Chromosaponin I stimulates the sugar taste receptor cells of the blowfly, Phormia regina

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, stimulates the growth of roots in a variety of plants. In the present work, we introduce CSI as a sugar taste substance for the blowfly, Phormia regina. The blowfly has taste chemosensilla on the labellum. The sensory receptor cells in the chemosensillum are highly specialized for the tastes of sugar, salt and water, respectively. Application of CSI induced the feeding response of blowflies including full proboscis extension. CSI also induced impulses of the sugar taste receptor cell in the LL-type sensillum. The optimum concentration of CSI in these responses was 0.1 mM which is much lower than that of sucrose. Based on the comparison of dose-response relationships, CSI is 100 times more effective than sucrose in stimulating the sugar taste receptor cells. CSI-induced impulses appeared after a significant latency compared with sucrose. As far as we know, this is the first report describing that a natural saponin induces sugar responses in insects. CSI is a unique saponin because of its bifunctional property in plants and insects.     

Biochemical and physiological evidence that calmodulin is involved in the taste response of the sugar receptor cells of the blowfly, Phormia regina

The gustatory system is essential for almost all animals. However, the signal transduction mechanisms have not yet been fully elucidated. We isolated labellar chemosensilla from blowfly, Phormia regina, and purified calcium binding proteins from the water soluble fraction. The most abundant calcium-binding protein was calmodulin. To investigate the role of calmodulin in taste transduction, electrophysiological responses were recorded with the calmodulin inhibitor, W-7. When we stimulated the labellar chemosensillum with sucrose plus W-7, a dose-dependent decrease of impulse frequency was observed when the concentration was <50 microM. In addition, when W-7 at 50 microM or higher concentration was added, an initial short-term impulse generation from the sugar receptor cell was observed, but this was followed by a silent period. When the sensillum was stimulated with W-7 plus a membrane-permeable cGMP analog, dibtyryl-cGMP or 8-bromo-cGMP, impulses of the sugar receptor cell were induced but the frequency was decreased. By the sidewall-recording method, we observed that the receptor potential induced by sucrose stimulation was decreased by W-7 in the sugar receptor cell, and corresponded with a disappearance of impulses. These data strongly suggest that the cGMP-gated channel generating receptor potential in the sugar receptor cell requires calmodulin for its gating.     

Intrinsic nitric oxide regulates the taste response of the sugar receptor cell in the blowfly, Phormia regina

The taste organ in insects is a hair-shaped taste sensory unit having four functionally differentiated contact chemoreceptor cells. In the blowfly, Phormia regina, cGMP has been suggested to be a second messenger for the sugar receptor cell. Generally, cGMP is produced by membranous or soluble guanylyl cyclase (sGC), which can be activated by nitric oxide (NO). In the present paper, we electrophysiologically showed that an NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO), an NO donor, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC 7) or an NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) specifically affected the response in the sugar receptor cell, but not in other receptor cells. PTIO, when introduced into the receptor cells in a sensillum aided by sodium deoxycholate (DOC, pH 7.2), depressed the response of sugar receptor cells to sucrose but did not affect those of the salt or water receptor cells. NOC 7, given extracellularly, latently induced the response of sugar receptor cells; and L-NAME, when introduced into the receptor cells, depressed the response of sugar receptor cells. The results clearly suggest that NO, which may be produced by intrinsic NOS in sugar receptor cells, participates in the transduction cascade of these cells in blowfly.     

An artificial sweetener stimulates the sweet taste in insect: dual effects of glycyrrhizin in Phormia regina.

Glycyrrhizin, found in the root of licorice (Glycyrrhizia glabra), has been used extensively as a non-sugar sweetener for humans and also as a medicine. As far as we know, the present work is the first report describing that a non-sugar sweetener for humans induces a sweet taste in insects. In behavioural experiments, we found that glycyrrhizin induced the feeding response, including full proboscis extension in the blowfly, Phormia regina. Glycyrrhizin also induced impulses of the sugar receptor cell in the labellar chemosensillum, which is highly specialized for the tastes of sugars and nucleotides. The optimum concentration of glycyrrhizin was 3.0 mM, which is much lower than that of sucrose. It has been established that multiple receptor sites, the pyranose receptor site (P site) and the furanose receptor site (F site), are present in the sugar receptor cell of the blowfly and the fleshfly. The inhibitors specific to the P site, starch and PCMB (p-chloromercuribenzoate), partially inhibited glycyrrhizin-induced responses but not levan (an inhibitor to the F site), indicating that the P site on the sugar receptor cell is involved in the glycyrrhizin action but not the F site. When 30 s stimulation with 3.0 mM glycyrrhizin was repeated with an interval of 3--10 min, the impulse frequency to the second stimulus was higher than that to the first one and doubled within 6 min. The first stimulus lasting longer than 10 s potentiated the impulse generation and reduced the adaptation rate during the second stimulus. These results suggest that, in addition to the action via the P site, an additional mechanism, possibly in the signal transduction cascade of the sugar receptor cell, may be involved in the action of glycyrrhizin.     

