Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Taste Modulators are Tools to Gain a Better Insight into Specific Sensitivity of Chemoreceptors in Blowflies
Department of Experimental Biology, Section of General Physiology, University of Cagliari, Cittadella Universitaria di Monserrato, SS. 554 Km 4.500, I-09042 Monserrato (CA), Italy
Correspondence to be sent to: Anna Liscia, e-mail: Liscia{at}unica.it
Key words: blowfly, electrophysiology, Na-cyclamate, sugar, sweet, taste, taste modulators
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Introduction |
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 Top Introduction Materials and methodsResultsDiscussionReferences |
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One of the questions arising in studying taste transduction is whether natural sweets are distinguished from sweeteners as the former are the major source of metabolic energy. Blowflies are a convenient preparation to study chemoreception, given their relatively simplicity, availability and the vast body of related work published. The labellar chemosensory system of blowflies consists of sensilla housing four chemoreceptive neurons, named ‘salt’, ‘sugar’, ‘water’ and ‘deterrent’ cells after their best recognized stimuli. While spike activity from these sensory neurons is readily recordable, dendrite membranes remain difficult to access (Murakami and Kijima, 2000
),
making the mechanism(s) of transduction little understood at the membrane level. However,
information can be obtained by studying the effects on the spike activity of specific
pharmacological modulators used in association with chemical stimuli.
The sugar receptor cell is capable of detecting a broad variety of substances, such
as pyranose or furanose sugars, as well as amino acids and proteins. On the other hand,
Ahamed et al. (2001) found
that the sweetener glycyrrhizin activates the ‘pyranose site’ of the
‘sugar’ cell membrane in Phormia regina, while Na-saccharine
stimulates the ‘deterrent cell’ in Protophormia terraenovae
(Liscia et al., 2004).
Calcium ions appear to be in many ways involved in the transduction mechanisms of various tastants. We therefore decided to investigate the role of calcium in the chemoreception mechanism(s) of sugars and sweeteners. In the present study we have specifically investigated the reception mechanism of a natural pyranose sugar (sucrose) and a commonly used sweetener for humans (Na-cyclamate) by addressing such issues as the involvement of Ca2+ cascade and/or of Ca2+ channels in the sugar transduction mechanism.
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Materials and methods |
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TopIntroductionMaterials and methodsResultsDiscussionReferences |
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The spike activity from the chemosensory cells was recorded by means of the tip-recording technique (Hodgson et al., 1955
) in labellar chemosensilla of Protophormia terraenovae. The
electrophysiological recordings were pass-band filtered (10–1000 Hz), digitized
through a Metrabyte DAS-16 A/D converter (10 000 points/s) and stored on disk for
computer analysis. Spikes in the discharges were sorted out by means of the S.A.P.I.D.
Tools software (Smith et al.,
1990). Spike analysis was applied to the first second of the discharges
skipping the first 30 ms from the onset of stimulation. Taste stimuli
Concentrations of 1–100 mM Na-cyclamate were used in the dose–response relationship, while 200 mM sucrose and 50 mM Na-cyclamate were applied alone or added with the following modulators.
Modulators
The following compounds were added to the stimulating solutions: amiloride (inhibitor of the ENaC superfamily of ion channels); W-7 (calmodulin antagonist and inhibitor of Na-activated cation channels); EGTA (Ca2+ chelator); Mibefradil (blocker of the Ca2+ T-type channel); SK&F-96365 (inhibitor of receptor mediated Ca2+ influx).
All stimuli were administered in a blind sequence at the concentration of 0.1 mM except for EGTA (1 mM).
Statistical analysis
Significant differences were calculated by means of Student’s t-test (Statistical software package) with a 95% confidence level.
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Results |
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TopIntroductionMaterials and methodsResultsDiscussionReferences |
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Mean spike firing frequency values ± SE recorded from labellar chemosensilla in response to 1–100 mM Na-cyclamate are shown in Figure 1. A clear dose-response is seen for the ‘sugar’ cell and an inverse correlation for the ‘water’ cell. Since at the highest concentration tested (100 mM) the response of the ‘salt’ cell was relatively high, 50 mM was adopted as the test concentration to further study the stimulating effect of Na-cyclamate on the ‘sugar’ cell.
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Figure 2A shows that both amiloride and EGTA decrease the response to Na-cyclamate but not to sucrose; in contrast, W-7 decreases the response to sucrose but not to Na-cyclamate. Mibefradil decreases the responses to both stimuli, particularly affecting the tonic portion of the discharge (200–1000 ms; Figure 2B). SK&F-96365 inhibits the stimulating effectiveness of both sucrose and Na-cyclamate.
