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Effects of sulphhydryl reagents on the labellar sugar receptor of the fleshfly
7. Ins& Phyriol., 1972.Vol. 18,pp. 1845to 1855. PergawuvaPress. P&d
in cleut Britain
EFFECTS OF SULPHHYDRYL REAGENTS ON THE LABELLAR SUGAR RECEPTOR OF THE FLESHFLY ICHIRO
SHIMADA, AK10 SHIRAISHI, HIROMASA and HIROMICHI MORITA
KIJIMA,
Department
of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan (Received 17 L?ecember 1971)
Abstract-A 5 to 7 min treatment with PCMB dissolved in the phosphate buffer deeply depressed the response of the rugar receptor. The depression was easily recovered by subsequent treatment with fi-mercaptoethanol and tcystein in the buffer, but not with the buffer alone. The depression was charaaerized by a greater decrease in the maximum response (I&) rather than by a change in the apparent Michaelis constant (ZQ. Thus, PCMB was concluded to react with sulphhydryl groups in one or more components, perhaps proteins, of the sugar receptor system, and was suggested to react at a difkent site from the receptor site and to block a change in the receptor membrane permeability. INTRODUCTION
IT HASBEENproposed that the first step in the stimulation of the chemoreceptor is a reversible complex formation between stimulants and the receptor (BEIDLER, 1954). Moreover, it has been assumed implicitly that this complex formation results in a conformational change in the receptor molecule leading either directly, or through some further steps, to permeability changes in the receptor membrane. DASTOLIand PRICE (1966) isolated a ‘sweet sensitive protein’ from bovine tongues. This was one of the earliest attempts to study directly the properties of the receptor substances. KARLINand BARTLES(1966) showed the presence of sulphhydryl groups and disulphide bonds in the acetylcholine receptor of the electroplax of EIectrophorus electi and concluded that one of the components of the receptor was a protein. More recently, PARISIet UZ.(1971) have isolated the proteolipid from the electroplax and have shown a biophysical response to acetylcholine when the proteolipid is embedded in artificial bilayered lipid membranes. As to the insect sugar receptor, some information about the structure of the stimulants has recently been accumulated using both behavioural and electrophysiological methods. Little is known, however, about the structure and the substance of the receptor itself (DIXHIER, 1955; HANAMORI et al., 1971; JAKINOvIcH et al., 1971). HANSEN(1969) proposed that an or-glucosidasewas the receptor protein for disaccharides. Though there were some parallelisms between the Michaelis constants of the ol-glucosidase and behavioural thresholds of the sugar receptor of 1845
1846 ICHIROSHIMA~A, Axlo SHIRA~~HI, HIROMA~A KIJIMA,ANDHIROMICHIMORITA the fly, no direct proof supporting his working hypothesis has yet been given (cf. AMAKAWA et al., 1972). The insect sugar receptor, however, has some advantages for the study of the receptor substance compared with other chemoreceptors such as pharmacological receptors. To find a clue to the receptor substance, several treatments such as modification, labelling, digestion, solubilization, immunological test and so on, must be aimed, first of all, at the exact region of the receptor in question. In contrast to the pharmacological receptors, the contact chemoreceptor organs of flies are hair-like sensilla of the basiconica type. There are two lumens, outer and inner, in the chemosensory hair. The chemosensory cells send dendrites up to the tip through the inner lumen of the hair. There, the terminals of the dendrites contact outside stimulant solutions through a small pore (less than 1 p in dia.) (LARSEN,1962; ADet al, 1965). Therefore, treatments can be primarily confined to a small region around the terminals of the dendrites (chemoreceptor membrane). Moreover, the impulse is initiated near the cell body at the base of the hair, and is propagated antidromically toward the hair tip as well as orthodromically to the central nervous system. The impulse frequency is proportional to the receptor potential in the stationary phase, while the shape of the impulse reflects the site of impulse initiation and dendritic impulse conduction (MORITA, 1959; WOLBARSHTand HANSON, 1965 ; MORITA AND YAMASHITA,1966). From these characteristics it is possible to determine which region is affected by the experimental treatment. In many species of flies, electrophysiological investigations have revealed that four dendrites of the sensory cells in a single labellar hair correspond to sugar, water, salt receptors, and the ‘second salt receptor’, respectively (HODGSONand ROEDER, 1959; EVANS and MELLON, 1962; STEIN-T, 1965; DETHIER and HANSON, 1968). In order to pursue the direct study of the structure and the substance of the receptor, we can adopt a technique of chemical modification, one of the most powerful methods popular in protein chemistry. By behavioural assay KOYAMAand KURIHARA(1971) examined the effects of several reagents on the taste response of the fleshfly, which react with specific amino acid residue in a protein molecule. Using electrophysiological methods, we worked on the effects of the sulphhydryl reagent, p-chloromercuribenzoate (PCMB), on the response of the sugar receptor to sucrose and the effect of some other regaents on it. We will report here the results and will discuss the character of the depression. MATERIALS AND METHODS The fleshfIy, Boettcherisca pmgrina, 4 to 6 days old was used in the experiments. The chemosensory hairs used were of the largest type and located at the outer margin of the labellum. A side wall recording was employed (MORITA, 1959; MORITA and YAMASHITA,1959). An isolated head was mounted on a piece of platinum wire which served as an indifferent electrode and a glass microcapillary
EFFECTS OF SULPHHYDRYL REAGENTS ON THE FLESHFLY
1847
electrode, filled with Waterhouse’s saline (BUCK, 1953), was impaled into the outer lumen of the sensory hair, while stimulus solutions were applied to the tip of the hair with another glass capillary. As the impulse frequency can be considered to be proportional to the receptor potential in the stationary phase (MORITA and YAMASHITA,1966), the magnitude of response was defined as the number of impulses during a period from 0.15 to 0.35 set after beginning the stimulus. The duration of stimulation was less than O-5 sec. The usual interval between stimuli by sucrose solutions was about 3 min unless otherwise stated. The ambient temperature in the course of the experiments was 23 + O*S”C. Relative humidities of the experimental room were maintained at 62 to So% throughout this work, and did not change more than 5% during any series of the experiments. The treatment of the receptor with sulphhydryl reagents was performed in the same way as in the stimulation using a glass capillary filled with the solutions. All solutions for the treatment were made up in l/15 M phosphate buffer (pH 7*4), and left to stand for 30 min at room temperature (23°C) before use. A stimulus substance was dissolved in redistilled water. PCMB dissolved in 1 ml of 1 N NaOH was neutraliied with 1 N HCI and then diluted with phosphate buffer (pH 7.4). PCMB was used within half a day after being dissolved and stocked at 4”C, since it was unstable in the solution, and its effect was decreased over half a day. PCMB and L-cystein were purchased from Sigma Chemical Co. (St Louis, MO.). N-Ethyhnaleimide (NEM), 1,4-dithiotbreitol (D’IT), and kmercaptoethanol were of a special grade from Wako Chemical Industries Ltd, Japan.
