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The black scorpion Heterometrus longimanus: Pharmacological and biochemical investigation of the venom
Toslow~, Vol . 31, No. 10, pQ. 1303-1314, 1993. Printed in Cm~t Britain .
0041-0101 J93 56 .00 + .00 © 1993 Pmpmoo Pros Ltd
THE BLACK SCORPION HETEROMETRUS LONGIMANUS: PHARMACOLOGICAL AND BIOCHEMICAL INVESTIGATION OF THE VENOM MATTHEW
C. E. GwEe,' Pl~elt T.-H. WoxG,l P. GOPALAKRISHNARONE? L. S. Cl->Enx' and KERwnv S. Y. Low'
Venom and Toxin Research Group, Departments of Pharmacology and ZAnatomy, Faculty of Medicine, National University of Singapore, Lower Kent Ridge Road, Singapore 0511 (Received 30 November 1992 ; accepted 22 April 1993)
M. C. E. Gw>~, P. T.-H. Woxc, P. Gop~L,~KRISi-n~rnKOxE, L. S. Cz~ax and K. S. Y. Low. The black scorpion Heterometrus longimanus: pharmacological and biochemical investigation of the venom. Toxicon 31, 1301314, 1993.-Documentation on the biological activity (including the lethality) of the venom (BSV) from the black scorpion Heterometrus longimm~us is lacking. We have investigated the effects of BSV on adrenergic transmission using the rat isolated anococcygeus muscle (Acm), since the venom from several species of scorpions causes peripheral sympathetic nerve stimulation with enhanced adrenergic responses. The catecholamine content in BSV was also measured by HPLC. The effects of phentolamine (5 ~M), guanethidine (5 ~M), desipramine (1 .5 pM), tetrodotoxin (2 pM) and reserpine pretreatment in vivo (5 mg/kg s.c. x 24 hr and 5 mg/kg i.p. x 3 hr) on contractile responses of the rat Acm to field stimulation, crude BSV (2-10 p1 in 6 ml bath), noradrenaline (3 pM), tyramine (10-15 pM), carbachol (2-3 ~M) and potassium chloride (50-75 mM) were investigated. BSV mimicked the agonist actions of noradrenaline (NA) by acting directly on postjunctional a-adrenoceptors in the anococcygeus muscle . The LDso of crude BSV injected i.v. into mice was 0.13 ml per kg mouse. Sequential ultrafiltration of the crude BSV revealed the presence of a substance of low mol. wt which mediates the postjunctional a-agonist actions of BSV. HPLC measurements confirmed the presence of noradrenaline (NA; mean concentration of 1 .8 t 0.3 mM) in BSV; the dopamine concentration (mean of 31 f 4 ~M) was 60-fold lower than that of NA, whereas adrenaline was not detected in all the 15 samples investigated. Thus, the presence of NA in BSV can account for the postjunctional a-agonist actions of the venom in the Acm. INTRODUCTION
species of scorpions including Buthus tamulus (or Mesobuthus tamulus, Indian red scorpion), Leirus quinquestriatus, Centroides sculpturatus and Tityus serrulatus have been well documented as causing potentially lethal envenomation in humans (especially in children) and in experimental animals (GUERON and OVSYSHI~üIIt, 1984; KARL and YEOLEKAR, 1986; B~w~sx~R and B~w~+sKnR, 1989). The biochemistry and biological activity of the venoms and toxins from the different species of scorpions have been widely SEVERAL
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studied (see COURAUD and JovEx, 1984; EL-ASMAR, 1984 ; EL-AYEB and DELORI, 1984; GuERON and OVSYSHCHER, 1984 ; HASSAN, 1984; PossnNl, 1984; SIMARD et al. 1992). However, little is known about the biological properties, including the potential toxicity, of the venom from the black (Asian forest) scorpion, Heterometrus longimanus, commonly found in several regions in the tropics including Indonesia, Malaysia and the Phillippines (HULL-Wn .LIAMS, 1988). We have therefore investigated the pharmacological activity of the black scorpion venom (BSV): firstly, we were interested to determine whether BSV had any effect on adrenergic transmission using the rat isolated anococcygeus muscle (Acm) (Gu.LFSp>E, 1972, 1980), since the venoms from several species of scorpions have been reported to cause peripheral sympathetic nerve stimulation and increased catecholamine release resulting in enhanced adrenergic responses, especially of the cardiovascular system (GUERON and OVSYSHt.iIER, 1984 ; RAMACHANDRAN et al., 1986; MURTHY and VAKIL, 1988). On the basis of the pharmacological profile of BSV identified in this study, the ca.techolamine content in BSV was also measured biochemically. MATERIALS AND METHODS Dregs mid chemicals Drugs and chemicals used in this study were obtained from the sources indicated: adrenaline bitartrate, carbachol (carbamylcholine chloride), deaipramine hydrochloride, dopamine hydrochloride, ethylenediaminetetraoetic acid disodium salt (EDTA), guanethidine monosulfate, noradrenaline bitartrate, octane sulfonic acid (sodium salt), tetrodotoxin and tyramine (4-hydroxyphenethylamine) hydrochloride (Sigma Chemicals, St Louis, MO, U.S .A .); chloroaatic acid (Fluke, Switzerland); tetrahydrofuran (Merck, F.R .G .). All drugs were prepared as stock solutions (noradrenaline in 2'/° ascorbic acid) and diluted in Krebs just before use in experiments with the anocoocygeus muscle . Black seorplon venom (BSVJ
For extraction of venom, a scorpion was placed in a l'erspex restrainer box which allows only the tail (metasoma) to protrude . The tail is firmly held with a pair of forceps which acts as an electrode, and another pinlike electrode is placed just at the base of the sting. Electrical shocks (rectangular pulses of l50 V, l meet duration and frequency of 10 Hz) were then delivered, using a Grass stimulator (model S88) with an isolation unit . The ejected venom (few drops to 0.1 ml) was collected in small polythene vials, pooled (0 .5-1 ml) together and kept frozen at -20°C. When required for use, the venom was slowly thawed at 5°C and the resulting solution was used for investigation. Lethality of crude BSY in mice The t n,° values for HSV were determined by i.v . injection of the fresh crude venom into the caudal vein of
male Swiss albino mice weighing 20 f 2 g. Four mice were tested with 0.1 ml of crude BSV and with 0.1 ml of various dilutions of BSV in 0.9% w/v saline up to a dilution of 1 : 50. Anococcygeus muscle
The anoooccygcus muscles from male Sprague-Dawley rats weighing 280-350 g were isolated and mounted in 6m1 organ bath (Grt~+~, 1972) containing Krebs solution of the following composition: NaCI (118mM); KCl (4.8); KH=PO, (1 .2), CaC1 2 (2 .5); NaHC03 (25) ; MgSO, (2.4) and n{+}glucose (l1) . The solution was maintained at 37°C and aerated with 5% carbon dioxide in oxygen. The preparation was allowed to equilibrate for about 305 min with changes of Krebs solution at 15 min intervals. Motor responses of the muscle were then elicited by electrical field stimulation (20-30 V, IO Hz for 10 sec, 1 meet pulse width) every 2 min and recorded on a Grass polygraph via a force-displacement transducer (model FT 03). In each experiment, motor responses (control) were first obtained for about 30 min via field stimulation (FS) . Submaximal contractile responses of the muscle to 3 leM noradr+enaline (NA), 10-15 leM tyramine (Tyr), 2-10 pl black scorpion venom (HSV), 2-3leM carbachol (CCh) and SO-75 mM potassium chloride (KCl) were then obtained using 90 sec drug contact time and a 10 min interval between drug washout and the next drug exposure; FS was then resumed and, when motor responses were stable, S teM phentolamine was added to the organ bath for 30 min to product blockage of the motor responses; FS was then stopped and NA, Tyr, HSV, CCh and KCl
