High-affinity uptake of [3H]serotonin in cultured neurones of the cockroach Periplaneta americana.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 12:253-266 (1 989) High-Affinity Uptake of [3H]Serotonin in Cultured Neurones of the Cockroach Periplaneta arnericana I s abel Bermudez a n d D a v i d J. Beadle School of Biological and Molecular Sciences, Oxford Polytechnic, Headington, Oxford, England Cultured central neurones from the American cockroach, Periplaneta arnericana, have been used to investigate the uptake of [3H]serotonin. The neurones accumulate [3H]serotonin from the extracellular medium by both a highand a low-affinity system. The activity of the high-affinity mechanism i s decreased by low temperature and metabolic poisons, and i s dependent on sodium and chloride ions. Both depolarising levels of external potassium ions and veratridine decrease the high-affinity uptake system, suggesting i t i s influenced by the transmembrane potential. The pyrethroid insecticides, deltamethrin and permethrin, enhance the inhibitory effect of veratridine. Pyrethroid enhancement is completely blocked by tetrodotoxin, and neither pyrethroid affects the uptake system in the absence of veratridine. Avermectin BA I is a powerful inhibitor of the high-affinity uptake system, and its effect i s blocked by picrotoxin. High-affinity uptake of [3H]serotonin i s inhibited by imipramine and amitriptyline; desipramine has no significant effect o n this uptake. The activity of the high-affinity system is also reduced by 8-hydroxy-dipropylaminotetralin, a-methyl-serotonin, and l-(3-chloropheny1)piperazine. Dopamine, noradrenaline, octopamine, and the formamidine insecticides, chlordimeform and demethylchlordimerform, are moderate inhibitors of the high-affinity uptake system. The formamidine effect is not blocked by tetrodotoxin or picrotoxin. Key words: avermectin, formamidine, ion-channel blocker, neurotoxicant, pesticide, pyrethroid, sodium gradient The authors thank CIBA-GEIGY Ltd., Basel, for financial support during the course of this work. Received July 18,1989; accepted December 18,1989. Address reprint requests to David J. Beadle, School of Biological and Molecular Sciences, Oxford Polytechnic, Headington, Oxford OX3 OBP, England. 0 1989 Alan R. Liss, Inc. 254 Bermudez and Beadle INTRODUCTION There is now strong experimental evidence that 5HT" is a neurotransmitter in the central nervous system of vertebrates [I] and molluscs . This contrasts with the situation in the CNS of insects where a transmitter or modulator role for 5HT has not yet been fully established. However, several lines of investigation suggest this may be the case. 5HT and 5HT-immunoreactive neurones are distributed widely in the insect central nervous system [3-121. Serotonergic neurones appear early in development [7,13] and some persist throughout postembryonic life , thus suggesting that SHT may influence the development of the insect CNS. Radioligand binding studies have identified at least one type of high-affinity binding site for 5HT in brain homogenates of Locusta  and Drosophila , and in frozen sections of the honeybee brain . A few studies have also shown that 5HT affects the electrical activity of central neurones in several insect species [17-191. In vertebrates  and molluscs [21-231, the possession of a high-affinity sodium-and energy-dependent SHT uptake system is considered to be a major characteristic of serotonergic neurones. It is thought that re-uptake is the principal mechanism of inactivation of synaptically released 5HT in both molluscs [21,24]and vertebrates [20,25]. Recent work in our laboratory has shown that cockroach central neurones in culture possess a sodium-dependent uptake system for 5HT . Rapid uptake of 5HT has also been reported to occur in isolated cockroach nerve cords . In an attempt to characterise the insect 5HT uptake system more fully we have continued investigating some of its properties in cockroach cultured neurones. In the present work we describe the ionic and pharmacological specificities of the cockroach high-affinity 5HT uptake system. The effect of various neurotoxicants and pesticides on this uptake system is also reported. MATERIALS AND METHODS Chemicals [3H]5HT(28.5-30.4 Ci/mmol) was obtained from Dupont (UK) Ltd., Herts, England. The