1.12% fig; Univemfigy OVERDUE FINES: 25¢ per aw per item RETURNING LIBRARY MATERIALS: Phce in book return to remove charge from c1 rculat1on records PYRETHROID INHIBITION OF NEURAL ATPaseS BY John Marshall Clark II A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1981 ABSTRACT PYRETHROID INHIBITION OF NEURAL ATPases By John Marshall Clark 11 Various ATPases present in an axon-rich, membrane preparation from the retinal nerves of the squid, Loligo pealei, were studied. In each case, the activity specific to each enzyme can be recognized by the different activity in the presence and absence of specific ion(s). Other criteria used in classifying these ATPases are: apprOpriate ATP substrate concentrations (e.g., Km), temperature dependence, protein concentration, and the effect of inhibitors such as ouabain, CN-, EGTA, EDTA, ruthenium red and lanthanum. As a result, six distinctly recognizable ATPase systems have been found; (1) ouabain insensitive Na+-K+ ATPase, (2) ouabain sensitive Na+-K+ ATPase, (3) 2 2+ ATPase, (4) (332+ ATPase, (5) Mg2+ ATPase and (6) a nerve myosin. Ca +-Mg The biochemical process by which various pyrethroid insecticides affect membrane-bound ATPase activities of the squid nervous system was then examined. Of the six recognized ATPases, only Ca2+-stimulated ATPases are seen to be clearly sensitive to pyrethroid inhibition. These enzymes are Ca2+ ATPase, and Cam—Mg2+ ATPase. Both were shown to be located in subcellular fractions of the retinal nerve and Optic lobe of the squid. It was found that pyrethrin and its closely related synthetic analog, allethrin, primarily inhibit Ca2+ ATPase, whereas highly modified pyrethroids such as cypermethrin and decamethrin mainly inhibit Ca2+-Mg2+ ATPase. Permethrin, which is considered John Marshall Clark 11 to possess structural similarities to both naturally-occurring pyrethrins and highly modified pyrethroids, was found to have an intermediate property in terms 2 2 of its inhibitory potency to both Ca2+ and Ca +-Mg + ATPase. An effort was made to relate calcium regulation resulting from inhibition 2+-stimulated ATPases to the electrOphysiological symptoms induced of these Ca both in the axonic and synaptic regions of the nervous system by these insecticides. . 2+ 2+ 2+ . . . For “118 purpose, Ca ATPase and Ca —Mg ATPase were identified by similar means in the microsomal and synaptosomal fractions of the brain of the American cockroach, Periplaneta americana. Both enzymes were found to be 2 highly sensitive to the action of pyrethroid insecticides. Ca + ATPase was clearly more sensitive toward the action of allethrin when compared to the more highly modified compounds such as cypermethrin. Conversely, cypermethrin was 2+-Mg2+ ATPase activity than was determined to be more inhibitory to Ca allethrin. Overall, the synaptic preparation appeared to be the more sensitive site of pyrethrin action. This was particularly true in the case of 2+-Mg2+ ATPase which was the most sensitive enzyme nonmitochondrial Ca examined. These i_n vitro findings have been substantiated by in vivo experiments 2+-stimulated ATPases were where similar pyrethroid inhibition patterns of Ca observed in roaches which elicited pyrethroid poisoning symptoms. More importantly, i_n_v_i\_Ig inhibition occurred in the presence of a similar amount of insecticide which had been determined to cause a substantial amount of inhibition _i_r_1 vitro. By utilizing specific modifiers of ion flux, it was determined that at least a proportion of the ATPase activity may be related to Ca2+ transport. An effort 2 was made to correlate Ca +-stimulated ATPase activity which was clearly John Marshall Clark II 2 sensitive to pyrethroid action and Ca + transport mechanisms which control Ca2+ regulation in the nerve and hence its excitability. The author would like to dedicate this work to Professor Carolyn Burdick, Brooklyn College of the City University of New York, without whose help and continual scientific generosity this project would never have been completed. ii ACKNOWLEDGMENTS The author wishes to express his deep appreciation to Professor Fumio Matsumura for so many things that it is impossible to adequately summarize them here. In particular, the author would like to thank Mrs. Teruko Matsumura and her wonderful family for always providing such genuine hospitality and good feeling in their home wherever that may be. John Clark is especially grateful to Ms. Katie Pointer for containing her cosmic spirit, particularly during periods of complete lunar light. The author also wishes to acknowledge Mrs. Alice Ellis for her excellent assistance in the preparation of this dissertation. This project was supported by the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, research grant E801963: Toxic Effects of Chlorinated and Pyrethroid Pesticides. iii TABLE OF CONTENTS LIST OF TABLES . . . . .................... LIST OF FIGURES ..................... CHAPTER I. ATPase IN THE MEMBRANE PREPARATION FROM THE RETINAL NERVE OF THE SQUID, LOLIGO PEALEI . . INTRODUCTION ...................... MATERIALS AND METHODS ................. Animals . ...................... Chemicals ....................... Preparations of ATPase Fractions ............. ATPase Assay . . . . . . . .............. RESULTS . ....................... Ouabain Insensitive Np+-§$+ ATPase ............ Ouabain §ensitive NQ+-K ATPase ....... . . . . . Totfi‘l Ca 2; and Mg ATPase .............. Ca2+-Mg ATPase . . . ................ Ca AT se ............... . . . . . . Other Ca2+-stimulated ATPases ............. Total Mg ATPases .................. Myosin-type ATPases .................. DISCUSSION............ ...... CHAPTER 11. PYRETHROID INHIBITION OF NEURAL ATPases or THE SQUID, LOLIGO PEALEI .............. INTRODUCTION ...................... MATERIALS AND METHODS ................. Animals ..... . . . . . .............. Preparation of ATPase Fractions ............ ATPase Assay . . . . . ................ Chemicals ....................... iv vii 39 39 41 41 41 42 43 RESULTS............. .......... Survey of ATPase Sensitivity to Allethrin . . . . . . . . . 1) Ouabain Sensitive Na‘k-K+ ATPase . . . . . . . 2) Myosin-type ATPase 4. .+ ........... 3) Ouabain lingensitive Na -K ATPase . . . . . . . 4) T033} Mg ATPase . . . . ........ . . 5) Ca2+ ATgase . .......... . . . . . 6) Ca -Mg ATPase . ........ . . . . . 7) Axoplasmic ATPases . . ........... DISCUSSION . . . . . . ............. . . . . . CHAPTER HI. PYRETHROID INHIBITION OF NEURAL ATPases OF THE AMERICAN COCKROACH, PERIPLANETA AMERICANA INTRODUCTION. . . . . . . . ............... MATERIALS AND METHODS . ............ . . . . Animals............ ............ Preparation of Insect Brain Fractions ....... . . . . Determination of ATPase Activity ...... . . . . . . ATPase Inhibition Studies . ............... Determination of Permethrin Levels and Effects in Various Insect Brain Fractions, InVivoStudies.................... QWStudies.................... Chemicals . . . . . . . ................ RESULTS ...... . . . . .......... . ..... Characterization of Insect Brain ATPases . ........ Determination of Pyrethroid Action on ATPases oftheInsectBrain . Influence of Agents Modifying Ionic Fluxes Across Biomembranes . . . . . . . . . . .......... DISCUSSION............. ....... LISTOFREFERENCES..................... 44 44 44 47 49 49 49 49 60 62 66 66 68 68 68 69 70 71 71 72 73 73 78 91 99 107 10 11 12 13 14 LIST OF TABLES Standard Buffer Compositions . . . . . . . ..... . . . . . Effect of ATP Concentration on the Activity of Na+-K+ ATPase in the Presence of Ouabain and ON . . . . . . . . . . . Effects of Various Inhibitors on ATPase Activities . . . ..... Percent Inhibition of a High Affinity, 10-6M-C 2+ ATPase byVariousPyrethroid Esters . . . . . . . . . . . . . . . . . Percent Inhibition of Ca2+—Mg2+ Stimulated ATPase Activity byVariousPyrethroid Esters. . . . . . . . . . . . . . . . . . Percent Inhibition of 1 mM-Ca2+ ATPase Activity from Squid Optic Lobe Synaptosomes by Various Pyrethroid Esters ...... Percent Inhibition of Ca2+-Mg2+ ATPase Activity from Squid Optic Lobe Synaptosomes by Various Pyrethroid Esters . . . . . . Effect of Allethrin on 1 mM-Ca2+ ATPase and Caer-Mg2+ ATPase Activities Found in the Axoplasm of the Giant Axon oquuid,Loligopealei...... ..... Standard Buffer Compositions . . . . . . . . . . . ...... Distribution of ATPase Activities in Microsomal and Synaptosomal Fractions of the Insect Brain . . . . . ...... Influence of Cellular Inhibitors onzMitocfiipndrial Mg2+ ATPase and Nonmitochondrial Ca -Mg ATPase Activities from Disrupted Synaptosomes of the Insect Brain . . . . . . . . . Effects of l_n_ Vivo Administered Permethrin on Microsomal and Synaptosomal ATPase Activities of the Insect Brain . . . . . . . Binding of 14C-Permethrin to Insect Brain Fractions under In Vivo and E Vitro Conditions . . . .............. Influegge of Agents Modifying Na+, K+, or Ca2+ Flux on Ca -stimulated ATPase Activities from Microsomal and Synaptosomal Fractions of the Insect Brain . . . . ....... vi 11 26 55 56 58 59 61 74 75 79 89 90 LIST OF FIGURES Effect of Na+ concentration on the level of Na+-K+ ATPase activity in the presence (dotted line) and absence (solid line) of ouabain infihe squid retinal nerve3290,000 g precipitate as determined by Pi production (i.e., y- P-ATP hydrolysis). The