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were each tested in turn in the presence of phentohuaine as described. Contractile reaPonsea to CCh and KCl bdore and after the addition of 1 pM atropine for 30 min were also obtained. In separate acts of experiments, aubmaximal contractile responses of the muscle to FS, NA, Tyr, BSV, CCh and KCl were obtained in the absence (control) or presence of 5 pIVI guanethidine, 2ftM tetrodotoxin or 1 .5 pIVi deaipramine using the same procedure as for phentohunine. In another set of experiments, contractile responses to FS, NA, Tyr, HSV, CCh aced KCl were obtained from muscles isolated from rats pretreated with reserpine (5 mg/kg s.c . x 24 hr and S mg/kg i.p. x 3hr). A high concentration (100 ~of tyramine was used for this leaf set of experiments to ensure the effectiveness of rseerpine pretreatment . Contractile responses to NA, Tyr, BSV, CCh and KC1 under the various conditions of study wen measurod and expressed as g-tension aced tabulated (see Tables 1 and 2) . BSV ultrafrJtrate
Ultrafiltration of HSV was carried out using centricon microconcentrators (Centricon-10, Amicon Division, WR Grace dt Co ., CT, U.S.A .) with 10,000 mol. wt cutoff Microooncentratora with mol. wt cutoff of less than 10,000 could not be used, owing to extensive clogging of pores that prevented filtration of the venom. BSV 0.3 ml was transferred to the sample reservoir and centrifuged at 4500a for 90 min at 5°C and the ultrafiltrate (F,) was collected; distilled water to give a Snal volume of 0.3 ml was then added to the retentate with thorough mixing and the suspension was centrifuged es described and the ulttafiltrate (F~ was collected. The procedure was repeated to obtain one more sample of ultrafiltrate (F~. The retentate wes then recovered by inverting the microoonoentrator and centrifuging at 1000a for 2 min; distilled water up to 0.3 ml was then added to the rstentate was thorough mixing of the suspension (S). Two different samples of BSV were investigated . Contractile respot>aea of the Acm to 2 ~1 and 4 pl of BSV and to equivalent volumes of each sample of filtrate (F Fr F~ and suspension of retentate (S) veers then obtained as described. The contractile responses (B-tension) of the Acm to F Fy F~ and S from the two samples of BSV tested were expressed as a peroentage of the corresponding reaPonaea to BSV. DetectJon of catecholruninu
Venom collected in a polythene vial was diluted 500 times by volume with deionized water and then filtered through cellulose acetate filter (pore size 0.2 Jnn) . The catecholamine content in the filtrate was determined without further extraction ; in some ~further dïlution was necessary. NA, dopamine (DA) aced adrenaline (Adr) were measured by iaocratic high performance liquid chromatography (HPLC) with dual channel electrochemical detection. A HPLC system from Hioanalytical Systems (BAS 200A) was used with a phase II ODS column (100 x 3.2 mm, 3 pm) and a Hewlett-Packard integrator (HP3396 Stries In . Both channels of the electrochemical detector were set at an applied potential of f750mV, but one channel was set at a gain of 10 nA (full scale) while the other at 1 nA . The detection limit on the high gain channel was 20 pg. The mobile phase contained chloroscetic acid (75 mIul], ethylenediaminetehaacetic acid (disodium salt, 0.7 mM), 1-octanesulphonic acid (sodium salt, 0.2 gR) and tetrahydrofuran (0.5%), pH 3.1 . The flow rate was 1 ml/min. Under t6eae experimental conditions, the retention times were approximately 2.8 min for NA, 4.1 for Adr and 10.5 for DA. The identities of the two peaks detected for the BSV samples were confirmed by co~lution with standard NA and DA .
RESULTS
Figure 1 shows that 5 ~M phentolamine completely blocked the contractile responses of the rat isolatod anococcygeus muscle to field stimulation (FS), 3 ~M noradrenaline (NA) and 10-15 uM tyramine (Tyr); contractile responses to the 2-10 ~1 black scorpion venom (BSV) were markedly blocked (91 f 2%) as well ; the quantitative data are shown in Table 1 . The contractile response to 2-3 ~M carbachol (CCh) was slightly blocked (19 f 3%) by phentolamine but it was completely blocked by 1 ~M atropine; the response to 50-75 mM KC1 was partially blocked (28 f 5%) by phentolamine but it was unaffected by atropine . During wash-out of each agonist, the contracted muscle relaxed readily and completely . Guanethidine (5 ~cM) completely blocked the contractile response of the muscle to FS only, whereas the responses to NA and BSV were potentiated by 56 f 15% and 62 t 18%, respectively ; responses to Tyr and CCh were unaffected by gtiartethidine; the response to
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'1 FIG . 1 . EFF8CIS OF PE~NTOLAI~IINE .
Relevant segments of tracing from a typical experiment showing contractile responses of the anococcygeus muscles to FS (20-30 V, 10 Hz x 10 sec, 1 msec pulse width), NA 3 pM (~), Tyr 10 pM (/), BSV 8 pl (Q), Ch 3 uM (/) and KC1 50 mM (p) in the (a) absence (control) or (b) presence of phentolamine 5 pM (j -. ). Each agonist was washed out at ( j). Quantitative data for the various agonist responses are given in Table l .
KC1 was slightly increased (13 t 3%) (see Fig. 2 and Table 1). Tetrodotoxin (TTx) (2 pM) also completely blocked the contractile responses of the muscle to FS only, but generally did not affect the responses to all the other agonists used, as shown in Fig. 3 and Table 1 . Desipramine 1 .5 pM completely blocked the contractile responses of the muscle to Tyr, whereas the peak responses to FS, NA and BSV were potentiated by 16 t 3%, 79 f 11 and 96 f 14%,respectively; the response to CCh was generally not affected by desipramine . However, the response to KC 1 was slightly enhanced and accompanied by a slower relaxation rate, presumably as a wnsequence of transmitter NA release following depolarization of the neuronal membrane (GII130N and Pot,t .ocK, 1973) (see Fig. 4 and Table 1). The figure also shows that, in the presence of desipramine, the muscle contracted by FS, NA and BSV also relaxed at a slower rate, which can be attributed to the block of NA reuptake into the nerve terminals by desipramine. Figure 5 shows that the anococcygeus muscle isolated from rats pretreated with reserpine (5 mg/kg s.c. x 24 h and 5 mg/kg i.p. x 3 hr) did not contract to FS and to the high concentration (100 pM) of Tyr used, whereas NA, BSV, CCh an KC1 produced contractile responses of the muscle which were significantly potentiated by 72%, 62%, 18% and 11% respectively, as shown in Table 2. The ultrafiltrates (2~ ~1) of BSV, FI, F~, F3, and the suspension of retentate (S) produced contractile responses of the Acm which were 93.3% (93.2, 93.3), 30.1 % (21 .9, 38.3), 11 .1 % (5 .5, 16.7) and 3.3% (0, 6.6), respectively, of the mean responses produced by 2 ~1 and 4 gel of the two different samples of BSV tested; the figures in parentheses are the range of values obtained for the filtrates and S from the two samples of BSV. Contractile responses of the Acm to all the filtrates and S, like the response to BSV, were completely
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T~t~ 1. CONIRACTQE Rt?sPOtvses oF rt~ ner ~sou~o errococcsrasus utusctE ro NA, TYR, BSV, CCH ~tvo KCl nv rm (a) ~zt9exc~ (coxtna.) oR (b) rxr~cE oF t~ro~nmve, cueNert~u~, retaoooroxuv oR t~rx~emve i?hentolamine 5 kM (n = 6) NA (3 ~eM) (a) (b) Tyr (10-15 ~I) (a) (b) BSV (2-10 pL (a) (b) CCH (3 pM) (a) (b) KCl (SO-75 pM) (a) (b)
2.SOt0.04 (0) 2.60t0.10 (0) 2.(0t0.20 0.17f0 .04 "" (91.4 f2.2 °/"l) 2.SOt0.06 2.OOt0.08' (I8.9t3 .0%1) 2.SOt0.09 1 .70t0.08" (28.4f5 .01%1)
Response (8-tension) Guanethidine Tetrodotoxin 2 pM (n = 6) 2 ~M (n = 4) 2.23t0.11 3.43f0.24"" (56 .3 t 14 .6%t) 2.37t0.09 2.47 t0.19 2.48t0.19 3.88f0.28"" (61.7t I8.2%T) 2.35t0.14 2.35t0.19 2.38t0.04 2.68t0.08' (12.7f3 .4%j)
2.33f0.09 2.63t0.17 2.33f0.12 2.73f0.09 1 .98t0.09 1 .93t0.10
Desipramine 1.5 pM (n = 6) 2.35t0.08 4.18f0.20" (79.2 t 10 .6%j) 2.31t0.17 (0) 2.08t0.10 4 .05t0.26'" (96.2t 13.5%j)
2.45t0.03 2.15t0.06
2.52t0.17 2.40t0.20
2.40t0.13 2.53t0.13
2.57t0.15 3.28t0.23
Each value is the mean t S.E .M . Figures in parentheses show percentage (%)inhibition (j) or potentiation (j) of contractile responses of the muscle ; (0) = complete inhibition of responses. Significantly different (' "P < 0.01 ; "P < 0.05) from corresponding control value (paired t-test).