following vertebrate serotonergic drugs were obtained from Semat Technical Ltd., Herts, England: MDL 72222 (an antagonist of the 5HT3 receptor), 8-OH-DPAT (a 5HTIA receptor agonist), a-met5HT (an agonist of the 5HT2 receptor), and mCPP (a 5HTIBreceptor agonist). AMP, DSMP, SHT, IMP, LS, PTX, TTX, and VTN were from Sigma, England. AVM, CDM, DCDM, LS, MDL 72222, and the pyrethroids DMT and PMT were dissolved in approximately 0.1% dimethylsulfoxide or less. In control experiments 0.1-1% dimethylsulfoxide produced no effect on [3H]5HTuptake. *Abbreviations used: ACH = acetylcholine; AMP = amitriptyline; AVM = avermectin B,A; CDM = chlordimeform; CNS = central nervous system; mCPP = 1-(3-~hlorophenyl)piperazine; CU saline = sodium-free saline; DCDM = demethylchlordimeform; DMT = deltamethrin; DSMP = desipramine; GABA = y-aminobutyric acid; 5HT = serotonin; IMP = imipramine; LS = lysergic acid; MDL72222 = 3-tropanyl-3,5-dichlorobenzoate; a-met-5HT = a-methylserotonin; 8-OH-DPAT = ( t -8-hydroxydipropyl-aminotetralin; PMT = permethrin; PTX = picrotoxin; TTX = tetrodotoxin; TU saline = sodium-containing saline; VTM = veratridine. Serotonin Uptake by Insect Neurones 255 t3H15HT Uptake Studies Cell culture. Neuronal cultures were prepared from the cerebral ganglia of 22-23 day-old embryos of Periplanetu americana cockroaches as described in detail elsewhere . The cultures were maintained at 29°C for 20-22 days prior to use for [3H]5HTuptake studies. I3H15HT uptake. A single culture dish was used as an assay unit. Cultures were washed twice, preincubated at 29°C for 10 min in TU saline, and then incubated for a standard period of 4 min (2 to 25 min for time course experiments) in TU saline containing various concentrations of [3H]5HT(0.28-2.86 KCi per culture dish). In all cases a series of parallel assays containing the same amount of radiolabelled 5HT but performed in CU saline was used to access sodium-independent uptake, and these values have been substracted from all experimental results. After incubation with [3H]5HT, all cultures were washed rapidly three times (approximately 30 s in all) with ice-cold CU saline. The cultures were then dissolved in 1 ml of Lowry’s solution C  containing 1%sodium dodecyl sulfate  and protein content was determined according to Lowry et al. . Due to limitations in the amount of material (30-60 pg protein per culture dish), an aliquot (0.8 ml) of the final Lowry solution was counted for radioactivity in Hyonic Fluor scintillant (Packard Inst. Co, Berks, England) following protein estimation. Counting efficiency was approximately 48%. The effect of metabolic inhibitors, drugs, and neurotoxicants on [3H]5HT uptake was examined by including these substances in both the preincubation and incubation media. TU saline was composed of 200 mM NaC1,3.1 mM KC1,9 mM CaC12, 10 KM pargyline (an inhibitor of monoamine oxidase), 0.1% ascorbic acid, 5 mM and HEPES, pH 7.2. Ascorbic acid was added to all assays because of its positive effect on [3H]5HTstability . In some experiments the potassium concentration was increased to either 10 or 20 mM. In making a chloride-free medium, NaCl was replaced with sodium isethionate, and KC1 and CaC12were replaced by potassium acetate and calcium acetate respectively. The CU saline was obtained by replacing NaCl with choline chloride. 13H15HT autoradiography. After incubation and washing the cultures were prepared for autoradiography. After the final wash the cultures were fixed in situ in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2 containing 0.25 M sucrose at 4°C for 30 min, washed twice in buffer, airdried, and coated with Ilford K5 nuclear emulsion diluted 1:l with distilled water. After 3 weeks exposure at 4°C the autoradiographs were developed in Kodak D19 for 5 min at 20°C and fixed in Ilfofix before examination with bright-field optics. Positive and negative chemography control cultures were included in each batch of autoradiographs prepared. Unlabelled cultures provided a check for positive chemography and a measure of background radioactivity. A further culture exposed to brief flashes of light after coating with emulsion was used to assess the extent of any negative chemography and the integrity of the applied emulsion layer. Statistical analysis. The effect of