Molecular and pharmacological properties of insect biogenic amine receptors: lessons from Drosophila melanogaster and Apis mellifera

In the central nervous system (CNS) of both vertebrates and invertebrates, biogenic amines are important neuroactive molecules. Physiologically, they can act as neurotransmitters, neuromodulators, or neurohormones. Biogenic amines control and regulate various vital functions including circadian rhythms, endocrine secretion, cardiovascular control, emotions, as well as learning and memory. In insects, amines like dopamine, tyramine, octopamine, serotonin, and histamine exert their effects by binding to specific membrane proteins that primarily belong to the superfamily of G protein-coupled receptors. Especially in Drosophila melanogaster and Apis mellifera considerable progress has been achieved during the last few years towards the understanding of the functional role of these receptors and their intracellular signaling systems. In this review, the present knowledge on the biochemical, molecular, and pharmacological properties of biogenic amine receptors from Drosophila and Apis will be summarized. Arch.     

Neurochemicals and respiratory control during development

During ontogeny, the central nervous system undergoes neuronal growth, regression, and remodeling. The development of neurotransmitter and modulator systems is a plastic process with individual temporal characteristics for each system. These characteristics include the synthesis, degradation, or uptake of neurochemicals and, largely independently, the appearance of their receptors. Message transmission during ontogeny is compounded by the variable development of these systems and by the coexistence and cofunction among these chemicals. Nine neurochemical systems are discussed: adenosine, gamma-aminobutyric acid, opioids, prostaglandins, serotonin, progesterone, substance P, thyrotropin-releasing hormone, and the catecholamines. The possible role of each of these in natural perinatal respiratory control is evaluated according to predetermined criteria. These include the presence of a substance system in respiratory-related regions, physiologically appropriate changes in its concentration in these regions, elicitation of respiratory effects by agonists and antagonists, and abolition with an antagonist of the effect of a substance during its presumed activation by a physiological process. It is suggested that excessive levels of suppressant neuromodulators or an imbalance among neurochemicals can partly explain the special features of respiratory control in the perinatal period.     

Neuroactive steroids.

Neuroactive steroids are natural or synthetic steroids that rapidly alter the excitability of neurons by binding to membrane-bound receptors such as those for inhibitory and (or) excitatory neurotransmitters. The best-studied neuroactive steroids are a series of sedative-hypnotic 3 alpha-hydroxy ring A-reduced pregnane steroids that include the major metabolites of progesterone and deoxycorticosterone, 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydroDOC), respectively. These 3 alpha-hydroxysteroids do not interact with classical intracellular steroid receptors but bind stereoselectively and with high affinity to receptors for the major inhibitory neurotransmitter in brain, gamma-amino-butyric acid (GABA). Biochemical and electrophysiological studies have shown that these steroids markedly augment GABA-activated chloride ion currents in a manner similar (but not identical) to that of anesthetic barbiturates. Several steroids have also been observed to have convulsant or proconvulsant properties, including the synthetic amidine 3 alpha-hydroxy-16-imino-5 beta-17-azaandrostan-11-one (RU5135) and the natural sulfate esters of pregnenolone and dehydroepiandrosterone. Several of these have been shown to be bicuculline or picrotoxin-like GABAA receptor antagonists. Examples of steroids that alter neuronal excitability rapidly by augmenting or inhibiting excitatory amino acid receptor-mediated responses have also been reported. Recently, allopregnanolone and allotetrahydroDOC have also been measured in brain and plasma where their levels have been shown to fluctuate in response to stress and during the estrous and menstrual cycles of rats and humans, respectively. Although the major fraction of allopregnanolone in tissue, including brain, is of adrenal and/or ovarian origin, appreciable levels of allopregnanolone can still be measured in the brains of adrenalectomized and/or oophorectomized animals. Receptor-active neurosteroids may represent an important class of neuromodulators that can rapidly alter central nervous system excitability via novel nongenomic mechanisms.     

Heterogeneity of receptor immunoreactivity at synapses of glycine-utilizing neurons.

