- Email: [email protected]
Vorticella—a model for chemopharmacodynamic action on smooth muscle
Comp. gen. Pharmac., 1972 , 3, 363-37 TM [Scientechnica (Publishers) Ltd.]
363
VORTICELLA--A MODEL FOR CHEMOPHARMACODYNAMIC ACTION ON SMOOTH MUSCLE M. M A R A N , R. H I M E L S T E I N , Am) S. D I K S T E I N * Department of Applied Pharmacology, School of Pharmacy, Hebrew University, Jerusalem, Israel
(Received 4 May, I972 ) ABSTRACT i. Vorticella contracture is a unique model for studying non-specific chemical stimulus (chemopharmacodyrmmic action). 2. The classic agonists have no activity up to x raM, except for low sensitivity towards nicotine. We could not find evidence for the presence of the adenyl cyclase or the prostaglandin systems. 3" On the other hand, the usual non-specific stimulators are active. They include oxidizing agents with low redox potential, such as phenazine methosulphate or menadione; glutatione oxidizing agents, such as diamide, KCN, N-tert.-butyl-5-methylisoxazolium. Papaverine is inhibitory, whereas ouabain has no effect on the phenazine methosulphate-induced contractures.
EACH time a pharmacological agonist induces a physical phenomenon, like the contraction of a muscle, the following series of events is assumed to occur: (a) the chemical molecule hits the external side of the cell (' target a r e a ' ) , causing (b) a chain of reactions known as the ' r e c e p t o r m e c h a n i s m ' , and finally (c) producing the intracellular liberation of a Ca ~+ ion or increasing its permeability to initiate the response. T h e role which the Ca ~+ ion plays is that of a coupler, i.e., it couples together the stimulation of the m e m b r a n e with the response, which in our case is the contraction of myofibriles (Dikstein, 1969a ). In the multicellular models common in pharmacology a serious problem of intracellular diffusion exists. Some consequences are: the different metabolic condition of the cells on the surface of the preparation, as distinct from those inside it; a different environment for the cells on the surface of the preparation as distinct from those inside it; uneven distribution of the activity of the cells. None of these problems exists in a unicellular preparation. *Reprints requests to be addressed to Dr. Dikstein.
One such preparation common in pharmacological research today is the muscle of the barnacle (Ashley, I971), which is not easy to obtain under our laboratory conditions. Vorticella, on the other hand, can be cultured in unlimited quantities. Moreover, it has been published that the Vorticella reacts in cyclic contractures to oxidizing materials, which m a y hint at a biochemical process (Dikstein, 1969b ). I t is also known that other Peritrich ciliates react like the Vorticella to electrical and mechanical stimulation (Sugi, i96o ). T h e fact that the contractions of Vorticella are dependant on Ca 2+ has been known for a long time (Koltzoff, 1926 ). A similar fact has been established with regard to glycerinated preparations (Levine, i 956; Townes and Brown, I965). It is also known that the Vorticella contains ATPase (Levine, 196oa), essential sulphydril groups (Levine, I96ob), and myofilaments (Sotelo and TrujilloCenoz, I959). The morphology of the Vorticella is very well known, (Randall and Hopkins, 1962 ; Reid, I967; Amos, i97o), though little is known about the pharmacological specificity of the contractures. A quantitative system for determining
Comp. gen. Pharmac.
MAR.AN AND OTHERS
364
the effect of drugs was i n t r o d u c e d b y Sleigh (i97o), in Spirostomum. H e d e t e r m i n e d the n u m b e r of contractures per m i n u t e for a n u m b e r of cells at different times a n d used
I t has recently b e e n established, b y m e a n s of i n t r a c e l l u l a r injection of aequorine, that the coupling factor i n Spirostomum is the Ca *+ i o n - - a s in the case with muscle (Etienne, : 97o). Here we were faced b y the p r o b l e m of whether to work with a Vorticella i n which the
Table I,--STANDARD CULTUREC O N D I T I O N S VorticeUa
FIG. I .--Schematic drawing of VorticeUa ( × 145)' Reprinted from Drugs and Cholinergic Mechanisms in the CARS', E. Heilbronn and A. Winter (Eds.), p. 483 . Research Institute of National Defenee, Stockholm I97O.' Table ]/.--ABILITY
OF
FOR
CONSTITUENTS OF STOCK SOLUTION
MOLARITY
g. per IOO ml.