ions, metabolic inhibitors, drugs, and neurotoxicants on [3H]5HTuptake was assessed for significance against appro- Bermudez and Beadle 256 priate controls by Student’s two-tailed t-tests. The uptake kinetic constants (K, and V,,) and their respective standard errors were calculated according to Wilkinson . RESULTS After 20-22 days in vitro neuronal cultures of Peviplaneta arnericana rapidly accumulated [3H]5HTfrom the extracellular medium when incubated in TU saline. The kinetics of the accumulation of [3H]5HTinto neuronal cultures was determined by incubating the cultures for 4 min at 29°C with various concentrations of [3H]5HT(Fig. 1A). Analysis of the data by Lineweaver-Burk plot (Fig. 1B) revealed a ”high-affinity” system with an affinity constant (K,) of 0.23 ? 0.02 pM and a maximal velocity of uptake (V,,,) of 8.17 ? 0.32 pmol/min/mg prot., and a ”low-affinity” uptake with a K, of 2.14 ? 0.25 pM and a V,, of 27.91 & 4.0 pmol/min/mg prot. (Fig. 1B). The K, value of the high-affinity system was comparable to the K, reported for its vertebrate counterpart (0.1-0.8 pM) [33,34], whereas the K, of the low-affinity system was much lower than the K, values reported for the vertebrate low-affinity system (7-9 pM) [33-351. The V,,, values of the high- and low-affinity systems were lower than their vertebrate counterparts (0.6-2 pmol/min/g and 8-26 pmol/min/g respectively) [33,34]. In order to measure the relative preponderance of the high- and low-affinity uptake systems at different [3H]5HTconcentrations, the velocites of both transport systems were calculated from the Michaelis-Menten equation v = V,, [S]/ K, + [S] at various substrate concentrations. As Table 1shows, the relative preponderance of both systems changed with increasing [3H]5HT concentrations. At lower concentrations (0.1 to approxi- B 10 Km023?D02pM 0 4 -1 v 5 V 02 I K, , ,V , / 05 S 1 2 1 4 + 0 . 2 5 pM 27.9 1 t 4.0 prnol/rnin/rng 5 10 1 S Fig. 1. A : Accumulation of [3H]5HTby cockroach neurones in vitro by using increasing concentrations of [3H]5HT. Incubation and processing of all samples were as detailed in Materials and Methods. Data are the results of three experiments performed in triplicate. V (initial velocity of uptake) i s in pmol/min/mg protein and s (substrate concentration) i s in pM. B: Doublereciprocal plot of the above data. Serotonin Uptake by Insect Neurones 257 TABLE 1. Relative Velocity of [3H]5HT Accumulation by the High- and Low-Affinity Uptake Systems at Varying 13H]5HTConcentrations* V (pmol/min/mg prot) High-affinity uptake Low-affinity uptake [3H]H5HT 0.1 ?.2 2.5 3.8 5.6 5.9 6.4 6.6 0.: 0.6 0.8 1 1.2 2.4 5.3 6.1 7.6 8.9 Total uptake 3.7 6.2 10.9 12.0 14.0 15.5 *Kinetic constants for the dual-affinity uptake system were substituted in the Michaelis-Menten equation, v = V,,, x SIK,, + S. mately 0.5 pM) the velocity of the high-affinity system exceeded that of the low-affinity component, while at higher concentrations (higher than 0.6 pM) the reverse was true. Thus, at 0.1 and 0.2 pM the high-affinity system would be expected to account for 68% and 61% of the total accumulated [3H]5HT, whilst at about 1 pM low-affinity transport would be expected to account for 57%. At about 0.6 pM one would expect both systems to contribute about equally to 13H]5HTuptake. Because at 0.1 pM accumulation of [3H]5HToccurred predominantly via the high-affinity transport component we used that substrate concentration to analyse further the high-affinity system. High-affinity accumulation of [3H]5HTinto neuronal cultures was temperaturedependent (Fig. 2). At 29°C uptake was linear for about the first 5 min of incubation and then gradually plateaued. Comparatively little uptake occurred at 29°C 5 - - L 1 10 15 Incubation time Iminutes) - 0°C 0 1 I 20 25 Fig. 2. Accumulation of [3H]5HTinto cockroach neuronal cultures at two different temperatures ptotted as a function of incubation time. Uptake is expressed a5 dpm of I3H]5HTaccumulated per culture. The points represent the means of data from two experiments performed in triplicate. 