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Effect of insecticidal cyclic phosphorothionates on adenylate cyclase and phosphodiesterase
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
38, 186-195 (1%)
Effect of Insecticidal Cyclic Phosphorothionates Cyclase and Phosphodiesterase AKINORI
HIRASHIMA,KAZUHIKO
Department of Agricultural
on Adenylate
OYAMA, AND MORIFUSA ETO
Chemistry, Kyushu University, Fukuoka 812, Japan
Received June 6, 1990; accepted July 31, 1990 Octopamine (0.1 and 1 mM) stimulated the adenylate cyclase prepared from Periplaneta americana ventral nerve cords (615 and 1112% relative to the control). D-( -)-ZAmino-l-phenylethanol (APE) was more potent than 2-amino-l-(4-fluorophenyl)ethanol (an octopamine agonist) and L-(+)-APE in stimulating the adenylate cyclase. 2-Methoxy-S-phenyl-1,3,2-oxazaphospholidine 2-sulfide (5PMOS) derived from DL-(+)-APE did not activate adenylate cyclase at 0.75 and 7.5 mM (91 and 95% relative to the control) but suppressed the octopamine (0.1 mM) potency to 268% at 1 m&f relative to the control. Salithion (Zmethoxy-4&1,3,2-benzodioxaphosphorin 2-sulfide) at 0.1 m&f, fenitrothion (dimethyl 3-methyl-4nitrophenyl phosphorothionate), 2-amino-1-(2,3dimethoxy)phenylethanol, 5-PMOS, and other oxazaphospholidines at 1 m&f showed similar phenomena. l-Naphthyl-, 2-naphthyl-, 4-ethylphenyl-, and 4-isopropylphenyloxazaphospholidine derivatives at 0.1 mkf reduced the octopamine potency at 0.1 mM more severely than the octopan-&e-receptor antagonists fchlordimeform and cyproheptadine) at 1 mM. 5-(2,3-Dimethoxyphenyl)-2-methoxy-1,3,2-oxazaphospholidine 2-sulfide (Ki = 107.9 p&f), fenitrothion (Ki = 37.3 t&f), and 3-isobutyl-l-methylxanthine (Ki = 1.5 t&f) reduced the phosphodiesterase activity of beef heart in a competitive manner with respect to cyclic adenosine 3’,5’-monophosphate (CAMP). At 50 p&f, salithion, salioxon (2-methoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide), and other oxazaphospholidines reduced phosphodiesterase activity. Hence, D-(-)-APE could be an agonist, and 5-PMOS and salithion analogs and fenitrothion could be partial antagonists to the octopamine receptor. The increased level of whole-body CAMP of Musca domestica and Tribolium castaneum larvae treated with these phosphorothionates is due to reduction of phosphodiesterase activity. 0 1990 Academic Press, Inc.
rived from L-leucine by stabilizing the Since the insecticidal six-membered cy- structure through introduction of a nitrogen clic phosphorothionate salithion was dis- atom into the ring system (2). In oxazacovered in our laboratory (l), we have phospholidines, a hydrophobic substituent searched for new five-membered cyclic at the 4-position generally has enhanced the phosphorothionates and found insecticidal insecticidal activity more than that at the activities in 4-isobutyl-2-methoxy-1,3,2oxazaphospholidine 2-sulfide (iBMOS)’ deINTRODUCTION
’ Abbreviations used: iBMOS, 4-isobutyl2-methoxy-1,3,2-oxazaphospholidine 2-sulfide; 5PMOS, 2-methoxy-5-phenyl-1,3,2-oxazaphospholidine t-sulfide; APE, 2-amino-1-phenylethanol; CAMP, cyclic adenosine 3’,5’-monophosphate; 5RMOS, 5-substituted 2-methoxy-1,3,Zoxazaphospholidine 2-sulfide; AChE, acetylcholinesterase; KSCP, potassium saligenin cyclic phosphorothionate; EGTA, ethylene glycol bis(B-aminoethyl ether) N,N,N’,N’tetraacetic acid; ATP, adenosine 5’-triphosphate; GTP, guanosine 5’-triphosphate; IBMX, 3-isobutyl-1-methylxanthine; l-NMOS, 2-methoxy-
5-(1-naphthyl)-1,3,2-oxazaphospholidine 2-sulfide; DMOS, 5-(2,3-dimethoxyphenyl)-2-methoxy1,3,2-oxazaphospholidine 2-sulfide; AFPE, Z-aminol-(4fluorophenyl)ethanol; FMOS, 5-(4-fluorophenyl)2-methoxy-1,3,2-oxazaphospholidine 2-sulfide; ADPE, 2-amino-1-(2,3-dimethoxyphenyl)-ethanol; MMOS, 2-methoxy-S-(4-methylphenyl)-1,3,2-oxazaphospholidine 2-sulfide; 2-NMOS, 2-methoxy5-(2-naphthyl)-1,3,2-oxazaphospholidine 2-sulfide; EMOS, 5-(4-ethylphenyl)-2-methoxy-1,3,2-oxazaphospholidine 2-sulfide; iPMOS, 5-(4-isopropylphenyl)-2-methoxy-1,3,2-oxazaphospholidine 2-sultide; cGMP, cyclic guanosine 3’,5’-monophosphate; NSCP, sodium saligenin cyclic phosphorothionate.
186 0048-3575/90 $3.00 Copyright 0 1990 by Academic Press. Inc. AU rights of reproduction in any form reserved.
PHOSPHOROTHIONATES,
ADENYLATE
5-position, except when the substituent is a phenyl group (3). 2-Methoxy-5-phenyl1,3,2-oxazaphospholidine 2-sulfide (5 PMOS) derived from an octopamine analog, 2-amino-1-phenylethanol (APE), has been found to have a stronger insecticidal activity than salithion and iBMOS at lethal concentrations by topical application to Muscu domestica female adults (3-5). Dietary salithion and 5-PMOS have been found to reduce larval weight gain, reduce trehalase activity, and increase the level of whole-body CAMP of M. domestica (6-8) and Tribolium custaneum (7-9) at sublethal concentrations. Increased levels of CAMP could be due to either the activation of adenylate cyclase, an enzyme that catalyzes the biosynthesis of CAMP, or the inhibition of phosphodiesterase, an enzyme that catalyzes the hydrolysis of CAMP. In order to optimize these biological activities, numerous structural modifications were made, and in order to understand quantitatively the dependence of activity upon the substituent, the structure-activity relationships of salithion analogs and 5-substituted 2methoxy-1,3,2-oxazaphospholidine 2sulfide (S-RMOS) were analyzed (10, 11), including anti-AChE activity, M. domesticu insecticidal activity by topical application, and T. custuneum larval growth inhibitory activity, which could be due to perturbation of monoamine-related production of CAMP. However, the relationship of these biological phenomena, if any, has not been clarified yet. Hence, this paper deals with the effects of salithion and 5-RMOS on the adenylate cyclase in homogenates of ventral nerve cords of Peripluneta umericunu, the determination of the cause of the increased level of whole-body CAMP in M. domestica and T. castaneum larvae treated with these cyclic phosphorothionates, and the further elucidation of the effect of altered CAMP level on various physiologicd phenomena. MATERIALS
Chemicals.
