taste trans duct ion mechanism

8/8/2019 Taste Trans Duct Ion Mechanism

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T a s t e T r a n s d u c t io n M e c h a n i s mSimilar Effects of VariousM odifications of G ustatory Receptorson Neu ral R esponses to C hem ical andElectrical Stimulation in The FrogM A K O T O K A S H I W A Y A N A G I , K I Y O N O R I Y O S H I I ,Y O N O S U K E K O B A T A K E , an d K E N Z O K U R I H A R AFro m t h e F ac u l t y o f Ph a rm ac e u t i c a l S c ie n ce s, H o k k a i d o U n i v e rs it y , S ap p o ro 0 6 0 , J ap a n

A B S T R A C T R e s p o n s e s i n t h e f ro g g l o s s o p h a r y n g e a l n e r v e in d u c d b y e l e c tr i c als t i m u l a t i o n o f t h e t o n g u e w e r e c o m p a r e d w i t h t h o s e in d u c e d b y c h e m i c a ls t i m u l i u n d e r v a r i o u s c o n d i t i o n s . ( a ) A n o d a l s t i m u l a t i o n i n d u c e d m u c h l a r g e rr e s p o ns e s t h a n c a t h o d a l s t i m u l a t i o n , a n d a n o d a l s t i m u l a t i o n o f th e t o n g u ea d a p t e d t o 5 m M M g C I 2 p r o d u c e d m u c h l a r g e r re s p o ns e s t h a n s t i m u l a t i o n w i t ht h e t o n g u e a d a p t e d t o 1 0 m M N a C I a t e q u a l c u r r e n t i n t e n s it i es , as c h e m i c a ls t i m u l a t i o n w i t h M g C I 2 p r o d u c e d m u c h l a r g e r r e s p on s e s t h a n s t i m u l a t i o n w i t hN a C I a t e q u a l c o n c e n t r a t i o n . (b ) T h e e n h a n s i v e a n d s u p p r e s s iv e e f fe c t s o f 8-a n i l i n o - l - n a p h t h a l e n e s u l f o n a t e , N i C I 2 , a n d u r a n y l a c e t a t e o n t h e r e s p o n s e s t oa n o d a l c u r r e n t w e r e s i m i l a r t o t h os e o n t h e r e s p o n s e s to c h e m i c a l s t i m u l a t i o n .(c) A n o d a l s t i m u l a t i o n o f th e t o n g u e a d a p t e d t o 5 0 m M C a C I 2 r e s u l te d i n al a r g e r e s p o n s e , w h e r e a s a p p l i c a t i o n o f 1 M C aC 1 2 t o t h e t o n g u e a d a p t e d t o 50m M C a C I 2 p r o d u c e d o n l y a s m a l l r e s po n s e . T h is , t o g e t h e r w i t h t h e o r e t i c a lc o n s i d e r at i o n s , s u g g e s te d t h a t t h e a c c u m u l a t i o n o f s a lt s o n t h e t o n g u e s u r f a c e i sn o t t h e c a u s e o f t h e g e n e r a t i o n o f th e r e s p o n s e t o a n o d a l c u r r e n t . ( d) C a t h o d a lc u r r e n t s u p p r e s s e d t h e r e sp o n s e s i n d u c e d b y 1 m M C a C I 2 , 0 .3 M e t h a n o l , a n dd i s t i ll e d w a t e r . (e ) T h e a d d i t i o n o f E G T A o r C a - c h a n n e l b l o c k e r s (C d C1 2 a n dv e r a p a m i l ) t o t h e p e r f u s i n g s o l u t i o n f o r t h e l i n g u a l a r t e r y r e v e r s i b l y s u p p r e s s e db o t h t h e r e s po n s e s to c h e m i c a l s t i m u l u s ( N a C I ) a n d t o a n o d a l c u r r e n t w i t h 1 0m M N a C I . ( f ) W e a s s u m e f r o m t h e re s u lt s o b t a i n e d t h a t e l e c t ri c a l c u r r e n t f r o mt h e m i c r o v i l l u s m e m b r a n e o f a t a s t e ce l l t o t h e s y n a p t i c a r e a s u p p l i e d b y a n o d a ls t i m u l a t i o n o r i n d u c e d b y c h e m i c a l s t im u l a t i o n a c t i v a t e s t he v o l t a g e - d e p e n d e n tC a c h a n n e l a t t h e s y n a p t i c a r e a .I N T R O D U C T I O N

I t h a s b e e n k n o w n s i n c e t h e t i m e o f V o l t a t h a t e l e c t ri c a l s t i m u l a t i o n o f t h eh u m a n t o n g u e e v o k e s t a s t e s e n s a t i o n . N u m e r o u s s t u d i es o n " e l e c t r ic a l t a s t e "p e r f o r m e d p s y c h o p h y s i c a l l y r e v e a l e d t h e c h a r a c t e r i s ti c s o f e l e c t ri c a l ta s t eJ . GE N. PHYSXOL.9 Th e R ockefel ler Univers i ty P ress 9 0022-1295/81/09/0259/17 $1 .00 259Volu me 78 Sep te mb er 1981 259-275

Published September 1, 1981


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260 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 78 9 1981(Bujas, 1971 and 1977). To explain the psychophysical data, a n umb er ofhypotheses as described below have been a dvan ced (Bujas, 1971 and 1977):(a) Electrical taste is the result of an adequate stimulation of taste receptorsby some specific prod ucts of the electrolysis of the saliva. (b) Electrical tas te isinduced by direct stimulation of the gustatory nerve with electric current. (c)The current directly provokes taste receptors. Despite numerous studies, it isnot yet kno wn which hyp othesis is correct. For fur ther studies on a mechan ismof electrical taste, an electrophysiological techni que seems to be a useful tool.However, only a limited n umb er o f electrophysiological works (Pfaffman,1941; S mith a nd Bealer, 1975; Pfa ffm ann and Pritchar d, 1980) have beendone and no systematic studies have been carried out as far as we know.

In a previous paper (Aiuchi et al., 1976), we proposed the followinghypothesis for a taste transdu ction me chanis m: adso rption of chemical stimulion the microvillus mem bra ne o f a taste cell depolarizes the membr anepotent ial at the microvillus membr ane, which induces an electric curren t fromthe microvilli to the syna ptic area of the taste cell to produ ce nerve impulses.If this hypothesis is correct, it would be expected that an electric current fromthe tongue surface to the back side of the tongue elicits gustatory responsessimilar to those induced by chemical stimulation. Thus, electrical stimulationseems to be a useful tool for elucidating the taste transduction mechanism.In this study, the function of the frog gustatory receptors was modified byvarious reagents and the effects of the modifications on the glossopharyngealnerve responses to chemical and electrical stimulation were compared. Wefound that various modifications of electrical and chemical stimulation pro-duce responses that are quite similar to each other. In addition, electricalstimulation of the tongue was performed while the lingual artery was perfusedwith artificial solutions containing Ca-channel blockers to inhibit the releaseof a chemical transmitter from taste cells; and we found that responses toelectrical stimulation, as well as those to chemical stimulation, are reversiblysuppressed under this condition. Discussion is made on a taste transductionmecha nism as well as a mechan ism o f electrical taste.

