Octopamine receptor subtypes and their modes of action

Octopamine receptor subclasses were first proposed to explain differences in the pharmacological profiles of a range of physiological responses to octopamine obtained in the extensor-tibiae neuromuscular preparation of the locust. Thus, OCTOPAMINE₁ receptors which inhibit an endogenous myogenic rhythm, increase intracellular calcium levels. Also OCTOPAMINE₂ receptors which modulate neuromuscular transmission in this preparation, increase the level of adenylate cyclase activity. The current status of this classification is reviewed by examining the pharmacology of responses to octopamine in a range of preparations. It is concluded that the distinction between OCTOPAMINE₁ and OCTOPAMINE₂ receptor types is still valid, but that OCTOPAMINE₂ receptors exhibit some tissue specific variations. Studies on a clonedDrosophila octopamine/tyramine (phenolamine) receptor are discussed and illustrate many of the difficulties presently encountered in making a definitive classification of octopamine receptors. These include the possibilities that single receptors may activate multiple second messenger systems and that different agonists may differentially couple the same receptor to different second messenger systems.     

Site-directed mutagenesis studies on the Drosophila octopamine/tyramine receptor

The cloned Drosophila octopamine/tyramine receptor can be coupled to second messenger pathways in an agonist-specific fashion by the endogenously occurring biogenic amines, octopamine and tyramine, when expressed in Chinese hamster ovary cells. We have mutated to alanine a range of receptor amino acids that could potentially form hydrogen bonds with the beta-hydroxyl group of octopamine based on homologies with alpha- and beta-adrenergic receptor subtypes. After stable expression of the mutant receptors in CHO cells we have compared the ability of octopamine and tyramine to displace [(3)H]yohimbine binding to membrane fractions from the mutant cell lines with their ability to modulate adenylyl cyclase activity in intact cells. The results suggest that none of the mutated amino acids residues, at least in isolation, are likely to be involved in interactions with the beta-hydroxyl group of the octopamine side chain. It is possible that amino acids not mutated in the present study are somehow involved in this interaction. Alternatively, it is also possible that the beta-hydroxyl group of the octopamine side chain is capable of interacting with more than one of the amino acids mutated in the present study.     

Characterization of a Prawn OA/TA Receptor in Xenopus Oocytes Suggests Functional Selectivity between Octopamine and Tyramine

Here we report the characterization of an octopamine/tyramine (OA/TA or TyrR1) receptor (OA/TA_Mac) cloned from the freshwater prawn, Macrobrachium rosenbergii, an animal used in the study of agonistic social behavior. The invertebrate OA/TA receptors are seven trans-membrane domain G-protein coupled receptors that are related to vertebrate adrenergic receptors. Behavioral studies in arthropods indicate that octopaminergic signaling systems modulate fight or flight behaviors with octopamine and/or tyramine functioning in a similar way to the adrenalins in vertebrate systems. Despite the importance of octopamine signaling in behavioral studies of decapod crustaceans there are no functional data available for any of their octopamine or tyramine receptors. We expressed OA/TA_Mac in Xenopus oocytes where agonist-evoked trans-membrane currents were used as readouts of receptor activity. The currents were most effectively evoked by tyramine but were also evoked by octopamine and dopamine. They were effectively blocked by yohimbine. The electrophysiological approach we used enabled the continuous observation of complex dynamics over time. Using voltage steps, we were able to simultaneously resolve two types of endogenous currents that are affected over different time scales. At higher concentrations we observe that octopamine and tyramine can produce different and opposing effects on both of these currents, presumably through the activity of the single expressed receptor type. The pharmacological profile and apparent functional-selectivity are consistent with properties first observed in the OA/TA receptor from the insect Drosophila melanogaster. As the first functional data reported for any crustacean OA/TA receptor, these results suggest that functional-selectivity between tyramine and octopamine is a feature of this receptor type that may be conserved among arthropods.     

Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors

Summary Insect octopamine receptors are G-protein coupled receptors. They can be coupled to second messenger pathways to mediate either increases or decreases in intracellular cyclic AMP levels or the generation of intracellular calcium signals. Insect octopamine receptors were originally classified on the basis of second messenger changes induced in a variety of intact tissue preparations. Such a classification system is problematic if more than one receptor subtype is present in the same tissue preparation. Recent progress on the cloning and characterization in heterologous cell systems of octopamine receptors from Drosophila and other insects is reviewed. A new classification system for insect octopamine receptors into “α-adrenergic-like octopamine receptors (OctαRs)”, “β-adrenergic-like octopamine receptors (OctβRs)” and “octopamine/tyramine (or tyraminergic) receptors” is proposed based on their similarities in structure and in signalling properties with vertebrate adrenergic receptors. In future studies on the molecular basis of octopamine signalling in individual tissues it will be essential to identify the relative expression levels of the different classes of octopamine receptor present. In addition, it will be essential to identify if co-expression of such receptors in the same cells results in the formation of oligomeric receptors with specific emergent pharmacological and signalling properties.     

Characterisation of AmphiAmR4, an amphioxus (Branchiostoma floridae) α2-adrenergic-like G-protein-coupled receptor

Little is known about the evolutionary relationship between vertebrate adrenergic receptors and invertebrate octopamine and tyramine receptors. The complexity of the adrenergic signalling system is believed to be an innovation of the vertebrate lineage but the presence of noradrenaline has been reported in some invertebrate species. The cephalochordate, amphioxus (Branchiostoma floridae), is an ideal model organism for studying the evolution of vertebrate GPCRs, given its unique position at the base of the chordate lineage. Here, we describe the pharmacological characterisation and second messenger coupling abilities of AmphiAmR4, which clusters with α₂-adrenergic receptors in a phylogenetic tree but also shares a high sequence similarity to invertebrate octopamine/tyramine receptors in both BLAST and Hidden Markov Model analyses. Thus, it was of particular interest to determine if AmphiAmR4 displayed similar functional properties to the vertebrate α₂-adrenergic receptors or to invertebrate octopamine or tyramine receptors. When stably expressed in Chinese hamster ovary (CHO) cells, noradrenaline couples the receptor to both the activation of adenylyl cyclase and to the activation of the MAPKinase pathway. Pharmacological studies with a wide range of agonists and antagonists suggest that AmphiAmR4 functions as an α₂-adrenergic-like receptor when expressed in CHO cells.     

Pharmacological characterization of a tyramine receptor from the southern cattle tick,Rhipicephalus (Boophilus) microplus

The southern cattle tick (Rhipicephalus (Boophilus) microplus) is a hematophagous external parasite that vectors the causative agents of bovine babesiosis or cattle tick fever, Babesia bovis and B. bigemina, and anaplasmosis, Anaplasma marginale. The southern cattle tick is a threat to the livestock industry in many locations throughout the world. Control methods include the use of chemical acaricides including amitraz, a formamidine insecticide, which is proposed to activate octopamine receptors. Previous studies have identified a putative octopamine receptor from the southern cattle tick in Australia and the Americas. Furthermore, this putative octopamine receptor could play a role in acaricide resistance to amitraz. Recently, sequence data indicated that this putative octopamine receptor is probably a type-1 tyramine receptor (TAR1). In this study, the putative TAR1 was heterologously expressed in Chinese hamster ovary (CHO-K1) cells, and the expressed receptor resulted in a 39-fold higher potency for tyramine compared to octopamine. Furthermore, the expressed receptor was strongly antagonized by yohimbine and cyproheptadine, and mildly antagonized by mianserin and phentolamine. Tolazoline and naphazoline had agonistic or modulatory activity against the expressed receptor, as did the amitraz metabolite, BTS-27271; however, this was only observed in the presence of tyramine. The southern cattle tick's tyramine receptor may serve as a target for the development of anti-parasitic compounds, in addition to being a likely target of formamidine insecticides.     

Expression of octopaminergic receptor genes in 4 nonneural tissues in female Nicrophorus vespilloides beetles

Octopamine regulates the function of many tissues and physiological processes in invertebrates. The expression of octopamine receptor genes has been examined in multiple tissue types in several different insect orders. However, little work has addressed this issue in Coleoptera. Most studies characterize individual genes in different tissue types, but here we describe the expression of 6 octopamine receptor genes in thoracic musculature, oviducts, Malpighian tubules, and fat body of female Nicrophorus vespilloides beetles to characterize both different genes and different tissues within a single study. We then compare the gene expression profiles found in this beetle to other insects to examine the extent to which expression profiles are conserved across insects. We also examine the relative involvement of octopamine verses octopamine/tyramine receptors based on receptor gene expression in each tissue to help elucidate if tyramine plays a role in the regulation of these tissues. We find a high degree of overlap in the expression profile of the 6 genes examined in the thoracic musculature, a moderate amount for the oviducts, and divergent profiles for Malpighian tubules and fat body. Based on expression difference in receptor subtypes, our results also support the suggestion that tyramine is a biogenic amine with physiological actions separate from octopamine.     