AND
METHODS
Chlordimeform [N’-(Cchloro-
CYCLASE,
AND
PHOSPHODIESTERASE
187
o-tolyl)-l\r,N-dimethylformamidinel was prepared by reacting 2-amino-5-chlorotoluene with dimethylformamide in the presence of phosphorus oxychloride (12). L-( +)- and D-(-)-APES were obtained from L-( + )- and D-( -)-mandelic acid, respectively (4). Salithion (1), iBMOS (2), and other oxazaphospholidines (10) were synthesized by reacting methyl phosphorodichloridothionate with saligenin, Lleucinol (2-amino-4-methyl-1-pentanol), and the corresponding amino alcohols, respectively. Salithion enantiomers (>98% ee) were prepared by the proline ester method (13) and potassium saligenin cyclic phosphorothionate (KSCP) by demethylating salithion with potassium dimethyldithiocabamate (14). Salioxon was obtained by m-chloroperbenzoic acid oxidation of salithion (5). Fenitrothion (96.5% pure) was a gift from Sumitomo Chemical Co. Ltd. (Takarazuka, Japan). Octopamine [2amino-1-(4-hydroxyphenyl)ethanol], theophylline (1,3-dimethyixanthine), eserine (physostigmine), EGTA, and cyproheptadine [4-(SH-dibenzo[u,d]cyclohepten5-ylidene)-1-methylpiperidiene] were purchased from Nacalai Tesque Inc. (Kyoto, Japan), ATP, GTP, IBMX, and CAMP from Sigma Chemical Co. Ltd. (St. Louis, MO), 3’,5’-cyclic nucleotide phosphodiesterase (EC 3.1.4.17>, adenosine deaminase (EC 3.5.4.4), and alkaline phosphatase (EC 3.1.3.1) from Boehringer-Mannheim, GmbH Biochimica (Mannheim, West Germany), and RIA kit “Yamasa” (Code YSI7701) from Yamasa Shoyu Co. (Chiba, JaPan). Bioassay. The dietary effect of the test compounds on T. castaneum larval growth was measured according to a previously published method (5, 7-10, 15). Twelve fourth instar T. custaneum larvae weighing 1.O ~fr 0.1 mg each were introduced into each test vial of five replicates, along with 1.5-g portions of diet. The vials were held at 30°C for 2 days to determine weight gains and CAMP level. Measurement
of whole-body
CAMP
188
HIRASHIMA,
level. The level of T. castaneum
OYAMA,
wholebody cAMP was determined by using a radioimmunoassay according to a previously published method (7,8). Last instar T. castaneum larvae, 2.0 2 0.2 mg each, which had been fed on treated diet at 30°C for 2 days, were homogenized in cold 6% trichloroacetic acid at 4°C to give 10% (w/v) homogenate. The homogenate was centrifuged at 3000 rpm and 4°C for 10 min, the pellet being resuspended in the same buffer and recentrifuged. The combined supernatants were washed three times with 3 vol of diethyl ether saturated with water. To 100~plaliquots of the aqueous extract were added 5 pJ of triethylamine and 45 p,l of succinyl anhydride solution in dioxane (44 mg/ml). After the mixture was kept at room temperature for 10 min, 350 l.~lof 0.3 M imidazole buffer (pH 6.5) was added, and diluted to an appropriate concentration (1 mg of larvae/ml) with the buffer containing 1% triethylamine, 9% succinyl anhydride solution, and 20% distilled water. The CAMP level was determined with an aid of RIA kit “Yamasa” (Code YSI-7701). Adenylate cyclase assay. Ventral nerve cords of adult cockroaches (P. americana) were homogenized (15 mg/ml) in 6 mM Tris-maleate buffer (pH 7.4) using a chilled glass-Teflon Potter-Elvehjem tissuegrinding tube (16). This homogenate was diluted to a volume of 15 ml in 6 mM Trismaleate, and centrifuged at 120,OOOgand 4°C for 20 min. The supernatant was used for a phosphodiesterase source, the pellet being resuspended by homogenization in 15 ml of the buffer and again centrifuged at 120,OOOg and 4°C for 20 min. The resulting pellet (PZ fraction) was resuspended in a volume of 6 mM Tris-maleate equivalent to the starting amount. The adenylate cyclase activity was measured by Nathanson’s procedure under optimal conditions (16) in test tubes containing (in 0.3 ml) 80 mM Trismaleate @H 7.4), 10 mM theophylline, 8 mM M&l,, 0.1 m&f GTP, 0.5 mM EGTA, 2 mM ATP, 0.06 ml of P, fraction, and compound solutions to be tested, which were
AND
ET0
prepared (9) by dissolving the compounds in 0.5 ml of acetone, and then adding 2 drops (50 pg) of Tween 80 and 99.5 ml of water. Appropriate solvent controls were run in parallel. The enzyme reaction (5 min at 30°C) was initiated by adding ATP, stopped by heating at 90°C for 2 min, and then centrifuged at 1OOOgfor 15 min to remove the insoluble material. The CAMP level in the supernatant was measured by a radioimmunoassay (7, 8) and protein concentration was determined by the Lowry method (17). For reversibility studies (Table 2), the nerve cords (9 mg/ml) were first preincubated with 1 n&f IBMX in the presence or absence of 100 Q4 I-NMOS in cockroach saline at 30°C for 20 min, and centrifuged at IOOOgand 4°C for 10 min. The precipitate was homogenized in 1 ml of Tris-maleate buffer (pH 7.4), and centrifuged twice at 120,OOOgand 4°C for 20 min as mentioned above to remove soluble I-NMOS and produce P2 fraction. This fraction was assayed for adenylate cyclase activity in the absence or the presence of additional octopamine. Five replicates of 20 fourth instar T. castaneum larvae were treated with dietary 80 ppm of 5-PMOS or 6 ppm of DMOS at 30°C for 2 days. Acetone extracts were prepared (8) by homogenizing the feeding larvae in cold acetone (50 mg/ml), and the homogenate was centrifuged at 3000 rpm and 4°C for 10 min. The supematant fraction was diluted (10 mg/ml) with a solvent system including 0.5 ml of acetone, 99.5 ml of water, and 50 pg of Tween 80, and was used for adenylate cyclase assay as above. Phosphodiesterase inhibition assay. For the phosphodiesterase assay in homogenates of cockroach nerve cords, the adenylate cyclase assay was modified slightly by using the supernatant instead of P2fraction. The reaction mixture consisted of (in 0.3 ml) 80 mM Tris-maleate (pH 7.4), 8 mM MgCl,, 0.5 mJ4 EGTA, 0.06 ml of the supernatant fraction, and a compound solution to be tested. For the control, the su-
PHOSPHOROTHIONATES,
ADENYLATE
pernatant was heated at 90°C for 2 min before the reaction. The enzyme reaction (5 min at 30°C) was initiated by adding the compound solution, stopped by heating at 90°C for 2 min, and then centrifuged at 1OOOg for 15 min to remove the insoluble material. The CAMP level in the supernatant was measured as above. Beef heart phosphodiesterase activity was assayed according to the BoehringerMannheim procedure (18) with a slight modification. Test compounds in a O.l-ml suspension (acetone-water-Tween 80), prepared as above, were preincubated with 0.05 ml of phosphodiesterase (0.711 U/ml), 0.1 ml of magnesium sulfate (82 mM), 0.01 ml of adenosine deaminase (400 U/ml), 0.04 ml of alkaline phosphatase solution (I500 U/ml), and 2.7 ml of 0.1 M glycylglycine buffer (pH 7.5) at 25°C for 10 min. The remaining phosphodiesterase activity was determined by initiating the reaction with the addition 0.1 ml of CAMP (1.35 mM) to the preincubated mixture. The decrease in absorbance per minute (AA) at 265 nm was continuously recorded against a water blank containing test compound in E units at 25°C with a Shimadzu UV-2100 spectrophotometer and the activity was calculated by the equation v = 3.1 x AAl(e x 0.05 x 1) U/ml, where eZe5 = 8.1 mmol-’ liter cm-‘. The enzyme activity was linear up to 1 min and 80 kg protein/ml (1 U/ml). The concentration of the compound is expressed as that present in the preincubation medium. RESULTS
AND
DISCUSSION
Effect upon Octopamine-Sensitive Adenylate Cyclase
At 0.1 and 1 mM, octopamine has shown a maximum activity for activation of adenylate cyclase prepared from cockroach nerve cord (19-21), hemocyte (22-24), corpus cardiacurn (25), fat body (26), and brain homogenate (27). In our experiments, octopamine (0.1 and 1 mM) stimulated adenylate cyclase (615 and 1112% relative to the
CYCLASE,
AND
PHOSPHODIESTERASE
189
control) prepared from ventral nerve cords of P. americana (Table 1). The absence of the 4-hydroxy group substantially reduces potency: APE is over 10 times less potent than octopamine in stimulating adenylate cyclase activity in homogenates of cockroach brain (27). However, AFPE, an octopamine analog in which fluorine has been substituted for the 4-hydroxy group, is a potent agonist to octopamine receptor (27, 28). D-(-)-APE (0.1 and 1 mM) was more potent (337 and 380% relative to the control) than 0.1 mM AFPE (299% relative to the control) and L-( +)-APE (102 and 123% relative to the control) at 0.1 and 1 mM in stimulating adenylate cyclase (Table 1)) suggesting that response is stereoselective for the ~-(-)-isomer. This result agrees with previous reports of the agonistic potency of octopamine enantiomers and insecticidal activity of 5-PMOS optical isomers: naturally occurring I+( - )-octopamine is more potent than the L-( + )-enantiomer in stimulating adenylate cyclase (27); (R&Z,)and (&,S,)-5-PMOSs derived from D-( -)-APE have been more potent insecticides than (S&Z,)- and&S,)isomers prepared from the L-(+)-APE (4). Neither 5-PMOS derived from DL-(f)APE at 0.75 and 7.5 mM (91 and 95% relative to the control) nor FMOS derived from AFPE at 0.1 mM (83% relative to the control) activated adenylate cyclase. However, they suppressed the octopamine (0.1 mM) potency to 268% (S-PMOS) at 1 mM and 272% (FMOS) at 0.1 mM relative to the control, respectively (Table 1). Table 1 also shows that salithion, fenitrothion, 2,3dimethoxyphenyl analogs (ADPE and DMOS) of APE and 5-PMOS, and other oxazaphospholidines had a similar tendency. The acetone extracts of T. castaneum larvae treated with dietary 80 ppm of 5-PMOS or 6 ppm of DMOS did not stimulate the adenylate cyclase. These results suggest that D-(-)-APE and AFPE are agonists, but 5-PMOS and salithion analogs and fenitrothion are partial antagonists to octopamine receptor except the (4-methyl-
