ca(2+) stores were depleted in the absence of Ca(2+) influx through voltage-gated channels. The phasic contraction to phenylephrine was also abolished by niflumic acid even in Ca(2+)-free Krebs solution. This showed that the response depended on intracellular Ca(2+) release stimulated directly by depolarization, resulting from opening of Ca(2+)-activated Cl(-) channels, but did not require Ca(2+) influx. In support of this, K(+)-induced phasic contractions were also produced in Ca(2+)-free Krebs solution. The phenylephrine but not K(+)-induced phasic contractions in Ca(2+)-free Krebs solution were inhibited by ryanodine or cyclopiazonic acid. This would be consistent with Ca(2+) release from more superficial intracellular stores (affected most by these agents), probably by inositol 1,4,5-trisphospate, being required to stimulate the phenylephrine depolarization.     

Citric acid and quinine share perceived chemosensory features making oral discrimination difficult in C57BL/6J mice

Evidence in the literature shows that in rodents, some taste-responsive neurons respond to both quinine and acid stimuli. Also, under certain circumstances, rodents display some degree of difficulty in discriminating quinine and acid stimuli. Here, C57BL/6J mice were trained and tested in a 2-response operant discrimination task. Mice had severe difficulty discriminating citric acid from quinine and 6-n-propylthiouracil (PROP) with performance slightly, but significantly, above chance. In contrast, mice were able to competently discriminate sucrose from citric acid, NaCl, quinine, and PROP. In another experiment, mice that were conditioned to avoid quinine by pairings with LiCl injections subsequently suppressed licking responses to quinine and citric acid but not to NaCl or sucrose in a brief-access test, relative to NaCl-injected control animals. However, mice that were conditioned to avoid citric acid did not display cross-generalization to quinine. These mice significantly suppressed licking only to citric acid, and to a much lesser extent NaCl, compared with controls. Collectively, the findings from these experiments suggest that in mice, citric acid and quinine share chemosensory features making discrimination difficult but are not perceptually identical.     

Electrophysiological responses of the chemoreceptor neurones in the antennal taste sensilla to plant alkaloids and glucosides in a granivorous ground beetle

The electrophysiological response of chemoreceptor neurones from the antennal chaetoid taste sensilla of the omnivorous ground beetle Pterostichus oblongopunctatus to several plant alkaloids and glucosides is investigated. A quinine‐sensitive neurone responding to quinine and quinine hydrochloride is found, most probably related to the granivorous feeding habit of P. oblongopunctatus. The response to quinine hydrochloride is concentration‐dependent at 0.001–50 mm , with the response threshold at 0.01 mm and a maximum rate of firing of 67 spikes/s at 50 mm . The stimulatory effect of caffeine is very weak, where the firing rate increases by only 1.4 spikes/s at a concentration of 10 mm compared with that evoked by a control stimulus. In addition, both quinine and quinine hydrochloride strongly inhibit spike production by the salt‐ and pH‐sensitive neurones when presented in mixtures with 10 mm NaCl. Several tested plant secondary compounds (i.e. salicin, sinigrin, caffeine and nicotine), which have only little or no effect on the firing rate of the quinine‐sensitive neurone, greatly reduce the responses of the salt‐ and pH‐sensitive neurones. The results of the present study suggest that the antennal taste sensilla of P. oblongopunctatus may detect plant defensive compounds both through the activation of a quinine‐sensitive neurone and via peripheral inhibition of other chemoreceptor neurones of the taste sensillum.     

Stretch-elicited calcium responses in the intact mouse thoracic aorta

Stretch-elicited intracellular calcium ([Ca(2+)](i)) changes in individual smooth muscle cells in a ring of aorta were measured simultaneously with the force developed by the ring. A phasic increase in [Ca(2+)](i) was observed in 30% of the cells and a sustained one in 10%. Depletion of intracellular calcium store by thapsigargin and caffeine decreased phasic and increased sustained calcium responses. The inhibition of calcium entry either by stretching the aorta in a calcium-free medium or by the inhibition of stretch-activated, non-selective cationic channels by 5 microM GsMtx-4 toxin, decreased the proportion of sustained [Ca(2+)](i) responses but increased transient responses. In this condition, a third of the cells responded to stretch by a bursts of [Ca(2+)](i) spikes. The decrease of calcium influx triggered the generation of burst of calcium spikes after the application of stretch steps to the vascular wall. We conclude that progressive recruitment of smooth muscle cells is the mechanism underlying the force-generating part of the myogenic response. Two types of stretch-elicited calcium responses were observed during the recruitment of the smooth muscle cells. One was a phasic calcium discharge generated by the sarcoplasmic reticulum. The second was a tonic response produced by the activation of the stretch-sensitive cationic channels allowing extracellular Ca(2+) entry.     

