- Email: [email protected]
Effect of tetrodotoxin on electrically-induced release of autonomic mediators in the rabbit sinoatrial node
Life Sciences Vol . 9, Part I, pp. 383-393, 1970. Printed in Great Britain
Pergamon Press
EFFDCT OF TSTRODOT07CIN ON ELHCTRICALLY-h®UCBD RSLSASB OF AUTONO~QC 1~DIATORB IF THE RABBIT SINOATRIAL NOD$* Daniel B. Jackson** Department of Pharmacology, School of Medicine, University of Washington, Seattle, Kaehingtoa 98105
(Received 30 June 1989; in final form 18 January 1970)
Electrorelease (gB) is defined to be the release of neurotransmitter substances flram the terminals of autonomic neurons in response to electrical stimulation using a utal extracellular electrode .
8 suggests that Blt of neurotransmitters Eras the
~cperimental evidence
autonomic neurone is the aiaoatrial node of the rabbit depends on excitation of the postganglionic choliaergic and adrenergic a:oas .
It has been shown
that the following pharmacologic agents block electrically-induced acetylcholine release by acting at the intraaodal post-gaoglionic choliaergic nerve 8 terminals : hemicholinium and morphine 5 . Likerise, it has been shown that the following agents block electrically-induced norepinephriae release by acting at the iatraaodal poetganglionic adrenergic nerve terminals : guanethidine and bretyliun
2
.
It hoe been shorn that 3 x 10~ g~ml tetrodotozin (TTX) blocky tranrmural excitation (rectangular pulses of 1 msec duration, and 6~min .) of iatraatral neurone in the guinea pig ileum; it vas suggested that TTX blocked the release 7 of acetylcholiae frost the serve terminals . There is a study using esrdiac tissue in rhich TT7C (5 x 10-7 g~ml) abolished both the guinea pig right atrial response to nicotine and the response *Funds for anisals sad drugs were supplied by the National Heart Institute, postdoctoral fellowship F2-fQ~35,~71-01, and the National Institutes of Health, grant HS-0'r321-05 . **Department of General Pharmcology, Abbott Lboratories, N rth Chicago, Illinois 60064 ( Present address and reprint requests .
383
384
TETRODOTOXIN AND ELECTRORELEASE
Vol. 9, No. 7
to tranemural stimulation (3o sec pulse, 5 cycles sec, 0.3 mace)
j
.
In the
present study, the rabbit atrium ras used to study the effects of TTX on ER and on chronotropic responses to exogenous norepiaephrine (NE) . Studies have described the interactions of TTX and NE on the frog atrium and sinus venosus 1 .
It hen also béen shown that
conduction in the
rabbit right atrium, but not is the SA node, is blocked by TTX 10 g~ml) .
6 (1 x lÔ
TT7C is a toxin which, at low concentrations, can specifically block
conduction in the axosal portion of nerve cells xithout affecting (a) cholisergic or adrenergic receptors or (b) nerve terminals . The purpose of this study ie to quantitatively examine the effect of TTX an electrically-induced aeurotre,namitter release from the terminals of the morons in the midst of the rabbit ainoatrial nodal tissue .
It has been shown
that by electrically stinulatisg the 8A node, one can elicit a biphaaic chronotropic response in the right atrium of the rabbit 9 .
The ER response is bi-
phaaic because there is first s decrease, then as increase in beating rate . The initial slowing is called the choliaergic electroreleaae reaponae (CB
Is addition, it was inportast to observe the effects of
TTX oa the chronotropic responses to exogenous HS .
It has been suggested that
j . TT)C potentiates the chronotropic sad isotropic response to exogenous AE
A detailed study of the SA nodal inaervatioa is needed, both as to the degree of blockade of electrically-induced release of neurotransmitters, and as to the character of the changes in responses of the adrenergic receptors to AE is the TTJC-treated SA node preparation.
Biaoatrial node-right atrial preparations were isolated from 14 rabbits reining 2 1Cg.
The preparations were mounted in a 10 ec Plexiglas bath and
placed under 1 g diastolic tension by means of a Grass P'POj force-displacement transducer .
The tissue ran perflised at j5' C with standard nutrient solution
Vol. 9, No . 7
TETRODOTOXIN AND ELECTRORELEASE
of the following composition (in millimoles~liter) : CaC1 2, 2 .2 ; NaHC09,
11 .9 ;
sed with a mixture of
dextrose, 11 .1 .
9590 2-5$ C02.
FaCl,
385
145 .5 ; KCl, 5.4 ;
The solution was continuously gas-
A period of sixty minutes was alloxed for
equilibration of each preparation. The postganglionic neurone in the region of the SA node were stimulated for
5
sec with rectangular electrical pulses with a duration of 0 .1 to 0 .3
meet ; a stimulus voltage amplitude 10~, above threshold of the intranodal nerves was produced by a Tektronix type 161 pulse generator.