M A T E R I A L S A N D M E T H O D S

Animal sAdult bullfrogs, Ran a catesbeiana,weighing 260-300 g, were used in these experiments.For the perfusion of the lingual artery, frogs obtained in the winter were used, sincethey exhibited stable responses under perfusing conditions at this time.

Recording o f G ustatory N erve Act iv it iesThe responses to chemical stimuli and electric current were recorded from theglossopharyngeal nerves. The method of the preparation of the glossopharyngealnerves and recording of the nerve activity were the same as those described in aprevious paper (Kashiwagura et al., 1980). The nerve impulses were integrated withan electronic integrator with time constant of 0.3 s.



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KASHIWAYANAGIET AL. TasteTransduttion Mechanism in the Frog 261Chemical Stimulation

C h e m i c a l s t i m u l a t i o n w a s c a r r i e d o u t e s s en t i a l ly a s d e s c r i b e d i n a p r e v i o u s p a p e r( K a m o e t al ., 1 9 78 ). S t i m u l a t i n g s o l u t i o n s w e r e a p p l i e d t o t h e t o n g u e w i t h a f l o w r a t eo f 2 m l / s a f t e r 2 0 m M N a C 1 h a d p e r f u se d t h e t o n g u e w i t h t h e s a m e f l o w r a t e .

Electrical StimulationE l e c t ri c a l s t i m u l a t i o n i n m o s t e x p e r i m e n t s w a s c a r r i e d o u t b y s u p p l y i n g a c o n s t a n tc u r r e n t ( 0. 7 m A ) t o th e f r o g t o n g u e w i t h a n e l e c t r o ni c s t i m u l a t o r ( M S E - 3 R ; N i h o nK o d e n K o g y o , T o k y o ) a n d a n i s o la t in g un i t ( M S E - J M ; N i h o n K o d e n K o g y o ) . T h ef r o g t o n g u e w a s p l a c e d i n a c h a m b e r f i l le d w i t h a n a d a p t i n g s o l u t io n . O n e p l a t i n u me l e c t r o d e ( e l e c t r o d e I ) f o r e l e c t r i ca l s t i m u l a t i o n w a s i m m e r s e d i n a n a d a p t i n g s o l u t i o na n d a n o t h e r p l a t i n u m e l e c t ro d e ( e l ec t ro d e 2 ) w a s p l a c e d o n t h e b a c k s i de o f t h e r o o tp a r t o f t h e t o n g u e w h e r e t h e t o n g u e w a s n o t i m m e r s e d i n a s o l u ti o n , S i m i l a r r e su l tsw e r e o b t a i n e d w h e n e l e c tr o d e 1 w a s p l a c e d i n d i re c t c o n t a c t w i t h t h e t o n g u e s u r f a c ei n s t e a d o f i m m e r s i n g i t i n a n a d a p t i n g s o l u ti o n . W h e n a n a d a p t i n g s o l u ti o n o f l o wc o n d u c t a n c e s u c h a s d i s t i ll e d w a t e r w a s u s e d , e l e c t r o d e 1 w a s p l a c e d i n d i re c t c o n t a c tw i t h t h e t o n g u e s u r f a c e . E l e c t r i c c u r r e n t t h a t f l o w e d f r o m e l e c t r o d e 1 t o e l e c t r o d e 2a n d t h a t f r o m e l e c t r o d e 2 t o el e c t ro d e 1 a r e r e f e r re d t o a s a n o d a l a n d c a t h o d a Ic u r r e n t , r e s p e c t i v e l y .

8-Anilino- l-naphthalenesulfonate ( A N S) TreatmentT h e t r e a t m e n t o f t h e f r o g t o n g u e w i t h A N S w a s c a r ri e d o u t a s d es c r ib e d i n a p r e v i o usp a p e r ( K a s h i w a g u r a e t a l ., 1 97 7): T h e t o n g u e w a s i n c u b a t e d i n 1 m M A N S s o l u ti o na t 5 ~ f o r 2 m i n a n d t h e A N S s o l u t io n w a s w a s h e d a w a y b y f lo w i n g 2 0 m M N a C Is o l u t i o n a t 2 0 ~ o n t h e s u r f a c e o f t h e t o n g u e f o r 2 r a i n w i t h a f l o w r a t e o f 2 m l / s . F o rc h e m i c a l s t i m u l a t i o n , a s t i m u l a t i n g s o l u t i o n ( 0 .4 M N a C I s o l u t i o n ) w a s a p p l i e d t o t h et o n g u e a t t h e s a m e f l o w r a t e . F o r e l e c t r i ca l s t i m u l a t i o n , a n o d a l c u r r e n t w a s s u p p l i e dt o t h e t o n g u e a d a p t e d t o 1 0 m M N a C I s o l u t io n a f t e r t h e A N S t r e a t m e n t .

Effect o f Uranyl AcetateF o r c h e m i c a l s t i m u l a t i o n , t h e t o n g u e w a s a d a p t e d t o 2 0 - r a M N a C I s o l u t io n c o n t a i n i n gu r a n y l a c e t a t e o f v a r i o u s c o n c e n t r a t i o n s f o r 2 r a i n a n d s t i m u l a t i n g s o l u ti o n s c o n t a i n i n gu r a n y l a c e t a t e o f t h e s a m e c o n c e n t r a t i o n a s t h e a d a p t i n g s o l u t i o n w e r e a p p l ie d . F o re l e ct r ic a l s t i m u l a t i o n , a n o d a l c u r r e n t w a s s u p p l ie d t o t h e t o n g u e a d a p t e d t o 1 0 - r a mN a C I s o l u ti o n o r 5 - m M M g G l ~ s o l u ti o n c o n t a i n i n g u r a n y l a c e t a t e o f v a r io u s c o n c e n -t r a t i o n s .

Perfusion of the Lingual ArteTyP e r f u s i o n o f t h e l i n g u a l a r t e r y w a s c a r r i e d o u t e s s e n t i a l ly a s d e s c r i b e d b y M o r i m o t oa n d S a t e ( 19 7 5) : A p o l y e t h y l e n e t u b e w a s c a n n u l a t e d i n t o t h e l i n g ua l a r t e r y a n dR i n g e r ' s s o lu t i o n ( 11 2 m M N a C I , 3 .4 m M K C I , 0 .2 m M C a C I 2 , 3 .6 m M M g S O 4 , 2 . 5m M N a H C O 3 , p H 7 .2 ) c o n t a i n i n g 1 0 U o f s o d i u m h e p a r i n w a s p e rf u s e d t h r o u g h t h et u b e i n t o t h e a r t e r y b y u s i n g a p e r i s ta l t i c p u m p ( S J - I 2 1 5 ; M i t s u m i S c i e n t i f ic , I n c. ,T o k y o ) a t a r a t e o f 0 .1 m l / m i n . T h e p e r f u s e d s ol u t io n w a s d r a i n e d t h r o u g h t h e v e i na t t h e b o t t o m o f t h e t o n g u e . D u r i n g p e r f u s i n g , th e r e s p o n se t o 1 m M C a C I 2 w a sm e a s u r e d a s a r e f er e n c e re s p on s e . A f t e r b l o o d w a s c o m p l e t e l y e l i m i n a t e d f r o m t h ev e i n a n d s t i m u l a t i o n b y 1 m M C a C I 2 c a m e t o g i v e a c o n s t a n t r e s p o ns e , t he c o n t r o lr e s p o n se w a s r e c o rd e d . A d d i t i o n o f C a - c h a n n e l b l o c ke r s w a s p e r f o r m e d b y s w i t c h in gt h e p e r f u s i n g s o l u t i o n t o t h e R i n g e r ' s s o l u t i o n c o n t a i n i n g t h e b l o c k e r s .