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0.05) with respect to pure
compounds. (B) Time course (spikes/100 ms intervals) of the ‘sugar’
cell in response to sucrose and sucrose + mibefradil. |
Discussion |
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TopIntroductionMaterials and methodsResultsDiscussionReferences |
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Our results show that Na-cyclamate predominantly stimulates the same sensory cell (‘sugar’) as sucrose. However, evidence is provided that the two stimuli act through different transduction mechanisms: in fact, differences in spike firing frequency were found between sucrose and Na-cyclamate, when various pharmacological modulators were added to these stimuli in the test solution. The response to Na-cyclamate seems to be largely mediated by an Na+ influx across amiloride-sensitive channels (ENaC) and, to a lesser extent, by an amiloride-insensitive component (AIC) which could account for the relative sensitivity to EGTA, Mibefradil and SK&F-96365, thus implying the involvement of Ca2+ in the transduction mechanism.
Instead, the sucrose response is amiloride insensitive, as previously reported
(Liscia et al., 1997), but is
inhibited by W-7 (Liscia et al.,
2002), thus suggesting a different pathway. A Na+-activated
cation channel could be in fact opened by an early Na+ influx through
sugar-activated ionotrophic channels such as those described by
Murakami and Kijima (2000) in the
fleshfly. W-7 interacts either from the outside of the membrane and/or from the inside by
inhibiting Ca2+-modulated cascades (Laver et al., 1997;
Zhainanarov et al., 1998).
Ion influx would mainly be made of Na+ and Ca2+, both of
which reinforce channel activation, thus sustaining spread of depolarization.
Similar Na+-activated W-7-inhibited cation channels have been
reported in the antennal olfactory dendrites of lobsters (Zhainanarov et al., 1998, 2001). Mibefradil
strongly reduced the tonic portion of the discharge (200–1000 ms; Figure
2B), and this suggests an effect on
the Na+-activated cation channel but no influence on the early
Na+ current entering via the ionotrophic channel.
In conclusion, we found that sucrose and Na-cyclamate activate separate transduction pathways on the same chemosensory ‘sugar’ cell in the labellar sensilla of the blowfly P. terraenovae. Besides, the different effects exerted by the various modulators tested point to an involvement of Ca2+ in the transduction mechanism of both sucrose and Na-cyclamate.
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References |
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TopIntroductionMaterials and methodsResultsDiscussionReferences |
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Ahamed, A., Tsutumi, S., Ozaki, M. and Amakawa, T. (2001) An artificial sweetener stimulates the sweet taste in insect: dual effects of glycyrrhizin in Phormia regina. Chem. Senses, 26, 507–515.
Hodgson, E.S., Lettvin, J.Y. and Roeder, K.D. (1955) Physiology of primary chemoreceptor unit. Science, 122, 417–418.
Laver, D.R., Cherry C.A. and Walker N.A. (1997) The action of calmodulin antagonist W-7 and TFP and of calcium on the gating kinetics of the calcium activated large conductance potassium channel of the chara protoplasmic drop: a substate –sensitive analysis. J. Membr. Biol., 26, 263–274.
Liscia, A., Solari, P., Majone, R., Tomassini Barbarossa, I. and Crnjar, R. (1997) Taste reception mechanism in the blowfly: evidence of amiloride-sensitive and insensitive recepror sites. Physiol. Behav., 62, 875–879.[CrossRef][Medline]
Liscia, A., Crnjar, R., Masala, C., Sollai, G. and Solari, P. (2002) Sugar reception in the blowfly: a possible Ca++ involvement. J. Insect Physiol., 48, 693–699.[CrossRef][ISI][Medline]
Liscia, A., Crnjar, R., Masala, C., Sollai, G. and Solari, P. (2004) Saccharin stimulates the ‘deterrent’ cell in the blowfly: behavioral aqnd electrophysiological evidence. Physiol. Behav. , 80, 647–646.[CrossRef][Medline]
Murakami, M. and Kijima, H. (2000) Trasduction ion channels directly gated by sugars on the insects ion channel. J. Gen. Physiol., 115, 455–466.
Smith, J.J.B.., Mitchell, B.K., Rolseth, B.M., Whitehead, A.T. and Albert, P.J. (1990) SAPID tools: microcomputer programs for analysis of multi-unit nerve recordings. Chem. Senses, 13, 253–270.
Zhainanarov, A., Doolin, R., Herlihy J-D. and Ache, B.W. (1998) Sodium-gated cation channel implicated in the activation of lobster olfactory receptor neurons. J. Neurophysiol., 79, 1349–1359.
Zhainanarov, A., Doolin, R., Herlihy J-D. and Ache, B.W. (2001) Odor-stimulated phosphatidylinositol 3-kinase in lobster olfactory receptor cells. J. Neurophysiol., 85, 2537–2544
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