Tyramine as a possible neurotransmitter/neuromodulator at the spermatheca of the African migratory locust, Locusta migratoria

Tyramine-like immunoreactivity was identified in neurons of the VIIIth abdominal ganglion and in axons projecting to the spermatheca of adult females of Locusta migratoria. Tyramine-like immunoreactive processes were also found throughout all regions of the spermatheca and tyramine-like immunoreactive bipolar or multipolar neurons were present on the spermathecal sac. HPLC coupled with electrochemical detection revealed more tyramine than octopamine present in spermathecal tissue. Electrical stimulation of the ventral ovipositor nerve resulted in a significant increase in calcium-dependent release of tyramine from the spermatheca. Both tyramine and octopamine increase the frequency and basal tonus of spermathecal contractions in a dose-dependent manner, with octopamine having a lower threshold. When tyramine is applied along with a half maximal octopamine dose, there is an additive effect on contractions of the spermatheca with slight synergistic effects at lower doses of tyramine. High concentrations of tyramine (10(-4)M) stimulated increases in cyclic AMP levels of the spermatheca; an effect blocked by phentolamine. Phentolamine has a higher affinity (and thus a lower IC(50) value congruent with5.6x10(-8)M) than yohimbine (IC(50) congruent with1.1x10(-4)M) in reducing tyramine-induced spermathecal contractions. Taken together, these results suggest that tyramine may be a co-transmitter with octopamine at the spermatheca, with both neuroactive chemicals acting on an octopamine receptor.     

Identification and Function of Octopamine and Tyramine Conjugates in the Limulus Visual System

Major metabolites of octopamine and tyramine in the Limulus nervous system are identified here as gamma-glutamyl octopamine and gamma-glutamyl tyramine. We show that these conjugates are normal products of amine metabolism in Limulus, and that they are normally present in octopamine-rich Limulus tissues. The synthesis of these conjugates is not restricted to nervous tissue, but the highest activity of gamma-glutamyl amine synthetase was measured in the CNS. Our interest in these molecules stems from our previous observations which showed that they were synthesized and stored in, and released from, the efferent fibers to Limulus eyes which modulate the sensitivity of the eyes to light. Here we provide direct evidence for the release of the conjugates from Limulus eyes in response to depolarization, and that gamma-glutamyl octopamine can increase the sensitivity of the lateral eye to light. Our observations lend support to the hypothesis that gamma-glutamyl octopamine may serve as an intercellular messenger in the Limulus visual system.     

A comparison of the signalling properties of two tyramine receptors from Drosophila

In invertebrates, the phenolamines, tyramine and octopamine, mediate many functional roles usually associated with the catecholamines, noradrenaline and adrenaline, in vertebrates. The α‐ and β‐adrenergic classes of insect octopamine receptor are better activated by octopamine than tyramine. Similarly, the Tyramine 1 subgroup of receptors (or Octopamine/Tyramine receptors) are better activated by tyramine than octopamine. However, recently, a new Tyramine 2 subgroup of receptors was identified, which appears to be activated highly preferentially by tyramine. We examined immunocytochemically the ability of CG7431, the founding member of this subgroup from Drosophila melanogaster, to be internalized in transfected Chinese hamster ovary (CHO) cells by different agonists. It was only internalized after activation by tyramine. Conversely, the structurally related receptor, CG16766, was internalized by a number of biogenic amines, including octopamine, dopamine, noradrenaline, adrenaline, which also were able to elevate cyclic AMP levels. Studies with synthetic agonists and antagonists confirm that CG16766 has a different pharmacological profile to that of CG7431. Species orthologues of CG16766 were only found in Drosophila species, whereas orthologues of CG7431 could be identified in the genomes of a number of insect species. We propose that CG16766 represents a new group of tyramine receptors, which we have designated the Tyramine 3 receptors.     

