PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE ” ’Tl 76% MSU Is An Affirmative Action/Equal Opportunity lmttution EFFECTS OF CALCIUM, CALMODULIN AND PYRETHROIDS ON PROTEIN PHOSPHORYLATION PROCESSES IN THE NERVOUS SYSTEM OF THE SUSCEPTIBLE AND THE KDR-RESISTANT INSECTS AND IN THE RAT BRAIN by Petros Charalambous A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1989 ABSTRACT EFFECTS OF CALCIUM, CALMODULIN AND PYRETHROIDS ON PROTEIN PHOSPHORYLATION PROCESSES IN THE NERVOUS SYSTEM OF THE SUSCEPTIBLE AND THE KDR-RESISTANT INSECTS AND IN THE RAT BRAIN by Petros Charalambous To study the effects of DDT and pyrethroid insecticides on calcium and calmodulin sensitive protein phosphorylation processes, a DDT-resistant strain of the German cockroach was first selected using permethrin. By the tenth generation of selection the selected strain (VDLSP) had developed a resistance of 18-fold to the selecting agent. The major factors responsible for the permethrin resistance in the VDLSP appear to be the reduced sensitivity of the target site, kdr (knockdown resistance), and to a lesser extent enhanced metabolic detoxification particularly an increased mixed-function oxidase activity. By comparing phosphorylation activities of isolated, lysed synaptic membranes, it was concluded that the stimulatory effect of calcium on protein phosphorylation was the same in the susceptible and kdr-resistance strains. Calmodulin, however, significantly increased the level of phosphorylation of the two subunits of calcium/calmodulin dependent protein kinase (CCPK) in the susceptible insects, but not in the lair-resistant insects. IR-deltamethrin inhibited both the total protein phosphorylation and the autophosphorylation of the two subunits of CCPK in both susceptible and resistant strains. In intact synaptosomes depolarization induced by veratridine or "high K+" in the presence of IR-deltamethrin significantly increased the total level of endogenous protein phosphorylation only in the susceptible strain. The effects of pyrethroids and DDT on the (at—subunit protein of the rat brain sodium channel were studied, by using partially purified, unpurified (i.e., intact synaptosomes), native and exogenously added CAMP dependent protein kinases. Radioautograms of SDS-polyacrylamide gel-electrophoresis of labeled phosphoroproteins showed that the (IL-subunit of the voltage sensitive sodium channel protein is the only 260 Kd phosphorytable protein present in intact and lysed synaptosomal preparations. Phosphorylation of the a-subunit was induced by depolarization, and this process was inhibited by the biologically active IR-deltamethrin and DDT, but not by IS-deltamethrin, the biologically inactive enantioner of deltamethrin or DDE. Phosphorylation of the a-subunit using lysed synaptosomal membranes was also inhibited by IR-deltamethrin and DDT. In summary, the neural calcium/calmodulin dependent protein kinase from the Mr- resistant insects appears to be different from the one found in the corresponding susceptible insects. The major differences appears to be in the mechanism by which this enzyme is activated by calmodulin. The resistant enzyme is less sensitive toward bovine calmodulin. One of the substrates of this protein kinases appears to be the a-subunit of the sodium channel. ACKNOWLEDGEMENTS Conducting this study has been a very challenging and enlightening experience. I am especially grateful to Dr. Fumio Matsumura for his valuable suggestions and guidance and for the financial support during the course of my studies. Appreciation is extended to the other members of my guidance committee, Dr Matthew J. Zabik, Dr. James Miller, Dr. William Mattson and Dr. Alfred Haug for their guidance and instructions through the course of study. I thank all the members of Professor Matsumura's group past and present and all the people who have been helpful to my efforts on these studies. I thank also, the goverment of Cyprus, the IAEA, the National Academy of Science for providing me the support for the two-year training. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES GENERAL INTRODUCTION CHAPTER I. STUDIES ON THE DEVELOPMENT OF RESISTANCE, AND CROSS- RESISTANCE SPECTRUM IN PERMETRIN-RESISTANT BLATTELLA GERMANICA ABSTRACT INTRODUCTION MATERIALS AND METHODS Insects Chemicals Surface contact method (continuous exposure) Topical application method Injection Method RESULTS Development of resistance to permethrin in the laboratory effects of synergists on the expression of resistance in the VDLSP strain Cross-resistance spectrum DISCUSSION REFERENCES CHAPTER II. EFFECTS OF CALCIUM, CALMODULING AND PYRETHROIDS ON PROTEIN PHOSPHORYLATION PROCESSES IN THE NERVOUS SYSTEM OF DDT AND PYREI‘HROID RESISTANT AND SUSCEPTIBLE INSECTS ABSTRACT INTRODUCTION MATERIALS AND METHODS Insects Chemicals PAGE viii \O\O\OO\UI4> 10 10 11 15 15 21 27 29 31 32 33 36 36 36 Preparation of lysed synaptosomal membranes ( = lysed membranes ) Preparation of intact synaptosomes Preparation of calmodan from the susceptible and the resistant strains of the German cockroach Preparation of lysed membranes for “Ca” and 3H-1R-de1tamethrin binding to calmodan Phosphorylation of membrane proteins Phosphorylation of intact synaptosomes Binding of 45Ca2+ and 3H-lR-deltamethrin to calmodan using calmodulin antibody Electophoresis and autoradiography RESULTS Effects of calcium concentration on protein phosphorylation in lysed membrane preparations Effects of exogeneously added bovine carmoduline on membrane protein phosphorylation Effects of lR-deltamethrin ROS-4864 and trifluoperazine on membrane protein phosphorylation Effects of depolarizing agents on the endogenous phosphorylation activities in the intact synaptosomes of the German cockroach Studies on the qualitative differences between susceptible and resistant calmoduline DISCUSSION REFERENCES CHAPTER III. MODIFICATION OF PHOSPHORYLATION ACTIVITIES ON THE RAT BRAIN SODIUM CHANNEL BY PYRETHROIDS AND DDT ABSTRACT INTRODUCTION MATERIALS AND METHODS Chemicals Preparation of synaptosomal fraction (P2) Phoshorylation by endogeneous ATP and protein kinases DEAE-sephadex chromatography WGA- sepharose column chromatography Rephoshorylation by exogeneous CAMP dependent protein kinase iv 37 40 4O 4O 40 41 41 42 43 43 50 '61 62 68 75 78 81 82 83 85 85 85 88 88 89 89 Purification of the sodium channel from rat brain 89 Brain membrane preparation 90 Solibilization of sodium channel 90 Ion exchange chromatography 91 Hydroxyl apatite chromatography 91 Lectin chromatography 92 Electrophoresis and autoradiography 92 Radioiodination of sea Anemone toxin II (AT'X-II) 93 ATX-II coupling 93 32P—phoshorylation of lysed synaptic membranes 94 RESULTS 95 DISCUSSION 109 REFERENCES 1 12 GENERAL DISCUSSION 1 15 TABLE LIST OF TABLES CHAPTER I. 1. Selection of the German cockroach (VDLSP) strain‘1 by permethrin using a surface contact method. Development of resistance to permethrin in the VDSLP strain of the German cockroach during selection in the laboratory. Susceptibility levels of the susceptible (CSMA) and the resistant (VDLSP F3) strain of the German cockroach to several insecticides with and without synergist (combination of topical application and surface contact method). Susceptibility levels of the susceptible (CSMA) and the resistant (VDLSP F3) strain of the German cockroach to pyrethroid insecticides, with and without piperonyl butoxide (combination of topical application and surface contact method). Susceptibility levels of the susceptible (CSMA) and the resistant (VDLSP F3) strain of the German cockroach to various neuroactive agents (cross-resistance, injection method). Susceptibility levels (knockdown) of the susceptible (CSMA) and the resistant (VDLSP F3) strain of the German cockroach to permethrin and IR-deltamethrin by a surface contact method. Susceptibility levels of the susceptible (CSMA) and the resistant (VDLSP F10) the German cockroach to IR-deltamethrin, with and without piperonyl butoxide. CHAPTER II. 1. Susceptibility levels of resistant and susceptible strains of the German cockroach and the housefly against permethrin , (cis: trans ratio 35:65) using a surface contact method. Effects of changes in calcium concentration on total protein phosphorylation in lysed membrane preparations of susceptible and kdr-resistant German cockroaches in the presence of 21.3 nM of calmodulin. 45 Effects of changes in calmodulin concentrations on autophosphorylation of the two subunits of calcium/calmodulin dependent protein kinase in the synaptosomal lysed membrane preparations from the susceptible and the kdr-resistant German cockroach strains (radioscanned). Effects of changes in calmodulin concentrations on autophosphorylation of the two subunits of calcium/calmodulin vi PAGE 18 19 20 22 23 24 25 55 dependent protein kinase in the synaptosomal lysed membrane preparations from the susceptible and the kdr-resistant housefly strains (radioscanned). Densitometric readings of the band intensities of the total 32P- phosphorylated proteins from intact synaptosomes of susceptible and kdr-resistant German cockroaches, treated with various depolarizing agents as shown in Fig. 3 legend. The proteins were first phosphorylated (non-radioactive) by endogenous protein kinases. After st0pping the reaction, unphosphorylated proteins remaining were phsophorylated using 32P-ATP and exogenously added protein kinase A (holoenzyme). Stimulatory effect of different sources of calmodulin on protein phosphorylation of isolated lysed membranes from the susceptible and lair-resistant strain of the German cockroach. Levels of 45Ca2+-bound soluble proteins reacting to the goat antibody preparation against bovine calmodulin found in the lysed synaptosomal preparations from the susceptible and kdr-resistant insects. Levels of 3H—lR-deltamethrin bound soluble proteins reacting to the goat antibody preparation against bovine calmodulin found in the lysed membrane preparations from the susceptible and kdr-resistant German cockroach. CHAPTER III. 1. Densitometric readings of the band intensities of the 32P- phosporylated sodium channel from intact synaptosomes treated with various depolarizing conditions as shown in Fig. 3 legend. The proteins were phosphorylated (nonradioactive) by endogenous protein kinases. After stopping the reaction remaining unphosporylated proteins were phosphorylated using 32P- -7 ATP and exogenously added protein kinase A. Densitometric readings of the band intensities of the 32P- phosphorylated sodium channels from intact synaptosomes treated with various concentrations of deltamethrin. The lane numbers correspond to those shown in Fig. 4. The results are expressed in % radioactivity found at 260 Kd band of the total radioactivities per lane. (97 Kd - ). Averages of two independent experiments (see Fig. 4 for more details). Note that depolarization is expected to cause a decline in phosphorylation. 56 67 70 71 72 101 104 LIST OF FIGURES FIGURE CHAPTER I. 1. 2. The chemical structures of the chemicals used for this study. The development of permethrin resistance in a DDT-resistant strain (VDLSP) of the German cockroach during selection under laboratory conditions. CHAPTER II. A. 1a.. 1b. 2a. 2b. The chemical structures of the chemicals used for this study Radioautogram of 32P-labeled phosphoproteins of lysed membranes analyzedon 10% SDS-polyacrylamide gel-electrophoresis (SDS- PAGE). The membranes were obtained from the susceptible German cockroaches,and labeled using 7-32P-ATP and the endogenous calcium/calmodulin-dependent protein kinase (CCPK) in the presence of 21.3 nM of calmodulin. Lane (1) control (standard incubation) without Ca2+ (0.2 mM EGTA), (2) with Ca2+ 10'5M, (3) with Ca2+ 104M, (4) with Ca2+ 10'3M, (5) with Ca2+ 10'2M, (6) same as 2 (replicate) (7) same as 3, (8) same as 4, (9) same as 5. Std is where a molecular weight standard protein mixture (nonlabeled) was added for molecular weight determination. Radioautogram of 32P-labeled phosphoproteins of lysed membranes from the kdr resistant German cockroaches analyzed on 10% SDS- PAGE. The proteins were labeled using 7-32P-ATP and the endogenous (CCPK in the presence of 21.3nM of calmodulin. Lane (1) control without Ca2+ (0.2mM EGTA), (2) with Ca2+ 10'5M, (3) with Ca2+ 104M (4) with Ca2+ 10'3M, (5) with Ca2+ 10-2M, (6) same as 2, (7) same as 3, (8) same as 4, (9) same as 5. Radioautogram of 10% SDS-PAGE of 32P-labeled phosphoproteins from the lysed membranes of the susceptible German cockroaches. The proteins were labeled using 7-32P-ATP and the endogenous CCPK in the presence of 0.3 mM Ca2+. Lane (1) control, without exogenously added bovine calmodulin, (2) the same in the presence of 5.3 nM calmodulin (3) 10.6 nM calmodulin, (4) 21.3 nM calmodulin in the presence of lR-deltamethrin 10‘3M (7) with 10.6 mM calmodulin and lR-deltamethrin 10'5M (8) with 10.6 nM calmodulin and ROS-4864 10'5M, (g) with 10.6 nM calmodulin and nifluoperazine 105M. Radioautogram of 10% SDS-PAGE of 32P—labeled phosphoproteins from the lysed membranes of the kdr-resistant German cockroaches. The proteins were labeled with 7-32P-ATP and the endogenous PAGE 12 16 38 46 48 51 3a. 3b. CCPK in the presence of 0.3 mM Ca2+. Lane (1) control, without exogenously added bovine calmodulin, (std) standard protein mixture (nonlabeled), (2) with 5.3 nM Calmodulin, (3) 10.6 nM calmodulin, (4) 21.3 nM calmodulin, (5) 42.3 nM calmodulin, (6) with 10.6 nM calmodulin in the presence of lR-deltamethrin 10'3M, (7) with 10.6 nM calmodan and lR-deltamethrin 10‘6M (8) 10.6 nM calmodan and ROS-4864 10'5M, (9) 10.6 nM calmodulin and trifluoperazine 105M. Effects of changes in calmodulin concentrations on autophosphorylation of the two subunits of calcium/calmodulin dependent protein kinase in the synaptosomal lysed membrane preparations from the susceptible and the kdr-resistant German cockroch (radioscanned). The bars show the 2*. SD. Effects of changes in calmodulin concentrations on autophosphorylation of the two subunits of calcium/calmodulin dependent protein kinase in the synaptosomal lysed membrane preparations from the susceptible and the kdr-resistant housefly (radioscanned). The bars show the j-_ SD. Radioautogram/SDS-PAGE of 32P-phosphoproteins from intact synaptosomes of the susceptible German cockroaches treated with various neuroactive agents in situ. After the treatments the synaptosomes were dissolved in 0.1% SDS, heated for 2 min, chilled to 0'C, diluted using 1% Triton X-100 to block the action of SDS, and labeled with 7-32P-ATP and the exogenously added CAMP-dependent protein kinase (holoenzyme). Lane (1) control, no treatment, (Std) standard protein mixture (nonlabeled), (3) treated with lR—deltamethrin 1043M, (4) treated with 8-bromo-cAMP, (5) treated with veratridine 104M, (6) treated with veratridine and IR- deltamethrin 10'10M, (7) treated with veratridine