blocked by phentolamine (5 ~M). The i.v. LD w of BSV was found to be 0.13 ml per kg body weight of mouse. The concentration of NA detected in the venom from 15 scorpions ranged from about 0.4 to 4.5 mM with a mean of 1 .8 t 0.3 mM. The concentration of DA ranged from about 8 to 58 pM with a mean of 31 f 4 ~M, only 1.7% of that of NA. Adr was not detected in all the BSV samples tested . After the first collection of BSV, the 15 scorpions were divided into three groups of five. BSV was collected 7, 14 or 21 days later. The NA and DA contents were expressed as a percentage of the values obtained previously (on first collection) for the same five scorpions . NA contents on days 7 and 14 were significantly reduced to about 30% and 75% of control, respectively . On day 21, the NA content was no longer significantly different from control. In contrast, DA content on day 7 was more than four-fold higher than the control level and then it decreased back to control levels on days 14 and 21 (Fig. 6). DISCUSSION
The contractile responses of the anococcygeus muscle to FS, NA and Tyr are due to activation of postjunctional a-adrenoceptors present in the muscle : FS causes the release of NA which produces the motor (excitatory) responses obtained (GILLFSPIE, 1972, 1980),
M. C. E. GWEE et d.
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hio. 2. Et+t+acrs of ao~xertnnuvE . Relevant segments of tracing from a typical experiment showing contractile responses of the anoooaygeus muscle to FS (20-30 V, 10Iiz x 10 sec, 1 msec pulse width), NA 3 pM (~), Tyr 15 pM ("), BSV 10 pl (p), CCh 3 pM (") and KC1 75 mM (p) in the (a) absence (control) or (b) presence of guanethidine 5pM (j -. ). Each agonist was washed out at (j) . Quantitative data for the various agonist responses are given in Table 1.
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Ftia. 3. ErPeccs oa~ israonoTO~. Relevant segments of tracing from a typical experiment showing contractile responses of the anococcygeus muscle to FS (20-30 V, 10 Hz x 10 sec, 1 cosec pulse width), NA 3 pM (~), Tyr 10 pM ("), BSV 6 pl (Q), CCh 2 pM (") and KCl 50 mM (p) in the (a) absence (control) or (b) presence of tetrodotoxin 2 pM (j -. ). Each agonist was washed out at (j) . Quantitative data for the various agonist responses are given in Table 1.
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s ~.
L~ FIG . 4. EPFECIS OF DI~IPRA6rINE .
Relevant segments of tracing from a typical experiment showing contractile responses of the anoooocygeus muack to FS (20-30 V, 10 Hz x 10 sec, I cosec pulse width), NA 3 ~M (~), Tyr is plvl ("), HSV s~l (p), CCh 3 pM (") and KCI 7s mM (p) in the (a) absence (control) or (b) presence of desipramine Ls pM (j ~ ). Each agoniat was washed out at (j) . Quantitative data for the various agonist responses are given in Table 1.
whereas Tyr acts indirectly via the discharge of NA from the adrenergic nerve terminals (GERBER and N>FS, 1990 ; TRENDELENBURG, 1991). The contractile response of the muscle to CCh is mediated via stimulation of postjunctional muscarinic receptors which are widely distributed in the anocoocygeus muscle (G>z .LESr~, 1980). The response of the anococcygeus muscle to KC1 has been attributed to direct depolarization of the smooth muscle membrane as well as to the release of NA from the adrenergic nerve terminals as a consequence of neuronal depolarization (GB3SON and Por r..ocK, 1973). The agonist action of BSV in mediating the contractile (adrenergic) response of the anocoocygeus muscle closely resembled the action of NA: firstly, blockade of the BSV-induced response by the selective a-adrenoceptor antagonist, phentolamine, shows
FS
s~ FKI . s. EFFI~CIS of IIF~tFnva rxErAr.~r~rr. Rekvant segments of tracing from a typical experiment showing contractile responses of the anocoaygeus mueck to FS (20-30 V, 10 Hz x 10 sec, 1 cosec pule width), Tyr 100 pM ("), NA 3 ~I (~), BSV 3 pl (Q), CCh 2pM (") and KCI 50 mM (D); muscle tisue was isolated from rats pretreated with reserpine (s mg/kg e.c . x 24 hr and s mg/kg i.p . x 3 hr). Each agoniet was washed out at (j). Quantitative data for the various agoniet responses are given in Table 2.
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TARIE 2. EFPECIS OF RE~ItPINE l'REI'REATAICNT ON CONTRACI7LE RE4PONgF3 OF TF~ RAT ISOI.ATPD ANOCOCCYOI?US UtU3CLE TO NA, TYR, BSV, CCH AND KCl
Control
Response (g-tension) Pretreatmentt
(n = 4)
% Potentialion
2 .35 f 0.04 2 .40 t 0.07 2 .16f0.08 2 .45 f 0.06 2 .45 f 0.05
4 .03 f 0 .32*' (0) 3 .SOt0 .33 ** 2 .88 f 0 .24* 2 .73 f 0 .05*
71 .5 62 .0 17 .6 11 .4
(n = 22
NA (3 pM) Tyr (100 uM) BSV(2-10p1) CCH (2-3 pM) KCl (50-75 mM)
Each value is the mean f S.E .M . The control mean is calculated from the pooled individual values obtained for each condition of study shown in Table 1 . (0) = no response . Significantly different (**P < 0.01 ; *P < 0.0~ from the corresponding control value (group t-teat) . $Reserpine pretreatment: 5 mg/kg s.c . x 24 hr and 5 mg/kg i .p . x 3 hr .
that the agonist action of BSV was mediated via stimulation of postjtmctional aadrenoceptors in the muscle either directly, or, indirectly, via the release of NA from adrenergic nerve terminals. However, BSV could not have caused stimulation of the adrenergic nerve to release transmitter NA since, like the response to exogenous NA, the response to BSV was not sensitive to blockade either by TTx, a selective blocker of voltage-sensitive sodium channels (NAAAHA.CFir, 1974 ; CATTIItALL, 1980; R1TCfue, 1980), or by guanethidine, a typical adrenergic neurone blocker (GERBER and N>FS, 1990). Furthermore, the agonist action of BSV in the anococcygeus muscle also could not have been due to the indirect discharge of NA from the adrenergic nerve terminals, since muscle from reserpine-treated and non-treated rats exhibited similar responses to BSV. Reserpine treatment would have caused depletion of vesicular NA in the adrenergic nerve terminals of the muscle (GERBER and N>ES, 1990), as evidenced by a lack of response of the muscle to FS and the high concentration of Tyr, both of which are entirely dependent upon the
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~aya FIO. 6. NA AND DA wxceNrRAnoNS IN BSV AsreR eLecrRICAI, srutvunoN. Changes in NA (~) and DA (/) concentrations in BSV at various times after total discharge of venom induced by electrical stimulation on day 0. Results are expressed as percentage of control concentrations measured on day 0 for the same scorpion . *Statistically different (P < 0.05) from control as calculated by Student's t-teat for paired data .