190
HIRASHIMA,
OYAMA, TABLE
AND
ET0
1
Nonadditive Effects of Octopamine and Various Compounds upon the Adenylate Cyclase Activity in Homogenates of Cockroach Nerve Cords Increase CAMP Additive Control Octopamine
Cone
AFPE ADPE 5-PMOS 1-NMOS ZNMOS
FMOS MMOS EMOS
iPMOS
DMOS Salithion Fe&o&ion Cyproheptadine
Eserine Acetone extract control 5-PMOS (SO ppm) acetone extract’ DMOS (6 ppm) acetone extract’
100
loo0 100 loo0 100 loo looo 750 7500 10 100 1 10 100 loo loo 1 10 100 1 10 loo 750 7500 loo loo 1000 1 10 loo loo0 100
Octopamine D-(-)-APE L-( + )-APE
AFPE DMPE 5-PMOS l-NMOS ZNMOS FMOS MMOS EMOS
(100 piU) 1ooo
loo0 loo 1ooo moo 100 100 100 100 100
(pmoUmin/mg of protein)
56 212 345 623 189 213 57 69 167 86 81 51 54 63 37 48 58 39 47 54 38 36 46 44 40 44 52 52 46 55 39 34 40 36 30 51 60 56 59
10 100 1000
D-(-)-APE L-( + )-APE
(p&f)
in the adenylate
+ Additive 202 3 14 197 174 150 54 61 153 305 69
(55-57) (199-226) (269-420) (598-648) (178-200) (205-220) (55-60) (48-90) (166-168) (57-l 15) (74-88) (49-53) (48-60) (62-64)
cyclase
activity”
Relative to the control 100 379 615 1112 337 380 102 123 298 154 144 91 95 113
(98-102) (355-403)’ (473-750)8 (1068-1157)h (311357) (366-393)f (98-107) (86-161pd (296-300) (102-205pd (132-157pd (88-95) (86-107) (111-114)
66,’
(48-48) (57-59) (36-42) (45-48) (52-55) (37-39) (36-36) (46-46) (42-47) (40-41) (42-47) (5G54) (48-56) (42-49) (53-58) (3-3) (33-34) w4 (30-41) (28-32) (49-52) (58-62) (48-63) (55-64)
85 104 70 83 96 68 63 82 79 72 78 92 92 82 99 70 60 72 63 53 91 107 100 105
(85-85)bsc (102-105) (64-75)b (80-86)b,C (93-98) (66-70)b (63-63)b (82-82)b*C (75-84)b.C (71-73)b (75-84)bnc (89-%) (86-100) (75-88)b,c (95-104) (64-77)b (59-61)b (71-73)b (54-73)b (50-57)b (88-93) (104-111) (86-l 13) (98-l 14)
(198-205) (250-378) (193-201) (155-192) (150-150) (424) (52-69) (142-W) (297-313) (65-72)
360 561 352 3 10 268 97 108 272 544 122
(354366)’ (W75)8 (345-359)’ (277-343) (268-268) (75-l 18) (93-123) (254-293) (53&559)8 (1 16-129pd
PHOSPHOROTHIONATES,
ADENYLATE
TABLE
CYCLASE,
AND
191
PHOSPHODIESTERASE
l-Continued Increase in the adenylate cyclase activitp
Additive iPMOS DMOS Salithion Fenitrothion Chlordimeform Cyproheptadine Eserine
Cone (piU)
CAMP (pmol/min/mg of protein)
Relative to the control
100 1000 100 1000 1000 1000 100
78 (77-79) 174 (16U81) 168 (168-168) 217 (168-265) % (91-101) 88 (87-88) 341 (329-353)
139 (13W4l)d -3 10 (296-323) 300 (300-300) 387 (300-473)’ 171 (163-180)d 156 (155-157)d 609 (588-630)8
Octopamine (10 Qf) + Additive 10 197 (188-208) 1 190 (182-198) 10 156 (156-156) 1 160 (152-167) 150 (148-153) 10 1 182 (176-189) 10 193 (193-193) 1 150 (148-153) 69 (66-72) 10 1 132 (132-132)
352 (336-371)’ 339 (325-354yf 279 (279-279) 2-NMOS 280 (27 l-298) 268 (264-273) EMOS 325 (31k338Y9 345 (345-345)’ iPMOS 268 (264-273) 123 (l&129)‘.’ Cyproheptadine 236 (236-236)d,’ ___.’ The adenylate cyclase activity of P. americana was measured according to Nathanson’s procedure and the CAMP levels were measured by a radioimmunoassay. The data are the average of duplicates, range values being shown in parentheses. U Differ significantly from each other at P = 0.05 according to Duncan’s multiple range test (33). ’ A single measurement. j The acetone extract of the treated T. castaneum larvae at 30°C for 2 days was obtained by homogenization in ice-cold acetone followed by centrifugation at 3000 rpm and 4°C. I-NMOS
phenyl)oxazaphospholidine derivative MMOS. L-(+)-APE, MMOS, and eserine (an AChE inhibitor) showed little agonistic and antagonistic effects. 1-Naphthyl- (l-NMOS), 2-naphthyl- (2NMOS), 4-ethylphenyl- (EMOS), and (4-isopropylphenyl)oxazaphospholidine (iPMOS) derivatives at 0.1 mM reduced the octopamine potency at 0.1 m&I in stimulating adenylate cyclase more severely than 1 mM chlordimeform, a partial antagonist to the octopamine-receptor in the nerve cord of P. americana (20), and cyproheptadine, a potent, if not the most potent (29, 30), antagonist to the octopamine receptor of P. americana nerve cord (20, 21), hemocyte (22, 24), and brain homogenate (27). However, at the lower concentrations of 10 and 1 PM the antagonistic effect of these oxazaphospholidines to octopamine receptor was not significant. Figure 1 shows the antago-
nistic effect of I-NMOS, fenitrothion, and cyproheptadine, all of which caused significant inhibition of basal adenylate cyclase activity at concentrations of lo-80 PM. l-
I
Antagonist
Concentration(uM)
FIG. 1. Effect of I-NMOS, fenitrothion, and cyproheptadine on ventral nerve cord adenylate cyclase activity in the absence or presence of a fmed (la0 CCM) concentration of octopamine (OC71. In the control, the adenylate cyclase activity was 511 k 2 pmoll minlmg of protein. Data are the average of duplicates and the range is shown by vertical bars.
192
HIRASHIMA,
OYAMA,
NMOS at 80 @4 reduced the effect of 100 ELMoctopamine to 23% of maximal activity, whereas fenitrothion and cyproheptadine suppressed the activity to 58 and 16%, respectively. Thus, I-NMOS, fenitrothion, and cyproheptadine displayed characteristics suggesting that they are antagonists of octopamine-activated adenylate cyclase lacking any significant agonist activity. Table 2 shows that washing removed nearly all of the inhibitory activity of lNMOS. In other words, octopaminestimulated activity after preincubation with l-NMOS and then washing was similar to the octopamine-stimulated activity of enzyme not preincubated with l-NMOS. These data suggest that l-NMOS bind reversibly to the nerve cord octopamine receptor. Similar reversibility has been also reported for formamidines (16). Effect upon Phosphodiesterase
AND ET0 TABLE 3 Effect of Various Compounds upon the Phosphodiesterase Activity in Homogenates of Cockroach Nerve Cords
Compound (cone, 100 CLM) None (N) I-NMOS 2-NMOS DMOS Fenitrothion Theophylline Control (C)
CAMP (pmoUmin/mg of protein) 81 (77-85) 76 (75-77) 72 (60-84) 81 (75-88) 114 (109-118) 127 (118-136) 183 (162-203)
Phosphodiesterase inhibitory activity” relative to the control m 0 -5 -9 0 33 45 loo
(-4 to 4) (-6 to -4) (-21 to 4) (-6 to 7) (27 to 36) (36 to 54) (79 to 120)
a Phosphodiesterase inhibitory activity was measured according to Nathanson’s procedure of adenylate cyclase assay using the supematant instead of P, fraction with a modification and calculated by the equation (A - N)/(C - N) X 100 (%) relative to the control (C), where A and N are CAMP levels with and without test compound, respectively. In the control (C), the supematant was heated at 90°C for 2 mitt before the reaction. The data are the average of duplicates and range values are shown in parentheses.
Against phosphodiesterase in homogenates of P. americana nerve cords, theophylline and fenitrothion at 0.1 mM had an inhibitory activity of 45 and 33% relative to itory activity at 0.1 mM (Table 3). At 50 the control, respectively, although l- l&f, salioxon, MMOS, KSCP, salithion, 5NMOS, 2-NMOS, and DMOS had no inhib- PMOS, iBMOS, DMOS, iPMOS, EMOS, KSCP (100 @4), fenitrothion, 2-NMOS, lNMOS, and phosphodiesterase inhibitor TABLE 2 IBMX inhibited 12, 14, 14, 16, 20, 21, 23, Reversibility of Receptor-Mediated Inhibition of 25, 31, 36, 52, 55, 57, and 92% of the phosCockroach Nerve Cord Adenylate Cyclase phodiesterase activity of beef heart relative by I-NMOS to the control, respectively (Table 4). They Postwashing adenylate reduced T. castaneum larval growth and incyclase activity0 creased whole-body CAMP level slightly at (prnol/min/mg of protein) sublethal concentrations. Hence, the inOctopamine creased whole-body CAMP level, which Basal Pretreatment UC@PM) seems to correlate with the inhibition of 28 (25-31) 428 (426A29) No drug AChE, of treated T. castaneum and M. do454 (42-81) I-NMOS (100 m 36 (35-36) - mestica larvae with dietary 5-PMOS, iBMOS, and salithion enantiomers in the o Cockroach ventral nerve cords (9 mglml) were first preincubated (left column) with 1 m&f IBMX in previous reports (7, 8) is due to the reducthe presence or the absence of 100 p,M l-NMOS and tion of the phosphodiesterase activity, alwashed twice as described in text. The Pz fraction was though other possibilities remain to be clarretested (the right two columns) for basal and octoified, e.g., interaction with dopamine- and/ parnine-stimulated adenylate cyclase activity. Values effects are the average of duplicates, while the numbers in or 5-hydroxytryptamine-mediated on CAMP production; interaction with parentheses represent the range.