Graded chrono-
tropic responses were achieved by electrical stimulation at 2 min intervals at freouencies of 1, 2,
5,
10, 20,
50,
and 100 cycles sec .
The interval between spontaneous beats was recorded with an interval meter consisting of a Tektronix 162 waveform generator, a transistorized free running linear voltage ramp generator sad a Tektronix type 161 pulse generator. The voltage ramp was reset from the pulse generator by extracellular action potentials which were amplified with as Argonaut differential preamplifier and power supply .
One chs.nael of a Sanborn
150
calibrated for 100 to 1000 meet intervals .
direct-writing oscillograph was When longer intervals were expect-
ed, the attenuator was adjusted accordingly to extend the range up to
5000
asst .
The drugs used in this study were TTX (lyophilized as 1 mg of toxin in mg sodium citrate buffer, pH 4 .8), chloride .
5
norepiaephrine bitartrate, and acetylcholiae
Stock'solutions of TTX and ACh were prepared were prepared with de
ionized distilled water .
NE was prepared just prior to each experiment .
After the first dose-response was obtained using incremental concentrations of NE, the preparation was washed five times with fresh solution .
Then,
after equilibrium with the fresh nutrient solution was established (20 minutes),
either .06 ml of TTX or
.06 ml of solution was added to the 10 ml bath .
Ten minutes later, the eecond dose-response was obtained using incremental concentrations of NE .
388
TETRODOTOIÜN AND ELECTRORELEA3E
Vol. 9, No. 7
RBBUL'19 The effect of TTX on CSiR " Mg . 1 shows the per cent increase in beat interval plotted versus the above mentioned range of annulus frequencies .
1000 500
100 ~ ô ~o b O
I
2 5 10 20 50 Frequency of stimulation (Pulses/sec)
100
FIG. 1 Increnae in frequency of stimulation of the SA node of the rabbit atrium sad the dependent per cent increase in beat interval, indicating the cholinergic electrorelease response CERR . Control curve, and curves with two concentrations of tetrodotoxin . The per cent increase in beat interval is a aeasure of the earlier choliaergic phase of the response CERR . .06 ml TTX wan put into the bath .
After control ER responses were obtained, Ten minutes later, the stimulus-response
Vol . 9, No. 7
TETRODOTOQCIN AND ELECTRORELEA9E
387
TTX (T z 10~ g/=1) decreased ttu CAiR to 80~, of control at 5,
ras obtained .
10 and 20 pulses/sec ; at loner or higher frequencies of stieulatian, this concentration of TTX decreased the CSR to 40~ of control. reduced CIItR to about 2~ of the control response . reduce CBiR .
TTZ (2 z 10~ g/al)
TTE (3 z 10~ g/si) did not
lf~erical values are shorn in Table l . TABT.E 1
psr çent ~ncr easa in beat iaterval(C~it ~ Conceatratians ot TT1( . g/sti 8ti>talus lh~equency cSrclea aeç
Control
T z 10~
1
T.4 ± i.T
3 "T ± 0"6
-
2
T.6±1.8
5 .4~1.2
-
5
15.8 ~ 3.2
10 .0 ~ 3.0
-
10
49.2 ± 13.4
39 " 2 * 14 .9
0.8 ~ o.i
20
184 .8 ..31 .5
~38 .1 r 46.9
~ 0.2
50
453 .4 ~ 82 .0
215 .6 f 53" 2
2.3 ± i.0
100
y9T " 4 ~ 64 .1
206 .2 ' 61.5*
2.2 * 1.0
(ns6)
(nsl4)
2 z 10"8
i.s
(ns4)
* 8lgnificaat difference P t .O5 The effect of TTX oa A&tR .
lig. 2 share the per amt decrease is beat
interval plotted versus the increasing t5requancies of stirulation ibr AIItR . The per cent decrease in beat interval is a abasura of the later adrenergic phase of the response (AIItR) . applied after addition of TTX .
The iraquency range of atiwli rare again iTX (T z 10~ g/al) decreased the I1IItR 85~ ;
this indicates a greater sensitivity of the I1IItR phase tàaa the CHtR phue at
2, 5 sad 10 cycles/sec .
TTIt (2 z 10-8 g/al) reduced AIItR to 0.5~ of control .
lhaierical values are shorn in Table 2.