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262 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y 9 V O L U M E 7 8 9 1 9 8 1Chemicals

ANS was purchased f rom Eas tman Kodak Co . , Roches t e r , N . Y. and e thy l eng lyco l -b i s - ( f l- aminoe thy l e the r )-N,N ' , - t e t r aaee ti c ac id (EGTA) was purchased f rom D oj indoL a b o r a t o r y , K u m a m o t o , J a p a n . U r a n y l a c e t a t e a n d s o d i u m h e p a r in w e r e p u r c h a s e df r o m W a k o P u r e C h e m i c a l C o . , O s a k a , J a p a n . V e r a p a m i l w a s k i n d l y s u p p l i e d b yE i sa i Co . , Tokyo .Al l t he expe r imen t s we re ca r r ied o u t a t 200C.R E S U L T S

A n o d a l s t i m u l a t io n o f f r o g t o n g u e t h a t w a s r i ns e d t h o r o u g h l y w i t h d i s ti ll e dw a t e r d i d n o t i n c r e a s e a c ti v i ti e s o f t h e g l o s s o p h a r y n g e a l n e r v e , b u t s t i m u l a t i o no f t h e t o n g u e a d a p t e d t o s a lt s o l u t i o n s g r e a t l y i n c r e a s e d t h e a c t iv i ti e s. F i g . 1 As h o w s t h e s u m m a t e d r e sp o n s es i n d u c e d b y a n o d a l c u r r e n t w h e n t h e t o n g u e i sa d a p t e d t o v a r i o u s sa l t s o l u t io n s . S o l u t i o n s o f 1 0 m M 1 :1 t y p e s al ts ( N a C l ,c h o l in e c h lo r id e , a n d t e t r a e t h y l a m m o n i u m c h lo r i de ) a n d o f 5 m M 2 :1 t y p es a l t ( M g C 1 2 ) w e r e c h o s e n a s a d a p t i n g s o l u t i o n s b e c a u s e t h e s e s a l t s e l i c i t o n l ys m a l l r e s p o n s e s i n t h e g l o s s o p h a r y n g e a l n e r v e a n d , m o r e o v e r , t h e r e s p o n s e sw e r e e a s i ly a d a p t e d t o t h e s p o n t a n e o u s l ev e l. A s s e e n f r o m t h e f i g u r e , t h em a g n i t u d e o f t h e r e s p o n s e s v a r i e d w i t h i o n s p e c ie s i n t h e a d a p t i n g s o l u t i o ne v e n t h o u g h t h e t o n g u e i s a d a p t e d t o s o l u t i o n s c o n t a i n i n g s a lt s t h a t a r ee l e c tr o c h e m i c a l ly e q u i v a l e n t . F o r e x a m p l e , t h e r e s p o n s e o f t h e t o n g u e a d a p t e dt o 5 m M M g C 12 is m u c h l a rg e r t h a n t h a t o f t h e t o n g u e a d a p t e d t o 1 0 m Ms a lt s o f m o n o v a l e n t c a t i o n s a t e q u a l c u r r e n t i n t e n s it y . T h e a v e r a g e r a t i o o ft h e m a g n i t u d e o f t h e r e sp o n s e w i t h 5 m M M g C I 2 to t h a t w i t h 1 0 m M N a C I ,w h i c h w a s o b s e r v e d w i t h e i g h t f r o gs , w a s 3 . 6 _ 0 . 8 fo r t h e p e a k r e s p o n s e a n d3 .8 _ 0 . 5 fo r th e r e s p o n s e 2 0 s a f t e r o n s e t o f s t i m u l a t i o n . T h i s t e n d e n c yc o i n c i d e s w i t h t h a t o f t h e r e s p o n s e s t o c h e m i c a l s t i m u l a t i o n w h e r e t h er e s p o n s e s t o N a C 1 a n d M g C I 2 a r e c o m p a r e d a t e q u a l e l e c t r o c h e m i c a l e q u i v -a l e n t : t h e m a g n i t u d e o f t h e r e s p o n s e to 0 . 2 M M g C I 2 is m u c h l a r g e r t h a n t h a tt o 0 . 4 M N a C I ( F ig . 1 B ) o r t h a t t o 0 .1 M M g C 1 2 is m u c h l a r g e r t h a n t h a t t o0 . 2 M N a C 1 . T h e a v e r a g e r a t i o o f t h e m a g n i t u d e o f t h e r e s p o n s e t o 0 . 2 MM g C 1 2 t o t h a t t o 0 . 4 M N a C I , w h i c h w a s o b s e r v e d w i t h s e v e n f r o g s, w a s 3 . 0 0 . 7 f o r t h e p e a k r e s p o n s e a n d 4 . 2 0 . 5 f o r t h e r e s p o n s e 2 0 s a f t e r o n s e t o fs t i m u l a t i o n . F i g . 1 C s h o w s t h e m a g n i t u d e o f t h e r e s p o n s e s t o a n o d a l a n dc a t h o d a l c u r r e n t o f v a r i o u s i n te n s i ti e s w h e n t h e t o n g u e is a d a p t e d t o 1 0 m MN a C I a n d 5 m M M g C 1 2. T h e r e s p o n s e s t o a n o d a l c u r r e n t i n c r e a s e w i t hi n c r e a s i n g c u r r e n t i n t e n s i t y a n d t h e r e s p o n s e s w i t h 5 m M M g C 1 2 a r e m u c hg r e a t e r t h a n w i t h 1 0 m M N a C I a t a ll in t e n si t ie s . T h e r e s p o n s e s t o c a t h o d a lc u r r e n t a r e m u c h l es s t h a n t o a n o d a l c u r r e n t . T h e c u r r e n t i n t e n s i t y r e q u i r e df o r i n d u c t i o n o f e l e c tr i c re s p o n s e s in t h e f r o g is m u c h h i g h e r t h a n t h a t i n t h er a t ( B u j a s , 1 9 7 1; P f a f f m a n n a n d P r i t c h a r d , 1 9 80 ). T h e f r o g t o n g u e is m u c hm o r e t e n d e r a n d c o n t a in s m o r e w a t e r t h a n t h e r a t to n g u e a n d h e n c e m o s tc u r r e n t a p p l i e d t o t h e f r o g t o n g u e m a y p a s s t h r o u g h t is s u e s o t h e r t h a n t a s t ece l l s .A s s h o w n i n a p r e v i o u s p a p e r ( K a s h i w a g u r a e t al ., 1 9 7 7 ), t r e a t m e n t o f t h ef r og to n g u e w i t h A N S l e a d t o g r ea t e n h a n c e m e n t o f t h e responses t o sa l t