Locusofd?7tgac~
RESULTS
The response of the labellar sugar receptor of the fleshfly to 0.1 M sucrose was depressed after treatment with O-5 mM PCMB. Fig. l(A) shows the record of the control response of the sugar receptor to 0.1 M sucrose before PCMB treatment. The shape of the impulse is biphasic, namely the impulses are initially positive, then abruptly become negative, and then return to the base line. Fig. l(B) shows the record in the stimulation of the sugar receptor by 0.1 M sucrose after treatment with 1 mM PCMB for 5 min. The depression due to PCMB was easily recovered by treatment with thiol compounds such as 5 n&I p-mercaptoethanol and 1OmM L-cystein. Fig. l(C) shows the record of the response to 0.1 M sucrose after treatment with 10 mM L-cystein for 3 min. The recovery was almost complete compared with the control response. After PCMB treatment the impulse frequency was clearly depressed in comparison with the control response, but no change was seen in the shape of the impulse. It may therefore be concluded that PCMB affected neither impulse initiation nor dendritic impulse conduction, but affected the primary processes of chemoreception occurrin g in the receptor membrane at the hair tip. This was true in all the results reported here regarding chemical modifications.
1848 ICHIROSNIMADA,AKIO SHIRAISHI, HIROMA~A KIJIMA,ANDHIROMICHI MORITA
ImVl 100 m SW
FIG. 1. Records of control, depression, and recovery experiments. (a) Controi response to 0.1 M sucrose before treatment with O-5mM PCMB. (b) Response to O-1 M sucrose after PCMB treatment. Cc) Response to 0.1 M sucrose after recovery by 10 mM L-cystein.
The fleets of FCMB on the sugar receptor Treatment of the hair with 05 mM PCMB in l/15 M phosphate buffer (pH 7.4) for 5 to 7 mm reduced subsequent responses of the sugar receptor to O-1 M sucrose to 30.4 per cent + 11-O (n = 13, where n is the number of experiments) of the control response. Duration of the PCMB treatment was critical. The depression after PCMB treatment for 3 min was about 40 to 60 per cent and the receptor recovered spontaneously after 30 min, while treatment for more than 10 min gave mostly 100 per cent depression without spontaneous recovery. Phosphate buffer (l/IS M) alone had no effect on the sugar receptor even if applied for more than 10 min. Excitability
of the salt receptor was also depressed, but the water receptor
was usually not affected by PCMB treatment.
PCMB itself evoked no impulse discharge but it depressed the spontaneous in the salt receptor within 30 set from the start of the treatment. After a long PCMB treatment, for more than 10 min, high frequency discharge of impulses in the salt receptor often appeared, continuing for some time before an abrupt cessation and leaving no spontaneous impulses. Phosphate buffer (l/15 M) itself evoked no response of the salt receptor. dis&arge
Recovery by ~mercaptoethanol and t-cysteirz The depression of the sugar receptor was reversed rapidly by treatment with either 5 mM p-mercaptoethanol or 10 mM L-cystein in the phosphate buffer at pH 7.4 for 3 mm (n = 13 and 6, respectively). The recovery in both reagents was almost complete. After the recovery in cystein, however, a discharge of impulses
EFF?XTS OF SULPHHYDRYL
1849
REAGENTS ON THE FLESHFLY
P: 0.5 mM PCMB 8: VI5 M phosphate
P
0
-3 0
20
buffer
I
L
40
60
min
FIG. 2. Course of depression by PCMB treatment. The figure is the tracing of the record of a single experiment. All stimulations are performed with 0.1 M sucrose solution throughout this experiment. P, treatment with O-5 mM PCMB (PH 7.4); B, treatment with & M phosphate buEer (pH 7.4). Length of lines above P and B indicates the time of treatment.
R: Woterhouee’s roiine M: 5mM fl- mercoptwthonol
20 t
(0)
C: 10 mM’ L-cysiein
c
P 0
P 20
T 40
60
min.
FIG. 3. Recovery by fl-mercaptoethanol and L-cystein from the depression due to PCMB. (a) P, 0.5 m.M PCMB; R, Waterhouse’s saline; M, 5 mM j?mercaptoethanol (pH 7.4). (b) The same as in (a), but C indicates treatment with 10 mM L-cystein (pH 7.4). Other details are the same as in Fig. 2.
1850 ICHIRO SHIMADA, AXIO SHIRAISHI, HIROMASA
KIJIMA,ANDHIROMICWI MORITA
often continued after the end of stimulation for a few hundred msec. Even when the depression was 100 per cent, it was often reversed by the treatment. This was not the case when PCMB treatment was so prolonged as to evoke high frequency impulses in the salt receptor (see above). Under these conditions the sugar receptor also appeared to have been seriously injured and was irreversibly depressed. The ej$ect of PCMB treatment on the response-concentration relationship in the stimulation by sucrose According to BEIDLER(1954) and MORITA (1969), the magnitude of response (R) is related to the concentration of taste stimulus (C) by the equation where R,,, is the maximum response magnitude and Kb is the concentration of the stimulant, at which the magnitude of response is one-half of the maximum. The equation indicates a straight line relationship with slope, l/G, and intercept on the abscissa of - Kb. It has been shown for the chemosensory hair of the largest type in the fleshfly labellum that the response of the sugar receptor to sucrose is described by the equation mentioned above (MORITA and YAMAGIITA, 1966). The effects of PCMB treatment on the relationship between C/R and C are shown in Fig. 4 compared with those before the treatment. Each hair was
J x
./
i
Molar
concmtration,
0
04
0.2
C
FIG. 4. &idler’s plot of the reaponae to sucrose before and after treatment with 05 mM PCMB for 5 min. Each graph indicates the result of each independent series of experiments. 0 -0,
before treatment; x -
x , after treatment.