TYRA-2 (F01E11.5): a Caenorhabditis elegans tyramine receptor expressed in the MC and NSM pharyngeal neurons

Tyramine appears to regulate key processes in nematodes, such as pharyngeal pumping, and more complex behaviors, such as foraging. Recently, a Caenorhabditis elegans tyramine receptor, SER-2, was identified that is involved in the TA-dependent regulation of these processes. In the present study, we have identified a second C. elegans gene, tyra-2 (F01E11.5) that encodes a tyramine receptor. This is the first identification of multiple tyramine receptor genes in any invertebrate. Membranes from COS-7 cells expressing TYRA-2 bind [(3)H]tyramine with high affinity with a K(d) of 20 +/- 5 nM. Other physiologically relevant biogenic amines, such as octopamine and dopamine, inhibit [(3)H]tyramine binding with much lower affinity (K(i)s of 1.55 +/- 0.5 and 1.78 +/- 0.6 microM, respectively), supporting the identification of TYRA-2 as a tyramine receptor. Indeed, tyramine also dramatically increases GTPgammaS binding to membranes from cells expressing TYRA-2 (EC(50) of 50 +/- 13 nM) and the TA-dependent GTPgammaS binding is PTX-sensitive suggesting that TYRA-2 may couple to Galpha(i/o). Based on fluorescence from tyra::gfp fusion constructs, TYRA-2 expression appears to be exclusively neuronal in the MC and NSM pharyngeal neurons, the AS family of amphid neurons and neurons in the nerve ring, body and tail. Taken together, these results suggest that TYRA-2 encodes a second Galpha(i/o)-coupled tyramine receptor and suggests that TA-dependent neuromodulation may be mediated by multiple receptors and more complex than previously appreciated.     

Tyramine injections reduce locust viability

In the locust nervous system, tyramine is the direct precursor for octopamine synthesis and, as an octopamine analogue, it can activate octopamine receptors. Furthermore, the identification of specific tyramine receptors in Locusta migratoria and Drosophila melanogaster suggests that it is an important transmitter or modulator candidate. In this paper, we report that repeated tyramine injections reduced the viability of last instar larvae of Locusta and Schistocerca. In addition, a retardation of the last ecdysis was observed as a sublethal effect of the repeated tyramine treatment. Moreover, egg deposition by adult females was also retarded and/or drastically reduced. These effects show similarity to sublethal effects described for certain "insecticidal" octopamine receptor agonists, such as formamidines and phenyliminoimidazolidines. Since certain formamidine compounds were also shown to be agonists for the cloned tyramine receptors, it cannot be excluded that some lethal or sublethal consequences of tyramine administration are the result of an interaction with specific tyramine receptors.     

Tyramine receptor (SER-2) isoforms are involved in the regulation of pharyngeal pumping and foraging behavior in Caenorhabditis elegans

Octopamine regulates essential processes in nematodes; however, little is known about the physiological role of its precursor, tyramine. In the present study, we have characterized alternatively spliced Caenorhabditis elegans tyramine receptor isoforms (SER-2 and SER-2A) that differ by 23 amino acids within the mid-region of the third intracellular loop. Membranes prepared from cells expressing either SER-2 or SER-2A bind [3H]lysergic acid diethylamide (LSD) in the low nanomolar range and exhibit highest affinity for tyramine. Similarly, both isoforms exhibit nearly identical Ki values for a number of antagonists. In contrast, SER-2A exhibits a significantly lower affinity than SER-2 for other physiologically relevant biogenic amines, including octopamine. Pertussis toxin treatment reduces affinity for both tyramine and octopamine, especially for octopamine in membranes from cells expressing SER-2, suggesting that the conformation of the mid-region of the third intracellular loop is dictated by G-protein interactions and is responsible for the differential tyramine/octopamine affinities of the two isoforms. Tyramine reduces forskolin-stimulated cAMP levels in HEK293 cells expressing either isoform with nearly identical IC50 values. Tyramine, but not octopamine, also elevates Ca2+ levels in cells expressing SER-2 and to a lesser extent SER-2A. Most importantly, ser-2 null mutants (pk1357) fail to suppress head movements while reversing in response to nose-touch, suggesting a role for SER-2 in the regulation of foraging behavior, and fail to respond to tyramine in assays measuring serotonin-dependent pharyngeal pumping. These are the first reported functions for SER-2. These results suggest that C. elegans contains tyramine receptors, that individual SER-2 isoforms may differ significantly in their sensitivity to other physiologically relevant biogenic amines, such as octopamine (OA), and that tyraminergic signaling may be important in the regulation of key processes in nematodes.     

