The American lobster, Homarus americanus, contains morphine that is coupled to nitric oxide release in its nervous and immune tissues: Evidence for neurotransmitter and hormonal signaling.

OBJECTIVES: The study was designed to determine if morphine was present in lobster tissues. It was also important to determine, as in other animals, if its levels would change in response to stress. In this regard, it was also important to determine if lobster immune and neural tissues express the mu opiate receptor subtype, which was coupled to constitutive nitric oxide synthase derived nitric oxide release. METHODS: Homarus americanus were used in these experiments. Morphine was purified in lobster tissues via high pressure liquid chromatography coupled to UV detection. It was quantified via radioimmunoassay (RIA) and was identified via quadruple time of flight - mass spectrometry. Animals were subject to 2 forms of trauma, namely pereiopod-ablation or lipopolysaccaride (LPS) - injection, and morphine levels determined in nerve cord or hemolymph. Real-time nitric oxide production was determined via an amperometric probe. RT-PCR was used to determine the presence of a micro opiate receptor transcript. RESULTS: In Homarus americanus hemolymph and nerve cord morphine was found. RIA revealed morphine levels of 3.36 pg/mg +/ - 0.48 SEM (N=8) in nerve cord and 717.88 pg/ml +/ - 56.77 SEM (N=58) in hemolymph. In stressed (pereiopod-ablated or LPS-injected) animals, the endogenous morphine levels initially increased significantly by 24% for hemolymph and 48% for nerve cord. By day 5, the stressed and control values for endogenous morphine, in both tissues, was lower and non-distinguishable. In both hemocytes and neural cells, morphine, not met-enkephalin, stimulated constitutive nitric oxide release in a naloxone antagonizable manner, demonstrating a mu opiate receptor mediated phenomenon and suggesting the presence of the mu opiate receptor subtype, micro3, since it is opiate alkaloid selective and opioid peptide insensitive. RT-PCR revealed the presence of a micro opiate receptor transcript in Homarus neural and immune tissues, which exhibits a 100% sequence identity with its human counterpart. CONCLUSION: Taken together, after eliminating all sources of contamination, morphine is present in lobster tissues, potentially demonstrating hormonal and neurotransmitter functions that are involved in the animals' stress response.

Identification of a novel receptor for an invertebrate oxytocin/vasopressin superfamily peptide: molecular and functional evolution of the oxytocin/vasopressin superfamily.

Annetocin is structurally related to an OT (oxytocin)/VP (vasopressin) family peptide, which has been isolated from the earthworm Eisenia foetida and has been shown to induce OT-like egg-laying behaviour. We now report the identification of an endogenous AnR (annetocin receptor). The deduced AnR precursor displays high sequence similarity with OT/VP receptors. Genomic analysis of the AnR gene revealed that the intron-inserted position is conserved between the AnR gene and the mammalian OT/VP receptor genes. These results indicate that AnR and mammalian OT/VP receptors share a common ancestor gene. Administration of annetocin to the AnR expressed in Xenopus oocytes induced a calcium-dependent signal transduction. Reverse transcriptase-PCR analysis and in situ hybridization showed that the AnR gene is expressed specifically in the nephridia located in the clitellum region, although the nephridia are distributed throughout the worm body. This result suggests that annetocin induces egg-laying behaviour through its action on the nephridia. This is the first description concerning the functional correlation between an invertebrate OT/VP-related peptide and egg-laying behaviour.

Identification in Drosophila melanogaster of the invertebrate G protein-coupled FMRFamide receptor.