composition of other ions andz+incubation conditions remained unchanged (e.g., K = 40 mM8 Mg = 10 mM, Tris-HCl = 30 mM t pH 7.1 and ATP = 5.6 x 10 M._ Incubation was for 10 min at 30 C. Ouabain concentration was 10 M. Experimental variability reported as standard error. - - Effect of K+ concentration on the level of NaJr-K+ ATPase activity in _ he presence (dotted line) and absence+ (solid line) of guabain (10 M). Other ionic compositions wereé Na = 160 mM, Mg = 10 mM. Ail? concentration was 5.6 x 10 M. Ouabain concentration was 10 M. Experimental variability reported as standard error. . - Effect of the change in Na+zK+ ratio on the level of Na+-K+ ATPase activity in the presence (dotted line) and absence (solid line) of ouabain (10 M) and with EGTA (1 mM) plus ouabain (circle line). Otrgr incubation conditions were: Mg = 10 mM and ATP = 5.6 x 10 M. Experimental variability reported as standard error. - . - - Effect of temperature changes on Na‘F-K+ ATPase activity in4the presence (dotted line) and abs nce (solid line) of ouabain (IZQ M). Incubation conditions wergé Na = 160 mM, K = 40 mM, Mg = 10 mM, and ATP = 5.6 x 10 M. Experimental variability reported as Standard error. ' Q 0 1 Q o 0 Q c Q 0 o O Q o o o o o c Q o o 0 Effect of temperature changes on ouabain sensitive Na+-K+ ATPase activity plotted as the difference between the total activity minus the ouabain insensitive activity (i.e., A) at high ATP concentration (ATP = 10 4 Other incubation conditionszvere: Na = 160 mM, K =40 mM,Mg = 10 mM,andouabain=10 M. . . . . . ._ . . . 2+ . 2+ 2+ Effect of Ca concentratIon on the level of Tota Ca and Mg ATPase activity. 2cher ionic compositions were Na = 160 mM, _ = 160 mM, and Mg = 10 mM._4ATP concentration was 5.6 x 10 M. Ouabain concentration was 10 M. Experimental variability reported as standard error. . . . . . . . ............... Effect of temperature changes on Total Ca2+ and Mg2+ ATPase activity in presence (dotted line) and absence (solid lime) of EGTA (Lag, mM. Ionic compo§itions were: Na = 160 mM, K = 160 mM, Mg = 10 mM, and Ca = 0.2 mM. ATP concentration was 5.6 x 8 10 M. Experimental variability reported as standard error. - . . - vii 10 12 15 16 10 11 12 13 14 15 16 17 Effects of EGTA concentration changes on Total Ca2+ and Mg2+ ATPase activity. Ionic comaqsitions were: Na = 160 mM, K = 160 mM,_§'Ig = 10 mM, and Ca = 0.2 mM. ATP concentration was 5.6 x 10 M. Experimental variability reported as standard error. . . . Effect of Ca2+ concentration on the level of Cay-Mg2+ TPase activity. Other ionic compositions wgre: K = 160 mM, Mg = 10 mM. ATP concentration was 5.6x10 M. . . . . ....... Effect of Ca2+ concentration on Ca2+ ATPase Pi production (solid line) and phosphorylation (i Pi-E, dotted line)+ activity. Other ionic compositions were: Na_8 = 160 mM, K = 160 mM. ATP concentration was 5.6 x 10 M. Experimental variability reported as standard error. ...... . . . . ......... . . . . Effect of Na+ concentration on Ca2+ ATPazsg activity. Other ionic compositions were: K 38 160 mM, Ca = 0.2 mM. ATP concentration was 5.6 x 10 M. Experimental variability reported as standard error. .................. . . . . . . Effect of K+ concentration on Ca2+ ATPgse activity. Other ionic compositions were: Na 38160 mM, Ca = 0.2 mM, and ATP concentration was 5.6 x 10 M. Experimental variability reported as standarderror................... ...... Effect of Ca2+zgoncentration on Li+ and K+ stimulated ATPase activity (i.e., Ca + ATPase like). Other ionic compositions were_g_.i = 160 mM and K = 160 mM. ATP concentration was 5.6 x 10 M. Experimental variability reported as standard error. . . . ..... Effect o£+Li+ concentration on Mg2+ plus K+ stimulated (dotted line), and Ca plus K stimulate?+ (solid line) ATPase activiaies. Concentration of ions were: Mg = 10 mM, K = 40 mM, and Ca = 2 w, K = 160 mM, respectively. ATP concentration was 5.6 x 10 M in each case. Experimental variability reported as standard error... Effect of Mg2+ concentration +on total Mg2+ ATPase activity. Other ionic compositions were: N38 = 160 mM, K = 160 mM. ATP concentration was 5.6 x 10 M. KCN concentration was 2 mM. Experimental variability reported as standard error. . ....... Effect of Na+ concentration+on total Mg2+ ATE‘ase activity. Other ionic compositions were: K_8 = 160 mM, Mg = 10 mM. ATP concentration was 5.6 x 10 M and KCN concentration was 2 mM. Experimental variability reported as standard error --------- Effect of lanthanum concentration on total Mg2+ ATPase aglivity. Incubation conditions were: Na = 160 mM, K =_§60 mM, Mg = 10 mM, and ATP concentration was 5.6 x 10 M Experimental variability reported as standard error. - - - - - - . viii 17 18 20 21 22 23 25 27 28 .29 18 19 20 21 22 23 24 Effect of Mg2+ concentration on myosin-type ATPasg activity. Other incubation_gonditions were: K = 60 mM, ATP = 10 M (dotted line) or 5.6 x 10 M (solid line), and EGTA = 0.5 mM. Experimental variability reported as standard error. . . . . . . . . . . . . . . Effect of Ca2+ concentration on nerye myosin-type ATPas_e_3activity. Other incubation_ onditions were: K = 60 mM, ATP = 10 (dotted line) or 5.6 x 10 M (solid line), and EGTA = 0.5 mM. Experimental variability reported as standard error ............... Effect of K+ concentration on nerve rayosin-type ATPase activiéy. Other incubation condit'ons were: Mg = 1 mM, ATP = 10 M (dotted line) or 5.6 x 10 M (solid line), EGTA = 0.5 mM, EDTA = 2.5 mM. Experimental variability reported as standard error. . . . . . Development of stable pyrethroid esters. Compounds unstable under field conditions are given in the left-hand column. Compounds more stable under field conditions are given in the right-hand column. . . Effect of (+)-trans allethrin on ouabain sensitive Nafik-K+ ATPa e (N+a pump) as determined as the difference (M) between total a J: K ATPase activity (-ouabain) and ouabain insensitive Na -K ATPase activity (+ ouabain). Enzyme source was the 90,000 g fraction of the squid retinal axon preparatigg. ATP concentration was 10 M. Ouabain concentratipn was 10 1V5.+ Other incubation conditions were: Na =160 mM, K =40 mM, Mg =10 mM in 30 mM Tris-HCl at pH 7 .1. Experimental variability is reported as standard error (: S.E.) of mean values of at least two separate experiments. - - Effect of (+)—trans allethrin on nerve myosin ATPase activity as determined as the difference (A-A) between the activity in the presence (+ EDTA) and absence (- EDTA) of EDTA. Enzyme source was the 90,000 g fract_i§>n of the squid retinal axon preparation. ATP concentration was 10 M. O abain concentfption was 10 . Other incubation conditions were: K =600 mM, Mg =1 mM, EDTA=2.5 mM in 30 mM Tris-HCl at pH 7.1. Experimental variability is reported as : S.E. of mean values of at least two separate experiments. - - - Effects of (+)—trans allethrin onbpuabain insensitive Natlg ATPase (A), total nqnmitcbgrhondrial Mg ATPase (B), 1 mM-Ca ATPase (C), and Ca Mg ATPase (D) activities. Enzyme source was the 90,000 g fraction of the fluid retinal axon preparation. ATP concentration was 5.6 x 10 M.2+Other incubation conditions were: (A)—Na =21+60 mM, K =40 mM, Mg =10 nlM; (B)—Na =1§0 mM, K =160 mN§z+ Mg =10 mM, KCN=2 mM; (C)-Na =160 mM, =160 mM, free Ca2+=1 mM, EDTA=1 mM; (D)-K =160 mM, Mg =10 mM, free Ca =0.01 mM, ethyleneglycol-bis-(B -aminoethylether) N,N'- tetraacetic acid (EGTA)=0.5 mM. All buffers were prepared @1430 mM Tris-HCl at pH 7.1. Ouabain concentration was 10 M. Experimental variability is reported as + S.E. of an values of at least two separate experiments. Nomifial free Ca concentrations were established at reported levels by the buffering method described by Portzehl et a1. (1964). The specific activities reported for both + ix 3O 31 32 45 46 48 25 26 27 28 29 30 Ca2+ ATPase and Cart-Mg2+ ATPase refer to only Ca2+—stimulated ECtIVIty. Q Q o o o 0 Q o o o 9 o c o o o o o o o o o o o o 0 Inhibition of 1 mM-Ca2+ATPase activity by various pyrethroid esters. All pyrethroid congfntrations were adjusted to give a fi a1 assay concentration of 10 M. Percent inhibition refers to C_ - stimulated activity only. ATP concentration was 5.6 x 10 M. Ouabain concentration was 10 M. gther incubation conditions were: Na =160 mM, K =160 mM, free Ca =1 mM and EDTA=1 mM in 30 mM Tris-HCl at pH 7.1. Experimental variability is reported as i S.E. of mean values of at least two separate experiments. Nominal free Ca concentration was established at reported levels by the buffering method described by Portzehl et al. (1964). . . . . . . . Effect of Ca2+ concentrat'on on Ca2+ ATPase activity in the presence and absence of 10 M (+)-tran52anethrin. The 96 inhibition reported refers to 96 inhibition of Ca -stimulated activity only. Enzyme source was the 90,000 g fraction of the squ'hd retinal axon preparation. ATP cggcentration was 5.6 x 10 M. Ouabain concentration was 10 M. Other incubation conditions were: Na =160 mM, K =160 mM, and EGTA=1.25 mM, in 30 mM Tris-HCI at pH 7 .1. Experimental variability is reported as : S.E. of mean valufs of at least two separate experiments. Nominal free Ca concentrations were established at reported levels by the buffering method described by Portzehl et al. (1964). . . . . . . . . . . . ATPase activity in the Effect of Ca2+ concentration on Ca2+ _ presence ( O- —O) and absence ( H) of 10 M permethrin. The