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Secretory effect of the venom of the scorpion Tityus trinitatis on rat pancreatic slices
Tosfeo~, 1977, VoL 13, yp" ~41~6. Paraamon Pras. Prlnted iu C3reat Brl4tn
SECRETORY EFFECT OF THE VENOM OF THE SCORPION TITYUS TRINITATIS ON RAT PANCREATIC SLICES H. $ANKARAN, C. BAx~rxol.o~w, O. FrrzGERALn and K. F. McG>~lv>:~r Department of Medicine and Therapeutics, University College, Dublin 4, Ireland (Acceptedforpubllcatlon 28 March
1977)
H. S~xxwn~x, C. BARTHOLOMEW, O. FrrzGete~+r~ andK. F. McGEexsr . Secretory effect of the venom of the scorpion Tltyus trlnltatis on rat pancreatic slices . Toxlcon 15, 441446, 1977 .A linear concentration response relationship was found for amylase release from rat pancreatic slices by venom of the scorpion 7Ytyus trlnitatis. Maximum release of amylase was caused by 20 Rg venom per ml of medium. Acetylcholine, used as a standard stimulant had a maximum effect at 3 x 10- ' M. Atropine partially blocked both venom and acetylcholine induced release of amylase. Nicotine, hexamethonium and tubocurarine did not affect this stimulated amylase release, nor did they alter the non~timulated (basal) release of amylase. Physostigmine potentiated the venom stimulated amylase release but had no effect on nonstimulated release. These observations suggest that the venom exerts its secretory effect through a cholinergic mechanism which may involve muscarinic receptors. INTRODUCITON
Tim vsnloM of the scorpion, Tityus trinitatis causes acute pancreatitis in man (WATERMAN, 1938 ; Poox-KING, 1963 ; BAlt~rxol.oi~w, 1970). In anaesthetised dogs this vencm caused an increase in both volume and enzyme output from the pancreas (BARTHOLOMEW et al., 1974). The venom also caused exocrine secretion in the totally isolated perfused canine pancreas (BARTHOLOMEW et al., 1976). An isolated preparation of the canine sphincter of Oddi contracted in response to the venom (SANKARAN et al., 1976). These investigations on the aetiology of venom-induced pancreatitis have been carried out employing dif%rent techniques (organ bath experiments and whole animal or perfused organ) where it was not possible to standardise the stimulatory effect of the venom on amylase release with acceptable accuracy . Mouse pancreatic fragments have been employed to study the sequence of events leading to amylase release by acetylcholine (ACh) (MATTHEWS et al., 1973) and to characterise the nature of receptors in the acinar cells (WILLIAMS, 1975). It was therefore thought that tissue slices might not only overcome the difficulty in standardising the venom effect but also might give an insight into the venom-acinar cell interaction . Hence, the present investigation was aimed to investigate the concentration-response relationship for the stimulatory action of the venom in rat pancreatic slices, and to examine scme aspects of the mode of amylase release by the venom. MATE~tIATS AND MBTHODS Sprague-Dawley rats (150-200 g), fasted for 20 hr (with water ad lib.), were killed by a blow on the head and the pancreas was quickly removed and trimmed. The adhering fatty material was removed by blunt dissection . The tissue (roughly 400 mg per gland) was cut into 4 slices of 100 mg each and suspended in physiological salt solution (pH 7"4) of following composition (mM) NaCI 103, KCI 4~7, CaCI, 256, MgCI, 1 "13, NaHCO, 25, NaH,PO, 1"15, rrGlucose 2"8, Na Pyruvate 4"9, Na Fumarate 2"7, Na Glutamate 4"9 and equilibrated with 9S ~ 0, and S ~ CO, (MwTrm?ws et al ., 1973). Tr~sylol® (ROHBERECFrr and C~iRL4ToP~, 1971) 500 i.u./ml of medium was used to prevent protein breakdown in the incubate and tissue homogenate. Following 3 washings with medium the pancreatic slices were incubated fo a 10 min period to 441
442
H. SANKARAN, C. BARTHOLOMEW, O. FITZGßRALD and K. F. McGEENEY
remove blood adhering to the tissue and amylase which had leaked from the damaged tissue . Each 100 mg piece of tissue was sliced into 25 mg slices and the tissue slices pooled . Tissue slices (100 mg) in preincubation vessels were gassed with 95 ~ O, and 5 ~ CO, and incubated for 30 min at 37 °C in a metabolic shaker at 120 oscillations per min. The slices were washed twice with 3~0 ml vol of fresh medium, blotted and transferred to incubation vessels containing 3~0 ml medium or 3~0 ml medium plus additive . The tissue slices were then re~quilibrated with 95 ~ O, and 5 ~ CO and incubated as described above for 30 Inin . At the end of incubation the tissue slices were removed by filtration on a nylon-mesh filter . Medium (2~0 ml) was used to rinse the incubation vessel and to wash the tissue on the filter . The 5 ml filtrate (incubate) was stored at 4°C for amylase assay. The tissue was re-suspended in 2 ml of medium and homogenized with an UltraTurrax . The homogenization tube was further rinsed with 3 ml of medium and added to the homogenate . The homogenate volume was made up to 10 ml with medium and centrifuged at 1500 x g at 4°C for 10 min and amylase assayed in the supernatant . The amylase levels of the incubates and the corresponding homogenates were estimated by the Phadebastablet method (CESKA et al., 1969). The sum of the amylase levels in the incubate and the corresponding tissue homogenate gave the total