CaC12.2H20 KC1 NaHCO3 MgC12.6H20
IO-1 10--2 iO--1 IO--2
i "47 0"074 o.84 o-~o3
Preparation of the egg solution: Three g. yolk of hard-boiled egg were triturated in IOO ml. H20. The solution was boiled for 5 minutes and filtered through cotton-wool. Culture medium: One ml. each of the stock solutions was taken. Ten ml. of the egg-yolk extract and water were added up to a total of ioo ml. Four mg. of Complan (trade mark Glaxo Ltd., Great Britain) were added to this solution.
DIFFERENT AGENTS TO INDUCE CYCLIC Vorticella CONTRACTURES
NAME
APPROXIMATE MINIMALCONCENTRATION
Nicotine Cyclic-AMP Dibutyryl-cyclic-AMP Acetylcholine Prostaglandin E: and E2 Adrenaline Noradrenaline Histamine Serotonin Theophyline Caffeine
the data o b t a i n e d as criteria for the m e d i c a m e n t s ' influence. W e too a d o p t e d this method. Sleigh himself n o t e d the similarity b e t w e e n Spirostomum a n d VorticeUa (Sleigh, x969) in their response to the influence of p h a r m a c o l o g i c a l materials.
3" I o- aM No No No No No No No No No
effect effect effect effect effect effect effect effect effect
up up up up up up up up up
to to to to to to to to to
t I I I i I I I I
mM mM mM mM mM mM mM mM mM
Very weak effect at ~ m M
i m p o r t a n c e of the Ca 2+ has b e e n established only indirectly, or with Spirostomum, where the coupling m e c h a n i s m of the Ca 2+ has been established b e y o n d d o u b t (Etienne, 197o). After some hesitation we decided to proceed with Vorticella, for the same reasons for which
365
MODEL FOR CHEMOPHARMACODYNAMIC ACTION
I972, :3
The chemicals were obtained from Sigma (St. Louis, Missomi, U.S.A.), or from Calbiochem (San Diego, California). A fresh stock solution of drugs was prepared each day and was carefully neutralized. Where the drug was insoluble in water the stock solution was dissolved in acetone, the final concentration of acetone in the experiment never being more thanoq per cent. Observation of the VorticeUa was made at room temperature (about 2o ° C.) under a 4 o x binocular microscope (Carl Zeiss, Germany) and the results were expressed according to Sleigh (I97o). Using 3-5 Vorticellaein three different plates the standard error of the mean was never more than -t-o'5 contractures per Vorticellaper minute. The highest concentration of any drug tested was arbitrarily MATERIALS AND M E T H O D S limited at zo-3M. Inhibition studies were carried Vorticella was cultured in standard conditions as out by ~o-minute preincubations at room temperadescribed in Table I, at 2~-24 ° C. in non-sterile ture with the inhibitor. conditions.
we h a d originally preferred it to Spirostomum, n a m e l y (Fig. I ) : - a. T h e Vorticella is fixed to the b o t t o m of the c u l t u r i n g plate, while Spirostomum moves a b o u t which makes observation more difficult. b. I n contrast to Spirostomum, n o ciliac are present i n the c o n t r a c t i n g p a r t of Vorticella. c. W h i l e i n Spirostomum the whole cell contracts, c o n t r a c t u r e i n Vorticella is limited to a specific p a r t of the cell, the stem, which is a few h u n d r e d gm. i n length.
s"e'" "-~ •
12 t-
z a
L T//
=
}8 •
0
/~
:~4,., /"" x ter" 0Z u
""x
z 10
:°l-I ~ II
%"-
I "--.--.---r, ~1 0
I 2
1
T ~
~
I 3
I 4
I 5
1 6
7
8
/
°
/
ti/
'', "-.-o
7 ""1
~e
u 9
10
M I N U TES
Fro. 2.--The influence of nicotine; O, zo-aM; A, 3 × I°-4M"
0
1
2
3
/,,
5
6
7
8
9
10
MINUTES influence of isoxazolium per-
FIG. 3.--The chlorate (N-tert.-butyl-5-methylisoxazolium +C104-); 0 , Io-aM; A, zo-4M.
2
RESULTS I. EFFECT OF NEUROTRANSMITTERS AND RELATED MATERIALS tt~
°i 8
0
1
2
3
4 5 6 7 8 9 10 MINUTES FIO. 4 . - - T h e influence of adenosine derivatives. 0 , ATP Io-4M; A, ADP io-4M.