EFFECTS
OF SULPHHYDRYL.
RRAGRNTS
ON
THE
1851
FLRMFL.Y
stimulated first with a series of concentrations of sucrose, then treated with 1 mM PCMB (for 5 min), washed with buffer, and finally stimulated with a second series of concentrations of sucrose. Repeated stimulation with sucrose of 0.5 M or higher concentrations sometimes made subsequent response unstable and therefore the range of concentration was limited below 0.2 M. Sometimes a high concentration (1 M) was tested only at the end of the second series. As shown in Fig. 4, the main effect of PCMB treatment was rather a decrease of R,,, than an increase of Kb. This tendency was observed in six examples out of nine, though values of & and Kb were fairly variable. PCMB as an inhibitor PCMB mixed in sucrose solutions inhibited the response of sugar receptor to sucrose. The interval between stimuli was about 3 min. The control response to 0.1 M sucrose was found to be unchanged in the lapse of interval time after the test with stimuli mixed with 0.5 mM PCMB whose duration was less than 0.5 sec. Thus, the modification effect of PCMB mixed in sucrose solutions, treated for O-5 set, was negligible after the interval of 3 min during which depression with the treatment, if any, might have been recovered. Fig. 5 shows that the type of inhibition was rather non-competitive. R, was decreased while K, was unchanged (n = 9).
2
0
4x-
. Sucrose mixed with PCMB
e
Sucrose only
z 9 E ‘T 2 I.0 %? x z u 001
O-05
0.1
Molar concentration,
C
FIG. 5. Beidler’s plot in stimulations by sucrose and mixed with PCMB. sucrose only (pH 7.4); l +, sucrose mixed with 0.5 mM PCMB
O(pH 7.::
The ejfect of NEM on the sugar receptor N-Ethylmaleimide had no remarkable effect on the excitability of the sugar receptor. Application of 4 mM NEM (pH 7.0) for 10 min showed no depression (n = 2). Treatment with 4 mM NEM for 15 min and 40 mM NEM for 15 min gave a temporary depression of the response to sucrose (a = 4 and 5, respectively) (Fig. 6). The depression was recovered spontaneously to the control level within
1852 ICHIROSHIMADA, AKIO SHIRAISHI, HIROMASA KIJIMA,ANDHIROMICHI MORITA
30 min. The phosphate buffer itself showed a slight temporary depression after 15 min treatment. Therefore, NEM gave a very weak depression in contrast to PCMB. 20-
1 40
I 20 min
I 0
0
FIG. 6. The effect of NEM on the sugar receptor. N, 4 mM NEM treatment (pH 7.0) for 15 min. Other detaila are the ssme as in Fig. 2.
The eflect of DTT m the sugar receptor The depression due to 1,4_dithiothreitol (DTT) also recovered spontaneously (Fig. 7). Immediately after the application of 10 mM DTT for 20 min a distinguishable depression could be observed, but it was recovered almost to the control level within 30 min (n = 3). As the phosphate buffer also gave a temporary depression, the depression by DTT was fairly weak.
0
I
I
20
0
,
40
min
FIG. 7. The. effect of DTT treatment (pH 7.4) for 15 min. Other details are the same as in Fig. 2. DISCUSSION
PCMB treatment resulted in a decrease of the depolarization of the sugar receptor membrane stimulated by sucrose. This effect was due to the reaction of PCMB with some component of the cell membrane of the sugar receptor at
RFlmcrs
OF -RYL
RRAOENTS ON THE FLESHFLY
1853
the hair tip. This is supported by the results shown in Fig. 1. That is, little
change is seen in the shape of the impulse after PCMB treatment which resulted in a marked depression in the impulse frequency. PCMB will first react with available sulphhydryl groups for which it is highly specific (BOYER, 1954). The depression due to PCMB was quite different in several points from that by heavy metals which were considered to be less specific. For instance, treatment with 1 n&I HgCl,, for only 0.5 set, resulted in the marked depression in the sugar receptor (unpublished data), while PCMB treatment did not show any depression for such a short time. The bond formed by PCMB in our experiments must dissociate very slowly since there is no great change in the depression after washing with the phosphate buffer for 10 min. Complete reversibility of the depression due to PCMB treatment by sulphhydryl compounds does indicate also that the site of the reaction is accessible to lipophobic molecules such as cystein, and that the components with which PCMB reacts are not irreversibly denatured. By behavioural assay, KOYAMA and KURIHARA(1971) showed the complete elimination of the taste response in the fleshfly by PCMB treatment for 1 hr. But with such a long treatment PCMB might penetrate into the cell and react with the proteins other than those at the cell surface of the receptor system. This is suggested by our observation that the depression of single hair discharge after PCMB treatment for such a long time could not be reversed by treatment with either p-mercaptoethanol or by cystein. The depression by PCMB treatment is characterized by a decrease in R, rather than an increase in & (Fig. 4). Meanwhile, the type of inhibition by PCMB mixed in the sucrose solution is non-competitive (Fig. 5). If we assume two steps in the reaction of PCMB, namely, PCMB is first adsorbed to the site of action (a reversible complex formation) and then makes bonds with the sulphhydryl groups at the site (bond formation), Fig. 5 represents the adsorption step because the control response to O-1M sucrose did not change before and after the test with the sucrose solution mixed with PCMB. On the other hand, Fig. 4 shows the state after PCMB makes bonds with the sulphhydryl groups at the site of action. At the adsorption step PCMB behaves as a non-competitive inhibitor (Fig. 5), so it is adsorbed to a differentsite from the sucrose binding site (receptor site). If two steps of the reaction occur at the same site of