difference curve (A- -A) is reported as the percentage of activity remaining of an untreated control value which was taken to be e al to 10096. The specific activity of the ATPase refers to Ca - stimulated activity of microsomal and intact synaptosomal tissue fractions. Effect of Ca2+ concentration on nonmitochondrial Ca2+-Mg2: TPase activity in the presence (0- -O) and absence (H) of 10 M permethrin. The difference curve (A- -A) is reported as the percentage of activity remaining of an untreated control value which was taken to He equal to 10096. The specific activity of the ATPase refers to Ca -stimulated activity of microsomal and disrupted synaptosomal tissue fractions. , , , . . . . . . . . . . . . . . the presence —-I) and absence (O——O) of 10 M permethrin. Mitochondrial ATPase activity was determined as that activity sensitive to mitochondrial poisons (see Methods). The difference curve (A- -A) is reported as the percentage of activity remaining of an untreated control value which was taken to be equal to 100%. The specific activity of the ATPase refers to the activity of the disrupted synaptosomal tissue fraction. . . . . . . . . . . . . . . . . . Inhibition of Ca2+ ATPase activity by three pyrethroid esters; allethrin (H), permethrin (O-- O) and cypermethrin (A- -A). ATPase activity is reported on a relative basis as the percentage of Effects of Ca2+ concentration on mitochondrial ATgase activity in X 50 52 53 81 82 83 31 32 33 34 35 activity remaining qu+an untreated control value takeQ+as equal to 10096 for each Ca concentration examined. Ca -stimulated activity was examined from microsomal and intact synaptosomal tissuefractions....................... . Inhibition of Carr-Mg2+ ATPase activity by three pyrethroid esters; allethrin (H), permethrin (O--O) and cypermethrin (A- -A). ATPase activity is reported on a relative basis as the percentage of activity regiTaining of an untreated control value taken as equal to 10096. Ca -stimulated activity was utilized from microsomal and disrupted synaptosomal tissue fractions. Total and nonmitochondrial activities are defined in text, see Methods. - - - . . - - - - - - - Inhibition of Ca2+-stimulated ATPases by monensin (H) and orthovanadate (O——O). ATPase activity is reported on a relative basis as the percentage of activity remaining of an untreated control value taken as equal to 100% for each enzyme system examined. Ca -stimulated activity+ of microsomal tissue fractions was examined. Ca Mg ATPase activity refers only to nonmitochondrial activity. . - - - . . . . . . . . . . . . . . . Effect of ruthenium red cfpcentration on Ca2+ ATPase activity at three concentrations of Ca ; 1 mM (O—O), 50 u M (O- -O) and 0.5 u M H). ATPase activity is reported on a relative basis as the percentage of activity remaining of an untreated control value talfien as equal to 10096 for each Ca concentration examined. Only Ca - stimulated activity was examined for ruthenium red sensitivity in both microsomal and intact synaptosomal tissue fractions. . . . . . Efffgt of ruthenium red concentration on nonmitochondrial Ca2+- Mg ATPase activity. ATPase activity is reported on a relative basis as percentage of activity remaining+ of an untreated control value taken as equal to 100%. Only Ca -stimulated activity was examined for ruthenium red sensitivity in both microsomal and disrupted synaptosomal tissue fractions. Open circles (0) indicate stimulation of sample value above untreated control. The number in parentheses indicate the percentage of stimulation. . . . . . . . . Effect of La3+ concentration on Ca2+-stimulated ATPase activity. ATPase activity is reported on a relative basis as the percentage of activity remaining on untreated cqqtrol value taken as equal to 10096 forzgach enzyme system and Ca concentratjgn examined. Only Ca -stimulated activity was examined for La sensitivity in both microsomal tissue fractions. Open circles (0) indicate stimulation of sample value above untreated control. Thez+num9ers in parentheses indicate the percentage of stimulation. Ca Mg ATPase activity reportedisnonmitochondrial. . . . . . . . . . . . . . . . . . . xi 86 87 93 95 97 98 CHAPTER I ATPases IN THE AXON-RICH MEMBRANE PREPARATION FROM THE RETINAL NERVE OF THE SQUID, LOLIGO PEALEI INTRODUCTION Squid axons have been extensively studied by physiologists for their electrophysiological properties, and yet, surprisingly very little data are available regarding their biochemical characteristics. The major reason for this lack of information is that the giant axons which are usually used for electrophysiological experiments have only a small ratio of the actual axonic membrane to other neural matters, and are unsuited for biochemical studies (Ritchie, 1973). It was originally found by Canessa—Fisher et al. (1967) that by using a large Chilean squid species, the retinal nerve (Optical nerve) of the squid consists of small axonic fibers with a favorable axon to glial cell ratio (10:1). Such small axons have relatively large membrane surface areas which facilitate biochemical studies. An earlier report (Matsumura, 1977) showed that such retinal nerve preparations from the most commonly studied squid species in North America, Loligo pealei, had high ATP—dependent membrane phosphorylation activities which were greatly influenced by the ionic environment of the assay medium. Because of the requirement of ATP as the substrate, most of the above phosphorylation activities have been considered to be related to ATPases. In this context, ATPase is defined as an enzyme which utilizes ATP as a substrate and results in a phosphorylated form of the enzyme itself. Dephosphorylation is required prior to binding another ATP molecule as substrate. Protein kinase and phosphoprotein phosphatase activities were not determined in the present study 1 2 due to previous reports that such activities were not susceptible to inhibition by chlorinated hydrocarbon insecticides (Doherty and Matsumura, 1975). Information on ATPases in the squid axonic membrane is incomplete, the only enzyme which has been extensively studied being Na+-K+ ATPase (Brinley and Mullins, 1968; Sjodin, 1974) which has been acknowledged to work as a Na+ pump. Recently there has been an upsurge of interest in the regulatory mechanisms for Ca2+ (for example DiPolo, 1974; Mullins and Brinley, 1975) and Mg2+ (de Weer, 1976) which require ATP as their energy source. Clearly, there must be other ATPases than Na‘L-K+ ATPase in the squid axon. The present study was undertaken to gather more information on such ATPases. MATERIALS AND METHODS Animals. The North American species of squid, Loligo gealei, was used in the preparation of the cell-free membrane fraction of retinal nerves which comprises the assay material used throughout this study. All squids were captured and kept alive until ready for use at the Marine Biological Laboratory at Woods Hole, Massachusetts during the summer months of 1977 and 1978. Squid dissection and storage techniques for nerve tissue have been previously reported (Matsumura, 1977). Approximately 40 mg (wet wt) of retinal nerve tissue was obtained from each squid. Chemicals. A tetraethylammonium salt of gamma (y)-32P-1abeled adenosine triphosphate (ATP), purchased from New England Nuclear, Boston, Massachusetts, was utilized as the substrate for all assays and had a starting specific activity of approx 26.2 mCi/u mol. The ionic strengths of individual assay buffers were adjusted with the chloride salts of sodium, potassium, magnesium, calcium and lithium (all reagent grade). Potassium cyanide was used for the tests on the effects of CN-. Lanthanum chloride (La3+) was obtained from Alfa Products, Beverly, Massachusetts, and activated charcoal (Norite 3 brand) was purchased from the University of Wisconsin Stores, Madison, Wisconsin. The following chemicals were all purchased from Sigma Chemical Company, St. Louis, Missouri: ouabain, ethyleneglycol—bis-N,N'—tetracetic acid (EGTA), ethylenediamine tetraacetic acid (EDTA), ruthenium red, the sodium salt of adenosine diphosphate (ADP, Grade III), bovine serum albumin (A4378), and dithiothreitol (DTT). All agents to be tested were prepared in the corresponding buffer of the assay and unless otherwise stated, all inhibition experiments were carried out by preincubation of the agent for 10 min at 25°C prior to the addition of [Y3ZP] ATP substrate. Preparations of ATPase Fractions. When membrane preparations were needed, homogenates of retinal nerves were made in 750 mM sucrose plus 1 mM EDTA and 0.1 mM dithiothreitol (DTT) at 40 mg/ml by using a hand-operated glass-glass homogenizer at 0°C. The homogenate was diluted to a final concentration of 250 mM sucrose with distilled-deionized water at 0°C and centrifuged at 8000 g for 10 min at 4°C in a swing bucket rotor (SW 25.1, Spinco). The supernatant was saved at 0°C and the pellet resuspended in an equal volume of 250 mM sucrose and centrifuged as before. The supernatants were combined and centrifuged at 90,000 g for 2.5 hr at 4°C. After discarding the supernatant, the pellet was resuspended into the original volume of 750 mM sucrose containing 0.1 mM DTT and stored frozen. This 90,000 g fraction was used exclusively in all enzymatic assays unless otherwise indicated. Protein determination was by the method of Lowry et al. (1951). ATPase Assay. The extent of inorganic phosphate (Pi) production from ATP was determined as follows: to 0.9 ml of 30 mM Tris-HCl buffer (at pH 7.1 unless stated otherwise) with various ion combinations, 0.1 ml of the above nerve preparations was added. The ionic compositions of various buffers used are summarized in Table 1. Various inhibitors were added to the enzyme-buffer .38: .3 Ho Boston .823? 