Cholecystokinin in the central nervous system of the sea lamprey Petromyzon marinus: precursor identification and neuroanatomical relationships with other neuronal signalling systems.

Cholecystokinin (CCK) is a neuropeptide that modulates processes such as digestion, satiety, and anxiety. CCK-type peptides have been characterized in jawed vertebrates and invertebrates, but little is known about CCK-type signalling in the most ancient group of vertebrates, the agnathans. Here, we have cloned and sequenced a cDNA encoding a sea lamprey (Petromyzon marinus L.) CCK-type precursor (PmCCK), which contains a CCK-type octapeptide sequence (PmCCK-8) that is highly similar to gnathostome CCKs. Using mRNA in situ hybridization, the distribution of PmCCK-expressing neurons was mapped in the CNS of P. marinus. This revealed PmCCK-expressing neurons in the hypothalamus, posterior tubercle, prethalamus, nucleus of the medial longitudinal fasciculus, midbrain tegmentum, isthmus, rhombencephalic reticular formation, and the putative nucleus of the solitary tract. Some PmCCK-expressing neuronal populations were only observed in adults, revealing important differences with larvae. We generated an antiserum to PmCCK-8 to enable immunohistochemical analysis of CCK expression, which revealed that GABA or glutamate, but not serotonin, tyrosine hydroxylase or neuropeptide Y, is co-expressed in some PmCCK-8-immunoreactive (ir) neurons. Importantly, this is the first demonstration of co-localization of GABA and CCK in neurons of a non-mammalian vertebrate. We also characterized extensive cholecystokinergic fibre systems of the CNS, including innervation of habenular subnuclei. A conspicuous PmCCK-8-ir tract ascending in the lateral rhombencephalon selectively innervates a glutamatergic population in the dorsal isthmic grey. Interestingly, this tract is reminiscent of the secondary gustatory/visceral tract of teleosts. In conclusion, this study provides important new information on the evolution of the cholecystokinergic system in vertebrates.

Opsin evolution in the Ambulacraria.

Opsins--G-protein coupled receptors involved in photoreception--have been extensively studied in the animal kingdom. The present work provides new insights into opsin-based photoreception and photoreceptor cell evolution with a first analysis of opsin sequence data for a major deuterostome clade, the Ambulacraria. Systematic data analysis, including for the first time hemichordate opsin sequences and an expanded echinoderm dataset, led to a robust opsin phylogeny for this cornerstone superphylum. Multiple genomic and transcriptomic resources were surveyed to cover each class of Hemichordata and Echinodermata. In total, 119 ambulacrarian opsin sequences were found, 22 new sequences in hemichordates and 97 in echinoderms (including 67 new sequences). We framed the ambulacrarian opsin repertoire within eumetazoan diversity by including selected reference opsins from non-ambulacrarians. Our findings corroborate the presence of all major ancestral bilaterian opsin groups in Ambulacraria. Furthermore, we identified two opsin groups specific to echinoderms. In conclusion, a molecular phylogenetic framework for investigating light-perception and photobiological behaviors in marine deuterostomes has been obtained.

International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB1 and CB2.

There are at least two types of cannabinoid receptors (CB(1) and CB(2)). Ligands activating these G protein-coupled receptors (GPCRs) include the phytocannabinoid Δ(9)-tetrahydrocannabinol, numerous synthetic compounds, and endogenous compounds known as endocannabinoids. Cannabinoid receptor antagonists have also been developed. Some of these ligands activate or block one type of cannabinoid receptor more potently than the other type. This review summarizes current data indicating the extent to which cannabinoid receptor ligands undergo orthosteric or allosteric interactions with non-CB(1), non-CB(2) established GPCRs, deorphanized receptors such as GPR55, ligand-gated ion channels, transient receptor potential (TRP) channels, and other ion channels or peroxisome proliferator-activated nuclear receptors. From these data, it is clear that some ligands that interact similarly with CB(1) and/or CB(2) receptors are likely to display significantly different pharmacological profiles. The review also lists some criteria that any novel "CB(3)" cannabinoid receptor or channel should fulfil and concludes that these criteria are not currently met by any non-CB(1), non-CB(2) pharmacological receptor or channel. However, it does identify certain pharmacological targets that should be investigated further as potential CB(3) receptors or channels. These include TRP vanilloid 1, which possibly functions as an ionotropic cannabinoid receptor under physiological and/or pathological conditions, and some deorphanized GPCRs. Also discussed are 1) the ability of CB(1) receptors to form heteromeric complexes with certain other GPCRs, 2) phylogenetic relationships that exist between CB(1)/CB(2) receptors and other GPCRs, 3) evidence for the existence of several as-yet-uncharacterized non-CB(1), non-CB(2) cannabinoid receptors; and 4) current cannabinoid receptor nomenclature.