Table 11 shows the effect of some neurot r a n s m i t t e r s , ' second messengers ', a n d related agents. I t c a n be seen that in the highest c o n c e n t r a tion tested n o n e of the agents, except nicotine, h a d a n y effect. T h e effect of nicotine is shown i n Fig. 2. Because of the activity of nicotine, we decided to try a synthetic c o m p o u n d , c a p a b l e of irreversibly comb i n i n g with the a n i o n i c sites of receptors: N-tert.-butyl-5-methylisoxazolium perchlorate
366
"I"I"\ =,2 _
I
z
\
Pharmac.
'~,~
I
.[/,., ',,
lo
;o m 113 Lid fie --1
Comp.gen.
MARAN AND OTHERS
x
- F//o:, \ ',
8
(J
\ 41, I I
I
I
I
,
2
3
/,,
5
I
I
6
7
t
=
I
8
9
10
0
M INUTES
FIG. 5 . - - T h e influence of phenazine methosulphate (PMS). O, Io-SM; O , Io-*M; £ , Io-3M.
1
2
3
4 5 6 7 8 9 10 MINUTES FIG. 6 . - - T h e influence o f KC1 o n p h e n a z i n e methosulphate-induced contractures. O , PMS x o - 4 M ; O , P M S I o - 4 M after i n c u b a t i o n for i o m i n u t e s in xo m M KC1; A, s a m e , b u t in 2o m M KC1. t
16
=E
6
pn," o ;>
14 Z
i
"~" 12
0-
u~ 14J
er O
p-
2
•... 10 tt)
Fz 0 ¢.3
w
t,e
},-- 8 U < e,o
v.. 6
•
Z O u
0
I
2
3
4
5
6
7
8
9
10
MINUTES
FIG. & - - T h e effect of KsFe(CN)6 (O, I ° - 3 M ; zo-4M) and K,Fe(CN)6 (A, Io-SM; A, Io-'M).
O,
%o.~.
°~ ~°
°~
( M i t t a g a n d T o r m a y , I97I ) (Fig. 3)- I t was f o u n d to be m o r e active t h a n nicotine. ~2. EFFECT OF ADENOSINE DERIVATIVES
Fig. 4 shows the effect o f A D P a n d A T P . A M P h a d no effect even at i r a M . 0
t
2
3
4
5
6
7
8
9
10
MINUTES
FIG. 7.--The
influence of menadione; Io-SM; O, Io-4M.
O,
3" EFFECT OF OXIDIZING AGENTS
Fig. 5 shows the effect o f p h e n a z i n e m e t h o s u l p h a t e ( P M S ) in different concentrations.
MODEL FOR CHEMOPHARMACODYNAMICACTION
I972, 3
This material was chosen later as the reference material at xo-4M. The replacement of the external solution by KCI caused very little
367
PMS has a very low redox potential and is able to accept electrons from either flavoprotein of sulphydril groups. We, therefore,
i
14
n,
12
>°6
Z
D ~ o
i
U') M.I
.,. 10
mz,
i "f "O~,~O ~ ~ , ~ l
pC.) <[ ,,y
/
2
O
>
\
Z O tJ
8
IM ¢ i
~
I
1
2
3
!
I
!
l
I
I
I
4
5
6
7
8
9
10
MINUTES
Fro. 9.--The effect of diamide, O, I x Io-*M; O, 3 × io-*M.
E:I/ 10 I-
0
6
U "
4
O t,) ~O
o.
1
2
3
4 5 6 MINUTES
7
8
9
10
FIG. I I.--Inhibition by preincubation with Panthesine of the contractions induced by Io-*M PMS. O, lo-4M PMS; O, Io-4M PMS after incubation in to-aM Panthesine. change, except at concentrations which were far above the tonicity of the usual culture media (see Table I) of Vorticella (Fig. 6). Other oxidizing agents are also effective; e.g., Fig. 7 shows the effect of menadione; Fig. 8 shows KsFe(CN)6. From Fig. 8 it is clear that the oxidized form is, indeed, the one which is active.