258 Berrnudez and Beadle 0°C (Fig. 2). The transport system for L3H]5HTalso appeared to be dependent on metabolic energy since both cyanide and iodoacetamide reduced [3H]5HT uptake significantly (Fig. 3). Ouabain, an inhibitor of the NA+-K+-ATPase, reduced uptake by about 76% (Fig. 3). As shown in Figures 3 and 4, high-affinity uptake of [3H]5HTwas almost completely abolished in the absence of sodium ions. Autoradiographs of cultures incubated in TU saline containing labelled 5HT revealed that only some of the cells present in the cultures were heavily labelled. The differential labelling of the cells suggests that high-affinity uptake of [3H]5HTinto insect neurones may be a selective property. This suggestion is further supported by the observation that unlabelled and labelled cells are in close proximity (Fig. 4A, B). Accumulation of [3H]5HTinto the cultured neurones was completely eliminated by CU medium, as shown by the absence of silver grains even over densely populated regions of the cultures (Fig. 4C). Chloride-free saline reduced [3H]5HTaccumulation by almost 90% (Fig. 3). Increasing potassium concentrations significantlyreduced [3H]5HTuptake (Fig. 3). In view of the depolarizing effect of the K + concentrations studied on cultured cockroach neurones  it would appear that reduced transmembrane potentials decrease uptake of [3H]5HT.The high-affinity, sodium-dependent uptake of choline , GABA , and L-glutamate  into cockroach cultured neurones is also inhibited by high concentrations of K + . The results of the ion-substitution studies suggested that ion-channel selective neurotoxicants may interfere with the high-affinity transport of 5HT. We therefore tested the effect of several ion-channel toxic agents on [3H]5HTuptake. VTN is an alkaloid known to activate voltage-dependent sodium channels in mouse [39,40]and fish brain synaptosomes and to inhibit choline uptake 7.0.2 a 24.3 a I l t l a 62?9 40t4 b a 3654 2 7 2 5 a a Fig. 3. Effect of ions and metabolic inhibitors on high-affinity accumulation of I3H]5HT in cultures of cockroach central neurones. Control incubation saline was as described in Materials and Methods. Vertical bars represent the standard error of the determined mean. Student's two-tailed t'-tests: a P < 0.002; b P < 0.02. Serotonin Uptake by insect Neurones 259 Fig. 4. [3H]5HTautoradiography. A and B: Cockroach neurones in culture that were incubated in TU saline (sodium-containing saline) supplemented with 0.1 pM L3H]5HT.The arrows show cells devoid of [3H]5HTlabelling. C:A neuronal culture after incubation in 0.1 pM L3H15HT in the absence of sodium ions. 260 Bermudez and Beadle in rat hippocampal synaptosomes . In cockroach cultured neurones, VTN reduced uptake of [3H]5HTsignificantly at the two concentrations tested (Fig. 5). This inhibitory effect of VTN was also observed in Caf-free medium (data not shown), which is known to inhibit VTN-evoked release of radiolabelled material . Addition of TTX (a blocker of voltage-dependent sodium channels) to the incubation medium had no effect by itself on [3H]5HTuptake, but did block the VTN induced-decreased in [3H]5HTuptake (Fig. 5). AVM, an anthelmintic  and insecticidal  macrocyclic lactone, has been shown to block transmission at arthropod neuromuscular junctions by increasing GABA-mediated C1- permeability  and to increase C1- conductance in cockroach neurones in vitro . In the present study, inclusion of AVM to the incubation medium caused a marked decrease in [3H]5HTaccumulation (Fig. 5). AVM was more effective in inhibiting [3H]5HTuptake than the other pesticides studied (Table 2). The inhibitory effect of AVM was blocked by PTX but not by TTX (Fig. 6), thus indicating that the AVMinduced decrease in [3H]5HT accumulation was probably due to a specific action of AVM upon C1- channels. The formamidine pesticides, CDM and DCDM, were moderate inhibitors M (Table 2), of the uptake of [3H]5HT(Fig. 5). DCDM, with an IC50of 1 X was about six times more potent than CDM ICs0 = 6.3 x M; Table 2). Neither TTX nor PTX had any effect on the formamidine-induced reduction of [3H]5HT uptake (Fig. 6). DMT and PMT, two pyrethroid pesticides known to interact with voltagedependent sodium channels , had no significant effect on the accumulation of [3H]5HT (Fig. 5). However, incubation of the cultured neurones with both VTN and either DMT or PMT produced a decrease in [3H]5HTuptake 1 1L 1 1 T h 5 In u X t- c& h . 7 -B c t 0 0 00 f 11 5 0 N h f 5 u Y) v 7 F> X z c- 69 f 9 C + u T > 95 92 f 11 ns h f 0 u I- h 0 t 4 ns z 4,-u 9 4 t 9 6 2 1 40.1 6 ns ns a v u c k I- B Q 46. 