The role of octopamine in locusts and other arthropods

The biogenic amine octopamine and its biological precursor tyramine are thought to be the invertebrate functional homologues of the vertebrate adrenergic transmitters. Octopamine functions as a neuromodulator, neurotransmitter and neurohormone in insect nervous systems and prompts the whole organism to “dynamic action”. A growing number of studies suggest a prominent role for octopamine in modulating multiple physiological and behavioural processes in invertebrates, as for example the phase transition in Schistocerca gregaria. Both octopamine and tyramine exert their effects by binding to specific receptor proteins that belong to the superfamily of G protein-coupled receptors. Since these receptors do not appear to be present in vertebrates, they may present very suitable and specific insecticide and acaricide targets.     

Cloning, expression and functional analysis of an octopamine receptor from Periplaneta americana

Octopamine regulates multiple physiological functions in invertebrates. The biological effects of octopamine and the pharmacology of octopamine receptors have been extensively studied in the American cockroach, Periplaneta americana. This paper reports the cloning of the first octopamine receptor from Periplaneta americana. A cDNA encoding a putative 7 transmembrane receptor was isolated from the head of Periplaneta americana. The encoded protein contains 628 amino acids and has sequence similarity to other biogenic amine receptors. This protein was expressed in COS-7 cells for radioligand binding studies using the antagonist 3H-yohimbine. Competitive binding comparing biogenic amines that could potentially function as endogenous ligands demonstrated this receptor had the highest affinity for octopamine (Ki = 13.3 microM) followed by tyramine, dopamine, serotonin and histamine. Octopamine increased both cAMP levels (EC50 = 1.62 microM) and intracellular concentrations of calcium through the receptor expressed in HEK-293 cells. Tyramine increased levels of both of these second messengers but only at significantly higher concentrations than octopamine. The cAMP increase by octopamine was independent of the increase in calcium. Competitive binding with antagonists revealed this receptor is similar to Lym oa1 from Lymnaea stagnalis. The data indicate that this cDNA is the first octopamine receptor cloned from Periplaneta americana and therefore has been named Pa oa1.     

A new family of insect tyramine receptors

The Drosophila Genome Project database contains a gene, CG7431, annotated to be an "unclassifiable biogenic amine receptor." We have cloned this gene and expressed it in Chinese hamster ovary cells. After testing various ligands for G protein-coupled receptors, we found that the receptor was specifically activated by tyramine (EC(50), 5x10(-7)M) and that it showed no cross-reactivity with beta-phenylethylamine, octopamine, dopa, dopamine, adrenaline, noradrenaline, tryptamine, serotonin, histamine, and a library of 20 Drosophila neuropeptides (all tested in concentrations up to 10(-5) or 10(-4)M). The receptor was also expressed in Xenopus oocytes, where it was, again, specifically activated by tyramine with an EC(50) of 3x10(-7)M. Northern blots showed that the receptor is already expressed in 8-hour-old embryos and that it continues to be expressed in all subsequent developmental stages. Adult flies express the receptor both in the head and body (thorax/abdomen) parts. In addition to the Drosophila tyramine receptor gene, CG7431, we found another closely related Drosophila gene, CG16766, that probably also codes for a tyramine receptor. Furthermore, we annotated similar tyramine-like receptor genes in the genomic databases from the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera. These four tyramine or tyramine-like receptors constitute a new receptor family that is phylogenetically distinct from the previously identified insect octopamine/tyramine receptors. The Drosophila tyramine receptor is, to our knowledge, the first cloned insect G protein-coupled receptor that appears to be fully specific for tyramine.     