388
TETRODOTOXIhT AND ELECTRORELEASE
Frequency of
Vol . 9, No. 7
stimulation (Pulses%sec)
Fig. z Increase in h~equency of stisu~ation of the 8A gods of the rabbit atri~an and the dependent per cent decrease in beat interval, indicating the adremerg~electrorelesse response (A~iR) . Control curve, and~ourv xith txo coacentrstions of tetrodotoxia . The effeçts of 7~on rat~ca~ntrscti~e force of~tal~eousl~besting right atria .
BYperiments were done to determine xhether TTIC had gay effect on
either the rate of besting of the SA nodal-right atrial preparation, or oa the force of contraction . A concentration of 2 z 10-8 8~a1 TTX, xhich cospletely blocked the response to electrical stiaulation (Blt) had no effect on either the beat frequency, or on the force of contraction. Although 1 z 10- 6 g/nl TTX did decrease the force of contraction, the beat frequency xas unchanged . Time of onset of TTZ action .
IIi responses to a stimulus frequency of .
100 cycles/sec xere observed for the entire period during xhich TTX blockade developed .
Vol . 9, No. 7
TETRODOTORIId AND ELECTRORELEASE
S89
TAHZE 2 Per çe nt decrease is beat interval iA~tR) . Stimulus Frequency Cycl es/sec
Concentrations of TTX, a/ml
i x 10~
Control
2 x 10-8
1
0.~ ~ 0.4
0.03
~ o
-
2
2.4 + o.q
o.03
+o
-
5
q .q ± 0 .9
0 .4 ± 0.3*
-
l0
20 .9 ± 2.4
1.3 ~ 0.6*
-
20
26 .5 ~ O.q
4 .0 t 1.2*
-
50
29 .8 ~ 0.8
Z .o +
l .3*
1.6 ~ 0 .2
lo0
30 .2 t 5.2
5 .4 ± 0 .9*
1.6 ~ 0 .2
(a s 14)
(n
s
6)
(n
s
4)
* Sigsificeat difference P ~ "OS The onset of blockade of C~tR and A~ïR by TTX occurred rapidly ; eight minutes after TTX (2 x 10'8 g~ml), CgiIt ran 80~ blocked while A&tR was 99f blocked . Hoth C~tR and AERR were 100
blocked in ten mfnutes .
Ro response was observed
with repeated tests of 100 cycles sec during a sixty minute period after caste plete blockade of FR had been achieved with 2 x 10-8 g/ml T17C . Comparison of TTX blockade and fbC-3 blocàsde . The pharmacologic action of hemicholiaium depends on nerve stimulation 8 .
In order to determine rheth-
er TTX blocàsde of IIt depends on neuronal stimulation, the SA nodal stimulating electrode was relocated is the nodal region after TTX (2 x 10-8 g~ml) blockade was +~xi~.l . ras observed .
At other locations is the region of the SA node, blockade of &t The ~t response could be revered by washing the preparations
with fresh rrutrient solution . The effect of exogenous aorepinephrine . other investigators 3 that TTX (5 x 10
-ï
To test the observations of
g~ml) potentiates the chronotropic
990
TETRODOTOXIII AND ELECTRORELEASE
Vol . 9, No. 7
efTect of exogenous 1fE, experiaenta rere done in order to measure the positive chronotropic response to AE is the presence of TTX prea mce of TTX
(2 z 10'8
(2 x 10' 8
g/ml) .
In the
g~ml), the per cent increase is me was not diff-
erent team the per cent increase is the presence of the nutrient solution (Student's paired t teat, p < "05) "
Aa is shown in Tables
3
and
4,
there is
ao significant difference between control respoasea and reapoasee in the acme prepsntions treated with either TT1C or mitrient solution .
Bach prepsntion
ras its own control.
r cmt iacreaee in nte _+
S .E . in ~
Coatrol
NE Concentntion (g~ml)
4)
TTX (2 z 10-8
1 z lô8 3 z lô 8
44 .4 + 10.q
22.6 + 6.6
60 .q * 11.8
41 .3 ± T" 5
1 z 10' 7
68.5 + 12.3
45 .0 + 10 .8
3 z 10' 7
Ti.O ± 13.9
49 .q ± 2q .4
i x 10 6
86.8 + 14 .1
48 .1 ± 14 .4
g~ml)
TABLE 4 Per cent increase in me ± B .E . (n ~~ Control
ëS Canceatntion (g~ml) 1 x 10' 8
32 .4 ± 15.0
12.3 t 4 .0
lo'e
41.8 ± 23 .0
1q.6 ± 8.0
1 x l0' 7
55" 4 + 25 .2
23.1 ± 9 .6
3 x
58 " 7
2T " 5
30"9 _+ 9 " 5
62 .q i 25 " 9
34"5 ± 9 " 5
3 x
1Ô
T
i z 10' 6
a
Putrient Solution°'
As control for addition of TTX
(2 x
3
10' 8
g/ml),
shown in Tnble
3.