0
1
2
3
4 5 6 MINUTES
•
°
7
8
9
10
FIG. io.--Inhibition by tetrazolium salt (~,3,5triphenyl-2H-tetrazolium chloride) (compare with Fig. 5). O, PMS Io-4M after incubation for io minutes in io-*M tetrazolium; O, PMS Io-SM after incubation for Io minutes in Io-SM tetrazolium.
decided to test a ' s p e c i f i c ' intracellular glutathione oxidizing agent--Diamide (Kosower and Kosower, I969). Fig. 9 shows its effectivity. To demonstrate further the similarity to other models, we present the blocking effect of a tetrazolium salt (Fig. Io). It clearly blocks the action of PMS, in a manner analogous to the blocking ability on acetylcholine and histamine action (Chauhan and Basuray, I969).
4" EFFECT OF NON-SPECIFIC BLOCKINGAGENTS
Fig. I I shows the full inhibition by a local anaesthetic called ' Panthesine ' at I m M on the contracture induced by I o - 4 M PMS.
Comp. gen Pharmac.
MARAN AND OTHERS
368
Fig. 12 shows the inhibition by papaverine, and the lack of inhibition by ouabain, on the contractures induced by PMS. It can be seen that papaverine causes approximately 5 ° per cent inhibition at Io-4M.
Fig. 13 shows that I m M K C N alone causes contractures, at the same time inhibiting PMS action. Both effects are interesting, since on the glycerinated preparation I m M K C N has no effect whatsoever (Amos, 197o ). 5" EFFECT ON ~-~EAVY META LS
12
Lanthanides are known to cause stimulus on other preparations (Sanborn and Langer,
A
,o/\\\
,o
Z
i_
z
-
s
o
\
g
g4
3
U
g
0
1
2
3
4 5 6 MINUTES
7
8
9
tO
FIG. 12.--The effect of papaverine and ouabain on the contractions induced by xo-*M PMS. O, Io-4M PMS after incubation for zo minutes in io-4M papaverine; A, zo-4M PMS after incubation for io minutes in Io-4M ouabain.
.z
i
8
W
m
Z 0 U
2
3
4 5 6 MINUTES
7
8
9
10
Fro. I3.--The effect of KCN. O, Io-3M KCN; O, io4-M PMS after incubation in io-3M KCN; &, Io-4M KCN; A, io-4M PMS after incubation in io-*M KCN.
197o), probably acting by ATPase inhibition (Schatzman and Tschabold, I971). Fig. I4 shows that both La s+ and Pb *+ are able to induce contractures.
or
U
I
[ I°~
",
2I - i i ~ o
0
1
2
",..
3
4 5 6 MINUTES
7
8
9
10
FIG. z4.--The effect of heavy metals. O, io-4M lanthanum nitrate; A, Io -SM plumbous nitrate; A, io-~M plumbous nitrate.
DISCUSSION The results show that the Vorticella is very suitable for the study of non-specific chemical stimulus. There is a lack of reaction to classic neurotransmitters and agonists (Table H), with the exception of a low sensitivity to nicotine (Fig. 2); and a lack of reaction to agents presumed to act through the adenyl cyclase system (Table II). On the other hand, the stimulus by non-specific agents is present, such as (x) oxidizing agents, especially those with low redox potential (Figs. 5, 7, 8) (Dikstein, Tal, and Sulman, I965; Hamburger, Dikstein, and Sulman, 1967; Dikstein,
37 °
MARAN AND OTHERS
Protozoology, Third Int. Cong. Protozool., Leningrad, p. 165. Leningrad: Nauka. SL~mH, M. A. (197o), ' Some factors affecting the excitation of contraction in Spirostomum ', Acta Protozool., 7, 335-352. SOTELO, R., and TRUJILLO-CENoz, O. (I959), ' The fine structure of an elementary contractile system ', 07. biophys, biochem. Cytol., 6, 126-127. Suoi, H. (I96o), ' Propagation of contraction in the stalk muscle of Carchesium ', Fac. Sci. Univ. Tokyo, IV, 8, 6o3-615 . TowNEs, M., and BROWN, D. (1965), ' T h e involvement of pH, adenosine triphosphate,
calcium and magnesium in the contraction of the glycerinated Vorticella ', 07. call comp Physiol., 65, 261-269. VALETT~, G., and LECLAm, M. (I 97°), ' Sensibilization par les inhibiteurs mdtaboliques de l'intestin isol6 de rat aux effets des Sympathomim6tiques ', 07. Pharmac., Paris, x, 445-456.