Neurons often contain, and probably release, more than one neuroactive substance that may have diverse or opposite actions on the postsynaptic cell. It remains unexplained how these neurons utilize their multiple neuroactive substances while maintaining appropriate resolution of neurotransmitter functions. Here, we have examined the ultrastructural localization of glycine receptors by using a monoclonal antibody directed to the intracellular domain of the strychnine-sensitive glycine receptor. We have found that glycine receptors are only localized to 56% of the synapses made by presumed 'glycinergic' (more accurately, glycine-utilizing) amacrine cells in the turtle retina. The remaining synapses made by these same boutons show no evidence of glycine receptors. As there is no evidence to suggest the presence of a second type of glycine receptor, these data indicate that only a portion of the postsynaptic sites contacted by the glycine-utilizing neurons can respond to glycine. They also suggest that a neuron containing multiple neuroactive substances can selectively affect postsynaptic elements by means of heterogeneous receptor localization.     

Modulation of glutamate receptor functions by glutathione

In addition to its well-known antioxidant effects, glutathione apparently has an additional double role in the central nervous system as a neurotransmitter and neuromodulator. A number of recent neurochemical, neuropharmacological and electrophysiological studies have yielded evidence on both functions. As an excitatory neurotransmitter, glutathione depolarizes neurons by acting as ionotropic receptors of its own which are different from any other excitatory amino acid receptors. As a neuromodulator, it displaces ionotropic glutamate receptor ligands from their binding sites and regulates calcium influx through N-methyl-D-aspartate receptor-governed ionophores. In brain slices glutathione has been shown to regulate the release of other transmitters, e.g., gamma-aminobutyrate and dopamine, mediated by N-methyl-D-aspartate receptors. In the present article, we review recent findings on the neuromodulatory actions of glutathione and discuss possible physiological and pathophysiological consequences.     

CNS melanocortin system involvement in the regulation of food intake

Accumulating evidence indicates that the central melanocortin (MC) system plays a key role in the regulation of food intake and energy balance. This evidence includes findings that either spontaneous genetic mutations or targeted gene deletions that impair melanocortin signaling cause disrupted food intake and body-weight control. In addition, expression of the mRNA that encodes the endogenous agonists and antagonists for CNS melanocortin receptors is regulated by changes in energy balance and body-adiposity signals. Finally, administration of both natural and synthetic ligands to MC receptors produces changes in food intake. The data collectively suggest a critical role for melanocortin signaling in the control of energy balance.     

Endothelin involvement in respiratory centre activity.

To evaluate the role of endothelin (ET) in respiratory homeostasis we studied the effects of the ET(A) and ET(B) receptor blocking agent bosentan on respiratory mechanics and control in seven anaesthetised spontaneously breathing pigs, for 180 min after single bolus administration (20 mg/kg i.v.). The results show that the block of ET receptors induced a significant increase in compliance and decrease in resistance of the respiratory system, entailing a significant reduction of diaphragmatic electromyographic activity, without affecting the centroid frequency of the power spectrum. Bosentan administration induced a significant increase in tidal volume (V(T)), accompanied by a significant decrease in respiratory frequency, without any significant change in pulmonary ventilation, CO(2) arterial blood gas pressure or pH. Since the relationship between V(T) and inspiratory time remained substantially constant after bosentan administration, the changes in respiratory pattern appear to be the result of an upward shift in inspiratory off-switch threshold. Both inspiratory and expiratory times during occluded breathing were increased by block of ET receptors, suggesting also a central respiratory neuromodulator effect of ET. In conclusion the present results suggest that the block of ET receptors in spontaneously breathing pigs exerts a role on mechanical properties of the respiratory system as well as on peripheral and central mechanisms of breathing control.     