A genomic view of the sea urchin nervous system.

The sequencing of the Strongylocentrotus purpuratus genome provides a unique opportunity to investigate the function and evolution of neural genes. The neurobiology of sea urchins is of particular interest because they have a close phylogenetic relationship with chordates, yet a distinctive pentaradiate body plan and unusual neural organization. Orthologues of transcription factors that regulate neurogenesis in other animals have been identified and several are expressed in neurogenic domains before gastrulation indicating that they may operate near the top of a conserved neural gene regulatory network. A family of genes encoding voltage-gated ion channels is present but, surprisingly, genes encoding gap junction proteins (connexins and pannexins) appear to be absent. Genes required for synapse formation and function have been identified and genes for synthesis and transport of neurotransmitters are present. There is a large family of G-protein-coupled receptors, including 874 rhodopsin-type receptors, 28 metabotropic glutamate-like receptors and a remarkably expanded group of 161 secretin receptor-like proteins. Absence of cannabinoid, lysophospholipid and melanocortin receptors indicates that this group may be unique to chordates. There are at least 37 putative G-protein-coupled peptide receptors and precursors for several neuropeptides and peptide hormones have been identified, including SALMFamides, NGFFFamide, a vasotocin-like peptide, glycoprotein hormones and insulin/insulin-like growth factors. Identification of a neurotrophin-like gene and Trk receptor in sea urchin indicates that this neural signaling system is not unique to chordates. Several hundred chemoreceptor genes have been predicted using several approaches, a number similar to that for other animals. Intriguingly, genes encoding homologues of rhodopsin, Pax6 and several other key mammalian retinal transcription factors are expressed in tube feet, suggesting tube feet function as photosensory organs. Analysis of the sea urchin genome presents a unique perspective on the evolutionary history of deuterostome nervous systems and reveals new approaches to investigate the development and neurobiology of sea urchins.

The phylogenetic distribution and evolutionary origins of endocannabinoid signalling.

The endocannabinoid signalling system in mammals comprises several molecular components, including cannabinoid receptors (e.g. CB1, CB2), putative endogenous ligands for these receptors [e.g. anandamide, 2-arachidonoylglycerol (2-AG)] and enzymes involved in the biosynthesis and inactivation of anandamide (e.g. NAPE-PLD, FAAH) and 2-AG (e.g. DAG lipase, MGL). In this review we examine the occurrence of these molecules in non-mammalian organisms (in particular, animals and plants) by surveying published data and by basic local alignment search tool (BLAST) analysis of the GenBank database and of genomic sequence data from several vertebrate and invertebrate species. We conclude that the ability of cells to synthesise molecules that are categorised as "endocannabinoids" in mammals is an evolutionarily ancient phenomenon that may date back to the unicellular common ancestor of animals and plants. However, exploitation of these molecules for intercellular signalling may have occurred independently in different lineages during the evolution of the eukaryotes. The CB1- and CB2-type receptors that mediate effects of endocannabinoids in mammals occur throughout the vertebrates, and an orthologue of vertebrate cannabinoid receptors was recently identified in the deuterostomian invertebrate Ciona intestinalis (CiCBR). However, orthologues of the vertebrate cannabinoid receptors are not found in protostomian invertebrates (e.g. Drosophila, Caenorhabditis elegans). Therefore, it is likely that a CB1/CB2-type cannabinoid receptor originated in a deuterostomian invertebrate. This phylogenetic information provides a basis for exploitation of selected non-mammalian organisms as model systems for research on endocannabinoid signalling.

Localisation of cannabinoid receptor 1 in rat dorsal root ganglion using in situ hybridisation and immunohistochemistry.