Key Word Index: Vorticdla, chemopharmacodyamics, nicotine, papaverine, Ca 2+, myoneme contraction.
363
VORTICELLA--A MODEL FOR CHEMOPHARMACODYNAMIC ACTION ON SMOOTH MUSCLE M. M A R A N , R. H I M E L S T E I N , Am) S. D I K S T E I N * Department of Applied Pharmacology, School of Pharmacy, Hebrew University, Jerusalem, Israel
(Received 4 May, I972 ) ABSTRACT i. Vorticella contracture is a unique model for studying non-specific chemical stimulus (chemopharmacodyrmmic action). 2. The classic agonists have no activity up to x raM, except for low sensitivity towards nicotine. We could not find evidence for the presence of the adenyl cyclase or the prostaglandin systems. 3" On the other hand, the usual non-specific stimulators are active. They include oxidizing agents with low redox potential, such as phenazine methosulphate or menadione; glutatione oxidizing agents, such as diamide, KCN, N-tert.-butyl-5-methylisoxazolium. Papaverine is inhibitory, whereas ouabain has no effect on the phenazine methosulphate-induced contractures.
EACH time a pharmacological agonist induces a physical phenomenon, like the contraction of a muscle, the following series of events is assumed to occur: (a) the chemical molecule hits the external side of the cell (' target a r e a ' ) , causing (b) a chain of reactions known as the ' r e c e p t o r m e c h a n i s m ' , and finally (c) producing the intracellular liberation of a Ca ~+ ion or increasing its permeability to initiate the response. T h e role which the Ca ~+ ion plays is that of a coupler, i.e., it couples together the stimulation of the m e m b r a n e with the response, which in our case is the contraction of myofibriles (Dikstein, 1969a ). In the multicellular models common in pharmacology a serious problem of intracellular diffusion exists. Some consequences are: the different metabolic condition of the cells on the surface of the preparation, as distinct from those inside it; a different environment for the cells on the surface of the preparation as distinct from those inside it; uneven distribution of the activity of the cells. None of these problems exists in a unicellular preparation. *Reprints requests to be addressed to Dr. Dikstein.
One such preparation common in pharmacological research today is the muscle of the barnacle (Ashley, I971), which is not easy to obtain under our laboratory conditions. Vorticella, on the other hand, can be cultured in unlimited quantities. Moreover, it has been published that the Vorticella reacts in cyclic contractures to oxidizing materials, which m a y hint at a biochemical process (Dikstein, 1969b ). I t is also known that other Peritrich ciliates react like the Vorticella to electrical and mechanical stimulation (Sugi, i96o ). T h e fact that the contractions of Vorticella are dependant on Ca 2+ has been known for a long time (Koltzoff, 1926 ). A similar fact has been established with regard to glycerinated preparations (Levine, i 956; Townes and Brown, I965). It is also known that the Vorticella contains ATPase (Levine, 196oa), essential sulphydril groups (Levine, I96ob), and myofilaments (Sotelo and TrujilloCenoz, I959). The morphology of the Vorticella is very well known, (Randall and Hopkins, 1962 ; Reid, I967; Amos, i97o), though little is known about the pharmacological specificity of the contractures. A quantitative system for determining
Comp. gen. Pharmac.
MAR.AN AND OTHERS
364
the effect of drugs was i n t r o d u c e d b y Sleigh (i97o), in Spirostomum. H e d e t e r m i n e d the n u m b e r of contractures per m i n u t e for a n u m b e r of cells at different times a n d used
I t has recently b e e n established, b y m e a n s of i n t r a c e l l u l a r injection of aequorine, that the coupling factor i n Spirostomum is the Ca *+ i o n - - a s in the case with muscle (Etienne, : 97o). Here we were faced b y the p r o b l e m of whether to work with a Vorticella i n which the
Table I,--STANDARD CULTUREC O N D I T I O N S VorticeUa
FIG. I .--Schematic drawing of VorticeUa ( × 145)' Reprinted from Drugs and Cholinergic Mechanisms in the CARS', E. Heilbronn and A. Winter (Eds.), p. 483 . Research Institute of National Defenee, Stockholm I97O.' Table ]/.--ABILITY
OF
FOR
CONSTITUENTS OF STOCK SOLUTION
MOLARITY
g. per IOO ml.