all~ylase content. The secretory response of the tissue was expressed as a percentage of the total amylase content. The response to a stimulant was indicated by the increase in this figure . Investigation of the effect of cholinergic agonist and antagonists on venom-induced amylase release was carried out as follows. After preincubation, 100 IIIg of tissue slices were transferred to vessels containing medium alone (basal release), 3 x 10 - ' M ACh~hloride, venom (201Ig/ml), 3 x 10'' M ACh~hloride plus cholinergic antagonist, venom (20 Wg/ail) plus cholinergic antagonist, or medium containing the highest concentration of cholinergic agonist or antagonist . Acetylcholine chloride, physosdgmine, nicotine, hexamethonium bromide, atropine and D-tubocurarine chloride pentahydrate were supplied by Sigma Chemicals, London, U.K . Trasylol® was obtained from Bayer. All the chemicals used were of analer quality. Venom obtained from the scorpion Tityus trinitatis by electrical stimulation was stored desia:ated at - 20°C . RESULTS
(a) Acetylcholine stimulated amylase release
The tissue slices were incubated with medium containing ACh (3 x 10 - ° M-3 x 10 - a M). The results presented in Fig. 1 show that the peak response was observed at 3 x 10- ' M . 15
°
ô â
10
0 9
//
a 0
5
v 0 T Q
0
3x10
_s
3a10
e
M acetylcholine
3alÔ ~
3a10
s
3i10_5
chloride
CONCENTRATION-RESPONSE RELATIONSHIP BETWEEN ACh IN INCUBATION MEDIUM AND AMYLASE OUTPUT BY TAE PANCREATIC SLICES . Data are presented as amylase secreted (~ of total) by 100 mg (wet wt) of rat pancreatic slices in modified Krebs-bicarbonate medium containing ACh~hloride. Each point is mean f S.E .M . No. of observations is indicated in circles.
FIO . 1 .
Scorpion Venom on Amylase Release .
443
(b) Effect of venom on amylase output
Tissue slices were incubated with medium containing crude venom at concentrations ranging from 5 to 100 Fig per ml of incubation medium. The venom stimulated release of amylase from the tissue slices was linear up to a venom concentration of 20 ~g/ml. Although venom at 50 and 100 Fig per ml of medium stimulated the tissue slices to release amylase over the basal level, the stimulated output was well below the maximal effect produced by 20 4tg/ml (Fig. 2).
Venom ( yg/ml )
Fta . 2 . VENOM-IIdDUCED AMYLASE OUTPUT . Amylase secreted (as ~ of total) by 100 mg wet wt of rat pancreatic slices in modified Krebs-bicarbonate medium containing different concentrations of venom . Each point is mean f S .E .M . Number of observations is indicated in circles. Amylase output plotted against venom concentration on the same scale as that for ACh (Fig. 1) to show the similarity between the two curves .
To investigate the mode of amylase release by the venom, the effect of atropine and other cholinergic antagonists and agonista on venom-induced amylase release was studied. In all these studies, the effect of cholinergic agonists or antagonists on ACh-induced amylase release was carried out in parallel, for comparison. The concentration producing the maximal amylase release from the tissue slices was chosen as the concentration of the venom or ACh for subsequent studies. Atropine (3 x 10-° M) did not have an effect on the nonstimulated release of amylase by the tissue slices, while 3 x 10-° and 1~5 x 10-° M concentrations produced an inhibition of the venom or ACh induced amylase release by 80 and over 50~ respectively. The ~ figures for atropine inhibition was calculated by comparing the net amylase release figures in presence and absence of atropine for ACh or venom and expressing the difference between the two in each case as a ~ inhibition of that of the net value for stimulated release (Table 1). The effect of hexamethonium, nicotine and tubocurarine on secretion is shown in Tables 2, 3 and 4. These agents did not have an effect on the basal release of amylase by the tissue slices. No alteration in the ACh or venom-induced amylase release was observed in presence ofthese drugs. ' Physostigmine Sulphate (Eserine), 3 x 10 -° M did not alter the basal release of amylase, but potentiated ACh (3 x 10- ° M) or venom (5 pg/ml}-induced amylase release. Thepotentiation was 70 and 57~ respectively . As Frserine is a cholinergic potentiating agent the concentration of ACh or venom selected for this aspect of the study was below the concentration that produced the peak effect . In the case of venom the concentration of 5 ltg/ml was the lowest concentration on the linear part of the concentration-response curve. The results are given in Table 5.
444
H. SANKARAN, C. BARTHOLOMBW, O. FITZGERALD and K. F. McGEENEY TABLE I . EFFECT OF ATROPINE
Agent Medium (Basal) ACh (3 x 10 - ' M)
Amylase secreted ( ~ of total) by 100 mg wet wt of rat pancreatic slices in presence of atropino 0 3x10-'M 1"5x10-'M
6~5 f 0"8 (~ "14 "4 f 0"5 (~ *
6"9 ~ 1"4 (3) b7"8 f 02 (3)
~9 "9 f 1"2 (~*
Medium (Basal) 8"5 f 0~4 (9) 8"7 f Oß (~ Venom(20tIg/ml) "15~9 ~ 0" 2 (~* b9 "8 f 1~1 (~ ~12"4 f 0"S (9)* Values are mean f S.E .M . * Denotes significance (calculated using Student's t-test) of difference from the respective basal value in each group (P values < 0"05). a and b (P < 0"001), a and c (P < 0"01), b and c are not significantly different. Number of observations shown in parentheses .
