Venoms and enzymes: effects on permeability of isolated single electroplax

Tostcon, 1973, Voi. 11, pp. 149-154. Pergsrrwn Prtsa. Printed in Great Britain

VENOMS AND ENZYMES : EFFECTS ON PERMEABILTTY OF ISOLATED SINGLE ELECTROPLAX P. Ros>?rtr>ntG Section of Pharmacology aad ToxtooloQy, University of Connecticut, School of Pharmacy, Scoria, Conn., 06268, U.S.A. (Accepted jo~Pirb/ftaattoe

is AIIgILTt 1972)

AMiset-The isolated single electroplax was mounted betwaea two pools of fluid, one initiai~ non-radioactive, the other containing'~Ccholine. Treated cells wereexposedfor ZO min to 1 mg per ml conxntrations ofvarious eazymes aad veaoms. Cottonmouth moocasia venom (C~ caused a htmdred-fold il>crease in the oantrol level (0.01 per neat per 10 min) ofpenetration. Add-boiled CMV, phtupholipase C and lipase caused only a 2- to 3-fold iliabase in peaetiatioa while the following treatments had no effect on permeability : all~aline-boiled CMV, phosP~P~e D. l~yaluronidase, dlymotrypsin, trypsin, veaa~m hamino acid oxidase sad phosphodiaetelaae, Pined cobra neurotoxm (0"2 mg/mn, oetyltrimatliylammonium bromide, e.,entrlaiofdea atallpttQatea, Notedtta acirtatua, Heloa~rnea %tOlr%difllt and Ngja nrya vemoma . The taxies of acid-boiled CMV is assoetiated with extensive phospholipid splitting, therefore, it does not appear that intact phospholipids are necessary for membrane permeability. The oomponemt of uabor7ed CMV which increases permeability is not haown. IT rtes

INTRODUCTION

been suggested that phospholipids are responsible for the membrane resistanex and capacitance characteristics of bioelextrically excitable tissues (Torres,1960 ; Nwi:waesfu and Tortes, 1964). However, the squid giant axon exposed to venom phospholipase A (PhA) or bacterial phospholipase C (PhC) can maintain its normal permeability properties even in the presenex of extensive phospholipid splitting and loss of phospholipid phosphorus from then membrane (RoBLNrEite3 and COxnrtEe, 1968; RUBENBERCi, 1970). I thought it of interest to extend these studies to a synaptic containing preparation, since it has been suggested that phospholipids may be involved in the permeability alterations induced by transmitter agents (WeTxuvs, 1965 ; Dulexrr.r, et al., 1969 ; i .AAAAwa~ et al., 1963 ; i .~AA "AtizR and LSCIIT, 1965 ; I:exrterEe, 1968). The isolated single electroplax from the electric cel, which was used in this study, has thousands of synapses per all. It was noted that the post-synaptic potential and action potential of this preparation are blocked when more than ona-third of the phosphatidyl choline of the cell was hydrolyzed (HARTELS and Ro3~TrErtG, 1972). Stimulation of the cell and exposure to sextylcholine also increased 32P and t~C incorporation into phosphatidylinositol and phosphatidyl choline (unpublished observations) . It appears therefore that phospholipids might be involved in the molecular events associated with excitation of this synaptic containing preparation. It is also of interest to know whether the resting membrane permeability of this preparation is also controlled by phospholipids ; this is the subject of the present study. 7n27CON 1D7a YoL !I.