In this study we used in situ hybridisation and double-labelling immunohistochemistry to characterise cannabinoid receptor 1 (CB(1)) expression in rat lumbar dorsal root ganglion (DRG) neurons.Approximately 25% of DRG neurons expressed CB(1) mRNA and displayed immunoreactivity for CB(1). Sixty-nine percent to 82% of CB(1)-expressing cells were also immunoreactive for neurofilament 200, indicative of myelinated A-fibre neurons, which tend to be large- and medium-sized DRG neurons (>600 microm(2)). Approximately 10% of CB1-expressing cells also expressed transient receptor potential vanilloid family ion channel 2 (TRPV2), the noxious heat-transducing channel found in medium to large lightly myelinated Adelta-fibre DRG neurons. Seventeen percent to 26% of CB(1)-expressing cells co-stained using Isolectin B4, 9-10% for calcitonin gene-related peptide and 11-20% for transient receptor potential vanilloid family ion channel 1 (TRPV1), predominantly markers of small non-myelinated C-fibre DRG neurons (<600 microm(2)). These findings suggest that whilst a wide range of DRG neuron phenotypes express CB(1), it is predominantly associated with myelinated fibres.

Comparative analysis of fatty acid amide hydrolase and cb(1) cannabinoid receptor expression in the mouse brain: evidence of a widespread role for fatty acid amide hydrolase in regulation of endocannabinoid signaling.

Fatty acid amide hydrolase (FAAH) catalyses hydrolysis of the endocannabinoid arachidonoylethanolamide ("anandamide") in vitro and regulates anandamide levels in the brain. In the cerebellar cortex, hippocampus and neocortex of the rat brain, FAAH is located in the somata and dendrites of neurons that are postsynaptic to axon fibers expressing the CB(1) cannabinoid receptor [Proc R Soc Lond B 265 (1998) 2081]. This complementary pattern of FAAH and CB(1) expression provided the basis for a hypothesis that endocannabinoids may function as retrograde signaling molecules at synapses in the brain [Proc R Soc Lond B 265 (1998) 2081; Phil Trans R Soc Lond 356 (2001) 381] and subsequent experimental studies have confirmed this [Science 296 (2002) 678]. To assess more widely the functions of FAAH in the brain and the potential impact of FAAH activity on the spatiotemporal dynamics of endocannabinoid signaling in different regions of the brain, here we have employed immunocytochemistry to compare the distribution of FAAH and CB(1) throughout the mouse brain, using FAAH(-/-) mice as negative controls to validate the specificity of FAAH-immunoreactivity observed in wild type animals. In many regions of the brain, a complementary pattern of FAAH and CB(1) expression was observed, with FAAH-immunoreactive neuronal somata and dendrites surrounded by CB(1)-immunoreactive fibers. In these regions of the brain, FAAH may regulate postsynaptic formation of anandamide, thereby influencing the spatiotemporal dynamics of retrograde endocannabinoid signaling. However, in some regions of the brain such as the globus pallidus and substantia nigra pars reticulata, CB(1) receptors are abundant but with little or no associated FAAH expression and in these brain regions the spatial impact and/or duration of endocannabinoid signaling may be less restricted than in regions enriched with FAAH. A more complex situation arises in several regions of the brain where both FAAH and CB(1) are expressed but in a non-complementary pattern, with FAAH located in neurons and/or oligodendrocytes that are proximal but not postsynaptic to CB(1)-expressing axon fibers. Here FAAH may nevertheless influence endocannabinoid signaling but more remotely. Finally, there are regions of the brain where FAAH-immunoreactive neurons and/or oligodendrocytes occur in the absence of CB(1)-immunoreactive fibers and here FAAH may be involved in regulation of signaling mediated by other endocannabinoid receptors or by receptors for other fatty acid amide signaling molecules. In conclusion, by comparing the distribution of FAAH and CB(1) in the mouse brain, we have provided a neuroanatomical framework for comparative analysis of the role of FAAH in regulation of the spatiotemporal dynamics of retrograde endocannabinoid signaling in different regions of the brain.

The neurobiology and evolution of cannabinoid signalling.