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current (mA)F IG U RE l . (A a n d B ) S u m m a t e d r e s p o n s e s o f t h e f r o g g l o s s o p h a r y n g e a l n e r v et o a n o d a l c u r r e n t ( ,4 ) a n d c h e m i c a l s t i m u l i ( B ) . F o r e l e c t r ic a l s t i m u l a t i o n , t h et o n g u e w a s a d a p t e d t o 1 0 m M N a C 1 , 10 m M c h o l i n e c h l o r id e , 1 0 m Mt e t r a e t h y l a m m o n i u m c h l o r id e , ( T E A ) , a n d 5 m M M g C l z . B ar s a t th e b o t t o m o fe a c h r e c o r d r e p r e s e n t d u r a t i o n o f a p p l i c a t i o n o f a n o d a l c u r r e n t o r c h e m i c a ls t im u l i . ( C ) R e l a t i v e m a g n i t u d e o f th e p e a k r e s p o n se s t o e le c t ri c c u r r e n t a s af u n c t i o n o f c u r r e n t i n t e n s i t y . E a c h p o i n t i n t h e f i g u r e i s t h e a v e r a g e v a l u e o f t h ed a t a o b t a i n e d w i t h t h r e e f r o gs . C ), a n o d a l s t i m u l a t i o n o f t h e t o n g u e a d a p t e d t oI 0 m M N a C I ; I--1, a n o d a l s t i m u l a t i o n o f t h e t o n g u e a d a p t e d t o 5 m M M g C 12 ;0 , c a t h o d a l s t i m u l a t i on o f th e t o n g u e a d a p t e d t o 1 0 m M N a C I ; m , c a th o d a ls t i m u l a t i o n o f t h e t o n g u e a d a p t e d t o 5 m M M g C I 2 ; R e s p o n s e s ( R ) w e r ec a l c u l a t e d r e l a ti v e t o th e r e s p o n s e t o 1 . 1 - m A a n o d a l c u r r e n t w i t h 5 m M M g C 1 2.



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264 THE JOUR NAL OF GENER AL PHYSIOLOGY 9 VOLUME 78 9 1981

stimuli. Fig. 2A shows that a chemical response to 0.4 M NaCl is greatlyenhanced after ANS treatment. The ANS trea tment also greatly increased theresponse to anod al cur rent (Fig. 2 B) when the responses before and after ANStreatment are compared at equal current intensity.

The response to 100 mM NaC1 was greatly enhanced by the presence of 1mM NiCIz whereas that to 100 mM LiCI was only slightly increased (Kash-iwagur a et al., 1978). The electrical response of the t ongue a dap ted to 10 mMNaCI was also greatly enha nced by the presence of 1 mM NiCI2, whereas theresponse of the ton gue ada pte d to 10 mM LiCI was not pronounced . In Fig.3, the ma gni tud e of the response to 100 mM NaC1 and that to anodal curr entwith 10 mM NaC1 are plotte d against the NiC12 conce ntrat ion where responses(Fig. 3, R) are calculated relative to respective responses at 10 mM NiC12.Here, current intensity is fixed at 0.7 mA. Both responses to the chemicalstimulus an d to anoda l curren t are increased with an increase of NiC12

bA ) Chemicat$ t i mu ta t i o n

lo s

St imutat ing 04M N a Q 0./,M N aQso l u t i o n

B) ElectdcatSt imutat ion

E l e c E l e c .Adapt ing 10mlvl NaQ lOrn ld NaO_so l u t i o nFIGURE 2. Summa ted responses of the frog glossopharyngeal nerve to 0.4 MNaCI (A) and 0.7-mA anodal current with 10 mM NaC1 (B) as determinedbefore (a) and after (b) the tongue was treated with 1 mM ANS.

concentration. The response to anodal current is larger than that to thechemical stimulus in the low-concentration range of NiCI2; because anodalcurrent with 10 mM NaCI induces appreciable responses even in the absenceof NiC12, but 100 mM NaC1 indu ces onl y a very small response.The addition of uranyl acetate to a st imulating solution greatly affectedboth responses to chemical stimuli and anodal current. Fig. 4 shows themagni tude s of the responses to the chemical stimuli (0.4 M NaC1 and 0.2 MMgCI2) an d electric current o f consta nt inten sity (0.7 mA) as a function ofuranyl acetate concentration where the magni tude of each response in theabsence of uran yl acetat e is taken as a unit in the ordinate. Both curves forchemical responses to 0.4 M N aCl and electrical responses with 10 mM NaC1show a peak at -1 0 -s M urany l acetate, whereas both curves for chemicalresponses to 0.2 M MgC:I2 an d e lectrical responses with 5 mM MgCl2 show nopeak an d th e responses decrease monotoni call y with an increase of urany lacetate c oncen trati on >3 X 10 -8 M.



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KASHIWAYANAGI ET A L. Taste Transduction Mechanism in the Frog 265The above results indicate that the effects of various modifications of frog

gustatory receptors on the responses to anodal current are quite similar tothose on the chemical responses. There is a possibility that an electricalresponse is induced by salts accumulated on the tongue surface by iontopho-resis. This possibility was checked by the experiments shown in Fig. 5. Asshown in Fig. 5 A, the ma gni tud e of the response to CaC12 shows a max im umresponse at ~ 2 mM and decreases with a furt her increase of CaC12 concentra -tion. As expected fro m the above relation, ap plicatio n of 0.1 M and 0.5 MCaCI2 to the tongue adapted to 50 mM CaCI2 brought about no response.Fig. 5 B shows a typica l record wher e 1 M CaCI2 was applied, a t th e poi ntindicated by an arrow, to the tongue that h ad been ad apted to 50 mM CaC12,

Elec t r i ca l S t imu la t i on o1 .0 Chem ica l S t imu la t i on 9 ~ , , ) ~ - , - ~ " - 9/ / /

0.5 / /

/[ 9 I 1 Jo -7 -6 -5 -4 -3 -2l og [ N iC I2 ] (M)

FIGURE 3. Relative magnitude of the peak responses to 100 mM NaCI (I ) andto anodal current (O) as a function of logarithmic concentration of NiCla.Anodal current (0.7 mA) was supplied to the tongue adapted to 10 mM NaCIcontaining various concentrations of NiC12. Responses (R) were calculatedrelative to respective responses at 10 mM NiCI2. Each point in the figure is theaverage value of the data obtained with three frogs.indic atin g tha t 1 M CaCI~ brou ght a bout onl y a small response. On th e otherhan d, electrical stimulat ion of the ton gue ad apt ed to 50 mM CaC12 gave alarge response as shown in Fig. 5 C. This suggests that accu mula tion of saltson the tongu e surface is not the cause of generati on of the response to anodalcurrent. All results shown in Fig. 5 were confirmed with four frogs.

In the above experiments, the frog tongue was stimulated by anodal current.As shown in Fig. 1 C, cath odal stimul ation induc ed onl y small responses. InFig. 6, 1 mM CaCI2, 0.3 M ethanol, and distilled water were first applied tothe tongue a nd cathodal current was then applied after the responses inducedby the chemic al stimuli appr oach ed the steady-st ate level. Th e responses weresuppressed by catho dal current, and with cessation of the cathodal current,the responses were recovered. Simila r results were obta ined with four frogs.