149

P. ROSENBERß MATERIALS AND METHODS

The method of isolating single electroplax cells from the Sachs organ of the electric eel Electrophorus electricus, was the same as previously described (BARTFi c and ROSENBERG, 1972). The single cell ( ~ 10 x 4 x 0~2 mm) is mounted in a special chamber between two pools of Eel Ringers solution (pH 7) of the following composition (mM) : NaCI, 160; KCI, 5 ; CaCl2, 2 ; MgC12, 2 ; NaH2P04, 0"3 ; NaZHP04, 1 "2 ; and glucose 10. The innervated membrane of the cell which contains about 50,000 synapses is placed directly against a window (5 x 1 mm) punched out of a sheet of nylon, and is held in place by a nylon grid which is pushed against the non-innervated membrane. Compounds dissolved in one pool could pass to the other pool only through the cell. The non-innervated membrane of the cell was exposed to a 3"5 ml pool of Eel Ringers containing the radioactive material . Every ten minutes 0" 1 ml samples were taken from the 1 "5 ml pool bathing the innervated membrane ; replacing it with 0"1 ml of non-radioactive Ringers solution and making approP riete corrections in calculations of radioactivity . In most of our studies we used [N-methyl-i4C] choline chloride (3"8 mC/mM) as an example of a lipid insoluble, impermeable compound. The tertiary analogue of choline [2-i4C] dimethylaminoethanol (~ 1"5 mC/mM) was used as an example of a lipid soluble and permeable compound . Sufficient non-radioactive choline or dimethylaminoethanol was added to each pool to give a final concentration of 10_a M. The radioactivity in the large pool opposite the non-innerrated membrane was determined prior to the beginning of each experiment and ranged between 900,000 and 1 " 1 million counts pcr min per ml. The response of each cell to direct and indirect stimulation was checkod prior to the beginning of each experiment, and only those cells with normal sized action potentials were used. The usual proeodun was to mount the cell, add the radioactive material to the large pool and obtain two 10 min control readings from the small pool in order to make sure that there were no leaks. In two experiments in which there wen obvious leaks, the radioactivity found in the small pool in 10 min was much greater than that found in any of our other experiments, even after 90 min. The enzymes, venoms or other tnatments wen then added to both pools for 20 min abler which the pools were rinsed with fresh eel Ringers solution and fresh radioactive material was added to the large pool. Samples wen then taken from the small pool at 10 min intervals for another 40 or 50 min. Aquasol (New England Nuclear Corp.) liquid scintillator fluid was added to each sample and they wen counted in a Packard (Mode13375) Tri-Garb liquid scintillation spectrometer . All results wen expressed as per cent penetration, that is, the radioactivity per ml in the small pool expnssod as a percentage of that in the large pool. The acid- and alkaline-boiled solutions of cottonmouth moccasin venom wen prepared as previously described (RosEtlsatG and PODLF~RI, 1962). Measurements of phospholipase A activity wen made by titrating the liberation of free fatty acids frôm egg yolk (Dor..E, 1956). The radioactive materials were obtained from the New England Nuclear Corporation, Boston, Mass . The other venoms and enzymes used and their sources are as follows : Naja naja, Crotabus admnanteus and Agkistrodon piscivorus (cottonmouth moccasin) venomsRoss Allen Reptile Institute, Silver Springs, Florida; Heloderma horridu»r-Miami Serpentarium, Miami, Florida ; Notechis scutatus-L. Light and Co., Colabrook, England ; Apis mell~ca (bee venom}-Nutritional Biochemicals Corp. Cleveland, Ohio; phospholipase D sigma Chemical Co., St. Louis, Missouri ; phospholipase C, hyaluronidase, trypsin and lipase (wheat germ}-Mann Research Lab., New York, N.Y. ; chymotrypsin-Miles Lab. Kankakee, Illinois ; lysolecithin-Pierce Chem . Co., Rockford, Illinois ; phosphodiesterase TOXICON1973 Yol. Il.

Venoms on Electrophuc

151

(venom) and 1-amino acid oxidase (venom~Wortlvngton Biochemical Corp., Freehold, New Jersey ; I am especially grateful to Dr. C. C. Yang, Kaohsiung, Taiwan for his generous gift of a purified sample of cobra neurotoxin. RFC [Jl,'f$

The control penetration of l4C choline averaged only slightly greater than 001 per cent per 10 min period (Fig. 1). Even this may not represent actual penetration of choline but may be due to trace contamination with its tertiary analogue . The penetration of the tertiary

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IHBISOIATBD

All data ere p~ted as mesas f steadard errors and are each based upon 3 to 6 experiments : ~Pt far the 10 and 20 min control chokers vah~es which are the pooled values from all of the experiments and are based upon 30 experiments for each value, and the NT points which are oath only based upon one experiment. CMV = cottonmouth moccasin venom; H+ and oH- - spa ana alkaline bodes CMV (1 m~mn reapectivdy. rrr - purißed cobraneurotoxin. Pendration of -- - DMAE, - Choline.

analogue of choline (dimethylaminoethanol), between 20 and 50 min, was about 100 times that of choline. We obtained almost the same levels of control penetration if the cells were reversed in the holder, with the innervated membrane being exposed to the large radioactive pool. Cottonmouth moccasin venom (CMV) increased penetration of choline almost 100-fold, wherase acid-boiled CMV only caused a 2- to 3-fold increase in penetration, and alkalino-boiled CMV had no effect. Our measurements of PhA activities of these prepararoxrcovr~s votrr.