PHOSPHOROTHIONATES,
ADENYLATE
CYCLASE,
TABLE Effect
193
AND PHOSPHODIESTERASE
4
of Various Compounds on T. castaneum Larval Weight after Treatment and Effect on rhe Phosphodiesterase
Gain and Whole-Body CAMP Level Acrivify of Beef Heart in Vitro
2 Days
Relative to the control” (%) Compound 5-PMOS I-NMOS 2-NMOS MMOS EMOS iPMOS DMOS Salithion (R)-( + )-Salithion (S)-( - )-Salithion Salioxon KSCP iBMOS Fenitrothion IBMX
Dietary cone (pm) 80 30 30 10 10 10 10 10 4 40 500 80 5 10 100
AWeight * SE 19 + 35 -t 17 2 17 t 422 33 * 39 t 43 k 46 2 100 2 32 k 86 + 40 5 108 2
CAMP
5b 5 6 1 2 2 6’ 6’ 3f,8 ?ib 3b 6b 3’
137 (131-143)b.C 126 (105-148) 102 (81-122) 104 (102-107) 136d 106 (98-l 14) 141 (119-163) 99 (88-l 10) 99 (98-loon/ 105 (103-108)‘,J 130 (128-132)b 132 (131-133)b 184 (183-185)b 140 (133-147)b
Phosphodiesterase activity 80 43 45 86 69 75 77 84
-
88 86 (64)h 79 48 8
a Phosphodiesterase activity was determined according to the method of Boehringer-Mannheim with 50 p,M test compounds and calculated by the equation v = 3.1 X AA& X 0.05 X 1) U/ml solution, where AA is the decrease in absorbance per minute at 265 nm and e265 = 8.1 mmol - ’ liter cm - ’ Larval weight gain is the average of 5 replicates of 12 fourth instar larvae and the CAMP data are the average of duplicates, range values being shown in parentheses. The dietary compound concentrations (ppm) are for larval weight gain and CAMP level measurements. In the control, the larval weight gain, CAMP level, and the phosphodiesterase activity were 0.536 * 0.008 mg, 0.59 2 0 pmoVmg of larvae, and 0.406 f 0.045 U/ml, respectively. ’ Cited from Ref. (7). ’ Measured 3 days after treatment. * A single measurement. e Cited from Ref. (9). f Cited from Ref. (8). B Measured as sodium salt (NSCP). h Figure in parentheses is activity measured with 100 p.M KSCP.
monoamine catabolism through N-acetyl transferase. According to Bodnaryk (31), organophosphates appear to affect the level of cGMP indirectly through the following sequence of events: inhibition of AChE; accumulation of acetylcholine; stimulation of guanylate cyclase by acetylcholine; elevation of cGMP. Secondary interactions in the nervous system resulting from elevated cGMP levels may also lead to altered CAMP Ieveis, but such interactions have not been determined yet in insects. Kinetic studies on the CAMP hydrolysis by beef heart phosphodiesterase revealed a K, of 48 l.& and a V,,, of 0.83 U/ml (Fig.
2). Fenitrothion (20, 40, and 60 FM), DMOS (75 and 100 ~L,M), and IBMX (OS, 1, and 4 CLM) increased lu, to 70, 111, 125,80, 89,65, 109, and 154 PM, respectively, with little effect on V,,,, indicating that these compounds inhibited CAMP phosphodiesterase in a competitive manner: Ki values for DMOS, fenitrothion, and IBMX were 107.9, 37.3, and 1.5 l&f, respectively, according to Fig. 2. Thus, DMOS and fenitrothion might inhibit the phosphodiesterase in a similar manner with the classical phosphodiesterase inhibitor such as IBMX, which interacts directly with the catalytic site on phosphodiesterase and
194
HIRASHIMA,
-2 -1
I 2 3 4 lIENIl R
-100
OYAMA,
[ OYOSII/N
AND
100
ET0
-30
IFmlrathionlR
30
60 6OuN
40
i
w
I
I/lcANPI A
2 3 4 Y IO-’
I
-2 -1
IilclNPI
1 2 3 4
2. Double-reciprocal plot of the inhibition IBMX, DMOS, and fenitrothion. FIG.
competitively interferes with the binding of the cyclic nucleotide substrates (32). In order to clarify the relationship, if any, between the increased whole-body CAMP level and the reduced trehalase activity leading to the larval weight gain suppression (59), more detailed experiments are in progress.
au
x 10-z
4.
5.
6.
7.
REFERENCES
1. M. Eto, Y. Kinoshita, T. Kato, and Y. Oshima, Saligenin cyclic methyl phosphate and its thiono analogue: New insecticides related to the active metabolite of tri-o-cresyl phosphate, Nature (London) 200, 171 (1%3). 2. M. Eto, A. Hirashima, S. Tawata, and K. Oshima, Structure insecticidal activity relationship of five-membered cyclic phosphoramidates derived from amino acids, .I. Chem. Sot. Japan 5, 705 (1981). 3. S.-Y. Wu, A. Hirashima, R. Takeya, and M. Eto,
I
I/lcANPI R
2 3 4 x IO-”
of CAMP phosphodiesterase
ACKNOWLEDGMENTS
We thank Dr. Masahiko Kuwahara of the National Food Institute in Japan for the supply of T. castuneum, Dr. Makoto Mizunami of the Faculty of Science at Kyushu University for donating the cockroaches (P. americana), and our colleague Yutaka Yoshii for technical assistance. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.
-2 -I
8.
9.
from beef heart by
Synthesis and insecticidal activity of oxazaphospholidines, oxathiaphospholanes and thiazaphosphohdmes, Agric. Biol. Chem. 52,291l (1988). S.-Y. Wu, A. Hirashima, R. Takeya, and M. Eto, Synthesis and insecticidal activity of optically active 2-methoxy-5-phenyl-1,3,2-oxazaphospholidine 2-sulfide, Agric. Biol. Chem. 53, 165 (1989). A. Hirashima, I. Ishaaya, R. Ueno, Y. Ichiyama, S.-Y. Wu, and M. Eto, Biological activity of optically active salithion and salioxon, Agric. Biol. Chem. 53, 175 (1989). A. Hirashima, K. Oyama, R. Ueno, I. Ishaaya, and M. Eto, Carbohydrate enzymes in house fly Musca domestica: Biochemical properties and toxicological signiticance, J. Fat. Agric., Kyushu Univ. 34, 37 (1989). A. Hirashima, R. Ueno, K. Oyama, I. Ishaaya, and M. Eto, Effect of five-membered cyclic phosphorothionates on larval growth, trehalase, digestive enzymes, acetylcholinesterase, and cyclic adenosine 3’,5’-monophosphate level of Tribolium castaneum and Musca domestica, Pestic. Biochem. Physiol. 35, 127 (1989). A. Hirashima, R. Ueno, K. Oyama, H. Koga, and M. Eto, Effect of salithion enantiomers on larval growth, carbohydrases, acetylcholinesterase, adenylate cyclase activities and cyclic adenosine 3’,5’-monophosphate level of Musca domestica and Tribolium castaneum, Agric. Biol. Chem. 54, 1013 (1990). A. Hirashima, I. Ishaaya, R. Ueno, K. Oyama, and M. Eto, Effect of salithion enantiomers on
PHOSPHOROTHIONATES,
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
ADENYLATE
the trehalase system and on the digestive protease, amylase, and invertase of Tribolium castaneum, Pestic. Biochem. Physiol. 34, 205 (1989). A. Hirashima, Y. Yoshii, K. Kumamoto, K. Oyama, and M. Eto, Structure-activity studies of 2-methoxy-1,3,2-oxazaphospholidine 2sulfides against Musca domestica and Tribolium castaneum, J. Pesticide Sci., in press. A. Hirashima and M. Eto, Quantitative structureactivity studies of cyclic phosphorothionates and phosphates, J. Fat. Agric., Kyusyu Univ., in press. H. Bredereck, R. Gompper, K. Klemm, and H. Rempfer, Reaktionen von SLureamid-Acylhalogenid-Addukten: Darstellung substituierten Amidine und Amidrazone, Chem. Ber. 92, 837 (1959). S.-Y. Wu, A. Hirashima, M. Eto, K. Yanagi, E. Nishioka, and K. Moriguchi, Synthesis of highly pure enantiomers of the insecticide salithion, Agric. Biol. Chem. 53, 157 (1989). M.-Y. Eto, M. Iio, H. Omura, and M. Eto, Synthesis of a new phosphorylating agent, 2methylthio-4H-1,3,2-benzodioxaphosphorin 2oxide (MTBO) from its thiono isomer, J. Fat. Agric., Kyushu Univ. 22, 25 (1977). I. lshaaya and J. E. Casida, Dietary TH 6040 alters composition and enzyme activity of housefly larval cuticle, Pestic. Biochem. Physiol. 4, 484 (1974). J. M. Nathanson and E. J. Hunnicutt, NDemethylchIordimeform: A potent agonist of octopamine-sensitive adenylate cyclase, Mol. Pharmacol. 20, 68 (1981). 0. A. Lowry, N. J. Rosebrough, A. L. Farr, and R. L. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265 (1951). J. Keesey, (Ed.), Phosphodiesterase (PDE), in “Method Sheets of Enzyme Determination,” p. 20, Boehringer-Mannheim Biochemicals, Indianapolis, IN 1987. J. A. Nathanson and P. Greengard, Octopaminesensitive adenylate cyclase: Evidence for a biological role of octopamine in nervous tissue, Science 180, 308 (1973). J. W. Gole, G. L. Orr, and R. G. H. Downer, Interaction of formamidines with octopaminesensitive adenylate cyclase receptor in the nerve cord of Periplaneta americana, Life Sci. 32, 2939 (1983). R. G. H. Downer, J. W. D. Gole, and G. L. Orr, Interaction of formamidines with octopamine-, dopamine- and 5-hydroxytryptamine-sensitive
CYCLASE,
22.