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availability of endogenous NA for their agonist actions. The potentiation of the contraofile responses to NA, BSV, CCh and KC1 by reserpine pretreatment can be attributed to the development of non-specific supersensitivity of the muscle tissue to the compounds used (GnxB>~ and N~s, 1990). The contractile responses of the anocoocygeus muscle to Tyr was, as expected, abolished by desipramine which is attributable to the blockade of Tyr transport (via uptake-1) into its site of action in the adrenergic nerve terminals (TRENDELENBURG, 1991). However, the contractile responses of the muscle to NA and BSV were markedly potentiated by 79 t 11 % and 96 ± 14% by desipramine and by 56 t 15% and 62 ± 18 %, respectively, by guanethidine, a phenomenon that can be attributed to blockade of the uptake-1 process for catecholamines (G>:1tBnx and N>~s, 1990 ; Tx~vnsr..ExsuxG, 1990). Thus, the NA-like fraction of BSV probably also undergoes uptake-1 into the adrenergic nerve terminals and it is not dependent on such an uptake to mediate its agonist action . Our results provide the first substantial evidence that BSV can act directly on postjunctional a-adrenoceptors to produce contractile responses of the rat isolated anococcygeus muscle; the a-agonist action of BSV in the rat anococcygeus muscle therefore closely mimics the action of NA. Moreover, the a-agonist activity of BSV was present almost entirely in the ultrafiltrate obtained after dialysis of the crude venom, since equal volumes of the first filtrate (F,) and crude BSV taken from the same sample produced almost equiactive contractile responses of the Acm which were sensitive to phentolamine blockade . Thus, the a-agonist activity of BSV cannot be due to the presence of a large protein molecule; instead, a substance of smaller mol. wt, possibly NA itself, is responsible for mediating the a-agonist actions of BSV in the Acm. HPLC measurements confirmed the presence of NA in the BSV: the high concentration (mean of 1 .8 mM) of NA found in BSV can therefore account for the marked postjunctional a-agonist action of BSV in the rat Acm. Thus, 3-10 p1 of crude BSV (equivalent to 5-18 nmol of NA, calculated on the basis of the mean NA concentration of 1 .8 mM) produced contractile responses of the Acm which were comparable to those produced by NA 3 pM (i.e. 18 nmol in a 6 ml bath). The DA concentration, however, was 60-fold lower than that of NA, suggesting that catecholamine is synthesized via the usual pathway from tyrosine with DA as one of the intermediates . The venom of the honey bee, Apis meilifica, has also been reported to contain catecholamines (BANKS et al., 1976; OWEN and BRIDGFS, 1982), although the reported contents were far less than that detected in the BSV in the present study. The NA and DA contents in the bee venom have also been found to have age-dependent and seasonal variations (OweN and BitlncES, 1982). It is also possible that BSV may exhibit the same types of variations which can thus explain the presented observed 7- to 11-fold individual variation in the concentration of catecholamines . After complete discharge of the venom on the first collection, it was observed that the NA content recovered up to 75% of the mean control value within 2 weeks; recovery was statistically complete in the 3rd week . In contrast, DA content was markedly elevated only on day 7 when the NA content was only about 30% of control . This may reflect extremely active synthesis due to markedly increased tyrosine hydroxylase activity caused by loss of feedback product inhibition by NA. The 2-week period required for a 75% recovery of the NA content seems long, but this long recovery period may be a direct result of marked depletion of the venom gland caused by the electrical method employed for venom extraction, an unlikely condition in the event of natural venom discharge, for scorpions are known to be economical in the use of venom (CLOUDSLEY-Txo~soN, 1992).
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In conclusion, our results clearly show that the black scorpion venom has marked postjunctional a-agonist action in the anococcygeus muscle, which is mediated by noradrenaline present in the venom. Acknowkdgenrent-This work was supported by research grants RP 870357 8c RP 890321 from the National University of Singapore. REFERENCES Buvrcs, B. E. C., H~uvsoN, J. M. and Suact.tae, N. M. (1976) The isolation and identification of noradrenaline and dopamine from the venom of the honey bee, Apir mellifica. Toxicon 14, ! 17-125 . B~wesrute, H. S. and Bew~a, P. H. (1989) Stings by red scorpooa (Buthotus laminas) in Maharashtra State, India: a clinical study. Traps. R. Soc. trop. Med. Hyg. 83, 858-860. CATIt~IALL, W. A. (1980) Neurotoxins that act on voltage-sensitive sodium channels. .! . Rev. Pharmac. Toxicol. 20, 15--43 . Cwunsr~r-THOIIPSON, J. L. (1992) Scorpions. Biologist 39, 206-210. Covrutm, F. and Jovtflt, E. (1984) Mechanism of action of scorpion toxins . Handbook Notwal Toxins 2, 659-678. Et.-Asw+x, M. F. (1984) Metabolic effect of scorpion venom. Handbook Notwal Toxins 2, 551-575 . EL-AYE, M. and D>~.ont, P. (1984) Immunology and immunochemistry of scorpion neurotoxins . Handbook Notwal Toxins 2, 607-637. Gexet+x, J. G. and Nn3s, A. S. (1990) Antihypertensive agents and the drug therapy of hypeRension . In : Goodman and Gilmmi's The Pharmacological Basis of Therapeutks, pp . 784-813 (Gooo~x, Gu.~N, A., Ru .r,, T. W., Nm A. S. and T~Yt.on, P., Eds) . New York : Pergamon Press. Giesox, A. and Pot.t .oc[, D. (1973) The effects of drugs on the sensitivity of the rat anocoocygeus muscle to agonists . Br. J. Pharmac. 49, 506-513. Gar ~ravm, J. S. (1972) The rat anocoocygeus muscle and its response to nerve stimulation and to some drugs . Br. J. Pharmac. 45, 404-416. Gn.rrsrtE, J. S. (1980) The physiology and pharmacology of the anococcygeus muscle . Trends Pharmac. Sci. 1, 45357. Guertox, M. and OVSYSi1Cr~t, I. (1984) Cardiovascular effects of scorpion vtaoms . Handbook Notwal Toxins Z, 639-657. Hex, F. (1984) Production of scorpion antivenin. Handbook Notwal Toxins 2, 57706. Htn.trWaa.utis, V. (1988) How to Ifeep Scorpions. London : Fitzgerald. Ktitt, R. K. M. and YHOZ~a, M. E. (1986) Electrocardiographic changes is experimental myocarditis induced by scorpion (Buthus tamtthv) venom. Ind. Heart J. 38, 206-210. MtmrxY, K. R. K. and Veen., A. E. (1988) Elevation of plasma angiotensin levels in dogs by Indian red scorpion (Bather tamuhit) venom and its reversal by administration of insulin and tohuoGne. Irrd. J. med Res. ag,376-379 . Neawxi+sra, T. (1974) Chemicals as tools in the study of excitable membranes. Physiol. Res. 54, 81389. Oww, M. D. and BarooHS, A. R. (1982) CatechoLunines in honey bee (Apis mellljera L.) and various vespid (hymenoptera) venoms. Toxicon 20, 1075-1084. Pay L. D. (1984) Structure of scorpion toxins. Handbook Notwal Toxins 2, 513-549. Rf~cw~rrorurt, L. K., Aa+3ewet,, O. P., AcEtYVZxwx, K. E., Cxntrorunt, L., Vmtsato~n, J. R. and Getvatn.Y, D. K. (1986) Fractionation and biological activities of venom of the Indian scorpions ButhurtamLkrs and heterometrua bengaleaess. Ind. J. Biochem. Biophys. 23, 355-358. Rtic,~, J. M. (1980) Tetrodotoxin and saxitoxin and the sodium channels of excitable tissue . Trends Pharmac. Scl. 1, 275-279. Suwrn, J. M., Meves, H. and W~rr, D. D. (1992) Neurotoxins in venom from the North American scorpion Centrwoides sculptwatw Ewing. In: Notwal Toxlru : Toxkology, Chemistry and Sajety, pp . 236-263 (IC~.ea, R. F., Mt rrowe, N. B. and Tu, A. T., Eda). U.S.A .: Ahtlren. Taet~l~t+strrta, U. (1991) Functional aspects of the neuronal uptake of noradronaline. Trends Pharmac. Sci. 12, 334-337.