action, the effect of bond formation of PCMB may result in changed R,,, rather than &,. This agrees well with Fig. 4. It may, therefore, be suggested that PCMB reacta at a difIerent site from the receptor site and does not inhibit the specific interaction between sucrose and the sugar receptor site, but it may block a change in the receptor membrane permeability which immediately follows the specific interaction. However, an important question still remains as to whether the I& value in this case may or may not be simply related to the receptor-sucrose complex dissociation constant (MORITA,1969). We did not examine the effect of sucrose of a high concentration on PCMB treatment (receptor protection) though it seemed promising to determine the site of action of PCMB more to a certainty. It is because the application of sucrose of a high concentration for a fairly long time, for example,
1854 ICH~RO SHIMADA, AKIO SHIRAISHI,HIROMASAKIJIMA, AM) HIROMICHIMORITA 5 rnin, requires a long disadaptation time and thus reduces a subsequent response to sucrose, so that the effect of sucrose on PCMB treatment will become indistinguishable. In contrast to PCMB, NEM treatment had no remarkable effect on the sugar receptor. NEM was found to be less reactive towards certain sulphhydryl groups 1962). This might account for the weak than PCMB (cf. KATZ and MOMMAERTS, effect of NEM on the sugar receptor. DTT is a reagent designed to reduce disulphides but showed no great effect on the excitability of the sugar receptor, too. Therefore, disulphide bonds may not play an important role in the stimulation of the sugar receptor. These results, indicating that the presence of sulphhydryl groups in the sugar receptor system of the fleshfly, support the view that at least one of the components of this system is a protein. Acknowledgements-The authors thank Miss E. Kadota for preparing this manuscript and Drs. Kurihara and Jakinovich for sending their preprints. This work was supported in part by Scientific Research Fund from the Ministry of Education of Japan. REFERENCES ADAMSJ. R., HOLBERTP. E., and FORGASHA. J. (1965) Electron microscopy of the contact chemoreceptors of the stable fly, Stomoxys calitrans (Diptera: Muscidae). Ann. ent. SOC. Am. 58, 909-917. AMAKAWA T., KAWABATA K., KIJIMA H., and MORITA H. (1972) Isozymes of a-glucosidase in the proboscis and legs of flies. g. Inrsct PhysioZ. 18, 541-553. BEIDLER L. M. (1954) A theory of taste stimulation. g. gen. Physiol. 38, 133-139. BOYERP. D. (1954) Spectrophotometric study of the reaction of protein sulfhydryl groups with organic mercurials. g. Am. them. Sot. 76,4331-4337. BUCKJ. B. (1953) Physical properties and chemical composition of insect blood. In Insect Physiology (Ed. by K. D. ROBDER),147. Wiley, New York. DASTOLIF. R. and PRICE S. (1966) Sweet sensitive protein from bovine taste buds: isolation and assay. S&nce, Wash. 154,905-907. DETHIERV. G. (1955) The physiology and histology of the contact chemoreceptors of the blowfly. Quart. Z&v. Biol. 30, 348-371. DETHIBRV. G. and HANSoN F. E. (1968) Electrophysiological responses of the chemoreceptors of the blowfly to sodium salts of fatty acids. Proc. natn. Acad. Sci. U.S.A. 60, 1296-1303. EVANSD. R. and MELLON D., Jr. (1962) Electrophysiological studies of a water receptor associated with the taste sensilla of the blowtIy. J. gen. Physiol. 45, 487-500. HANAMORI T., SHIRAI~HIA., KIJIMA H., and MORITA H. (1971) Stimulation of labehar sugar receptor of the fleshfly by glucosides. 2. vergl. Physiol. 76, 11 S-124. HANSENK. (1%9) The mechanism of insect sugar reception, a biochemical investigation. In O&zction and Taste 3, 382. (Ed. by C. PFAFFMANN.) Rockefeller University Press, New York. HODGKINE. S. and ROBDERK. D. (1956) Electrophysiological studies of arthropod chemoreception. J. cell. camp. Physiol. 48, 51-75. JAKINO~ICHW., Jr., GOLDSTEIN I. J., VON BAUMGARTEN R. J., and AGFUNOPPB. W. (1971) Sugar receptor speci&ity in the fleshfIy, Sarcophuga bullata. Brain Res. 35, 369-378. KAFUN A. and BAWIZB~E. (1966). Effects of blocking sulfhydryl groups and of reducing disulfide bonds of the acetylcholine-activated permeability system of the electroplax. Biochim. biophys. Acto 126, 525-535.
EFFECTS
OF SuPHI-lYnRYLREAGENTS ON THE =?rFLY
KATZ A. M. and MOMMAERTS W. F. H. N. (1962) Sulfhydryl groups of actin.
1855 Biochiw.
biophys. Actu 65, 82-92. KOYAMAN. and K~RIHARAK. (1971) Modification by chemical reagents of proteins in the gustatory and olfactory organs of the Aeahfly and cockroach. J. Insect Physiol. 17, 2435-2440. LARSENJ. R. (1962) The fine structure of the labeilar chemosensory hairs of the blowfly, Phwmia regina. J. Insect Physiol. 8, 683-691. MORITA H. (1959) Initiation of spike potentials in contact chemosenaory hairs of insectIII. D.C. stimulation and generator potential of labellar chemoreceptor of Culliphoru.
J. cell. camp. Physiol. 54,189-204. MORXTAH. (1969) Electrical signs of taste receptor activity. In O&r&m and Taste 3, 370. .) Rockefeller University Press, New York. (Ed. by C. PFAFFMANN MOFUTAH. and YAMASHITAS. (1959) Generator potential of insect chemoreceptor. Stience, wash. l30, 922. MORITA H. and YAMASHITAS. (1966) Further studies on the receptor potential of chemoreceptors of the blowfly. Mem. Fat. Sci. Kyuahu Univ. Ser. E. (Biol.) 4,123-135. PARXSIM., RNAS E., and DE ROBERTISE. (1971) Conductance in lipidic membranes containing a ploteolipid from a Electrophorus. Science, Wush. 172, 56-57. STBINHARDTFL A. (1965) Cation and anion stimulation of the electrolyte receptors of the blowfly, Phonnia regina. Am. Zool. 5, 651-652. WOLBARSHTM. L. and HANSONF. E. (1965) Electrical activity in the chemoreceptors of the blowfly-III. Dendritic action potentials. J. gm. Physiol. 48, 673-683.