The plant Cannabis sativa has been used by humans for thousands of years because of its psychoactivity. The major psychoactive ingredient of cannabis is Delta(9)-tetrahydrocannabinol, which exerts effects in the brain by binding to a G-protein-coupled receptor known as the CB1 cannabinoid receptor. The discovery of this receptor indicated that endogenous cannabinoids may occur in the brain, which act as physiological ligands for CB1. Two putative endocannabinoid ligands, arachidonylethanolamide ('anandamide') and 2-arachidonylglycerol, have been identified, giving rise to the concept of a cannabinoid signalling system. Little is known about how or where these compounds are synthesized in the brain and how this relates to CB1 expression. However, detailed neuroanatomical and electrophysiological analysis of mammalian nervous systems has revealed that the CB1 receptor is targeted to the presynaptic terminals of neurons where it acts to inhibit release of 'classical' neurotransmitters. Moreover, an enzyme that inactivates endocannabinoids, fatty acid amide hydrolase, appears to be preferentially targeted to the somatodendritic compartment of neurons that are postsynaptic to CB1-expressing axon terminals. Based on these findings, we present here a model of cannabinoid signalling in which anandamide is synthesized by postsynaptic cells and acts as a retrograde messenger molecule to modulate neurotransmitter release from presynaptic terminals. Using this model as a framework, we discuss the role of cannabinoid signalling in different regions of the nervous system in relation to the characteristic physiological actions of cannabinoids in mammals, which include effects on movement, memory, pain and smooth muscle contractility. The discovery of the cannabinoid signalling system in mammals has prompted investigation of the occurrence of this pathway in non-mammalian animals. Here we review the evidence for the existence of cannabinoid receptors in non-mammalian vertebrates and invertebrates and discuss the evolution of the cannabinoid signalling system. Genes encoding orthologues of the mammalian CB1 receptor have been identified in a fish, an amphibian and a bird, indicating that CB1 receptors may occur throughout the vertebrates. Pharmacological actions of cannabinoids and specific binding sites for cannabinoids have been reported in several invertebrate species, but the molecular basis for these effects is not known. Importantly, however, the genomes of the protostomian invertebrates Drosophila melanogaster and Caenorhabditis elegans do not contain CB1 orthologues, indicating that CB1-like cannabinoid receptors may have evolved after the divergence of deuterostomes (e.g. vertebrates and echinoderms) and protostomes. Phylogenetic analysis of the relationship of vertebrate CB1 receptors with other G-protein-coupled receptors reveals that the paralogues that appear to share the most recent common evolutionary origin with CB1 are lysophospholipid receptors, melanocortin receptors and adenosine receptors. Interestingly, as with CB1, each of these receptor types does not appear to have Drosophila orthologues, indicating that this group of receptors may not occur in protostomian invertebrates. We conclude that the cannabinoid signalling system may be quite restricted in its phylogenetic distribution, probably occurring only in the deuterostomian clade of the animal kingdom and possibly only in vertebrates.

Neural control of muscle relaxation in echinoderms.

Smooth muscle relaxation in vertebrates is regulated by a variety of neuronal signalling molecules, including neuropeptides and nitric oxide (NO). The physiology of muscle relaxation in echinoderms is of particular interest because these animals are evolutionarily more closely related to the vertebrates than to the majority of invertebrate phyla. However, whilst in vertebrates there is a clear structural and functional distinction between visceral smooth muscle and skeletal striated muscle, this does not apply to echinoderms, in which the majority of muscles, whether associated with the body wall skeleton and its appendages or with visceral organs, are made up of non-striated fibres. The mechanisms by which the nervous system controls muscle relaxation in echinoderms were, until recently, unknown. Using the cardiac stomach of the starfish Asterias rubens as a model, it has been established that the NO-cGMP signalling pathway mediates relaxation. NO also causes relaxation of sea urchin tube feet, and NO may therefore function as a 'universal' muscle relaxant in echinoderms. The first neuropeptides to be identified in echinoderms were two related peptides isolated from Asterias rubens known as SALMFamide-1 (S1) and SALMFamide-2 (S2). Both S1 and S2 cause relaxation of the starfish cardiac stomach, but with S2 being approximately ten times more potent than S1. SALMFamide neuropeptides have also been isolated from sea cucumbers, in which they cause relaxation of both gut and body wall muscle. Therefore, like NO, SALMFamides may also function as 'universal' muscle relaxants in echinoderms. The mechanisms by which SALMFamides cause relaxation of echinoderm muscle are not known, but several candidate signal transduction pathways are discussed here. The SALMFamides do not, however, appear to act by promoting release of NO, and muscle relaxation in echinoderms is therefore probably regulated by at least two neuronal signalling systems acting in parallel. Recently, other neuropeptides that influence muscle tone have been isolated from the sea cucumber Stichopus japonicus using body wall muscle as a bioassay, but at present SALMFamide peptides are the only ones that have been found to have a direct relaxing action on echinoderm muscle. One of the Stichopus japonicus peptides (holothurin 1), however, causes a reduction in the magnitude of electrically evoked muscle contraction in Stichopus japonicus and also causes 'softening' of the body wall dermis, a 'mutable connective tissue'. It seems most likely that this effect of holothurin 1 on body wall dermis is mediated by constituent muscle cells, and the concept of 'mutable connective tissue' in echinoderms may therefore need to be re-evaluated to incorporate the involvement of muscle, as proposed recently for the spine ligament in sea urchins.