0041-0101 J93 56 .00 + .00 © 1993 Pmpmoo Pros Ltd
THE BLACK SCORPION HETEROMETRUS LONGIMANUS: PHARMACOLOGICAL AND BIOCHEMICAL INVESTIGATION OF THE VENOM MATTHEW
C. E. GwEe,' Pl~elt T.-H. WoxG,l P. GOPALAKRISHNARONE? L. S. Cl->Enx' and KERwnv S. Y. Low'
Venom and Toxin Research Group, Departments of Pharmacology and ZAnatomy, Faculty of Medicine, National University of Singapore, Lower Kent Ridge Road, Singapore 0511 (Received 30 November 1992 ; accepted 22 April 1993)
M. C. E. Gw>~, P. T.-H. Woxc, P. Gop~L,~KRISi-n~rnKOxE, L. S. Cz~ax and K. S. Y. Low. The black scorpion Heterometrus longimanus: pharmacological and biochemical investigation of the venom. Toxicon 31, 1301314, 1993.-Documentation on the biological activity (including the lethality) of the venom (BSV) from the black scorpion Heterometrus longimm~us is lacking. We have investigated the effects of BSV on adrenergic transmission using the rat isolated anococcygeus muscle (Acm), since the venom from several species of scorpions causes peripheral sympathetic nerve stimulation with enhanced adrenergic responses. The catecholamine content in BSV was also measured by HPLC. The effects of phentolamine (5 ~M), guanethidine (5 ~M), desipramine (1 .5 pM), tetrodotoxin (2 pM) and reserpine pretreatment in vivo (5 mg/kg s.c. x 24 hr and 5 mg/kg i.p. x 3 hr) on contractile responses of the rat Acm to field stimulation, crude BSV (2-10 p1 in 6 ml bath), noradrenaline (3 pM), tyramine (10-15 pM), carbachol (2-3 ~M) and potassium chloride (50-75 mM) were investigated. BSV mimicked the agonist actions of noradrenaline (NA) by acting directly on postjunctional a-adrenoceptors in the anococcygeus muscle . The LDso of crude BSV injected i.v. into mice was 0.13 ml per kg mouse. Sequential ultrafiltration of the crude BSV revealed the presence of a substance of low mol. wt which mediates the postjunctional a-agonist actions of BSV. HPLC measurements confirmed the presence of noradrenaline (NA; mean concentration of 1 .8 t 0.3 mM) in BSV; the dopamine concentration (mean of 31 f 4 ~M) was 60-fold lower than that of NA, whereas adrenaline was not detected in all the 15 samples investigated. Thus, the presence of NA in BSV can account for the postjunctional a-agonist actions of the venom in the Acm. INTRODUCTION
species of scorpions including Buthus tamulus (or Mesobuthus tamulus, Indian red scorpion), Leirus quinquestriatus, Centroides sculpturatus and Tityus serrulatus have been well documented as causing potentially lethal envenomation in humans (especially in children) and in experimental animals (GUERON and OVSYSHI~üIIt, 1984; KARL and YEOLEKAR, 1986; B~w~sx~R and B~w~+sKnR, 1989). The biochemistry and biological activity of the venoms and toxins from the different species of scorpions have been widely SEVERAL
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studied (see COURAUD and JovEx, 1984; EL-ASMAR, 1984 ; EL-AYEB and DELORI, 1984; GuERON and OVSYSHCHER, 1984 ; HASSAN, 1984; PossnNl, 1984; SIMARD et al. 1992). However, little is known about the biological properties, including the potential toxicity, of the venom from the black (Asian forest) scorpion, Heterometrus longimanus, commonly found in several regions in the tropics including Indonesia, Malaysia and the Phillippines (HULL-Wn .LIAMS, 1988). We have therefore investigated the pharmacological activity of the black scorpion venom (BSV): firstly, we were interested to determine whether BSV had any effect on adrenergic transmission using the rat isolated anococcygeus muscle (Acm) (Gu.LFSp>E, 1972, 1980), since the venoms from several species of scorpions have been reported to cause peripheral sympathetic nerve stimulation and increased catecholamine release resulting in enhanced adrenergic responses, especially of the cardiovascular system (GUERON and OVSYSHt.iIER, 1984 ; RAMACHANDRAN et al., 1986; MURTHY and VAKIL, 1988). On the basis of the pharmacological profile of BSV identified in this study, the ca.techolamine content in BSV was also measured biochemically. MATERIALS AND METHODS Dregs mid chemicals Drugs and chemicals used in this study were obtained from the sources indicated: adrenaline bitartrate, carbachol (carbamylcholine chloride), deaipramine hydrochloride, dopamine hydrochloride, ethylenediaminetetraoetic acid disodium salt (EDTA), guanethidine monosulfate, noradrenaline bitartrate, octane sulfonic acid (sodium salt), tetrodotoxin and tyramine (4-hydroxyphenethylamine) hydrochloride (Sigma Chemicals, St Louis, MO, U.S .A .); chloroaatic acid (Fluke, Switzerland); tetrahydrofuran (Merck, F.R .G .). All drugs were prepared as stock solutions (noradrenaline in 2'/° ascorbic acid) and diluted in Krebs just before use in experiments with the anocoocygeus muscle . Black seorplon venom (BSVJ
For extraction of venom, a scorpion was placed in a l'erspex restrainer box which allows only the tail (metasoma) to protrude . The tail is firmly held with a pair of forceps which acts as an electrode, and another pinlike electrode is placed just at the base of the sting. Electrical shocks (rectangular pulses of l50 V, l meet duration and frequency of 10 Hz) were then delivered, using a Grass stimulator (model S88) with an isolation unit . The ejected venom (few drops to 0.1 ml) was collected in small polythene vials, pooled (0 .5-1 ml) together and kept frozen at -20°C. When required for use, the venom was slowly thawed at 5°C and the resulting solution was used for investigation. Lethality of crude BSY in mice The t n,° values for HSV were determined by i.v . injection of the fresh crude venom into the caudal vein of
male Swiss albino mice weighing 20 f 2 g. Four mice were tested with 0.1 ml of crude BSV and with 0.1 ml of various dilutions of BSV in 0.9% w/v saline up to a dilution of 1 : 50. Anococcygeus muscle
The anoooccygcus muscles from male Sprague-Dawley rats weighing 280-350 g were isolated and mounted in 6m1 organ bath (Grt~+~, 1972) containing Krebs solution of the following composition: NaCI (118mM); KCl (4.8); KH=PO, (1 .2), CaC1 2 (2 .5); NaHC03 (25) ; MgSO, (2.4) and n{+}glucose (l1) . The solution was maintained at 37°C and aerated with 5% carbon dioxide in oxygen. The preparation was allowed to equilibrate for about 305 min with changes of Krebs solution at 15 min intervals. Motor responses of the muscle were then elicited by electrical field stimulation (20-30 V, IO Hz for 10 sec, 1 meet pulse width) every 2 min and recorded on a Grass polygraph via a force-displacement transducer (model FT 03). In each experiment, motor responses (control) were first obtained for about 30 min via field stimulation (FS) . Submaximal contractile responses of the muscle to 3 leM noradr+enaline (NA), 10-15 leM tyramine (Tyr), 2-10 pl black scorpion venom (HSV), 2-3leM carbachol (CCh) and SO-75 mM potassium chloride (KCl) were then obtained using 90 sec drug contact time and a 10 min interval between drug washout and the next drug exposure; FS was then resumed and, when motor responses were stable, S teM phentolamine was added to the organ bath for 30 min to product blockage of the motor responses; FS was then stopped and NA, Tyr, HSV, CCh and KCl
Black Scorpion Venom Activity
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were each tested in turn in the presence of phentohuaine as described. Contractile reaPonsea to CCh and KCl bdore and after the addition of 1 pM atropine for 30 min were also obtained. In separate acts of experiments, aubmaximal contractile responses of the muscle to FS, NA, Tyr, BSV, CCh and KCl were obtained in the absence (control) or presence of 5 pIVI guanethidine, 2ftM tetrodotoxin or 1 .5 pIVi deaipramine using the same procedure as for phentohunine. In another set of experiments, contractile responses to FS, NA, Tyr, HSV, CCh aced KCl were obtained from muscles isolated from rats pretreated with reserpine (5 mg/kg s.c . x 24 hr and S mg/kg i.p. x 3hr). A high concentration (100 ~of tyramine was used for this leaf set of experiments to ensure the effectiveness of rseerpine pretreatment . Contractile responses to NA, Tyr, BSV, CCh and KC1 under the various conditions of study wen measurod and expressed as g-tension aced tabulated (see Tables 1 and 2) . BSV ultrafrJtrate
