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Effects of cholinergic and anticholinergic drugs on the synthesis of silk fibroins of spiders
Comp. Biochem. Physiol., 1964, Vol. 12, pp. 465 to 470. Pergamon Press Ltd. Printed in Great Britain
E F F E C T S OF C H O L I N E R G I C A N D A N T I C H O L I N E R G I C D R U G S ON T H E S Y N T H E S I S OF S I L K F I B R O I N S OF S P I D E R S * D A V I D B. P E A K A L L t l)epartment of Pharmacology, State University of New York, Upstate Medical Center, Syracuse, New York (Received 13 J a n u a r y 1964 ; in revised f o r m 13 M a r c h 1964)
A b s t r a c t - - 1 . The rate of incorporation of C14-alanine into the silk fibroins of the spider (Araneus sericatus) was studied. Emptying the silk glands by pulling out the thread stimulates the gland to greater activity as measured by the rate of incorporation of alanine. 2. Physostigmine, carbachol and paraoxon all increased the rate of incorporation of alanine to about the same extent as emptying the gland. It is therefore considered likely that the activation of this gland is under the control of a cholinergic mechanism. 3. Attempts to depress the rate of synthesis with anticholinergic drugs were largely unsuccessful. INTRODUCTION THE starting point of the work described in this paper was the observation of Witt (1963) that physostigmine increases the amount of protein used in web formation. T h e present study was directed towards answering two questions raised by this observation. T h e first question was whether the rate of synthesis of web proteins is increased, or could the increase be due to squeezing more silk from the glands by smooth muscle contraction ? T h e second question was: Does physostigmine act as a cholinesterase blocking agent or does it act in some other way ? T h e effect of a range of drugs on silkworms and their thread production has been measured by T a m a n o & Kuriaki (1961). T h e y found that thread production was increased by pilocarpine and decreased by atropine. T h e silk gland of the spider is, in one respect, fundamentally different from that of the silkworm. I n the spider, silk is produced continuously throughout the life of the animal, while silkworms produce silk only in one single continuous cycle, silk production being largely confined to a 14 day period of their life. T h e effects of drugs on web building of spiders has been studied by Witt (1956, 1963). Hokin & Hokin (1959) have found that enzyme secretion in the pancreatic tissues of pigeons was increased by cholinergic agents. * This investigation was supported in part by a research grant PN-5234 from the National Institutes of Health, U.S. Public Health Service. t Supported by U.S. Public Health Service Institutional Grant. 31
465
466
DAVID B. PEAKALL
MATERIALS AND METHODS draneus sericatus, the species of spider used, were collected locally. Only females were taken as they have been found to be more regular in web building and the capacity of the silk glands may well be sex dependent. The thread proteins were isolated by pulling the silk from the living spider. The end of the thread was attached to a glass rod which was turned by a small motor. The spider was held by hand until all the silk had been rolled onto the glass rod. The spiders appear to be unaffected by this treatment and, if necessary, the procedure can be repeated every few hours to follow drug effects. Histological examination showed that the ampullate glands were emptied by this procedure. This emptying of the silk glands was always carried out 24 hr before the start of any experiment. This was to ensure, as far as possible, that the physiological condition was as uniform as possible. Previous work on web building has shown that the amount of thread used in webs is affected by the preceding sequence and frequency of web building (Breed et al., in press). Uniformly labeled C~I-alanine, 5 mc/kg body weight (Vold CAA-41, 87 mc/mM), was given orally at the start of the experiment. At a predetermined time afterwards, usually 6 hr, the silk glands of the spider were emptied again. The silk was digested for nitrogen determination and radioactive counting. The counts were made using a scintillation well counter; the background counts were about twenty per minute, which was never more than 15 per cent of the counts measured. The nitrogen was determined colorimetrically after digestion with selenium sulfuric acid, using Nessler's reaction. Full details are given in Breed et al. (in press). Physostigmine, 1 mg/kg (as physostigmine sulfate, Merck), was given orally in sugar water. The drug was given immediately preceding the labeled alanine. Two sets of experiments were made. In the first the glands of the spiders were re-emptied just before the start of the experiment. In the other no further emptying was done and the experiment started with the administration of