6 b 50: 6 b -f 90 t 15 ns z 93 t 3 ns Fig. 5. Effects of neurotoxicants on the accumulation of [3H15HTinto cultured cockroach neurones. The values are the means of four to five experiments. Vertical bars represent the standard error of the determined mean. Student's two-tailed t'-tests: a P < 0.002; b P < 0.02; c P < 0.05; n s no significant effect. Serotonin Uptake by Insect Neurones 261 TABLE 2. Effects of Pesticides on [3H]5HT Uptake by Cockroach Cultured Neurones+ ICSO (MI Incubation conditions AVM (0.1 pM) CDM (100pM) DCDM (10 pM) DMT + VTN (20 yM) PMT 1.9 X 6.3 x 1 0 - ~ 1.0 x 10-5 2.3 x 10-7 1.1 x + V T N (20 yM) *ICmvalues represent the concentration of pesticides causing 50% inhibition of [“HISHTuptake. T T T i X k- T f1 I>X +I- I- - 2 c C 8 % = 100 t 5 X II- f:< 38t3 a X I- a z Q Z z? I- 5 9423 87.13 ns ns 0 - 28t4 85.10 a ns 37.3 4928 a b Fig. 6. Effect of ion-channel blockers on pesticide-induced inhibition of [3HJ5HTuptake. The concentrations of channel blockers and pesticideswere as follows. PTX and l T X 5 yM; VTN 20 yM: AVM 0.1 pM; DTM and PMTI pM; CDM 100 yM; DCDM 10 yM. The values are the means of four to six experiments. Vertical bars represent the standard error of the determined mean. Student‘s two-tailed t‘-tests: a P < 0.002; b P < 0.02; ns no significant effect. that was greater than the inhibition caused by VTN alone (Fig. 5). The effect of pyrethroid-VTN was completely blocked by TTX (Fig. 6). PTX, a blocker of chloride channels, had not effect upon 13H]5HTuptake or the inhibitory effect of pyrethroid-VTN (Fig. 6). A variety of drugs was examined for effects on the accumulation of [3H]5HT. Unlabelled 5HT was the most potent inhibitor of uptake of [3H]5HT,reducing uptake by about 80% (Fig. 7). The other biogenic amines, dopamine, noradrenaline, and octopamine, were less potent, reducing uptake by only about 38-41% (Fig. 7). ACH, GABA, L-glutamate, and LS had no significant effect on [3H]5HT accumulation (Fig. 7). These data suggest, therefore, that the carrier mediating the high-affinity accumulation of [3H]5HTin cockroach neurones is specific for 5HT. This view was supported by experiments using tricyclic antidepressants. IMP and AMP are specific inhibitors of the vertebrate 5HT uptake system [47,48]. These two tricyclic antidepressants were the second most potent inhibitors of [3H]5HTaccumulation: they produced approximately 76% and 262 Bermudez and Beadle 11 1 1 cu cu cu a, 5 N 5 .t a - o Q _I 1 0 0 0 2 0 z v) J -% = 100 5 t 20. 3 a 60 ! 6 2 % 62 12 19 10 C c c !4 t 27. 1 5 a a I 0 < - 76 t 75 f 103 t 9 5 * 100 2 1 15 15 1 1 15 ns ns ns ns ns Fig. 7. Effects of drugs on a [3H]5HTaccumulation in cultures of cockroach brain neurones in culture. The concentration of all drugs was 10 pM. Values are the means of five to seven experiments. Vertical bars represent the standard error of the determined mean. Student's two-tailed t'-tests. 70% decrease in t3H]5HT uptake respectively (Fig. 7). In contrast, DSMP, a specific inhibitor of catecholamine uptake [47,48], was the least potent of the tricyclic antidepressants studied, being approximately twofold less potent than imipramine (Fig. 7). 8-OH-DPAT, mCPP, and a-met5HT were about equally effective in reducing [3H]5HTuptake (Fig. 7). In contrast, MDL 72222 had no significant effect upon [3H]5HTuptake (Fig. 7). Taken together, these data suggest that the insect high-affinity carrier for 5HT may share some structural specificities with the vertebrate 5HT1 and 5HT2 receptors. DISCUSSION A small population of central cockroach neurones in vitro accumulated 5HT from the extracellular medium via both high- and low-affinity uptake systems. Since the most potent inhibitors of the hgh-affinity uptake system were unlabelled 5HT and inhibitors of the vertebrate 5HT uptake system, the cockroach uptake mechanism appears to be specific for 5HT and pharmacologically similar to the vertebrate system. In both vertebrates  and molluscs [21-231 there is strong evidence that serotonergic neurones are specifically endowed with high-affinity uptake systems for 5HT. Therefore, it is possible that at least some of the cockroach neurones capable of accumulating 5HT from the extracellular medium were serotonergic cells. These results, therefore, support the view that 5HT may