Molecular cloning and functional characterization of the dopamine transporter from Eloria noyesi, a caterpillar pest of cocaine-rich coca plants

Cocaine is produced by coca plants as a chemical defense to deter feeding by insects. It has been shown that cocaine sprayed on tomato leaves reduces insect feeding, causes abnormal behaviors at low doses and kills feeding insects at doses equivalent to that in coca leaves [Nathanson, J.A., Hunnicutt, E.J., Kantham, L., Scavone, C., 1993. Cocaine as a naturally occurring insecticide. Proc. Natl. Acad. Sci. U. S. A. 90, 9645-9648.]. Most insects avoid coca leaves except the larvae of Eloria noyesi, a caterpillar pest of coca plants, which feeds preferentially on coca leaves. In the current study, we cloned and characterized the dopamine transporters (DATs) from caterpillars of E. noyesi (enDAT) and the silkworm, Bombyx mori (B. mori, bmDAT). The two insect DATs shared 88% amino acid sequence homology and functional similarity. Although enDAT and bmDAT showed the highest affinity for dopamine among endogenous amines, they were more sensitive to mammalian NET-selective inhibitors than to mammalian DAT-selective inhibitors. Despite a high cocaine content in the food source for E. noyesi, cocaine sensitivity of enDAT was similar to that of bmDAT, suggesting that mechanisms other than DAT insensitivity to cocaine, such as cocaine sequestration, might be responsible for cocaine resistance in this species. Given the significant differences in pharmacological profile from mammalian DATs, invertebrate DATs provide excellent tools for identifying regions and residues in the transporters that contribute to high-affinity binding of psychostimulants and antidepressants.     

Focused microwave-assisted extraction of cocaine and benzoylecgonine from coca leaves

Extraction of cocaine and benzoylecgonine from coca leaves was performed by focused microwave-assisted extraction (FMAE). Cocaine extraction was optimised with respect to the nature of the extracting solvent, the particle size distribution, the moisture of the sample, the applied microwave power and the radiation time. A central composite design was used to optimise the two latter parameters and to assess the robustness of the extraction method around the best conditions. FMAE generated extracts similar to those obtained by conventional solid-liquid extraction but in a more efficient manner, i.e. 30 s were sufficient to extract cocaine quantitatively from leaves. Analyses of cocaine and benzoylecgonine in coca leaves was carried out by capillary GC-FID and GC-MS for peak identification, as well as by capillary electrophoresis with UV detection.     

Identification of Erythroxylum taxa by AFLP DNA analysis

Erythroxylum coca, indigenous to the Andean region of South America, is grown historically as a source of homeopathic medicine. However, in the last century, cultivation of E. coca and several closely-related species for the production of illicit cocaine has become a major global problem. Two subspecies, E. coca var. coca and E. coca var. ipadu, are almost indistinguishable phenotypically; a related cocaine-bearing species also has two subspecies (E. novogranatense var. novogranatense and E. novogranatense var. truxillense) that are phenotypically similar, but morphologically distinguishable. The purpose of this research was to discover unique AFLP DNA patterns ("genetic fingerprinting") that characterize the four taxa and then, if successful, to evaluate this approach for positive identification of the various species of coca. Of seven different AFLP primer pairs tested, a combination of five proved optimal in differentiating the four taxa as well as a non-cocaine-bearing species, E. aerolatum. This method of DNA fragment separation was selective, and faster, for coca identification, compared with analyses based on flavonoid chemotaxonomy. Using the 5-primer AFLP approach, 132 known and unknown coca leaf accessions were evaluated. Of these, 38 were collected in 1997-2001 from illicit coca fields in Colombia, and all were genetically differentiated from coca originating in Peru and Bolivia. Based on the DNA profiling, we believe that the Colombian coca now represents a hybridization of E. coca var. ipadu. Geographical profiling within Colombia also seems feasible as new coca production areas are developed or new types of coca are introduced within traditional growing areas.     

Coca Leaf and Cocaine Addiction: Some Historical Notes

Coca-leaf habituation has affected millions of Andean natives for over 400 years. In the last half-century it has also involved millions more Malayans. Coca leaf, from which cocaine and extracts for some commercial carbonated soft drinks are obtained, remains relatively unknown by the medical and allied professions elsewhere. A review of the original medical, historical and other pertinent literature of the last 350 years illustrates the origins of the use of coca leaf, its spread, the isolation of cocaine and its first uses, as well as some of the euphoric and other effects of both substances.     