Insulin and insulin-like growth factor receptors in the nervous system

Insulin and the insulin-like growth factors (I and II) are homologous peptides essential to normal metabolism as well as growth. These peptide hormones are present in the brain, and, based on biosynthetic labeling studies as well as evidence for local gene expression, they are synthesized by nervous tissue as well as being taken up by the brain from the peripheral circulation. Furthermore, the presence of insulin and IGF receptors in the brain, on both neuronal and glial cells, also suggests a role for these peptides in the nervous system. Thus, these ligands affect brain electrical activity, either as neurotransmitters or as neuromodulators, altering the release and re-uptake of other neurotransmitters. The insulin and IGF-I and -II receptors found in the brain exhibit a lower molecular weight than corresponding receptors on peripheral tissues, primarily caused by alterations in glycosylation. Despite these alterations, both brain insulin and IGF-I receptors exhibit tyrosine kinase activity in cell-free systems, as do their peripheral counterparts. Brain insulin and IGF-I receptors are developmentally regulated, with the highest levels appearing in fetal or perinatal life. However, the altered glycosylation of brain receptors does not appear until late in fetal development. The receptors are widely distributed in the brain, but especially enriched in the circumventricular organs, choroid plexus, hypothalamus, cerebellum, and olfactory bulb. These studies on the insulin and IGF receptor in brain, add strong support to the suggestion that insulin and IGFs are important neuroactive substances, regulating growth, development, and metabolism in the brain.     

Actual data concerning the brain--immune system interface

After a brief presentation of the immune system as sensorial and effector organ, which recognizes and defends against cellular aggressions, the main psycho-neuro-endocrine components of immune reaction regulation and modulation will be shown. Both central nervous structures that control the hormonal emissions, the vegetative innervation of the lymphoid organs as well as the afferent neurohumoral pathways involved in the making of the self-regulating and neuromodulating circuits of the humoral and cellular immune responses will be mentioned. An important position will be held by the interrelations between the hypothalamus-pituitary-corticoadrenal gland, the sympathetic-parasympathetic efferent pathways and the chemical messengers (hormones, neurotransmitters, interleukins, neurotrophins) which make possible the bi-directional neuroimmune communication for maintaining the homeostatic balances on this third effector pathway, too. Also will be presented experimental proof concerning the ability of central neurons to secrete neuromodulator cytokines and the presence of specific receptors for the various neuroactive molecules within lymphoid organs and circulating lymphocytes. To close, the psychoemotional components of the neuro-immunomodulator circuits will be mentioned, using as examples the changes induced by stress generally and oxidative stress in particular.     

Glutathione and Signal Transduction in the Mammalian CNS

The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its gamma-glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose-dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.     

Chemokines: a new class of neuromodulator?

Chemokines are not only found in the immune system or expressed in inflammatory conditions: they are constitutively present in the brain in both glial cells and neurons. Recently, the possibility has been raised that they might act as neurotransmitters or neuromodulators. Although the evidence is incomplete, emerging data show that chemokines have several of the characteristics that define neurotransmitters. Moreover, their physiological actions resemble those of neuromodulators in the sense that chemokines usually have few effects by themselves in basal conditions, but modify the induced release of neurotransmitters or neuropeptides. These findings, together with the pharmacological development of agonists and antagonists that are selective for chemokine receptors and can cross the blood-brain barrier, open a new era of research in neuroscience.     

The role of CRH in behavioral responses to stress

Corticotropin-releasing hormone (CRH) and urocortin in the central nervous system affect behavior and can enhance behavioral responses to stressors. The action of CRH-related peptides is mediated through multiple receptors that differ markedly in their pharmacological profiles and anatomical distribution. Comparative pharmacology of CRH receptor agonists suggests that CRH, urocortin, sauvagine and urotensin consistently mimic, and CRH receptor antagonists consistently lessen, functional consequences of stressor exposure. Recently, important advances have been made in understanding the CRH system and its role in behavioral responses to stress by the development of specific CRH receptor antagonists, application of antisense oligonucleotides and development of transgenic mice lacking peptides and functional receptors. This review summarizes recent findings with respect to components of the CRH system and their role in stress-induced behavioral responses.     

Neuroactive steroids: new biomarkers of cognitive aging

Intensive studies in animals established that neuroactive steroids display neuronal actions and influence behavioral functions. We describe here investigations on the role of neuroactive steroids in learning and memory processes during aging and suggest their role as biomarkers of cognitive aging. Our work demonstrated the role of the steroid pregnenolone (PREG) sulfate as a factor underlying an individual’s age-related cognitive decline in animals. As new perspectives of research we argue that knowing whether neuroactive steroids exist as endogenous neuromodulators and modulate physiologically behavioral functions is essential. To this end, a new approach using the sensitive, specific, and accurate quantitative determination of neuroactive steroids by mass spectrometry seems to have potential for examining the role of each steroid in discrete brain areas in learning and memory alterations, as observed during aging.     