and IR- deltamethrin 10-6M, (8) treated with "high Id" (140 mM) in the presence of low Ca2+ concentration (10'6M), (9) same as 8 in the presence of lR-deltamethrin 10'10M (10) same as 8 in the presence of lR-deltamethrin 10'6 M. Note that in this mode of labeling with 7-32P-ATP the proteins heavily phosphorylated by the endogenous protein kinases are appearing as light bands and those unphosphorylated due to inhibition of the endogenous protein kinase by chemicals appear as dark (i.e., high intensity on the autoradiogram) bands. Radioautogram of SDS-PAGE of labeled phosphoproteins from intact synaptosomes of resistant German cockroaches treated with various agents in situ. Other experimental conditions and explanations are identical to those shown in Fig. 3a caption. Radioautogram of 5% SDS-PAGE of 32P-phosphoproteins from the lysed membranes of the susceptible (lanes 1-4) and the resistant (lanes 5-8) German cockroaches. The proteins were labeled with y- 32P-AT'P and the endogenous CCPK in the presence of 0.3 mM Ca“. Lane (1) control, (susceptible) without exogenously added bovine calmodulin, (2) 21.3 nM calmodan (3) 21.3 nM calmodulin 1X 53 57 59 63 65 and lR-deltamethrin 10'3M (4) 21.3 nM calmodan and ROS-4864 10'5M, (5) control (resistant) without exogenously added bovine calmodulin, (6) 21.3 nM calmodulin, (7) 21.3 nM calmodulin and lR-deltamethrin 103M, (8) 21.3 nM calmodulin and ROS-4864 10' 5M. Note that the bands appearing at 260 Kd most likely represent the tat-subunit of the sodium channel. Exogenously added bovine calmodulin clearly increased the level of phosphorylation on this protein and other proteins. CHAPTER III. A. 1. The chemical structures of the chemicals used for this study Radioautogram of 5% SDS polyacrylamide gel electrophoresis (PAGE) of partially purified sodium channel labeled with 7-32P- ATP and the catalytic subunit of CAMP dependent protein kinase. Lane (1) intact synaptosomes treated identical manner as 2, without 8-Br-CAMP treatment, (2) intact synaptosomes first treated with 8- Br-CAMP, lysed using 0.1% SDS, diluted with 1% Triton X-100 and 32P-phosphorylated using CAMP, CAMP-dependent protein kinase and 7-32P-ATP, (3) 32P-phosphorylated sodium channel purified from lysed membrane according to Hartshome and Catterall (1984) up to the wheat germ affinity column step and 32P- phosphorylated using catalytic subunit of CAMP-dependent protein kinase and y-3ZP-ATP, (4) same as 3 treated with 111M 1R- deltamethrin (84.4% inhibition of phosphorylation on the sodium channel) and (5) same treated with 0.1 mM veratridine (97.8% inhibition). SDS-PAGE/radioautogram of 125I-sea anemone toxin (125I-ATX) bound synaptic membrane proteins. After binding 125I-ATX was chemically immobilized using disuccinimidyl suberate as a crosslinking agent. (1) Lysed synaptic membrane proteins partially purified using Sephadex G-150 column after solubilization and (2) solubilized lysed synaptic membranes. Note: This experiment was conducted by Y. Ishikawa. Radioautogram of SDS PAGE of labeled phosphoproteins from intact synaptosomes treated with various agents in situ. After the treatments the synaptosomes were dissolved by heating with an SDS-containing solution, cooled, diluted using Triton X-100 to block actions of SDS, labeled with y -32P-ATP and CAMP dependent protein kinases and analyzed on SDS PAGE. 1) Control, no treatment, (2) treated with 8-Bromo-CAMP, (3) treated with 10' 4M veratridine, (4) 15 sec. treatment with A23187, (5) treated with high K+ for 15 sec, (6) same for 30 sec, (7) same for 1 min and (8) same for 5 min. Note that in this mode of labeling 32P - phosphorylation of the sodium channel is reduced, since it is already phosphorylated with the endogenous protein kinases in situ before the 32P-phosphorylation treatment. Std is where a molecular weight standard protein mixture was placed. X 73 86 96 98 102 Radioautogram of SDS PAGE of phosphoproteins from intact synaptosomes treated with IR and IS deltamethrin (see Fig. 3 and Table l for additional explanation). Lane (1) control 2) treated with 8-Br-CAMP, (3) treated with high K+ for 30 sec. in the presence of 10'10 M lR-deltamethrin, (4) same with 10‘9 M (5) same with 10'8 M, (6) 10'7 M, (7) 10'6 M, (8) same with 10'6 M of 1S- deltamethrin. Radioautogram of SDS PAGE of 32P-labeled lysed synaptosomal membrane sodium channel. Lane (1) treated with 10'4 M veratridine, (2) treated with 10‘6 M 1R deltamethrin (3) control, no treatment, (4) treated with 10-5 M DDT and (5) treated with 10-5 M DDE. Note: This experimnet was conducted by Y. Ishikawa. 105 107 GENERAL INTRODUCTION Resistance has been defined as "the development ability in a strain of a species to tolerate doses of toxicants which would prove lethal to the majority of individuals in a normal population of the same species." (Anonymous 1957). The development of resistance is dependent on genetic variability already present in a population or arising during the period of selection. (Oppenoorth, 1985). The phenomenon of resistance has grown exponentially since the first report of pesticide resistance (Melander 1914), and by the end of 1980, 428 insect and acarine species are resistant to insecticides and acaricides (Georghiou and Saito 1983). Resistance is a problem of economic importance. It has been estimated that resistance increased the costs of pest control in the US. for $133.09 million annually (Pimentel et al., 1980). Also, the synthesis and development of new pesticides becomes increasingly more expensive and difficult, and moreover the new pesticides are no more effective in controling pests, because of cross and multiple resistance. For example, by 1980, 10 species of insects developed resistance to all five major classes of insecticides (Georghiou 1981). While there are many ways insects are known to develop resistance to various insecticides, it is the kdr-resistance that attracts many entomologists' attention and concern. There are many reasons for this phenomenon. The most intriguing aspect of kdr-resistance is the phenomenon that it was originally found among insects against which DDT was used as a main insecticide, and yet after many years, since the time of DDT banning in the United States and in many parts of the world, it suddenly reappeared as a result of development and deployment with pyrethroid insecticides. The observation immediately indicates that first the kdr-factor must be a very stable gene among many insect populations and second both DDT chlorinated insecticides and pyrethroids must be attacking the same biological target, since the modification of the "the target" by these insects results in effective reduction of susceptibility to both groups of insecticides, despite the complete differences in their molecular structures. As will be shown in this thesis, many scientists have tried to understand how insects develop kdr-resistance and how those insecticides affect various animals including insects, but to date no satisfactory explanations have been offered. According to the results of genetical analysis, kdr-factor is resistance due to a single gene conferring resistance to the insects possessing it (e. g. 3000-fold resistance in the case of "super" kdr factor in the housefly). Therefore, it is likely that a simple biochemical Change rather than a series of complex modifications must have occurred to allow those resistant insects to tolerate these powerful insecticides. By many accounts, such a change usually does not involve increased metabolic activities and reduced penetration. The term "target insensitivity" has been commonly used to describe such cases, and kdr-resistance is said to be one of the best examples of this type of resistance; but yet, nobody has been able to explain what the actual "target" of these insecticides is. Many scientists have already made efforts to understand this problem but have so far failed What is interesting, however, is the fact that all kdr-insects have show distinct cross-resistance to agents known to affect calcium homeostasis. Because calcium is known to antagonize the actions of these insecticides at the level of neurons, as judged by electrophysiological studies, there is a possibility that such a cross-resistance phenomenon could be the key indication as to how these insects could withstand the action of such powerful insecticides in their nervous system. To investigate the possible relationship of changes in calcium homeostasis to resistance we have concentrated in three major areas. a) Development of pyrethroid resistant insect strains and studies on their cross-resistant characteristics. b) Studies on calcium and calmodulin sensitive biochemical systems in the nervous system of both kdr-resistant and susceptible insects. c) Studies of the biochemistry of the sodium channel which has been proposed to be the main "target" of DDT and pyrethroids. As such, the results of our investigations in this thesis are summarized in three distinct chapters. CHAPTER I STUDIES ON THE DEVELOPMENT OF RESISTANCE, AND CROSS-RESISTANCE SPECTRUM IN PERMETHRIN-RESISTANT BLATTELLA GERMANICA ABSTRACT A DDT—resistant strain of the German cockroach Blattella germanica L., was selected with permethrin (35:65 cis vs trans ratio) in the laboratory. By the tenth generation of selection, the selected strain (VDLSP) has developed a resistance of 18-fold to the selecting agent. The major factors responsible for the permethrin resistance in the VDLSP were the reduced sensitivity of the target site kdr (knockdown resistance), and to a lesser extent enhanced metabolic detoxification (increased mixed-function oxidase activity). The permethrin resistant strain has shown cross and multi-resistance to DDT, carbaryl, pyrethroids (type I and type II), and to several neuroactive agents for which the mechanism of toxicity or action is relatively well defined (e.g. calcium ionophore, theophylline, veratridine, pentylenetetrazole and ROS-4864). INTRODUCTION Pyrethroids constitute a group of highly active synthetic insecticides, with high insecticidal potency, low toxicity to mammals and relatively modest persistence in the ecosphere. These favorable toxicological and ecological properties have promoted the widespread application of pyrethroids in the control of disease vectors, ectoparasites and pests infesting important agricultural crops (Ruigt, 1985). One of the potential problems associated with the intensive, extensive and/or prolonged use of pyrethroids is the development of resistance to pyrethroids and to other insecticides by insects. Development of resistance is a problem of economic importance with the synthesis and development of new insecticides becoming increasingly more difficult. The mechanisms of pyrethroid resistance in different insect species could be related to the target site insensitivity (kdr-type resistance), metabolic differences (enhanced activity of detoxifying enzymes), and reduced penetration which works as a complementary mechanism (see Georghiou and Saito, 1983). A high level of resistance to DDT due to lowered nerve sensitivity (kdr-type resistance) was first found in the Orlando house fly strain (Milani and Travaglino, 1957). This type of resistance in the housefly is related to the kdr-gene on chromosome 3, causing resistance to DDT, pyrethrins and pyrethroids. The kdr-gene directly or indirectly affects the nerve which makes the site of action less vulnerable to these insecticides. The kdr type resistance in houseflies also confaers cross- resistance to type I and type II pyrethroids (DeVries and Georghiou, 1981). A DDT resistant strain of Boophilus microplus (Nolan et al., 1977) and a DDT-resistant mosquito strain (Prasittisuk and Busvine, 1977) have also shown cross-resistance to pyrethroids. The kdr-gene is also responsible for pyrethroid resistance in different insect species. Priester and Georghiou (1978) have reported a level of resistance higher than 4,000 fold to d-trans permethrin in larvae of Culex pipiens quinquefaciatus, and suggested that reduced sensitivity of the target site may be the primary source of resistance. Pyrethroid resistance in a strain of Spodoptera littoralis is correlated with decreased sensitivity of the central nervous system (Gammon, 1979). Scott and Matsumura (1981, 1983) have found no metabolic or peneu'ation differences between the DDT resistant and susceptible strains of Blattella germanica when l‘iC-permethrin was administered topically in vivo. Their studies have shown that the resistance to DDT is due to the target site insensitivity and that the kdr-factor confers high levels of resistance to pyrethroids in the German cockroach. There are two groups of detoxifying enzymes which metabolize pyrethroids: the mixed function oxidases (MFO) and esterases. Both groups of enzymes could play an important role in resistance to pyrethrins and synthetic pyrethroids (Farnham, 1973). The primary metabolism of insecticides and other xenobiotics is affected by several types of reactions, but oxidation by the mixed-function oxidases (MFO) is of importance and often plays a dominant role in determining the toxicity or biological activity of a given compound (Wilkinson, 1983). The microsomal mixed-function oxidases system is involved in the detoxification of every pyrethroid in mammals and of at least some pyrethroids in insects and fish (Casida et al., 1983). MacDonald et a1. (1985) found that enhanced oxidative metabolism appeared to be a major factor involved in resistance to permethrin in a strain of housefly, and that synergism by piperonyl butoxide(PB) reduced the resistant ratio from 97 to 15. Scott (1985) showed also that the mixed function oxidase system is responsible for the major part of the permethrin resistance in the resistant strain of housefly, and that PB reduced the resistant ratio from 5,946-fold to 32-fold. Nicolson and Miller (1985) reported that resistance to pyrethroids in field collected strains of the tobacco budworm Heliothis virescens, results from both metabolic and target site mechanisms. The primary importance of reduced penetration as a mechanism of resistance to pyrethroids may be in its supplementary action with other mechanisms of resistance. DeVries and Georghiou (1981), and Scott (1985) found that decreased cuticular penetration appears as a mechanism of resistance to pyrethroids in housefly. This mechanism of resistance confers very low levels of resistance to a variety of insecticides. (Plapp and Hoyer 1968) The objective of this work is to study the development of permethrin resistance in a strain of the German cockroach under laboratory selection with permethrin, to examine if reduced sensitivity of the target site kdr (knockdown resistance) confers resistance to permethrin and to other synthetic pyrethroids during selection, and to determine the cross- resistance spectrum using type I and