Experimental design in supercritical fluid extraction of cocaine from coca leaves

An optimisation procedure for the supercritical fluid extraction (SFE) of cocaine from the leaves of Erythroxylum coca var. coca was investigated by means of experimental design. After preliminary experiments where the SFE rate-controlling mechanism was determined, a central composite design was applied to evaluate interactions between selected SFE factors such as pressure, temperature, nature and percentage of the polar modifier, as well as to optimise these factors. Predicted and experimental contents of cocaine were compared and robustness of the extraction method estimated by drawing response surfaces. The analysis of cocaine in crude extracts was carried out by capillary GC equipped with a flame ionisation detector (GC-FID), as well as by capillary GC coupled with a mass spectrometer (GC-MS) for peak identification.     

Inter- and intra-specific variation among five Erythroxylum taxa assessed by AFLP

BACKGROUND: and Aims The four cultivated Erythroxylum taxa (E. coca var. coca, E. novogranatense var. novogranatense, E. coca var. ipadu and E. novogranatense var. truxillense) are indigenous to the Andean region of South America and have been cultivated for folk-medicine and, within the last century, for illicit cocaine production. The objective of this research was to assess the structure of genetic diversity within and among the four cultivated alkaloid-bearing taxa of Erythroxylum in the living collection at Beltsville Agricultural Research Center. METHODS: Amplified fragment length polymorphism (AFLP) fingerprinting was performed in 86 Erythroxylum accessions using a capillary genotyping system. Cluster analysis, multidimensional scaling (MDS) and analysis of molecular variance (AMOVA) were used to assess the pattern and level of genetic variation among and within the taxa. KEY RESULTS: A clear distinction was revealed between E. coca and E. novogranatense. At the intra-specific level, significant differentiation was observed between E. c. var. coca and E. c. var. ipadu, but the differentiation between E. n. var. novogranatense and E. n. var. truxillense was negligible. Erythroxylum c. var. ipadu had a significantly lower amount of diversity than the E. c. var. coca and is genetically different from the E. c. var. ipadu currently under cultivation in Colombia, South America. CONCLUSIONS: There is a heterogeneous genetic structure among the cultivated Erythroxylum taxa where E. coca and E. novogranatense are two independent species. Erythroxylum coca var. coca is most likely the ancestral taxon of E. c. var. ipadu and a founder effect may have occurred as E. c. var. ipadu moved from the eastern Andes in Peru and Bolivia into the lowland Amazonian basin. There is an indication of artificial hybridization in coca grown in Colombia.     

Coca chewing in prehistoric coastal Peru: dental evidence

In this study, we describe the dental health of four prehistoric human populations from the southern coast of Peru, an area in which independent archaeological evidence suggests that the practice of coca-leaf chewing was relatively common. A repeated pattern of cervical-root caries accompanying root exposure was found on the buccal surfaces of the posterior dentition, coinciding with the typical placement of coca quids during mastication. To further examine the association between caries patterning and coca chewing, caries site characteristics of molar teeth were utilized as indicators for estimating the likelihood of coca chewing for adults within each of the study samples. Likelihood estimates were then compared with results of a test for coca use derived from hair samples from the same individuals. The hair and dental studies exhibited an 85.7% agreement. Thus, we have demonstrated the validity of a hard-tissue technique for identifying the presence of habitual coca-leaf chewing in ancient human remains, which is useful in archaeological contexts where hair is not preserved. These data can be used to explore the distribution of coca chewing in prehistoric times. Simultaneously, we document the dental health associated with this traditional Andean cultural practice.     

Tissue distribution of cocaine in the pregnant rat

Cocaine hydrochloride was administered by single intraperitoneal (IP) doses to pregnant rats at day 18 or 19 of gestation. Plasma and tissue cocaine and norcocaine concentrations were measured by high-pressure liquid chromatography. Pharmacokinetic analysis of concentration versus time data showed rapid distribution of cocaine and its metabolite to maternal and fetal tissues. The area under the cocaine concentration versus time curve (AUC) in fetus compared to maternal plasma was 3.33. The half-life of cocaine in the maternal plasma and fetus was 46 and 55 minutes, respectively, similar to values reported for cocaine elimination half-life in human plasma. The order of cocaine concentrations was placenta greater than fetal liver greater than maternal heart greater than whole fetus greater than fetal brain greater than maternal brain = maternal plasma. Norcocaine concentrations were usually less than 20% of cocaine concentrations in plasma and tissues. These results support extensive fetal exposure to cocaine following administration to pregnant rodents. Pharmacodynamic studies of cocaine in pregnancy should consider the effects of the drug on the developing fetus.     