28.25 «dram a0 5 fits 822338 963 mcofiwbcoocoo + so out «o 20>»: 25. N O n m $333032 .Sv-oH x m can STE 0.63 20333850 ’3.me 2.8 5325 .+~wS mo 82a 5 com: 886.658 was +Nw0 SE H .NSOmS no 332: «.OmmS Es 22 <22 :22 Eggs 1. «2 25 a 5832 3C «sum EMIOH Swan—.630 O wH cm O AEH— 6.5 uCQEdZmOQSQC Adv 62:32 35-:_mo>S :25 S zox 222 x m; 5825 o 2 Q2 :2 2&5. $92 32. 9:: : 5% 82 1.: 222 x 2... 5325 N 9 22 c 22.2 Ado-+2 :25 : «Sm . 222 x 9m 582° N c c2 22 22.2 $8 «sow 222 x 2m 5825 S... o :2 a 8&5. + 8 88m 2 -2 x 2m 5825 c 2 92 a 692.... as; swam C1. .9.) l—— o I I g I l I an? IE 0 I 5 10 +3 . La concentration,mM Figure 17. Effect of lanthanum conqentration on total Mg2+ ATPase activity. Incubation conditions were: Na = 1%) mM, K = 160 mM, Mg - 10 mM, and ATP concentration was 5.6 x 10 M. Experimental variability reported as standard error. 30 Ol/U!3101d 5”/!d eIOmu [JILL] .coto 2%:me mm ootoooa 3:52.23 Eucostoaxm .26 m5 .1. <99“ EB goo: Sony 2 :3 x 9m .8 AME: 8303 2 13 u 9:. 5:: cm H x 8.83 98528 :23 9: .850 .3353 @393. momuufimomE co cofiwhcoocoo $92 no uootm .3 95me 2E.co:o:coocoo 92 +N 0— n , n. d o _ _ a ~ d _ _ u d _ a w _ O _ I :I L _ a _ W m H _ a / _ / \ \\\\ m _ \\\ _ will m m \\H .o «T \x I a m. _ H, \\\l\\ \ B. u .1/ _\\mw. nu I \ U u ,/ \\ o ”— //\\ I" w 1 u 31 Ol/Ulatold 5”/!d elawu UIUJ BEmEanxm 8303 825.3» A... .898 28:me mo 83..an 3:53.23 .EE 3. u <90m 98 A25 23$ 2 13 on ad .8 8c: 3 n 95. .32: cm H M ”8.83 £83688 538%85 .850 .3338 931333.: o>8= cm 8393838 no mo 383m .3 PSME +N EE.co:o:cmocoo+~oU U!UJO[/U!8]Ojd can/Id alowd 32 U!wOl/U!3101d 5”/!d enowu .88 38::me m: 83.88.: 325833 83:8Er8oxm .2E m.m n «whom .2E m5 n «ewm 8:: 28$ 2 3 x 9m 8 8:: .8383 2 :3 u 9:. .28 H u w2 883 m:o38:oo 138985 8:30 .3338 8 9:. 8831:3928 83+ :o 838.3838 1.x .8 383m .3 8.83m 2662858080 +0. coda— owo con 03 oo o d 2 ll / \L 53+ H r————?——-—.——--— // /// par/H“ \. 6—7 UILUOl/UlalOld art/Id elowd 33 is no ATPase activity due to heavy neurofilaments (HN F) in the absence of Mg2+ at this K+ concentration but report 5096 of optimal response in total HNF activity when assayed in 600 mM K+ plus 1 mM Mg2+. By utilizing this same assay method with our membrane preparation, it is seen (Figure 20) that addition of 2.5 mM EDTA to the 600 mM K+ buffer results in a 116% increase in ATPase activity over the level that is seen in the presence of 1 mM Mg2+. The above evidence clearly indicates the presence of a high-titer of brain myosin in our enzyme preparation. As to the neurofilaments of axoplasm, the above data would suggest the possibility of its presence in our preparation because of its overall similarity in ion sensitivity to the data obtained here. However, the specific activity of the neurofilament ATPase in the whole axoplasm is rather low in the order of 2 nmol/min/mg under optimum conditions whereas under these same conditions, the total ATPase activity of our membrane preparation was in the order of 200 nmol/min/mg protein. Therefore, even if it is present in our nerve preparation, its contribution cannot be more than 196 of the total ATPase activity. DISCUSSION Since ATP is a common substrate for many enzyme systems, it is most important to design methods to clearly distinguish each enzyme system from others before their functional roles can be disclosed. In the present study, it was decided that such classification is more clearly defined by first establishing a basal level of activity to which one compares any changes which occur when certain selective additions are made to the assay system. The properties of Na+-K+ ATPase have been described by many workers. This enzyme requires a precise ratio of Na+ and K+ in addition to Mg2+. Its sensitivity toward cardiac glycosides has also been well documented (e.g., Skou and Hillberg, 1969). Thus, the recognition of this enzyme in the squid axon 34 preparation has not been difficult (Brinley and Mullins, 1968). On the other hand, the presence of cardiac glycoside insensitive Na+-K+ ATPase in the squid nerve had been noticed by other workers (Sjodin, 1974) where as much as 5096 of the total Na+-K+ stimulated ATPase activity had been found to be insensitive to cardiac clycosides such as ouabain. In this study, we have also found such an enzyme system. Its properties can be studied either in the presence of 10-4M ouabain and/or at a low ATP concentration. Aside from substrate affinity, the optimum concentrations of Na+ and K+ were similar to those found for ouabain sensitive NaJr-K+ ATPase. The major difference between the two enzyme sytems is their temperature sensitivities (i.e., the ouabain sensitive Na+-K+ ATPase having a much higher temperature dependency). While the functional role of ouabain sensitive Na+-K+ ATPase is likely to be "Natpumping," that of the ouabain insensitive Na+-K+ ATPase remains unclear. Nevertheless, its recognition as a distinct entity is possible by its characteristic stimulatory response to the Na+ and K+ ratio over that of Mg2+ stimulation alone. Carr-Mg2+ ATPase is recognized as the difference in the ATPase activity 2 between the sum of Mg2+ stimulated plus Ca + stimulated activity and the total activity seen in the combined Carr-Mg2+ buffer. This method of detection is similar to the approach used by Robinson (1976) and Duncan (1976) but corrects also for nonspecific Ca2+ stimulation. There are two additional characteristics which aid the recognition of this enzyme; first, its high temperature dependency and second, its relatively low (lo-SM) requirement for Ca2+. As judged by the 2+ and Mg2+ ATPase system (Figure 8), the 2 level of activity of the Total Ca +-stimulated ATPase 2 enzyme occupies a significant portion of the total Ca complex. Its functional role could be construed as a "Ca +-pump," though its 2+ localization in specialized Ca sequestering components such as endoplasmic 35 reticulum is also possible (Henkart et al., 1978). Since this enzyme has a relatively low Km value (=10-8M) for ATP, a direct relationship to the above ATP-induced Ca2+ extrusion activity at this stage is uncertain. On the other hand, Baker and Glitsch (1973) observed a drastic decrease in the baseline Ca2+ efflux in the squid giant axon by the addition of apyrase which hydrolyzes ATP and thereby removes endogenous ATP. Also close similarities do exist in the amount of Ca2+ which is most efficiently extruded by active ATP hydrolysis (DiPolo, 1977) and which optimally activates our enzyme. Moreover, both these phenomena have a strict Mg2+ requirement. Thus, it is probable that there is an ATP-dependent Ca2+ effluxing mechanism operating even at a low ATP concentration. Properties of Ca2+ ATPase have been described by Lauter et a1. (1977) and recently by Matsumura and Ghiasuddin (1979) who have studied the characteristics of such an ATPase in the axonal preparation of the leg nerve fiber of the American lobster, Homarus americanus. The Ca2+ requiring ATPase recognized in the squid membrane preparation is similar to that from the lobster in many ways. Particularly important is its requirement of a relatively high concentration of Ca2+ (1 mM) and its low sensitivity to temperature (i.e., Q10 = 2.23). As judged by the similarity of the Ca2+ influence, it is possible that the Ca2+-ATPase which can accept Li+ (Baker et al., 1969) in place of Na+ (Figure 2+ ATPase. To detect the enzyme, it is necessary 13) is the same enzyme as Ca to remove Mg2+ by adding EDTA from the time of homogenization throughout the entire duration of enzyme preparation. The difference between the basal activity with the buffer composition of Na+ (160 mM), K+ (160 mM), Tris-HCl (30 mM) and that plus 1 mM of Ca2+ is regarded as the criterion for this enzyme. A critical proof is, however, still lacking that it is indeed an ecto—enzyme. 