Agent Medium (Basal) ACh (3 x 10-'M)
TABLE 2. EFFECT OF HSXAIIIR1IiONIUM Amylase secreted ( ~ of total) by 100 mg wet wt of rat pancreatic slices in presence of hexamethonium bromide 0 5 x 10-'M 2 x 10-' M 1 x 10 - 4 M
6"2 f 2"0 (6) 6"4 f 0~9 (6) 11 "9 f 1~6 (~* 13 "8 f lß (5)*
12" 6 ~ 2~1 (S)*
13 "8 f 2~7 (~*
Medium (Basal) a6 "2 ~ 1"1 (~ b8~4 ~ 1"7 (3) Venom (20tIg/ml) l2ß f Oß (3)* 150 f 03 (4)* 12 " 6 f 0"4 (~* 11 "4 ~ 1 "4 (3)* Values are mean f S.E.M . * Denotes significance (calculated using Student's t-test) of difference from the respectivo basal value in each group (P values < 0"02). a and b are not significantly different. Number of observations shown in parentheses .
Agent Medium (Basal) ACh (3 x 10 - ' M)
TABLE 3. EFFECT of NICOTINE Amylase secreted (~ of total) by 100 mg wet wt of rat pancreatic slices in presence of nicotine 0 3x10-'M 3x10- 'M 3x10-°M 3x10 - 'M
5"1 ~ 0"7 (9) 4"8 f 1~0 (~ 8"6 f 0"6 (~* 9~5 ~ 0"8 (~*
9"4 f 1~0 (~*
9"2 f 1" 1 (8)* 10"1 f 0"5 (~*
Medium (Basal) 6ß f P4 (13) 6"0 f 0"9 (8) Venom (20 tIg/ml) 9"5 f 0~7 (12)* 9"5 f 0"8 (14)* 10"6 f 0"8 (14)* 10"9 f 0"7 (13)* 10~0 f 1"5 (8)* Values are mean f S.E .M. * Denotes significance (calculated using Student's t-test) of difference from the respective basal value in each group (P values < 0"02). Number of observations shown in parentheses . TABLE
Agent Medium (Basal) ACh (3 x 10 -' M)
4.
EFFECT
of
TIteoCaRARIrtE
Amylase secreted (~ of total) by 100 mg wet wt of rat pancreatic slices in presence of D-tubocurarine 0 3 x 10 -5 M 3 x 10-' M 3 x l0 - ° M 3 x 10-° M 4"9 f 0"7 (4) 9"2 f 2ß (3)*
4~5 f 0"7 (S) 8"8 f 0"6 (~*
7ß f Oß (3)*
8"9 f 0"6 (4)*
9"2 f lß (3)*
Medium (Basal) 8"2 f 0"8 (9) 8"8 f 1"1 (~ Venom (201Ig/ml) 11 "2 ~ 0" 4 (~* 13~1 f 0"7 (~* 11 "1 f 0"7 (8)* 12 "6 f 12 (~* 11~8 f 1"6 (8)* Values are mean f S.E .M . * Denotes significance (calculated using Student's t-test) of difference from the respective basal value in each group (P values < 0" 02). Number of observations shown in parentheses . DISCUSSION
Scorpion venom (T. serrulatus and T. bahyetrsis) has a cholinergic action on smooth (DtNlz and VAi.>~axi, 1959 ; Dnvlz and TORxES, 1968) and cardiac muscle (CORRADO et al., 1968). The hypertensive effect of the venom of T. serrulatus was suggested to be mediated
Scorpion Venom on Amylase Release
Agent
445
T~ S. EFrec,-r oa manu$ Amylase secreted (~ of total) by 100 mg wet wt of rat pancreatic slices in presence of eserine 0 3 x 10 - ' M
Medium (Basal) 5~4 ~ 0~4 (9) 5~3 f 0~5 (4) AC;hh (3 x 10-9 M) 7~0 f 0~4 (4)* fi10~5 f l'6 (4)* Venom (5 pg/ml) 6'8 f 0'3 (~* fi8'7 f 0~5 (4)* Valuos ara mean f S.B .M . * Denotes significance (calculated using Student's t-test) of difference from the basal value (P values < 002) . fi Denotes significance of difference from the values for the corresponding stimulant without eserine. Number of observations shown in parentheses .