the physostigmine and alanine. Both groups consisted of an equal number of drugged spiders and controls. The effect of physostigmine on the rate of synthesis in time was studied by pulling the thread from the spider at various time intervals after the administration of physostigmine. Carbachol, 100 mg/kg (K & K Laboratories), was given orally. The spiders were not re-emptied at the start of the experiment, merely divided into drugged animals and controls. Paraoxon, (}.2 mg/kg (kindly supplied by Dr. O'Brien of Cornell University), dissolved in glycerine, was injected. Atropine, 1 g/kg (as atropine sulfate, Mallinckrodt), was given both orally and by injection. Homatropine, 5 g/kg (as homatropine hydrobromide, Merck), was given orally. Mecamylamine, 2 mg/kg (as mecamylamine hydrochloride, Merck), was given orally. RESULTS The results of the incorporation experiments with cholinergic drugs are summarized in Table 1. The results are given in counts per minute indicating the amount of radioactive material incorporated into the silk fibroins. The nitrogen
467
C H O L I N E R G I C DRUGS AND S I L K F I B R O I N SYNTHESIS
content is also given in some cases. However, in the experiments in which the spiders were not pre-pulled at the start of the experiment the nitrogen value would consist of silk produced in the 24 hr preceding the start as well as that produced during the experiment. T h u s the nitrogen figures would be diluted with old protein and cannot be compared directly with the radioactive counts. Since the TABLE
1--AMOUNT
OF I N C O R P O R A T I O N OF C 1 4 - A L A N I N E I N T O WEB P R O T E I N
~I~HE LABELED A L A N I N E WAS GIVEN AT THE START OF THE EXPERIMENT. FOR D E T E R M I N A T I O N S
Treatment
Unpulled, no drug Unpulled, physostigmine Unpulled, carbachol Unpulled, paraoxon Pulled, no drug Pulled, physostigmine
6 hr
DURING
6 hr.
T H R E A D WAS PUI.LED
AFTERWARDS
No. of spiders
Activity counts/min
Standard error of the mean
Micrograms nitrogen/100 mg body wt
Standard error of the mean
15 18 14 6 18 20
123 290 301 329 314 346
11.1 27'4 25"4 60-7 13"5 17"9
----15'0 16.2
----1"4 1.6
start of the experiment was the administration of labeled alanine, the counts per minute furnish a direct measure of the amount of new synthesis. For those experiments in which the silk glands were emptied immediately before the start of the experiment, direct comparison with the nitrogen value can be made. In these cases the nitrogen values are given. A comparison of spiders whose glands are emptied immediately before receiving the labeled alanine with those which were not emptied shows that the removal of thread from the gland stimulates more rapid synthesis of new protein; the rate of C 14 incorporation was more than doubled. In those spiders in which the silk was not pulled at the start of the experiment physostigmine stimulated the gland. Emptying the gland by pulling out thread, or by administration of physostigmine, stimulated the gland to synthesize new protein to approximately the same extent. No significant effect was noted, however, when physostigmine was given to a spider after the gland had been emptied by pulling the thread. T h u s three groups (pulled non-drugged, pulled physostigmine treated and unpulled physostigmine treated) all gave essentially similar amounts of new protein and these were all significantly increased (at the 1 per cent level) over the resting state. T h e r e is no variation in the specific activity (counts/min//zg nitrogen) between the pulled nondrugged and pulled physostigmine-treated animals. T h e effect of carbachol (Table 1, line 3) on the unemptied gland shows an increase (significant at the 1 per cent level) of protein synthesis to approximately the same extent as physostigmine. With paraoxon (Table 1, line 4) a considerable n u m b e r of spiders died. It was difficult to give a dose that was effective without killing the spider. T h e increase
468
DAWD B. PEAKALL
f o u n d was as g r e a t as w i t h p h y s o s t i g m i n e , b u t t h e v a r i a t i o n was m u c h l a r g e r a n d t h e low n u m b e r o f s p i d e r s s u r v i v i n g t h e e x p e r i m e n t s m e a n t t h a t t h e s a m p l e was r a t h e r small. T h e i n c r e a s e was significant o n l y at t h e 5 p e r c e n t level. TABLE
2--AMOUNT
OF I N C O R P O R A T I O N OF C I " I - A L A N I N E I N T O WEB P R O T E I N D U R I N G
T H E LABELED A L A N I N E YVAS GIVEN AT THE START OF THE EXPERIMENT. DETERMINATIONS
Treatment at start of experiment Physostigmine Physostigmine Physostigmine Physostigmine Physostigmine Physostigmine Gland emptied Gland emptied Gland emptied (;land emptied Carbachol (oral) Carbachol (oral)
Pretreatment if any Atropine (oral) Control Atropine (injected) Control Homatropine (oral) Control Atropine (oral) Control Mecamylamine Control Atropine(injected) Control
6 hr
6 hr.