We here describe the cloning and characterization of the functionally active Drosophila melanogaster (Drm) FMRFamide receptor, which we designated as DrmFMRFa-R. The full-length ORF of a D. melanogaster orphan receptor, CG 2114 (Berkeley Drosophila Genome Project), was cloned from genomic DNA. This receptor is distantly related to mammalian thyroid-stimulating hormone-releasing hormone receptors and to a set of Caenorhabditis elegans orphan receptors. An extract of 5,000 central nervous systems from the related but bigger flesh fly, Neobellieria bullata (Neb), was used to screen cells expressing the orphan receptor. Successive purification steps, followed by MS, revealed the sequence of two previously uncharacterized endogenous peptides, APPQPSDNFIRFamide (Neb-FIRFamide) and pQPSQDFMRFamide (Neb-FMRFamide). These are reminiscent of other insect FMRFamide peptides, having neurohormonal as well as neurotransmitter functions. Nanomolar concentrations of the Drm FMRFamides (DPKQDFMRFamide, TPAEDFMRFamide, SDNFMRFamide, SPKQDFMRFamide, and PDNFMRFamide) activated the cognate receptor in a dose-dependent manner. To our knowledge, the cloned DrmFMRFa-R is the first functionally active FMRFamide G protein-coupled receptor described in invertebrates to date.

Cloning and characterization of a new isoform of Choristoneura hormone receptor 3 from the spruce budworm.

Choristoneura hormone receptor 3 (CHR3) is a 20E (20-hydroxyecdysone)-induced delayed early gene that is homologous to Manduca hormone receptor 3 (MHR3), Drosophila hormone receptor 3 (DHR3), and Galleria hormone receptor 3 (GHR3). We recently cloned and characterized a cDNA that was copied from the 4.5 kb CHR3B mRNA. To isolate additional CHR3 isoforms, the Choristoneura fumiferana embryonic cDNA library was screened using CHR3B cDNA as a probe. Characterization and partial sequencing of 16 clones showed that one of them differed from the CHR3B in two regions. This cDNA (CHR3C) was completely sequenced; the sequence analysis showed that the longest open reading frame had 651 codons. The deduced amino acid sequence of this open reading frame contained all five domains that are typical for a steroid hormone nuclear receptor. The nucleotide sequence of CHR3C cDNA is identical to the nucleotide sequence of CHR3B cDNA except for two major differences in the A/B and D-domains. The CHR3C specific probes detected two mRNAs 5.4 kb (CHR3C), and 6.4 kb (CHR3D), which were present in the pupal stage. The CHR3C and CHR3D mRNAs are induced by the stable ecdysteroid analog RH-5992. The CHR3C protein also binds to the response element of the retinoic acid receptor-related orphan receptor.

Cloning and developmental expression of Choristoneura hormone receptor 3, an ecdysone-inducible gene and a member of the steroid hormone receptor superfamily.

Degenerate oligonucleotides and cDNA converted from Choristoneura fumiferana embryonic RNA were used in a polymerase chain reaction (PCR) procedure to isolate a 683 bp cDNA fragment. Comparison of the deduced amino acid sequence of this cDNA fragment showed that it was a region of an MHR3-like gene from C. fumeferana; we therefore named it Choristoneura hormone receptor 3 (CHR3). This CHR3 cDNA fragment was used as a probe to screen a C. fumiferana embryonic cDNA library. Twenty clones were isolated and two overlapping clones were sequenced. The longest open reading frame of CHR3 cDNA codes for 546 amino acids. The deduced amino acid sequence of this open reading frame contained all five regions typical of a steroid hormone nuclear receptor. The C domain showed the highest identity to Manduca hormone receptor 3 (MHR3), Drosophila hormone receptor 3 (DHR3) and Galleria hormone receptor 3 (GHR3). The A/B, D and E domains also showed significant amino acid similarity with MHR3, DHR3 and GHR3. The 683 bp CHR3 cDNA probe detected two mRNAs of 3.8 and 4.5 kb present during the ecdysteroid peaks for embryonic, larval, pupal and adult molts but were not detected during the intermolt periods. In sixth instar larvae, the 3.8 and 4.5 kb mRNA were detected in the epidermis, fat body and midgut tissues and the maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CHR3 mRNA was induced in 20-hydroxyecdysone treated CF-203 cells as well as in the midgut, fat body and epidermis of larvae that were fed the non-steroidal molting hormone agonist, RH-5992. In vitro transcription and translation of the CHR3 cDNA yielded a 61 kDa protein that bound to the retinoid related orphan receptor response element.