by ACh (CORRADO et al., 1974). Goa~z et al. (1975) reported stimulation of ACh release from rat brain cortical slices by tityustoxin (TsTX). The venom-induced (T. trinitatis) contraction of sphincter of Oddi was abolished by atropine (SANICARAN et al., 197 . These venom effects seem to be ACh-mediated. In the present study on the secretory effect of T. trinitatis, rat pancreatic slices have proven to be a suitable tool to study concentrationresponse effects of the venom on acinar cells. Although there is a significant variation in the basal (non-stimulated~alues between groups, the values within the groups for nonstimulated and stimulated release are compatible . Thus, ACh, employed in this study as a standard stimulant of pancreatic acinar cells, showed a concentration-response curve between 3 x 10 - ° and 3 x 10 -6 M with the peak effect at 3 x 10 - ' M (Fig. 1). This observation is strikingly similar to that Of Wrr .r .rAS,rc (1975) who employing mouse pancreatic fragments found the peak response at 3 x 10 - ' M. The venom-induced amylase release followed a similar pattern ; the concentration-response curve was linear up to 20 ~.g/ml. The amylase release obtained with 50 and 100 lAg venomJml of medium was consistently below the peak effect produced by 20 4tg/ml of medium. (Fig. 2.) The venom-induced amylase release seems to be mediated through a cholinergic mechanism. This is borne out by the observation that eserine potentiatea the venom and AChinduced amylase release. Cholinergic involvement in venom action is further demonstrated by the partial abolition of venom-induced amylase release with atropine . MAZ-n-n:ws et al. (1973) in their studies on ACh-induced membrane depolarisation, calcium e~ux and amylase release in mouse pancreas suggested the interaction of ACh with the muscarinic receptor of the cell followed by Na+ and Ca'+ flux, depolarization and amylase release. While discussing the nature of receptors in the acinar cells of mouse pancreatic fragments Wü .i .iAMC (1975) concluded that the cholinergic receptor mediating amylase release is muscarinic . Our studies indicate the absence of nicotinic receptors in rat pancreatic slices, as nicotine (3 x 10 -6 M) had no effect on either the basal release or ACh-induced release in agreement with the report of Wic.Lmhts (1975), nor did it affect the venom-induced amylase release. Hexamethonium and tubocurarine also had no effect on the basal release or the release stimulated by ACh or venom. These observations clearly rule out the possibility of the venom exerting its effect through the ganglion or at the nerve-organ level. This in combination with the partial inhibition of venom action by atropine and potentiation by eserine strongly suggests that the venom exerts its effect through the cholinoceptive receptors which are muscarinic in nature . Acknowledgements-We thank Mr. N. O'Corrrrox and Mr. A. Gtna.Ex for technical assistance. This work was supported by a grant from the Wellcome Trust, London .
446
H. SANKARAN, C. BARTHOLOMEW, O. FITZGERAI ;D and K. F. McGEENEY
REFERENCES B~erxoi.on~w, C. (1970) Acute scorpion pancreatitis in Trinidad . Br. med. J. 1, 666. B.~x~rxor.oMEw, C., Muxrxsr, J. J., McGsexEr, K. F. and FrrzGEtea~, O. (1974) Pancreatic response to Tityus trinitatis venom. W. Indime Med. J. 23, 41 . BARTHOLOI~~V, C., McGsexev, K. F., MuierxY, J. J., FrrzGenu.n, O. and Snxxexnrr, H. (1976) Experimental studies on the aetiology of acute scorpion pancreatitis. Br . J. Sung. 63, 807. Cesxw, M., Hui.~x, E. and Ixoer tKnx, B. G. A. (1969) A new method for determination of alpha amylase. Experientia 25, 555. Coxxnno, A. P., Ax~rorno, A. and Dixtz, C. R. (1968) Brazilian Scorpion venom (Tityus serrulatus), an unusual sympathetic post-ganglionic stimulant. J. Pharmac. exp. Ther.164, 253. Coxx~oo, A. P., Ricctorro Ns~ro, F. and Axrorno, A. (1974) The mechanism of the hypertensive effect of Brazilian Scorpion venom (7Styus serrulatus Lutz E. Mello) . Toxicon 12, 145. Dixiz, C. R. and Va~tu, V. (1959) Effects of a toxin present in a purified extract of telsons from the scorpion Tityus serrulatus on smooth muscle preparations, and in mice . Archs. tnt. Pharmacodyn . 7irér. 71, 1 . Dixiz, C. R. and Toes, J. M . (1968) Release of an acetylcholine-like substance from guinea pig ileum by scorpion venom. Toxicon S, 277. Gon~z, M. V., DiNtz, C. R. and Hnnaosa, T . S. (1975) A comparison of the effects of scorpion venom tityustoxin and Ouabain on the release of acetykholine from incubated slices of rat brain. I. Neurodttm. 24, 331. M~~rr~ws, E. K., P~xsox, E. H. and WILLiAMB, J. A. (1973) Pancreatic acinar cells. Acetylcholineinduced membrane depolarization, calcium eHiux and amylase release. J. Physiol. 234, 689. Poox-Knvo, T. (1963) Myoearditis from scorpion stings . Br. med. J. 1, 374. RoHaexecfrr, P. and C~srorxe, J. (1971) Secretion of hydrolases by perfused fragments of rat pancreas : effect of calcium. Am . J. Physiol. 220, 911. SnxKnnnx, H., FrrzGEtutu, O., BaRTxotoe~w, C. and McG~xEV, K. F. (1976) Action of the venom of 7Ytyus trinitatLs on the isolated sphincter of Oddi . Ir. J. med. Sci.145, 97. W~~R~N, J. A. (1938) Some notes on scorpion poisoning in Trinidad . 7}ans. R. Soc. trop . Med. Hyg. 31, 607. Wuu~tifs, J. A. (1975) An in vitro evaluation of possible cholinergic and adrenergic n~ceptors affecting pancreatic amylase secretion. Proc. Soc. exp. Biol . Med. ISO, 513 .