CaC12.2H20 KC1 NaHCO3 MgC12.6H20
IO-1 10--2 iO--1 IO--2
i "47 0"074 o.84 o-~o3
Preparation of the egg solution: Three g. yolk of hard-boiled egg were triturated in IOO ml. H20. The solution was boiled for 5 minutes and filtered through cotton-wool. Culture medium: One ml. each of the stock solutions was taken. Ten ml. of the egg-yolk extract and water were added up to a total of ioo ml. Four mg. of Complan (trade mark Glaxo Ltd., Great Britain) were added to this solution.
DIFFERENT AGENTS TO INDUCE CYCLIC Vorticella CONTRACTURES
NAME
APPROXIMATE MINIMALCONCENTRATION
Nicotine Cyclic-AMP Dibutyryl-cyclic-AMP Acetylcholine Prostaglandin E: and E2 Adrenaline Noradrenaline Histamine Serotonin Theophyline Caffeine
the data o b t a i n e d as criteria for the m e d i c a m e n t s ' influence. W e too a d o p t e d this method. Sleigh himself n o t e d the similarity b e t w e e n Spirostomum a n d VorticeUa (Sleigh, x969) in their response to the influence of p h a r m a c o l o g i c a l materials.
3" I o- aM No No No No No No No No No
effect effect effect effect effect effect effect effect effect
up up up up up up up up up
to to to to to to to to to
t I I I i I I I I
mM mM mM mM mM mM mM mM mM
Very weak effect at ~ m M
i m p o r t a n c e of the Ca 2+ has b e e n established only indirectly, or with Spirostomum, where the coupling m e c h a n i s m of the Ca 2+ has been established b e y o n d d o u b t (Etienne, 197o). After some hesitation we decided to proceed with Vorticella, for the same reasons for which
365
MODEL FOR CHEMOPHARMACODYNAMIC ACTION
I972, :3
The chemicals were obtained from Sigma (St. Louis, Missomi, U.S.A.), or from Calbiochem (San Diego, California). A fresh stock solution of drugs was prepared each day and was carefully neutralized. Where the drug was insoluble in water the stock solution was dissolved in acetone, the final concentration of acetone in the experiment never being more thanoq per cent. Observation of the VorticeUa was made at room temperature (about 2o ° C.) under a 4 o x binocular microscope (Carl Zeiss, Germany) and the results were expressed according to Sleigh (I97o). Using 3-5 Vorticellaein three different plates the standard error of the mean was never more than -t-o'5 contractures per Vorticellaper minute. The highest concentration of any drug tested was arbitrarily MATERIALS AND M E T H O D S limited at zo-3M. Inhibition studies were carried Vorticella was cultured in standard conditions as out by ~o-minute preincubations at room temperadescribed in Table I, at 2~-24 ° C. in non-sterile ture with the inhibitor. conditions.
we h a d originally preferred it to Spirostomum, n a m e l y (Fig. I ) : - a. T h e Vorticella is fixed to the b o t t o m of the c u l t u r i n g plate, while Spirostomum moves a b o u t which makes observation more difficult. b. I n contrast to Spirostomum, n o ciliac are present i n the c o n t r a c t i n g p a r t of Vorticella. c. W h i l e i n Spirostomum the whole cell contracts, c o n t r a c t u r e i n Vorticella is limited to a specific p a r t of the cell, the stem, which is a few h u n d r e d gm. i n length.
s"e'" "-~ •
12 t-
z a
L T//
=
}8 •
0
/~
:~4,., /"" x ter" 0Z u
""x
z 10
:°l-I ~ II
%"-
I "--.--.---r, ~1 0
I 2
1
T ~
~
I 3
I 4
I 5
1 6
7
8
/
°
/
ti/
'', "-.-o
7 ""1
~e
u 9
10
M I N U TES
Fro. 2.--The influence of nicotine; O, zo-aM; A, 3 × I°-4M"
0
1
2
3
/,,
5
6
7
8
9
10
MINUTES influence of isoxazolium per-
FIG. 3.--The chlorate (N-tert.-butyl-5-methylisoxazolium +C104-); 0 , Io-aM; A, zo-4M.
2
RESULTS I. EFFECT OF NEUROTRANSMITTERS AND RELATED MATERIALS tt~
°i 8
0
1
2
3
4 5 6 7 8 9 10 MINUTES FIO. 4 . - - T h e influence of adenosine derivatives. 0 , ATP Io-4M; A, ADP io-4M.