Vol . 9, No . 7
TETRODOTOXIId AND ELECTRORELEA3E
391
Since the responses to incremental concentrations of NB in the second doseresponse curves were leas then the responses during the first dose-reepa~nse curves, these differences are listed in Table 5 .
It ie apparent that these
differences result from the wash after the first NS done-response ourve .
Difference in per cent _increase in rate± 8 .$ . L ~4) 1fE Concentration (g~ml)
7R7C (2 z 10-8 gal)
Nutrient 8olutiona
1 x 10-8 j x lô8
-21 .8 + 11.9
-22 .5 ± ll .j
-19 "4 ± T "9
-24 .1 * 15 .0
7 j x lô
-21 .j ± 15 .6
-27 .8 ± 18 .0
i x l0-6
-jB .T ± 9 .1
-28 .2 ± 16 .9
a As control for aùdition of -8 g~ml) TTX (2 z 10 . DIBCUSSIOF The effect of TTX on poatgaoglionic chloinerglc and adrenergic electrorelease was studied in isolated rabbit einaatrial node preparatia~ns .
There
was a relatively greater blockade of A&iR than C~tR at T z 10~ gal TTX~
8 whereas at 2 x lÔ g~ml TTIC, CF
In four ezper-
invents with rabbit right atrium, TTX at a concentration of 2 z lÔ8 g/s~l did not potentiate the positive chronotropic effect of NE .
On the other hand, Bell j showed that thb positive chronotropic reepome to tranemural stimulation was blocked in the presence of TTX .
This finding U
392
TETRODOTOIÜN AND ELECTRORELEASE
Vol . ~ Na 7
supported by our results which show that AIItR in the rabbit atrium is blocked by 2 x l0-8 g~~ TTX. Thus, it appears that 2 x lÔ
8 gal TT7C effective~r blocks the release
of neurotransmitters form electrically stimulated intraoardiac nerves, but has no direct or indirect effect an either the cholinergic or adrmergic receptors in the region of the SA node .
In addition, contrary to what one observes in
denervation supersensitivity, TTX blockade of the intracardiac neurone does not potentiate the respoasivmese of the adrmergic receptors to exogenous liS. Sil!lfARY Tetrodotoxin (TTX) in the concentration of ~ x 10-9 g~ml reduces both cholinergic electrorelense reapoase (CIItR) sad adrmergic electrorelease respouse (AHtR) in iaolste8 rabbit einoatrial node preparations .
The effect of
this TTX concentration is 65~ greater on the adrenergic portion of the biphaaic response than an the cholinergic portion . There is no blxkade or potentiation by 2 x 10-8 g~ml TTX of the positive chronotropic responses of the einoatrial nodal preparatiaa to exogenous NE . It is evident that the biphasic chronotropic response to several frequencies of electrical stimulation of the intranodal cholinergic and adrmergic nerve fibers is dependent on the excitation of the axonal portions of these fibers . The differential blocàsde of the cholinergic and adrenergic electrorelease responses by s relatively lorr concentration of TT7C (2 x 10-8 g~ml) suggests a differential sensitivity of the choliaergic and sdrenergic components of the autonomic nervous system which inaervate the pacmoaker regions of the right atrium .
Whether this differential sensitivity of C&2R a~ A~tR to TTX (2 x 10-8
g~ml) is related to the greater involvement of Nâ in the ABitR than in the CFRR is as yet unknown .
Vol. 9, No. 7
TETRODOTOXIN AND ELECTRORELEA3E
393
The author thanks T . C . West sad F. F. Viaceazi for their ideas regarding the project and their aaaistaace with instruaentation . R~~tAIC~ 1. J " Acrves aad D . Erlij, Rature ?~; 1178 (1967) .
2. D . W. Aswry and T . C . West, J" Pharmacol. ezvtl. Therav . ~j: 14 (1962) . r 2 ; 96 0968y " 3" C " Bell, Brit. J. Phariacol. aod Chemotheray . j 4 . C . Y. 1Lao, Phara. Rev. 18 ; 99T (1966) "
5 " B . L. lCenaedy nad T. C . Weat, J" Pharmcol . e:otl . Therav . ~: 149 0967y . 6. J . W. lfoore aad T . Farsheshi, Fed . Proc . 26 : 1655 (196T) .
7" Y . Ogura, 'Y . llori aad Y . Wataaabe, J " Pharmcol . esDtl. Therav . 1~4 ; 8. F. F. Viacensi sad T . C. bleat, J. Pharmacol. ~; 349 0965) .
9 " T . C: West, e 8 cial zid asues of the Bsrt p. 81. ed . Paes de Carvslho, A ., BLsevier(19 1) . 10 . S . Yaasgiahi and T. Saao, Proc . Jsb. Aced . 42 ; 1194 (1966) .