SECRETORY EFFECT OF THE VENOM OF THE SCORPION TITYUS TRINITATIS ON RAT PANCREATIC SLICES H. $ANKARAN, C. BAx~rxol.o~w, O. FrrzGERALn and K. F. McG>~lv>:~r Department of Medicine and Therapeutics, University College, Dublin 4, Ireland (Acceptedforpubllcatlon 28 March
1977)
H. S~xxwn~x, C. BARTHOLOMEW, O. FrrzGete~+r~ andK. F. McGEexsr . Secretory effect of the venom of the scorpion Tltyus trlnltatis on rat pancreatic slices . Toxlcon 15, 441446, 1977 .A linear concentration response relationship was found for amylase release from rat pancreatic slices by venom of the scorpion 7Ytyus trlnitatis. Maximum release of amylase was caused by 20 Rg venom per ml of medium. Acetylcholine, used as a standard stimulant had a maximum effect at 3 x 10- ' M. Atropine partially blocked both venom and acetylcholine induced release of amylase. Nicotine, hexamethonium and tubocurarine did not affect this stimulated amylase release, nor did they alter the non~timulated (basal) release of amylase. Physostigmine potentiated the venom stimulated amylase release but had no effect on nonstimulated release. These observations suggest that the venom exerts its secretory effect through a cholinergic mechanism which may involve muscarinic receptors. INTRODUCITON
Tim vsnloM of the scorpion, Tityus trinitatis causes acute pancreatitis in man (WATERMAN, 1938 ; Poox-KING, 1963 ; BAlt~rxol.oi~w, 1970). In anaesthetised dogs this vencm caused an increase in both volume and enzyme output from the pancreas (BARTHOLOMEW et al., 1974). The venom also caused exocrine secretion in the totally isolated perfused canine pancreas (BARTHOLOMEW et al., 1976). An isolated preparation of the canine sphincter of Oddi contracted in response to the venom (SANKARAN et al., 1976). These investigations on the aetiology of venom-induced pancreatitis have been carried out employing dif%rent techniques (organ bath experiments and whole animal or perfused organ) where it was not possible to standardise the stimulatory effect of the venom on amylase release with acceptable accuracy . Mouse pancreatic fragments have been employed to study the sequence of events leading to amylase release by acetylcholine (ACh) (MATTHEWS et al., 1973) and to characterise the nature of receptors in the acinar cells (WILLIAMS, 1975). It was therefore thought that tissue slices might not only overcome the difficulty in standardising the venom effect but also might give an insight into the venom-acinar cell interaction . Hence, the present investigation was aimed to investigate the concentration-response relationship for the stimulatory action of the venom in rat pancreatic slices, and to examine scme aspects of the mode of amylase release by the venom. MATE~tIATS AND MBTHODS Sprague-Dawley rats (150-200 g), fasted for 20 hr (with water ad lib.), were killed by a blow on the head and the pancreas was quickly removed and trimmed. The adhering fatty material was removed by blunt dissection . The tissue (roughly 400 mg per gland) was cut into 4 slices of 100 mg each and suspended in physiological salt solution (pH 7"4) of following composition (mM) NaCI 103, KCI 4~7, CaCI, 256, MgCI, 1 "13, NaHCO, 25, NaH,PO, 1"15, rrGlucose 2"8, Na Pyruvate 4"9, Na Fumarate 2"7, Na Glutamate 4"9 and equilibrated with 9S ~ 0, and S ~ CO, (MwTrm?ws et al ., 1973). Tr~sylol® (ROHBERECFrr and C~iRL4ToP~, 1971) 500 i.u./ml of medium was used to prevent protein breakdown in the incubate and tissue homogenate. Following 3 washings with medium the pancreatic slices were incubated fo a 10 min period to 441
442
H. SANKARAN, C. BARTHOLOMEW, O. FITZGßRALD and K. F. McGEENEY
remove blood adhering to the tissue and amylase which had leaked from the damaged tissue . Each 100 mg piece of tissue was sliced into 25 mg slices and the tissue slices pooled . Tissue slices (100 mg) in preincubation vessels were gassed with 95 ~ O, and 5 ~ CO, and incubated for 30 min at 37 °C in a metabolic shaker at 120 oscillations per min. The slices were washed twice with 3~0 ml vol of fresh medium, blotted and transferred to incubation vessels containing 3~0 ml medium or 3~0 ml medium plus additive . The tissue slices were then re~quilibrated with 95 ~ O, and 5 ~ CO and incubated as described above for 30 Inin . At the end of incubation the tissue slices were removed by filtration on a nylon-mesh filter . Medium (2~0 ml) was used to rinse the incubation vessel and to wash the tissue on the filter . The 5 ml filtrate (incubate) was stored at 4°C for amylase assay. The tissue was re-suspended in 2 ml of medium and homogenized with an UltraTurrax . The homogenization tube was further rinsed with 3 ml of medium and added to the homogenate . The homogenate volume was made up to 10 ml with medium and centrifuged at 1500 x g at 4°C for 10 min and amylase assayed in the supernatant . The amylase levels of the incubates and the corresponding homogenates were estimated by the Phadebastablet method (CESKA et al., 1969). The sum of the amylase levels in the incubate and the corresponding tissue homogenate gave the total all~ylase content. The secretory response of the tissue was expressed as a percentage of the total amylase