Table 11 shows the effect of some neurot r a n s m i t t e r s , ' second messengers ', a n d related agents. I t c a n be seen that in the highest c o n c e n t r a tion tested n o n e of the agents, except nicotine, h a d a n y effect. T h e effect of nicotine is shown i n Fig. 2. Because of the activity of nicotine, we decided to try a synthetic c o m p o u n d , c a p a b l e of irreversibly comb i n i n g with the a n i o n i c sites of receptors: N-tert.-butyl-5-methylisoxazolium perchlorate
366
"I"I"\ =,2 _
I
z
\
Pharmac.
'~,~
I
.[/,., ',,
lo
;o m 113 Lid fie --1
Comp.gen.
MARAN AND OTHERS
x
- F//o:, \ ',
8
(J
\ 41, I I
I
I
I
,
2
3
/,,
5
I
I
6
7
t
=
I
8
9
10
0
M INUTES
FIG. 5 . - - T h e influence of phenazine methosulphate (PMS). O, Io-SM; O , Io-*M; £ , Io-3M.
1
2
3
4 5 6 7 8 9 10 MINUTES FIG. 6 . - - T h e influence o f KC1 o n p h e n a z i n e methosulphate-induced contractures. O , PMS x o - 4 M ; O , P M S I o - 4 M after i n c u b a t i o n for i o m i n u t e s in xo m M KC1; A, s a m e , b u t in 2o m M KC1. t
16
=E
6
pn," o ;>
14 Z
i
"~" 12
0-
u~ 14J
er O
p-
2
•... 10 tt)
Fz 0 ¢.3
w
t,e
},-- 8 U < e,o
v.. 6
•
Z O u
0
I
2
3
4
5
6
7
8
9
10
MINUTES
FIG. & - - T h e effect of KsFe(CN)6 (O, I ° - 3 M ; zo-4M) and K,Fe(CN)6 (A, Io-SM; A, Io-'M).
O,
%o.~.
°~ ~°
°~
( M i t t a g a n d T o r m a y , I97I ) (Fig. 3)- I t was f o u n d to be m o r e active t h a n nicotine. ~2. EFFECT OF ADENOSINE DERIVATIVES
Fig. 4 shows the effect o f A D P a n d A T P . A M P h a d no effect even at i r a M . 0
t
2
3
4
5
6
7
8
9
10
MINUTES
FIG. 7.--The
influence of menadione; Io-SM; O, Io-4M.
O,
3" EFFECT OF OXIDIZING AGENTS
Fig. 5 shows the effect o f p h e n a z i n e m e t h o s u l p h a t e ( P M S ) in different concentrations.
MODEL FOR CHEMOPHARMACODYNAMICACTION
I972, 3
This material was chosen later as the reference material at xo-4M. The replacement of the external solution by KCI caused very little
367
PMS has a very low redox potential and is able to accept electrons from either flavoprotein of sulphydril groups. We, therefore,
i
14
n,
12
>°6
Z
D ~ o
i
U') M.I
.,. 10
mz,
i "f "O~,~O ~ ~ , ~ l
pC.) <[ ,,y
/
2
O
>
\
Z O tJ
8
IM ¢ i
~
I
1
2
3
!
I
!
l
I
I
I
4
5
6
7
8
9
10
MINUTES
Fro. 9.--The effect of diamide, O, I x Io-*M; O, 3 × io-*M.
E:I/ 10 I-
0
6
U "
4
O t,) ~O
o.
1
2
3
4 5 6 MINUTES
7
8
9
10
FIG. I I.--Inhibition by preincubation with Panthesine of the contractions induced by Io-*M PMS. O, lo-4M PMS; O, Io-4M PMS after incubation in to-aM Panthesine. change, except at concentrations which were far above the tonicity of the usual culture media (see Table I) of Vorticella (Fig. 6). Other oxidizing agents are also effective; e.g., Fig. 7 shows the effect of menadione; Fig. 8 shows KsFe(CN)6. From Fig. 8 it is clear that the oxidized form is, indeed, the one which is active.