T H R E A D WAS P U L L E D FOR
AFTERWARDS
Standard Micrograms Standard No. of Activity error of nitrogen/100 error of spiders counts/min the mean mg body wt the mean 11 10 5 4 5 4 12 9 12 10 6 7
226 246 304 282 231 259 226 254 273 304 110 245
33"6 20.1 28.2 34"1 32"8 32.8 31"7 23'9 33'8 29.1 8.8 32.0
-----13"6 14.3 -----
------1'7 1-3 -----
T h e results of t h e i n c o r p o r a t i o n e x p e r i m e n t s w i t h a n t i c h o l i n e r g i c d r u g s are s u m m a r i z e d in T a b l e 2. T h e effects of a t r o p i n e were s t u d i e d on s p i d e r s w h o s e g l a n d s h a d b e e n s t i m u l a t e d b y p h y s o s t i g m i n e or b y e m p t y i n g t h e glands. B o t h oral a n d injection r o u t e s w e r e i n v e s t i g a t e d , a n d a l t h o u g h on occasions a slight d e c r e a s e of p r o t e i n s y n t h e s i s was n o t e d , this was not significant. T h e effects of h o m a t r o p i n e on p h y s o s t i g m i n e - s t i m u l a t e d s p i d e r s was also f o u n d n o t to b e significant. H o w e v e r , c a r b a c h o l s t i m u l a t i o n was b l o c k e d b y p r e t r e a t m e n t w i t h a t r o p i n e . T h e results w e r e significant at t h e 1 p e r c e n t level, b u t t h e e x p e r i m e n t s w e r e carried out during the winter and a considerable number of the experimental a n i m a l s d i d n o t give silk. T h e s e a n i m a l s w e r e n o t i n c l u d e d in t h e c a l c u l a t i o n s ; thus the data may not be strictly comparable with the other drug experiments w h i c h w e r e c a r r i e d o u t in t h e s u m m e r w h e n silk p r o d u c t i o n is m o r e regular. W i t h l n e c a m y l a m i n e , a g a n g l i o n i c b l o c k i n g agent, no r e d u c t i o n in t h e a m o u n t of i n c o r p o r a t i o n was n o t e d unless the dose was e n o u g h to p r e v e n t t h r e a d f r o m b e i n g d r a w n f r o m t h e spider. U n d e r t h e s e c o n d i t i o n s t h e a n i m a l was s l u g g i s h b u t s u r v i v e d . I n a n u m b e r of cases it was difficult to o b t a i n t h r e a d , b u t w h e n this was d o n e it was f o u n d to c o n t a i n as m u c h l a b e l e d m a t e r i a l as t h e controls. T h e rate of f o r m a t i o n of silk fibroin after e m p t y i n g of the silk g l a n d is s h o w n in Fig. 1 T h e g l a n d s were e m p t i e d at t h e b e g i n n i n g of t h e e x p e r i m e n t a n d t h e n r e p u l l e d a l t e r a given t i m e interval. T h e a m o u n t of increase o f s y n t h e s i s d u e to p h y s o s t i g m i n e is p l o t t e d against t i m e in Fig. 2. Since it was f o u n d t h a t e m p t y i n g
C H O L I N E R G I C DRUGS A N D S I L K F I B R O I N S Y N T H E S I S
40
30
20
10
I l0
0
I 20
I 30
I 40
I 50
FIG. 1. Rate of formation of silk fibroin after emptying the silk glands by pulling out thread. Data obtained by re-emptying after a given time and determining the amount of nitrogen in the pulled thread. Ordinate: amount of nitrogen in /xg/100 mg body weight. Abscissa: time in hours. Each point is the mean of 8-10 determinations. Standard deviation is represented by vertical line.