23. 24.
25.
26.
27.
28.
29.
30.
31. 32.
33.
AND
PHOSPHODIESTERASE
195
adenylate cyclase in the nerve cord of Periplaneta americana, Pestic. Sci. 16, 472 (1985). G. L. Orr, J. W. D. Gole, and R. G. H. Downer, Characterisation of an octopamine-sensitive adenylate cyclase in haemocyte membrane fragments of the American cockroach Periplaneta americana L., Insect Biochem. 15, 695 (1985). G. L. Orr and R. M. Hollingworth, Agonistinduced desensitization of an octopamine receptor, Insect Biochem. 20, 239 (1990). J. W. D. Gole, G. L. Orr, and R. G. H. Downer, Octopamine-mediated elevation of cyclic AMP in haemocytes of the American cockroach, Periplaneta americana L., Canad. J. Zool. 65, 1509 (1986). R. G. H. Downer, G. L. Orr, J. W. D. Gole, and I. Orchard, The role of octopamine and cyclic AMP in regulating hormone release from corpora cardiaca of the American cockroach, J. Insect Physiol. 30, 457 (1984). J. W. D. Gole and R. G. H. Downer, Elevation of adenosine 3’,5’-monophosphate by octopamine in fat body of the American cockroach, Periplaneta americana L., Comp. Biochem. Physiol. C: Comp. Pharmacol. 64, 223 (1979). A. J. Harmar and A. S. Horn, Octopaminesensitive adenylate cyclase in cockroach brain: Effects of agonists, antagonists, and guanylyl nucleotides, Mol. Pharmacol. 13, 512 (1977). J. A. Nathanson, Cyclic AMP synthesis and degradation: Possible targets for pesticide action, in “Membrane Receptors and Enzymes as Targets of Insecticidal Action” (J. M. Clark and F. Matsumura, Eds.), pp. 157-171, Plenum, New York, 1986. N. Minhas, J. W. D. Gole, G. L. Orr, and R. G. H. Downer, Pharmacology of [3H]mianserin binding in the nerve cord of the American cockroach, Periplaneta americana, Arch. Insect Biochem. Physiol. 6, 191 (1987). R. G. Downer, ‘Cockroaches as Models for Neurobiology: Applications in Biomedical Research” (Huber, Master, and Ras, Eds.). Vol. 2, pp. 103-124, CRC Press, Boca Raton. FL, 1989. R. P. Bodnaryk, The effects of pesticides and related compounds on cyclic nucleotide metabolism, lnsecr Biochem. 12, 589 (1982). W. C. Prozialeck and J. M. Roberts-Lewis, Development of selective inhibitors of calmodulindependent phosphodiesterase and adenylate cyclase, in “Design of Enzyme Inhibitors as Drugs” (M. Sandler and H. J. Smith, Eds.), pp. 650-697, Oxford Univ. Press, Oxford, 1989. D. B. Duncan, Multiple range and multiple Ftests, Biometrics 11, 1 (1955).
BIOCHEMISTRY
AND
PHYSIOLOGY
38, 186-195 (1%)
Effect of Insecticidal Cyclic Phosphorothionates Cyclase and Phosphodiesterase AKINORI
HIRASHIMA,KAZUHIKO
Department of Agricultural
on Adenylate
OYAMA, AND MORIFUSA ETO
Chemistry, Kyushu University, Fukuoka 812, Japan
Received June 6, 1990; accepted July 31, 1990 Octopamine (0.1 and 1 mM) stimulated the adenylate cyclase prepared from Periplaneta americana ventral nerve cords (615 and 1112% relative to the control). D-( -)-ZAmino-l-phenylethanol (APE) was more potent than 2-amino-l-(4-fluorophenyl)ethanol (an octopamine agonist) and L-(+)-APE in stimulating the adenylate cyclase. 2-Methoxy-S-phenyl-1,3,2-oxazaphospholidine 2-sulfide (5PMOS) derived from DL-(+)-APE did not activate adenylate cyclase at 0.75 and 7.5 mM (91 and 95% relative to the control) but suppressed the octopamine (0.1 mM) potency to 268% at 1 m&f relative to the control. Salithion (Zmethoxy-4&1,3,2-benzodioxaphosphorin 2-sulfide) at 0.1 m&f, fenitrothion (dimethyl 3-methyl-4nitrophenyl phosphorothionate), 2-amino-1-(2,3dimethoxy)phenylethanol, 5-PMOS, and other oxazaphospholidines at 1 m&f showed similar phenomena. l-Naphthyl-, 2-naphthyl-, 4-ethylphenyl-, and 4-isopropylphenyloxazaphospholidine derivatives at 0.1 mkf reduced the octopamine potency at 0.1 mM more severely than the octopan-&e-receptor antagonists fchlordimeform and cyproheptadine) at 1 mM. 5-(2,3-Dimethoxyphenyl)-2-methoxy-1,3,2-oxazaphospholidine 2-sulfide (Ki = 107.9 p&f), fenitrothion (Ki = 37.3 t&f), and 3-isobutyl-l-methylxanthine (Ki = 1.5 t&f) reduced the phosphodiesterase activity of beef heart in a competitive manner with respect to cyclic adenosine 3’,5’-monophosphate (CAMP). At 50 p&f, salithion, salioxon (2-methoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide), and other oxazaphospholidines reduced phosphodiesterase activity. Hence, D-(-)-APE could be an agonist, and 5-PMOS and salithion analogs and fenitrothion could be partial antagonists to the octopamine receptor. The increased level of whole-body CAMP of Musca domestica and Tribolium castaneum larvae treated with these phosphorothionates is due to reduction of phosphodiesterase activity. 0 1990 Academic Press, Inc.
rived from L-leucine by stabilizing the Since the insecticidal six-membered cy- structure through introduction of a nitrogen clic phosphorothionate salithion was dis- atom into the ring system (2). In oxazacovered in our laboratory (l), we have phospholidines, a hydrophobic substituent searched for new five-membered cyclic at the 4-position generally has enhanced the phosphorothionates and found insecticidal insecticidal activity more than that at the activities in 4-isobutyl-2-methoxy-1,3,2oxazaphospholidine 2-sulfide (iBMOS)’ deINTRODUCTION
’ Abbreviations used: iBMOS, 4-isobutyl2-methoxy-1,3,2-oxazaphospholidine 2-sulfide; 5PMOS, 2-methoxy-5-phenyl-1,3,2-oxazaphospholidine t-sulfide; APE, 2-amino-1-phenylethanol; CAMP, cyclic adenosine 3’,5’-monophosphate; 5RMOS, 5-substituted 2-methoxy-1,3,Zoxazaphospholidine 2-sulfide; AChE, acetylcholinesterase; KSCP, potassium saligenin cyclic phosphorothionate; EGTA, ethylene glycol bis(B-aminoethyl ether) N,N,N’,N’tetraacetic acid; ATP, adenosine 5’-triphosphate; GTP, guanosine 5’-triphosphate; IBMX, 3-isobutyl-1-methylxanthine; l-NMOS, 2-methoxy-
5-(1-naphthyl)-1,3,2-oxazaphospholidine 2-sulfide; DMOS, 5-(2,3-dimethoxyphenyl)-2-methoxy1,3,2-oxazaphospholidine 2-sulfide; AFPE, Z-aminol-(4fluorophenyl)ethanol; FMOS, 5-(4-fluorophenyl)2-methoxy-1,3,2-oxazaphospholidine 2-sulfide; ADPE, 2-amino-1-(2,3-dimethoxyphenyl)-ethanol; MMOS, 2-methoxy-S-(4-methylphenyl)-1,3,2-oxazaphospholidine 2-sulfide; 2-NMOS, 2-methoxy5-(2-naphthyl)-1,3,2-oxazaphospholidine 2-sulfide; EMOS, 5-(4-ethylphenyl)-2-methoxy-1,3,2-oxazaphospholidine 2-sulfide; iPMOS, 5-(4-isopropylphenyl)-2-methoxy-1,3,2-oxazaphospholidine 2-sultide; cGMP, cyclic guanosine 3’,5’-monophosphate; NSCP, sodium saligenin cyclic phosphorothionate.
186 0048-3575/90 $3.00 Copyright 0 1990 by Academic Press. Inc. AU rights of reproduction in any form reserved.