Sensory afferents and motor neurons as targets for nitric oxide in the locust.

In the adult locust, nitric oxide (NO) synthase is expressed in interneurons that innervate mechanosensory neuropils, indicating that NO may participate in mechanosensory processing. Here, we have identified potential neuronal targets of NO by localizing the expression and activity of soluble guanylyl cyclase (SGC), its principal molecular target in the nervous system. We used two complementary approaches, namely immunolocalization of SGC alpha-subunit (SGCalpha), and of cyclic GMP (cGMP) after exposure to an NO donor. The cell bodies, axons and central projections of thoracic exteroceptors, proprioceptors, auditory receptors, and chemoreceptors were strongly immunoreactive for SGCalpha. Strong SGCalpha immunoreactivity also occurred in all thoracic motor neurons, including their axon terminals. NO-donors induced a pattern of cGMP immunostaining that was similar to the distribution of SGCalpha, indicating that both sensory and motor neurons contain functional SGC. Therefore, NO may modulate both the input from these sensory neurons and the output of motor neurons. Although the expression of SGCalpha was highly consistent, NO donors did not always induce cGMP-staining in SGC-containing neurons, suggesting that SGC is coregulated by factors other than NO. Complementing previous reports in the visual and olfactory system, our results indicate a general role for NO-cGMP signaling in early sensory processing; diffusible signals may mediate a cross-adaptation or -sensitization within neural maps where similarly tuned neurons have adjacent projections, an anatomical arrangement shared by many sensory systems.

Localisation of cannabinoid receptors in the rat brain using antibodies to the intracellular C-terminal tail of CB.

The CB(1)-type cannabinoid receptor mediates physiologic effects of Delta(9)-tetrahydrocannabinol, the psychoactive ingredient of the drug marijuana. In this report, the authors analyse the expression of CB(1) in the rat brain by using antibodies to the C-terminal 13 amino acids of the receptor. Western blot analysis of rat brain membranes revealed a prominent immunoreactive band with a molecular mass ( approximately 53 kDa) consistent with that predicted for CB(1) from the rat cDNA sequence. In addition, however, less intense immunoreactive bands corresponding to glycosylated ( approximately 62 kDa) and putative N-terminally shorter ( approximately 45 kDa) isoforms of CB(1) were detected. The distribution of CB(1)-immunoreactivity in rat brain was similar to the distribution of binding sites for radiolabelled cannabinoids, with high levels of expression in the olfactory system, the hippocampal formation, the basal ganglia, the cerebellum, and the neocortex. This provides important evidence that CB(1) is likely to be largely responsible for mediating effects of cannabinoids in the brain. CB(1) immunoreactivity was associated with nerve fibre systems and axon terminals but was not detected in neuronal somata. This is consistent with the presynaptic inhibitory effects of cannabinoids on neurotransmitter release in the brain. Detailed immunocytochemical analysis of anatomically or functionally related regions of the brain revealed the location of CB(1) receptors within identified neural circuits. Determination of the cellular and subcellular location of CB(1) within known neuronal circuits of the brain provides an anatomic framework for interpretation of the neurophysiologic and behavioural effects of cannabinoids.

Cannabinoid CB(1) receptor expression in rat spinal cord.