80
60
40
e_+__+_-+
20
0
I IO
I 20
I 30
I 40
I 50
FxG. 2. Amount of increase of formation of silk fibroin after administration of 1 mg/kg physostigmine. Open circles represent data from animals given physostigmine. Closed circles represent controls. Ordinate: amount of nitrogen in /~g/100 mg body weight. Abscissa : time in hours. Standard deviation is represented by vertical line.
469
470
DAVID B. PEAKALL
the gland stimulates protein synthesis it was not practical to e m p t y the glands at the beginning of the experiment. T h e glands were, however, emptied 24 hr before the start of the experiment as before. T h e spiders were divided into two groups of five, one group were given physostigmine and the other were controls. Both sets of figures are plotted in Fig. 2 and the difference between t h e m is the increase of incorporation due to physostigmine. T h e results of the physostigmine experiments clearly answer the first question : the rate of synthesis of silk protein is about doubled by the administration of physostigmine. A similar increase in the rate of synthesis was found by emptying of the glands and by administration of carbachol. T h e actions of cholinesterase-blocking agents (physostigmine and paraoxon) and of a long-acting drug of acetylcholine type in stimulating synthesis suggest action through an acetylcholine-cholinesterase mechanism. However, work on isolated glands, and on animals with the central nervous system destroyed, will be necessary to determine whether these effects are direct or through the nervous system. Acknowledgement--The author is grateful to Dr. Peter N. Witt for advice and encouragement throughout the course of this work.
REFERENCES BREED A. I,., I,EV1NEV. D., PEAKALI,D. B. & WITT P. N. (1964) The fate of the intact orb web of the spider (Araneus diadematus). Behaviour. In press. ttOKIN M. R. & HoKIN I,. E. (1959) Studies of pancreatic tissues in vitro. Gastroenterology 36, 368-376. TAMANO N. & KURIAKIK. (1961) Applicability of the silkworm for pharmacological studies on the drugs acting on the nervous system, antimitotics and insecticides. Arch. Int. Pharmacodyn. 132, 49-59. Wrrw P. N. (1956) Die Wirkung yon Substanzen auf den Netzbau der Spinne als biologischer Test. Springer-Verlag, Heidelberg. WtTT P. N. (1963) Inter-relationships between web-building behavior and amount of thread material in the spider Araneus diadematus C1. Proc. X U I Intern. Congr. Zool. 2, 7.
E F F E C T S OF C H O L I N E R G I C A N D A N T I C H O L I N E R G I C D R U G S ON T H E S Y N T H E S I S OF S I L K F I B R O I N S OF S P I D E R S * D A V I D B. P E A K A L L t l)epartment of Pharmacology, State University of New York, Upstate Medical Center, Syracuse, New York (Received 13 J a n u a r y 1964 ; in revised f o r m 13 M a r c h 1964)
A b s t r a c t - - 1 . The rate of incorporation of C14-alanine into the silk fibroins of the spider (Araneus sericatus) was studied. Emptying the silk glands by pulling out the thread stimulates the gland to greater activity as measured by the rate of incorporation of alanine. 2. Physostigmine, carbachol and paraoxon all increased the rate of incorporation of alanine to about the same extent as emptying the gland. It is therefore considered likely that the activation of this gland is under the control of a cholinergic mechanism. 3. Attempts to depress the rate of synthesis with anticholinergic drugs were largely unsuccessful. INTRODUCTION THE starting point of the work described in this paper was the observation of Witt (1963) that physostigmine increases the amount of protein used in web formation. T h e present study was directed towards answering two questions raised by this observation. T h e first question was whether the rate of synthesis of web proteins is increased, or could the increase be due to squeezing more silk from the glands by smooth muscle contraction ? T h e second question was: Does physostigmine act as a cholinesterase blocking agent or does it act in some other way ? T h e effect of a range of drugs on silkworms and their thread production has been measured by T a m a n o & Kuriaki (1961). T h e y found that thread production was increased by pilocarpine and decreased by atropine. T h e silk gland of the spider is, in one respect, fundamentally different from that of the silkworm. I n the spider, silk is produced continuously throughout the life of the animal, while silkworms produce silk only in one single continuous cycle, silk production being largely confined to a 14 day period of their life. T h e effects of drugs on web building of spiders has been studied by Witt (1956, 1963). Hokin & Hokin (1959) have found that enzyme secretion in the pancreatic tissues of pigeons was increased by cholinergic agents. * This investigation was supported in part by a research grant PN-5234 from the National Institutes of Health, U.S. Public Health Service. t Supported by U.S. Public Health Service Institutional Grant. 31