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26 6 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 78 9 1981T h e r e is a p o s s i bi l i ty t h a t t h e r e s p o n se s t o e l e c tr i c c u r r e n t w e r e b r o u g h t b y

d i r e c t s t i m u l a t i o n o f t h e g u s t a t o r y n e r v e w i t h e l e c t ri c c u r r e n t . T o c h e c k t h isp o s s ib i l it y , t h e f r o g l i n g u a l a r t e r y w a s p e r f u s e d w i t h a r t if i c ia l R i n g e r ' s s o l u t i o na n d e l e c tr i c al s t i m u l a t i o n o f t h e t o n g u e w a s c a r r i e d o u t u n d e r t h e c o n d i t i o n

A S

0 "|-7

C h e m i c a l s t i m u l a t i o n

O ' - o ~ O

- 6 - 5 - 4l o g [ U Oz ( M )

0 0 ~ M h ~ O~, 0 2M l',4gO

~ o - . . ~ . E l e c t ri c a l s t i m u l a ti o n/ X o I o ~ ~ c t

0 .L , 0- 7 - 6 - 5 - 4log[UOz {M)

F IG U R E 4 . R e l a t i v e m a g n i t u d e o f th e p e a k r e sp o n s e s t o c h e m i c a l s t im u l i ( A )( 0.4 M N a C I a n d 0 .2 M M g C I 2 ) a n d a n o d a l c u r r e n t ( B ) w i t h 1 0 m M N a C I a n d5 m M M g C I 2 as a f u n c t io n o f l o g a r i t h m i c c o n c e n t r a t i o n o f u r a n y l a c e t a t e .R e s p o n s e s ( R ) w e r e c a l c u l a t e d r e l a t i v e t o r e s p e c t i v e r e s p o n s e s i n t h e a b s e n c e o fu r a n y l a c e t a t e . E a c h p o i n t i n t h e f i g u r e is t h e a v e r a g e v a l u e o f t h e d a t a o b t a i n e dw i t h t h r e e f r o g s .w h e r e r e l e a s e o f a c h e m i c a l t r a n s m i t t e r f r o m t a s t e c el ls w a s b l o c k e d . I n as e p a r a t e s t u d y , a w e s h o w e d t h a t e l i m i n a t i o n o f C a 2+ f r o m t h e p e r f u s i n gs o l u t i o n o r a d d i t i o n o f C a - c h a n n e l b l o c k e r s s u c h a s C d C I 2 , M n C I 2 , a n d

Nagahama e t a l . , manuscr ip t in p repara t ion .



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Y'~J~.SHIWAYANAOIET AL. T a s t e T r a n s d u ct i on M e c h a n i s m i n t h e F r o g 26 7

A) B) Chemical5t~nula~oe

t.0

n,.0- 5

20 s

- 6 - 5 - 4 - 3 - 2 - 1 - - 5 0m M C ~ 2[og lCaCl . ] (M) =: ,IM CaCI

C) Electrical5timul~ion

Elec.50mM CaCl2

F IG U R E 5 . ( A ) R e l a t i v e m a g n i t u d e o f p e a k r e sp o n s e s t o C aC 1 2 a s a f u n c t i o n o fi t s l o g a r i t h m i c c o n c e n t r a t i o n . R e s p o n s e s ( R ) w e r e c a l c u l a t e d r e l a t i v e t o t h er e s p o n se t o 3 m M C a C I 2 . E a c h p o i n t is t h e a v e r a g e v a l u e o f t h e d a t a o b t a i n e dw i t h f o u r f ro g s . ( B ) S u m m a t e d r e s p o n s e s to 1 M C a C I 2 a f t e r t h e t o n g u e w a sa d a p t e d t o 50 m M C a C1 2. 1 M C a C I 2 w a s s u p p li e d a t t h e p o i n t i n d i c a t e d b yt h e a r r o w i n t he f i g ur e . ( C ) S u m m a t e d r e s p o n se t o 0 . 7 - m A a n o d a l c u r r e n t a f t e rt h e t o n g u e w a s a d a p t e d t o 50 m M C a C I 2 . A n o d a l c u r r e n t w a s su p p l i e d a t t h ep o i n t i n d i c a t e d b y t h e a r r o w .

E l e c . E [ e c . E l e c .ImM CaCI2 0-3M EtOH Dist il led wate r

F IO U R E 6 . E f f e c t o f c a t h o d a l c u r r e n t o n th e s u m m a t e d r e p o ns e s i n d u c e d b yc h e m i c a l s t i m u l i . 0 . 7 - m A c a t h o d a l c u r r e n t w a s s u p p l i e d f o r t h e d u r a t i o n i n d i -c a te d b y s h o r t b a r s w h e n t h e r e s p o n s e s t o c h e m i c a l s t i m u l i (1 m M C a O l 2 , 0 .3 Me t h a n o l , a n d d i s t i l l e d w a t e r ) a p p r o a c h e d t o t h e s t e a d y - s t a t e l e v e l . T o s u p p r e s st h e w a t e r r e s p on s e , 1 0 m M N a C I w a s a d d e d t o 0 .3 M e t h a n o l .

v e r a p a m i l r e v e r s i b l y s u p p r e s s e d t h e g u s t a t o r y n e r v e r e s p o n se s t o s al ts , su g a r s ,a m i n o a c i d s , a n d d i s t i ll e d w a t e r . F i g . 7 A s h o w s t h a t a d e c r e a s e o f C a 2+c o n c e n t r a t i o n b y a d d i t i o n o f 1 m M E G T A t o a p e r f u s i n g so l u t i o n g r e a t l y



10/17

A ) a) R inger b)+ImMEGTA c) -EGTA

B )E l e ~

a) Rlnger

~ N a C t

101

Elec. 0.4M NK:I

b) +0.1ram CdCI

E lec . 0 .6 M NaCI

c ) - C d C l2

E lec 0~M NaCIC ) a ) R inger

1011

_},z~L _j . .~E l e ~ 0 . 4 4 q N IK : I

b) - I.04mM Verapa ml lE lec OJoMNaCI

c ) - Y e r a p a m l l

E lec . 0 .4MNaCI

10 s

Elec. 0-4M NaCt E lec . 0 .4M NaCI



11/17

K A S H I W A Y A N A G I E T A L . Taste Transduction Mechanism in the Frog 2 6 9s u p p r e s s e d b o t h r e s p on s e s to 0 .4 M N a C 1 a n d t o a n o d a l c u r r e n t w i t h 1 0 m MN a C I ; w h e n E G T A w a s r e m o v e d f r o m t h e p e r fu s i n g so l u ti o n , b o t h r e s p o n s esr e c o v e re d . A d d i t i o n o f C a - c h a n n e l b l o c k er s ( 0.1 m M C d C 12 a n d 0 .1 m Mv e r a p a m i l ) t o a p e r f u s i n g s o l u t i o n a ls o re v e r s i b l y s u p p r e s s e s b o t h t h e r e s p o n s et o N a C I a n d t h e r e s p o n s e t o a n o d a l c u r r e n t w i t h 1 0 m M N a C I , a s s h o w n i nF i g . 7 B a n d C . S i m i l a r r e s u lt s t o t h o s e s h o w n i n F ig . 7 w e r e o b t a i n e d w i t hf o u r f r o g s . T h e a b o v e r e s u l t s r u l e o u t t h e p o s s i b i l i t y t h a t t h e r e s p o n s e s t oa n o d a l c u r r e n t a r e i n d u c e d b y d i r e ct s t i m u l a t io n o f t h e g u s t a t o r y n e r v e w i t he l e c t r i c c u r r e n t .