36 In brief then, the effect of Ca2+ on various ATPase systems seems to fall into two groups; those in which Ca2+ stimulates at low concentrations, require Mg2+, and are highly temperature sensitive (Duncan, 1976; Robinson, 1976; DiPolo, 1977; Hasselback, 1978), and those that require higher levels of Ca+, are Mg2+ independent, and are relatively temperature insensitive (Matsumura and Ghiasuddin, 1979; Rosenblatt et al., 197 6; Lauter et al., 1977). As for the presence of myosin in the squid nerve, See and Metuzals (1976) have described its properties by biochemical and electron microscopic examination. The most characteristic aspects of this ATPase are the inhibitory effects of Mg2+ at high (they are tested at 560 mM) K+ concentrations and the subsequent stimulation of activity with addition of EDTA. Since such a property is extremely rare among ATPases, it is certainly a useful indicator of the presence of myosin. The specific activity of this ATPase is rather high (i.e., in the order of 100 nmol Pi released/min/mg protein at 560 mM K+ + 2 mM EDTA). This value is at least in the same order of magnitude as the one obtained in the current study under a similar experimental condition (K = 600 mM + 2.5 mM EDTA), indicating that the bulk of the ATPase activity under these conditions must be due to this enzyme. The presence of a high titer of myosin in the membrane preparation is interesting in the light of the recent discovery by Metuzals and Tasaki (1978) that the inner cell surface of the live and perfused giant axon shows a fine network of myosin probably cross-linked by actin. These workers associated the presence of such net-like structures to the excitability of the membrane. Indeed, the removal of the net-like structure by pronase resulted in the loss of excitability. In our preparation, the membranes have never been subjected to high ion concentrations until the time of assaying. Therefore, it is not surprising 37 that myosin which seems to be tightly bound to the inner membrane surface should be carried into the final preparation. There is no doubt that the total Mg2+ ATPase assay method must have activated many types of Mg2+, Na+ and K+ stimulated ATPases. It must be mentioned here that the purpose of this particular experiment was not to define a method to detect a specific enzyme system; rather it was to examine the total ATPase behavior which might be of help in the interpretation of some of the data which elecrophysiologists obtain when they study ATP dependent transport of radioactive ions. For instance, the data obtained by this method may be compared to that of de Weer's (1976) who studied ATP dependent, Mg2+ extrusion mechanisms. He found the optimum Mg2+ extrusion (i.e., hypothetical "Mg2+-pump") occurred at 10 mM Mg2+ concentration, the same value as the one obtained here. Moreover, the sensitivities to lanthanum of these two systems were identical. The Mg2+—pump is also dependent on external Na+ (Mullins and Brinley, 1975; de Weer, 1976), and most likely to internal K+. The overall similarity of the Mg2+-pump to the total Mg2+ ATPase studied here offers a future possibility of finding a specific ATPase responsible for the Mg2+-pump among the Mg2+-stimulated ATPases in the membrane preparation. Finally, it should be noted that no claim has been made that all these ATPases are located in or on the axonic membrane. That is, the possibility of contribution by other components such as glial cell fragments and axoplasmic material cannot be overlooked. Indeed, Fischer et a1. (1970) could not improve the purity of axonic material by further treatment of the membrane preparation (e.g., similar to the current preparation) by a discontinuous sucrose density gradient technique. Thus, the possibility exists that any of these ATPases could belong to some non-axonic component of the preparation. Also, in no cases is it implied that each enzyme system consists of a pure enzyme. The possibility of 38 the presence of several similarly behaving ATPases, or isozymes, in each system 2+_Mg2 cannot be overruled. Terms such as Ca + ATPase instead of a more generic Ca2+-Mg2+ ATPases system have been applied for the sake of simplicity. The presence of ion-stimulated ATPase systems in a variety of excitable tissue is well documented, and in the case of NafiL-K+ stimulated ATPases, much valuable data have been obtained since their discovery (Skou and Hillberg, 1969). However, as for other ATPases, their importance in the regulation of nerve activity has hardly been studied. In this regard, it is interesting to note that so far the only enzymatic systems known to regulate the ionic environment of a cell are ATPases, and, as such, possibilities do exist that some of the ATPases described here play very important nerve functions. In summary, as reported originally by Fischer et al. (1970) for Dosidicus gig_a_s and confirmed in Loligo pealei, the retinal nerve seems to be the biological material of choice due to its relatively negligible contamination by extracellular membranes and rather high axonal to supportive material ratio. Further support for the usefulness of this preparation has come from Pant et a1. (1979) who has demonstrated the actual transport of extracellular ATP across the squid axon. By using the axon-rich membrane preparation, several ATPases present in the retinal nerves of the squid, Lologo pealei, have been recognized. Though it is probable that there are more ATPases remaining in this preparation, the efforts here provide the initial starting material for future characterization. Such information should be of great interest to electrOphysiologists and biochemists alike who may eventually use the data to ascertain the functional roles of these enzymes. CHAPTER II PYRETHROID INHIBITION OF NEURAL ATPases OF THE SQUID, LOLIGO PEALEI INTRODUCTION Generally, it has been accepted that DDT and pyrethroids act directly on the nervous system causing disruption of normal ion permeabilities in the nerve membrane that are implicit in the generation and conduction of nerve impulses (Shanes, 1951; Wang et al., 1972). Narahashi (1976) has determined that it is the Na+ and to a lesser extent K+ permeabilities which are principally affected by DDT and pyrethroids. However, other ionic fluxes have also been found to be mandatory for nerve tissue to function prOperly, particularly that of Ca2+ (Frankenhaeuser and Hodgkin, 1957). It has been previously established (Matsumura and Clark, 1980) that the retinal nerve is a suitable material for future biochemical works concerning the functional roles of various membrane-bound enzymes. This was primarily because the axons of the Optic nerve have a rather high axonal to supportive tissue ratio as compared to the more often studied giant axon (Canessa—Fisher et al., 1967). By utilization of this axon-rich membrane preparation, several ATPases were recognized. The present study was undertaken to determine which, if any, are sensitive to inhibition by pyrethroid insecticides. It must be pointed out that so far the only biochemical systems known to regulate the ionic environment of a cell are ATPases. They are known to function as indispensable enzymes in neurons carrying out a variety of tasks necessary for normal nerve activities. For these reasons, a keen research interest has been focused on the inhibitory role of DDT on ATPase systems. 39 40 Brunnert and Matsumura (1969) demonstrated that both DDT and DDE bind proteins of various nerve components of rat brain and subsequently showed ATPase activity to be inhibited by these insecticides (Matsumura and Patil, 1969). In 1971, Matsumura and Narahashi (1971) reported a correlation between the degree of DDT inhibition of ATPases and the electrophysiological symptoms of DDT poisoning. Schneider (1975) showed that both DDT and allethrin inhibited (Na + K) ATPase but rejected this as a mechanism of DDT or pyrethroid action since the Na+ pump (ouabain sensitive) enzymes do not participate directly in the action potential. Doherty and Matsumura (1974) likewise reported that DDT inhibited the 32 P incorporation from y-labeled ATP into proteins from lobster nerve but showed also that such a decrease might be related to the inhibition of a ouabain insensitive Na+-K+ ATPase (Doherty and Matsumura, 1975). Desaiah, Cutkomp and Koch (1974) and Desaiah et al. (1975) have reported that a mitochondrial (oligomycin—sensitive), Mg2+ ATPase is sensitive to DDT and pyrethroids. In this context of ionic regulation, Matsumura (1972) has reported that brine shrimp, while relatively unaffected by either high levels of sodium chloride or DDT, were quite susceptible to this combination. In a related work using a species of fresh water alga, Bratterton et al. (197 2) showed, even in the presence of high levels of sodium chloride, that DDT-related growth inhibition could be 2+ addition. reversed by Ca The effect of DDT (or DDE) on egg shell thinning is well documented. Recently, Miller et al. (1976) demonstrated DDE inhibition of a Ca-ATPase (Ca- pump) which is responsible for active transport of this cation from the blood to the developing shell. In domestic fowl, which is resistant to the thinning aspect of DDE poisoning, no inhibition of this Ca-ATPase was found (EPA, 1975). 