465
466
DAVID B. PEAKALL
MATERIALS AND METHODS draneus sericatus, the species of spider used, were collected locally. Only females were taken as they have been found to be more regular in web building and the capacity of the silk glands may well be sex dependent. The thread proteins were isolated by pulling the silk from the living spider. The end of the thread was attached to a glass rod which was turned by a small motor. The spider was held by hand until all the silk had been rolled onto the glass rod. The spiders appear to be unaffected by this treatment and, if necessary, the procedure can be repeated every few hours to follow drug effects. Histological examination showed that the ampullate glands were emptied by this procedure. This emptying of the silk glands was always carried out 24 hr before the start of any experiment. This was to ensure, as far as possible, that the physiological condition was as uniform as possible. Previous work on web building has shown that the amount of thread used in webs is affected by the preceding sequence and frequency of web building (Breed et al., in press). Uniformly labeled C~I-alanine, 5 mc/kg body weight (Vold CAA-41, 87 mc/mM), was given orally at the start of the experiment. At a predetermined time afterwards, usually 6 hr, the silk glands of the spider were emptied again. The silk was digested for nitrogen determination and radioactive counting. The counts were made using a scintillation well counter; the background counts were about twenty per minute, which was never more than 15 per cent of the counts measured. The nitrogen was determined colorimetrically after digestion with selenium sulfuric acid, using Nessler's reaction. Full details are given in Breed et al. (in press). Physostigmine, 1 mg/kg (as physostigmine sulfate, Merck), was given orally in sugar water. The drug was given immediately preceding the labeled alanine. Two sets of experiments were made. In the first the glands of the spiders were re-emptied just before the start of the experiment. In the other no further emptying was done and the experiment started with the administration of the physostigmine and alanine. Both groups consisted of an equal number of drugged spiders and controls. The effect of physostigmine on the rate of synthesis in time was studied by pulling the thread from the spider at various time intervals after the administration of physostigmine. Carbachol, 100 mg/kg (K & K Laboratories), was given orally. The spiders were not re-emptied at the start of the experiment, merely divided into drugged animals and controls. Paraoxon, (}.2 mg/kg (kindly supplied by Dr. O'Brien of Cornell University), dissolved in glycerine, was injected. Atropine, 1 g/kg (as atropine sulfate, Mallinckrodt), was given both orally and by injection. Homatropine, 5 g/kg (as homatropine hydrobromide, Merck), was given orally. Mecamylamine, 2 mg/kg (as mecamylamine hydrochloride, Merck), was given orally. RESULTS The results of the incorporation experiments with cholinergic drugs are summarized in Table 1. The results are given in counts per minute indicating the amount of radioactive material incorporated into the silk fibroins. The nitrogen
467
C H O L I N E R G I C DRUGS AND S I L K F I B R O I N SYNTHESIS
content is also given in some cases. However, in the experiments in which the spiders were not pre-pulled at the start of the experiment the nitrogen value would consist of silk produced in the 24 hr preceding the start as well as that produced during the experiment. T h u s the nitrogen figures would be diluted with old protein and cannot be compared directly with the radioactive counts. Since the TABLE
1--AMOUNT
OF I N C O R P O R A T I O N OF C 1 4 - A L A N I N E I N T O WEB P R O T E I N
~I~HE LABELED A L A N I N E WAS GIVEN AT THE START OF THE EXPERIMENT. FOR D E T E R M I N A T I O N S
Treatment
Unpulled, no drug Unpulled, physostigmine Unpulled, carbachol Unpulled, paraoxon Pulled, no drug Pulled, physostigmine
6 hr
DURING
6 hr.