Pergamon Press
EFFDCT OF TSTRODOT07CIN ON ELHCTRICALLY-h®UCBD RSLSASB OF AUTONO~QC 1~DIATORB IF THE RABBIT SINOATRIAL NOD$* Daniel B. Jackson** Department of Pharmacology, School of Medicine, University of Washington, Seattle, Kaehingtoa 98105
(Received 30 June 1989; in final form 18 January 1970)
Electrorelease (gB) is defined to be the release of neurotransmitter substances flram the terminals of autonomic neurons in response to electrical stimulation using a utal extracellular electrode .
8 suggests that Blt of neurotransmitters Eras the
~cperimental evidence
autonomic neurone is the aiaoatrial node of the rabbit depends on excitation of the postganglionic choliaergic and adrenergic a:oas .
It has been shown
that the following pharmacologic agents block electrically-induced acetylcholine release by acting at the intraaodal post-gaoglionic choliaergic nerve 8 terminals : hemicholinium and morphine 5 . Likerise, it has been shown that the following agents block electrically-induced norepinephriae release by acting at the iatraaodal poetganglionic adrenergic nerve terminals : guanethidine and bretyliun
2
.
It hoe been shorn that 3 x 10~ g~ml tetrodotozin (TTX) blocky tranrmural excitation (rectangular pulses of 1 msec duration, and 6~min .) of iatraatral neurone in the guinea pig ileum; it vas suggested that TTX blocked the release 7 of acetylcholiae frost the serve terminals . There is a study using esrdiac tissue in rhich TT7C (5 x 10-7 g~ml) abolished both the guinea pig right atrial response to nicotine and the response *Funds for anisals sad drugs were supplied by the National Heart Institute, postdoctoral fellowship F2-fQ~35,~71-01, and the National Institutes of Health, grant HS-0'r321-05 . **Department of General Pharmcology, Abbott Lboratories, N rth Chicago, Illinois 60064 ( Present address and reprint requests .
383
384
TETRODOTOXIN AND ELECTRORELEASE
Vol. 9, No. 7
to tranemural stimulation (3o sec pulse, 5 cycles sec, 0.3 mace)
j
.
In the
present study, the rabbit atrium ras used to study the effects of TTX on ER and on chronotropic responses to exogenous norepiaephrine (NE) . Studies have described the interactions of TTX and NE on the frog atrium and sinus venosus 1 .
It hen also béen shown that
conduction in the
rabbit right atrium, but not is the SA node, is blocked by TTX 10 g~ml) .
6 (1 x lÔ
TT7C is a toxin which, at low concentrations, can specifically block
conduction in the axosal portion of nerve cells xithout affecting (a) cholisergic or adrenergic receptors or (b) nerve terminals . The purpose of this study ie to quantitatively examine the effect of TTX an electrically-induced aeurotre,namitter release from the terminals of the morons in the midst of the rabbit ainoatrial nodal tissue .
It has been shown
that by electrically stinulatisg the 8A node, one can elicit a biphaaic chronotropic response in the right atrium of the rabbit 9 .
The ER response is bi-
phaaic because there is first s decrease, then as increase in beating rate . The initial slowing is called the choliaergic electroreleaae reaponae (CB
Is addition, it was inportast to observe the effects of
TTX oa the chronotropic responses to exogenous HS .
It has been suggested that
j . TT)C potentiates the chronotropic sad isotropic response to exogenous AE
A detailed study of the SA nodal inaervatioa is needed, both as to the degree of blockade of electrically-induced release of neurotransmitters, and as to the character of the changes in responses of the adrenergic receptors to AE is the TTJC-treated SA node preparation.
Biaoatrial node-right atrial preparations were isolated from 14 rabbits reining 2 1Cg.
The preparations were mounted in a 10 ec Plexiglas bath and
placed under 1 g diastolic tension by means of a Grass P'POj force-displacement transducer .
The tissue ran perflised at j5' C with standard nutrient solution
Vol. 9, No . 7
TETRODOTOXIN AND ELECTRORELEASE
of the following composition (in millimoles~liter) : CaC1 2, 2 .2 ; NaHC09,
11 .9 ;
sed with a mixture of
dextrose, 11 .1 .
9590 2-5$ C02.
FaCl,
385
145 .5 ; KCl, 5.4 ;
The solution was continuously gas-
A period of sixty minutes was alloxed for
equilibration of each preparation. The postganglionic neurone in the region of the SA node were stimulated for
5
sec with rectangular electrical pulses with a duration of 0 .1 to 0 .3
meet ; a stimulus voltage amplitude 10~, above threshold of the intranodal nerves was produced by a Tektronix type 161 pulse generator.