The effects of amiloride on the labellar taste receptor cells of the fleshfly Boettcherisca peregrina

Amiloride is known to inhibit the taste response of vertebrates to salt by blocking the amiloride-sensitive sodium channel. In this study, we investigated electrophysiologically the effect of amiloride on the taste response of the fleshfly Boettcherisca peregrina. When 0.5 mM amiloride was included in taste solutions, the response of the salt receptor cell (salt response) to sodium chloride (NaCl) was not depressed but those of the sugar receptor cell (sugar responses) to sucrose, glucose, fructose, l-valine (l-Val) and l-phenylalanine (l-Phe) were strongly depressed. An inhibitory effect of amiloride on the concentration-response relationship for both sucrose and l-Phe was clearly revealed, but not at high concentrations of sucrose. After pretreatment of a chemosensory seta with 0.15 mM amiloride for 10 min, the salt response to NaCl was not affected. On the other hand, the sugar responses to sucrose, fructose, l-Val and l-Phe were depressed just after amiloride pretreatment. The sugar response to adenosine 5’-diphosphate (ADP) mixed with 0.5 mM amiloride was not depressed, but the response to ADP alone was depressed after amiloride pretreatment. It was therefore observed that amiloride depressed the responses to all stimulants that react with each of the receptor sites of the sugar receptor cell.     

Inositol 1,4,5-trisphosphate transduction cascade in taste reception of the fleshfly, Boettcherisca peregrina

The role of an inositol 1,4,5-trisphosphate (IP3)-mediated transduction cascade in the response of taste receptor cells of the fleshfly Boettcherisca peregrina was investigated by using the following reagents: neomycin (an inhibitor of IP3 production), U73122 (an inhibitor of phospholipase C), adenophostin A (an agonist of the IP3-gated channel), IP3, ruthenium red (a blocker of the IP3-gated channel), and 2-aminoethoxydiphenylborate (2-APB; an antagonist of the IP3-gated channel). For introduction into the receptor cell, the reagents were mixed with a detergent, deoxycholate (DOC). After treatment with neomycin + DOC or U73122 + DOC, the response of the sugar receptor cell to sugars was depressed compared with responses after treatment with DOC alone. During the treatment of adenophostin A + DOC, the response of the sugar receptor cell was elicited. After treatment with IP3 + DOC, the response of the sugar receptor cell to sugars and to amino acids was apparently enhanced. When taste stimuli were administered in the presence of ruthenium red or 2-APB, the response of the sugar receptor cell to glucose were inhibited. The expression of genes for substances involved in the IP3 transduction cascade, such as G protein alpha subunit (dGqalpha), phospholipase C (norpA), and IP3 receptor (itpr), were examined in the taste receptor cell of the fruitfly Drosophila melanogaster by using the pox-neuro70 mutant (poxn70), which lacks taste receptor cells. The expressed levels of dGqalpha and itpr in the tarsus of poxn70 mutant flies were reduced compared with those of wild-type flies. These results suggest that the IP3 transduction cascade is involved in the response of the sugar receptor cell of the fly.     

Sugar reception in the blowfly: a possible Ca(++) involvement

The present study investigates the effects of W-7 (a calmodulin antagonist involved in the Ca⁺⁺ cascade) on the response of the ‘sugar’ and ‘water’ cells of labellar chemosensilla in the blowfly Protophormia terraenovae to stimulation with sucrose or fructose. In order to ascertain whether Ca⁺⁺ conductance is involved, the effects of EGTA, one of the most used Ca⁺⁺ chelating agent, and of SK&F-96365, an inhibitor of receptor mediated calcium influx, were also studied. Our electrophysiological data indicate that W-7 addition strongly depresses the ‘sugar’ chemoreceptor response to both sugars and in the case of sucrose stimulation also influences adaptation rate. The Ca⁺⁺ chelator has no significant effects on the response of the ‘sugar’ cell following stimulation with sucrose, but lowers fructose stimulating effectiveness. In the presence of SK&F-96365 both sucrose and fructose responses are inhibited. A possible transduction mechanism for sugar reception is discussed.     