Cocaine and Cinnamoylcocaine Content of Erythroxylum Species

The dried leaves of 31 species and two varieties of the genusErythroxylum were quantitatively analysed for cocaine and bothcis- and trans-cinnamoylocaine, using a stable-isotope dilutionmethod and gas chromatography-mass spectrometry selected-ionmonitoring. Both cocaine and cinnamoylcocaine were detectedin all four varieties of cultivated coca (E. coca var. coca,E. coca var. ipadu, E. novogranatense var. novogranatense andE. novogranatense var.truxillense). Cinnamoylcocaines (cis andtrans) were found in much higher concentrations in both varietiesof E. novogranatense than in either variety of E. coca. Amazoniancoca (E. coca var. ipadu) contained consistently lower cocainelevels than the montane variety (E. coca var. coca). Twenty-ninewild species of Erythroxylum, selected to represent morphological,ecological and taxonomic diversity, were analysed for the samealkaloids; cocaine was detected in 13 neotropical species, representingfive sections of the genus. No cocaine was detected in the OldWorld species. Two wild species from Venezuela, E. recurrensand E. steyermarkii, contained cocaine levels comparable tothose found in the commercially cultivated species. Erythroxylaceae, Erythroxylum, coca, cocaine, cinnamoylcocaine, alkaloids, chemotaxonomy     

Responses of Quechua Indians to coca ingestion during cold exposure

Fourteen Quechua men chewed coca leaves as they were exposed to a mild cold stress of 15.5°C for two hours. When compared to their own responses in a similar exposure without coca, they showed lower finger and toe temperatures. This was attributed to a mild vasoconstriction induced by coca leaf chewing. During the second hour of cold, coca users also showed a more gradual decline in core temperature which could represent greater heat conservation.     

Pharmacologic approaches to the treatment of cocaine dependence.

When pharmacologic agents are considered in the treatment of cocaine addiction, the objective of such treatment--sustained abstinence--must be considered. Medication and medical approaches have been disappointing in the treatment of cocaine overdose. The central neurobiologic mechanism(s) involved in cocaine toxicity are poorly understood. Without a cocaine antagonist, pharmacologic approaches have been less than promising in preventing relapse. Various psychoactive medications have been tried in early cocaine abstinence, with some success.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.     

Effects of alcohol and coca on foot temperature responses of highland Peruvians during a localized cold exposure

Two groups of highland Quechua Indian males were tested under conditions of local foot exposure to cold air (0°C). Foot temperatures were monitored throughout the hour cold test and for 16 minutes recovery at room temperature (24°C). In the first group (age range 14–20 years), 29 subjects were tested while chewing coca leaves and while under control conditions. The second group (age range 20–50 years) of 25 subjects was tested while consuming 1.1 gm of ethyl alcohol per kilogram of body weight and again under control conditions. Both drugs (coca and alcohol) are habitually consumed by members of the native population. The mastication of coca leaves had no effect on foot skin temperatures. Alcohol consumption, however, elevated foot temperatures to between 4 and 6°C higher than control values at the end of 60 minutes of cold exposure. It is suggested that alcohol consumption gives the Indian a slight thermal advantage and increases levels of comfort during natural cold exposure.     

Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.     

Body fluid homeostasis and cardiovascular adjustments during submaximal exercise: influence of chewing coca leaves

 The present study was undertaken to determine the haematological and cardiovascular status, at rest and during prolonged (1 h) submaximal exercise (approximately 70% of peak oxygen uptake) in a group (n = 12) of chronic coca users after chewing approximately 50 g of coca leaves. The results were compared to those obtained in a group (n = 12) of nonchewers. At rest, coca chewing was accompanied by a significant increase in heart rate [from 60 (SEM 4) TO 76 (SEM 3) beats · min⁻¹], in haematocrit [from 53.2 (SEM 1.2) to 55.6 (SEM 1.1)%] in haemoglobin concentration, and plasma noradrenaline concentration [from 2.8 (SEM 0.4) to 5.0 (SEM 0.5) μmol · l⁻¹]. It was calculated that coca chewing for 1 h resulted in a significant decrease in blood [−4.3 (SEM 2.2)%] and plasma [−8.7 (SEM 1.2)%] volume. During submaximal exercise, coca chewers displayed a significantly higher heart rate and mean arterial blood pressure. The exercise-induced haemoconcentration was blunted in coca chewers compared to nonchewers. It was concluded that the coca-induced fluid shift observed at rest in these coca chewers was not cumulative with that of exercise, and that the hypovolaemia induced by coca chewing at rest compromised circulatory adjustments during exercise. Accepted: 29 October 1996     