in cleut Britain
EFFECTS OF SULPHHYDRYL REAGENTS ON THE LABELLAR SUGAR RECEPTOR OF THE FLESHFLY ICHIRO
SHIMADA, AK10 SHIRAISHI, HIROMASA and HIROMICHI MORITA
KIJIMA,
Department
of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan (Received 17 L?ecember 1971)
Abstract-A 5 to 7 min treatment with PCMB dissolved in the phosphate buffer deeply depressed the response of the rugar receptor. The depression was easily recovered by subsequent treatment with fi-mercaptoethanol and tcystein in the buffer, but not with the buffer alone. The depression was charaaerized by a greater decrease in the maximum response (I&) rather than by a change in the apparent Michaelis constant (ZQ. Thus, PCMB was concluded to react with sulphhydryl groups in one or more components, perhaps proteins, of the sugar receptor system, and was suggested to react at a difkent site from the receptor site and to block a change in the receptor membrane permeability. INTRODUCTION
IT HASBEENproposed that the first step in the stimulation of the chemoreceptor is a reversible complex formation between stimulants and the receptor (BEIDLER, 1954). Moreover, it has been assumed implicitly that this complex formation results in a conformational change in the receptor molecule leading either directly, or through some further steps, to permeability changes in the receptor membrane. DASTOLIand PRICE (1966) isolated a ‘sweet sensitive protein’ from bovine tongues. This was one of the earliest attempts to study directly the properties of the receptor substances. KARLINand BARTLES(1966) showed the presence of sulphhydryl groups and disulphide bonds in the acetylcholine receptor of the electroplax of EIectrophorus electi and concluded that one of the components of the receptor was a protein. More recently, PARISIet UZ.(1971) have isolated the proteolipid from the electroplax and have shown a biophysical response to acetylcholine when the proteolipid is embedded in artificial bilayered lipid membranes. As to the insect sugar receptor, some information about the structure of the stimulants has recently been accumulated using both behavioural and electrophysiological methods. Little is known, however, about the structure and the substance of the receptor itself (DIXHIER, 1955; HANAMORI et al., 1971; JAKINOvIcH et al., 1971). HANSEN(1969) proposed that an or-glucosidasewas the receptor protein for disaccharides. Though there were some parallelisms between the Michaelis constants of the ol-glucosidase and behavioural thresholds of the sugar receptor of 1845
1846 ICHIROSHIMA~A, Axlo SHIRA~~HI, HIROMA~A KIJIMA,ANDHIROMICHIMORITA the fly, no direct proof supporting his working hypothesis has yet been given (cf. AMAKAWA et al., 1972). The insect sugar receptor, however, has some advantages for the study of the receptor substance compared with other chemoreceptors such as pharmacological receptors. To find a clue to the receptor substance, several treatments such as modification, labelling, digestion, solubilization, immunological test and so on, must be aimed, first of all, at the exact region of the receptor in question. In contrast to the pharmacological receptors, the contact chemoreceptor organs of flies are hair-like sensilla of the basiconica type. There are two lumens, outer and inner, in the chemosensory hair. The chemosensory cells send dendrites up to the tip through the inner lumen of the hair. There, the terminals of the dendrites contact outside stimulant solutions through a small pore (less than 1 p in dia.) (LARSEN,1962; ADet al, 1965). Therefore, treatments can be primarily confined to a small region around the terminals of the dendrites (chemoreceptor membrane). Moreover, the impulse is initiated near the cell body at the base of the hair, and is propagated antidromically toward the hair tip as well as orthodromically to the central nervous system. The impulse frequency is proportional to the receptor potential in the stationary phase, while the shape of the impulse reflects the site of impulse initiation and dendritic impulse conduction (MORITA, 1959; WOLBARSHTand HANSON, 1965 ; MORITA AND YAMASHITA,1966). From these characteristics it is possible to determine which region is affected by the experimental treatment. In many species of flies, electrophysiological investigations have revealed that four dendrites of the sensory cells in a single labellar hair correspond to sugar, water, salt receptors, and the ‘second salt receptor’, respectively (HODGSONand ROEDER, 1959; EVANS and MELLON, 1962; STEIN-T, 1965; DETHIER and HANSON, 1968). In order to pursue the direct study of the structure and the substance of the receptor, we can adopt a technique of chemical modification, one of the most powerful methods popular in protein chemistry. By behavioural assay KOYAMAand KURIHARA(1971) examined the effects of several reagents on the taste response of the fleshfly, which react with specific amino acid residue in a protein molecule. Using electrophysiological methods, we worked on the effects of the sulphhydryl reagent, p-chloromercuribenzoate (PCMB), on the response of the sugar receptor to sucrose and the effect of some other regaents on it. We will report here the results and will discuss the character of the depression. MATERIALS AND METHODS The fleshfIy, Boettcherisca pmgrina, 4 to 6 days old was used in the experiments. The chemosensory hairs used were of the largest type and located at the outer margin of the labellum. A side wall recording was employed (MORITA, 1959; MORITA and YAMASHITA,1959). An isolated head was mounted on a piece of platinum wire which served as an indifferent electrode and a glass microcapillary
EFFECTS OF SULPHHYDRYL REAGENTS ON THE FLESHFLY
1847
electrode, filled with Waterhouse’s saline (BUCK, 1953), was impaled into the outer lumen of the sensory hair, while stimulus solutions were applied to the tip of the hair with another glass capillary. As the impulse frequency can be considered to be proportional to the receptor potential in the stationary phase (MORITA and YAMASHITA,1966), the magnitude of response was defined as the number of impulses during a period from 0.15 to 0.35 set after beginning the stimulus. The duration of stimulation was less than O-5 sec. The usual interval between stimuli by sucrose solutions was about 3 min unless otherwise stated. The ambient temperature in the course of the experiments was 23 + O*S”C. Relative humidities of the experimental room were maintained at 62 to So% throughout this work, and did not change more than 5% during any series of the experiments. The treatment of the receptor with sulphhydryl reagents was performed in the same way as in the stimulation using a glass capillary filled with the solutions. All solutions for the treatment were made up in l/15 M phosphate buffer (pH 7*4), and left to stand for 30 min at room temperature (23°C) before use. A stimulus substance was dissolved in redistilled water. PCMB dissolved in 1 ml of 1 N NaOH was neutraliied with 1 N HCI and then diluted with phosphate buffer (pH 7.4). PCMB was used within half a day after being dissolved and stocked at 4”C, since it was unstable in the solution, and its effect was decreased over half a day. PCMB and L-cystein were purchased from Sigma Chemical Co. (St Louis, MO.). N-Ethyhnaleimide (NEM), 1,4-dithiotbreitol (D’IT), and kmercaptoethanol were of a special grade from Wako Chemical Industries Ltd, Japan.
