Histamine Recycling Is Mediated by CarT,a Carcinine Transporter in Drosophila Photoreceptors

Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to β-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, we identified CG9317, an SLC22 transporter family protein, and named it CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals.     

The role of carcinine in signaling at the Drosophila photoreceptor synapse

The Drosophila melanogaster photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG) analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized inebriated protein. The inebriated gene (ine) encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. We also show that by feeding wild-type flies carcinine, we can duplicate components of mutant ine ERGs. Finally, we demonstrate that treatment with H₃ receptor agonists or inverse agonists rescue several components of the mutant ine ERG phenotype. Here, we provide pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. We also speculate that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H₃ receptor.     

Tyrosine and catecholamine levels in the hemolymph of tobacco hornworm larvae,Manduca sexta,parasitized by the braconid wasp,Cotesia congregata,and in the developing parasitoids

Parasitism of fifth instar Manduca sexta larvae by the gregarious parasitoid Cotesia congregata prevented normal storage of tyrosine in the hemolymph, whereas total tyrosine levels increased over eight times in the hemolymph of unparasitized larvae by day 4. Tyrosine glucoside, the hemolymph storage form of tyrosine and the precursor for pupal cuticle sclerotizing agents, was found only in trace amounts in parasitized larvae at the time of parasitoid emergence, but had increased to over 6 mM in hemolymph of unparasitized larvae. Concentrations of dopamine and N-β-alanyldopamine (NBAD), precursors for melanization and sclerotization of cuticle, respectively, had approximately doubled in the hemolymph of parasitized larvae by the day of parasitoid emergence, but not in unparasitized larvae. Catecholamine biosynthesis may be transiently stimulated for wound-healing, as black melanic pigmentation appeared around the wasp emergence holes in the host integument. C. congregata larvae accumulate tyrosine, dopamine, and NBAD by the time of emergence and cocoon spinning, either by direct uptake or by synthesis from precursors obtained from the host. NBAD increased in parasitoid larvae close to pupation, suggesting it functions as the main precursor for pupal cuticle tanning. Both dopamine and NBAD increased dramatically in pharate adult wasps just before eclosion and N-acetyldopamine (NADA) appeared for the first time. Dopamine was highest in concentration and total amount, and it can serve both as a precursor for black melanic pigmentation of adult wasp cuticle and for synthesis of NADA and NBAD, the precursors for cuticle sclerotization. Arch. Insect Biochem. Physiol. 38:193–201, 1998. © 1998 Wiley-Liss, Inc.     

Cuticular strength and pigmentation of rust-red and black strains of Tribolium castaneum: Correlation with catecholamine and β-alanine content

Rust-red wild and black mutant strains of the red flour beetle, Tribolium castaneum, were used to investigate temporal patterns of catecholamine and β-alanine content during sclerotization and pigmentation of adult cuticle and to relate these patterns to corresponding changes in cuticle resistance to puncture. Rust-red elytral cuticle sclerotized more rapidly than black cuticle until 6 days after adult eclosion when both became equal in puncture resistance. The cuticular concentrations of $\text{N-β-}\text{alanyldopamine}$ (NBAD), β-alanine and 3,4-dihydroxyphenylacetic acid (DOPAC) increased more rapidly in the rust-red strain than in the black strain during the first 7 days following adult eclosion. Conversely, cuticular dopamine increased more rapidly in black than in the red strain. Thus the rust-red pigmentation and rapid sclerotization appear to be related to the availability of β-alanine, $\text{N-β-}\text{alanyldopamine}$ and DOPAC. Melanization was prevented and rust-red pigmentation induced by injections of β-alanine or NBAD into newly ecdysed black mutant beetles. Crosses of the two strains generally had intermediate levels of cuticular dopamine and β-alanine, but the NBAD levels were similar to those of the rust-red strain. Dopamine, NBAD and DOPAC levels became similar in both black and rust-red strains about 6 days after adult ecdysis as did resistance to puncture. Therefore, dopamine appears to be directed initially into the melanin pathway in black adults due to a temporary lack of $\text{N-}\text{acylation}$ with β-alanine. After the melanization phase, dopamine is metabolized to sclerotization precursors eventually resulting in normal physical properties of the exoskeleton.     