T H R E A D WAS PUI.LED
AFTERWARDS
No. of spiders
Activity counts/min
Standard error of the mean
Micrograms nitrogen/100 mg body wt
Standard error of the mean
15 18 14 6 18 20
123 290 301 329 314 346
11.1 27'4 25"4 60-7 13"5 17"9
----15'0 16.2
----1"4 1.6
start of the experiment was the administration of labeled alanine, the counts per minute furnish a direct measure of the amount of new synthesis. For those experiments in which the silk glands were emptied immediately before the start of the experiment, direct comparison with the nitrogen value can be made. In these cases the nitrogen values are given. A comparison of spiders whose glands are emptied immediately before receiving the labeled alanine with those which were not emptied shows that the removal of thread from the gland stimulates more rapid synthesis of new protein; the rate of C 14 incorporation was more than doubled. In those spiders in which the silk was not pulled at the start of the experiment physostigmine stimulated the gland. Emptying the gland by pulling out thread, or by administration of physostigmine, stimulated the gland to synthesize new protein to approximately the same extent. No significant effect was noted, however, when physostigmine was given to a spider after the gland had been emptied by pulling the thread. T h u s three groups (pulled non-drugged, pulled physostigmine treated and unpulled physostigmine treated) all gave essentially similar amounts of new protein and these were all significantly increased (at the 1 per cent level) over the resting state. T h e r e is no variation in the specific activity (counts/min//zg nitrogen) between the pulled nondrugged and pulled physostigmine-treated animals. T h e effect of carbachol (Table 1, line 3) on the unemptied gland shows an increase (significant at the 1 per cent level) of protein synthesis to approximately the same extent as physostigmine. With paraoxon (Table 1, line 4) a considerable n u m b e r of spiders died. It was difficult to give a dose that was effective without killing the spider. T h e increase
468
DAWD B. PEAKALL
f o u n d was as g r e a t as w i t h p h y s o s t i g m i n e , b u t t h e v a r i a t i o n was m u c h l a r g e r a n d t h e low n u m b e r o f s p i d e r s s u r v i v i n g t h e e x p e r i m e n t s m e a n t t h a t t h e s a m p l e was r a t h e r small. T h e i n c r e a s e was significant o n l y at t h e 5 p e r c e n t level. TABLE
2--AMOUNT
OF I N C O R P O R A T I O N OF C I " I - A L A N I N E I N T O WEB P R O T E I N D U R I N G
T H E LABELED A L A N I N E YVAS GIVEN AT THE START OF THE EXPERIMENT. DETERMINATIONS
Treatment at start of experiment Physostigmine Physostigmine Physostigmine Physostigmine Physostigmine Physostigmine Gland emptied Gland emptied Gland emptied (;land emptied Carbachol (oral) Carbachol (oral)
Pretreatment if any Atropine (oral) Control Atropine (injected) Control Homatropine (oral) Control Atropine (oral) Control Mecamylamine Control Atropine(injected) Control
6 hr
6 hr.
T H R E A D WAS P U L L E D FOR
AFTERWARDS
Standard Micrograms Standard No. of Activity error of nitrogen/100 error of spiders counts/min the mean mg body wt the mean 11 10 5 4 5 4 12 9 12 10 6 7
226 246 304 282 231 259 226 254 273 304 110 245
33"6 20.1 28.2 34"1 32"8 32.8 31"7 23'9 33'8 29.1 8.8 32.0
-----13"6 14.3 -----
------1'7 1-3 -----
T h e results of t h e i n c o r p o r a t i o n e x p e r i m e n t s w i t h a n t i c h o l i n e r g i c d r u g s are s u m m a r i z e d in T a b l e 2. T h e effects of a t r o p i n e were s t u d i e d on s p i d e r s w h o s e g l a n d s h a d b e e n s t i m u l a t e d b y p h y s o s t i g m i n e or b y e m p t y i n g t h e glands. B o t h oral a n d injection r o u t e s w e r e i n v e s t i g a t e d , a n d a l t h o u g h on occasions a slight d e c r e a s e of p r o t e i n s y n t h e s i s was n o t e d , this was not significant. T h e effects of h o m a t r o p i n e on p h y s o s t i g m i n e - s t i m u l a t e d s p i d e r s was also f o u n d n o t to b e significant. H o w e v e r , c a r b a c h o l s t i m u l a t i o n was b l o c k e d b y p r e t r e a t m e n t w i t h a t r o p i n e . T h e results w e r e significant at t h e 1 p e r c e n t level, b u t t h e e x p e r i m e n t s w e r e carried out during the winter and a considerable number of