content. The response to a stimulant was indicated by the increase in this figure . Investigation of the effect of cholinergic agonist and antagonists on venom-induced amylase release was carried out as follows. After preincubation, 100 IIIg of tissue slices were transferred to vessels containing medium alone (basal release), 3 x 10 - ' M ACh~hloride, venom (201Ig/ml), 3 x 10'' M ACh~hloride plus cholinergic antagonist, venom (20 Wg/ail) plus cholinergic antagonist, or medium containing the highest concentration of cholinergic agonist or antagonist . Acetylcholine chloride, physosdgmine, nicotine, hexamethonium bromide, atropine and D-tubocurarine chloride pentahydrate were supplied by Sigma Chemicals, London, U.K . Trasylol® was obtained from Bayer. All the chemicals used were of analer quality. Venom obtained from the scorpion Tityus trinitatis by electrical stimulation was stored desia:ated at - 20°C . RESULTS
(a) Acetylcholine stimulated amylase release
The tissue slices were incubated with medium containing ACh (3 x 10 - ° M-3 x 10 - a M). The results presented in Fig. 1 show that the peak response was observed at 3 x 10- ' M . 15
°
ô â
10
0 9
//
a 0
5
v 0 T Q
0
3x10
_s
3a10
e
M acetylcholine
3alÔ ~
3a10
s
3i10_5
chloride
CONCENTRATION-RESPONSE RELATIONSHIP BETWEEN ACh IN INCUBATION MEDIUM AND AMYLASE OUTPUT BY TAE PANCREATIC SLICES . Data are presented as amylase secreted (~ of total) by 100 mg (wet wt) of rat pancreatic slices in modified Krebs-bicarbonate medium containing ACh~hloride. Each point is mean f S.E .M . No. of observations is indicated in circles.
FIO . 1 .
Scorpion Venom on Amylase Release .
443
(b) Effect of venom on amylase output
Tissue slices were incubated with medium containing crude venom at concentrations ranging from 5 to 100 Fig per ml of incubation medium. The venom stimulated release of amylase from the tissue slices was linear up to a venom concentration of 20 ~g/ml. Although venom at 50 and 100 Fig per ml of medium stimulated the tissue slices to release amylase over the basal level, the stimulated output was well below the maximal effect produced by 20 4tg/ml (Fig. 2).
Venom ( yg/ml )
Fta . 2 . VENOM-IIdDUCED AMYLASE OUTPUT . Amylase secreted (as ~ of total) by 100 mg wet wt of rat pancreatic slices in modified Krebs-bicarbonate medium containing different concentrations of venom . Each point is mean f S .E .M . Number of observations is indicated in circles. Amylase output plotted against venom concentration on the same scale as that for ACh (Fig. 1) to show the similarity between the two curves .
To investigate the mode of amylase release by the venom, the effect of atropine and other cholinergic antagonists and agonista on venom-induced amylase release was studied. In all these studies, the effect of cholinergic agonists or antagonists on ACh-induced amylase release was carried out in parallel, for comparison. The concentration producing the maximal amylase release from the tissue slices was chosen as the concentration of the venom or ACh for subsequent studies. Atropine (3 x 10-° M) did not have an effect on the nonstimulated release of amylase by the tissue slices, while 3 x 10-° and 1~5 x 10-° M concentrations produced an inhibition of the venom or ACh induced amylase release by 80 and over 50~ respectively. The ~ figures for atropine inhibition was calculated by comparing the net amylase release figures in presence and absence of atropine for ACh or venom and expressing the difference between the two in each case as a ~ inhibition of that of the net value for stimulated release (Table 1). The effect of hexamethonium, nicotine and tubocurarine on secretion is shown in Tables 2, 3 and 4. These agents did not have an effect on the basal release of amylase by the tissue slices. No alteration in the ACh or venom-induced amylase release was observed in presence ofthese drugs. ' Physostigmine Sulphate (Eserine), 3 x 10 -° M did not alter the basal release of amylase, but potentiated ACh (3 x 10- ° M) or venom (5 pg/ml}-induced amylase release. Thepotentiation was 70 and 57~ respectively . As Frserine is a cholinergic potentiating agent the concentration of ACh or venom selected for this aspect of the study was below the concentration that produced the peak effect . In the case of venom the concentration of 5 ltg/ml was the lowest concentration on the linear part of the concentration-response curve. The results are given in Table 5.
444
H. SANKARAN, C. BARTHOLOMBW, O. FITZGERALD and K. F. McGEENEY TABLE I . EFFECT OF ATROPINE
Agent Medium (Basal) ACh (3 x 10 - ' M)
Amylase secreted ( ~ of total) by 100 mg wet wt of rat pancreatic slices in presence of atropino 0 3x10-'M 1"5x10-'M
6~5 f 0"8 (~ "14 "4 f 0"5 (~ *
6"9 ~ 1"4 (3) b7"8 f 02 (3)
~9 "9 f 1"2 (~*
Medium (Basal) 8"5 f 0~4 (9) 8"7 f Oß (~ Venom(20tIg/ml) "15~9 ~ 0" 2 (~* b9 "8 f 1~1 (~ ~12"4 f 0"S (9)* Values are mean f S.E .M . * Denotes significance (calculated using Student's t-test) of difference from the respective basal value in each group (P values < 0"05). a and b (P < 0"001), a and c (P < 0"01), b and c are not significantly different. Number of observations shown in parentheses .