Beta-alanylation, a means for neutralization of histamine in the central nervous system of Carcinus maenas

Histamine neutralization in Carcinus maenas is not ensured by oxidation, methylation, or acetylation. After injecting labelled histamine, radioactive carcinine (beta-alanylhistamine), biosynthesized in the central nervous system, rapidly accumulates in the heart. This synthesis is intense and proportional to the amount of histamine injected; on the contrary, it is very low after injecting labelled beta-alanine, whatever the amount injected. Ten days after injecting [14C]histamine, the amounts of radioactive carcinine stocked in the heart remain high. When incubated in the presence of labelled carcinine, various Carcinus tissues are unable to metabolize it. Thus it appears that carcinine would be the catabolite of histamine in Carcinus maenas and that beta-alanylation would be a novel pathway for histamine neutralization. Since carcinine synthetase activity is very high in the central nervous system, this enzyme might neutralize not only neuronal histamine, but also possibly exogenous histamine; thus it would constitute an element of the blood-brain barrier.     

昆虫的黑化机理

昆虫的黑化是昆虫出现的一种体色变异。文章介绍工业污染、激素和温度等对昆虫黑化的影响、昆虫色素形成中多巴、多巴胺、N-β丙酰多巴胺(NBAD)和N-乙酰多巴胺(NADA)等色素前体的代谢、昆虫的黑化突变以及昆虫黑化的遗传。     

Constitutive expression and enzymatic activity of Tan protein in brain and epidermis of Ceratitis capitata and of Drosophila melanogaster wild-type and tan mutants

The present report shows a partial biochemical characterization and life cycle expression of N-??-alanyldopamine hydrolase (Tan protein) in Ceratitis capitata and Drosophila melanogaster. This enzyme catalyzes the hydrolysis of N-??-alanyldopamine (NBAD), the main tanning precursor of insect brown cuticles. It also plays an important role in the metabolism of brain neurotransmitters, recycling dopamine and histamine. In contrast to NBAD-synthase, Tan is expressed constitutively in epidermis and does not respond directly to microbial challenge. Immunodetection experiments showed the novel localization of NBAD-hydrolase in the embryo central neural system and in different regions of the adult brain, in addition to optic lobes. We sequenced and characterized Drosophila mutants tan¹ and tan³. The latter appears to be a mutant with normal expression in neural tissue but weak one in epidermis.     

Effects of altered catecholamine metabolism on pigmentation and physical properties of sclerotized regions in the silkworm melanism mutant

Catecholamine metabolism plays an important role in the determination of insect body color and cuticle sclerotization. To date, limited research has focused on these processes in silkworm. In the current study, we analyzed the interactions between catecholamines and melanin genes and their effects on the pigmentation patterns and physical properties of sclerotized regions in silkworm, using the melanic mutant melanism (mln) silkworm strain as a model. Injection of β-alanine into mln mutant silkworm induced a change in catecholamine metabolism and turned its body color yellow. Further investigation of the catecholamine content and expression levels of the corresponding melanin genes from different developmental stages of Dazao-mln (mutant) and Dazao (wild-type) silkworm revealed that at the larval and adult stages, the expression patterns of melanin genes precipitated dopamine accumulation corresponding to functional loss of Bm-iAANAT, a repressive effect of excess NBAD on ebony, and upregulation of tan in the Dazao-mln strain. During the early pupal stage, dopamine did not accumulate in Dazao-mln, since upregulation of ebony and black genes led to conversion of high amounts of dopamine into NBAD, resulting in deep yellow cuticles. Scanning electron microscope analysis of a cross-section of adult dorsal plates from both wild-type and mutant silkworm disclosed the formation of different layers in Dazao-mln owing to lack of NADA, compared to even and dense layers in Dazao. Analysis of the mechanical properties of the anterior wings revealed higher storage modulus and lower loss tangent in Dazao-mln, which was closely associated with the altered catecholamine metabolism in the mutant strain. Based on these findings, we conclude that catecholamine metabolism is crucial for the color pattern and physical properties of cuticles in silkworm. Our results should provide a significant contribution to Lepidoptera cuticle tanning research.     