PHOSPHOROTHIONATES,
ADENYLATE
5-position, except when the substituent is a phenyl group (3). 2-Methoxy-5-phenyl1,3,2-oxazaphospholidine 2-sulfide (5 PMOS) derived from an octopamine analog, 2-amino-1-phenylethanol (APE), has been found to have a stronger insecticidal activity than salithion and iBMOS at lethal concentrations by topical application to Muscu domestica female adults (3-5). Dietary salithion and 5-PMOS have been found to reduce larval weight gain, reduce trehalase activity, and increase the level of whole-body CAMP of M. domestica (6-8) and Tribolium custaneum (7-9) at sublethal concentrations. Increased levels of CAMP could be due to either the activation of adenylate cyclase, an enzyme that catalyzes the biosynthesis of CAMP, or the inhibition of phosphodiesterase, an enzyme that catalyzes the hydrolysis of CAMP. In order to optimize these biological activities, numerous structural modifications were made, and in order to understand quantitatively the dependence of activity upon the substituent, the structure-activity relationships of salithion analogs and 5-substituted 2methoxy-1,3,2-oxazaphospholidine 2sulfide (S-RMOS) were analyzed (10, 11), including anti-AChE activity, M. domesticu insecticidal activity by topical application, and T. custuneum larval growth inhibitory activity, which could be due to perturbation of monoamine-related production of CAMP. However, the relationship of these biological phenomena, if any, has not been clarified yet. Hence, this paper deals with the effects of salithion and 5-RMOS on the adenylate cyclase in homogenates of ventral nerve cords of Peripluneta umericunu, the determination of the cause of the increased level of whole-body CAMP in M. domestica and T. castaneum larvae treated with these cyclic phosphorothionates, and the further elucidation of the effect of altered CAMP level on various physiologicd phenomena. MATERIALS
Chemicals.
AND
METHODS
Chlordimeform [N’-(Cchloro-
CYCLASE,
AND
PHOSPHODIESTERASE
187
o-tolyl)-l\r,N-dimethylformamidinel was prepared by reacting 2-amino-5-chlorotoluene with dimethylformamide in the presence of phosphorus oxychloride (12). L-( +)- and D-(-)-APES were obtained from L-( + )- and D-( -)-mandelic acid, respectively (4). Salithion (1), iBMOS (2), and other oxazaphospholidines (10) were synthesized by reacting methyl phosphorodichloridothionate with saligenin, Lleucinol (2-amino-4-methyl-1-pentanol), and the corresponding amino alcohols, respectively. Salithion enantiomers (>98% ee) were prepared by the proline ester method (13) and potassium saligenin cyclic phosphorothionate (KSCP) by demethylating salithion with potassium dimethyldithiocabamate (14). Salioxon was obtained by m-chloroperbenzoic acid oxidation of salithion (5). Fenitrothion (96.5% pure) was a gift from Sumitomo Chemical Co. Ltd. (Takarazuka, Japan). Octopamine [2amino-1-(4-hydroxyphenyl)ethanol], theophylline (1,3-dimethyixanthine), eserine (physostigmine), EGTA, and cyproheptadine [4-(SH-dibenzo[u,d]cyclohepten5-ylidene)-1-methylpiperidiene] were purchased from Nacalai Tesque Inc. (Kyoto, Japan), ATP, GTP, IBMX, and CAMP from Sigma Chemical Co. Ltd. (St. Louis, MO), 3’,5’-cyclic nucleotide phosphodiesterase (EC 3.1.4.17>, adenosine deaminase (EC 3.5.4.4), and alkaline phosphatase (EC 3.1.3.1) from Boehringer-Mannheim, GmbH Biochimica (Mannheim, West Germany), and RIA kit “Yamasa” (Code YSI7701) from Yamasa Shoyu Co. (Chiba, JaPan). Bioassay. The dietary effect of the test compounds on T. castaneum larval growth was measured according to a previously published method (5, 7-10, 15). Twelve fourth instar T. custaneum larvae weighing 1.O ~fr 0.1 mg each were introduced into each test vial of five replicates, along with 1.5-g portions of diet. The vials were held at 30°C for 2 days to determine weight gains and CAMP level. Measurement
of whole-body
CAMP
188
HIRASHIMA,
level. The level of T. castaneum
OYAMA,
wholebody cAMP was determined by using a radioimmunoassay according to a previously published method (7,8). Last instar T. castaneum larvae, 2.0 2 0.2 mg each, which had been fed on treated diet at 30°C for 2 days, were homogenized in cold 6% trichloroacetic acid at 4°C to give 10% (w/v) homogenate. The homogenate was centrifuged at 3000 rpm and 4°C for 10 min, the pellet being resuspended in the same buffer and recentrifuged. The combined supernatants were washed three times with 3 vol of diethyl ether saturated with water. To 100~plaliquots of the aqueous extract were added 5 pJ of triethylamine and 45 p,l of succinyl anhydride solution in dioxane (44 mg/ml). After the mixture was kept at room temperature for 10 min, 350 l.~lof 0.3 M imidazole buffer (pH 6.5) was added, and diluted to an appropriate concentration (1 mg of larvae/ml) with the buffer containing 1% triethylamine, 9% succinyl anhydride solution, and 20% distilled water. The CAMP level was determined with an aid of RIA kit “Yamasa” (Code YSI-7701). Adenylate cyclase assay. Ventral nerve cords of adult cockroaches (P. americana) were homogenized (15 mg/ml) in 6 mM Tris-maleate buffer (pH 7.4) using a chilled glass-Teflon Potter-Elvehjem tissuegrinding tube (16). This homogenate was diluted to a volume of 15 ml in 6 mM Trismaleate, and centrifuged at 120,OOOgand 4°C for 20 min. The supernatant was used for a phosphodiesterase source, the pellet being resuspended by homogenization in 15 ml of the buffer and again centrifuged at 120,OOOg and 4°C for 20 min. The resulting pellet (PZ fraction) was resuspended in a volume of 6 mM Tris-maleate equivalent to the starting amount. The adenylate cyclase activity was measured by Nathanson’s procedure under optimal conditions (16) in test tubes containing (in 0.3 ml) 80 mM Trismaleate @H 7.4), 10 mM theophylline, 8 mM M&l,, 0.1 m&f GTP, 0.5 mM EGTA, 2 mM ATP, 0.06 ml of P, fraction, and compound solutions to be tested, which were
AND
ET0
prepared (9) by dissolving the compounds in 0.5 ml of acetone, and then adding 2 drops (50 pg) of Tween 80 and 99.5 ml of water. Appropriate solvent controls were run in parallel. The enzyme reaction (5 min at 30°C) was initiated by adding ATP, stopped by heating at 90°C for 2 min, and then centrifuged at 1OOOgfor 15 min to remove the insoluble material. The CAMP level in the supernatant was measured by a radioimmunoassay (7, 8) and protein concentration was determined by the Lowry method (17). For reversibility studies (Table 2), the nerve cords (9 mg/ml) were first preincubated with 1 n&f IBMX in the presence or absence of 100 Q4 I-NMOS in cockroach saline at 30°C for 20 min, and centrifuged at IOOOgand 4°C for 10 min. The precipitate was homogenized in 1 ml of Tris-maleate buffer (pH 7.4), and centrifuged twice at 120,OOOgand 4°C for 20 min as mentioned above to remove soluble I-NMOS and produce P2 fraction. This fraction was assayed for adenylate cyclase activity in the absence or the presence of additional octopamine. Five replicates of 20 fourth instar T. castaneum larvae were treated with dietary 80 ppm of 5-PMOS or 6 ppm of DMOS at 30°C for 2 days. Acetone extracts were prepared (8) by homogenizing the feeding larvae in cold acetone (50 mg/ml), and the homogenate was centrifuged at 3000 rpm and 4°C for 10 min. The supematant fraction was diluted (10 mg/ml) with a solvent system including 0.5 ml of acetone, 99.5 ml of water, and 50 pg of Tween 80, and was used for adenylate cyclase assay as above. Phosphodiesterase inhibition assay. For the phosphodiesterase assay in homogenates of cockroach nerve cords, the adenylate cyclase assay was modified slightly by using the supernatant instead of P2fraction. The reaction mixture consisted of (in 0.3 ml) 80 mM Tris-maleate (pH 7.4), 8 mM MgCl,, 0.5 mJ4 EGTA, 0.06 ml of the supernatant fraction, and a compound solution to be tested. For the control, the su-
PHOSPHOROTHIONATES,
ADENYLATE