Graded chrono-
tropic responses were achieved by electrical stimulation at 2 min intervals at freouencies of 1, 2,
5,
10, 20,
50,
and 100 cycles sec .
The interval between spontaneous beats was recorded with an interval meter consisting of a Tektronix 162 waveform generator, a transistorized free running linear voltage ramp generator sad a Tektronix type 161 pulse generator. The voltage ramp was reset from the pulse generator by extracellular action potentials which were amplified with as Argonaut differential preamplifier and power supply .
One chs.nael of a Sanborn
150
calibrated for 100 to 1000 meet intervals .
direct-writing oscillograph was When longer intervals were expect-
ed, the attenuator was adjusted accordingly to extend the range up to
5000
asst .
The drugs used in this study were TTX (lyophilized as 1 mg of toxin in mg sodium citrate buffer, pH 4 .8), chloride .
5
norepiaephrine bitartrate, and acetylcholiae
Stock'solutions of TTX and ACh were prepared were prepared with de
ionized distilled water .
NE was prepared just prior to each experiment .
After the first dose-response was obtained using incremental concentrations of NE, the preparation was washed five times with fresh solution .
Then,
after equilibrium with the fresh nutrient solution was established (20 minutes),
either .06 ml of TTX or
.06 ml of solution was added to the 10 ml bath .
Ten minutes later, the eecond dose-response was obtained using incremental concentrations of NE .
388
TETRODOTOIÜN AND ELECTRORELEA3E
Vol. 9, No. 7
RBBUL'19 The effect of TTX on CSiR " Mg . 1 shows the per cent increase in beat interval plotted versus the above mentioned range of annulus frequencies .
1000 500
100 ~ ô ~o b O
I
2 5 10 20 50 Frequency of stimulation (Pulses/sec)
100
FIG. 1 Increnae in frequency of stimulation of the SA node of the rabbit atrium sad the dependent per cent increase in beat interval, indicating the cholinergic electrorelease response CERR . Control curve, and curves with two concentrations of tetrodotoxin . The per cent increase in beat interval is a aeasure of the earlier choliaergic phase of the response CERR . .06 ml TTX wan put into the bath .
After control ER responses were obtained, Ten minutes later, the stimulus-response
Vol . 9, No. 7
TETRODOTOQCIN AND ELECTRORELEA9E
387
TTX (T z 10~ g/=1) decreased ttu CAiR to 80~, of control at 5,
ras obtained .
10 and 20 pulses/sec ; at loner or higher frequencies of stieulatian, this concentration of TTX decreased the CSR to 40~ of control. reduced CIItR to about 2~ of the control response . reduce CBiR .
TTZ (2 z 10~ g/al)
TTE (3 z 10~ g/si) did not
lf~erical values are shorn in Table l . TABT.E 1
psr çent ~ncr easa in beat iaterval(C~it ~ Conceatratians ot TT1( . g/sti 8ti>talus lh~equency cSrclea aeç
Control
T z 10~
1
T.4 ± i.T
3 "T ± 0"6
-
2
T.6±1.8
5 .4~1.2
-
5
15.8 ~ 3.2
10 .0 ~ 3.0
-
10
49.2 ± 13.4
39 " 2 * 14 .9
0.8 ~ o.i
20
184 .8 ..31 .5
~38 .1 r 46.9
~ 0.2
50
453 .4 ~ 82 .0
215 .6 f 53" 2
2.3 ± i.0
100
y9T " 4 ~ 64 .1
206 .2 ' 61.5*
2.2 * 1.0
(ns6)
(nsl4)
2 z 10"8
i.s
(ns4)
* 8lgnificaat difference P t .O5 The effect of TTX oa A&tR .
lig. 2 share the per amt decrease is beat
interval plotted versus the increasing t5requancies of stirulation ibr AIItR . The per cent decrease in beat interval is a abasura of the later adrenergic phase of the response (AIItR) . applied after addition of TTX .
The iraquency range of atiwli rare again iTX (T z 10~ g/al) decreased the I1IItR 85~ ;
this indicates a greater sensitivity of the I1IItR phase tàaa the CHtR phue at
2, 5 sad 10 cycles/sec .
TTIt (2 z 10-8 g/al) reduced AIItR to 0.5~ of control .
lhaierical values are shorn in Table 2.
388
TETRODOTOXIhT AND ELECTRORELEASE
Frequency of
Vol . 9, No. 7
stimulation (Pulses%sec)
Fig. z Increase in h~equency of stisu~ation of the 8A gods of the rabbit atri~an and the dependent per cent decrease in beat interval, indicating the adremerg~electrorelesse response (A~iR) . Control curve, and~ourv xith txo coacentrstions of tetrodotoxia . The effeçts of 7~on rat~ca~ntrscti~e force of~tal~eousl~besting right atria .