D I S C U S S I O N

O u r r e su l ts s h o w t h a t a n o d a l s t i m u l a t io n o f t h e f r o g t o n g u e i n d u c e s r e sp o n s e ss i m i l a r t o r e s p o n s e s t o c h e m i c a l s t im u l i , a l t h o u g h t h e f o r m e r a p p e a r s l i g h t l ym o r e t r a n s i e n t t h a n t h e l a tt e r . A s s h o w n b y F i g . 1 , t h e r e s p o n s e s t o a n o d a lc u r r e n t d e p e n d o n t h e i o n s p ec ie s i n a n a d a p t i n g s o l u t io n o f t h e t o n g u e . F o re x a m p l e , e l e c tr ic a l s ti m u l a t i o n w i t h 5 m M M g C I 2 b ri n g s a b o u t a m u c h l a rg e rr e s p o n s e t h a n w i t h 1 0 m M N a C 1 a t a l l c u r r e n t i n t e n s it i es . B e c a u s e M g 2+ h a sa l a r g e r S t o k e s ' r a d i u s t h a n N a + , M g 2 m a y b e l es s p e r m e a b l e t o t h e c e llm e m b r a n e t h a n N a F u r t h e r m o r e , e l e c tr ic a l s t i m u l a t io n w i t h 1 0 m M c h o l i n ec h l o r id e o r 1 0 m M t e t r a e t h y l a m m o n i u m c h l o r id e ( w h i c h m a y b a r e l y p e r m e a t et h e m e m b r a n e ) c a u s e s t h e r e s p o n s e s . T h e r e f o r e , t h e d i f f e r e n c e i n t h e m a g n i -t u d e o f t h e e l e c t ri c a l r e s p o n s es c a n n o t b e e x p l a i n e d i n t e r m s o f t h e d i f f e r e n c ei n p e r m e a b i l i t y o f c a t io n s t o t h e m e m b r a n e . I n t h e f ro g , M g C I~ a l w a y s i n d u c e sm u c h l a r g e r r e s p o n s e s t h a n N a C 1 a t e q u a l e l e c t r o c h e m i c a l e q u i v a l e n t s . T h i ss u g g e st s t h a t a c o m m o n m e c h a n i s m e x is ts b e t w e e n e l e c tr ic a l a n d c h e m i c a lr e s p o n s e s . T h e r e s u l t s d e s c r i b e d a b o v e a r e c o n s i s t e n t w i t h t h o s e r e p o r t e d b yP f a f f m a n n a n d P r i t c h a r d ( 1 98 0 ): w i t h e q u a l c u r r e n t i n te n s i ty , t h e r es p o n s e o fr a t c h o r d a t y m p a n i t o a n o d a l c u r r e n t w i t h N a C 1 w a s g re a t e r t h a n t h e r es p o n s ew i t h K C 1 , w h e r e a s a c h e m i c a l r e s p o n s e t o N a C 1 w a s g r e a t e r t h a n t o K C 1 i nt h e r a t .

I n a p r e v i o u s p a p e r ( K a s h i w a g u r a e t al ., 1 9 7 7 ), w e s u g g e s t e d t h a t t h et r e a t m e n t o f th e f r og t o n g u e w i t h A N S r e m o v e s C a 2+ f r o m t h e r e c e p t o rm e m b r a n e a n d t h e r e fo r e a c o n f o r m a t i o n a l c h a n g e o f t h e re c e p t o r d o m a i n s f o rs a lt s is e a s i ly i n d u c e d b y a d s o r p t i o n o f s a lt s to t h e d o m a i n s . T h e p r e s e n tr e s u lt s i n d i c a t e t h a t t h e r es p o n s e s t o a n o d a l c u r r e n t w i t h 1 0 m M N a C I a r ea l s o e n h a n c e d a f t e r A N S t r e a t m e n t . T h i s a l s o s u g g e s t s t h a t e l e c t r ic a l r es p o n s e sa r e i n d u c e d b y a m e c h a n i s m s i m i l a r to th a t f o r c h e m i c a l r e sp o n s e s .A f t e r o n e t r e a t m e n t w i t h A N S , f ro g t o n g u e e x h i b i ts e n h a n c e d r e sp o n s e s t os a lt s t i m u l i u n l e s s C a 2+ i s a p p l i e d t o t h e t o n g u e . O n t h e o t h e r h a n d , t h et o n g u e e x h i b i t s e n h a n c e d r e s p o n s e s in t h e p r e s e n c e o f N i C I2 , b u t w i t h r e m o v a lo f N iC 1 2 f r o m t h e t o n g u e s u r f a c e , r e s p o n s e s r e t u r n t o t h e o r i g i n a l l e v e l( K a s h i w a g u r a e t al ., 1 9 7 8 ). T h a t is , t h e e n h a n c e m e n t o f t h e r e s p o n s e s b yFIGURE 7. ( O p p o s i t e ) The summated re sponses t o t he chemica l s t imul i (0 .4 MNaCI) and anoda l cu r ren t (0 .7 mA) wi th 10 m M NaCI be fo re (a ) and 1 h a f t e r(b ) 1 m M EG TA (A) , 0 .1 m M CdC12 (B), and 0 .1 mM ve rapam i l (C) we readded to a perfusing solut ion for the l ingual a r tery . The records (c) a re there sponses a f t e r EGTA , CdCIz , and ve rapam i l we re e l im ina t ed f rom the pe r fus ingsolut ion.



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2 7 0 THE JOURNAL OF GENERAL PHYSIOLOGY * VOLUME 7 8 * 1981

NiC12 is not broug ht abou t by remo val of Ca 2+ from the receptor me mbran e.Prob ably NiC12 acts on the receptor d omains so that a conforma tiona l chang eis easily induced by adsorptio n of salts. As similar to the case of ANStreatment, electrical responses with NaCl are also enhanced by the presenceof NiC12.

The mechanism of action of uranyl acetate is unknown. The enhanced orsuppressed responses to salt stimuli in the presence of uranyl acetate recoverto the original level immediately after elimination of uranyl acetate from thetongue surface. Thus, uranyl acetate does not appear to penetrate taste cellsbut acts on the taste cell membrane. The enhanc ive and suppressive effects ofuranyl acetate on the electrical responses with NaCl are similar to those onchemical responses to NaC1 and the effects on the electrical responses withMgC12 are si milar to those on c hemic al responses to MgC12. Th us , responsesto electrical stimuli under various modifications are quite similar to chemicalstimuli in all cases examined in this study.

As described in the Int roduc tion, a numb er of hypotheses on a mechan ismof "electrical taste" have been advanced. The possibility that electrical tasteis the result of stimulation of taste receptors by specific products of theelectrolysis of the saliva can be ruled out by the present results which indicatethat the mag nitud e of the responses to anodal current is highly dependent onthe species of ions in an a dap tin g solution.

Ou r results have shown that a decrease of Ca 2+ concen tratio n in a perf usingsolution or addit ion of Ca ch annel blockers to the perfusing solution reversiblysuppresses both responses to the chemical stimulus and to anoda l current. Thissuggests that Ca 2+ is involv ed in the tra nsd uct ion process of electrical responsesas well as chemical responses, although we notice that some of the blockersmay act on the Na channel under certain conditions (Baker et al., 1973;Kosty uk and Krishtal, 1977; Nachshe n and Blaustein, 1979; Pel lmar andCarpenter, 1979). Thus, the present results rule out the possibility thatelectrical responses are brought abou t by direct stimula tion of the gusta torynerves with electric current.