A cannabinoid mechanism in relapse to cocaine seeking

Treatment of cocaine addiction is hampered by high rates of relapse even after prolonged drug abstinence. This relapse to compulsive cocaine use can be triggered by re-exposure to cocaine, by re-exposure to stimuli previously associated with cocaine or by exposure to stress. In laboratory rats, similar events reinstate cocaine seeking after prolonged withdrawal periods, thus providing a model to study neuronal mechanisms underlying the relapse to cocaine. The endocannabinoid system has been implicated in a number of neuropsychiatric conditions, including drug addiction. The active ingredient of marijuana, Delta9-tetrahydrocannabinol, activates the mesolimbic dopamine (DA) reward system and has rewarding effects in preclinical models of drug abuse. We report here that the synthetic cannabinoid agonist, HU210 (ref. 13), provokes relapse to cocaine seeking after prolonged withdrawal periods. Furthermore, the selective CB1 receptor antagonist, SR141716A (ref. 14), attenuates relapse induced by re-exposure to cocaine-associated cues or cocaine itself, but not relapse induced by exposure to stress. These data reveal an important role of the cannabinoid system in the neuronal processes underlying relapse to cocaine seeking, and provide a rationale for the use of cannabinoid receptor antagonists for the prevention of relapse to cocaine use.     

Cocaethylene: a neuropharmacologically active metabolite associated with concurrent cocaine-ethanol ingestion

High concentrations of cocaethylene (EC), the ethyl ester of benzoylecgonine, were measured in the blood of individuals who had concurrently used cocaine and ethanol. Since the powerful reinforcing effects of cocaine appear to be dependent on inhibition of dopamine reuptake in brain, we compared the effects of EC on the dopamine uptake system and its behavioral effects with those of cocaine. EC was equipotent to cocaine with respect to inhibition of binding of [3H]GBR 12935 to the dopamine reuptake complex, inhibition of [3H]dopamine uptake into synaptosomes and in its ability to increase extracellular dopamine concentration in the nucleus accumbens following its systemic administration to rats. Moreover, in rats, EC and cocaine each increased locomotor activity and rearing to the same extent following i.p. administration. In self-administration studies in primates, EC was approximately equipotent to cocaine in maintaining responding. The in vivo formation of this active, transesterified ethyl homolog of cocaine may contribute to the effects and consequences of combined cocaine and ethanol abuse.     

不同背景的同伴强化及催产素处理对雌性棕色田鼠可卡因条件位置偏爱的影响

同伴间的社会互作是一种天然奖赏,能够影响成瘾药物使用的敏感性。催产素（Oxytocin,OT)能够调节社会行为,并提高社会互作的奖赏价值。然而不同背景的同伴互作以及与OT合并使用是否对可卡因的奖赏效应有不同的影响尚不清楚。棕色田鼠(Microtus mandarinus)是一种单配制田鼠,个体间具有较复杂的社会行为。利用雌性棕色田鼠,我们首先检测了可卡因(20 mg/kg)单独强化以及与不同背景关系的同伴（熟性雌性、陌生雌性和陌生雄性）同时强化诱导的条件位置偏爱（conditioned place preference, CPP）及持续时间;其次检测了实验鼠外周注射OT (1 mg /kg)并给予不同背景同伴强化对可卡因CPP的影响。结果表明,可卡因单独强化时,实验鼠能够形成可卡因CPP并能持续至3周;用熟悉的雌性同伴强化时,实验鼠对可卡因CPP的维持时间缩短;用陌生的雌性或雄性同伴强化时可抑制可卡因CPP的形成。实验鼠注射OT后,用熟悉雌性或陌生雄性同伴分别强化时会抑制或反转可卡因CPP。这些结果表明不同背景关系的同伴强化对可卡因奖赏效应的影响不同。OT可促进同伴强化的奖赏价值,降低动物对可卡因的偏爱,且该效应因强化同伴的背景而不同。     

Effect of coca-leaf chewing on salivary progesterone assays

Although there is evidence for reduced fertility in Andean and Himalayan populations at higher altitudes, factors other than hypoxia may be primarily responsible. A valuable approach in the investigation of these fertility determinants is the use of salivary steroid assays. However, coca-leaf chewing—a ubiquitous practice among high altitude Andean populations—has negative consequences for the accurate measurement of ovarian steroids. This report evaluates the effects of coca-leaf chewing on assays of salivary progesterone. Study participants include naive and habitual users of coca leaf from La Paz and El Alto, Bolivia. Approximately 300 saliva samples were collected immediately before, during, and after coca-leaf chewing. The series includes samples with and without the alkaloid enhancer typically used by coca-leaf chewers. Coca chewing produces false salivary progesterone values that mimic luteal phase values. On the basis of this study, an appropriate protocol is developed for the collection of salivary samples in coca-leaf chewing populations. These results verify the feasibility of salivary assays, even for very difficult field conditions, and highlight the necessity of establishing suitable collection procedures before full field implementation of saliva sampling. © 1993 Wiley-Liss, Inc.