Locusofd?7tgac~
RESULTS
The response of the labellar sugar receptor of the fleshfly to 0.1 M sucrose was depressed after treatment with O-5 mM PCMB. Fig. l(A) shows the record of the control response of the sugar receptor to 0.1 M sucrose before PCMB treatment. The shape of the impulse is biphasic, namely the impulses are initially positive, then abruptly become negative, and then return to the base line. Fig. l(B) shows the record in the stimulation of the sugar receptor by 0.1 M sucrose after treatment with 1 mM PCMB for 5 min. The depression due to PCMB was easily recovered by treatment with thiol compounds such as 5 n&I p-mercaptoethanol and 1OmM L-cystein. Fig. l(C) shows the record of the response to 0.1 M sucrose after treatment with 10 mM L-cystein for 3 min. The recovery was almost complete compared with the control response. After PCMB treatment the impulse frequency was clearly depressed in comparison with the control response, but no change was seen in the shape of the impulse. It may therefore be concluded that PCMB affected neither impulse initiation nor dendritic impulse conduction, but affected the primary processes of chemoreception occurrin g in the receptor membrane at the hair tip. This was true in all the results reported here regarding chemical modifications.
1848 ICHIROSNIMADA,AKIO SHIRAISHI, HIROMA~A KIJIMA,ANDHIROMICHI MORITA
ImVl 100 m SW
FIG. 1. Records of control, depression, and recovery experiments. (a) Controi response to 0.1 M sucrose before treatment with O-5mM PCMB. (b) Response to O-1 M sucrose after PCMB treatment. Cc) Response to 0.1 M sucrose after recovery by 10 mM L-cystein.
The fleets of FCMB on the sugar receptor Treatment of the hair with 05 mM PCMB in l/15 M phosphate buffer (pH 7.4) for 5 to 7 mm reduced subsequent responses of the sugar receptor to O-1 M sucrose to 30.4 per cent + 11-O (n = 13, where n is the number of experiments) of the control response. Duration of the PCMB treatment was critical. The depression after PCMB treatment for 3 min was about 40 to 60 per cent and the receptor recovered spontaneously after 30 min, while treatment for more than 10 min gave mostly 100 per cent depression without spontaneous recovery. Phosphate buffer (l/IS M) alone had no effect on the sugar receptor even if applied for more than 10 min. Excitability
of the salt receptor was also depressed, but the water receptor
was usually not affected by PCMB treatment.
PCMB itself evoked no impulse discharge but it depressed the spontaneous in the salt receptor within 30 set from the start of the treatment. After a long PCMB treatment, for more than 10 min, high frequency discharge of impulses in the salt receptor often appeared, continuing for some time before an abrupt cessation and leaving no spontaneous impulses. Phosphate buffer (l/15 M) itself evoked no response of the salt receptor. dis&arge
Recovery by ~mercaptoethanol and t-cysteirz The depression of the sugar receptor was reversed rapidly by treatment with either 5 mM p-mercaptoethanol or 10 mM L-cystein in the phosphate buffer at pH 7.4 for 3 mm (n = 13 and 6, respectively). The recovery in both reagents was almost complete. After the recovery in cystein, however, a discharge of impulses
EFF?XTS OF SULPHHYDRYL
1849
REAGENTS ON THE FLESHFLY
P: 0.5 mM PCMB 8: VI5 M phosphate
P
0
-3 0
20
buffer
I
L
40
60
min
FIG. 2. Course of depression by PCMB treatment. The figure is the tracing of the record of a single experiment. All stimulations are performed with 0.1 M sucrose solution throughout this experiment. P, treatment with O-5 mM PCMB (PH 7.4); B, treatment with & M phosphate buEer (pH 7.4). Length of lines above P and B indicates the time of treatment.
R: Woterhouee’s roiine M: 5mM fl- mercoptwthonol
20 t
(0)
C: 10 mM’ L-cysiein
c
P 0
P 20
T 40
60
min.
FIG. 3. Recovery by fl-mercaptoethanol and L-cystein from the depression due to PCMB. (a) P, 0.5 m.M PCMB; R, Waterhouse’s saline; M, 5 mM j?mercaptoethanol (pH 7.4). (b) The same as in (a), but C indicates treatment with 10 mM L-cystein (pH 7.4). Other details are the same as in Fig. 2.
1850 ICHIRO SHIMADA, AXIO SHIRAISHI, HIROMASA
KIJIMA,ANDHIROMICWI MORITA
often continued after the end of stimulation for a few hundred msec. Even when the depression was 100 per cent, it was often reversed by the treatment. This was not the case when PCMB treatment was so prolonged as to evoke high frequency impulses in the salt receptor (see above). Under these conditions the sugar receptor also appeared to have been seriously injured and was irreversibly depressed. The ej$ect of PCMB treatment on the response-concentration relationship in the stimulation by sucrose According to BEIDLER(1954) and MORITA (1969), the magnitude of response (R) is related to the concentration of taste stimulus (C) by the equation where R,,, is the maximum response magnitude and Kb is the concentration of the stimulant, at which the magnitude of response is one-half of the maximum. The equation indicates a straight line relationship with slope, l/G, and intercept on the abscissa of - Kb. It has been shown for the chemosensory hair of the largest type in the fleshfly labellum that the response of the sugar receptor to sucrose is described by the equation mentioned above (MORITA and YAMAGIITA, 1966). The effects of PCMB treatment on the relationship between C/R and C are shown in Fig. 4 compared with those before the treatment. Each hair was
J x
./
i
Molar
concmtration,
0
04
0.2
C
FIG. 4. &idler’s plot of the reaponae to sucrose before and after treatment with 05 mM PCMB for 5 min. Each graph indicates the result of each independent series of experiments. 0 -0,
before treatment; x -
x , after treatment.