Competition of polysaccharides with sugars for the pyranose and the furanose sites in the labellar sugar receptor cell of the blowfly, Phormia regina

In the labellar sugar receptor cell of the blowfly, Phormia regina, soluble starch and dextran T500 inhibited the response to sucrose, to maltose or to glucose, but did not inhibit that to fructose. On the other hand, inulin inhibited the response to fructose, but did not inhibit that to sucrose. These results suggest that both soluble starch and dextran T500 compete with sucrose, with maltose or with glucose for the pyranose site (P site), and that inulin competes with fructose for the furanose site (F site) in a single sugar receptor cell. Each inhibition constant (K_i) was estimated to be 0.6–0.7% for soluble starch. about 4.5% for dextran T500, and about 1.3% for inulin.     

G-protein gamma subunit 1 is required for sugar reception in Drosophila

Though G-proteins have been implicated in the primary step of taste signal transduction, no direct demonstration has been done in insects. We show here that a G-protein gamma subunit, Ggamma1, is required for the signal transduction of sugar taste reception in Drosophila. The Ggamma1 gene is expressed mainly in one of the gustatory receptor neurons. Behavioral responses of the flies to sucrose were reduced by the targeted suppression of neural functions of Ggamma1-expressing cells using neural modulator genes such as the modified Shaker K+ channel (EKO), the tetanus toxin light chain or the shibire (shi(ts1)) gene. RNA interference targeting to the Ggamma1 gene reduced the amount of Ggamma1 mRNA and suppressed electrophysiological response of the sugar receptor neuron. We also demonstrated that responses to sugars were lowered in Ggamma1 null mutant, Ggamma1(N159). These results are consistent with the hypothesis that Ggamma1 participates in the signal transduction of sugar taste reception.     

Simultaneous chemical and electrical stimulation of labellar taste hairs of the blowfly Calliphora vicina

When recording from the tip of insect taste hairs, responses to chemical stimulation may be influenced by electrical currents, such as the preamplifier's input bias current. The effect of electrical currents on firing frequency of the salt receptor cell to KCl and NaCl stimulation was determined in labellar ‘aboral’ and ‘adoral’ taste hairs of the blowfly Calliphora vicina. Negative currents always decreased spike frequency, whereas positive currents either increased it, or did not change it significantly. Spike frequency changed less than 1% per 5 × 10^?11 A.A consistent picture of the electrophysiology of blowfly taste hairs is given. It includes a distal pore, present in the dendrite-free lumen of the hair. It abandons the concept of a generator current that transmits excitation from the distal, chemoreceptive part of the taste cell dendrite to the action potential generator in or near the taste cell body. The experimental results are interpreted on the basis of this picture. It is concluded that the ‘electrophoretic effect’ of the electrical current is very small. Thus, the measured effect should mainly be due to a ‘direct effect’ of electrical current on electrically excitable structures in the salt receptor cell, particularly in its dendrite.     

Cross-adapted sugar responses in the mouse taste cell

1. Intracellular recordings of mouse taste cell responses were made using a glass micro-electrode filled with Procion yellow dye solution. 2. Six sugars (sucrose, maltose, lactose, glucose, galactose and fructose) produced the depolarization responses. 3. Gustatory cross adaptation between sugars was determined. When the taste cell was pre-adapted with one of the six sugars, the other five sugars, cross adapted, produced depolarization, hyperpolarization or null responses. 4. From these observations, it is suggested that there are multiple sugar receptor sites on the receptor membrane of the mouse taste cell.     

Inositol 1,4,5‐trisphosphate transduction cascade in taste reception of the fleshfly,Boettcherisca peregrina

The role of an inositol 1,4,5‐trisphosphate (IP₃)‐mediated transduction cascade in the response of taste receptor cells of the fleshfly Boettcherisca peregrina was investigated by using the following reagents: neomycin (an inhibitor of IP₃ production), U73122 (an inhibitor of phospholipase C), adenophostin A (an agonist of the IP₃‐gated channel), IP₃, ruthenium red (a blocker of the IP₃‐gated channel), and 2‐aminoethoxydiphenylborate (2‐APB; an antagonist of the IP₃‐gated channel). For introduction into the receptor cell, the reagents were mixed with a detergent, deoxycholate (DOC). After treatment with neomycin + DOC or U73122 + DOC, the response of the sugar receptor cell to sugars was depressed compared with responses after treatment with DOC alone. During the treatment of adenophostin A + DOC, the response of the sugar receptor cell was elicited. After treatment with IP₃ + DOC, the response of the sugar receptor cell to sugars and to amino acids was apparently enhanced. When taste stimuli were administered in the presence of ruthenium red or 2‐APB, the response of the sugar receptor cell to glucose were inhibited. The expression of genes for substances involved in the IP₃ transduction cascade, such as G protein α subunit (dGqα), phospholipase C (norpA), and IP₃ receptor (itpr), were examined in the taste receptor cell of the fruitfly Drosophila melanogaster by using the pox‐neuro⁷⁰ mutant (poxn⁷⁰), which lacks taste receptor cells. The expressed levels of dGqα and itpr in the tarsus of poxn⁷⁰ mutant flies were reduced compared with those of wild‐type flies. These results suggest that the IP₃ transduction cascade is involved in the response of the sugar receptor cell of the fly. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 66–83, 2002     