BYperiments were done to determine xhether TTIC had gay effect on
either the rate of besting of the SA nodal-right atrial preparation, or oa the force of contraction . A concentration of 2 z 10-8 8~a1 TTX, xhich cospletely blocked the response to electrical stiaulation (Blt) had no effect on either the beat frequency, or on the force of contraction. Although 1 z 10- 6 g/nl TTX did decrease the force of contraction, the beat frequency xas unchanged . Time of onset of TTZ action .
IIi responses to a stimulus frequency of .
100 cycles/sec xere observed for the entire period during xhich TTX blockade developed .
Vol . 9, No. 7
TETRODOTORIId AND ELECTRORELEASE
S89
TAHZE 2 Per çe nt decrease is beat interval iA~tR) . Stimulus Frequency Cycl es/sec
Concentrations of TTX, a/ml
i x 10~
Control
2 x 10-8
1
0.~ ~ 0.4
0.03
~ o
-
2
2.4 + o.q
o.03
+o
-
5
q .q ± 0 .9
0 .4 ± 0.3*
-
l0
20 .9 ± 2.4
1.3 ~ 0.6*
-
20
26 .5 ~ O.q
4 .0 t 1.2*
-
50
29 .8 ~ 0.8
Z .o +
l .3*
1.6 ~ 0 .2
lo0
30 .2 t 5.2
5 .4 ± 0 .9*
1.6 ~ 0 .2
(a s 14)
(n
s
6)
(n
s
4)
* Sigsificeat difference P ~ "OS The onset of blockade of C~tR and A~ïR by TTX occurred rapidly ; eight minutes after TTX (2 x 10'8 g~ml), CgiIt ran 80~ blocked while A&tR was 99f blocked . Hoth C~tR and AERR were 100
blocked in ten mfnutes .
Ro response was observed
with repeated tests of 100 cycles sec during a sixty minute period after caste plete blockade of FR had been achieved with 2 x 10-8 g/ml T17C . Comparison of TTX blockade and fbC-3 blocàsde . The pharmacologic action of hemicholiaium depends on nerve stimulation 8 .
In order to determine rheth-
er TTX blocàsde of IIt depends on neuronal stimulation, the SA nodal stimulating electrode was relocated is the nodal region after TTX (2 x 10-8 g~ml) blockade was +~xi~.l . ras observed .
At other locations is the region of the SA node, blockade of &t The ~t response could be revered by washing the preparations
with fresh rrutrient solution . The effect of exogenous aorepinephrine . other investigators 3 that TTX (5 x 10
-ï
To test the observations of
g~ml) potentiates the chronotropic
990
TETRODOTOXIII AND ELECTRORELEASE
Vol . 9, No. 7
efTect of exogenous 1fE, experiaenta rere done in order to measure the positive chronotropic response to AE is the presence of TTX prea mce of TTX
(2 z 10'8
(2 x 10' 8
g/ml) .
In the
g~ml), the per cent increase is me was not diff-
erent team the per cent increase is the presence of the nutrient solution (Student's paired t teat, p < "05) "
Aa is shown in Tables
3
and
4,
there is
ao significant difference between control respoasea and reapoasee in the acme prepsntions treated with either TT1C or mitrient solution .
Bach prepsntion
ras its own control.
r cmt iacreaee in nte _+
S .E . in ~
Coatrol
NE Concentntion (g~ml)
4)
TTX (2 z 10-8
1 z lô8 3 z lô 8
44 .4 + 10.q
22.6 + 6.6
60 .q * 11.8
41 .3 ± T" 5
1 z 10' 7
68.5 + 12.3
45 .0 + 10 .8
3 z 10' 7
Ti.O ± 13.9
49 .q ± 2q .4
i x 10 6
86.8 + 14 .1
48 .1 ± 14 .4
g~ml)
TABLE 4 Per cent increase in me ± B .E . (n ~~ Control
ëS Canceatntion (g~ml) 1 x 10' 8
32 .4 ± 15.0
12.3 t 4 .0
lo'e
41.8 ± 23 .0
1q.6 ± 8.0
1 x l0' 7
55" 4 + 25 .2
23.1 ± 9 .6
3 x
58 " 7
2T " 5
30"9 _+ 9 " 5
62 .q i 25 " 9
34"5 ± 9 " 5
3 x
1Ô
T
i z 10' 6
a
Putrient Solution°'
As control for addition of TTX
(2 x
3
10' 8
g/ml),
shown in Tnble
3.