152

P. ROSENBERG TAHIE 1. rbl4blRATION OP [N-MEI11YIr1~ CHOIdAII. IN ISOLATED EIECIROPLAX 10'

0'Ol f0'001(30)

20'

30'

40'

a02 0" 03 a05 f0'003(30) f0'002(6) ~0-01(S) Naja n~ja Notechis JcstatuJ

c~ma,~rdes

JCiliptfOiOlffJ Heloderma horridum ~p~ >n<~~ ~MH l-AA oxiaase Phosphoaieatterase xyaluroniaase Trypsin Chymotrypsin Phoapholipasc c Phospholipase n

so'

Penetration

60'

70'

80'

90'

0'06 0'07 a08 f0'01(s) ~0" Ol(S) f0'Ol(3) a06 0'08 all 0'06 0 " 07 a08 so3 o'oa o'oa

0'11 a13 fa02(3) ~0'02(3) 0 " l4 0'ls 0'l0 al2 o~06

0'06

0'06

0'07

0'07

o-os o-os

o-06 o'06

aos am o'06

o-os o~os

o~

o'lo am

o-ll o'm

all aos

o~03 o-os 0~07 o'06 ; 0'l2 am ; 0'13 o"os; o-l2 ao9 : a13 o~07 o~os alo all o-06

o-lo al9 o-26 o~os; o~ o~; o-la o-lo; o"os o-06 am

o'09

o-30 0'20 o-la ; a22 o-2a ;

o-os

sao

All treatmeata were applied at a concentration of 1 mg/ml between the 20 and 40 min interval of time . The 10 sad 20 min control values represent the summed ava~ages for all axperimenta . The control pea cent penetration values are preseated as means f standard errors with the number of experiments indicated in parenthoais. All other values ere single expernneats . CTMH = cetyl trimethylammoniutn bromide ; 1-AA aucidase = 1 amino add oxldase .

tions showed that the acid-boiled preparation had at least 80 per cent of the enzymatic activity of the unboiled preparation, whereas the alkalino-boiled venom had no measurable activity ; results which are in agreement with previous findings (IIUGHFS 1935 ; BRAGANCA and QvASrs~., 1952; 1VIAGEB and TxonmsoN, 1960 ; RosEr~ExG and Na, 1963). Because of limited material we were only able to perform one experiment with purified cobra neurotoxin (0~2 mg/mn which appeared to decrease the penetration of choline. Some of the other enzymes and venoms used are shown in Table 1 . PhC, which splits off the phosphorylated bases from phospholipids, increased penetration to about the same extent as acid-boiled CMV. A preparation of lipase from wheat germ also appeared to slightly increase penetration. None of these effects are as great as observed with unheated CMV (Fig. 1). I also tested the effect of lysolecithin, the detergent produced by the action of PhA on lecithin, since some effects previously attributed to splitting of phospholipids by venom are now known to be due to production of lysophosphatides within the membrane (Rt788IVBEttG and CONDRBA, 1968) . The results, however, were for unknown reasons erratic, two experiments showing about a ten-fold increase in penetration while in two experiments there was no effect on penetration. Stimulating the electroplax cell either directly or indirectly at 10 times per sec for 20 min had no effect on the control penetration of choline. Helodernta horridton, PhD, lipase, 1-amino acid oxidase, hyaluronidase trypsin, chymotrypsin and alkaline-boiled CMV had no effect on the action potential during their 20 min of exposure to the cell. In contrast, the following treatments blocked conduction : cobra neurotoxin, hyaluronidase, Certtruroides JculpturatuJ venom, Naja ltaja venom, lysolecithin, cetyltrimethylammonium bromide, CMV and acid-boiled CMV. Similar observations with some of these drugs had been reported previously (B~ut~s and Ras»>~ta, 1972). T~OYICON 1973 Yot. 11.