Impaired Glucose Metabolism in Mice Lacking the Tas1r3 Taste Receptor Gene

The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+) inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3^tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-). Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain.     

Partial rescue of taste responses of alpha-gustducin null mice by transgenic expression of alpha-transducin

The transduction of responses to bitter and sweet compounds utilizes guanine nucleotide binding proteins (G proteins) and their coupled receptors. Alpha-gustducin, a transducin-like G protein alpha-subunit, and rod alpha-transducin are expressed in taste receptor cells. Alpha-gustducin knockout mice have profoundly diminished behavioral and electrophysiological responses to many bitter and sweet compounds, although these mice retain residual responses to these compounds. Alpha-gustducin and rod alpha-transducin are biochemically indistinguishable in their in vitro interactions with retinal phosphodiesterase, rhodopsin and G protein betagamma-subunits. To determine if alpha-transducin can function in taste receptor cells and to compare the function of alpha-gustducin versus alpha-transducin in taste transduction in vivo, we generated transgenic mice that express alpha-transducin under the control of the alpha-gustducin promoter in the alpha-gustducin null background. Immunohistochemistry showed that the alpha-transducin transgene was expressed in about two-thirds of the alpha-gustducin lineage of taste receptor cells. Two-bottle preference tests showed that transgenic expression of rod alpha-transducin partly rescued responses to denatonium benzoate, sucrose and the artificial sweetener SC45647, but not to quinine sulfate. Gustatory nerve recordings showed a partial rescue by the transgene of the response to sucrose, SC45647 and quinine, but not to denatonium. These results demonstrate that alpha-transducin can function in taste receptor cells and transduce some taste cell responses. Our results also suggest that alpha-transducin and alpha-gustducin may differ, at least in part, in their function in these cells, although this conclusion must be qualified because of the limited fidelity of the transgene expression.     

The Effects of G Protein Modulators on the Labellar Taste Receptor Cells of the Fleshfly (Boettcherisca peregrina)

A nonhydrolyzable G protein activator (guanosine 5'-O-(3-thiotriphosphate); GTPgammaS) and a G protein inhibitor (guanosine 5'-O-(2-thiodiphosphate); GDPbetaS) were introduced into the labellar taste receptor cells of the fleshfly by treatment of their receptive membranes beneath the tip opening of the chemosensory hair with each reagent in 0.03% deoxycholate solution for 4 min. After treatment with GTPgammaS, the responses of the sugar receptor cell to D-glucose, D-fructose, L-phenylalanine and L-valine and that of the salt receptor cell to cyclic AMP were markedly enhanced, compared with those after treatment with deoxycholate alone. Treatment with GDPbetaS depressed these responses. These results strongly suggest that the responses are mediated by G protein. However, the response of the salt receptor cell to NaCl was not affected by treatment with either GTPgammaS or GDPbetaS, and thus the response to NaCl clearly is not elicited through a G protein-regulated mechanism. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Adaptation-promoting effect of IP3, Ca2+, and phorbol ester on the sugar taste receptor cell of the blowfly, Phormia regina.

The fly has a receptor cell highly specialized for the taste of sugars. We introduced inositol 1,4,5-trisphosphate (IP3), Ca2+, or a phorbol ester, 12-deoxyphorbol 13-isobutylate 20-acetate (DPBA), into the cell and investigated their effects on the response to sucrose. The sugar receptor cell generates impulses during constant stimulation with sucrose, but the impulse frequency gradually declines as the cell adapts to the stimulus. Thus, this gradual reduction of the impulse frequency is a direct manifestation of adaptation of the cell. These reagents accelerated the gradual reduction of the impulse frequency, although the initial impulse frequency was little affected. In contrast to these reagents, glycoletherdiamine-tetraacetate (EGTA) retarded the gradual reduction of the impulse frequency. Moreover, when IP3 and DPBA were applied together, the gradual reduction of the impulse frequency was more accelerated than when either IP3 or DPBA was applied. When IP3 and EGTA were applied together, however, the accelerating effect of IP3 tended to be canceled. Based on these results, we hypothesized that an intracellular cascade involving inositol phospholipid hydrolysis, intracellular Ca2+ mobilization, and protein kinase C-mediated phosphorylation promotes adaptation of the sugar receptor cell.     