act centrally in the insect nervous system [3-121. The ionic, energy, and structural requirements of the high-affinity uptake of 5HT were comparable to those of vertebrates  and molluscs ; 5HT Serotonin Uptake by Insect Neurones 263 uptake was temperature dependent and inhibited by metabolic poisons, and therefore likely to be an active transport system. The ionic requirements of the high-affinity transport of 5HT make it susceptible to inhibition by neurotoxicants that act on sodium and chloride channels. The finding that VTN and AVM are potent inhibitors of sodium-dependent uptake of [3H]5HT is important because it contributes to the elucidation of the mechanism underlying sodium-dependent transport of systems. The inhibitory effects of pyrethroids indicate that the 5HT uptake system might be a secondary target for ion-channel selective pesticides in insects, but we cannot say whether or not it is a useful insecticide target until the importance of serotonergic synapses in the neural function of the insect has been assessed. The VTN-pyrethroid interaction was interesting because it illustrates at the channel level a mode of synergistic action, and therefore raises the possibility of improving the performance of pyrethroid insecticides. The inhibitory effect of TTX on the VTN-induced decrease of [3H]5HTuptake shows that the action of VTN on [3H]5HTuptake was caused by activation of voltage-dependent sodium channels. It is unlikely that the VTN-induced inhibition of uptake was caused by a stimulation of [3H]5HTrelease since the same levels of uptake were found in calcium-free medium. Since the opening of voltage-dependent sodium channels would cause a depolarizing change in the sodium electrochemical gradient the inhibitory effect of VTN supports the view that the sodium transmembrane gradient is the driving force in sodiumdependent transport processes . The inhibitory effects of increased external K + and ouabain-induced inhibition of Na+-K+-ATPase on [3H]5HT are also consistent with the sodium gradient hypothesis because both K+ and the sodium pump are required for maintaining the sodium gradient across the membrane. The inhibitory effect of C1-free incubation saline on the uptake of 5HT was comparable to its effect on other neurotransmitter uptake systems present in vertebrates  and invertebrates [38,50]. The r81e of chloride ions in neurotransmitter high-affinity transport systems has not been fully clarified. It has been suggested that C1- may help to preserve electrical neutrality during transport . In cockroach cultured neurones this may not be the case since the effect of AVM on 5HT uptake suggests that a subpopulation of insect neurones grown in culture conditions might contain high levels of chloride ions in the resting state. In these cells, activation of chloride channels by AVM or challenge with a C1-freeexternal environment would result in an outward movement of chloride ions which may be sufficient to cause a depolarisation of the neurones. As shown by the inhibitory effect of VTN and elevated levels of external K+, depolarisation of the cells leads to a depression of 5HT uptake. The VTN-pyrethroid-induced inhibition of [3H]5HTuptake was blocked by TTX but not by PTX, thus showing that the action of pyrethroid-VTN on the transport of [3H]5HTwas specificallymediated via the voltage-dependent sodium channels. Since pyrethroids are known to slow the inactivation and thus closure of sodium channels the VTN-pyrethroid interaction, which has also been observed in the uptake of sodium ions by vertebrate synaptosomes  suggests that pyrethroids might exert their action by prolonging the open time of sodium channels activated by VTN. 264 Bermudez and Beadle The formamidines, CDM and DCDM, were poor inhibitors of 5HT uptake. These compounds also appear to have very little effect on the uptake of 5HT by cockroach nerve cords , thus suggesting that inhibition of 5HT uptake is a secondary action of these pesticides in cockroach neurones. Since CDM and DCDM interact with 5HT- and dopamine-sensitive sites in the cockroach CNS in addition to their effects on the insect octopamine receptors [52-541, the effect of CDM and DCDM on 5HT uptake may have involved an interaction with the 5HT carrier. 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