Venons on Electroplax

153

DISCUSSION

PhA alone is not responsible for the marked increase in permeability produced by CMV (Fig. 1). The acid-boiled venom has almost the same PhA activity as the unboiled venom and yet is only weakly active. We had shown previously that this concentration (1 mg/ml) of acid-boiled CMV split 89 per cent of lecithin, 76 per cent of phosphatidyleth,~ngjamine and 98 per cent of phosphatidylserine in the isolated single electroplax (BARTIIS and RosElvBEltc, 1972). We also found similar amounts of splitting produced by PhC and Naja raja venom (unpublished observations). The results with acid-boiled CMV and with PhC indicate that extensive phospholipid splitting causes a small increase in gross membrane permeability, however, it does not explain the marked effect observed with unboiled CMV. It is quite interesting and unexpected that almost all of the phospholipids of the eel electroplax can be split with only a small alteration is gross membrane permeability being observed. The presence of intact phospholipids does not appear essential to the maintenance of normal membrane permeability . It is not likely that a non-enzymatic venom neurotoxin is rosponsible for the marked increase in membrane permeability. Venom neurotoxin is not destroyed by boiling at an acid pH (LARSHN and Woll~, 1968 ; M1a.DRUI~, 196 and so should still have been present in acid-boiled CMV. ïn addition, Nafa raja venom (which contains a neurotoxin) and a purified neurotoxin were both ineffective in altering membrane permeability. CMV contains many enzymes including phosphodiesterase, 1-amino acid oxidase, hyaluronidase and proteolytic enzymes resembling trypsin and chymotrypsin ; none of which increased permeability (T'able 1). The ability of CMV to increase permeability to 14C labelled choline as noted in this present study, ie paralleled by its ability to increase exposure of electroplax cholinesterase to its substrate acetylcholine (Raearts>utG and DEl-rBARN, 1964). Similar results were found with lobster and squid nerve and frog muscle. We had not, however, investigated which component of the venom was responsible for allowing acetylcholine to more effectively reach the enzyme . I do not know what heat sensitive component of CMV is responsible for markedly increasing permeability to t4C choline. None of the venom components we tried could explain the action of unboilod CMV. A combination of two or more venom components may be necessary for producing the increase in permeability. Ack~wwledganxnts-Thanks are extended to Mrs. CscaJS C3""~"~.vo for exoellmt technical sasistaaoe. This work was supported in part by a great from the National l:natitutea of HealW (NS09008) . REFERENCES BNereta, E. end Roswsvna, P. (1972) Correlation betweenelectrical activity and splitting of phospholipids by snake venom in the single eledr+oplax. J. Nruroclrear .l9,1251 . Bx~a~ca, B. M. and Ques~,, J. H. (1952) Action of snake venom on acetylcholiae synthesis in brain Nature, Load.169, 695. Do~tB, V. P. (1956) A relation betweennoa~aterifled fatty acids in plasma and the mdabolsm of glucose. 1. clia. larrst. 36, 150. Dug, J., eeaurro, J. T. and Fh~>ß., R. O. (1969) Aatylcholine action-bloche~ical aspects. Sclmae 166, 862. Hvom?s, A. (1935) The action of snake venom on surface films. Bloduar. l. 29, 437. L~atussa, M. c . (1968) Ttansynapttc stimulation of phosphatidylinositol metabolism in :y+mpathetic naaons in situ . l. Nturoc!>em .16, 803. L~xru~, M, ß. and L.atcsr, W. S. (1%5) Metabolism of phoaphatidyl inositol and other lipids in active neurons of sympathetic ganglia and other peripheral nervous tisanes. J. Narrodunr.12,1. r-Nenwe~, M. C3., IC~sux, J. D. and Lscxr, W. S. (1%3) Effects a~f temparatuc+e, cak~um sad activity on phoapholipid metabolism in a sympathetic ganglion. J. Nrranelrsm.l0, 549. 7nYICON 1973

YaJ. ll .

154

P. ROSENBERG

Lexseiv, P. R, and Wourn, J. (1968) The tôxic proteins of cobra venom. Biochem. Pharrnac . 17, 503. MeaEe, W. L. and TxoaesoN, R. H. S. (1960) The estimation of phoapholipase Aactivity in aqueous systems. Biochem. l. 77, 526. Ms.nntnb, B. S. (1965). The actions of snake venons on nerve and muscle. The pharmacology of phospholipase A and of polypeptide toxins. Plwrmac. Rev. 17, 393. Nenet~esFn, T. aad Toeua, J. M. (1964) Properties of axon membrane as atTected by cobra venom, digitonin and professes. Am. J. Physlol. 207, 1441 . RossneEao, P. (1970) Function of phospholipids in axons: depletion of membrane phosphoras by treatment with phospholipase C. Toxlcon S, 235. Itoset~neo, P. and Corroxae, E. (1968) Maintenance of axonal conduction and membrane permeability in presence of extensive phoapholipid splitting. Bfoclrem. Plrasrrac.l7, 2033. Raswe~ea, P, and DgrraeRx, W:D. (1964) Increased cholinesterase activity of intact cells caused by snake venons. Bdoehan. Pharnnc.l3, 1137. Itosar~ta, P. and Na, R. Y. (1963) Factors in veaoms leading to block of axonal conduction by curare . Btod~im. bfophys. Acts, 75, 116. Rauaa, P, asti Po~ni1~, T. R. (1962) Block of conduction by aoetylcholine and d-tubocmarine after treatment of squid axon with cottonmouth moccasin venom. l. Phanaoc . exp . 71~er.137, 249. Tosses, J. M. (1960) Farther studies on the nature of the excitable membrane . l. gcn. phyriol. 43, 57. Wez~mvs, J. C. (196 Pharmacological receptors and general permeab~7ity phenomeas in cell membranes. 1. tJxor. Biol. 9, 37.

7nY1CON 1973 VoL ll .