0
1
2
3
4 5 6 MINUTES
•
°
7
8
9
10
FIG. io.--Inhibition by tetrazolium salt (~,3,5triphenyl-2H-tetrazolium chloride) (compare with Fig. 5). O, PMS Io-4M after incubation for io minutes in io-*M tetrazolium; O, PMS Io-SM after incubation for Io minutes in Io-SM tetrazolium.
decided to test a ' s p e c i f i c ' intracellular glutathione oxidizing agent--Diamide (Kosower and Kosower, I969). Fig. 9 shows its effectivity. To demonstrate further the similarity to other models, we present the blocking effect of a tetrazolium salt (Fig. Io). It clearly blocks the action of PMS, in a manner analogous to the blocking ability on acetylcholine and histamine action (Chauhan and Basuray, I969).
4" EFFECT OF NON-SPECIFIC BLOCKINGAGENTS
Fig. I I shows the full inhibition by a local anaesthetic called ' Panthesine ' at I m M on the contracture induced by I o - 4 M PMS.
Comp. gen Pharmac.
MARAN AND OTHERS
368
Fig. 12 shows the inhibition by papaverine, and the lack of inhibition by ouabain, on the contractures induced by PMS. It can be seen that papaverine causes approximately 5 ° per cent inhibition at Io-4M.
Fig. 13 shows that I m M K C N alone causes contractures, at the same time inhibiting PMS action. Both effects are interesting, since on the glycerinated preparation I m M K C N has no effect whatsoever (Amos, 197o ). 5" EFFECT ON ~-~EAVY META LS
12
Lanthanides are known to cause stimulus on other preparations (Sanborn and Langer,
A
,o/\\\
,o
Z
i_
z
-
s
o
\
g
g4
3
U
g
0
1
2
3
4 5 6 MINUTES
7
8
9
tO
FIG. 12.--The effect of papaverine and ouabain on the contractions induced by xo-*M PMS. O, Io-4M PMS after incubation for zo minutes in io-4M papaverine; A, zo-4M PMS after incubation for io minutes in Io-4M ouabain.
.z
i
8
W
m
Z 0 U
2
3
4 5 6 MINUTES
7
8
9
10
Fro. I3.--The effect of KCN. O, Io-3M KCN; O, io4-M PMS after incubation in io-3M KCN; &, Io-4M KCN; A, io-4M PMS after incubation in io-*M KCN.
197o), probably acting by ATPase inhibition (Schatzman and Tschabold, I971). Fig. I4 shows that both La s+ and Pb *+ are able to induce contractures.
or
U
I
[ I°~
",
2I - i i ~ o
0
1
2
",..
3
4 5 6 MINUTES
7
8
9
10
FIG. z4.--The effect of heavy metals. O, io-4M lanthanum nitrate; A, Io -SM plumbous nitrate; A, io-~M plumbous nitrate.
DISCUSSION The results show that the Vorticella is very suitable for the study of non-specific chemical stimulus. There is a lack of reaction to classic neurotransmitters and agonists (Table H), with the exception of a low sensitivity to nicotine (Fig. 2); and a lack of reaction to agents presumed to act through the adenyl cyclase system (Table II). On the other hand, the stimulus by non-specific agents is present, such as (x) oxidizing agents, especially those with low redox potential (Figs. 5, 7, 8) (Dikstein, Tal, and Sulman, I965; Hamburger, Dikstein, and Sulman, 1967; Dikstein,
37 °
MARAN AND OTHERS
Protozoology, Third Int. Cong. Protozool., Leningrad, p. 165. Leningrad: Nauka. SL~mH, M. A. (197o), ' Some factors affecting the excitation of contraction in Spirostomum ', Acta Protozool., 7, 335-352. SOTELO, R., and TRUJILLO-CENoz, O. (I959), ' The fine structure of an elementary contractile system ', 07. biophys, biochem. Cytol., 6, 126-127. Suoi, H. (I96o), ' Propagation of contraction in the stalk muscle of Carchesium ', Fac. Sci. Univ. Tokyo, IV, 8, 6o3-615 . TowNEs, M., and BROWN, D. (1965), ' T h e involvement of pH, adenosine triphosphate,
calcium and magnesium in the contraction of the glycerinated Vorticella ', 07. call comp Physiol., 65, 261-269. VALETT~, G., and LECLAm, M. (I 97°), ' Sensibilization par les inhibiteurs mdtaboliques de l'intestin isol6 de rat aux effets des Sympathomim6tiques ', 07. Pharmac., Paris, x, 445-456.
Key Word Index: Vorticdla, chemopharmacodyamics, nicotine, papaverine, Ca 2+, myoneme contraction.