type II pyrethroids, non-pyrethroid insecticides and various neuroactive agents, which the mechanism of toxicity or action is relatively well defined. MATERIALS AND METHODS Insects The strains of the German cockroach Blattella germanica L., used in this study were CSMA (Chemical standard of Manufactures association) and VDLSP. CSMA is a susceptible strain originally obtained from the Wisconsin Alumni Research Foundation (WARF). The VDLS strain resistant to DDT (Ghiasuddin et al., 1981), was further selected with permethrin to yield VDLSP strain which has been reared continuously in our laboratory. The original VDLS strain had shown a low level of resistance to permethrin using a preliminary surface contact method, and, therefore, was selected with permethrin initially for three generations. The population of the third generation (F3) was used to complete bioassays to determine whether kdr (knockdown resistance) confers resistance to permethrin, and to study the cross-resistance spectrum. Selection processes were accomplished by placing 100-200 sixth instar-nymphs in each 500 m1 mason jar coated with a uniform residual layer of the desired dose of permethrin using 1 ml of acetone as a vehicle. Food and water were continuously provided after the initial 24 hours. The survivors (Table 1) were placed in a rearing jar and after their emergence allowed to mate. The offspring were selected for subsequent generations using the same method. Chemicals The chemicals used for this study were obtained from the following manufacturers or sources: allethrin 98.3% (EPA), permethrin 91% (Penick, cis: trans ratio 35:65), 10 fenvalerate 99.4% (Shell), cyperrnethrin 94.5% (EPA), IR-deltamethrin 99.6% (Roussel Uclaf), DDT 99% (Aldrich), carbaryl 98.8% (Union Carbide Co.), ROS-4864 (Fluka Chemical Co.). Veratridine, calcium ionophore (A23187), theophylline, pentylenetetrazole were purchased from Sigma Chemical Co. Pyperonyl butoxide (PB) 99% was obtained from EPA, and Chlorfenethol 99% was a gift from Dr. VoJazoglou. The structures of the above chemicals are shown in Figure 1. Surface contact method (continuous exposure) For this test the inner surface of 500 ml mason jars were coated with a uniform layer of pesticide using 1.0 ml of acetone in which the pesticide was dissolved. After complete evaporation of acetone 25 adult male cockroaches were placed in each test jar. Triplicates of each test concentration were made. One control for each test was run in a jar treated only with 1.0 m1 of acetone. Food (a cube of Purina Dog Chow) and water were provided after 24 hours. The mortality was recorded at regular intervals until 100% mortality was observed. A cockroach was considered dead when it was non-responsive to touch. Topical application method For synergism studies, the synergist in 1 ul of acetone was applied to the abdominal sternum, one hour before exposure to an insecticide or to a neuroactive agent. Twenty five adult male cockroaches treated with synergist were placed in each test jar in triplicates treated by surface contact method as above. One control in each test was run in a jar treated only with 1.0 ml of acetone, placing twenty five male adults cockroaches pretreated with the corresponding synergist. 11 Piperonyl butoxide (PB) was used as an inhibitor of MFO, diethyl maleate as an inhibitor of glutathione transferase and Chlorfenethol as an inhibitor of DDTases. Injection method Chemicals used in this method were dissolved in dimethylsulfoxide (DMSO), so that the appropriate concentration could be delivered in 0.25 [.11 of the solution. Injection was directed into the body cavity through the plura of the abdomen using a 1.0 ul Hamilton microsyringe. 12 Fig. 1. The chemical structures of the chemicals used for this study E101: Permethrin Cypermethrin at =1, Fenvalerate lR- deltamethrin 13 Fig. 1. Continued ,0 CH2CH2CH3 CH2 0 CH2 _ [OCH2CH2] 2 _ OC4H9 Piperonylbutoxide if ..O§O.. Chlorfenethol CszochH=CHCOZC2H5 Diethyl maleate Fig. 1. Continued OCH3 CH30 Pentelenetetrazole Theophylline R05- 4864 RESULTS Development of resistance to permethrin in the laboratory The VDLS strain showed a substantial increase in resistance by the tenth generation. The levels of permethrin used and the degree of selection process for each generation is shown in Table 1. By comparing the resistance ratio and the slope for every generation (Table 2), it was concluded that the resistance in the selected strain (VDLSP) rose rather gradually to the fourth generation of selection. By the sixth generation it became apparent from the increased resistance ratio (i.e., 5.4 at F4 and 9.0 at F6) and from the decreased slope (i.e., 5.44 at F4 and 2.99 at F6) that resistance was developing (Figure 2). It was also noticed that it was not possible to kill all cockroaches of the finally selected F10 generation under the experimental protocol. Effects of synergists on the expression of resistance in the VDLSP strain The results of tests using synergists (Table 3) showed that permethrin resistance in the German cockroach is affected by agents known to inhibit mixed-function oxidases (MFO). However, in neither case was resistance to permethrin completely eliminated, indicating the possible presence of the reduced sensitivity of the target site (i.e., kdr factor). The significant increase of permethrin toxicity itself is an evidence that the mixed function oxidase system is responsible for a part of the permethrin resistance in VDLSP strain. In bioassay using diethyl maleate, an inhibitor of glutathione transferase, the toxicity of permethrin in both susceptible and resistant strains was not altered by the synergist (T able 15 16 Fig. 2. The development of permethrin resistance in a DDT-resistant strain (VDLSP) of the German cockroach during selection under laboratory conditions. (smoH) emu Probit of % mortality 01 001 r 0001 18 Table 1. Selection of the German cockroach (VDLSP) straina by permethrin using a surface contact method. Selected Number of Nymphs Selecting Dose Morality Generation Selected mg/Jar % l 200 1.5 75 2 200 2.2 80 3 200 3.0 85 4 200 4.5 90 5 200 5.0 75 6 100 5.5 65 7 100 5.5 83 8 100 6.0 76 9 200 6.4 90 10 200 7.0 85 aStrain was selected earlier by DDT and permethrin. 19 Table 2. Development of resistance to permethrin in the VDSLP strain of the German cockroach during selection in the laboratory. Selected LT50 Confidence Resistance Tested Values Intervals Slope Ratio at (hrs) I-Tsoa Starting Generation (VDLS) 25.7 22.0-29.9 4.28 1.9 F2 37.4 35.0-39.8 3.81 2.8 F3 67.6 64.3-70.9 5.80 5.0 F4 72.3 685-75 .9 5.44 5.4 F6 121.1 1127-1322 2.99 9.0 F3 166.6 154.4- 179.9 2.84 12.3 F10 251.5 2160-3092 1.74 18.6 CSMA 13.5 11.5-15.3 7.33 -- (susceptible) aResistance ratio. LT50 of resistant strain/LT50 of susceptible strain. 20 Table 3. Susceptibility levels of the susceptible (CSMA) and the resistant (VDLSP F3) strain of the German cockroach to several insecticides with and without synergist (combination of topical application and surface contact method). Suan Insecticide Dose/Jar Synergista CSMA VDLSP (mg) LT50b SRc LT5O° SRc RRd Permethrin 0.45 None 13.5 - 68.6 - 5.1”“ 0.45 Piperonyl butoxide 13.0 1.0 32.5 2.1* 2.5“ 0.45 None 1 1.9 - 50.1 - 4.2" 0.45 Diethylmaleate 1 1 .8 1.0 48.1 1.0 4.0" DDT 12.0 None 27.7 - >140b - >5 .1 12.0 Piperonyl butoxide 3 1. l 0.9 >140 - >5. 1 12.0 Chlorfenethol 27.5 1 .0 > 140 - >5.1 Carbaryl 18.0 None 18.0 - >140 - >78 18.0 9.0 2.0“ 10.7 - 1.2 Piperonyl butoxide alSynergist applied topically 1 hr before exposure to insecticide with 30 ug/roach of piperonyl butoxide and clorfenethol, respectively, and 20 ug/roach of diethylmaleate. 1’Data are expressed in terms of time needed to kill 50% of the population. °Synergistic ratio. LT50 without synergist/LT50 with synergist. dResistance ratio: LT50 of resistant strain/LT50 of susceptible strain. eWithout mortality after 140 hrs of exposure to insecticide. *Significantly different at P = 0.05 level (student's t-test). 21 3). These results suggest that glutathione transferase is not involved in resistance to permethrin in the VDLSP strain. Cross-resistance spectrum In this study the resistant strain was found to show cross-resistance to three major classes of insecticides DDT, carbaryl (carbamates), and pyrethroids (type I and type II), as well as to several neuroactive agents (Tables 3, 4, 5). DDT cross-resistance appears to be mainly due to kdr-type resistance because piperonyl butoxide and Chlorfenethol had no effect on DDT toxicity in both susceptible and resistant strains (Table 4). Carbaryl cross- resistance was likely due to the increased mixed-function oxidases activity in the resistant strain, since piperonyl butoxide markedly suppressed the resistance to this compound. The results of a cross-resistance study to four pyrethroids are shown in Table 4. Despite generally the low levels of cross-resistance to type II pyrethroids one can reach the conclusion that there is a significant difference between these two strains in their susceptibilities to this class of chemicals. It is interesting to note that the resistance ratio to the three type II pyrethroids was higher when the insects were treated with pipeperonyl butoxide than that obtained without the synergist. Additionally, the resistant strain has shown cross-resistance to several neuroactive agents (Table 5), although it has never been exposed to these chemicals. The level of cross-resistance was highest for the calcium ionophore (A23187), and for theophylline confirming the results already published from this laboratory (Ghiasuddin et al., 1981; Rashatwar and Matsumura, 1985 ). The calcium ionophore is known to increase the intracellular calcium concentration by increasing the plasma membrane permeability specifically to calcium and thereby increasing membrane protein phosphorylation. Theophylline also increases the intracellular calcium concentration by releasing sequestered calcium from storage sites. 22 Table 4. Susceptibility levels of the susceptible (CSMA) and the resistant (VDLSP F3) strain of the German cockroach to pyrethroid insecticides, with and without piperonyl butoxide (combination of topical application and surface contact method). Sufln Pyrethroid Dose/Jar Piperonyl CSMA VDLSP butoxidea Insecticide (11g) LTSOb SRc LT50 SRc RRd Permethrin 450 - 13.5 68.6 5.7’ 450 + 13.0 1.0 32.5 2.1' 2.5'“ Allethrin 1200 - 15.2 35.2 2.3“ 1200 + 13.4 1.1 16.6 2.1" 1.2 IR—deltamethrin 12 - 48.6 79.2 1 .6'" 12 + 29.0 1.7' 60.7 1.3 2.1" Fenvalerate 500 - 30.4 50.4 1 .6“ 500 + 15.8 1.9" 31.7 1.6" 2.0' Cypermethrin 200 - 40.3 71.5 1.8* 200 + 14.6 2.7* 31.9 2.2* 2.2"' aPiperonyl butoxide applied t0pica11y 1 hr before exposure to insecticide with 30 jig/roach. bData are expressed in terms of time needed to kill 50% of the population. “Synergistic ratio: LT50 without synergist/LT50 with synergist. dResistance ratio: LT50 of resistant strain/LT50 of susceptible strain. *Significantly different at P = 0.05 level (student's t-test). 23 Table 5. Susceptibility levels of the susceptible (CSMA) and the resistant (VDLSP F3) strain of the German cockroach to various neuroactive agents (cross-resistance, injection method). Dose/ Neuroactive Roach CSMA Confidence VDLSP Confidence Agent (11 g) LT50 Interval LT50 Interval RRb Injected A23187 10 2.3 2.7-3.7 25.3 21.4-29.6 87" Pentylenetetrazole 40 1 1.0 9.0-13.3 31.4 26.7-35.8 28* Veratridine 0.2 3.8 31.0-50.0 96.0 76.0- 140.0 2.5“ ROS-4864 20 33.5 26.9-40.8 54.0 43.5-69.9 1.6 Theophylline 25 6.3 4.5280 41.5 30.6-59.5 66* ‘Data are expressed in terms of time needed to kill 50% of the population. bResistance ratio: LTSO of resistant strain/LT50 of susceptible strain. ’Significantly different at P = 0.05 level (student's t-test). 24 Table 6. Susceptibility levels (knockdown) of the susceptible (CSMA) and the resistant (VDLSP F3) strain of the German cockroach to permethrin and Ill-deltamethrin by a surface contact method. Sumn Pyrethroid Dose/Jar CSMA VDLSP Resistance Insecticide (11g) KTSO“ KT50 Ratio Permethrin 450 19.2 155.4 8.1 lR-deltamethrin 12 26.9 255.6 9.5 “Data are expressed in terms of time (in min) needed to knockdown 50% of the population. 25 Table 7. Susceptibility levels of the susceptible (CSMA) and the resistant (VDLSP F10) the German cockroach to III-deltamethrin, with and without piperonyl butoxide. Strains Piperonyl LDSO Synergistic Resistance butoxide Ratiob Ratio“ CSMA - 18.1 :1: 0.4 + 14.8 i 1.6 1.2 VDLSP - 2740* i 6.4 14.9 + 290.3“ 2*: 6.4 1.0 17.8 “Data are expressed in terms of dose (pg/jar) needed to kill 50% of the population. bSynergistic ratio: LD50 without synergist/LD50 with synergist. “Resistance ratio: LD50 of resistant strain/LD50 of susceptible strain. *LDSO value significantly different from the susceptible strain at P s 0.01 level (Student's t- test). 26 The levels of cross-resistance were intermediate for pentylenetetrazole and veratridine, but the cross-resistance to ROS-4864 was marginal. Veratridine is known to induce steady inward current of sodium (depolarization) by opening sodium channels and thereby trigger influx of Ca2+ into neurons (Catterall, 1980). Pentylenetetrazole also promotes an increase in intrasynaptosomal Ca2+ release and thereby stimulates phosphorylation of synapsin I (Onozuka et al., 1987). ROS-4864 is known to inhibit the endogenous calcium and calmodulin stimulated membrane protein phosphorylation (DeLorenzo 1981). 