EFFECTS
OF SULPHHYDRYL.
RRAGRNTS
ON
THE
1851
FLRMFL.Y
stimulated first with a series of concentrations of sucrose, then treated with 1 mM PCMB (for 5 min), washed with buffer, and finally stimulated with a second series of concentrations of sucrose. Repeated stimulation with sucrose of 0.5 M or higher concentrations sometimes made subsequent response unstable and therefore the range of concentration was limited below 0.2 M. Sometimes a high concentration (1 M) was tested only at the end of the second series. As shown in Fig. 4, the main effect of PCMB treatment was rather a decrease of R,,, than an increase of Kb. This tendency was observed in six examples out of nine, though values of & and Kb were fairly variable. PCMB as an inhibitor PCMB mixed in sucrose solutions inhibited the response of sugar receptor to sucrose. The interval between stimuli was about 3 min. The control response to 0.1 M sucrose was found to be unchanged in the lapse of interval time after the test with stimuli mixed with 0.5 mM PCMB whose duration was less than 0.5 sec. Thus, the modification effect of PCMB mixed in sucrose solutions, treated for O-5 set, was negligible after the interval of 3 min during which depression with the treatment, if any, might have been recovered. Fig. 5 shows that the type of inhibition was rather non-competitive. R, was decreased while K, was unchanged (n = 9).
2
0
4x-
. Sucrose mixed with PCMB
e
Sucrose only
z 9 E ‘T 2 I.0 %? x z u 001
O-05
0.1
Molar concentration,
C
FIG. 5. Beidler’s plot in stimulations by sucrose and mixed with PCMB. sucrose only (pH 7.4); l +, sucrose mixed with 0.5 mM PCMB
O(pH 7.::
The ejfect of NEM on the sugar receptor N-Ethylmaleimide had no remarkable effect on the excitability of the sugar receptor. Application of 4 mM NEM (pH 7.0) for 10 min showed no depression (n = 2). Treatment with 4 mM NEM for 15 min and 40 mM NEM for 15 min gave a temporary depression of the response to sucrose (a = 4 and 5, respectively) (Fig. 6). The depression was recovered spontaneously to the control level within
1852 ICHIROSHIMADA, AKIO SHIRAISHI, HIROMASA KIJIMA,ANDHIROMICHI MORITA
30 min. The phosphate buffer itself showed a slight temporary depression after 15 min treatment. Therefore, NEM gave a very weak depression in contrast to PCMB. 20-
1 40
I 20 min
I 0
0
FIG. 6. The effect of NEM on the sugar receptor. N, 4 mM NEM treatment (pH 7.0) for 15 min. Other detaila are the ssme as in Fig. 2.
The eflect of DTT m the sugar receptor The depression due to 1,4_dithiothreitol (DTT) also recovered spontaneously (Fig. 7). Immediately after the application of 10 mM DTT for 20 min a distinguishable depression could be observed, but it was recovered almost to the control level within 30 min (n = 3). As the phosphate buffer also gave a temporary depression, the depression by DTT was fairly weak.
0
I
I
20
0
,
40
min
FIG. 7. The. effect of DTT treatment (pH 7.4) for 15 min. Other details are the same as in Fig. 2. DISCUSSION
PCMB treatment resulted in a decrease of the depolarization of the sugar receptor membrane stimulated by sucrose. This effect was due to the reaction of PCMB with some component of the cell membrane of the sugar receptor at
RFlmcrs
OF -RYL
RRAOENTS ON THE FLESHFLY
1853
the hair tip. This is supported by the results shown in Fig. 1. That is, little
change is seen in the shape of the impulse after PCMB treatment which resulted in a marked depression in the impulse frequency. PCMB will first react with available sulphhydryl groups for which it is highly specific (BOYER, 1954). The depression due to PCMB was quite different in several points from that by heavy metals which were considered to be less specific. For instance, treatment with 1 n&I HgCl,, for only 0.5 set, resulted in the marked depression in the sugar receptor (unpublished data), while PCMB treatment did not show any depression for such a short time. The bond formed by PCMB in our experiments must dissociate very slowly since there is no great change in the depression after washing with the phosphate buffer for 10 min. Complete reversibility of the depression due to PCMB treatment by sulphhydryl compounds does indicate also that the site of the reaction is accessible to lipophobic molecules such as cystein, and that the components with which PCMB reacts are not irreversibly denatured. By behavioural assay, KOYAMA and KURIHARA(1971) showed the complete elimination of the taste response in the fleshfly by PCMB treatment for 1 hr. But with such a long treatment PCMB might penetrate into the cell and react with the proteins other than those at the cell surface of the receptor system. This is suggested by our observation that the depression of single hair discharge after PCMB treatment for such a long time could not be reversed by treatment with either p-mercaptoethanol or by cystein. The depression by PCMB treatment is characterized by a decrease in R, rather than an increase in & (Fig. 4). Meanwhile, the type of inhibition by PCMB mixed in the sucrose solution is non-competitive (Fig. 5). If we assume two steps in the reaction of PCMB, namely, PCMB is first adsorbed to the site of action (a reversible complex formation) and then makes bonds with the sulphhydryl groups at the site (bond formation), Fig. 5 represents the adsorption step because the control response to O-1M sucrose did not change before and after the test with the sucrose solution mixed with PCMB. On the other hand, Fig. 4 shows the state after PCMB makes bonds with the sulphhydryl groups at the site of action. At the adsorption step PCMB behaves as a non-competitive inhibitor (Fig. 5), so it is adsorbed to a differentsite from the sucrose binding site (receptor site). If two steps of the reaction occur at the same site of