Transduction ion channels directly gated by sugars on the insect taste cell

Insects detect sugars and amino acids by a specialized taste cell, the sugar receptor cell, in the taste hairs located on their labela and tarsi. We patch-clamped sensory processes of taste cells regenerated from the cut end of the taste hairs on the labelum of the flashfly isolated from the pupa approximately 20 h before emergence. We recorded both single channel and ensemble currents of novel ion channels located on the distal membrane of the sensory process of the sugar receptor cell. In the stable outside-out patch membrane excised from the sensory processes, we could repeatedly record sucrose-induced currents for tens of minutes without appreciable decrease. An inhibitor of G-protein activation, GDP-beta-S, did not significantly decrease the sucrose response. These results strongly suggested that the channel is an ionotropic receptor (a receptor/channel complex), activated directly by sucrose without mediation by second messengers or G protein. The channel was shown to be a nonselective cation channel. Analyses of single channel currents showed that the sucrose-gated channel has a single channel conductance of approximately 30 pS and has a very short mean open time of approximately 0.23 ms. It is inhibited by external Ca(2+) and the dose-current amplitude relation could be described by a Michaelis-Menten curve with an apparent dissociation constant of approximately 270 mM. We also report transduction ion channels of the receptor/channel complex type directly gated by fructose and those gated by L-valine located on the sensory process.     

Effects of ATP and cAMP on salt and sugar (responses of chemosensilla in the blowfly)

• 1.1. The addition of cAMP to stimulating solutions of NaCl, fructose (furanose sugar), sucrose, or glucose (pyranose sugars) decreases the responsiveness of labellar chemosensilla in Phormia.
• 2.2. The addition of ATP, while decreasing the responsiveness to NaCl or fructose enhances the responsiveness to glucose and sucrose.
• 3.3. The inhibiting effect of ATP on NaCl or fructose responses is suppressed by GDPßS, an inhibitor of adenylate cyclase (and thus of cAMP synthesis); moreover GDPßS further enhances the increase in response due to ATP when added to the sucrose or glucose solutions.
• 4.4. Results suggest a possible involvement of cAMP and ATP in the taste reception mechanism in the blowfly.

     

Transduction pathways mediated by second messengers including cAMP in the sugar receptor cell of the blow fly: study by the whole cell clamp method

The whole cell clamp method was directly applied to the sensory receptor neurons isolated from the adult labellar hair of the blow fly Phormia regina to locate the signal transduction pathways mediated by second messengers. First, the cAMP-mediated transduction pathway was examined to specify its location in the sugar receptor cell. Sugar receptor cell was identified by recording inward current flow under the voltage clamp applying sucrose solution to the surface of the taste neurons. When cyclic nucleotides, such as cGMP and cAMP, were introduced into the sugar receptor cell, inward current was observed (cGMP, 70pA; cAMP, 300pA at 350microM). Inhibitors and activators for the second messengers (GDPbetaS and forskolin) and non-cyclic nucleotides were also examined. Second, non-nucleotide second messengers (IP3 and Ca2+) were examined. The sugar receptor cell was activated when it was injected with IP3 or Ca2+. All the obtained results suggest that the cAMP-mediated signal transduction pathway plays a major role in the sugar receptor cell. The possibility of other transduction pathways mediated by IP3 or Ca2+ was not excluded.     

Irreversible inhibition of the labellar sugar receptor of the blowfly, Phormia regina, by periodate-activated starch

Activated starch bearing aldehyde groups, which is prepared by oxidation of soluble starch with sodium periodate (NaIO₄) and binds amino groups strongly, irreversibly inhibited the responses of taste receptors in the blowfly, Phormia regina. After highly activated starch (oxidized with 0.2 M NaIO₄) was applied through the tip opening of the chemosensillum, the responses of the sugar, salt and water receptors were nonspecifically depressed. The chemoreceptor membranes were nonspecifically protected by all the sugars tested against the treatment with highly activated starch, but in the case of the response to fructose, the membrane was specifically protected by fructose. Mildly activated starch (oxidized with 0.04 M NaIO₄), however, selectively depressed the sugar response. The depression depended on the pH of the solution containing the mildly activated starch. After treatment at pH 8.6, the response to sucrose, maltose or glucose was not depressed at all, but that to fructose or galactose was depressed. After treatment at pH 9.0, however, the responses to all sugars were depressed. Such depression of the sugar response lasted for 20 h or more after treatment.