Vol . 9, No . 7
TETRODOTOXIId AND ELECTRORELEA3E
391
Since the responses to incremental concentrations of NB in the second doseresponse curves were leas then the responses during the first dose-reepa~nse curves, these differences are listed in Table 5 .
It ie apparent that these
differences result from the wash after the first NS done-response ourve .
Difference in per cent _increase in rate± 8 .$ . L ~4) 1fE Concentration (g~ml)
7R7C (2 z 10-8 gal)
Nutrient 8olutiona
1 x 10-8 j x lô8
-21 .8 + 11.9
-22 .5 ± ll .j
-19 "4 ± T "9
-24 .1 * 15 .0
7 j x lô
-21 .j ± 15 .6
-27 .8 ± 18 .0
i x l0-6
-jB .T ± 9 .1
-28 .2 ± 16 .9
a As control for aùdition of -8 g~ml) TTX (2 z 10 . DIBCUSSIOF The effect of TTX on poatgaoglionic chloinerglc and adrenergic electrorelease was studied in isolated rabbit einaatrial node preparatia~ns .
There
was a relatively greater blockade of A&iR than C~tR at T z 10~ gal TTX~
8 whereas at 2 x lÔ g~ml TTIC, CF
In four ezper-
invents with rabbit right atrium, TTX at a concentration of 2 z lÔ8 g/s~l did not potentiate the positive chronotropic effect of NE .
On the other hand, Bell j showed that thb positive chronotropic reepome to tranemural stimulation was blocked in the presence of TTX .
This finding U
392
TETRODOTOIÜN AND ELECTRORELEASE
Vol . ~ Na 7
supported by our results which show that AIItR in the rabbit atrium is blocked by 2 x l0-8 g~~ TTX. Thus, it appears that 2 x lÔ
8 gal TT7C effective~r blocks the release
of neurotransmitters form electrically stimulated intraoardiac nerves, but has no direct or indirect effect an either the cholinergic or adrmergic receptors in the region of the SA node .
In addition, contrary to what one observes in
denervation supersensitivity, TTX blockade of the intracardiac neurone does not potentiate the respoasivmese of the adrmergic receptors to exogenous liS. Sil!lfARY Tetrodotoxin (TTX) in the concentration of ~ x 10-9 g~ml reduces both cholinergic electrorelense reapoase (CIItR) sad adrmergic electrorelease respouse (AHtR) in iaolste8 rabbit einoatrial node preparations .
The effect of
this TTX concentration is 65~ greater on the adrenergic portion of the biphaaic response than an the cholinergic portion . There is no blxkade or potentiation by 2 x 10-8 g~ml TTX of the positive chronotropic responses of the einoatrial nodal preparatiaa to exogenous NE . It is evident that the biphasic chronotropic response to several frequencies of electrical stimulation of the intranodal cholinergic and adrmergic nerve fibers is dependent on the excitation of the axonal portions of these fibers . The differential blocàsde of the cholinergic and adrenergic electrorelease responses by s relatively lorr concentration of TT7C (2 x 10-8 g~ml) suggests a differential sensitivity of the choliaergic and sdrenergic components of the autonomic nervous system which inaervate the pacmoaker regions of the right atrium .
Whether this differential sensitivity of C&2R a~ A~tR to TTX (2 x 10-8
g~ml) is related to the greater involvement of Nâ in the ABitR than in the CFRR is as yet unknown .
Vol. 9, No. 7
TETRODOTOXIN AND ELECTRORELEA3E
393
The author thanks T . C . West sad F. F. Viaceazi for their ideas regarding the project and their aaaistaace with instruaentation . R~~tAIC~ 1. J " Acrves aad D . Erlij, Rature ?~; 1178 (1967) .
2. D . W. Aswry and T . C . West, J" Pharmacol. ezvtl. Therav . ~j: 14 (1962) . r 2 ; 96 0968y " 3" C " Bell, Brit. J. Phariacol. aod Chemotheray . j 4 . C . Y. 1Lao, Phara. Rev. 18 ; 99T (1966) "
5 " B . L. lCenaedy nad T. C . Weat, J" Pharmcol . e:otl . Therav . ~: 149 0967y . 6. J . W. lfoore aad T . Farsheshi, Fed . Proc . 26 : 1655 (196T) .
7" Y . Ogura, 'Y . llori aad Y . Wataaabe, J " Pharmcol . esDtl. Therav . 1~4 ; 8. F. F. Viacensi sad T . C. bleat, J. Pharmacol. ~; 349 0965) .
9 " T . C: West, e 8 cial zid asues of the Bsrt p. 81. ed . Paes de Carvslho, A ., BLsevier(19 1) . 10 . S . Yaasgiahi and T. Saao, Proc . Jsb. Aced . 42 ; 1194 (1966) .