Temporal and spatial expression patterns of two G-protein coupled receptors inDrosophila melanogaster

Temporal and spatial expression patterns of a muscarinic acetylcholine receptor (Acr60C) and an octopamine/tyramine receptor (Octyr) were determined inDrosophila melanogaster using quantitative Northern analysis andin situ hybridization to tissue sections. Expression of mRNA encoding both of these G-protein coupled receptors peaks initially in 18 to 21 hour embryos following the formation of the mature larval nervous system. Levels of mRNA then decline during larval stages, rising to a second peak in 3 to 4-day-old pupae after a period of major nervous system reorganization. The muscarinic acetylcholine receptor mRNA is expressed throughout the cortical regions of the central nervous system in adults and embryos. Particularly high levels of expression of Acr60C are observed in cell bodies adjacent to the antennal lobes, suggesting a major role for this muscarinic receptor in the processing of olfactory information. In contrast, the octopamine/tyramine receptor mRNA is distributed diffusely throughout the adult brain, with patches of signal concentrated in the cortex of the dorsal protocerebrum near the mushroom bodies. These patches may represent individual cells expressingOctyr receptors.     

A tyramine receptor gene mutation causes a defective olfactory behavior in Drosophila melanogaster

We characterized molecular profiles of a new olfactory mutant line, honoka (hono), which was found among 500 viable P-element insertion lines screened first by 5-bromo-4-chloro-3-indrolyl-beta-D-galactopyranoside (X-gal) staining on the third segment of the antenna, and then by behavioral assays to several pure chemicals.The behavioral responses of hono mutants to repellents such as ethyl acetate (EA), benzaldehyde (BZ) and 4-methylcycrohexanol (MCH), were reduced compared with those of a control strain. The location of the P-element insertion was determined to be about 100bp) upstream of the first exon of the tyramine receptor gene. The level of 3.6kb tyramine receptor mRNA expression was reduced in hono compared with that of wild-type flies. The tyramine receptor cDNA hybridized to the chromosomal division 79C-D, the same locus as the P-element insertion point in hono, and not to 99A-B, previously reported by Arakawa et al. (1990. Neuron 2, 343-354).Electrophysiological responses to octopamine and tyramine were examined by measuring the excitatory junctional potential (EJP) amplitude from larval body-wall muscles of the hono mutant. The hono was impaired with responding to tyramine, while displaying normal response to octopamine. These results indicate that tyramine has a functional role in the Drosophila olfactory system as a neurotransmitter or a neuromodulator, and hono is the first tyramine receptor mutant. This study provides the first step toward understanding of the molecular genetics of tyramine-mediated neural functions in Drosophila.     

Differential effects of octopamine and tyramine on the central pattern generator for <Emphasis Type="Italic">Manduca</Emphasis> flight

The biogenic amine, octopamine, modulates a variety of aspects of insect motor behavior, including direct action on the flight central pattern generator. A number of recent studies demonstrate that tyramine, the biological precursor of octopamine, also affects invertebrate locomotor behaviors, including insect flight. However, it is not clear whether the central pattern generating networks are directly affected by both amines, octopamine and tyramine. In this study, we tested whether tyramine affected the central pattern generator for flight in the moth, Manduca sexta. Fictive flight was induced in an isolated ventral nerve cord preparation by bath application of the octopamine agonist, chlordimeform, to test potential effects of tyramine on the flight central pattern generator by pharmacological manipulations. The results demonstrate that octopamine but not tyramine is sufficient to induce fictive flight in the isolated ventral nerve cord. During chlordimeform induced fictive flight, bath application of tyramine selectively increases synaptic drive to depressor motoneurons, increases the number of depressor spikes during each cycle and decreases the depressor phase. Conversely, blocking tyramine receptors selectively reduces depressor motoneuron activity, but does not affect cycle by cycle elevator motoneuron spiking. Therefore, octopamine and tyramine exert distinct effects on the flight central pattern generating network.     

Characterization of the tyraminergic system in the central nervous system of the locust,Locusta migratoria migratoides

Tyramine occurs in the central nervous system (CNS) of the migratory locust,Locusta migratoria migratoides. The distribution of tyramine within the CNS does not parallel that of octopamine. Tyramine is synthesised from tyrosine in the presence of tyrosine decarboxylase. A second decarboxylase in the CNS is active against 5HTP and DOPA. The locust ganglia incorporate tyramine by high- and low-affinity uptake processes that appear to be independent of dopamine and octopamine. Depolarisation of the locust ganglia by high potassium concentration results in calcium-dependent release of incorporated [³H]tyramine.