Biosynthesis and metabolism of histamine in the central nervous system of Carcinus maenas

The central nervous system of Carcinus maenas synthesizes radioactive histamine when incubated in the presence of [14C] histidine and pyridoxal-5' phosphate. This biosynthesis increases linearly as a function of the amount of enzyme and the incubation time. It is not effected by heart, muscle or hepatopancreas extracts nor by haemolymph. Thus histamine appears to be synthesized mainly in the nervous system. The latter is also the seat of carcinine (beta-alanylhistamine) biosynthesis. Since carcinine seems to be a product of histamine neutralization, histamine metabolism should take place in its entirety in the nervous system. Thus histamine appears to be implicated in the neuronal activity of Carcinus. Different areas of the crustacean central nervous system: brain, eyestalks and thoracic ganglionic mass biosynthesize and metabolize histamine. Thus they all could contain sites of action for histamine. The nervous systems of two other Decapodes, Cancer and Astacus also effect histamine biosynthesis but don't metabolize it into carcinine.     

Mise en Evidence de la Carcinine Synthétase, une Nouvelle Enzyme Catalysant le Métabolisme de l''Histamine dans le Systeme Nerveux Central de Carcinus maenas

Carcinine biosynthesis was induced in vitro from its two components, beta-alanine and histamine. The reaction was catalyzed by muscle, heart, and CNS extracts from Carcinus maenas. The specific activity of the enzyme, carcinine synthetase, was 15 times higher in CNS than in other organs. Only CNS extracts induced biosynthesis of carcinine from histidine, and only in the presence of pyridoxal-5'-phosphate. Hence the seat of carcinine biosynthesis seems to be the CNS. It is highly probable that in the CNS, histidine is transformed into histamine, which is then catabolized into carcinine. The latter would then be transported and accumulated in the cardiac tissue. Thus histamine--the metabolism of which takes place totally within the CNS--would be implicated as a participant in the neuronal activity of Carcinus maenas. Carcinine synthetase is a soluble enzyme that requires the presence of ATP, beta-alanine, and histamine. Mg2+ and dithiothreitol are also essential for activity. Optimum pH is approximately 7.6. Carcinine synthetase differs from carnosine synthetase and gamma-glutamylhistamine synthetase in that it does not catalyze synthesis of beta-alanylhistidine or gamma-glutamylhistamine.     

Biosynthesis of carcinine (beta-alanyl-histamine) in vivo

Carcinine was biosynthesized by Carcinus maenas from [14C]beta-alanine, [14C] histidine and [14C] histamine. Since carnosine (beta-alanyl-histidine) could not be detected in crab tissues, biosynthesis of carcinine could only be effected by direct coupling of beta-alanine and histamine resulting from histidine decarboxylation. Biosynthesis of carcinine was weak when [14C]beta-alanine and [14C] histidine were used as precursors. On the contrary when [14C] histamine was used, synthesis was important. Thus carcinine appears to be a product of histamine catabolism. After injecting [14C] histamine, radioactive carcinine was concentrated mainly in the heart and nervous system; nonmetabolized [14C] histamine was recovered mainly in the latter. The nervous system might therefore be the seat of carcinine biosynthesis and thus the site of action of histamine.     

Existence of carcinine, a histamine-related compound, in mammalian tissues

Carcinine (beta-alanylhistamine) was synthesized in vitro from histamine and beta-alanine. It was detected quantitatively using an HPLC method previously described for the quantification of the related compounds histamine, histidine, carnosine and 3-methylhistamine. Carcinine was identified in several tissue of the rat, guinea pig, mouse and human, and was then shown to be metabolically related in vivo to histamine, histidine, carnosine and 3-methylhistamine through radioisotopic labeling. The results demonstrate that carcinine may be concurrently quantitated using the same HPLC method as that used to measure histamine, histidine, carnosine and 3-methylhistamine. These findings suggest a role for carcinine in the carnosine-histidine-histamine metabolic pathway and in the mammalian physiologic response to stress.     