0111 031 mi: COT DISCUSSION Previously it has been shown by Scott and Matsumura (1983) that VDLS strain possesses kdr factor which confers the resistance to both DDT and type I pyrethroids. This strain, however, showed only a marginally significant resistance to type II pyrethroids. Furthermore, they did not show any increased level of mixed-function oxidases. The results of the current study have clearly established that by selecting the same strain further with permethrin it is possible to develop a high level of resistance to permethrin. One of the reasons why the permethrin selected strain, now designated as VDLSP, is more tolerant to several insecticides and neuroactive agents is apparently the increased mixed function oxidase levels as compared to the original strain VDLS. This conclusion is based mainly on the action of piperonyl butoxide, a synergist known to work by competitively inhibiting mixed-function oxidases (Wilkinson, 1983). Another piece of evidence supporting this conclusion is the high level of cross-resistance shown by VDLSP toward carbaryl. Nevertheless the results of the current study also indicate that the major resistance mechanism of VDLSP toward DDT and pyrethroids is the kdr factor as judged by the fact that toward those compounds piperonyl butoxide was not as effective in reducing the VDLSP level of cross-resistance (Table 3, 4). Particularly significant are the data on DDT against which piperonyl butoxide showed no synergistic action (Table 3). Another set of evidence, indicating the importance of the kdr factor, is the phenomenon that the VDLSP strains' cross-resistance to pyrethroids expresses itself better, when the criterion of the bioassay was "knockdown" instead of "mortality" (Table 6). By using the "knockdown" criterion we could show a rather high level of cross-resistance to IR-deltamethrin, a typical type II pyrethroid against which the kdr-factor usually expresses 27 '1: m. it M ft: m: 28 only modest levels of cross-resistance to this species (Scott and Matsumura, 1983). The above reasoning is a logical extension of the original observation made by Milani and Travaglino (1957), who described kdr as "knockdown" resistance factor. Also supporting the above conclusion is the observation that VDLSP strain is highly cross-resistant to A23187 and theophylline. It has been originally noted by Ghiascuddin et a1. (1981) and confirmed by Rashatwar and Matsumura (1985) that the original VDLS strain shows cross- resistance to agents affecting calcium regulation, particularly to A23187. Rashatwar et a1 (1987) have found that Skdr strain of the housefly also shows significant cross-resistance to A23187 and other calcium regulating agents. Thus it is reasonable to assume that kdr- type resistance is directly or indirectly associated with reduced nerve sensitivity toward these agents. The meaning of such cross-resistance is not known at this time. However, since it extends to both calcium mimicking elements lanthanum-ruthenium (Ghiasuddin et al. 1981) against which mixed-function oxidases or other detoxification systems do not work, one may be able to rule out the possibility of metabolic factors contributing. Since this cross- resistance phenomenon is observed for many agents interfering with calcium regulation and involving different modes of action, it is likely that the site of modification in the resistant insects is a vital system commonly utilized by many systems such as Ca2+- binding site, calmodulin calcineurin Ca2+-sensitive protein kinases, etc. Elucidation of the nature of such an important interaction site is urgently needed to understand the mechanism by which these neurotoxic pesticides work and by which these insects develop such spectacular resistance to them. REFERENCES Anonymous (1957). World Health Organization, The expert committee on insecticides, 7th report, WHO Technical report series N o. 125. Casida J.E., D.W. Gammon, A.H. Glickman and LI. Lawrence (1983). Mechanisms of selective action of pyrethroid insecticides. Ann. Rev. Pharmacol. Toxicol. 23: 413-438 Catterall W.A. (1980). Neurotoxins that act on voltage-sensitive sodium channels in excitable membrances. Ann. Rev. Pharmacol Toxicol. 20: 15-43 Devries DH. and GP. Georghiou (1981). Decreased nerve sensitivity and decreased cuticular penetration as mechanisms of resistance to pyrethroids in a lR-transpermethrin- selected strain of the house-fly. Pestic. Biochem. Physiol. 15: 234-241. Del..orenzo R.J., S. Burdette and J. Holdemess (1981). Benzodiazepine inhibition of the calcium-calmodan protein kinase system in brain membrane. Science 213: 546-548. Farnham N .W. (1973). Genetics of resistance of pyrethroid-selected houseflies, Musca domestica L. Pestic. Sci. 7(4): 513-520. Gammon D.W. (1979). Pyrethroid resistance in a strain of Spodoptera littorallis is correlated with decreased sensitivity of CNS in vitro. Pestic. Biochem. Physiol. 2: 53-62. Georghiou GP. (1981). The occurence of resistance to pesticides in arthropods-an index of cases reported through 1980. FAO, Rome, 179 pp. Georghiou GP. and T. Saito (eds.) (1983). In Pest Resistance to Pesticides, pp. 1-809. Plenum Press, New York. Ghiasuddin S.M., A.A. Kadous and F. Matsumura (1981). Reduced sensitivity of a Ca- ATPase in the DDT-resistant strains of the German cockroach. Comp. Biochem. Physiol. 68C: 15-20. MacDonald R.S., K.R. Solomon, G.A. Surgeoner and RH. Harris (1985). Laboratory studies on the mechanisms of resistance to permethrin in a field selected strain of house flies. Pestic. Sci. 16: 10-16. Melander AL. (1914). Can insects become resistant to sprays? J. Econ. Entomol. 7: 167- 173. Milani R. and A. Travaglino (1957). Ricerche genetiche sulla resistenze a1 DDT in Musca domestica concatenazione del gene kdr (knockdown resistance) due mutanti morfologigi. Riv. Parasitol. 18: 199. Oppenoorth F.J. (1985). Biochemistry and genetics of insecticide resistance. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, 16, vol. 19 (Kerkut and Gilberts, eds.), pp. 731. Pimentel D., D. Andow, R. Dyson-Hudson, D. Gallahan, S. Jacobson, M. Irish, 8. Kroop, A. Moss, 1. Schenker, M. Shepard, T. Tompson and B. Vinzant (1980). 29 30 Environmental and social costs of pesticides: a preliminary assessment. Oikos 34: 126- 140. Plapp F.W. Jr. and RF. Hoyer (1968). Possible pleoitropism of a gene confering resistance to DDT, and DDT analogs, and pyrethrins in the housefly and Culex tarsalis. J. Econ. Entomol. 61: 761-765. Priester T.M. and GP. Georghiou (1978). Induction of high resistance to permethrin in Culex pipiens quinquefasciatus. J. Econ. Entomol. 71: 197-200. Rashatwar S. and F. Matsumura (1985). Reduced calcium sensitivity of the sodium channel and the sodium Na+/Ca"’ exchange system in the kdr-type, DDT and pyrethroid resistant German cockroach, Blattella germanica. Comp. Biochem. Physiol. 81c(l): 97- 103. Rashatwar S., F. Ishikawa and F. Matsumura (1987). Difference in calcium sensitivities of the sodium transporting systems in the nervous system of susceptible and kdr-resistant houseflies, Musca domestica L. Comp Biochem. physiol. 88c: 165-170. Ruight G.S.F. (1985). Pyrethroids. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, 16, vol. 12 (Kerkut and Gilberts, eds.), pp. 183-282. Scott J.G. and F. Matsumura (1981). Characteristics of a DDT-induced case of cross resistance to permethrin in Blattella germanica. Pestic. Biochem. Physiol. 16: 31-27. Scott LG. and F. Matsumura (1983). Evidence of two types of toxic actions of pyrethroids on susceptible and DDT-resistant German cockroaches. Pestic. Biochem. Physiol. 19: 141-150. Scott J.G. (Ph.D. thesis) (1985). The biochemistry, physiology and genetics of permethrin resistance in the housefly, Musca domestica L., pp. 41-71, University of California, Riverside. Wilkinson CF. (1983). The role of mixed function oxidases in insecticide resistance: in Pest Resistance to Pesticides (Georghiou and Saito, eds.). pp. 175-205. CHAPTER II EFFECTS OF CALCIUM, CALMODULIN AND PYRETHROIDS ON PROTEIN PHOSPHORYLATION PROCESSES IN THE NERVOUS SYSTEM OF DDT AND PYRETHROID-RESISTANT AND SUSCEPTIBLE INSECTS 31 ABSTRACT The effects of calcium, calmodulin and lR-deltamethrin on protein phosphorylation processes were investigated using lysed synaptosomal preparations isolated from the nervous system of the susceptible and kdr-resistant German cockroaches and houseflies. By comparing phosphorylation activities of isolated, lysed synaptic membranes, it was concluded that the stimulatory effect of calcium alone on protein phosphorylation was the same in the preparations from the susceptible and the kdr-resistant strains of the German cockroach. However, calmodulin, added in the presence of Ca2+, significantly increased the level of phosphorylation of the two subunits of calcium/calmodulin dependent protein kinase (CCPK) from the susceptible strains, but its effects on the same enzymes in the kdr- resistant strains of the German cockroach and housefly were much less. lR-deltamethrin at 103M inhibited both the total protein phosphorylation and the phosphorylation on the two subunits of CCPK in the susceptible and kdr-resistant strains of the German cockroach. Depolarization induced in intact synaptosomes by veratridine or "high K+" in the presence of lR-deltamethrin at 10'10 M and 10‘6 M had the effect of significantly increasing the total level of endogenous protein phosphorylation in the susceptible strain, but the increase was not significant in the kdr-resistant strain of the German cockroach. 32 INTRODUCTION Extracellular signals or the first messengers (e.g. neurotransmitters, hormones, nerve impulse) arriving at the cell surface produce many of their biological responses by increasing intracellular concentrations of the second messengers (cyclic AMP, cyclic GMP, calcium and inositol polyphosphates) within the target cells. In neural systems the effects of the second messengers appear to be achieved through the activation of specific CAMP- CGMP- and calcium-dependent protein kinases (Nestler and Greengard, 1984). When a protein kinase transfers the negative charged phosphate group from ATP to the hydroxyl group of serine, threonine or tyrosine of the substrate proteins, they change their function presumably by changing their conformation (Browning et al., 1985), leading to changes in metabolism, neurotransmitters biosynthesis and release, neuronal excitability, neuronal growth, differentiation and morphology (see Nestler and Greengard, 1984). The kdr (knock-down resistance) factor is one of the major genes responsible for DDT- and pyrethroid-resistance in several insect species (Georghiou and Saito, 1983). This mechanism was first found in the Orlando housefly strain (Milani and Travaglino, 1957). This type of resistance in the housefly is related to the kdr- gene on chromosome 3, causing resistance to DDT, pyrethrins (Farnham, 1973) and pyrethroids (Sawicki, 1973). The kdr-gene directly or indirectly affects the nervous system which makes the site of action less vulnerable toward these insecticides. Tsukamoto et a1. (1965) found that kdr- factor was responsible for reduced sensitivity of the nervous system toward DDT; the observation later confirmed by Miller et al. (1983). The kdr-type DDT-resistance in the housefly has been shown to cause some degree of cross-resistance to pyrethroids (DeVries and Georghiou, 1981). A DDT-resistant strain of Boophilus microplus (Nolan et al., 33 34 pyrethroids (DeVries and Georghiou, 1981). A DDT-resistant strain of Boophilus microplus (Nolan et al., 1977), and a DDT-resistant mosquito strain (Prasittisuk and Busvine, 1977) have also shown cross-resistance to pyrethroids. In addition to the housefly, kdr-type resistance has been found in Culex pipiens quinquefaciatus (Priester and Georghiou, 197 8), in strain of Spodoptera littoralis (Gammon, 1979) in Blattella germanica (Matsumura, 1971; Scott and Matsumura, 1983) and in the predatory mite Amblyseiusfallacis (Scott et al., 1983). The resistance phenomena due to the kdr factor are now attracting attention and concern because of the possible threat to the usefulness and the effectiveness of newly developed pyrethroids. The mechanism of kdr-resistance has been studied by many workers, but so far, no single biochemical cause appears to explain the mechanism of this type of resistance in insects. Salgado et a1. (1983) have suggested that kdr-type resistance is due to a modified sodium channel. Binding studies with 14C-DDT and 14C- permethrin to the housefly central nervous system have led to the conclusion that pyrethroids affect a putative "receptor" and that kdr-resistant houseflies have fewer receptors than the susceptible ones (Chang and Plapp, 1983). Ghiasuddin et a1. (1981) found that the DDT-resistant strain of German cockroach has an altered Ca-ATPase with a lower affinity to Ca2+ than that from the susceptible strain. It has been reported (Rashatwar and Matsumura 1985; Rashatwar et al., 1986) that kdr resistant German cockroaches and houseflies exhibit cross-resistance to calcium mimics or agents affecting calcium regulatory mechanism, and that the degree of stimulation of the Ca2+-ATPase by exogenously added calcium was much higher in the two susceptible strains than in the DDT-resistant strains. Furthermore, the tendency for calcium insensitivity was more pronounced in Skdr than in kdr strain of housefly. Meanwhile, it has been well established that the main target site of DDT and pyrethroids is the sodium channel, where they cause prolongation of depolarization induced Na+ inflow (Narahashi, 1985). Our recent study results (Ishikawa et al., 1989) 35 have shown that the rat brain sodium channels are phosphorylated during depolarization and such biochemical processes is inhibited by lR-deltamethrin and DDT. Additional observations corroborate with the above background investigations. Costa and Catterall (1982) have shown that phosphorylation of the alpha-subunit of the sodium channel by CAMP-dependent protein kinase is indirectly or directly related to the sodium transporting (i.e., gate operating) mechanism. The same workers (1984ab) have also shown that such phosphorylation processes are carried out by a CAMP-dependent protein kinase and protein kinase C, a calcium sensitive protein kinase. Matsumura (1986, 1988) demonstrated that DDT and pyrethroids affect protein phosphorylation processes particularly those involved in calcium regulatory processes. In view of the above findings we have studied the nature of target site insensitivity or knock down resistance (kdr), by investigating strain differences in calcium and calmodulin sensitivity with regard to specific actions of pyrethroids on protein phosphorylation processes, using synaptosomal preparations from the nervous system of the susceptible and kdr-resistant strains of the German cockroach and the housefly. MATERIALS AND METHODS Insects German cockroach (Blattella germanica) VDLSP, a kdr-resistant strain, and CSMA a susceptible strain, were used for this study. They have been reared in our laboratory for several years. The resistant strain has been selected from the original strain of VDLS (Telford and Matsumura, 1970) for 10 generations with technical permethrin (mixture of cis- and trans-permethrin). The degree of resistance to permethrin is shown in Table 1. Housefly (M usca domestica) The kdr strain, a resistant strain to DDT, was obtained from Dr. T. Miller, University of California, Riverside. The susceptible strain (WHO) has been reared in our laboratory for 4 years. All houseflies were reared on CSMA fly medium (larvae) and on sugar-milk (adults). The degree of resistance to permethrin is also shown in Table 1. Chemicals [7-32P] adenosine triphosphate (3,000 Ci/mmol) and 45Ca2+ (300 Ci/mmol) were purchased from Amersham. Unlabeled adenosine triphosphate (ATP disodium salt), bovine brain calmodulin, antibody to calmodulin (deve10ped in goat against purified bovine 36 37 brain calmodulin as an immunogen), bovine serum albumin, 8-bromo-cyclic AMP (8-Br- CAMP) veratridine, CAMP and CAMP-dependent protein kinase holoenzyme were obtained from Sigma Chemical Co. lR-deltamethrin and 3H-lR-deltamethrin were gifts from Roussel Uclaf. ROS-4864 was purchased from Fluka Chemicals, and trifluoperazine was from Boehringer. DEAE-sephadex was a product of Pharmacia, Inc. All Chemicals used were products with highest purity available. The structures of the above chemicals are shown in Figure A. Preparation of lysed synaptosomal membranes ( = lysed membranes) Preparation, homogenization and centrifugation procedures were basically identical to the method of Breer (1981). The heads of adult male German cockroaches and houseflies were homogenized (10 heads/ml) in 20 ml of 0.25 M sucrose, containing 0.1M Tris/HCl buffer pH 7.4, 0.1mM phenylmethyl sulfonylfluoride (PMSF), and lmM iodoacetamide, using a glass homogenizer. Using a teflon-glass homogenizer rotated at 1,200 rpm, the initial preparation was further homogenized, and centrifuged at 1.000 g for 10 min, using a Sorvall centrifuge with SS-34 rotor. The supernatant was filtered through glasswool, and centrifuged at 15,000g for 45 min, using the same centrifuge. The pellet obtained was resuspended in 10 ml (20 heads/ml) of SmM Tris/HCI buffer pH 7.4 containing the two same protease inhibitors as above. After keeping the suspension at 0°C for 15 min it was centrifuged at 100,000 g for 30 min using a Beckman centrifuge with a Ti 70 rotor at 4'C. The pellet was resuspended in 3 ml of 50mM PIPES buffer pH 7.4, containing SmM MgClz, 0.2mM ethyleneglycol (aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA) (i.e., minus calcium) or 0.2mM EGTA plus 0.5 mM CaC12 (i.e., plus 0.3mM calcium), lmM dithiothreitol (DTT) and the two protease inhibitors. The resuspended pellet was divided into small aliquots, stored at -20'C for no longer than one month and used for phosphorylation studies. 