Ultrafiltration of HSV was carried out using centricon microconcentrators (Centricon-10, Amicon Division, WR Grace dt Co ., CT, U.S.A .) with 10,000 mol. wt cutoff Microooncentratora with mol. wt cutoff of less than 10,000 could not be used, owing to extensive clogging of pores that prevented filtration of the venom. BSV 0.3 ml was transferred to the sample reservoir and centrifuged at 4500a for 90 min at 5°C and the ultrafiltrate (F,) was collected; distilled water to give a Snal volume of 0.3 ml was then added to the retentate with thorough mixing and the suspension was centrifuged es described and the ulttafiltrate (F~ was collected. The procedure was repeated to obtain one more sample of ultrafiltrate (F~. The retentate wes then recovered by inverting the microoonoentrator and centrifuging at 1000a for 2 min; distilled water up to 0.3 ml was then added to the rstentate was thorough mixing of the suspension (S). Two different samples of BSV were investigated . Contractile respot>aea of the Acm to 2 ~1 and 4 pl of BSV and to equivalent volumes of each sample of filtrate (F Fr F~ and suspension of retentate (S) veers then obtained as described. The contractile responses (B-tension) of the Acm to F Fy F~ and S from the two samples of BSV tested were expressed as a peroentage of the corresponding reaPonaea to BSV. DetectJon of catecholruninu
Venom collected in a polythene vial was diluted 500 times by volume with deionized water and then filtered through cellulose acetate filter (pore size 0.2 Jnn) . The catecholamine content in the filtrate was determined without further extraction ; in some ~further dïlution was necessary. NA, dopamine (DA) aced adrenaline (Adr) were measured by iaocratic high performance liquid chromatography (HPLC) with dual channel electrochemical detection. A HPLC system from Hioanalytical Systems (BAS 200A) was used with a phase II ODS column (100 x 3.2 mm, 3 pm) and a Hewlett-Packard integrator (HP3396 Stries In . Both channels of the electrochemical detector were set at an applied potential of f750mV, but one channel was set at a gain of 10 nA (full scale) while the other at 1 nA . The detection limit on the high gain channel was 20 pg. The mobile phase contained chloroscetic acid (75 mIul], ethylenediaminetehaacetic acid (disodium salt, 0.7 mM), 1-octanesulphonic acid (sodium salt, 0.2 gR) and tetrahydrofuran (0.5%), pH 3.1 . The flow rate was 1 ml/min. Under t6eae experimental conditions, the retention times were approximately 2.8 min for NA, 4.1 for Adr and 10.5 for DA. The identities of the two peaks detected for the BSV samples were confirmed by co~lution with standard NA and DA .
RESULTS
Figure 1 shows that 5 ~M phentolamine completely blocked the contractile responses of the rat isolatod anococcygeus muscle to field stimulation (FS), 3 ~M noradrenaline (NA) and 10-15 uM tyramine (Tyr); contractile responses to the 2-10 ~1 black scorpion venom (BSV) were markedly blocked (91 f 2%) as well ; the quantitative data are shown in Table 1 . The contractile response to 2-3 ~M carbachol (CCh) was slightly blocked (19 f 3%) by phentolamine but it was completely blocked by 1 ~M atropine; the response to 50-75 mM KC1 was partially blocked (28 f 5%) by phentolamine but it was unaffected by atropine . During wash-out of each agonist, the contracted muscle relaxed readily and completely . Guanethidine (5 ~cM) completely blocked the contractile response of the muscle to FS only, whereas the responses to NA and BSV were potentiated by 56 f 15% and 62 t 18%, respectively ; responses to Tyr and CCh were unaffected by gtiartethidine; the response to
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M . C . E. GWEE et nl .
s ~.
'1 FIG . 1 . EFF8CIS OF PE~NTOLAI~IINE .
Relevant segments of tracing from a typical experiment showing contractile responses of the anococcygeus muscles to FS (20-30 V, 10 Hz x 10 sec, 1 msec pulse width), NA 3 pM (~), Tyr 10 pM (/), BSV 8 pl (Q), Ch 3 uM (/) and KC1 50 mM (p) in the (a) absence (control) or (b) presence of phentolamine 5 pM (j -. ). Each agonist was washed out at ( j). Quantitative data for the various agonist responses are given in Table l .
KC1 was slightly increased (13 t 3%) (see Fig. 2 and Table 1). Tetrodotoxin (TTx) (2 pM) also completely blocked the contractile responses of the muscle to FS only, but generally did not affect the responses to all the other agonists used, as shown in Fig. 3 and Table 1 . Desipramine 1 .5 pM completely blocked the contractile responses of the muscle to Tyr, whereas the peak responses to FS, NA and BSV were potentiated by 16 t 3%, 79 f 11 and 96 f 14%,respectively; the response to CCh was generally not affected by desipramine . However, the response to KC 1 was slightly enhanced and accompanied by a slower relaxation rate, presumably as a wnsequence of transmitter NA release following depolarization of the neuronal membrane (GII130N and Pot,t .ocK, 1973) (see Fig. 4 and Table 1). The figure also shows that, in the presence of desipramine, the muscle contracted by FS, NA and BSV also relaxed at a slower rate, which can be attributed to the block of NA reuptake into the nerve terminals by desipramine. Figure 5 shows that the anococcygeus muscle isolated from rats pretreated with reserpine (5 mg/kg s.c. x 24 h and 5 mg/kg i.p. x 3 hr) did not contract to FS and to the high concentration (100 pM) of Tyr used, whereas NA, BSV, CCh an KC1 produced contractile responses of the muscle which were significantly potentiated by 72%, 62%, 18% and 11% respectively, as shown in Table 2. The ultrafiltrates (2~ ~1) of BSV, FI, F~, F3, and the suspension of retentate (S) produced contractile responses of the Acm which were 93.3% (93.2, 93.3), 30.1 % (21 .9, 38.3), 11 .1 % (5 .5, 16.7) and 3.3% (0, 6.6), respectively, of the mean responses produced by 2 ~1 and 4 gel of the two different samples of BSV tested; the figures in parentheses are the range of values obtained for the filtrates and S from the two samples of BSV. Contractile responses of the Acm to all the filtrates and S, like the response to BSV, were completely
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T~t~ 1. CONIRACTQE Rt?sPOtvses oF rt~ ner ~sou~o errococcsrasus utusctE ro NA, TYR, BSV, CCH ~tvo KCl nv rm (a) ~zt9exc~ (coxtna.) oR (b) rxr~cE oF t~ro~nmve, cueNert~u~, retaoooroxuv oR t~rx~emve i?hentolamine 5 kM (n = 6) NA (3 ~eM) (a) (b) Tyr (10-15 ~I) (a) (b) BSV (2-10 pL (a) (b) CCH (3 pM) (a) (b) KCl (SO-75 pM) (a) (b)
2.SOt0.04 (0) 2.60t0.10 (0) 2.(0t0.20 0.17f0 .04 "" (91.4 f2.2 °/"l) 2.SOt0.06 2.OOt0.08' (I8.9t3 .0%1) 2.SOt0.09 1 .70t0.08" (28.4f5 .01%1)
Response (8-tension) Guanethidine Tetrodotoxin 2 pM (n = 6) 2 ~M (n = 4) 2.23t0.11 3.43f0.24"" (56 .3 t 14 .6%t) 2.37t0.09 2.47 t0.19 2.48t0.19 3.88f0.28"" (61.7t I8.2%T) 2.35t0.14 2.35t0.19 2.38t0.04 2.68t0.08' (12.7f3 .4%j)
2.33f0.09 2.63t0.17 2.33f0.12 2.73f0.09 1 .98t0.09 1 .93t0.10
Desipramine 1.5 pM (n = 6) 2.35t0.08 4.18f0.20" (79.2 t 10 .6%j) 2.31t0.17 (0) 2.08t0.10 4 .05t0.26'" (96.2t 13.5%j)
2.45t0.03 2.15t0.06
2.52t0.17 2.40t0.20
2.40t0.13 2.53t0.13
2.57t0.15 3.28t0.23
Each value is the mean t S.E .M . Figures in parentheses show percentage (%)inhibition (j) or potentiation (j) of contractile responses of the muscle ; (0) = complete inhibition of responses. Significantly different (' "P < 0.01 ; "P < 0.05) from corresponding control value (paired t-test).