Localization of mu-opioid receptor 1A on sensory nerve fibers in human skin

Opioid peptides are endogenous neuromodulators that play a major role in the nociceptive pathway by interacting with opioid receptors. So far, four opioid receptors (micro-, delta-, kappa-, orphan-receptor) have been cloned with a wide distribution in the central and peripheral nervous system. In the present study, we give first evidence for the presence of the micro-opioid receptor (MOR) isoform 1A in nerve fibers of human skin. Immunohistochemical analysis revealed MOR immunoreactivity to be present in dermal and epidermal nerve fibers. Double-immunofluorescence staining revealed that MOR is present on calcitonin gene-related protein (CGRP)-positive sensory nerve fibers, while autonomic nerves of blood vessels, hair follicles, or skin glands were negative. In diseased skin such as psoriasis vulgaris, atopic dermatitis, and prurigo nodularis, distribution of MOR 1A immunoreactivity was similar to that of normal skin. These findings expand our knowledge about a direct regulatory role of cutaneous opioid receptors in the skin. Thus, peripheral cutaneous opioid receptors may be involved in the transmission of pain and pruritus, respectively. This is supported by previous observation that opioid receptor antagonists may significantly diminish experimentally evoked histamine-induced itch of the skin. Together, our findings contribute to the understanding of the high antipruritic potency of opioid receptor antagonists in various skin and systemic diseases.     

Purinergic receptors and synaptic transmission in enteric neurons

Purines such as ATP and adenosine participate in synaptic transmission in the enteric nervous system as neurotransmitters or neuromodulators. Purinergic receptors are localized on the cell bodies or nerve terminals of different functional classes of enteric neurons and, with other receptors, form unique receptor complements. Activation of purinergic receptors can regulate neuronal activity by depolarization, by regulating intracellular calcium, or by modulating second messenger pathways. Purinergic signaling between enteric neurons plays an important role in regulating specific enteric reflexes and overall gastrointestinal function. In the present article, we review evidence for purine receptors in the enteric nervous system, including P1 (adenosine) receptors and P2 (ATP) receptors. We will explore the role they play in mediating fast and slow synaptic transmission and in presynaptic inhibition of transmission. Finally, we will examine the molecular properties of the native receptors, their signaling mechanisms, and their role in gastrointestinal pathology.     

Persistence of eupnea and gasping following blockade of both serotonin type 1 and 2 receptors in the in situ juvenile rat preparation.

In severe hypoxia or ischemia, normal eupneic breathing is replaced by gasping, which can serve as a powerful mechanism for "autoresuscitation." We have proposed that gasping is generated by medullary neurons having intrinsic pacemaker bursting properties dependent on a persistent sodium current. A number of neuromodulators, including serotonin, influence persistent sodium currents. Thus we hypothesized that endogenous serotonin is essential for gasping to be generated. To assess such a critical role for serotonin, a preparation of the perfused, juvenile in situ rat was used. Activities of the phrenic, hypoglossal, and vagal nerves were recorded. We added blockers of type 1 and/or type 2 classes of serotonergic receptors to the perfusate delivered to the preparation. Eupnea continued following additions of any of the blockers. Changes were limited to an increase in the frequency of phrenic bursts and a decline in peak heights of all neural activities. In ischemia, gasping was induced following any of the blockers. Few statistically significant changes in parameters of gasping were found. We thus did not find a differential suppression of gasping, compared with eupnea, following blockers of serotonin receptors. Such a differential suppression had been proposed based on findings using an in vitro preparation. We hypothesize that multiple neurotransmitters/neuromodulators influence medullary mechanisms underlying the neurogenesis of gasping. In greatly reduced in vitro preparations, the importance of any individual neuromodulator, such as serotonin, may be exaggerated compared with its role in more intact preparations.     