38 Fig. A. The chemcal su'uctures of the chemicals used for this study NH2 1015 kN/ N 0 o ‘o——-p-———-o on H O Adenosine 3':5'- monophosphate NHz N U ,_.. N N 0 o -()—-p——O OH ll 0 8-Bromoadenosine-3',5'- monophosphate NH2 N N CDC > a" a" i‘ " " o— 51— o— fi—o—fi— 0 0 O O O OH OH Adenosine triphosphare (ATP) 39 Fig. A. continued H CN B’ (l) o > K020 I) H 1R- deltamethrin R05- 4864 $H2CH2CH2—N N—CHa CCU“? Trifluoroperazine Ell 40 Preparation of intact synaptosomes Intact synaptosomes were obtained by the above method to the step of centrifugation at 15,000 g using Sorvall centrifuge. The pellet was resuspended in 2ml of high Na buffer (140 mM NaCl, 5mM KCl, 3mM CaClz, 1.5mM MgClz, lOmM glucose, 20 mM HEPES/Tris pH 7.4) and used for endogenous phosphorylation. Preparation of calmodulin from the susceptible and the resistant strains of the German cockroach The suspension of synaptosomes (P2) fraction obtained from 200 heads of the adult male German cockroach was subjected to osmotic shock by homogenization and sonication in 1 ml of 5mM Tris/HCI pH 7.4, 0.1mM PMSF, and kept on ice for 15 min to optimize cell lysing. The homogenates were centrifuged on an Eppendorf centrifuge for 20 min at 16,000 g. The suppematant was collected, heated for 5 min at 90-100°C, stored at -20’C, and used as a source of calmodulin. Preparation of lysed membranes for “Ca“ and 3H-1R-deltamethrin binding to calmodulin Lysed synaptosomes for studies in 45Ca2+ and 3H-lR-deltamethrin binding to calmodulin were obtained as described above to the step of centrifugation at 15,000 g using Sorvall centrifuge. The pellet was resuspended in 2 ml of 5mM Tris/HCl pH 7 .4, 0.1mM PMSF, lmM iodoacetamide pretreated with Chelex-100 and was stored at -20"C and used as a source of lysed membrane. Phosphorylation of membrane proteins Phc b} 1 dti “is can 41 Phosphorylation experiments were conducted basically according to the method of DeLorenzo et a1. (1981). The standard reaction mixture (final volume, 100 pl) contained 70 pg membrane proteins, 50mM PIPES buffer pH 7.4, 5mM MgClz, 0.2mM EGTA (i.e., minus calcium) or 0.2mM EGTA plus 0.5mM CaClz (i.e., plus 0.3mM calcium) lmM D'I'l‘, 0.1mM PMSF, lmM iodoacetamide, and bovine brain calmodulin (3 to 100 units, 0.0125 pg to 4 pgfrncubation or 1.33 to 42.5 nM final concentration). The test Chemicals were added with 1 pl ethanol or DMSO (control received only the same volume of ethanol or DMSO), and the system was preincubated for 10 min at room temperature (24°C). A 10 pl aliquot of y—32P- ATP solution (2.7 pCi and 1.2 x 10'7M cold disodium ATP) in distilled water was added to start the phosphorylation reaction which was maintained at room temperature for 1 min. The reaction was stopped by the addition of 80 pl of electrophoresis sample treatment buffer (0.125m Tris/HCl pH 6.8, 4% SDS, 20% glycerol and 10% mercaptoethanol with bromphenol blue) and boiled for 2 min. Phosphorylation of intact synaptosomes Phosphorylation by endogenous ATP and protein kinases, and 32P-phosphorylation by exogenously added 7-32P-ATP and CAMP-dependent protein kinases were carried out as described in Chapter III. Binding of 45Ca2+ and 3H-1R-deltamethrin to calmodan using calmodulin antibody Membrane proteins (700 pg) in 550 pl of 5mM Tris/HCl pH 7.4, 0.1 mM PMSF, was sonicated for l min. Chelex-100 was added to the mixture, vortexed, and then centrifuged for 1 min at 1,500 g using a clinical centrifuge. 500 pl of the supernatant was taken out and incubated with 4 pCi of 45Ca2+ or 1.3 pCi of 3H-lR-deltamethrin for 30 min at 42 room temperature. After incubation 50 pl of the mixture was added to an Eppendorf tube in which calmoduhn antibody in 10 pl aliquot was added to the mixture and incubated at 4°C for two hrs. A suspension of protein A-sepharose CL-4B containing 6.25 mg in 100 pl of 30mM Tris/HCl pH 7.8 was added to the mixture vortexed and kept at 4°C for 30 min. The mixture was centrifuged on an Eppendorf centrifuge for l min at 16,000 g and the supernatant discarded. The pellet was resuspended and washed three times with 500 p1 of 30mM Tris/HCl pH 7.8. The immunocomplex was solubilized by addition of 400 pl of 2% sodium dodecyl sulfate (SDS) and boiled at 90-100°C for 2 min. After centrifugation for 2 min as before, aliquots of 350 pl each were taken for radioassay, using a liquid scintillation spectrophotometer (Beckmans instrument model LS 5801). Elecuophoresis and autoradiography SDS-polyacrylamide gel-electrophoresis was developed according to the method of Laemmli (1970). Protean H electrophoresis apparatus (Bio-Rad Lab) with 1.5mm spacers, 5% and 10% separating gel, 3% and 5% stucking gel were used. Other conditions were identical to the ones supplied by the manufacturer. The gels were stained with Coomassie brilliant blue R-250, destained, dried over thick filter papers using a vacuum drier (Hoefer Scientific), autoradiograms developed with x—ray films (Kodak x-omat AR-S), and radioscanned using an Ambis model (Automated Microbiology Systems, Inc. San Diego, CA). RESULTS Effects of calcium concentration on protein phosphorylation in lysed membrane preparations. Since calcium plays an essential role in protein phosphorylation (Browning et a1, 1985), and various protein phosphorylation processes have been implicated in important neural functions such as neurotransmitter biosynthesis and release, postsynaptic potentials, and ion Channel conductance (Nestler and Greengard 1984), the effects of calcium on protein phosphorylation were first examined using lysed membrane preparations from the susceptible and kdr-resistant strains of the German cockroach, according to the method of DeLorenzo et a1. (1981). The results (Fig. 1a and 1b, Table 2) showed that calcium at lmM and 10mM significantly increased the level of membrane protein phosphorylation. At calcium concentrations of 10 and 100 pM the level of protein phosphorylation was about the same level as in the treatment with 0.2mM EGTA, (minus calcium). Calcium at an Optimum concentration (lmM) in the presence of calmodulin stimulated the endogenous phosphorylation of several membrane proteins of which molecular weights were 38, 48, 52, 64, 70, 77 and 86 kd (Fig. 1). The phosphoproteins with molecular weight approximately 52 kd and 64 kd are likely the autophosphorylated subunits of calcium/calmodulin-dependent protein kinase II (CCPK). Walaas et al. (1983) have previously found that autophosphorylated subunits of CCPK are prominent substrates for this enzyme in the rat brain. Autophosphorylation of the enzyme subunits appears to increase its affinity to calmodulin. (Shields et al., 1984). Autophosphorylation of CCPK has also been documented earlier by several workers (Ahmad et al., 1982; Fukuanga et al., 43 Table l. Susceptibility levels of resistant and susceptible strains of the German cockroach and the housefly against permethrin , (cis: trans ratio 35:65) using a surface contact method. Species and Strains LD50 Resistance ratio Blattella gennam'ca CSMA 135.6 i 9.2 VDLSP 2882.0 i 267.6“ 21.3“ Musca domestica WHO 5.6 i 1.77 kdr 44.7 i 12.5“ 7.9“ "LD50 value significantly different from the susceptible strains at P3001 level (Student's t-test) “Resistance Ratio: LD50 of resistant strain/LDSO of susceptible strain 45 Table 2. Effects of changes in calcium concentration on total protein phosphorylation in lysed membrane preparations of susceptible and kdr-resistant German coclcroaches in the presence of 21.3 nM of calmodulin. Total protein phosphorylation (cpm/lane radioscanned) 70pg protein Calcium“ Concentrations Susceptible strain Resistant strain 0(EGTA 0.2 mM) 21515i 0 19719:l:0 10-5M 21197 i1718 19451 i 1997 104M 21189 i 26 19283 i 579 10'3M 47333 :t 168" 47916 i 2520" 10-2M 41900 i 7517“ 37476 i 4801“ Results are expressed as means i SD. Means with asterisks are significantly different from the corresponding Ca2+ free values at P < 0.05 and P < 0.01 level according to Duncan's multiple range test after one-way ANOVA in completely random design experiment. According to the same test, the means between suscpetible and resistance strain are not significantly different at P < 0.05 level. “Free Ca2+ concentrations, maintained using Caz+-EGTA buffering system (Portzehl et al., 1964). 46 Fig. la. Radioautogram of 32P-labeled phosphoproteins of lysed membranes analyzed on 10% SDS-polyacrylamide gel-elecuophoresis (SDS-PAGE). The membranes were obtained from the susceptible German cockroaches, and labeled using 7-32P-ATP and the endogenous calcium/calmodulin-dependent protein kinase (CCPK) in the presence of 21.3 nM of calmodulin. Lane (1) control (standard incubation) without Ca2+ (0.2 mM EGTA), (2) with Ca2+ 10'5M, (3) with Ca2+ 104M, (4) with Ca2+ 10'3M, (5) with Ca2+ 10‘2M, (6) same as 2 (replicate) (7) same as 3, (8) same as 4, (9) same as 5. Std is where a molecular weight standard protein mixture (nonlabeled) was added for molecular weight determination. 47 an. rue :- ' e .6 ‘I e .- ” «esxd - .23 I" CCPK» ”at—- ‘l- - <45Kd ‘~-“—-—~‘~ «341m ~—- —" Std1 2 3 4 5 6 1 a 9 Fig.1a. 48 Fig. lb. Radioautogram of 32P-labeled phosphoproteins of lysed membranes from the kdr resistant German cockroaches analyzed on 10% SDS-PAGE. The proteins were labeled using y—32P-ATP and the endogenous (CCPK in the presence of 21.3nM of calmodulin. Lane (1) control without Ca2+ (0.2mM EGTA), (2) with Ca“ 10'5M, (3) with Ca2+ 104M (4) with Ca2+ 10‘3M, (5) with Ca2+ 10'2M, (6) same as 2, (7) same as 3, (8) same as 4, (9) same as 5. 49 b at <66Kd m ”nu-t ‘ CCPK: LT: . <45Kd «34Kd Fig.1b. 50 1982). The stimulatory effect of exogenously added calcium in the presence of a constant concentration of calmodulin (21.3 nM or 50 unit/tube) was similar between the preparations from the susceptible and kdr resistant suains of the German cockroach, (Fig. 1a and 1b; Table 2). Effects of exogenously added bovine calmodulin on membrane protein phosphorylation In view of the known roles of calmodan in mediating the effects of calcium over a large number of enzymes and active proteins, (see Stull et al., 1986), its effect on membrane protein phosphorylation was studied, by using lysed membrane preparations from susceptible and kdr-resistant strains of the German cockroach and the housefly. The results indicated (Fig. 2a and 2b; Fig. 4) that the addition of different levels of calmodan 5.3 to 42.5 nM (12.5 - 100 units/tube) in the presence of 0.3 mM calcium increased the total level of membrane protein phosphorylation in both the susceptible and the kdr- resistant strain of the German cockroach. Especially calmodan increased the intensity of two bands at about 52 and 64 kd, which are likely to be the subunits of calcium/calmodulin dependent protein kinase themselves. The greatest average increase in phosphorylation was in the two major autophosphorylated bands (213%) for the susceptible strain and (117.5%) for the kdr-resistant strain in treatments with 42.5 nM (100 unit/tube) of calmodulin (Table 3). To confirm the above qualitative and quantitative data the same experiments were repeated, using preparations from the susceptible and the kdr-resistant housefly. The results shown in Table 4 and Fig. 5 indicate the same tendency. The addition of different levels of calmodulin significantly increased the level of phosphorylation of the two subunits of CCPK in the susceptible strain, but the increase in the level of phosphorylation was not significant in the kdr-resistant strain. The greatest average increase in phosphorylation of the two autophosphorylated bands was 245% for the susceptible strain and 151% for the resistant strain (Table 4). 51 Fig. 2a. Radioautogram of 10% SDS-PAGE of 32P-labeled phosphoproteins from the lysed membranes of the susceptible German cockroaches. The proteins were labeled using 7-32P-ATP and the endogenous CCPK in the presence of 0.3 mM Ca2+. Lane (1) control, without exogenously added bovine calmodulin, (2) the same in the presence of 5.3 nM calmodulin (3) 10.6 nM calmodulin, (4) 21.3 nM calmodulin in the presence of IR- deltamethrin 10'3M (7) with 10.6 mM calmodulin and lR-deltamethrin 10'6M (8) with 10.6 nM calmodulin and ROS-4864 10'5M, (g) with 10.6 nM calmodulin and trifluoperazine 10' 5M. 52 “Han-"Fm” . _, -r.‘ CCPK: :=B=== ~--._--_-‘_ Fig.23. <66 Kd j<45xd <34 Kd Std 53 Fig. 2b. Radioautogram of 10% SDS-PAGE of 32P-labeled phosphoproteins from the lysed membranes of the kdr-resistant German cockroaches. The proteins were labeled with 7-32P-ATP and the endogenous CCPK in the presence of 0.3 mM Ca2+. Lane ( 1) control, without exogenously added bovine calmodulin, (std) standard protein mixture (nonlabeled), (2) with 5.3 nM calmodulin, (3) 10.6 nM calmodulin, (4) 21.3 nM calmodulin, (5) 42.3 nM calmodulin, (6) with 10.6 nM calmodulin in the presence of IR- deltamethrin 10'8M, (7) with 10.6 nM calmodulin and lR-deltamethrin 10'5M (8) 10.6 nM calmodulin and ROS-4864 105M, (9) 10.6 nM calmodulin and trifluoperazine 105M. 54 ——-—-D.-‘ - —-——-—.--¢ ‘ ‘GBKd CCPK' -~—- --'—" *” "'" - d- — - ‘45Kd ~. ”a----—.’ -34“ 1 Std 2 3 4 5 G 7 8 9. Fig.2d. 55 Table 3. Effects of changes in calmodulin concentrations on autophosphorylation of the two subunits of calcium/calmodulin dependent protein kinase in the synaptosomal lysed membrane preparations from the susceptible and the kdr-resistant German cockroach strains (radioscanned). Protein phosphorylation % from control Calmodufin con in nM Susceptible strain Resistant strain 0.0 100.0 3: 0“ 100.0 i 0“ 1.3 128.9 i 5.0“ 69.5 i 3.5“ 2.7 133.0 i 24.0“ 89.7 :i: 15.9“ 5.3 157.0 i 12.5“ 106.5 :1: 12.0“ 10.6 186.7 :1: 53.2“ 117.0 1: 38.2“ 21.3 206.0 i 13.9“ 117.5 :1: 10.6“ 42.5 213.0 :1: 48.1“ 117 .5 i 20.5“ Results are expressed as means :1: SD. Means with a are significantly different from those bearing b or the control at the P < 0.05 level according to Duncan's multiple range test after one-way ANOVA in completely random design experiment. Means with b are not significantly different among themselves at P5005 level according to the same test. 56 Table 4. Effects of changes in calmodulin concentrations on autophosphorylation of the two subunits of calcium/calmodulin dependent protein kinase in the synaptosomal lysed membrane preparations from the susceptible and the kdr-resistant housefly strains (radioscanned). Protein phosphorylation % from control Calmodulin con in nM Susceptible strain Resistant strain 0.0 100: 0b 100 i 0“ 1.3 141 i 17“ 88 i 2“ 2.7 151:1:33“ 114: 13“ 5.3 184 :l: 33“ 119 i- 12“ 10.6 195 i 65“ 135 :i: 25“ 21.3 245i57“ 151:32“ 42.5 212 :l: 86“ 143 i 9“ Results are expressed as means :i: SD. Means with a are significantly different from those bearing b or the control at the P < 0.05 level according to Duncan's multiple range test. Means with b are not significantly different among themselves at P < 0.05 level according to the same test. 57 Fig. 4. Effects of changes in calmodulin concentrations on autophosphorylationof the two subunits of calcium/calmodulin dependent protein kinase in the synaptosomal lysed membrane preparations from the susceptible and the kdr-resistant German cockroch (radioscanned). The bars show the i SD. 58 Protein Phosphorylation (Percent of Control) moo N001 ‘00 HH l——-4 . Jr - G d d - d do no we no mo 02303.3 00383320: Aamzoaofigaoccmzoa IT 958266 93:: ll 38.22: 93.: 59 Fig. 5. Effects of changes in calmodulin concentrations on autophosphorylationof the two subunits of calcium/calmodulin dependent protein kinase in the synaptosomal lysed membrane preparations from the susceptible and the kdr-resistant housefly (radioscanned). The bars show the :1; SD. 60 Protein Phosphorylation (Percent of Control) woo Moo 1 ._oo do n J . . 1 no mo no 02308.3 Canoes—«mac: szoao_om\3occm=oa mo 113.1 33.32.: 935 ll. @5313? 93.: 61 Effects of lR-deltamethrin, ROS-4864 and trifluoperazine on membrane protein phosphorylation The effects of 1R-deltamethrin, ROS-4864 and trifluoperazine on protein phosphorylation reactions were studied using lysed synaptosomal membrane preparations from the susceptible and the kdr-resistant strains of the German cockroach, in the presence of 0.3mM calcium and 10.6 mM calmodulin (25 units/tube or 10.6 nM/tube). Under the standard conditions, (10 min preincubation of the test chemicals and 1 min phosphorylation at 24°C ) R-deltamethrin at 10'3M caused 16.3% reduction of the total protein phosphorylation. The corresponding percentages of reduction were 33.4 and 35.6 for ROS-4864, and trifluoperazine in the susceptible strain, respectively. Under the same conditions, the reduction of the total protein phosphorylation, in the resistant strain was 21.4% for lR-deltamethrin, 22.5% for ROS-4864 and 39.4% for trifluoperazine. The reduction of phosphorylation on the autophosphorylated subunits of CCPK was 23, 65 and 71.2% in treatments with 1R-deltamethrin, ROS-4864 and trifluopenzine, respectively in the susceptible strain. The corresponding figures for the kdr-resistant strain were 27.9, 66.4, and 66%, respectively. These same chemicals caused reduction of phosphorylation of the membrane proteins of molecular weight 70 and 86 Kd (Fig. 2a and 2b). The last phosphoprotein is most likely synapsin I (phosphorylated at site II) which has been found to be one of the best substrates for the brain calcium/calmodulin-dependent kinase II (N aim et al., 1985). Trifluoperazine is known to be a potent inhibitor of calmodulin (Cheung 1980; Weiss and Levin, 1978). ROS-4864 is known to inhibit the endogenous calcium and calmodulin stimulated membrane protein phosphorylation (DeLorenzo et al., 1981). 62 Effects of depolarizing agents on the endogenous phosphorylation activities in the intact synaptosomes of the German cockroach To study the effects of depolarizing agents on the endogenous phosphorylation activities in the intact synaptosomes, the method developed by Costa and Catterall (1982) was adopted. Under the standard conditions (10 min preincubation with lR-deltamethrin, 1 min endogenous phosphorylation, 10 min exogenous 32P-phosphorylation) depolarization of the synaptosomal membranes, by "high K+", or by veratridine caused a significant increase in the total level of endogenous protein phosphorylation in the susceptible strain, but not in the resistant strain. Particularly significant was the increase in intensities of labeling on phosphoproteins with molecular weight 49, 55, 86 and 105 Kd (Fig. 3a and 3b; Table 5). It must be noted here that by this method the radioautographic results are expressed as the reverse of those shown in Figs. land 2; i.e., the inhibition of protein phosphorylation appears as an increase in the band intensities and stimulation in phosphorylation as reduction. The level of endogenous phosphorylation was the highest in the susceptible strain at 1 min depolarization, using veratridine 104M in the presence of lR-deltamethrin at 10'10M and 105M (65% and 64%, respectively). Depolarization using "high K+" buffer (140 mM KCl) in the presence of lR-deltamethrin at 10*10M and 1045M increased the level of total phosphorylation significantly over the control (42.3% and 27%, respectively) only in the susceptible strain. There was also a marked increase of phosphorylation in treatments with 8-bromo-CAMP and veratridine alone, but the difference was not significant from the control in both susceptible and resistant strain (Table 5). Under the non-depolarized conditions, lR-deltamethrin at 1043M inhibited the endogenous phosphorylation in the susceptible and the kdr-resistant strain by 22% and 13%, respectively. However, these values were not statistically different from the control levels. 63 Fig. 3a. Radioautogram/SDS-PAGE of 32P-phosphoproteins from intact synaptosomes of the susceptible German cockroaches treated with various neuroactive agents in situ. After the treatments the synaptosomes were dissolved in 0.1% SDS, heated for 2 min, chilled to 0°C, diluted using 1% Triton X-100 to block the action of SDS, and labeled with 7-32P-ATP and the exogenously added CAMP-dependent protein kinase (holoenzyme). Lane ( 1) control, no treatment, (Std) standard protein mixture (nonlabeled), (3) treated with lR-deltamethrin lO-“M, (4) treated with 8-bromo-CAMP, (5) treated with veratridine 104M, (6) treated with verauidine and lR-deltamethrin 1010M, (7) treated with veratridine and lR-deltamethrin 10-6M, (8) treated with "high Id" (140 mM) in the presence of low Ca2+ concentration (10'5M), (9) same as 8 in the presence of IR- deltamethrin 10'10M (10) same as 8 in the presence of lR-deltamethrin 10'“ M. Note that in this mode of labeling with 7-32P-ATP the proteins heavily phosphorylated by the endogenous protein kinases are appearing as light bands and those unphosphorylated due to inhibition of the endogenous protein kinase by chemicals appear as dark (i.e., high intensity on the autoradiogram) bands. 64 <1 16 Kd * 97Kd < 66Kd - 45Kd 1 Std 2 3 4 5 6 7 8 9 Fig.3a. 65 Fig. 3b. Radioautogram of SDS-PAGE of labeled phosphoproteins from intact synaptosomes of resistant German cockroaches treated with various agents in situ. Other experimental conditions and explanations are identical to those shown in Fig. 3a caption. Fig.3d. 66 416Kd < 97Kd ‘ 66Kd < 45Kd He...» m. Uozmzeaoao Hana—3mm em :5 ESQ 58.55% 0». 5o 8:: uuwéromeroQ—fig @353. $08 382 $328083 0». E8836; Ea $73383 0035: 80.9.8038. .328 $3. $508 mag—Emam amass mm .992: E Em. m Emma. .25 3233 £20 ma" 38332.33 Soaémagoaév 3. gnome—5% @383 5:38. >52 mnoeemsm =5 Renae? cseromeronfia 908:5 Hoammaam £08 ermoeroQEnoa 55m 335% Ed oxomaaocma. women 3085 5:30 > cacaoanmaov. 67 5:0 20. Hex: em em 8&8035 mesa mm 3 Unto—mammaos Ba 3 m. m 9:2 "82338 m. m5”: w. mg? L 0038. gazing—“588 5o H o6 3o H cc N uwtanu—SBQE: HNN H Hue :w H 3.. w m-w~oBo-o>Z=u mm H No,U ma H 3: e ZO<> 5 neat—90¢. 8:33 90am: 931303. cam: W+ Gammon 75 87.— W9. arcs. Om?” E3. 68 Studies on the qualitative differences between susceptible and resistant calmodulin The above studies have shown that there is a differential response between synaptosomal CCPK from the susceptible and the resistant strains toward exogenously added bovine calmodulin. To understand the meaning of such a phenomenon, crude calmodan containing supernatant preparations were obtained from the German cockroach strains and, along with bovine calmodulin, used as activators of CCPK in the resistant and the susceptible preparations (lysed membranes). The results (Table 6) indicate that both S calmodulin and bovine calmodulin are better activators of CCPK from the susceptible insects than that from the resistant ones. On the other hand, for CCPK from the resistant strain, R calmodulin was slightly better or equally effective as S calmodulin. To further ascertain the qualitative differences between these calmodulins, we have tested their immunological reactivities toward an antibody against bovine calmodulin. For this purpose, the osmotically lysed synaptosomal preparations (i.e., containing both cytosol and membranes) were incubated either with “Ca“ or with 3H-lR-deltamethrin. An aliquot of the antibody suspension was added to the system, incubated and the immunocomplex was coupled to protein A-Sepharose, precipitated, washed and the antigens were released using sodium dodecylsulfate solution and by heating. The results (Table 7) showed that 45Ca2+-bound calmodulins from these strains reacted differently toward the antibody. Interestingly R calmodan from B. germanica was more reactive than S calmodulin from the same species. The relationship was reverse in the case of M. domestica. When 3H-lR-deltamethrin-bound cockroach calmodulins were tested (Table 8), it became apparent that the S calmodan has the ability to bind with a higher level of 3H-lR-deltamethrin than R calmodulin despite the fact that the latter appears to have a lower affinity to this antibody than the former. One important question remaining is whether the (rt-subunit of the sodium channel may be phosphorylated as a result of action of CCPK. We have tested this by using lysed 69 membranes and exogenously added bovine calmodulin . The results (Fig. 6) indicate that calmodulin increased phosphorylation on the 260 Kd protein and that lR-deltamethrin had the property to decrease phosphorylation on this band as well as other proteins (e. g. the very high molecular weight protein). 70 flea—e a. mass—Sea. 0209 on. 93.2.02 8:83 em 8553:: 3 @383 38332533 0m $238 €an 395338 :03 :5 «88320 25 €78mmmaan were: em Sn 09.33 8338:. $385 3830Q533 A830 3588338 wotm e885 magnesia mam: ”amaze: 2.35 mesa... om H03: H08— 03383: 3830333 >503830Q533 38303.58: >So3om30Q§mo= we MOMMA H E 33 H om T33 H 5o 38 H 3 we :33 H 5m poem H 3 Humow H EA 35 H 3 we 80% H 3— mmmo H 8 ~33 H h: mmmm H 3 ace—3855 $05.8 man. 380320 33. 53803:: $228 35. amaze: 3.3: owe/3." cannons—m: 71 Table 7. Levels of 45Ca2+-bound soluble proteins reacting to the goat antibody preparation against bovine calmodulin found in the lysed synaptosomal preparations from the susceptible and kdr-resistant insects. Ca45 bound (dprnl70 ug proteins) Species Susceptible strain Resistant strain B. germanica 107.7 + 12.3 135.6 + 17.5 M. domestica 480.3 + 92.8 407.2 + 112.8 In each case control was taken as 100 72 Table 8. Levels of 3H--1R-de1tamethrin bound soluble proteins reacting to the goat antibody preparation against bovine calmodulin found in the lysed membrane preparations from the susceptible and kdr-resistant German cockroach. 3H-lR-deltamethrin (dpml70 ug proteins) Treatments Susceptible strain Resistant strain Control (without antibody) 11286 i- 1260 9389 i 228 Addition of antibody 15146 i 1994 12153 :t 375 .— 73 Fig. 6. Radioautogram of 5% SDS-PAGE of 32P-phosphoproteins from the lysed membranes of the susceptible (lanes 1-4) and the resistant (lanes 5-8) German cockroaches. The proteins were labeled with 7-32P-ATP and the endogenous CCPK in the presence of 0.3 mM Ca2+. Lane (1) control, (susceptible) without exogenously added bovine calmodulin, (2) 21.3 nM calmodulin (3) 21.3 nM calmodulin and lR-deltamethrin 10'3M (4) 21.3 nM calmodulin and ROS—4864 10'5M, (5) control (resistant) without exogenously added bovine calmodulin, (6) 21.3 nM calmodulin, (7) 21.3 nM calmodulin and IR- deltamethrin 10'3M, (8) 21.3 nM calmodulin and ROS-4864 10‘5M. Note that the bands appearing at 260 Kd most likely represent the a-subunit of the sodium channel. Exogenously added bovine calmodulin clearly increased the level of phosphorylation on this protein and other proteins. 