action, the effect of bond formation of PCMB may result in changed R,,, rather than &,. This agrees well with Fig. 4. It may, therefore, be suggested that PCMB reacta at a difIerent site from the receptor site and does not inhibit the specific interaction between sucrose and the sugar receptor site, but it may block a change in the receptor membrane permeability which immediately follows the specific interaction. However, an important question still remains as to whether the I& value in this case may or may not be simply related to the receptor-sucrose complex dissociation constant (MORITA,1969). We did not examine the effect of sucrose of a high concentration on PCMB treatment (receptor protection) though it seemed promising to determine the site of action of PCMB more to a certainty. It is because the application of sucrose of a high concentration for a fairly long time, for example,
1854 ICH~RO SHIMADA, AKIO SHIRAISHI,HIROMASAKIJIMA, AM) HIROMICHIMORITA 5 rnin, requires a long disadaptation time and thus reduces a subsequent response to sucrose, so that the effect of sucrose on PCMB treatment will become indistinguishable. In contrast to PCMB, NEM treatment had no remarkable effect on the sugar receptor. NEM was found to be less reactive towards certain sulphhydryl groups 1962). This might account for the weak than PCMB (cf. KATZ and MOMMAERTS, effect of NEM on the sugar receptor. DTT is a reagent designed to reduce disulphides but showed no great effect on the excitability of the sugar receptor, too. Therefore, disulphide bonds may not play an important role in the stimulation of the sugar receptor. These results, indicating that the presence of sulphhydryl groups in the sugar receptor system of the fleshfly, support the view that at least one of the components of this system is a protein. Acknowledgements-The authors thank Miss E. Kadota for preparing this manuscript and Drs. Kurihara and Jakinovich for sending their preprints. This work was supported in part by Scientific Research Fund from the Ministry of Education of Japan. REFERENCES ADAMSJ. R., HOLBERTP. E., and FORGASHA. J. (1965) Electron microscopy of the contact chemoreceptors of the stable fly, Stomoxys calitrans (Diptera: Muscidae). Ann. ent. SOC. Am. 58, 909-917. AMAKAWA T., KAWABATA K., KIJIMA H., and MORITA H. (1972) Isozymes of a-glucosidase in the proboscis and legs of flies. g. Inrsct PhysioZ. 18, 541-553. BEIDLER L. M. (1954) A theory of taste stimulation. g. gen. Physiol. 38, 133-139. BOYERP. D. (1954) Spectrophotometric study of the reaction of protein sulfhydryl groups with organic mercurials. g. Am. them. Sot. 76,4331-4337. BUCKJ. B. (1953) Physical properties and chemical composition of insect blood. In Insect Physiology (Ed. by K. D. ROBDER),147. Wiley, New York. DASTOLIF. R. and PRICE S. (1966) Sweet sensitive protein from bovine taste buds: isolation and assay. S&nce, Wash. 154,905-907. DETHIERV. G. (1955) The physiology and histology of the contact chemoreceptors of the blowfly. Quart. Z&v. Biol. 30, 348-371. DETHIBRV. G. and HANSoN F. E. (1968) Electrophysiological responses of the chemoreceptors of the blowfly to sodium salts of fatty acids. Proc. natn. Acad. Sci. U.S.A. 60, 1296-1303. EVANSD. R. and MELLON D., Jr. (1962) Electrophysiological studies of a water receptor associated with the taste sensilla of the blowtIy. J. gen. Physiol. 45, 487-500. HANAMORI T., SHIRAI~HIA., KIJIMA H., and MORITA H. (1971) Stimulation of labehar sugar receptor of the fleshfly by glucosides. 2. vergl. Physiol. 76, 11 S-124. HANSENK. (1%9) The mechanism of insect sugar reception, a biochemical investigation. In O&zction and Taste 3, 382. (Ed. by C. PFAFFMANN.) Rockefeller University Press, New York. HODGKINE. S. and ROBDERK. D. (1956) Electrophysiological studies of arthropod chemoreception. J. cell. camp. Physiol. 48, 51-75. JAKINO~ICHW., Jr., GOLDSTEIN I. J., VON BAUMGARTEN R. J., and AGFUNOPPB. W. (1971) Sugar receptor speci&ity in the fleshfIy, Sarcophuga bullata. Brain Res. 35, 369-378. KAFUN A. and BAWIZB~E. (1966). Effects of blocking sulfhydryl groups and of reducing disulfide bonds of the acetylcholine-activated permeability system of the electroplax. Biochim. biophys. Acto 126, 525-535.
EFFECTS
OF SuPHI-lYnRYLREAGENTS ON THE =?rFLY
KATZ A. M. and MOMMAERTS W. F. H. N. (1962) Sulfhydryl groups of actin.
1855 Biochiw.
biophys. Actu 65, 82-92. KOYAMAN. and K~RIHARAK. (1971) Modification by chemical reagents of proteins in the gustatory and olfactory organs of the Aeahfly and cockroach. J. Insect Physiol. 17, 2435-2440. LARSENJ. R. (1962) The fine structure of the labeilar chemosensory hairs of the blowfly, Phwmia regina. J. Insect Physiol. 8, 683-691. MORITA H. (1959) Initiation of spike potentials in contact chemosenaory hairs of insectIII. D.C. stimulation and generator potential of labellar chemoreceptor of Culliphoru.
J. cell. camp. Physiol. 54,189-204. MORXTAH. (1969) Electrical signs of taste receptor activity. In O&r&m and Taste 3, 370. .) Rockefeller University Press, New York. (Ed. by C. PFAFFMANN MOFUTAH. and YAMASHITAS. (1959) Generator potential of insect chemoreceptor. Stience, wash. l30, 922. MORITA H. and YAMASHITAS. (1966) Further studies on the receptor potential of chemoreceptors of the blowfly. Mem. Fat. Sci. Kyuahu Univ. Ser. E. (Biol.) 4,123-135. PARXSIM., RNAS E., and DE ROBERTISE. (1971) Conductance in lipidic membranes containing a ploteolipid from a Electrophorus. Science, Wush. 172, 56-57. STBINHARDTFL A. (1965) Cation and anion stimulation of the electrolyte receptors of the blowfly, Phonnia regina. Am. Zool. 5, 651-652. WOLBARSHTM. L. and HANSONF. E. (1965) Electrical activity in the chemoreceptors of the blowfly-III. Dendritic action potentials. J. gm. Physiol. 48, 673-683.