41 Similarly, Huddard et al. (1974) reported that DDT caused almost complete inhibition of Ca2+ uptake by sarcoplasmic reticulum i_n gm. Matsumura and Ghiasuddin (1979) have found an extremely DDT-sensitive Ca-ATPase in the axonic preparation from the walking leg nerves of the American lobster. On the basis of Ca2+ interaction with the excitability of the axonic membrane, these authors proposed a working hypothesis that this Ca- ATPase is responsible for maintaining the surface Ca2+ level. Inhibition of this 2+ on the outer surface of the Ca-ATPase by DDT results in a depletion of Ca axon leading to a lowering of stimulus threshold. There have been a number of indications that there is a common poisoning mechanisms between DDT and pyrethroids. It is then the goal of this investigation to determine which ionic parameters are significantly affected by pyrethroids in various subcellular fractions of the squid nervous system. By employing the same organism used by electrophysiologists for studying the basic electrical properties of the nerve membrane, it is hoped to provide a logical biochemical link between the mode of action of pyrethroids and other similarly acting neurotoxins and the observed physiological symptoms of poisoning. MATERIALS AND METHODS Animals. The North American species of squid, Loligo pealei, used in this work were captured and kept alive until ready for use at the Marine Biological Laboratory at Woods Hole, Massachusetts in the summer months of 1979 and 1980. They were dissected alive and both retinal nerve (axons) and optic lobes were separately collected. Dissection and storage techniques for nerve tissue have been previously reported (Matsumura, 197 7). Preparation of ATPase Fractions. Preparation of retinal microsomes (axonal) was exactly as previously reported by Matsumura and Clark (1980). Synaptosomal preparation from squid optic lobe (i.e., pinched-off synaptic nerve 42 terminals of afferent sensory fibers) was as reported by Pollard and Pappas (1979). Optic lobes were removed from the squid and homogenized as a 10% (w/v) solution in 1 M sucrose, 1 mM ethylenediamine tetraacetic acid (EDTA) and 0.1 mM dithiothreitol (DTT) using a loose fitted glass-glass homogenizer (Ten Broeck). The homogenate was centrifuged at 20,000 g for 60 min at 4°C. The suspended pellicle was collected by aspiration with a pasteur pipet, and resuspended into 750 mM sucrose and 0.1 mM DTT. The axoplasm was also examined for its ATPase content. After decapitation, the hindmost giant axon from the stellate ganglion was dissected from the mantle in flowing seawater and cleaned under a dissecting microscope. The axoplasm was carefully extruded using a microroller and pooled in 750 mM sucrose, 1 mM EDTA and 0.1 mM DTT. Preparation of the microsomal fraction was exactly as for the retinal axons reported above. These fractions were used exclusively in all enzymatic assays unless otherwise indicated. All enzymes were stable to storage at -14°C except Carr-Mg2+ ATPase which was prepared fresh and used immediately. Protein was determined by the method of Lowry et al. (1951) and all assays were performed at a standard 10 u g protein per assay tube. ATPase Assay. The tetraethylammonium salt of gamma (y )-32P-labeled adenosine triphosphate (ATP), purchased from New England Nuclear, Boston, Massachusetts, was utilized as the substrate for all assays and had a starting specific activity of approximately 26.4 mCi/u mol. Approximately 3 x 105 dpm were used per assay tube. The extent of ATPase activity was determined as the 32Pi production) during incubation with amount of ATP hydrolysis (i.e., inorganic the enzyme source in selective ionic buffers. All water utilized in experimentation was first steam distilled and then sequentially passed through organic removal and mixed-bed deionizing systems. 43 Water quality was assessed by its conductivity which was measured as ppm of NaCl equivalents. At no time was the water conductivity allowed to exceed 0.1 ppm. Assay conditions, 32Pi workup and standard buffer conditions were exactly as previously reported by Matsumura and Clark (1980). Chemical. Pyrethroid compounds were obtained as gifts from various sources; pyrethrin (I and II) [PYrethrin I, 2-methyl-4-oxo-3-(2,4-pentadienyl)-2- cyclopenten-l-yl 2,2—dimethyl-3-(2-methyl-1-propenyl) cyclopropanecarboxyl- ate], [pyrethrin II, 2-methyl—4—oxo-3-(2,4-pentadienyl)—2-cyclopenten-l-yl 3-(3- methoxy-Z-methyl-3-oxo-1-propenyl)-2,2-dimethyl—cyclopropanecarboxylate] , resmethrin [5-benzyl-3-furylmethyl (:)—cis, trans—chrysanthemate] and phenothrin [3-phenoxybenzyl (:)—cis, trans 2,2-dimethyl-3—(2-methyl—1—propenyl) cyclopropanecarboxylate] from U. S. Environmental Protection Agency, Health Effects Research Laboratory, Environmental Toxicology Division, Research Triangle Park, North Carolina, (+)-trans allethrin [3-a11y1-2-methyl-4-oxocyclo- pent-2—enyl—(+)—trans-chrysanthemate of (:)—allethrolone] from Dr. T. Narahashi, Department of Pharmacology, Northwestern University, Chicago, Illinois, kadethrin [3-phenoxybenzyl(:)-cis-3-(1,2-ene-thiolactone)~2,2-dimethyl- cyclopropanecarboxylate] from Dr. K. Osawa, University of California, Berkeley, California, permethrin [3-phenoxybenzyl(:)—cis, trans-3-(2,2-dichloro- vinyl)-2,2-dimethylcycloprOpanecarboxylate] from FMC, Agricultural Chemicals Division, Middleport, New York, cypermethrin [(:)-a-cyano-3-phenoxybenzyl(:)— cis, trans-3-(2,2—dichlorovinyl)—2,2—dimethylcycloprOpanecarboxylate] from Shell Bioscience Laboratory, Sittingbourne Research Center, Sittingbourne, Kent, United Kingdom, decamethrin [(S)-a-cyano-3-phenoxybenzyl-cis-—(1R,3R)—2,2- dimethyl-3-(2,2—dibromovinyl) cyclopropanecarboxylate] from Dr. L. Ruzo, Division of Entomology and Parasitology, University of California, Berkeley, California and fenvalerate [(SH:-cyano-3-phenoxybenzyl-2-(4-chlorophenyl) iso- 44 valerate] from Shell Development Company, Modesto, California. All of the remaining biochemicals were purchased from Sigma Chemicals, St. Louis, Missouri. RESULTS Figure 21 illustrates the structures of the nine pyrethroid insecticides selected for this study. They range from the naturally-occurring esters, pyrethrin (I and 11), produced by the flower Chrysanthemum cinerariaefolium to one of the more highly synthetic analogues, fenvalerate. The remaining compounds were chosen as examples of major chemical modifications made during the evolution to more environmentally resilient components better suited for agricultural usage. Survey of ATPase Sensitivity to Allethrin. Because of the similarity of structure to naturally-occurring pyrethrins and the availability of electrophysiological study results, allethrin was chosen as a model pyrethroid by which we gauged the sensitivity of six ATPases recognized in the retinal axon. These were as described earlier by Matsumura and Clark (1980): (1) ouabain sensitive NaJr-K+ ATPase (Na+-pump), (2) ouabain insensitive Na‘L-K+ ATPase (maintenance ATPase), (3) total Mg2+ ATPase (nonmitochondrial Mg2+ ATPase), (4) Ca2+ ATPase (surface Ca2+-sequesterer), (5) CaZJF-Mg2+ ATPase (Ca2+- pump), and (6) nerve myosin (contractile protein of the inner wall of axolemma). The effect of allethrin on ouabain sensitive Na+-K+ ATPase determined as the difference in ATPase activity in the presence and absence of ouabain at a high ATP (10-4M) concentration is reported in Figure 22. As indicated by its specific activity, the ouabain sensitive Na‘I-K+ ATPase is quite active accounting for some 5096 of the total activity in the retinal nerve preparation. However, Na+ pump enzyme is completely insensitive to allethrin over the concentration range examined. In fact, this enzyme shows an overall stimulatory 45 . .521,sz o ' Permethrin 1r .Eygb Cyk ermethrin O \ Allethrin ~ 810 ( lo ° 1; Decamethrin 0 F216 {01,6420 , Fenvalerate Phenothrln ,0 Figure 21. Development of stable pyrethroid esters. Compounds unstable under field conditions are given in the left-hand column. Compounds more stable under field conditions are given in the right-hand column. Kadethrin 4L = T .E {/0 - 9. i l l\ \ 3 __ \ ._c_ \ - -ouobain o o E l a. -_ a) E \ 2 A ._ \A n. / \ ... E \\\ A I \L o ‘\ ’0 \ f;— I "' 1‘ +ouobain '6 O a m o a. .2 L I J_ 7 L l ! o—{rfi a 7 6 5 4 "log Allethrin concentration,M Fi re 22. Effect of (+)-trans allethrin on ouabain sensitive Nail-K+ AT ase (Na pump) as determined as the difference (A-A) bet een total Na -K ATPase activity (—ouabain) and ouabain insensitive Na -K ATPase activity (+ ouabain). Enzyme source was the 90,000 g ffaction of the squid retinal axon preparation. ATP concentration was 10' . Ouabain +concentratioré+was 10 M. Other incubation conditions were: Na =160 mM, K =40 mM, Mg =10 mM in 30 mM Tris-HCI at pH 7.1. Experimental variability is reported as standard error (: S.E.) of mean values of at least two separate experiments. 47 effect at 10-4M allethrin resulting in a 31.2% increase in activity over the control values (i.e., no allethrin treatment). Because ouabain, a specific inhibitor of the Na+ pump, has been shown to cause none of the electrical disturbances characteristic of DDT and pyrethroid poisoning, the Na+ pump has not been considered as a probable site of action (Matsumura, 1970). This data supports this claim. However, the overall stimu- lation of Na+ pump activity may be important in view of the recent work by Narahashi (1979), who reported that DDT and pyrethroids effect Na+- inactivation mechanisms of the Na+-K+gate during action potential generation. An increase in internal Na+ concentration would be expected to cause increased Na+ pump activity as it works to remove excess Na+. The other of the two ATPases which is activated only at high ATP concentration is nerve myosin. Myosin is recognized as the difference in ATPase 3M ATP as activity in the presence and absence of EDTA in 600 mM K+ at 10- reported by See and Metuzals (1976). As illustrated in Figure 23, nerve myosin is present in relatively high amounts. However, the ATPase activity of nerve myosin is uneffected by allethrin over the concentration range examined. As with the case of ouabain sensitive Nair-K+ ATPase, there is a significant increase in myosin activity (21.8%) at high allethrin concentration (10‘4M). Metuzals and Tasaki (1978) have reported that nerve myosin is closely associated with the inner surface of the squid axolem ma as a supporting network. Besides these structural considerations, they also believe that myosin may be involved in gating mechanisms during action potential generation in the axon. Our work indicates that although myosin is present in rather high amounts, it is not a likely candidate for the site of action of the pyrethroids. Nevertheless, it still may be implicated in the overall toxicological scheme of pyrethroid 4 _. I J. c I +EDTA / .E .h—‘\T 9- l/1 1 2 3 T/ 1;; . O a. l g I“EDTA ‘l' \ I /,o\\\ ‘- E 2 _ 0” J. \\o —-""" I 3 A // E //j: \ / :r T,”/ A 2: f I? 1 _ ‘J O O m U ,‘1‘. < . I 1 J 1 1. o—Ifi 8 7 6 5 4 ”'09 Allethrin concentration,M Figure 23. Effect of (+)-trans allethrin on nerve myosin ATPase activity as determined as the difference (A-A) between the activity in the presence (+ EDTA) and absence (- EDTA) of EDTA. Enzyme source was the 90,000 g graction of the squid retinal axon preparation. ATP concentration was 10 . Ouabain concentration was 10 . Other incubation conditions were: K =600 mM, Mg =1 mM, EDTA=2.5 mM in 30 mM Tris-HCI at pH 7.1. Experimental variability is reported as : S.E. of mean values of at least two separate experiments. 