74 <260 <205 ‘97 ~45 Fig.6- DISCUSSION The results of the current study indicate that there is an intrinsic difference between the kdr-resistant insects and the susceptible counterparts in the properties of the nervous system to respond to agents that affect Ca2+ binding and/or transporting processes. Such an observation agrees well with the previous study results from this laboratory that these kdr-resistant insects show cross-resistance in vivo to many agents which are known to affect calcium homeostasis (Ghiasuddin et al., 1981; Rashatwar and Matsumura, 1985; Rashatwar et al., 1987). Among the agents, the one which elicited the largest degree of cross-resistance in both B. germanica and M. domestica was A23187, a calcium ionophore. The action of this agent in the synapse is to increase the intracellular concentration of Ca2+ in the presynaptic terminal resulting in activation of CCPK of which action absolutely requires the participation of calmodulin(Browning et al., 1985). Earlier it was shown that DDT and several pyrethroids are potent inhibitors of calmodulin itself (Rashatwar and Matsamura, 1985). These workers used phosphodiesterase as a bioassay tool for the functional form of bovine calmodulin, and concluded that inhibition of calmodan dependent phosphodiesterase activity by these pesticides is totally explainable on the basis of their interaction with calmodulin, and not with the enzyme proper. Thus, if we assume that the major site of action of these agent is calmodulin, the reason why a modification on this peptide (e.g. to bind less with these pesticides) should become a selective advantage to those insects. The evidence accumulated in the current study that there are some qualitative differences in calmodan between the kdr and the corresponding susceptible strain in both species is supportive of the above hypothesis. It is also logical to assume that there must 75 76 be some interstrain difference in the property of the calmodulin binding site of CCPK and other calmodulin-requiring systems. Both bovine and S calmodulin were less effective in stimulating R—CCPK than S-CCPK. The double reciprocal plotting (Lineweaver—Burke plot) of the data shown in Fig. 4 revealed that bovine calmodulin stimulation lines for S and R do not intersect at the same point on the Y-axis, rather both intersecting at a point on the X-axis, indicating that the R-CCPK probably has an equivalent number of calmodulin binding sites as the S-CCPK, but the affinity of the former to bovine calmodulin is much lower than that of the latter. One major question we could not answer adequately in the current study was the relationship between calmodulin and the sodium channel. That is, since it is well recognized that the sodium channel is the main site of attack by DDT and pyrethroids (see Narahashi, 1985), and since the kdr insects have been shown to show cross-resistance to agents affecting sodium channels (Chang and Plapp, 1983; Rashatwar et al., 1987), it is expected that the sodium channels of the kdr insects are qualitatively or quantitatively different from those found in the susceptible counterparts. If one accepts the geneticists' analyses that kdr is a single gene conferring the DDT/pyrethroid resistance (Milani and Travaglino, 1957; Farnham, 1973), it is difficult to reconcile with the idea that there are at least two differences (i.e. calmodan and the sodium channel) between the resistant and the susceptible insect. One possibility is that calmodulin also participates in the operation of sodium channels in a certain capacity. Matsumura and Clark (1985) have observed some effect of calmodulin in the operation of isolated sodium channel incorporated into artificial liposomes. Also, the (at-subunit of the sodium channel containing the central channel is a good substrate for various protein kinases (Costa and Catterall, 1984). Furthermore, we have previously shown that the rat brain sodium channel (oz-subunit) is phosphorylated by endogenous protein kinases of which activities are elevated by increasing intracellular Ca2+, and that such biochemical process is inhibited by lR-deltamethrin and DDT. (Ishikawa et 77 al., 1989). However, the functional meaning of phosphorylation of the a—subunit is not totally understood. Moreover, so far all the evidence for participation of calmodulin on its operation is indirect. Therefore, this aspect requires much more in depth studies to prove or disprove such a relationship. Another possibility, entirely different from the above, is that the kdr insects have a much higher "resting" level of internal and external Ca2+ concentrations than the susceptible counterparts. If such is the case, all neural components which are normally controlled by changing concentration of calcium must reduce their affinities and sensitivities towards Ca2+. Since externally applied Ca2+ is known to antagonize actions of both DDT (Matsumura and Narahashi, 1971) and pyrethroids (Gammon, 1979) the above change, if true, could explain the reduced sensitivity of the nervous system of the kdr insects to these pesticides. However, at this stage, we do not have a direct evidence indicating the above change in the kdr insects. In conclusion, we have shown that there are qualitative differences between calmodulins found in the kdr insects and those of the susceptible counterparts in two insect species. There appears to be also similar interstrain differences in calmodulin binding properties of CCPK occurring in concert with the above change. REFERENCES Ahmad Z., DePaoli-Roach A.A. and Roach R]. (1982) Purification and characterization of a rabbit liver calmodulin-dependent protein kinase able to phosphorylate glycogen synthase. J. Biol. Chem. 257, 8348-8355. Breer H. (1981) Characterization of synaptosomes from the central nervous system of insects. Neurochemistry International, Vol. 3, No. 2, pp. 155-1963. Browing M.D., Huganir R. and Greengard P. (1985) Protein phosphorylation and neuronal function. J. Neurochem. 45, 11-23. Chang CR, and Plapp F.W. Jr. (1983) DDT and pyrethroids: receptor binding in relation to knockdown resistance (kdr) in the housefly. Pestic. Biochem. Physiol. 20, 86-91. Cheung W.Y. (1980) Calmodulin plays a pirotal role in cellular regulation. Science 207, 19-27. Costa M.R.C. and Catterall W.A. 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(1986) On inhibitory action of DDT and pyrethroids on ATP-utilizing, calcium transporting system in the neuronal tissues. In membrane receptors and enzymes as targets of insecticide action. 79: Clark, J.M. and F. Matsumura (eds.), Plenum Press, N.Y., pp. 173-178. Matsumura F. (1988) Deltamethrin induced changes in choline transport and phosphorylation activities in synaptosomes from the optic lobe of squids, Loligo pealei. Comp. Biochem. Physiol. 89C, 179-183. Matsumura F. and Clark J.M (1985) Investigations on the suitability of using nerve membrane fragments incorporated into artificial liposomes and a method for the study of pesticidal action on sodium channel activity. Neurotoxicology 6, 271-288. Matsumura F. (1971) Studies of biochemical mechanism of resistance in strains of the German cockroach. Proc. 2nd Pesticide Chem. Congr. 2, 95-116. Matsumura F. and Narahashi T. (1971) ATPase inhibition and electrophysiological change caused by DDT and related neuroactive agents in lobster nerve. Biochem Pharmacol. 20, 825-837. Miller T.A., Saglado V.L. and Irvings SN. (1983). The kdr factor in pyrethroid resistance. In: Pest Resistance to pesticides (G.P. Georghiou and T. Saito, Eds.), pp. 353-366, Plenum Press, New York. Milani R. and Travaglino A. (1957) Ricerche genetiche sulla resistanze a1 DDT in Musca domestica concatenazione del gene kdr (knockdown resistance) con due mutanti morfologigi. Riv. Parasitol 18, 199. Nestler E.J. and Greengard P. (1984) Protein Phosphorylation in the nervous system. PP. 1-382, John Wiley & Sons, New York. Naim A.C., Hugh C., Hemmings JR. and Greengard P. (1985) Protein kinases in the brain. Ann. Rev. Biochem. 54, 931-976. Nolan J., Roulston WJ. and Wharton RH. (1977) Resistance of synthetic pyrethroids in a DDT-resistant strain of Boophilus microplus, Pestic. Sci. 8, 484-486. Portzehl H., Caldwell RC. and Ruegg J .C. (1964) The dependence of contraction and relaxation of muscle fibers from the crab Maja squinado on the internal of free calcium ion. Biochem. Biophys. Acta 79, 581-591. 80 Prasittisuk C. and Busvine JR. (1977) DDT-resistant mosquito strains with cross- resistance to pyrethroids. Pestic. Sci. 8, 527-533. Priester T.M. and Georghiou GP. (1978) Induction of high resistance to permethrin in Culex pipiens quinquefasciatus. J. Econ. Entomol 71, 197-200. Rashatwar S., Ishikawa Y. and Matsumura F. (1987). Difference in calcium sensitivities of the sodium transporting systems in the nervous system of susceptible and kdr-resistant houseflies, musca domestica L. Comp. Biochem. Physiol. Vol. 88C. 165-170. Rashatwar S. and Matsumura F. (1985) Reduced calcium sensitivity of the sodium channel and the sodium Na“‘/Ca+ exchange system in the kdr-type, DDT and pyrethroid resistant German cockroach, Blattella germanica. Comp. Biochem. Physiol. 81C, 97- 103. Salgado V.L., Irving SN. and Miller TA. (1983) Depolarization of motor nerve terminals by pyrethroids in susceptible and kdr-resistant houseflies. Pestic. Biochem. Physiol. 20, 100-114. Sharma R.K. (1982) Cyclic nucleotide control of protein kinases. Prog. Nucleic Acid. Res. Mol. Biol. 27, 233-288. Sawicki RM. (1973) Recent advances in the study of the genetics of resistance in the housefly, Musca domestica. Pestic. Sci. 4, 501-512. Scott LG. and Matsumura F. (1983) Evidence of two types of toxic actions of pyrethroids on susceptible and DDT-resistant German cockroaches. Pestic. Biochem. Physiol. 19, 141-150. Scott J.G., Croft B.A. and Wagner SA. (1983) Studies on the mechanism of permethrin resistance in Amblyser'usfallacis (Acarina: Phytoseildae) relative to previous insecticide use on apple. J. Econ. Entomol. 76, 6-10. Stull J .T., Nunnally M.H. and Michnoff CH. (1986) Calmodulin-Dependent Protein Kinases. In The Enzymes. Vol. XVII 114-159. (P.D. Boyer, Eds.), Vol. XVH. pp. 113-159, Academic Press. Telford J.N. and Matsumura F. (1970) Dieldrin binding in subcellular nerve components of cockroaches. An electron microscopic and autoradiographic study. J. Econ. Entomol. 63, 795-800. Tsukamoto M., Narahashi T. and Yamasaki T. (1965) Genetic control of low nerve sensitivity to DDT insecticide-resistant houseflies. Botyukagaku. 30, 128-132. Walaas S.I., Nairn A.C. and Greengard P. (1983). Regional distribution of calcium and cyclic AMP-regulated protein phosphorylation systems in mammalian brain. Particulate Systems. J. Neurosci. 3, 291-301. CHAPTER III MODIFICATION OF PHOSPHORYLATION ACTIVITIES ON RAT BRAIN SODIUM CHANNEL BY PYRETHROIDS AND DDT 81 ABSTRACT The effects of pyrethroids and DDT on the or-subunit protein of the rat brain sodium channel were studied by using both native and exogenously added CAMP dependent protein kinases. For this purpose the sodium channel was partially purified using the method of Hartshome and Catterall (1984), and 32P-phosphorylated using 7-32P-ATP and exogenously added catalytic subunit of CAMP-dependent protein kinase. By comparing phosphorylation patterns of the partially purified with unpurified (i.e., intact synaptosomes) preparations using SDS-PAGE, it was concluded that the or-subunit of the voltage-sensitive sodium channel protein is the only 260Kd phosphorylatable protein present in intact and lysed synaptosomal preparations. Phosphorylation of the tit-subunit was induced by depolarization, and this process was inhibited by 10'6 to 10'10 M concentrations of the biologically active lR-deltamethrin, but not by IS-deltamethrin, the biologically inactive enantiorner of deltamethrin. DDT produced a similar effect, but only at 10'5 M concentration. Lysed synaptosomal membranes, were used to study the direct effects of DDT and deltamethrin on the Phosphorylation state of the a-subunit, which were similar to the effects produced by depolarization of intact synaptosomes. 82 INTRODUCTION The voltage sensitive sodium channels play a central role in the generation of an action potential, which is associated with the sodium current in a wide variety of electrically excitable tissues such as nerve, muscle and heart (Costa et al., 1982). There are three separate neurotoxin receptor sites on the sodium channel that have been well characterized. Neurotoxin receptor site 1 binds tetrodotoxin ('ITX) and saxitoxin (STX) which block the entry of sodium through the sodium channel (Ritchie and Rogart, 1977; Hartshome and Catterall, 1981; Catterall, 1976). Neurotoxin receptor site 2 binds the toxins verauidine, batrachotoxin, aconitine and grayanotoxin, which induce steady inward of sodium current (depolarization) by opening sodium channels and preventing inactivation leading to persistent activiation (Catterall, 1980). Neurotoxin receptor site 3 binds the polypeptide toxins of the North African scorpoins, Leiurus quinquestriatus and Androctonus austrialis as well as sea anemone toxins from Anemonr'a sulcata. These toxins act synergistically with neurotoxin receptor site 2, by inhibiting inactivation of voltage sensitive sodium channels causing persistent activation (Catterall, 1980). Similarly, a synergy of action has been demonstrated by electrophysiological and sodium influx techniques, between veratidine or batrachotoxin or grayanotoxin and pyrethroids as well as between polypeptide neurotoxins and pyrethroids (Jacques et al., 1980). These workers have shown that there exist receptor sites for pyrethroids on the sodium channel and that they are distinct from the other toxin receptors. The analysis of the polypeptides present in the sodium channel from rat brain revealed a major component with Mr~260,000 and that has been designated as the 0t- subunit. In rat brain preparations there are two additional major components b1 with 83 f" 84 Mr~39,000 and b2 with Mr~37,000, which with the tat-subunit constitute about 90% of the total Na channel protein (Hartshome et al., 1984). It has been shown by Costa et al., (1982); and Costa and Catterall (1984), that the a—subunit of the sodium channel from rat brain is rapidly and selectively phosphorylated by cAMP-dependent protein Kinase, in both lysed and intact synaptosomes and that such activities are influenced by agents which are known to affect sodium channel operations. Pyrethroids are one of the major classes of insecticides with high insecticidal potency and relatively low toxicity to mammals and therefore are successfully marketed (Elliot, 1977). One of their major action site appears to be the sodium channel (Lund and N arahashi, 1982; Yamamoto etal., 1983; Vijverberg and deWeille, 1985; Narahashi, 1985). These insecticides induce a marked prolongation of the sodium permeability during the action potential, leading to an increased negative-after potential which can be accompanied by repetitive after discharges. It has been pointed out that DDT acts in a similar manner on the sodium channel, particularly on the sensory neurons, where characteristic repetitive discharges are pronounced by both groups of chemicals (Lund and Narahashi, 1983; Lund, 1985). Jacques et al., (1980) showed that in mouse neuroplastome cells pyrethroids by themselves do not stimulate a discernible 22Na+ entry through the sodium channel but can stimulate ”Na" entry when used in combination with specific toxins for the gating system of the sodium channel, like batrachotoxin, veratridine dihydrograyanotoxin II or polypeptide toxins like sea anemone and scorpion toxins Meanwhile, our research team has shown that DDT and some pyrethroids affect membrane protein phosphorylation processess (Matsumura 1986, 1987). In view of their potent actions on the sodium channel and on protein kinases, we have examined their effects on phosphorylation of the a—subunit of the rat brain sodium channel. MATERIALS AND METHODS Chemical 8-Bromo-cyclic-AMP (8-Br—cAMP), veratridine, CAMP, A23187, and cAMP dependent protein kinase (holoenzyme and catalytic subunit), were obtained from Sigma Chemical Co. (St. Louis). 