Biogenic amines and the control of neuromuscular signaling in schistosomes

Biogenic amines are small cationic monoamines that function broadly as neurotransmitters and/or neuromodulators in every animal phylum. They include such ubiquitous substances as serotonin, dopamine and invertebrate-specific phenolamines (tyramine, octopamine), among others. Biogenic amines are important neuroactive agents in all the flatworms, including blood flukes of the genus Schistosoma, the etiological agents of human schistosomiasis. A large body of evidence spanning nearly five decades identifies biogenic amines as major modulators of neuromuscular function in schistosomes, controlling movement, attachment to the host and other fundamental behaviors. Recent advances in schistosome genomics have made it possible to dissect the molecular mechanisms responsible for these effects and to identify the proteins involved. These efforts have already provided important new information about the mode of action of amine transmitters in the parasite. Moreover, these advances are continuing, as the field moves into a post-genomics era, and new molecular tools for gene and protein analysis are becoming available. Here, we review the current status of this research and discuss future prospects. In particular, we focus our attention on the receptors that mediate biogenic amine activity, their structural characteristics, functional properties and "druggability" potential. One of the themes that will emerge from this discussion is that schistosomes have a rich diversity of aminergic receptors, many of which share little sequence homology with those of the human host, making them ideally suited for selective drug targeting. Strategies for the characterization of these important parasite proteins will be discussed.     

Mechanisms of GABAA receptor assembly and trafficking: implications for the modulation of inhibitory neurotransmission

Fast synaptic inhibition in the brain is largely mediated by ionotropic GABA receptors, which can be subdivided into GABAA and GABAC receptors based on pharmacological and molecular criteria. GABAA receptors are important therapeutic targets for a range of sedative, anxiolytic, and hypnotic agents and are implicated in several diseases including epilepsy, anxiety, depression, and substance abuse. In addition, modulating the efficacy of GABAergic neurotransmission may play a key role in neuronal plasticity. Recent studies have begun to reveal that the accumulation of ionotropic GABAA receptors at synapses is a highly regulated process that is facilitated by receptor-associated proteins and other cell-signaling molecules. This review focuses on recent experimental evidence detailing the mechanisms that control the assembly and transport of functional ionotropic GABAA receptors to cell surface sites, in addition to their stability at synaptic sites. These regulatory processes will be discussed within the context of the dynamic modulation of synaptic inhibition in the central nervous system (CNS).     

Neurochemical mechanisms in inhibitory effects of stimulation of the periaqueductal gray matter on high-threshold reflex activity of the reticular formation

A study was made of microinjections of antagonists of various neuromodulators on the dynamics of inhibition of the spino-bulbo-spinal reflexes which were evoked by stimulation of the central gray matter (PAG) in rats anesthetized with chloralose. Injections were made into the reticular gigantocellular nucleus (GN), which is the basic supraspinal center of this reflex. Administering methysergide (a blocker of serotonin receptors) was accompanied by significant (two to four times) diminution of inhibition evoked by PAG stimulation with a short, high-frequency series of stimuli. Long inhibition caused by long, rhythmic stimulation of the PAG was diminished less significantly: from 6–10 to 2.5–4 min. When the opiate receptors of the GN neurons were blocked with naloxone, duration of inhibition was reduced by two to five times. The most clearly expressed diminution of both types of inhibitions was noted with injections of haloperidol, an antagonist of catecholamines. Our data indicate that evidently all of these neuromediator (neuromodulator) systems participate in inhibition of high-threshold, reflex activity of the reticular formation evoked by stimulation of the PAG, but their participation in this process is unequal.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 455–463, July–August, 1991.     

Effect of putative neuromodulators on rhythmic buccal motor output in Lymnaea stagnalis

The effects of a variety of neuromodulator substances on rhythmic motor output and activity in neurons in the feeding circuitry of Lymnaea stagnalis were examined. Each neuromodulator produced a unique combination of effects at different levels in the network: i.e., pattern-generating interneurons (N1, N2, and N3), an identified higher-order interneuron (cerebral giant cell, CGC), and buccal motoneurons. 5-Hydroxytryptamine, acetylcholine, and FMRFamide all inhibited rhythmic motor activity. However, this was achieved in different ways. Dopamine changed the nature of rhythmic activity from one in which N2 interneuronal activity was predominant ("N2 rhythm") to a feeding rhythm. Dopamine was the only substance capable of activating the feeding rhythm. Activity in the CGC was increased by 5-hydroxytryptamine, dopamine, and acetylcholine and reduced by FMRFamide. Differential responses in buccal motoneurons were also observed. The results are discussed in relation to previous work on other species and also in terms of the selection of different patterns of motor output by neuromodulators.