There is a possibility that salts accumulated on the tongue surface byiontophoresis stimulate the receptors. The results shown in Fig. 5, however,suggest that accu mula tio n of salts by iontophoresis is not a mai n cause ofelectrical responses. Whether or not salts are accumulated on the tonguesurface by iontophoresis can be subjected to theoretical consideration; theactual system of electrical stimulat ion is rathe r comple x for theoretical treat-ment. It is not known which ions carry the electric current through the cellmembrane. Ions contained in the mucus on the surface of the tongue as wellas ions in an adapting solution must be taken into consideration as currentcarriers across the membranes, especially when the tongue is adapted to asolution of salts having imper meable ions. For theoretical analysis, a simplifiedmodel system is presented in the Appendix where concentration polarizationoccurring at the membr ane-s olutio n interface as electric current flows thro ughthe membrane is analyzed theoretically in a system where the two aqueoussolutions of l: l-t ype electrolyte are separate d by a negatively charged mere-



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KASHIWAYANAGIEl" AL. Ta ste Transduction Mechanism in the Frog 27 Ib r a h e . T h e t h e o r e t i c a l a n a l y s i s i n d i c a t e s t h a t t h e c o n c e n t r a t i o n a t t h e m e m -b r a n e - s o l u t i o n i n t e r fa c e a t t h e a n o d e s i d e is l o w e r t h a n t h e c o n c e n t r a t i o n i nt h e b u l k s o l u ti o n . I f t h e t h e o r e t i c a l t r e a t m e n t d e s c r i b e d i n t h e A p p e n d i x isa p p l i c a b l e t o t h e e l e c t ri c a l s t i m u l a t i o n o f t h e t o n g u e , t h e c o n c e n t r a t i o n o fs a lt s a t t h e t o n g u e s u r f a c e - s o l u ti o n i n t e r f a c e b e c o m e s l o w e r t h a n t h a t i n a na d a p t i n g s o l ut i o n d u r in g a n o d a l s t i m u l a t i o n a n d b e c o m e s h i g h er d u r i n gc a t h o d a l s t i m u l a t i o n . T h e r e f o r e , i t is u n l i k e l y t h a t r e s p o n se s t o a n o d a l c u r r e n ta r e p r o d u c e d b y s al ts a c c u m u l a t e d o n t h e t o n g u e s u rf ac e . O n t h e o t h e r h a n d ,t h e s m a l l r e s p o n se s p r o d u c e d b y c a t h o d a l s t i m u l a t i o n ( se e F i g. ! C ) m a y h a v eb e e n i n d u c e d b y sa l ts a c c u m u l a t i o n o n t h e t o n g u e s u r fa c e .

I n a p r e v i o u s p a p e r ( A i u c h i e t a l ., 1 9 7 6 ), w e p r o p o s e d a h y p o t h e t i c a lm e c h a n i s m f o r t a s t e t ra n s d u c t i o n . T h e r e s p o n s e s t o e l e c t ri c c u r r e n t m i g h t b ee x p l a i n e d s i m i la r ly . F i g . 8 s h ow s a s c h e m a t i c d i a g r a m i l l u s tr a t i n g t h e m e c h -

~ ,,, ,~ A ) M i c r o v i t t u s o u tk,~I/ A membrane ?

\ . _ k rf area

- J - v cR c

VB+Vc-VAiB= RB+Rc+RAFIGURE 8 . A h y p o th e t ica l m o d e l an d eq u iv a len t c i r cu it i l lu s t r a t in g a mec h a-n ism o f g en era t io n o f g u s ta to ry n erv e r esp o n ses to ch emica l s t imu l i a n d e lec t r iccu r r en t . No ta t io n s in th e f ig u re a r e d escr ib ed in th e tex t .

a n i s m o f t h e r e s p o n s e s t o c h e m i c a l s t i m u l i a n d t o e l ec t r ic c u r r e n t . H e r e , A i st h e m i c r o v i ll u s m e m b r a n e o f t a s t e ce l l, B t h e s y n a p t i c a r e a , a n d C t h e o u t e rs u r f a c e o f e p i t h e l i a l c e ll . V a, V b, a n d Vc r e p r e s e n t t h e p o t e n t i a l d i f f e r e n c e a t A ,B , a n d C in t h e f i g u re . R a , R b , a n d R c r e p r e s e n t t h e e l e c t r i c r e s i s t a n c e a t A , B ,a n d C , r e s p e c t i v e l y . A s d e s c r i b e d i n t h e f i g u r e , a n e l e c t r i c c u r r e n t , ib , is af u n c t i o n o f o n l y V~ a n d R ,, i f o t h e r v a l u e s a r e u n c h a n g e d d u r i n g c h e m i c a ls t i m u l a t i o n . A p p l i c a t i o n o f c h e m i c a l s t i m u l i t o t h e t o n g u e s u r f a c e d e c re a s e st h e m e m b r a n e p o t e n t i a l a t t h e m i c r o v i l l u s m e m b r a n e , V ~, w h i c h i n c r e a se st h e e l e c t r ic c u r r e n t , i t,. A d e c r e a s e i n t h e m e m b r a n e r e s is t a n c e o f th e m i c r o -v i l l us m e m b r a n e , R ~ , w i l l a l so i n c r e a s e i b. T h e c u r r e n t , ib , d e p o l a r i z e s t h es y n a p t i c a r e a o f t h e t a s t e c e ll , w h i c h o p e n s t h e v o l t a g e - d e p e n d e n t C a c h a n n e l



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272 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 78 9 1981

and induces Ca influx from intercellular medium into the taste cell. This Cainflux will lead to a release of a chemical tra nsmitter. If, instead of chemicalstimulation, the electric current, ib, is supplied to the taste cell by anodalstimulation of the tongue, response similar to that induced by chemicalstimulation will be induced. Cathodal current cancels the electric currentproduced by chemical stimuli to the tongue surface and thereby suppressesthe responses induced by chemical stimuli.

Chemic al stimu latio n of the frog tongue by 0.2 M MgCI2 elicited a muc hlarger response than that by 0.4 M NaC1. This suggests that the number ofthe receptor domains whose conformation is changed by adsorptin of Mg2+ islarger than that by adsorption of Na under the condition employed a nd /orthe extent of the con formational change induced by adsorption of Mg 2+ islarger th an that by Na +. A similar relation may hold in the condit ion ofelectrical stimulation where the tongue is adapted to 5 mM MgCI2 and 10mM NaCI, alth ough concentr ations of both salts are one-fortieth of those forchemical stimulation. The conformational changes of the receptor domainsmay lead to easier flow of the electric current across the taste cell membraneand then a noda l stimu lation of the tongu e adap ted to 5 mM MgCI~ will elicita larger response than that to 10 mM NaC1. This explanation suggests thatanodal stimulation to 5 mM MgC12 induces a larger current across the tastecell membranes than with 10 mM NaCI at equal voltage. One may considerthat the above explanation is not consistent with the experimental resultswhich show that an imposed identical current (0.7 mA) has different effectswith differ ent salts perfusing the tongue. However, it should be noted that theratio of area occ upied by taste cells to the total surface area o f the t ongue isextreme ly small, thus, most of the curren t app lied to the tongu e flows thro ughother areas than the taste cells. The experimental results, therefore, do notrule out the possibility that more current may flow through the taste cellswith 5 mM MgC12 than with 10 mM NaC1, even when the identical currentsare applied to the tongue. However, the above mechanism is still highlyspeculative and further study will be needed to confirm the mechanism.