Alternative Tasks of Drosophila Tan in Neurotransmitter Recycling Versus Cuticle Sclerotization Disclosed by Kinetic Properties

Upon a stimulus of light, histamine is released from Drosophila photoreceptor axonal endings. It is taken up into glia where Ebony converts it into β-alanyl-histamine (carcinine). Carcinine moves into photoreceptor cells and is there cleaved into β-alanine and histamine by Tan activity. Tan thus provides a key function in the recycling pathway of the neurotransmitter histamine. It is also involved in the process of cuticle formation. There, it cleaves β-alanyl-dopamine, a major component in cuticle sclerotization. Active Tan enzyme is generated by a self-processing proteolytic cleavage from a pre-protein at a conserved Gly-Cys sequence motif. We confirmed the dependence on the Gly-Cys motif by in vitro mutagenesis. Processing time delays the rise to full Tan activity up to 3 h behind its putative circadian RNA expression in head. To investigate its pleiotropic functions, we have expressed Tan as a His₆ fusion protein in Escherichia coli and have purified it to homogeneity. We found wild type and mutant His₆-Tan protein co-migrating in size exclusion chromatography with a molecular weight compatible with homodimer formation. We conclude that dimer formation is preceding pre-protein processing. Drosophila tan¹ null mutant analysis revealed that amino acid Arg²¹⁷ is absolutely required for processing. Substitution of Met²⁵⁶ in tan⁵, on the contrary, does not affect processing extensively but renders it prone to degradation. This also leads to a strong tan phenotype although His₆-Tan⁵ retains activity. Kinetic parameters of Tan reveal characteristic differences in K_m and k_cat values of carcinine and β-alanyl-dopamine cleavage, which conclusively illustrate the divergent tasks met by Tan.     

Catecholamine-beta-alanyl ligase in the medfly Ceratitis capitata

Dopamine (DA) and norepinephrine (NE) derivatives play an important role in the sclerotization and pigmentation of insect cuticles by serving as precursors for cuticular cross-linking. Protein preparations from prepupae of the medfly, Ceratitis capitata, were able to conjugate beta-alanine with DA producing N-beta-alanyldopamine (NBAD) or with NE, synthesizing N-beta-alanylnorepinephrine (NBANE). The latter reaction has been demonstrated for the first time. Apparent kinetic parameters were obtained for both substrates, DA (V(max)=30.7+/-6.0 pmol min(-1) mg(-1); K(m)=29.5+/-3.5 microM) and NE (V(max)=16.1+/-6.6 pmol min(-1)mg(-1); K(m)=89.0+/-8.3 microM). The same protein seems to be responsible for both enzymatic activities, judging from several criteria like identical behavior under heat inactivation as well as identical Mg2+ and Mn2+ dependent stimulation and Co2+ inhibition. Furthermore, the melanic mutants niger of C. capitata and ebony(4) of D. melanogaster, known to be defective for NBAD synthase, were also unable to synthesize NBANE. The protein preparation acylated tyrosine with much less efficiency, to produce sarcophagine (beta-alanyltyrosine). Strikingly, extracts from the melanic mutants were unable to synthesize sarcophagine. Our results strongly suggest that the enzymatic activity previously known as NBAD synthase is in fact a novel catalytic protein showing broad substrate specificity. We propose to identify it as catecholamine-beta-alanyl ligase.     

Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae

Laccase-2 is a highly conserved multicopper oxidase that functions in insect cuticle pigmentation and tanning. In many species, alternative splicing gives rise to two laccase-2 isoforms. A comparison of laccase-2 sequences from three orders of insects revealed eleven positions at which there are conserved differences between the A and B isoforms. Homology modeling suggested that these eleven residues are not part of the substrate binding pocket. To determine whether the isoforms have different kinetic properties, we compared the activity of laccase-2 isoforms from Tribolium castaneum and Anopheles gambiae. We partially purified the four laccases as recombinant enzymes and analyzed their ability to oxidize a range of laccase substrates. The predicted endogenous substrates tested were dopamine, N-acetyldopamine (NADA), N-β-alanyldopamine (NBAD) and dopa, which were detected in T. castaneum previously and in A. gambiae as part of this study. Two additional diphenols (catechol and hydroquinone) and one non-phenolic substrate (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) were also tested. We observed no major differences in substrate specificity between the A and B isoforms. Dopamine, NADA and NBAD were oxidized with catalytic efficiencies ranging from 51 to 550 min^?1 mM^?1. These results support the hypothesis that dopamine, NADA and NBAD are endogenous substrates for both isoforms of laccase-2. Catalytic efficiencies associated with dopa oxidation were low, ranging from 8 to 30 min^?1 mM^?1; in comparison, insect tyrosinase oxidized dopa with a catalytic efficiency of 201 min^?1 mM^?1. We found that dopa had the highest redox potential of the four endogenous substrates, and this property of dopa may explain its poor oxidation by laccase-2. We conclude that laccase-2 splice isoforms are likely to oxidize the same substrates in vivo, and additional experiments will be required to discover any isoform-specific functions.     

The Drosophila ninaC locus encodes two photoreceptor cell specific proteins with domains homologous to protein kinases and the myosin heavy chain head

The fruit fly Drosophila melanogaster has been extensively used to identify genes required for photoreceptor cell function. We show that the ninaC gene, originally isolated as a Drosophila visual mutation with an electrophysiological phenotype, encodes two novel cytoskeletal proteins. We identified the DNA sequences encoding the ninaC gene by rescuing the electrophysiological phenotype using P-element-mediated germ line transformation. The ninaC locus is expressed as two extensively overlapping mRNAs encoding proteins of 1135 and 1501 amino acids. Both proteins contain a putative protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain and are spatially restricted to photoreceptor cells.     

N‐β‐alanyldopamine metabolism,locomotor activity and sleep in Drosophila melanogaster ebony and tan mutants

Drosophila melanogaster Meigen mutants for N‐β‐alanyldopamine (NBAD) metabolism have altered levels of NBAD, dopamine and other neurotransmitters. The ebony¹ mutant strain has very low levels of NBAD and higher levels of dopamine, whereas the opposite situation is observed in the tan¹ mutant. Dopamine is implicated in the control of movement, memory and arousal, as well as in the regulation of sleep and wakefulness in D. melanogaster. N‐β‐alanyldopamine, which is best known as a cuticle cross‐linking agent, is also present in nervous tissue and has been proposed to promote locomotor activity in this fly. The daily locomotor activity and the sleep patterns of ebony¹ and tan¹ mutants are analyzed, and are compared with wild‐type flies. The tan¹ mutant shows reduced locomotor activity, whereas ebony¹ shows higher levels of activity than wild‐type flies, suggesting that NBAD does not promote locomotor activity. Both mutants spend less time asleep than wild‐type flies during night‐time; ebony shows more consolidated activity during night‐time and increased sleep latency, whereas tan is unable to consolidate locomotor activity and sleep in either phase of the day. The daily level of NBAD‐synthase activity is measured in vitro using wild‐type and tan¹ protein extracts, and the lowest NBAD synthesis is observed at the time of higher locomotor activity. The abnormalities in several parameters of the waking/sleep cycle indicate some dysfunction in the processes that regulates these behaviours in both mutants.     

A glial variant of the vesicular monoamine transporter is required to store histamine in the Drosophila visual system

Unlike other monoamine neurotransmitters, the mechanism by which the brain's histamine content is regulated remains unclear. In mammals, vesicular monoamine transporters (VMATs) are expressed exclusively in neurons and mediate the storage of histamine and other monoamines. We have studied the visual system of Drosophila melanogaster in which histamine is the primary neurotransmitter released from photoreceptor cells. We report here that a novel mRNA splice variant of Drosophila VMAT (DVMAT-B) is expressed not in neurons but rather in a small subset of glia in the lamina of the fly's optic lobe. Histamine contents are reduced by mutation of dVMAT, but can be partially restored by specifically expressing DVMAT-B in glia. Our results suggest a novel role for a monoamine transporter in glia that may be relevant to histamine homeostasis in other systems.