49 poisoning due to its apparent requirement of interaction with actin—like proteins during contractility which is Cay-regulated. Figure 24 illustrates the specific activities of the remaining four ATPases identified at a low ATP concentration (5.6 x 10-8M) in the squid retinal axons. Their sensitivity to allethrin at various concentrations is also reported in Figure 24. Figure 24-line A shows the ATPase activity of the ouabain insensitive Na+- K+ ATPase, an enzyme originally reported in the giant axon by Sjodin (1974), but whose function still remains unclear. Because of its low temperature—sensitivity and stable storage characteristics, it is not believed to be a metabolic pump like the Na+ pump described above. However, as with the Na+ pump enzyme, the ouabain insensitive Na+-K+ ATPase is relatively uneffected by allethrin, showing only slight inhibition (4.196) at the highest allethrin concentration examined (WM). 2 The total Mg + ATPase activity has been included to examine the effect of pyrethroids on nonmitochondrial, Mg2+-stimulated ATPase systems. As shown in Figure 24-line B, this enzyme is relatively active in the axon—rich preparation, 4 but shows only a 25.796 reduction in ATPase activity in the presence of 10- M allethrin. In this work, Mg2+-stimulated mitochondrial ATPase activity was suppressed by addition of 2 mM CN-, and therefore, its sensitivity towards allethrin was not determined. In this context, it should be noted that the major Ca2+ sequestering mechanism under physiological conditions in both the axon (Henkart et al., 1978) and synaptic nerve terminal (Blaustein et al., 1978) has been determined as endoplasmic reticulum and not the mitochondria. Figure 24-line C and line D shows the two Ca2+-stimulated ATPases 2 2 examined for their pyrethroid sensitivity; Ca + ATPase and Ca +—Mg2+ ATPase 2 (Ca2+-pump), respectively. Ca + ATPase activity was determined as the portion 50 1.5 — 1.0“- ’ \ ATPase activity, nmoles Pi / mg protein/10 min O .\. Hra J 1 i\L t o a 7 6 5 4 “log Allethrin concentration,M Figure 24. Effects of (+)-trans allethrin on ouabain insensithéq Na+-K+ ATPase (A),2+totalznonmitochondrial Mg ATPase (B), 1 mM-Ca ATPase (C), and Ca Mg ATPase (D) activities. Enzyme source was the 90,000 g fragtion of the squid retinal axon preparation. ATP concentration +was 5.6 x 10 2 . Other incubation conditions were: (AhNa =160 mM, K =40 mM, Mg+ =10 mM; (Bl-Na =160 mM, K =2§0 mM, Mg =10 mM, KCN=2 mM; (C)- Na2§160 mM, K =160 , free Ca =1 mM, EDTA=1 mM; (D)-K =160 mM, Mg =10 mM, free Ca =0.01 mM, ethyleneglycol—bis—( -aminoethy1ether) N,N'-tetraacetic acid (EGTA)=0.5 mM. All buffers were4prepared in 30 mM Tris-HCl at pH 7.1. Ouabain concentration was 10 M. Experimental variability is reported as i 8.25;. of mean values of at least two separate experiments. Nominal free Ca concentrations were established at reported levels by the buffering method describe};r by Portzehl et $1., (1364). The specific activitiris reported for both Ca ATPase and Ca -Mg ATPase refer to only Ca -stimulated activity. 51 of activity stimulated in the presence of 1 mM Ca2+ over a basal level of activity (i.e., activity in the absence of Ca2+, see Matsumura and Clark, 1980). CaZJr-Mg2+ ATPase activity was determined at a relatively lower Ca2+ concentration (IO-SM) and in the presence of Mg2+. Also, CaZIL-Mg2+ ATPase activity is measured only in fresh microsomal preparations whereas Ca2+ ATPase is measured only in once—frozen preparations, which completely isolate these two activities due to their separate storage sensitivities. It is interesting to note 2 that the Ca2+-Mg + ATPase, which is believed to be similar to the Ca2+-pump enzyme reported in nerve tissue and purified from sarcoplasmic reticulum, has the same high temperature-sensitivity of a metabolically active pump, whereas 2 the Ca + ATPase is very temperature insensitive both in its overall activity and storage characteristics (Matsumura and Clark, 1980). As seen in Figure 24-line 2+ ATPase activity is inhibited over the entire concentration range of 4 2+_Mg2+ C, Ca allethrin resulting in some 51.396 inhibition at 10' M allethrin. Ca ATPase activity was also found to be very sensitive to allethrin inhibition causing 84.3% reduction in activity at 10-4M. Because of its apparent greater 2 contribution to the total Ca +-stimulated ATPase activity in the retinal axon (7296 of total Ca2+ activity, see Figure 24), Ca2+ ATPase was examined first in more detail. 2 The percent inhibition of Ca + ATPase activity by various pyrethroids, all at a concentration of 10-4M, is illustrated in Figure 25. These compounds are ranked as to their level of inhibition with the most inhibitory on top and the least towards the bottom. The natural ester, pyrethrin, was found to be the most 2+ inhibitory causing a 69.396 decrease in Ca ATPase activity followed by allethrin and permethrin. To determine which of the stimulating inorganic ions most affect 2 pyrethroid inhibition, the Na+, K+, and Ca + concentrations were varied in turn. 52 Pyreth. i—-—4 m Alleth. e s 'u i: a o Perm. r———————I a. E 8 Resm. F-t Fenval. t——--i .0 2 Deco. H .r: t o ‘- Cyperm. H > a. Kad. H L J I J J l 1 A ‘0 20 30 40 50 60 70 % Inhibition Figure 25. Inhibition of 1 mM-Ca2+ATPase activity by various pyrethroid esters. All pyrethroiq concentrations were adjusted to give §+ final assay concentration of 10 M. Percent inhibition_8refers to Ca -stimulated activity 4only. ATP concentration was 5.6 x 10 M. Ouabain concentration was 10 . Other incubation conditions were: Na =160 mM, K =160 mM, free Ca =1 mM and EDTA=1 mM in 30 mM Tris-HCl at pH 7.1. Experimental variability is reported as + .E. of mean values of at least two separate experiments. Nominal free Ca concentration was established at reported levels by the buffering method described by Portzehl et al. (1964). 53 ”3" I — 100 c T. E 8 1.4 lA/ Q .E __ / g Q) I '6 OH // i ‘- 1 - / V D. ,A c: 0> — " .9. § '1’ :5 0- .6)- 5- E -°-’ / E g EflIfL-I /II+)ollethrin -‘ 3'2 z .2; , I/E — 10 LP 1 1 1 1 1 a? 1 o 165 162 1 2 20 2+ . Ca concentration,mM Figure 26. Effect of Caz:4 concentration on Ca2+ ATPase activity in the presence and absence of 10 2M (+)-trans allethrin. The 96 inhibition reported refers to 96 inhibition of Ca -stimulated activity only. Enzyme source was the 90,000 g fraction of_8the squid retinal axon preparatign. ATP concentration was 5.6 x 10 M, Ouabain cqncentration was 10 M. Other incubation conditions were: Na =160 mM, K =160 mM, and EGTA=1.25 mM, in 30 mM Tris-HCI at pH 7.1. Experimental variability is reported as i $.12?“e of mean values of at least two separate experiments. Nominal free Ca concentrations were established at reported levels by the buffering method described by Portzehl et al. (1964). 54 Na+ and K+ ions were found to have a modest effect on allethrin inhibition of Ca2+ ATPase activity (Clark and Matsumura, unpublished results), but Ca2+ was found to have a significant effect. In Figure 26, the Ca2+ concentration has 8M to 20 mM in the presence and absence of 10-4M been varied from 10- allethrin. The difference curve (i.e., the difference in values between untreated control and allethrin-treated samples reported as 96 inhibition) reveals a highly allethrin-sensitive Ca2+ ATPase apparent only at low Ca2+ concentrations (10- 6M). This high affinity Ca2+ ATPase was examined for sensitivity toward other pyrethroids. Although the concentration range of each compound has been expanded '8 to 10'4M), the same group of pyrethroids reported in Figure 25 was (10 incubated with this high affinity Ca2+ enzyme. The results of this experiment are reported in Table 4. The pyrethroids are ranked again as to their inhibitory characteristics with the top compounds most inhibitory, the bottom ones least. Under these conditions, all pyrethroids tested were found to be more efficient inhibitors of this ATPase as compared to the previous experiment (Figure 25) with pyrethrin and allethrin causing almost complete inhibition of Ca2+- stimulated activity. At the same time, it is noteworthy that the order of ranking of the compounds is the same that was reported for the 1 mM-Ca2+ ATPase. Because of the obvious importance of overall Ca2+ regulation in the 2 2 nervous system, the Ca +-Mg + ATPase of the retinal axon was also examined. The pattern of pyrethroid inhibition was studied using the same compounds and concentration range as above. The results of this experiment are reported in Table 5. The pyrethroids have been ranked as before. On the whole, these 2 compounds are more inhibitory on CaZ‘F-Mg2+ ATPase than on Ca + ATPase as judged by their apparent 150 values (compare Tables 4 and 5). However, the most 55 63? 