the experimental a n i m a l s d i d n o t give silk. T h e s e a n i m a l s w e r e n o t i n c l u d e d in t h e c a l c u l a t i o n s ; thus the data may not be strictly comparable with the other drug experiments w h i c h w e r e c a r r i e d o u t in t h e s u m m e r w h e n silk p r o d u c t i o n is m o r e regular. W i t h l n e c a m y l a m i n e , a g a n g l i o n i c b l o c k i n g agent, no r e d u c t i o n in t h e a m o u n t of i n c o r p o r a t i o n was n o t e d unless the dose was e n o u g h to p r e v e n t t h r e a d f r o m b e i n g d r a w n f r o m t h e spider. U n d e r t h e s e c o n d i t i o n s t h e a n i m a l was s l u g g i s h b u t s u r v i v e d . I n a n u m b e r of cases it was difficult to o b t a i n t h r e a d , b u t w h e n this was d o n e it was f o u n d to c o n t a i n as m u c h l a b e l e d m a t e r i a l as t h e controls. T h e rate of f o r m a t i o n of silk fibroin after e m p t y i n g of the silk g l a n d is s h o w n in Fig. 1 T h e g l a n d s were e m p t i e d at t h e b e g i n n i n g of t h e e x p e r i m e n t a n d t h e n r e p u l l e d a l t e r a given t i m e interval. T h e a m o u n t of increase o f s y n t h e s i s d u e to p h y s o s t i g m i n e is p l o t t e d against t i m e in Fig. 2. Since it was f o u n d t h a t e m p t y i n g
C H O L I N E R G I C DRUGS A N D S I L K F I B R O I N S Y N T H E S I S
40
30
20
10
I l0
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FIG. 1. Rate of formation of silk fibroin after emptying the silk glands by pulling out thread. Data obtained by re-emptying after a given time and determining the amount of nitrogen in the pulled thread. Ordinate: amount of nitrogen in /xg/100 mg body weight. Abscissa: time in hours. Each point is the mean of 8-10 determinations. Standard deviation is represented by vertical line.
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FxG. 2. Amount of increase of formation of silk fibroin after administration of 1 mg/kg physostigmine. Open circles represent data from animals given physostigmine. Closed circles represent controls. Ordinate: amount of nitrogen in /~g/100 mg body weight. Abscissa : time in hours. Standard deviation is represented by vertical line.
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DAVID B. PEAKALL
the gland stimulates protein synthesis it was not practical to e m p t y the glands at the beginning of the experiment. T h e glands were, however, emptied 24 hr before the start of the experiment as before. T h e spiders were divided into two groups of five, one group were given physostigmine and the other were controls. Both sets of figures are plotted in Fig. 2 and the difference between t h e m is the increase of incorporation due to physostigmine. T h e results of the physostigmine experiments clearly answer the first question : the rate of synthesis of silk protein is about doubled by the administration of physostigmine. A similar increase in the rate of synthesis was found by emptying of the glands and by administration of carbachol. T h e actions of cholinesterase-blocking agents (physostigmine and paraoxon) and of a long-acting drug of acetylcholine type in stimulating synthesis suggest action through an acetylcholine-cholinesterase mechanism. However, work on isolated glands, and on animals with the central nervous system destroyed, will be necessary to determine whether these effects are direct or through the nervous system. Acknowledgement--The author is grateful to Dr. Peter N. Witt for advice and encouragement throughout the course of this work.
REFERENCES BREED A. I,., I,EV1NEV. D., PEAKALI,D. B. & WITT P. N. (1964) The fate of the intact orb web of the spider (Araneus diadematus). Behaviour. In press. ttOKIN M. R. & HoKIN I,. E. (1959) Studies of pancreatic tissues in vitro. Gastroenterology 36, 368-376. TAMANO N. & KURIAKIK. (1961) Applicability of the silkworm for pharmacological studies on the drugs acting on the nervous system, antimitotics and insecticides. Arch. Int. Pharmacodyn. 132, 49-59. Wrrw P. N. (1956) Die Wirkung yon Substanzen auf den Netzbau der Spinne als biologischer Test. Springer-Verlag, Heidelberg. WtTT P. N. (1963) Inter-relationships between web-building behavior and amount of thread material in the spider Araneus diadematus C1. Proc. X U I Intern. Congr. Zool. 2, 7.