Agent Medium (Basal) ACh (3 x 10-'M)
TABLE 2. EFFECT OF HSXAIIIR1IiONIUM Amylase secreted ( ~ of total) by 100 mg wet wt of rat pancreatic slices in presence of hexamethonium bromide 0 5 x 10-'M 2 x 10-' M 1 x 10 - 4 M
6"2 f 2"0 (6) 6"4 f 0~9 (6) 11 "9 f 1~6 (~* 13 "8 f lß (5)*
12" 6 ~ 2~1 (S)*
13 "8 f 2~7 (~*
Medium (Basal) a6 "2 ~ 1"1 (~ b8~4 ~ 1"7 (3) Venom (20tIg/ml) l2ß f Oß (3)* 150 f 03 (4)* 12 " 6 f 0"4 (~* 11 "4 ~ 1 "4 (3)* Values are mean f S.E.M . * Denotes significance (calculated using Student's t-test) of difference from the respectivo basal value in each group (P values < 0"02). a and b are not significantly different. Number of observations shown in parentheses .
Agent Medium (Basal) ACh (3 x 10 - ' M)
TABLE 3. EFFECT of NICOTINE Amylase secreted (~ of total) by 100 mg wet wt of rat pancreatic slices in presence of nicotine 0 3x10-'M 3x10- 'M 3x10-°M 3x10 - 'M
5"1 ~ 0"7 (9) 4"8 f 1~0 (~ 8"6 f 0"6 (~* 9~5 ~ 0"8 (~*
9"4 f 1~0 (~*
9"2 f 1" 1 (8)* 10"1 f 0"5 (~*
Medium (Basal) 6ß f P4 (13) 6"0 f 0"9 (8) Venom (20 tIg/ml) 9"5 f 0~7 (12)* 9"5 f 0"8 (14)* 10"6 f 0"8 (14)* 10"9 f 0"7 (13)* 10~0 f 1"5 (8)* Values are mean f S.E .M. * Denotes significance (calculated using Student's t-test) of difference from the respective basal value in each group (P values < 0"02). Number of observations shown in parentheses . TABLE
Agent Medium (Basal) ACh (3 x 10 -' M)
4.
EFFECT
of
TIteoCaRARIrtE
Amylase secreted (~ of total) by 100 mg wet wt of rat pancreatic slices in presence of D-tubocurarine 0 3 x 10 -5 M 3 x 10-' M 3 x l0 - ° M 3 x 10-° M 4"9 f 0"7 (4) 9"2 f 2ß (3)*
4~5 f 0"7 (S) 8"8 f 0"6 (~*
7ß f Oß (3)*
8"9 f 0"6 (4)*
9"2 f lß (3)*
Medium (Basal) 8"2 f 0"8 (9) 8"8 f 1"1 (~ Venom (201Ig/ml) 11 "2 ~ 0" 4 (~* 13~1 f 0"7 (~* 11 "1 f 0"7 (8)* 12 "6 f 12 (~* 11~8 f 1"6 (8)* Values are mean f S.E .M . * Denotes significance (calculated using Student's t-test) of difference from the respective basal value in each group (P values < 0" 02). Number of observations shown in parentheses . DISCUSSION
Scorpion venom (T. serrulatus and T. bahyetrsis) has a cholinergic action on smooth (DtNlz and VAi.>~axi, 1959 ; Dnvlz and TORxES, 1968) and cardiac muscle (CORRADO et al., 1968). The hypertensive effect of the venom of T. serrulatus was suggested to be mediated
Scorpion Venom on Amylase Release
Agent
445
T~ S. EFrec,-r oa manu$ Amylase secreted (~ of total) by 100 mg wet wt of rat pancreatic slices in presence of eserine 0 3 x 10 - ' M
Medium (Basal) 5~4 ~ 0~4 (9) 5~3 f 0~5 (4) AC;hh (3 x 10-9 M) 7~0 f 0~4 (4)* fi10~5 f l'6 (4)* Venom (5 pg/ml) 6'8 f 0'3 (~* fi8'7 f 0~5 (4)* Valuos ara mean f S.B .M . * Denotes significance (calculated using Student's t-test) of difference from the basal value (P values < 002) . fi Denotes significance of difference from the values for the corresponding stimulant without eserine. Number of observations shown in parentheses .