35:8 .83 8353 933.5 .3 :oflstESm $359.0 I .mucoatoaxo 3233. 02:. «6.on 3 89¢ .898 28:me + 2on 2: mm 83298 0.8 momwucooaom .3328 923 33255 5:5 @835 3388 .Ho 3332... 3.8.5. umEBESTéwO .3 85335 Hcooaoa md @3335 93 San—n .3685 .3 «0 Boston Sousa <9Qm\<90m so a 3 833.62“ cozwbcoocoo +Nw0 mob $613 .3330“ 392.4 +mw0 525.3 .32 homo .c H ode m.H.H H 5.3 .m H «Hm. 93 H mam m. H H 93 5.233% m. H H13. 92 Mean c.mH H H.mv .93 H wém m.oH «45+ 5.538.696 m.H H H.Hm m; H Hém H.NH H m.mm 5.3.1... 93 m... H mém EEHoEwooQ H.H H 9mm :6 H mHo Hana H 5.3 .3. H Hév m.H H «.3 5.585an as H cém H. H H 93 H.mH H mHe 9H H ode 9m H 93+ 326358 9m H «.3 .m Hmém v.5 H ”.3 m6 H Hi? omHH H 93+ EEHoEmom m. H H 35 o. H H v.3 9m H 9%. Hum H «.mm m.HH H 93 S.EHoEHom c.H H 93 «H dem m5 H 5mm .3 H 19” .N H H.MH :25on «J + 93 ma + 33 9H + 92. m5. + :6” w.mH + 53 EEHmEHnH 7.: 9-2 93 TE $3 25358 :5 53955280 23mm 22:36.3 36:3, E 66m: + 60-2 -2 6.32:? 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S: H 8:3 3 H 8.9. £282.33 2: 2: 3 + 92. 5.." + :8 83 + 8.? 5.2828: a T: 1: 8-: Hg: 8-2: 2:6 Eco 3.5 8385:8880 8.88m 5858:.8 835.85 .3 85083853 83 380 Baum 29: 8528:. 8&9... 882.88 8 8522: 888: .N. 2.89 60 It should be noted however that in both the axon and synaptosome, Ca2+ ATPase is the major Ca2+—stimulated enzyme examined. This ATPase is responsible for approximately 7296 of the total Ca2+ activity (i.e., the 2 summation of the specific activities of Ca2+ ATPase and Ca +-Mg2+ ATPase) in the axon and some 64% in the synaptosome (see apprOpriate footnotes, Tables 6 and 7). However, the total Ca2+ activity in the synaptosome is only 6696 of that found in the axon. When one compares the specific activity of the CaZWL-Mg2+ ATPase in the synaptosome versus the axon, one finds that the synaptosome has 1596 less of this enzyme activity than the axon. Yet, when one examines the a2+ ATPase activity in a similar manner, the synaptosome has 4196 less activity 2+ 2 C than the latter. Therefore, the Ca + ATPase appears to be relatively 2 -Mg more important in overall Ca + regulation in the synaptosome as compared to the axon. Since ATP is a common substrate for many biochemical reactions, the axoplasm of the giant axon of the squid was examined to determine what, if any, contamination it might be contributing to our microsomal preparations. It was subsequently found that the axoplasm did indeed contain a modest ATPase activity, some of which was Ca2+-stimulated (Table 8). However, the Ca2+ 2+_Mg2+_ ATPase activity was only 2196 of that found in the axon and the Ca stimulated activity was only 17% of the axonal level. Nevertheless, neither of these two Ca2+-stimulated ATPases in the axoplasm showed any sensitivity to allethrin up to concentrations of 10-4 M. In fact, allethrin showed consistent increases in activity in both basal and Ca2+-treated samples. Such a response is quite different from that reported in the microsomal fraction of retinal axons and synaptosomes of synaptic nerve terminals from the optic lobe. In both these preparations, allethrin only inhibited Ca2+-stimulated activity and was never seen to effect basal activity. 61 .3333: 35 .<.zo .835 {.30 98 2:53am: .3 b90395 @258 83332. :82 .Aoocoaotmv rod couflsfibmfimwo EB :33 .393 .33?on 35.3.5 «do ooamw 9.65 8 .3 «o Boston 6832? 8.2.5 \ E q 3;- Q 8 '5 E lOO” _‘ ’4‘ I I q ‘3 // ~10 ‘ 8 a 1 1 (E 5. 4m J l I l 2.; <1 .3 Synaptosomal ~90 E :, O. " ‘ o 2" a': " ° ' 3 300 _ *5 =. 3 8° 0 w Control 200— ,+ ‘50 IOO- ; y/O _ )/ +1 Perm ,0 8884/11 7 6543 -Log Co" Concentration. M Figure 28. Effect of Ca2 concentration on nonmitochondrial Ca2+ fig ATPase activity in the presence (0- -O) and absence (H) of 10 permethrin. The difference curve (A- -A) is reported as the percentage 01% activity remaining of an untreated control value which was takefito be equal to 10096. The specific activity of the ATPase refers toCa -stimulated activity of microsomal and disrupted synaptosomal tissue fractions. 83 8 Control 400 — J /. 8 Mitochondrial ATPase Activity (pmole Pi/mg protein/l0 min) I 'k \ °/. of Control (A--A,A--A) §ynoptosomal ICC” " 1411 J l l l “Uri O 7 6 5 4 3 -Log Coz’ Concentration, M Figure 29. Effects of Ca2+ concentration on mitochondrialéATPase activity in the presence (I— —I) and absence (O—-O) of 10 M permethrin. Mitochondrial ATPase activity was determined as that activity sensitive to mitochondrial poisons (see Methods). The difference curve (A- -A) is reported as the percentage of activity remaining of an untreated control value which was taken to be equal to 10096. The specific activity of the ATPase refers to the activity of the disrupted synaptosomal tissue fraction. 84 similar means in the presence and absence of 10—6M permethrin. The results are illustrated in Figure 29. In the absence of Ca2+ (i.e., basal Mg2+ ATPase 2 activity), permethrin caused a 42% reduction in mitochondrial Mg + ATPase activity. This is similar to the results of Desaiah et a1. (1975). However, 2+ -7 additions of low levels of Ca (10 -10-6M) caused a decrease in control mitochondrial ATPase activity which was not reflected apparently in the treated samples (i.e., Ca2+ inhibition not permethrin inhibition). On further Ca2+ addition (10-5-10-3M), there was an observable Ca2+-stimulation of mitochondrial ATPase activity which is sensitive to permethrin inhibition. These results may be of interest because the Ca2+-sequestering aspect of mitochondria has been shown to be inhibitory to the ATP-generating system of mitochondria. Generation of ATP from ADP in the mitochondria has been shown -ATPase or F to be related to the F1 factor (i.e., F synthase) which has been 2+ 1 1 determined as identical to the forward reaction of the oligomycin sensitive Mg ATPase activity of the intact mitochondria (Leninger, 1977). Furthermore, it has been shown that the half saturation value for Ca2+ uptake by the 5 2 mitochondria is in the range of 10- M Ca + and that at least a proportion of this energy-linked uptake of Ca2+ is supported by ATP hydrolysis which is oligomycin sensitive (Malmstrom and Carafoli, 1979). At this point, it may be proper to speculate that the absence of increased ATPase activity in the presence of increasing Ca2+ concentration in the synaptosomal fraction (illustrated in Figure 2 28) may be due to the presence of some endogenous Ca + buffering system which 2 is capable of stabilizing the free Ca + concentration at a level lower than the 2+ load (e.g., < 10-5M). Whether or not such buffering is due to imposed Ca mitochondria, calcium binding proteins on some other organelle has not been ascertained at the time. 85 2+ ATPase and nonmitochondrial Ca2+--Mg2+ ATPase Nevertheless, both Ca have been shown to have rather high affinities for Ca2+ (Figures 27 and 28, respectively) and because Ca2+ regulation has been implicated as mandatory for proper nerve function, these ATPases were examined in more detail concerning their sensitivity towards pyrethroids. Figure 30 illustrates pyrethroid inhibition curves (i.e., allethrin, permethrin and cypermethrin) for Ca2+ ATPase at three Ca2+ concentrations in microsomal and synaptosomal preparations. Again, it is apparent that not only are these compounds more inhibitory at lower Ca2+ concentrations, but that they are also slightly more inhibitory when applied to synaptic preparations. In addition to this, allethrin is seen as consistently more inhibitory to Ca2+ ATPase activity than either permethrin or cypermethrin. This increased inhibition attributed to allethrin is apparent irregardless of which tissue fraction is examined. Similar experiments were performed on Ca2+-Mg2+ ATPase and the results 2+ 2+ are illustrated in Figure 31. The top two graphs represent Total Ca -Mg activity from both microsomal and synaptosomal fractions. The bottom two 2+ 2 graphs represent nonmitochondrial Ca -Mg + ATPase activity from the same two fractions, respectively. All three pyrethroids are more inhibitory to the nonmitochondrial activity compared with the total activity regardless of the preparation examined. It is also apparent from Figure 31 that Ca2+-Mg2+ ATPase activity is relatively more susceptible to the action of permethrin and cypermethrin than to allethrin. These results are quite similar to those previously reported for the squid (Clark and Matsumura, 1981a) where pyrethrin and its closely related synthetic analog, allethrin, primarily inhibited Ca2+ ATPase whereas highly modified pyrethroids such as cypermethrin mainly inhibit 2 Ca +-Mg2+ ATPase. 86 Microsomal __ §mgptosornal 8 5 8 8 5 l i Relative ATPase Activity, Co” ATPase E9 IO“ §$é54 -Log Pyrethroid Concentration, M Figure 30. Inhibition of Ca2+ ATPase activity by three pyrethroid esters; allethrin (H), permethrin (O— —O) and cypermethrin (A- -A). ATPase activity is reported on a relative basis as the percentage of activity rergaining of an untreated control yalue taken as equal to 10096 for each Ca concentration examined. Ca -stimulated activity was examined from microsomal and intact synaptosomal tissue fractions. 87 Microsomal Synaptosomal a) g; IOO —- E . ———