While evidence implicates the endogenous cannabinoid system as a novel analgesic target at a spinal level, detailed analysis of the distribution of the cannabinoid receptor CB(1) in spinal cord has not been reported. Here, immunocytochemical studies were used to characterize the CB(1) receptor expression in rat spinal cord. Staining was found in the dorsolateral funiculus, the superficial dorsal horn (a double band of CB(1) immunoreactivity (ir) in laminae I and II inner/III transition), and lamina X. Although CB(1)-ir was present in the same laminae as primary afferent nociceptor markers, there was limited colocalization at an axonal level. Interruption of both primary afferent input by dorsal root rhizotomy and descending input by rostral spinal cord hemisection produced minor changes in CB(1)-ir. This and colocalization of CB(1)-ir with interneurons expressing protein kinase C subunit gamma-ir suggest that the majority of CB(1) expression is on spinal interneurons. These data provide a framework and implicate novel analgesic mechanisms for spinal actions of cannabinoids at the CB(1) receptor.

Fatty acid amide hydrolase expression in rat choroid plexus: possible role in regulation of the sleep-inducing action of oleamide.

The enzyme fatty acid amide hydrolase (FAAH) catalyses hydrolysis of oleamide, a sleep-inducing lipid whose concentration in the cerebrospinal fluid (CSF) is elevated in sleep-deprived mammals. Previous studies have reported expression of FAAH by distinct populations of neurons in the rat brain. Here we demonstrate using immunocytochemical methods that FAAH is also expressed by non-neuronal epithelial cells of the rat choroid plexus. The choroid plexus is formed by invaginations of the pia mater into the ventricle cavities of the brain and an important function of the choroidal epithelium is to regulate production and composition of CSF. Therefore, the role of FAAH in epithelial cells of the choroid plexus may be to control the concentration of oleamide in the CSF and as such FAAH may exert an important regulatory role in shaping the duration and magnitude of the sleep-inducing effect of endogenously or exogenously derived oleamide.

A new perspective on cannabinoid signalling: complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain.

CB1-type cannabinoid receptors in the brain mediate effects of the drug cannabis. Anandamide and sn-2 arachidonylglycerol (2-AG) are putative endogenous ligands for CB1 receptors, but it is not known which cells in the brain produce these molecules. Recently, an enzyme which catalyses hydrolysis of anandamide and 2-AG, known as fatty acid amide hydrolase (FAAH), was identified in mammals. Here we have analysed the distribution of FAAH in rat brain and compared its cellular localization with CB1-type cannabinoid receptors using immunocytochemistry. High concentrations of FAAH activity were detected in the cerebellum, hippocampus and neocortex, regions of the rat brain which are enriched with cannabinoid receptors. Immunocytochemical analysis of these brain regions revealed a complementary pattern of FAAH and CB1 expression with CB1 immunoreactivity occurring in fibres surrounding FAAH-immunoreactive cell bodies and/or dendrites. In the cerebellum, FAAH was expressed in the cell bodies of Purkinje cells and CB1 was expressed in the axons of granule cells and basket cells, neurons which are presynaptic to Purkinje cells. The close correspondence in the distribution of FAAH and CB1 in rat brain and the complementary pattern of FAAH and CB1 expression at the cellular level provides important new evidence that FAAH may participate in cannabinoid signalling mechanisms of the brain.

Localization of soluble guanylyl cyclase alpha-subunit in identified insect neurons.

The distribution of soluble guanylyl cyclase in the brain of the locust Schistocerca gregaria was analysed using antisera to a highly conserved region (X-peptide) of the Drosophila soluble guanylyl cyclase alpha-subunit (SGCalpha). Analysis of locust brain and locust eye homogenates in Western blots using X-peptide antisera revealed specific staining of a approximately 65 kDa band, which is similar to the expected molecular mass for a SGCalpha-subunit. SGCalpha-immunoreactivity was localized in identified neuronal components of several sensory systems including photoreceptors of the compound eyes and ocelli, large ocellar interneurons, antennal mechanosensory neurons and olfactory interneurons. These neurons are putative targets for the gas nitric oxide which activates guanylyl cyclase activity in the locust brain.

An invertebrate G-protein coupled receptor is a chimeric cannabinoid/melanocortin receptor.

Analysis of a G-protein coupled receptor fragment isolated from the leech CNS reveals that it is a chimeric cannabinoid/melanocortin receptor. Two regions of the leech sequence display high levels of amino acid identity with mammalian cannabinoid receptors while a third region is 98% identical to part of the bovine adrenocorticotropic hormone receptor. The leech receptor may therefore resemble the putative ancestor of mammalian cannabinoid and melanocortin receptors.

Nitric Oxide Function in an Echinoderm.