blocked by phentolamine (5 ~M). The i.v. LD w of BSV was found to be 0.13 ml per kg body weight of mouse. The concentration of NA detected in the venom from 15 scorpions ranged from about 0.4 to 4.5 mM with a mean of 1 .8 t 0.3 mM. The concentration of DA ranged from about 8 to 58 pM with a mean of 31 f 4 ~M, only 1.7% of that of NA. Adr was not detected in all the BSV samples tested . After the first collection of BSV, the 15 scorpions were divided into three groups of five. BSV was collected 7, 14 or 21 days later. The NA and DA contents were expressed as a percentage of the values obtained previously (on first collection) for the same five scorpions . NA contents on days 7 and 14 were significantly reduced to about 30% and 75% of control, respectively . On day 21, the NA content was no longer significantly different from control. In contrast, DA content on day 7 was more than four-fold higher than the control level and then it decreased back to control levels on days 14 and 21 (Fig. 6). DISCUSSION
The contractile responses of the anococcygeus muscle to FS, NA and Tyr are due to activation of postjunctional a-adrenoceptors present in the muscle : FS causes the release of NA which produces the motor (excitatory) responses obtained (GILLFSPIE, 1972, 1980),
M. C. E. GWEE et d.
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s
ml.
l~l~l~J\l~ . . . . . .~
hio. 2. Et+t+acrs of ao~xertnnuvE . Relevant segments of tracing from a typical experiment showing contractile responses of the anoooaygeus muscle to FS (20-30 V, 10Iiz x 10 sec, 1 msec pulse width), NA 3 pM (~), Tyr 15 pM ("), BSV 10 pl (p), CCh 3 pM (") and KC1 75 mM (p) in the (a) absence (control) or (b) presence of guanethidine 5pM (j -. ). Each agonist was washed out at (j) . Quantitative data for the various agonist responses are given in Table 1.
s et. ro~
J~.J~.~~ '1
'1
°1
Ftia. 3. ErPeccs oa~ israonoTO~. Relevant segments of tracing from a typical experiment showing contractile responses of the anococcygeus muscle to FS (20-30 V, 10 Hz x 10 sec, 1 cosec pulse width), NA 3 pM (~), Tyr 10 pM ("), BSV 6 pl (Q), CCh 2 pM (") and KCl 50 mM (p) in the (a) absence (control) or (b) presence of tetrodotoxin 2 pM (j -. ). Each agonist was washed out at (j) . Quantitative data for the various agonist responses are given in Table 1.
Black Swrpion Venom Activity
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s ~.
L~ FIG . 4. EPFECIS OF DI~IPRA6rINE .
Relevant segments of tracing from a typical experiment showing contractile responses of the anoooocygeus muack to FS (20-30 V, 10 Hz x 10 sec, I cosec pulse width), NA 3 ~M (~), Tyr is plvl ("), HSV s~l (p), CCh 3 pM (") and KCI 7s mM (p) in the (a) absence (control) or (b) presence of desipramine Ls pM (j ~ ). Each agoniat was washed out at (j) . Quantitative data for the various agonist responses are given in Table 1.
whereas Tyr acts indirectly via the discharge of NA from the adrenergic nerve terminals (GERBER and N>FS, 1990 ; TRENDELENBURG, 1991). The contractile response of the muscle to CCh is mediated via stimulation of postjunctional muscarinic receptors which are widely distributed in the anocoocygeus muscle (G>z .LESr~, 1980). The response of the anococcygeus muscle to KC1 has been attributed to direct depolarization of the smooth muscle membrane as well as to the release of NA from the adrenergic nerve terminals as a consequence of neuronal depolarization (GB3SON and Por r..ocK, 1973). The agonist action of BSV in mediating the contractile (adrenergic) response of the anocoocygeus muscle closely resembled the action of NA: firstly, blockade of the BSV-induced response by the selective a-adrenoceptor antagonist, phentolamine, shows
FS
s~ FKI . s. EFFI~CIS of IIF~tFnva rxErAr.~r~rr. Rekvant segments of tracing from a typical experiment showing contractile responses of the anocoaygeus mueck to FS (20-30 V, 10 Hz x 10 sec, 1 cosec pule width), Tyr 100 pM ("), NA 3 ~I (~), BSV 3 pl (Q), CCh 2pM (") and KCI 50 mM (D); muscle tisue was isolated from rats pretreated with reserpine (s mg/kg e.c . x 24 hr and s mg/kg i.p . x 3 hr). Each agoniet was washed out at (j). Quantitative data for the various agoniet responses are given in Table 2.
131 2
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E . GWEE et al.
TARIE 2. EFPECIS OF RE~ItPINE l'REI'REATAICNT ON CONTRACI7LE RE4PONgF3 OF TF~ RAT ISOI.ATPD ANOCOCCYOI?US UtU3CLE TO NA, TYR, BSV, CCH AND KCl
Control
Response (g-tension) Pretreatmentt
(n = 4)
% Potentialion
2 .35 f 0.04 2 .40 t 0.07 2 .16f0.08 2 .45 f 0.06 2 .45 f 0.05
4 .03 f 0 .32*' (0) 3 .SOt0 .33 ** 2 .88 f 0 .24* 2 .73 f 0 .05*
71 .5 62 .0 17 .6 11 .4
(n = 22
NA (3 pM) Tyr (100 uM) BSV(2-10p1) CCH (2-3 pM) KCl (50-75 mM)
Each value is the mean f S.E .M . The control mean is calculated from the pooled individual values obtained for each condition of study shown in Table 1 . (0) = no response . Significantly different (**P < 0.01 ; *P < 0.0~ from the corresponding control value (group t-teat) . $Reserpine pretreatment: 5 mg/kg s.c . x 24 hr and 5 mg/kg i .p . x 3 hr .
that the agonist action of BSV was mediated via stimulation of postjtmctional aadrenoceptors in the muscle either directly, or, indirectly, via the release of NA from adrenergic nerve terminals. However, BSV could not have caused stimulation of the adrenergic nerve to release transmitter NA since, like the response to exogenous NA, the response to BSV was not sensitive to blockade either by TTx, a selective blocker of voltage-sensitive sodium channels (NAAAHA.CFir, 1974 ; CATTIItALL, 1980; R1TCfue, 1980), or by guanethidine, a typical adrenergic neurone blocker (GERBER and N>FS, 1990). Furthermore, the agonist action of BSV in the anococcygeus muscle also could not have been due to the indirect discharge of NA from the adrenergic nerve terminals, since muscle from reserpine-treated and non-treated rats exhibited similar responses to BSV. Reserpine treatment would have caused depletion of vesicular NA in the adrenergic nerve terminals of the muscle (GERBER and N>ES, 1990), as evidenced by a lack of response of the muscle to FS and the high concentration of Tyr, both of which are entirely dependent upon the
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~aya FIO. 6. NA AND DA wxceNrRAnoNS IN BSV AsreR eLecrRICAI, srutvunoN. Changes in NA (~) and DA (/) concentrations in BSV at various times after total discharge of venom induced by electrical stimulation on day 0. Results are expressed as percentage of control concentrations measured on day 0 for the same scorpion . *Statistically different (P < 0.05) from control as calculated by Student's t-teat for paired data .