Caffeine and excitation-contraction coupling in the guinea pig taenia coli

The effects of caffeine (0.2–10 mM) on the electrical and mechanical activities of guinea pig taenia coli were investigated with the double sucrose-gap method. Caffeine evoked a small tension with a latency of 20–30 sec, then phasic contraction developed and finally relaxation. The initial tension development also appeared in the Na-free solution without any marked changes in the membrane potential and membrane resistance. The phasic contraction disappeared in the Na-free solution. The relaxation in the presence of caffeine was accompanied by depolarization block of the spike generation. The minimum concentration of Ca ion needed to evoke the tension development by the caffeine was 10^-7 M. Caffeine also potentiated the twitch tension below a concentration of 5 mM either in the Na-free solution or at low temperature (5°C). NO₃ ^- and Br^- showed a similar response to caffeine on the potentiation of the twitch tension at low temperature.     

Aldosterone increases gustatory neural response to NaCl in frog

1. The effect of aldosterone on frog gustatory response was investigated by recording integrated responses of the whole glossopharyngeal nerve elicited by taste stimuli. 2. After aldosterone (1 microM) was perfused to the basolateral side of taste cells through the lingual artery, the gustatory neural response for a NaCl stimulus was greatly enhanced, but the gustatory responses for CaCl2, hydrochloric acid, quinine hydrochloride and galactose were not affected. 3. At 3 and 6 hr after the onset of aldosterone perfusion, the magnitudes of the responses for NaCl increased to 2.0 and 3.6 times the control, respectively. 4. These results suggest that aldosterone may regulate the gustatory responses for monovalent salts alone.     

Nitric oxide inhibits human and canine pulmonary vascular tone via a postjunctional, nonelectromechanical, cGMP-dependent pathway.

We examined nitric oxide mediated regulation of pulmonary arterial and venous smooth muscle (PASM and PVSM, respectively): whether this inhibition is mediated via prejunctional receptors on adrenergic nerve endings; whether NO is neuronally derived; the relationship between degree of inhibition and vessel size; and identification of the signalling mechanisms involved. Canine pulmonary vascular tissues were generally quiescent, while human PASM exhibited spontaneous phasic activity. The nitric oxide (NO) synthesis inhibitor Nomega-nitro-L-arginine (L-NNA; 10(-4) M) increased tone and enhanced phasic activity. Electrical field stimulation (EFS) evoked contractions were markedly enhanced by L-NNA in an endothelium-dependent fashion, and antagonized by the NO donor S-nitroso-N-acetylpenicillamine (SNAP; 10(-7) to 10(-5) M). 8-Bromo-cGMP mimicked the effects of SNAP on basal tone and EFS contractions, while an inhibitor of soluble guanylate cyclase mimicked those of L-NNA. While mechanical responses to exogenously added norepinephrine (10(-9)-10(-4) M) were also enhanced by L-NNA and suppressed by SNAP, EFS-evoked excitatory junction potentials were unaffected by SNAP. We conclude that, in human and canine PASM and PVSM, there is a tonic generation of NO originating within the endothelium that does not mediate a prejunctional effect, but which acts postjunctionally to activate a cGMP-dependent pathway within the smooth muscle.     

Sensory responses and axonal morphology of two different types of cerebral neurones in Helix pomatia L

1. The mechano- and chemosensory responses of two different types of cerebral neurones responding to food stimuli were studied by microelectrophysiological techniques. This was correlated with axonal morphology investigated by intracellular Co-lysine labelling. 2. The C4 neurone responded to food stimuli applied to the lip receptors by phasic (less than 30 sec duration), while the C14 neurone by an initial phasic and a subsequent tonic longer than 10 min duration) activity change. 3. The different sensory responses of the two neurones can partly be explained by the morphological differences found between them.