In vertebrates, nitric oxide (NO) is synthesized from L-arginine by NO synthase (NOS) and regulates relaxation of smooth muscle by activating the cyclic-GMP (cGMP) generating enzyme soluble guanylyl cyclase (SGC). Here we show that the NO-cGMP pathway mediates relaxation of the cardiac stomach in the starfish Asterias rubens. The NO-donors hydroxylamine, S-nitrosoglutathione (SNOG) and S-nitroso-N-acetylpenicillamine (SNAP) and the NOS substrate L-arginine cause relaxation of the cardiac stomach. The relaxing effect of SNAP is blocked by the SGC inhibitor 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and the relaxing effect of L-arginine is inhibited by ODQ and the NOS inhibitor Nw-monomethyl-L-arginine (L-NMMA). ODQ and methylene blue also cause contraction, which may be due to inhibition of the relaxing action of NO produced by cells in the cardiac stomach. These results suggest that NO is synthesized in the cardiac stomach and regulates relaxation by activating SGC. NO-cGMP-mediated relaxation of the cardiac stomach may be important during feeding in starfish where the relaxed stomach is everted through an oral opening and over the digestible parts of prey. The discovery of NO-cGMP-mediated relaxation in an echinoderm demonstrates that regulation of smooth muscle tone by this signaling pathway also occurs in animals other than vertebrates.

Distribution and action of SALMFamide neuropeptides in the starfish Asterias rubens.

The SALMFamides S1 and S2 are two structurally related neuropeptides that are present in starfish, and which share the C-terminal amino acid sequence SXLXFamide, where X is variable. To establish the distribution of S1 and S2 in starfish, we have raised antisera that recognise specifically the C-terminal pentapeptide sequence of either S1 or S2. Here we describe the production and characterisation of an S2-specific antiserum designated CLII. This antiserum, together with an S1-specific antiserum (BLII), has been used in a radioimmunoassay to measure S1 and S2 levels in extracts of body parts from the starfish Asterias rubens. High concentrations (250-400 pmol g-1) of both peptides were detected in the radial nerve cords of the nervous system and lower concentrations were detected in other body parts, including neuromuscular organs such as the tube feet, apical muscle and cardiac stomach. We have examined the pharmacological effects of S1 and S2 on the contractility of these three preparations. Neither S1 nor S2 influenced the tone of tube foot and apical muscle preparations but S2 caused relaxation of cardiac stomach preparations, antagonising the contracting action of acetylcholine.

Behavioral role for nitric oxide in chemosensory activation of feeding in a mollusc.

A role for the NO-cGMP pathway in mediating chemosensory activation of feeding is suggested by intense NADPH diaphorase staining observed in nerve fibers that project from sensory cells in the lips to the CNS and by the presence in the CNS of a NO-activated guanylyl cyclase. In preparations reduced to isolated lips and CNS, intracellular recordings were made from motoneurons driven by the interneurons of the central pattern generator (CPG) for feeding. Fictive feeding in such preparations can be recorded from these motoneurons following the application of sucrose to the lips. Sucrose activation of fictive feeding is inhibited by the NO scavenger hemoglobin, the NO synthase inhibitor N omega-Nitro-L-Arginine Methyl Ester (L-NAME) and by methylene blue, an inhibitor of guanylyl cyclase. Fictive feeding in isolated lip-CNS preparations can be activated without sucrose by superfusion of NO donor molecules such as SNAP and hydroxylamine and by the nonhydrolyzable analog of cGMP, 8-bromo-cGMP. The feeding CPG can also be activated centrally by depolarizing a modulatory interneuron, the slow oscillator (SO). When the CPG is activated in this way, fictive feeding is not susceptible to inhibition by hemoglobin, the most potent of the inhibitors of sucrose-activated fictive feeding. Behavioral experiments on intact snails confirm the findings from in vitro experiments and show that hemoglobin prevents feeding and methylene blue significantly delays the onset of feeding. These results indicate (1) that NO is a putative chemosensory transmitter in the snail L. stagnalis, (2) that the NO-cGMP pathway can mediate chemosensory activation of specific patterns of centrally generated behavior, (3) that NO is not involved in transmission within the central network of neurons responsible for the behavior, and more generally (4) that a freely diffusing and highly reactive gaseous signalling molecule can have restricted and specific behavioral functions.