Black Scorpion Venom Activity
131 3
availability of endogenous NA for their agonist actions. The potentiation of the contraofile responses to NA, BSV, CCh and KC1 by reserpine pretreatment can be attributed to the development of non-specific supersensitivity of the muscle tissue to the compounds used (GnxB>~ and N~s, 1990). The contractile responses of the anocoocygeus muscle to Tyr was, as expected, abolished by desipramine which is attributable to the blockade of Tyr transport (via uptake-1) into its site of action in the adrenergic nerve terminals (TRENDELENBURG, 1991). However, the contractile responses of the muscle to NA and BSV were markedly potentiated by 79 t 11 % and 96 ± 14% by desipramine and by 56 t 15% and 62 ± 18 %, respectively, by guanethidine, a phenomenon that can be attributed to blockade of the uptake-1 process for catecholamines (G>:1tBnx and N>~s, 1990 ; Tx~vnsr..ExsuxG, 1990). Thus, the NA-like fraction of BSV probably also undergoes uptake-1 into the adrenergic nerve terminals and it is not dependent on such an uptake to mediate its agonist action . Our results provide the first substantial evidence that BSV can act directly on postjunctional a-adrenoceptors to produce contractile responses of the rat isolated anococcygeus muscle; the a-agonist action of BSV in the rat anococcygeus muscle therefore closely mimics the action of NA. Moreover, the a-agonist activity of BSV was present almost entirely in the ultrafiltrate obtained after dialysis of the crude venom, since equal volumes of the first filtrate (F,) and crude BSV taken from the same sample produced almost equiactive contractile responses of the Acm which were sensitive to phentolamine blockade . Thus, the a-agonist activity of BSV cannot be due to the presence of a large protein molecule; instead, a substance of smaller mol. wt, possibly NA itself, is responsible for mediating the a-agonist actions of BSV in the Acm. HPLC measurements confirmed the presence of NA in the BSV: the high concentration (mean of 1 .8 mM) of NA found in BSV can therefore account for the marked postjunctional a-agonist action of BSV in the rat Acm. Thus, 3-10 p1 of crude BSV (equivalent to 5-18 nmol of NA, calculated on the basis of the mean NA concentration of 1 .8 mM) produced contractile responses of the Acm which were comparable to those produced by NA 3 pM (i.e. 18 nmol in a 6 ml bath). The DA concentration, however, was 60-fold lower than that of NA, suggesting that catecholamine is synthesized via the usual pathway from tyrosine with DA as one of the intermediates . The venom of the honey bee, Apis meilifica, has also been reported to contain catecholamines (BANKS et al., 1976; OWEN and BRIDGFS, 1982), although the reported contents were far less than that detected in the BSV in the present study. The NA and DA contents in the bee venom have also been found to have age-dependent and seasonal variations (OweN and BitlncES, 1982). It is also possible that BSV may exhibit the same types of variations which can thus explain the presented observed 7- to 11-fold individual variation in the concentration of catecholamines . After complete discharge of the venom on the first collection, it was observed that the NA content recovered up to 75% of the mean control value within 2 weeks; recovery was statistically complete in the 3rd week . In contrast, DA content was markedly elevated only on day 7 when the NA content was only about 30% of control . This may reflect extremely active synthesis due to markedly increased tyrosine hydroxylase activity caused by loss of feedback product inhibition by NA. The 2-week period required for a 75% recovery of the NA content seems long, but this long recovery period may be a direct result of marked depletion of the venom gland caused by the electrical method employed for venom extraction, an unlikely condition in the event of natural venom discharge, for scorpions are known to be economical in the use of venom (CLOUDSLEY-Txo~soN, 1992).
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M. C. E. GWEE et al.
In conclusion, our results clearly show that the black scorpion venom has marked postjunctional a-agonist action in the anococcygeus muscle, which is mediated by noradrenaline present in the venom. Acknowkdgenrent-This work was supported by research grants RP 870357 8c RP 890321 from the National University of Singapore. REFERENCES Buvrcs, B. E. C., H~uvsoN, J. M. and Suact.tae, N. M. (1976) The isolation and identification of noradrenaline and dopamine from the venom of the honey bee, Apir mellifica. Toxicon 14, ! 17-125 . B~wesrute, H. S. and Bew~a, P. H. (1989) Stings by red scorpooa (Buthotus laminas) in Maharashtra State, India: a clinical study. Traps. R. Soc. trop. Med. Hyg. 83, 858-860. CATIt~IALL, W. A. (1980) Neurotoxins that act on voltage-sensitive sodium channels. .! . Rev. Pharmac. Toxicol. 20, 15--43 . Cwunsr~r-THOIIPSON, J. L. (1992) Scorpions. Biologist 39, 206-210. Covrutm, F. and Jovtflt, E. (1984) Mechanism of action of scorpion toxins . Handbook Notwal Toxins 2, 659-678. Et.-Asw+x, M. F. (1984) Metabolic effect of scorpion venom. Handbook Notwal Toxins 2, 551-575 . EL-AYE, M. and D>~.ont, P. (1984) Immunology and immunochemistry of scorpion neurotoxins . Handbook Notwal Toxins 2, 607-637. Gexet+x, J. G. and Nn3s, A. S. (1990) Antihypertensive agents and the drug therapy of hypeRension . In : Goodman and Gilmmi's The Pharmacological Basis of Therapeutks, pp . 784-813 (Gooo~x, Gu.~N, A., Ru .r,, T. W., Nm A. S. and T~Yt.on, P., Eds) . New York : Pergamon Press. Giesox, A. and Pot.t .oc[, D. (1973) The effects of drugs on the sensitivity of the rat anocoocygeus muscle to agonists . Br. J. Pharmac. 49, 506-513. Gar ~ravm, J. S. (1972) The rat anocoocygeus muscle and its response to nerve stimulation and to some drugs . Br. J. Pharmac. 45, 404-416. Gn.rrsrtE, J. S. (1980) The physiology and pharmacology of the anococcygeus muscle . Trends Pharmac. Sci. 1, 45357. Guertox, M. and OVSYSi1Cr~t, I. (1984) Cardiovascular effects of scorpion vtaoms . Handbook Notwal Toxins Z, 639-657. Hex, F. (1984) Production of scorpion antivenin. Handbook Notwal Toxins 2, 57706. Htn.trWaa.utis, V. (1988) How to Ifeep Scorpions. London : Fitzgerald. Ktitt, R. K. M. and YHOZ~a, M. E. (1986) Electrocardiographic changes is experimental myocarditis induced by scorpion (Buthus tamtthv) venom. Ind. Heart J. 38, 206-210. MtmrxY, K. R. K. and Veen., A. E. (1988) Elevation of plasma angiotensin levels in dogs by Indian red scorpion (Bather tamuhit) venom and its reversal by administration of insulin and tohuoGne. Irrd. J. med Res. ag,376-379 . Neawxi+sra, T. (1974) Chemicals as tools in the study of excitable membranes. Physiol. Res. 54, 81389. Oww, M. D. and BarooHS, A. R. (1982) CatechoLunines in honey bee (Apis mellljera L.) and various vespid (hymenoptera) venoms. Toxicon 20, 1075-1084. Pay L. D. (1984) Structure of scorpion toxins. Handbook Notwal Toxins 2, 513-549. Rf~cw~rrorurt, L. K., Aa+3ewet,, O. P., AcEtYVZxwx, K. E., Cxntrorunt, L., Vmtsato~n, J. R. and Getvatn.Y, D. K. (1986) Fractionation and biological activities of venom of the Indian scorpions ButhurtamLkrs and heterometrua bengaleaess. Ind. J. Biochem. Biophys. 23, 355-358. Rtic,~, J. M. (1980) Tetrodotoxin and saxitoxin and the sodium channels of excitable tissue . Trends Pharmac. Scl. 1, 275-279. Suwrn, J. M., Meves, H. and W~rr, D. D. (1992) Neurotoxins in venom from the North American scorpion Centrwoides sculptwatw Ewing. In: Notwal Toxlru : Toxkology, Chemistry and Sajety, pp . 236-263 (IC~.ea, R. F., Mt rrowe, N. B. and Tu, A. T., Eda). U.S.A .: Ahtlren. Taet~l~t+strrta, U. (1991) Functional aspects of the neuronal uptake of noradronaline. Trends Pharmac. Sci. 12, 334-337.