pernatant was heated at 90°C for 2 min before the reaction. The enzyme reaction (5 min at 30°C) was initiated by adding the compound solution, stopped by heating at 90°C for 2 min, and then centrifuged at 1OOOg for 15 min to remove the insoluble material. The CAMP level in the supernatant was measured as above. Beef heart phosphodiesterase activity was assayed according to the BoehringerMannheim procedure (18) with a slight modification. Test compounds in a O.l-ml suspension (acetone-water-Tween 80), prepared as above, were preincubated with 0.05 ml of phosphodiesterase (0.711 U/ml), 0.1 ml of magnesium sulfate (82 mM), 0.01 ml of adenosine deaminase (400 U/ml), 0.04 ml of alkaline phosphatase solution (I500 U/ml), and 2.7 ml of 0.1 M glycylglycine buffer (pH 7.5) at 25°C for 10 min. The remaining phosphodiesterase activity was determined by initiating the reaction with the addition 0.1 ml of CAMP (1.35 mM) to the preincubated mixture. The decrease in absorbance per minute (AA) at 265 nm was continuously recorded against a water blank containing test compound in E units at 25°C with a Shimadzu UV-2100 spectrophotometer and the activity was calculated by the equation v = 3.1 x AAl(e x 0.05 x 1) U/ml, where eZe5 = 8.1 mmol-’ liter cm-‘. The enzyme activity was linear up to 1 min and 80 kg protein/ml (1 U/ml). The concentration of the compound is expressed as that present in the preincubation medium. RESULTS
AND
DISCUSSION
Effect upon Octopamine-Sensitive Adenylate Cyclase
At 0.1 and 1 mM, octopamine has shown a maximum activity for activation of adenylate cyclase prepared from cockroach nerve cord (19-21), hemocyte (22-24), corpus cardiacurn (25), fat body (26), and brain homogenate (27). In our experiments, octopamine (0.1 and 1 mM) stimulated adenylate cyclase (615 and 1112% relative to the
CYCLASE,
AND
PHOSPHODIESTERASE
189
control) prepared from ventral nerve cords of P. americana (Table 1). The absence of the 4-hydroxy group substantially reduces potency: APE is over 10 times less potent than octopamine in stimulating adenylate cyclase activity in homogenates of cockroach brain (27). However, AFPE, an octopamine analog in which fluorine has been substituted for the 4-hydroxy group, is a potent agonist to octopamine receptor (27, 28). D-(-)-APE (0.1 and 1 mM) was more potent (337 and 380% relative to the control) than 0.1 mM AFPE (299% relative to the control) and L-( +)-APE (102 and 123% relative to the control) at 0.1 and 1 mM in stimulating adenylate cyclase (Table 1)) suggesting that response is stereoselective for the ~-(-)-isomer. This result agrees with previous reports of the agonistic potency of octopamine enantiomers and insecticidal activity of 5-PMOS optical isomers: naturally occurring I+( - )-octopamine is more potent than the L-( + )-enantiomer in stimulating adenylate cyclase (27); (R&Z,)and (&,S,)-5-PMOSs derived from D-( -)-APE have been more potent insecticides than (S&Z,)- and&S,)isomers prepared from the L-(+)-APE (4). Neither 5-PMOS derived from DL-(f)APE at 0.75 and 7.5 mM (91 and 95% relative to the control) nor FMOS derived from AFPE at 0.1 mM (83% relative to the control) activated adenylate cyclase. However, they suppressed the octopamine (0.1 mM) potency to 268% (S-PMOS) at 1 mM and 272% (FMOS) at 0.1 mM relative to the control, respectively (Table 1). Table 1 also shows that salithion, fenitrothion, 2,3dimethoxyphenyl analogs (ADPE and DMOS) of APE and 5-PMOS, and other oxazaphospholidines had a similar tendency. The acetone extracts of T. castaneum larvae treated with dietary 80 ppm of 5-PMOS or 6 ppm of DMOS did not stimulate the adenylate cyclase. These results suggest that D-(-)-APE and AFPE are agonists, but 5-PMOS and salithion analogs and fenitrothion are partial antagonists to octopamine receptor except the (4-methyl-
190
HIRASHIMA,
OYAMA, TABLE
AND
ET0
1
Nonadditive Effects of Octopamine and Various Compounds upon the Adenylate Cyclase Activity in Homogenates of Cockroach Nerve Cords Increase CAMP Additive Control Octopamine
Cone
AFPE ADPE 5-PMOS 1-NMOS ZNMOS
FMOS MMOS EMOS
iPMOS
DMOS Salithion Fe&o&ion Cyproheptadine
Eserine Acetone extract control 5-PMOS (SO ppm) acetone extract’ DMOS (6 ppm) acetone extract’
100
loo0 100 loo0 100 loo looo 750 7500 10 100 1 10 100 loo loo 1 10 100 1 10 loo 750 7500 loo loo 1000 1 10 loo loo0 100
Octopamine D-(-)-APE L-( + )-APE
AFPE DMPE 5-PMOS l-NMOS ZNMOS FMOS MMOS EMOS
(100 piU) 1ooo
loo0 loo 1ooo moo 100 100 100 100 100
(pmoUmin/mg of protein)
56 212 345 623 189 213 57 69 167 86 81 51 54 63 37 48 58 39 47 54 38 36 46 44 40 44 52 52 46 55 39 34 40 36 30 51 60 56 59
10 100 1000
D-(-)-APE L-( + )-APE
(p&f)
in the adenylate
+ Additive 202 3 14 197 174 150 54 61 153 305 69
(55-57) (199-226) (269-420) (598-648) (178-200) (205-220) (55-60) (48-90) (166-168) (57-l 15) (74-88) (49-53) (48-60) (62-64)
cyclase
activity”
Relative to the control 100 379 615 1112 337 380 102 123 298 154 144 91 95 113
(98-102) (355-403)’ (473-750)8 (1068-1157)h (311357) (366-393)f (98-107) (86-161pd (296-300) (102-205pd (132-157pd (88-95) (86-107) (111-114)
66,’
(48-48) (57-59) (36-42) (45-48) (52-55) (37-39) (36-36) (46-46) (42-47) (40-41) (42-47) (5G54) (48-56) (42-49) (53-58) (3-3) (33-34) w4 (30-41) (28-32) (49-52) (58-62) (48-63) (55-64)
85 104 70 83 96 68 63 82 79 72 78 92 92 82 99 70 60 72 63 53 91 107 100 105
(85-85)bsc (102-105) (64-75)b (80-86)b,C (93-98) (66-70)b (63-63)b (82-82)b*C (75-84)b.C (71-73)b (75-84)bnc (89-%) (86-100) (75-88)b,c (95-104) (64-77)b (59-61)b (71-73)b (54-73)b (50-57)b (88-93) (104-111) (86-l 13) (98-l 14)
(198-205) (250-378) (193-201) (155-192) (150-150) (424) (52-69) (142-W) (297-313) (65-72)
360 561 352 3 10 268 97 108 272 544 122
(354366)’ (W75)8 (345-359)’ (277-343) (268-268) (75-l 18) (93-123) (254-293) (53&559)8 (1 16-129pd
PHOSPHOROTHIONATES,
ADENYLATE
TABLE
CYCLASE,
AND
191
PHOSPHODIESTERASE
l-Continued Increase in the adenylate cyclase activitp
Additive iPMOS DMOS Salithion Fenitrothion Chlordimeform Cyproheptadine Eserine
Cone (piU)
CAMP (pmol/min/mg of protein)
Relative to the control
100 1000 100 1000 1000 1000 100
78 (77-79) 174 (16U81) 168 (168-168) 217 (168-265) % (91-101) 88 (87-88) 341 (329-353)
139 (13W4l)d -3 10 (296-323) 300 (300-300) 387 (300-473)’ 171 (163-180)d 156 (155-157)d 609 (588-630)8
Octopamine (10 Qf) + Additive 10 197 (188-208) 1 190 (182-198) 10 156 (156-156) 1 160 (152-167) 150 (148-153) 10 1 182 (176-189) 10 193 (193-193) 1 150 (148-153) 69 (66-72) 10 1 132 (132-132)
352 (336-371)’ 339 (325-354yf 279 (279-279) 2-NMOS 280 (27 l-298) 268 (264-273) EMOS 325 (31k338Y9 345 (345-345)’ iPMOS 268 (264-273) 123 (l&129)‘.’ Cyproheptadine 236 (236-236)d,’ ___.’ The adenylate cyclase activity of P. americana was measured according to Nathanson’s procedure and the CAMP levels were measured by a radioimmunoassay. The data are the average of duplicates, range values being shown in parentheses. U Differ significantly from each other at P = 0.05 according to Duncan’s multiple range test (33). ’ A single measurement. j The acetone extract of the treated T. castaneum larvae at 30°C for 2 days was obtained by homogenization in ice-cold acetone followed by centrifugation at 3000 rpm and 4°C. I-NMOS
phenyl)oxazaphospholidine derivative MMOS. L-(+)-APE, MMOS, and eserine (an AChE inhibitor) showed little agonistic and antagonistic effects. 1-Naphthyl- (l-NMOS), 2-naphthyl- (2NMOS), 4-ethylphenyl- (EMOS), and (4-isopropylphenyl)oxazaphospholidine (iPMOS) derivatives at 0.1 mM reduced the octopamine potency at 0.1 m&I in stimulating adenylate cyclase more severely than 1 mM chlordimeform, a partial antagonist to the octopamine-receptor in the nerve cord of P. americana (20), and cyproheptadine, a potent, if not the most potent (29, 30), antagonist to the octopamine receptor of P. americana nerve cord (20, 21), hemocyte (22, 24), and brain homogenate (27). However, at the lower concentrations of 10 and 1 PM the antagonistic effect of these oxazaphospholidines to octopamine receptor was not significant. Figure 1 shows the antago-
nistic effect of I-NMOS, fenitrothion, and cyproheptadine, all of which caused significant inhibition of basal adenylate cyclase activity at concentrations of lo-80 PM. l-
I
Antagonist
Concentration(uM)
FIG. 1. Effect of I-NMOS, fenitrothion, and cyproheptadine on ventral nerve cord adenylate cyclase activity in the absence or presence of a fmed (la0 CCM) concentration of octopamine (OC71. In the control, the adenylate cyclase activity was 511 k 2 pmoll minlmg of protein. Data are the average of duplicates and the range is shown by vertical bars.