by ACh (CORRADO et al., 1974). Goa~z et al. (1975) reported stimulation of ACh release from rat brain cortical slices by tityustoxin (TsTX). The venom-induced (T. trinitatis) contraction of sphincter of Oddi was abolished by atropine (SANICARAN et al., 197 . These venom effects seem to be ACh-mediated. In the present study on the secretory effect of T. trinitatis, rat pancreatic slices have proven to be a suitable tool to study concentrationresponse effects of the venom on acinar cells. Although there is a significant variation in the basal (non-stimulated~alues between groups, the values within the groups for nonstimulated and stimulated release are compatible . Thus, ACh, employed in this study as a standard stimulant of pancreatic acinar cells, showed a concentration-response curve between 3 x 10 - ° and 3 x 10 -6 M with the peak effect at 3 x 10 - ' M (Fig. 1). This observation is strikingly similar to that Of Wrr .r .rAS,rc (1975) who employing mouse pancreatic fragments found the peak response at 3 x 10 - ' M. The venom-induced amylase release followed a similar pattern ; the concentration-response curve was linear up to 20 ~.g/ml. The amylase release obtained with 50 and 100 lAg venomJml of medium was consistently below the peak effect produced by 20 4tg/ml of medium. (Fig. 2.) The venom-induced amylase release seems to be mediated through a cholinergic mechanism. This is borne out by the observation that eserine potentiatea the venom and AChinduced amylase release. Cholinergic involvement in venom action is further demonstrated by the partial abolition of venom-induced amylase release with atropine . MAZ-n-n:ws et al. (1973) in their studies on ACh-induced membrane depolarisation, calcium e~ux and amylase release in mouse pancreas suggested the interaction of ACh with the muscarinic receptor of the cell followed by Na+ and Ca'+ flux, depolarization and amylase release. While discussing the nature of receptors in the acinar cells of mouse pancreatic fragments Wü .i .iAMC (1975) concluded that the cholinergic receptor mediating amylase release is muscarinic . Our studies indicate the absence of nicotinic receptors in rat pancreatic slices, as nicotine (3 x 10 -6 M) had no effect on either the basal release or ACh-induced release in agreement with the report of Wic.Lmhts (1975), nor did it affect the venom-induced amylase release. Hexamethonium and tubocurarine also had no effect on the basal release or the release stimulated by ACh or venom. These observations clearly rule out the possibility of the venom exerting its effect through the ganglion or at the nerve-organ level. This in combination with the partial inhibition of venom action by atropine and potentiation by eserine strongly suggests that the venom exerts its effect through the cholinoceptive receptors which are muscarinic in nature . Acknowledgements-We thank Mr. N. O'Corrrrox and Mr. A. Gtna.Ex for technical assistance. This work was supported by a grant from the Wellcome Trust, London .
446
H. SANKARAN, C. BARTHOLOMEW, O. FITZGERAI ;D and K. F. McGEENEY
REFERENCES B~erxoi.on~w, C. (1970) Acute scorpion pancreatitis in Trinidad . Br. med. J. 1, 666. B.~x~rxor.oMEw, C., Muxrxsr, J. J., McGsexEr, K. F. and FrrzGEtea~, O. (1974) Pancreatic response to Tityus trinitatis venom. W. Indime Med. J. 23, 41 . BARTHOLOI~~V, C., McGsexev, K. F., MuierxY, J. J., FrrzGenu.n, O. and Snxxexnrr, H. (1976) Experimental studies on the aetiology of acute scorpion pancreatitis. Br . J. Sung. 63, 807. Cesxw, M., Hui.~x, E. and Ixoer tKnx, B. G. A. (1969) A new method for determination of alpha amylase. Experientia 25, 555. Coxxnno, A. P., Ax~rorno, A. and Dixtz, C. R. (1968) Brazilian Scorpion venom (Tityus serrulatus), an unusual sympathetic post-ganglionic stimulant. J. Pharmac. exp. Ther.164, 253. Coxx~oo, A. P., Ricctorro Ns~ro, F. and Axrorno, A. (1974) The mechanism of the hypertensive effect of Brazilian Scorpion venom (7Styus serrulatus Lutz E. Mello) . Toxicon 12, 145. Dixiz, C. R. and Va~tu, V. (1959) Effects of a toxin present in a purified extract of telsons from the scorpion Tityus serrulatus on smooth muscle preparations, and in mice . Archs. tnt. Pharmacodyn . 7irér. 71, 1 . Dixiz, C. R. and Toes, J. M . (1968) Release of an acetylcholine-like substance from guinea pig ileum by scorpion venom. Toxicon S, 277. Gon~z, M. V., DiNtz, C. R. and Hnnaosa, T . S. (1975) A comparison of the effects of scorpion venom tityustoxin and Ouabain on the release of acetykholine from incubated slices of rat brain. I. Neurodttm. 24, 331. M~~rr~ws, E. K., P~xsox, E. H. and WILLiAMB, J. A. (1973) Pancreatic acinar cells. Acetylcholineinduced membrane depolarization, calcium eHiux and amylase release. J. Physiol. 234, 689. Poox-Knvo, T. (1963) Myoearditis from scorpion stings . Br. med. J. 1, 374. RoHaexecfrr, P. and C~srorxe, J. (1971) Secretion of hydrolases by perfused fragments of rat pancreas : effect of calcium. Am . J. Physiol. 220, 911. SnxKnnnx, H., FrrzGEtutu, O., BaRTxotoe~w, C. and McG~xEV, K. F. (1976) Action of the venom of 7Ytyus trinitatLs on the isolated sphincter of Oddi . Ir. J. med. Sci.145, 97. W~~R~N, J. A. (1938) Some notes on scorpion poisoning in Trinidad . 7}ans. R. Soc. trop . Med. Hyg. 31, 607. Wuu~tifs, J. A. (1975) An in vitro evaluation of possible cholinergic and adrenergic n~ceptors affecting pancreatic amylase secretion. Proc. Soc. exp. Biol . Med. ISO, 513 .