The mechanism by which ANS, NiCI2, and uranyl acetate enhance theresponses to certain species of salt stimuli and to anod al cu rrent is unkno wn.One possible explana tion is as follows: The tre atme nt o f the tong ue with ANSor the presence of NiC12 and urany l acetat e leads confor matio nal changes ofthe receptor do mains for certain species of salt stimuli and then electric currentwill flow more easily across the microvillus memb ran e, leading to en han cem entof the responses.

A P P E N D I X

The concentration polarization that occurs at the membrane-solution interface aselectric current flows has been analyzed theoret ically and experimental ly (Gregor andPeterson, 1964; Kobatake and Kamo, 1973). Here, we deal with a simple systemwhere the two aqueous solutions of 1:1-type electrolyte are separated by a membranebound between x .,, 0 and x = L when electric current is passed through the membrane(see Fig. 9). The membrane is assumed to be negatively charged like most biologicalmembranes. The stagnant layer of thickness, 8, is adjacent to the membrane surface



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KASHIWAYANAGIET AL. Tas te Transduction Mechanism in the Frog 27 3a n d t h e s o lu t io n p h a s e s i n th e c a t h o d e a n d a n o d e c o m p a r t m e n t s p l a c e d i n x < - 8a n d x > L + 8 , r e s p e c ti v e l y , h a v e a u n i f o r m c o n c e n t r a t i o n , C . T h e s a lt c o n c e n t r a t i o n sa t x -- 0 a n d x = L a r e d e n o t e d b y C ' a n d C " , r e s p e c t i v e ly . I f t h e v a l u e o f 6 isa s s u m e d t o b e s m a ll c o m p a r e d w i t h L a n d t h e n C ' a n d C " m a y b e e x p a n d e d i np o w e r s o f t h e r e l a t i v e t h i c k n e s s o f t h e s t a g n a n t l a y e r ( I, - - 6 / 2 L ) :

c ' = c + f (c )p ( l a )C " = C - f (C) p , (2 A)

w h e r e f ( C ) re p r e s e n t s t h e m a g n i t u d e o f t h e c o n c e n t r a t i o n p o l a r i z a t i o n .

a n o d e

-~ 0

M e m b r a n e ,i/= a t h o d e, , , 3 .

XL o bE l e c t r i c c u r r e n t

F I o u r E 9 . A s c h e m a t i c d i a g r a m i l l u s t r a ti n g c o n c e n t r a t i o n p o l a r i z a t i o n o f e le c -t r o ly t e s w h e n e l e c t ri c c u r r e n t f l ow s t h r o u g h a n e g a t i v e l y c h a r g e d m e m b r a n e .T h e m e m b r a n e is b o u n d b e t w e e n x = 0 a n d x -- L , a n d s t a g n a n t l a y e rs ar ep l a c e d b e t w e e n x < - 8 a n d x > L + 8 . C , C ' , a n d C " r e p r e s e n t e l e c t r o l y t ec o n c e n t r a t i o n s i n a b u l k s o l u t i o n : a t t h e m e m b r a n e - s o l u t i o n i n t e rf a c e , at t h ea n o d e s i d e , a n d a t t h e m e m b r a n e - s o l u t i o n i n t e r f a c e a t t h e c a t h o d e s i d e , r e s p e c -t i v e l y .

T h e a c t i v i t y ai a n d t h e m o b i l i t y u i o f i o n sp e c ie s ( i = + ) i n t h e m e m b r a n e a r er e p r e s e n t e d a s f o l l o w s :a + = a - + q ~ X , a - = C- (3 A )

u + C + = u ~ + q , X ) , u - C - = u ~H e r e , u ~ s t a n d s f o r t h e m o b i l i t y o f i - th i o n i n t h e b u l k s o l u t i o n , O X is t h e e f f e c t i v ef i xe d c h a r g e d e n s i ty . S e t t i n g u p t h e f lu x e q u a t i o n o f m o v a b l e i o n s in t h e m e m b r a n ep h a s e a n d a s s u m i n g t h e c o n d i t i o n o f s t e a d y s t a te , w e o b t a i n t h e f o l l o w i n g e x p r e s si o nf o r f (C ) ( K o b a t a k e a n d K a m o , 1 9 73 ):L u ~ + u ~f ( C ) = - R - - - T u ~ ~ ( r a ) / , ( 4 A )w h e r e I + a n d a s t a n d f o r t h e t r a n s f e r e n c e n u m b e r o f c a t i o n s r e l at i v e t o t h e l o c alc e n t e r o f m a s s i n t h e m e m b r a n e a n d t h a t i n t h e b u l k s o l u t io n . 1 + is d e f i n e d b y u + C + /( u+ C + + u - C - ) a n d a i s g i v e n b y u g , / ( u g . + u f l ) . I i s t h e e l e c t r i c c u r r e n t i n t e n s i t y .



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274 T H E JO U R N A L O F G E N E R A L PH Y SIO L O G Y 9 VOLUME 78 9 1981Eq. 4A can be rewritten as

f ( C ) = - ~ - - ~ u O u t u + + ( u - C - / C + ) u ~ + u ~ "The relat ion in Eq. 4A indicates that C - / C + is


17/17

KASHIWAYANAGIET AL. Taste Transduction Mechanism in the Frog 2 7 5N A CH SH EN , D . A . , an d M. P . BLA USTEXN . 1 9 77 . Th e e f fec t s o f so m e o rg an i c "c a l c i u m an t ag o n i s t s "

o n c a l c i u m i n f lu x i n p r e s y n a p t i c n e r v e t e r m i n a l s . Mol. Pharmacol. 1 6 : 5 7 9 - 5 8 6 .P E LL M A R, T . C . , a n d D . O . C A R PE N TE R . 1 97 9. V o l t a g e - d e p e n d e n t c a l c i u m c u r r e n t i n d u c e d b ys e r o t o n i n . Nature (Lond.). 2 7 7 : 4 8 3 - 4 8 4 .PFAFFMAN N , C . 1 9 41 . G u s t a t o ry a f fe r en t i m p u l se s . J. Cell . Comp. Physiol. 1 7 : 2 4 3 - 2 5 8 .PFAFFMAN N , C . , a n d T . PRITCH A RD . 1 9 80 . I o n sp ec i f i c i t y o f "e l e c t r i c t a s t e . " In O l f a c t i o n a n d

T a s t e V I I . H v a n d e r S t a r r e, e d i t o r. I n f o r m a t i o n R e t r i e v a l L t d . , L o n d o n . 1 7 5 -1 7 8 .S ~ I T H , D . V . , a n d S . L . B EA L E R. 1 97 5. S e n s i t i v i t y o f t h e r a t g u s t a t o r y s y s t e m t o t h e r a t e o f

s t i m u l u s o n s e t . Physiol. Beh av. 1 5 : 3 0 3 -3 1 4 .


taste trans duct ion mechanism

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