192
HIRASHIMA,
OYAMA,
NMOS at 80 @4 reduced the effect of 100 ELMoctopamine to 23% of maximal activity, whereas fenitrothion and cyproheptadine suppressed the activity to 58 and 16%, respectively. Thus, I-NMOS, fenitrothion, and cyproheptadine displayed characteristics suggesting that they are antagonists of octopamine-activated adenylate cyclase lacking any significant agonist activity. Table 2 shows that washing removed nearly all of the inhibitory activity of lNMOS. In other words, octopaminestimulated activity after preincubation with l-NMOS and then washing was similar to the octopamine-stimulated activity of enzyme not preincubated with l-NMOS. These data suggest that l-NMOS bind reversibly to the nerve cord octopamine receptor. Similar reversibility has been also reported for formamidines (16). Effect upon Phosphodiesterase
AND ET0 TABLE 3 Effect of Various Compounds upon the Phosphodiesterase Activity in Homogenates of Cockroach Nerve Cords
Compound (cone, 100 CLM) None (N) I-NMOS 2-NMOS DMOS Fenitrothion Theophylline Control (C)
CAMP (pmoUmin/mg of protein) 81 (77-85) 76 (75-77) 72 (60-84) 81 (75-88) 114 (109-118) 127 (118-136) 183 (162-203)
Phosphodiesterase inhibitory activity” relative to the control m 0 -5 -9 0 33 45 loo
(-4 to 4) (-6 to -4) (-21 to 4) (-6 to 7) (27 to 36) (36 to 54) (79 to 120)
a Phosphodiesterase inhibitory activity was measured according to Nathanson’s procedure of adenylate cyclase assay using the supematant instead of P, fraction with a modification and calculated by the equation (A - N)/(C - N) X 100 (%) relative to the control (C), where A and N are CAMP levels with and without test compound, respectively. In the control (C), the supematant was heated at 90°C for 2 mitt before the reaction. The data are the average of duplicates and range values are shown in parentheses.
Against phosphodiesterase in homogenates of P. americana nerve cords, theophylline and fenitrothion at 0.1 mM had an inhibitory activity of 45 and 33% relative to itory activity at 0.1 mM (Table 3). At 50 the control, respectively, although l- l&f, salioxon, MMOS, KSCP, salithion, 5NMOS, 2-NMOS, and DMOS had no inhib- PMOS, iBMOS, DMOS, iPMOS, EMOS, KSCP (100 @4), fenitrothion, 2-NMOS, lNMOS, and phosphodiesterase inhibitor TABLE 2 IBMX inhibited 12, 14, 14, 16, 20, 21, 23, Reversibility of Receptor-Mediated Inhibition of 25, 31, 36, 52, 55, 57, and 92% of the phosCockroach Nerve Cord Adenylate Cyclase phodiesterase activity of beef heart relative by I-NMOS to the control, respectively (Table 4). They Postwashing adenylate reduced T. castaneum larval growth and incyclase activity0 creased whole-body CAMP level slightly at (prnol/min/mg of protein) sublethal concentrations. Hence, the inOctopamine creased whole-body CAMP level, which Basal Pretreatment UC@PM) seems to correlate with the inhibition of 28 (25-31) 428 (426A29) No drug AChE, of treated T. castaneum and M. do454 (42-81) I-NMOS (100 m 36 (35-36) - mestica larvae with dietary 5-PMOS, iBMOS, and salithion enantiomers in the o Cockroach ventral nerve cords (9 mglml) were first preincubated (left column) with 1 m&f IBMX in previous reports (7, 8) is due to the reducthe presence or the absence of 100 p,M l-NMOS and tion of the phosphodiesterase activity, alwashed twice as described in text. The Pz fraction was though other possibilities remain to be clarretested (the right two columns) for basal and octoified, e.g., interaction with dopamine- and/ parnine-stimulated adenylate cyclase activity. Values effects are the average of duplicates, while the numbers in or 5-hydroxytryptamine-mediated on CAMP production; interaction with parentheses represent the range.
PHOSPHOROTHIONATES,
ADENYLATE
CYCLASE,
TABLE Effect
193
AND PHOSPHODIESTERASE
4
of Various Compounds on T. castaneum Larval Weight after Treatment and Effect on rhe Phosphodiesterase
Gain and Whole-Body CAMP Level Acrivify of Beef Heart in Vitro
2 Days
Relative to the control” (%) Compound 5-PMOS I-NMOS 2-NMOS MMOS EMOS iPMOS DMOS Salithion (R)-( + )-Salithion (S)-( - )-Salithion Salioxon KSCP iBMOS Fenitrothion IBMX
Dietary cone (pm) 80 30 30 10 10 10 10 10 4 40 500 80 5 10 100
AWeight * SE 19 + 35 -t 17 2 17 t 422 33 * 39 t 43 k 46 2 100 2 32 k 86 + 40 5 108 2
CAMP
5b 5 6 1 2 2 6’ 6’ 3f,8 ?ib 3b 6b 3’
137 (131-143)b.C 126 (105-148) 102 (81-122) 104 (102-107) 136d 106 (98-l 14) 141 (119-163) 99 (88-l 10) 99 (98-loon/ 105 (103-108)‘,J 130 (128-132)b 132 (131-133)b 184 (183-185)b 140 (133-147)b
Phosphodiesterase activity 80 43 45 86 69 75 77 84
-
88 86 (64)h 79 48 8
a Phosphodiesterase activity was determined according to the method of Boehringer-Mannheim with 50 p,M test compounds and calculated by the equation v = 3.1 X AA& X 0.05 X 1) U/ml solution, where AA is the decrease in absorbance per minute at 265 nm and e265 = 8.1 mmol - ’ liter cm - ’ Larval weight gain is the average of 5 replicates of 12 fourth instar larvae and the CAMP data are the average of duplicates, range values being shown in parentheses. The dietary compound concentrations (ppm) are for larval weight gain and CAMP level measurements. In the control, the larval weight gain, CAMP level, and the phosphodiesterase activity were 0.536 * 0.008 mg, 0.59 2 0 pmoVmg of larvae, and 0.406 f 0.045 U/ml, respectively. ’ Cited from Ref. (7). ’ Measured 3 days after treatment. * A single measurement. e Cited from Ref. (9). f Cited from Ref. (8). B Measured as sodium salt (NSCP). h Figure in parentheses is activity measured with 100 p.M KSCP.
monoamine catabolism through N-acetyl transferase. According to Bodnaryk (31), organophosphates appear to affect the level of cGMP indirectly through the following sequence of events: inhibition of AChE; accumulation of acetylcholine; stimulation of guanylate cyclase by acetylcholine; elevation of cGMP. Secondary interactions in the nervous system resulting from elevated cGMP levels may also lead to altered CAMP Ieveis, but such interactions have not been determined yet in insects. Kinetic studies on the CAMP hydrolysis by beef heart phosphodiesterase revealed a K, of 48 l.& and a V,,, of 0.83 U/ml (Fig.
2). Fenitrothion (20, 40, and 60 FM), DMOS (75 and 100 ~L,M), and IBMX (OS, 1, and 4 CLM) increased lu, to 70, 111, 125,80, 89,65, 109, and 154 PM, respectively, with little effect on V,,,, indicating that these compounds inhibited CAMP phosphodiesterase in a competitive manner: Ki values for DMOS, fenitrothion, and IBMX were 107.9, 37.3, and 1.5 l&f, respectively, according to Fig. 2. Thus, DMOS and fenitrothion might inhibit the phosphodiesterase in a similar manner with the classical phosphodiesterase inhibitor such as IBMX, which interacts directly with the catalytic site on phosphodiesterase and
194
HIRASHIMA,
-2 -1
I 2 3 4 lIENIl R
-100
OYAMA,
[ OYOSII/N
AND
100
ET0
-30
IFmlrathionlR
30
60 6OuN
40
i
w
I
I/lcANPI A
2 3 4 Y IO-’
I
-2 -1
IilclNPI
1 2 3 4
2. Double-reciprocal plot of the inhibition IBMX, DMOS, and fenitrothion. FIG.
competitively interferes with the binding of the cyclic nucleotide substrates (32). In order to clarify the relationship, if any, between the increased whole-body CAMP level and the reduced trehalase activity leading to the larval weight gain suppression (59), more detailed experiments are in progress.
au
x 10-z
4.
5.
6.
7.
REFERENCES
1. M. Eto, Y. Kinoshita, T. Kato, and Y. Oshima, Saligenin cyclic methyl phosphate and its thiono analogue: New insecticides related to the active metabolite of tri-o-cresyl phosphate, Nature (London) 200, 171 (1%3). 2. M. Eto, A. Hirashima, S. Tawata, and K. Oshima, Structure insecticidal activity relationship of five-membered cyclic phosphoramidates derived from amino acids, .I. Chem. Sot. Japan 5, 705 (1981). 3. S.-Y. Wu, A. Hirashima, R. Takeya, and M. Eto,
I
I/lcANPI R
2 3 4 x IO-”
of CAMP phosphodiesterase
ACKNOWLEDGMENTS
We thank Dr. Masahiko Kuwahara of the National Food Institute in Japan for the supply of T. castuneum, Dr. Makoto Mizunami of the Faculty of Science at Kyushu University for donating the cockroaches (P. americana), and our colleague Yutaka Yoshii for technical assistance. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.
-2 -I
8.
9.
from beef heart by
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10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
ADENYLATE
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CYCLASE,
22.
23. 24.
25.
26.
27.
28.
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AND
PHOSPHODIESTERASE
195
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