'y-32P-adenosine triphosphate (3,000 Ci/mmol was purchased from Amersham. Sea anemone toxin II (ATX-II) were from Calbio- chem Co. DEAE- sephadex and WGA-sepharose CL-4B were products of Pharmacia Inc. Deltamethrin (1 R-deltamethrin) and its inactive enantiomer IS-deltamethrin were generous gifts from ROUSSEL UCLAF. All other chemicals were commercial products with highest purity available. Preparation of synaptosomal fracu'on (P2) The P2 fraction was obtained using basically the method of Gray and Whittaker (1962). The rat brain was homogenized in a teflon-glass homogenizer rotated at 1,000 rpm in 20 ml 0.3 M sucrose, containing 5 mM sodium phosphate buffer pH 7.4, and 0.1 mM phenylmethyl sulfonylfluoride (PMSF). Homogenate was centrifuged at 1,000 g for 10 min using Sorvall centrifuge with SS-34 rotor. Supernatant was saved and the pellet was resuspended in the same volume of buffer, rehomogenized and centrifuged at 1,000 g for 10 min. The two supematants were combined and centrifuged at 15,000 g for 30 min. The pellet was resuspended in 8 ml of high Na buffer (140 mM NaCl, 5 mM KCl, 3 mM CaClz, 1.5mM MgClz, 10mM glucose, 20mM Hepes/Tris pH 7.4). 85 86 Fig. A. The chemical structures of the chemicals used for this study “*2 N N CI > N N 0 o ‘o—p—o OH H O Adenosine 3':5'- monophosphate NH2 “HEM k” N 0 o ‘o—p—o on ll 0 8-Bromoadenosine-3',5'- monophosphate NH; N \ N> 'k t i” t" " " o— t;— o— fi—o—fi— o 0 O O O OH OH Adenosine triphosphare (ATP) 87 Fig. A. Continued ocn3 C1130 o H .0" Br BF "’1 “elk OX0 0\0 H 1R- deltamethrin Br cc:3 DDT {HG cc:2 DDE 88 Phosphorylation by endogenous ATP and protein kinases Intact P2 fraction in high Na buffer was first incubated at 30°C for 30 min to equilibrate the phosphorylation-dephosphorylation process within the synaptosomes. To a microcentrifuge tube (1.5 ml), 250 pl of either high Na+ buffer (for non-depolarization condition) or high K+ buffer (for depolarization condition, 5 mM NaCl, 140 mM KCl, 3 mM CaClz, 1.5 mM MgC12, 20 mM Hepes/tris pH 7.4) was placed and chemicals to be tested were dissolved into this buffer. Two hundred and fifty ul of P2 fraction in high Na buffer was added to the tube to start the reaction, and after different incubation times (15 sec to 5 min) at room temperature (25°C), 50 ul, 1% SDS (final conc. 0.1%) was added, vortexed vigorously and boiled immediately for 2 min to stop all enzymatic reactions. For 8-Br-cAMP tests, P2 fraction, which had been preincubated as above, was incubated with this cyclic nucleotide analog at 30'C for an additional 10 min. After cooling, samples were centrifuged at 16,000 g for 5 min, and 500 ul of the supernatant was taken out and used for partial purification by DEAE sephadex and/or WGA sepharose column chromatography. For the application of this sample to WGA sepharose columns, Triton X-100 was added to a final concentration of 1% and then diluted ten times with buffer prior to application. DEAE-sephadex chromatography DEAE sephadex columns (0.5 ml) were made in 1 ml tuberculin syringe barrels and equilibrated with buffer A (100 mM KCl, 20 mM histidine/HCI pH 6.5, 0.1% Triton X- 100). Samples were mixed with the same volume of 50 mM histidine/HCI, pH 6.5, applied to the columns, washed with 10 ml of buffer A and eluted with 2 ml 400 mM KCl in buffer A. The eluate was either concentrated by ultrafiltration with Centricon 30 89 (Amicon Co., M.W. cutoff 30,000 Kd) according to the method provided by the company, or applied to WGA sepharose column directly. WGA-sepharose column chromatography WGA-sepharose (0.3 ml) was packed in 1 ml tuberculin syringe barrel, equilibrated and washed with 30 ml 0.15 M NaCl, 0.1% Triton X-100, 50 mM Hepes/I‘ris buffer pH 7.4. Samples were loaded and washed with 20 ml of the same buffer, and then eluted with 2 ml 100 mM N-acetylglucosamine in the same buffer. Eluate was concentrated by ultrafiltration with Centricon 30 and rephosphorylated as described below. Rephosphorylation by exogenous CAMP dependent protein kinase To the concentrate from Centricon 30 (Ca. 40 ul) was added 10 pl of 100 11M CAMP, 50 mM MgC12, and 30 ml solution of CAMP dependent protein kinase (=PKA, 15 ug). A 20 ul aliquot of y-32P-ATP solution (7 uCi in distilled water) was added to start phosphorylation and incubated at 30°C for 10 min. The reaction was stopped by the addition of 80 ul electrophoresis sample treatment buffer (0.125 M Tris/HCl pH 6.8, 4% SDS, 20% glycerol, and 10% mercaptoethanol, with bromphenol blue) and boiled for 2 min. The sample was directly applied to the electrophoresis without further treatment. Purification of the sodium channel from rat brain The sodium Channel from rat brain was purified by a combination of ion exchange chromatography (using DEAE-sephadex A-25-120), hydroxylapatite Chromatography (using Bio-Gel HTP) and lectin chromatography (using WGA-Sepharose) according to the method of Hartshome and Catterall (1984) with some slight modifications. 90 Brain membrane preparation Thirty rat brains were homogenized in a teflon- glass homogenizer rotated at 1000 rpm in 20 ml (per rat brain) of 0.32 M sucrose, 5 mM Tris/HCl pH 8.4, 0.1 mM phenylmethyl sulfonylflouride (PMSF), 1 mM phenanthroline, 1mMIodoacetamide and 1 mM pepstatin A. The four protease inhibitors were used to minimize the possibility of proteolytic degratation of the sodium Channel during its purification. Homogenates were centrifuged at 700 g for 10 min. using Sorvall centrifuge with SS-34rotor. The supematants were saved and the pellet was resuspended in 15 ml of the same solution per rat brain, rehomogenized and centrifuged at 700 g for 10 min. The two supematants were combined and centrifuged at 27,000 g for 40 min. The pellet (from each rat brain) was resuspended in 33 ml of 5mM T ris/HCl pH 9.2, 1 mM EDTA, and the four protease inhibitors, incubated on ice for 15 min and homogenized with glass Teflon-homogenizer. The lysed membrane homogenate was centrifuged at 27,000 g for 40 min and the pellet was resuspended in a final volume of 120 ml of solution containing 10mM HEPES/I‘ris pH 7.4, 200 mM KCl, and the four protease inhibitors. The resuspended pellet was stored at - 80'C for no longer than a month and used for purification. Solubilization of sodium channel A solution containing 10 mM HEPES/I‘ris pH 7.4, 5% Triton X-100, 0.5% phosphatidyl Choline, and the four protease inhibitors was added slowly in small increments to the resuspended lysed membrane of thirty rat brains, with constant mixing to yield 240 ml. The composition of this mixture was the following: 10.9 mg/ml membrane protein, 10 mM HEPES/Tris pH 7.4, 100 mM KCl, 2.5% Triton X-100, 0.25% phosphatidylocholine and the four protease inhibitors. This solution was stirred for 15 min 91 (0-4 °C) and centrifuged at 120,000 g for 50 min using 70 Ti rotor. The supernatant containing the soluble sodium channel was saved and kept at 0-4 'C for further purification. Ion exchange chromatography DEAE- Sephadex A-25- 120 was equilibrated with 120 mM KCl, 20 mM Histidine HCl/I'ris pH 6.5, 10 mM CaClz, 0.1% Triton X-100, and 0.025% phosphatidyl choline, and was packed (100 ml bed volume) in a 2.8 X 20 cm column. The resin was washed with 200 m1 (2 bed volumes) of equilibration buffer. For this purification step the membrane extract was adjusted to 10 mM CaC12 to improve stability, (Catterall et al., 1979), and the pH was lowered to 6.5 by the addition of 0.5 M histidine HCl/I‘ris pH 5.0. The membrane extract was loaded and was washed with 400 ml (4 bed volumes) of equilibration buffer, and then eluted with 200 ml (2 bed volumes) of 250 mM KCl, 20 mM Histidine HCl/I‘ris pH 6.5, 10 mM CaClz, 0.1 % Triton X-100, and 0.025% phosphatidyl choline. Hydroxylapatite chromatography Bio-Gel HTP was equilibrated with 250 mM KCl, 80 mM KH2PO4 pH 7.4, 0.1% Triton X-100, and 0.025% phosphatidyl choline and was packed (100 ml bed volume) in a 2.8 X 20 cm column. The resin was washed with 200 ml (2 bed volumes) of equilibration buffer. The pH of the eluate from DEAE-Sephadex was adjusted to 8.0 by the addition of 2 M Tris/HCl pH 8.9. The calcium was chelated by the addition of 100 mM disodium EDTA pH 8.0 to a final concentration of 11 mM. This step lowers the pH to approximately 7.4. 700 mM 1(1-12P04/KOH pH 7.4 was added to give a final concentration of 80 mM. The adjusted DEAE Sephadex eluate was applied and the column washed with 400 ml (4 92 bed volumes) of equilibration buffer and then eluted with 200 ml of elution buffer 100 mM KCl, 400 mM KHzPH4/KOH pH 7.4, 0.1% Triton X 100, and 0.025% phosphatidyl choline. Lectin chromatography WGA- Sepharose (6 ml) was packed in a 0.9 X 10 cm column, equilibrated and washed with 150 ml (25 bed volumes) of equilibration buffer 0.5 mM KCl, 20 mM HEPES/Tris pH 7.4, 0.1% Triton X-100 and 0.025% phosphatidyl choline. The eluate from the hydroxylapatite column was applied to WGA- Sepharose column and run through it at the rate of 1.0 ml/min. The resin was washed with 500 ml (85 bed volumes) of equilibration buffer and 10 more ml of the same buffer containing 10 mM CaClz. The column then was eluted with 30 ml (5 bed volumes) of 0.5 M KCl., 20 mM HEPES/Tris pH 7.4, 10 mM CaClz, 50 mM N-Acetyl glucosamine, ) 0.1% Triton-X and 0.025% phosphatidyl choline at the rate of 1 ml/min. The eluate was concentrated by ultrafiltration with centricon 30 (Amicon Co., M.W. cutoff 30,000) according to the method provided by the company. The purified fraction from lectin Chromatography phosphorylated and analyzed by SDS-PAGE gel electrophoresis. Electrophoresis and autoradiography SDS-polyacrylamide gel electrophoresis was developed according to the method of Laemli (1970). Protean H electrephoresis apparatus (Bio-Rad Lab.) with 1.5mm spacers, 5% separating gel, and 3% stucking gel were used. Other standard conditions used were identical to the ones supplied by the manufacturer. The gels were stained with Coomassie brilliant blue R-250, destained, dried over thick filter paper using a vacuum drier (Hoefer Scientific), and autoradiograms developed using x-ray films (Kodak X-omat AR-S). 93 Protein standards used. Myosin 205,000, b-galactosidase 116,000, phosphorylase b 97,400, Bovine albumine 66,000, and egg albumin 45,000. Radioiodination of Sea Anemone toxin 1] (ATX-II) ATX-II from Anemonia sulcata was radioiodinated as follows: toxin (10 pg in 100 pl 20 mM sodium phosphate buffer pH 7.0) and a IODO-BEAD (Pierce Co.), which had been prewashed with the phosphate buffer, were placed in a microcentrifuge tube and 1 mCi Na1251 was added. After 20 min incubation at room temperature, the liquid was transferred into a dialysis bag (M.W. cutoff 1,000, Spectra-Por 7), and dialysed against 20 mM sodium phosphate buffer pH 7.0 for 2 days with 4 changes of the buffer. Specific activity obtained was 1.6 Ci/mmol. ATX-II Coupling 1251-ATX-II was covalently coupled to sodium channel using disuccinimidyl suberate (DSS) as a crosslinker according to the method of Barhanin et al.,(1983) and Vincent et al., (1980). Synaptosomes (1.0 mg/ml, 200 ml) were prepared in Na-free buffer with 0.1% bovine serum albumin (BSA) (140 mM choline chloride, 5 mM KCl, 3 mM CaClz, 1.5 mM MgC12, 20 mM Hepes/tris pH 7.4 0.1% BSA), and incubated with 300,000 Cpm 125I-ATX-H and 100 trM veratridine for 40 min on ice. Synaptosomes were sedimented by centrifugation (30 sec, 16,000 g), washed once in the Na-free buffer without BSA, and resuspended at the initial concentration. DSS (7 mM in dimethyl sulfoxide) was added to a final concentration of 0.07 mM and the mixture was incubated for 15 min on ice. Glycine (20 mM) was added to neutralize excess crosslinker and the synaptosomes were sedimated by centrifugation and washed twice in the Na-free buffe. 94 32P-phosphorylation of Lysed Synaptic Membranes The effects of pesticides on the phosphorylation process in lysed synaptic membrane were studied by incubating 500 111 of membrane suspension containing 1.7 mg protein in 5 mM Tris/HCl buffer (with 1 mM EDTA, 1 mM EGTA and 0.1 mM PMSF) with a pesticide (added directly to each glass test tube already containing the membrane with 1 ul ethanol) for 5 min at 4'C, 7-32P-ATP (6 pCi per tube final conc. 6.7 nM) was added, incubated for l min at the same temperature, and the reaction was stopped using 50 ul of 1% SDS and boiling for 1 min. After cooling to O'C, 55 pl of 10% Lubrol PX was added, the system was incubated for 30 min to further solubilize the membranes, and centrifuged at 16,000 g for 5 min and 500 1.11 of the supernant was used for the electrophoresis experiment (see Fig. 5). RESULTS To verify the identity of the phosphorylated sodium channel we have first repeated the experiment of Costa et a1. (1982). For this purpose the rat brain synaptosomes were treated in an identical manner as described by Hartshome and Catterall (1984) to the step of WGA sepharose chromatography. The resulting, partially purified sodium channel was phosphorylated using 7 -32P-ATP in the presence and the absence of exogenously added catalytic subunit of CAMP-dependent protein kinase (PKA). The results of electrophoresis and radioautographic experiments indicate that there is only one 32P—labeled band with molecular weight corresponding to 260 Kd in the lane (Fig. 1). To ascertain that this phosphoprotein is the sodium channel, the intact synaptosomes were first reacted with 125I-ATX-II toxin, Chemically cross-linked, solubilized and the resulting soluble proteins were purified on sephadex column and analyzed on SDS polyacrylamide gel-electrophoresis. The 125I-ATX-II linked protein was detected using radioautography. The 125I-labeled band position was found to coincide with the 32P-labeled sodium channel, (Fig. 2). Effects of depolarization of the freshly prepared intact synaptosomes on the level of phosphorylation of the sodium channel were studied next. This was done by first depolarizing the synaptosomal membrane using high K+ or veratridine, stopping with SDS and, after dilution with Triton X-100 to reduce the protein denaturing effects of SDS, reacting with 'y-32P-ATP, CAMP and PKA as described by Costa and Catterall (1984). In this approach the proteins which were not phosphorylated at the time of phosphorylation by endogenous protein kinases appear as 32P- labeled band, while those phosphorylated by the endogenous kinases during depolarization do not go through 32P—phosphorylation. As 95 96 Fig. l. Radioautogram of 5% SDS polyacrylamide gel electrophoresis (PAGE) of partially purified sodium channel labeled with 7-32P-ATP and the catalytic subunit of CAMP dependent protein kinase. Lane (1) intact synaptosomes treated identical manner as 2, without 8-Br-CAMP treatment, (2) intact synaptosomes first treated with 8-Br-CAMP, lysed using 0.1% SDS, diluted with 1% Triton X-100 and 32P-phosphorylated using CAMP, CAMP-dependent protein kinase and 7-32P-ATP, (3) 32P-phosphorylated sodium Channel purified from lysed membrane according to Hartshome and Catterall (1984) up to the wheat germ affinity column step and 32P-phosphorylated using catalytic subunit of CAMP- dependent protein kinase and 7-32P-ATP, (4) same as 3 treated with luM lR- deltamethrin (84.4% inhibition of phosphorylation on the sodium channel) and (5) same treated with 0.1 mM veratridine (97.8% inhibition). 97 -