Effects of drugs interfering with the metabolism of octopamine on blood pressure of rats

1. p-Octopamine injected in lateral ventricle of conscious spontaneously hypertensive rats decreased systolic blood pressure (SBP). 2. Precursors of octopamine--tyrosine, tyramine and phenylethanolamine (PEA)--had the same effect. The administration of pargyline, a MAO inhibitor, which increased brain octopamine, resulted in a reduction of systolic blood pressure; and this decrease was greater after administration of octopamine precursors and PEA. 3. Similarly, drugs known to inhibit activity of phenylethanolamine N-methyl-transferase (PNMT) and to increase brain octopamine level such as SKF 64139 and DCMB decreased SBP. 4. p-Octopamine hypotension was not antagonized by piperoxan, yohimbine and prazosin, a relatively selective antagonist of post-synaptic alpha adrenoceptors. 5. These results suggest that octopamine may be involved in central blood pressure regulation, and the receptors sensitive to octopamine appeared to be distinct from those receptive to the catecholamines.     

Fluorimetric detection of octopamine in high-performance liquid chromatography and its application to the assay of dopamine β-monooxygenase in human serum

A high-performance liquid chromatographic procedure is described for the determination of octopamine. The method, which is based on the separation on a microparticulate bonded strong cation-exchange resin and measurement of the native fluorescence, has been applied to give a sensitive assay of dopamine β-monooxygenase (EC 1.14.17.1) activity in human serum with tyramine as the substrate. The procedure, which has been designed for use with an-automatic sampler, has a detection limit of about 50 pmoles of octopamine, and the analysis time is approximately 10 min per sample.     

Modulation of dopamine receptors in the Tapes clam by dextroamphetamine and phenylethanolamine

The mechanism underlying the modulation, by dextroamphetamine and compounds related to phenylethanolamine, of responses to dopamine and serotonin has been studied in the isolated ventricle and aortic bulb of the clam Tapes watlingi. Dextroamphetamine and phenylethanolamine but not cocaine and benztropine have the ability to unmask inhibitory responses to both dopamine and serotonin in the ventricle. Chlordimeform but not clozapine attenuates the inhibitory response to both dextroamphetamine and phenylethanolamine in concentrations which have little or no effect on the inhibitory response to dopamine in the ventricle. Phenylethanolamine, dextroamphetamine, phenylpropylolamine and p-chloro-phenylethanolamine but not octopamine or noradrenaline attenuate the contractile responses to both dopamine and serotonin in preparations of the quiescent aortic bulb. These data show that there are specific receptors for phenylethanolamine in the Tapes heart capable of modulating responses to dopamine and serotonin, and suggests that this biogenic phenethylamine can act as an environmental and physiological factor which may determine how the mollusc heart responds to dopamine.     

Isolation, cloning, and tissue expression of a putative octopamine/tyramine receptor from locust visceral muscle tissues

Octopamine has been shown to play major roles in invertebrate nervous systems as a neurotransmitter, neuromodulator, and neurohormone. Tyramine is the biochemical precursor of octopamine and its neuromodulatory role is now being investigated and clarified in invertebrates, particularly in insects. Both octopamine and tyramine mediate their actions via G protein-coupled receptors (GPCRs) and are believed to play important functions in the regulation of physiological processes in locust oviduct. Here we report the isolation, cloning, and tissue expression of a putative octopamine/tyramine receptor from the locust, Locusta migratoria. Degenerate oligonucleotides in PCR reactions were first used to obtain partial cDNA sequences and then these partial sequences were used in screens to obtain a full-length cDNA. The cloned cDNA is about 3.1 kb long and encodes a protein of 484 amino acid residues with typical characteristics of GPCRs including seven transmembrane domains and many signature residues. The amino acid sequence of the cloned cDNA displays sequence similarities with known GPCRs, particularly octopamine/tyramine receptors. Screening of the locust genomic DNA library resulted in isolation of a genomic DNA with the same size as the cDNA, indicating that the gene is intron-less. RT-PCR and Northern blot analyses revealed the expression of the receptor mRNA in brain, ventral nerve cord, oviduct, and midgut tissues. Southern blot analyses using EcoRI and HindIII restriction endonucleases recognized at least two distinct gene bands.     

OCTOPAMINE IN MAMMALIAN BRAIN: RAPID POST MORTEM INCREASE AND EFFECTS OF DRUGS

A modified enzyme radiochemical assay for octopamine, based upon the N-methylation of octopamine by the enzyme phenylethanolamine N-methyl transferase (S-Adenosyl-1-methionine: phenylcthanolamine N-methyl transferase EC 2.1.28), has been developed. [³H]Methyl-S-adenosyl-l- methionine was used as methyl donor, and the reaction products separated by thin-layer chromatography prior to liquid scintillation counting. The method had a sensitivity of about 100 pg, and was suitable for the measurement of endogenous octopamine levels in mammalian brain. Although the method could be used for the determination of phenylethanolamine with similar sensitivity, concentrations of this amine in brain were too low for routine measurement. Octopamine levels in the brains of a number of mammalian species were determined using this procedure. Concentrations of the amine in mouse brain were lower in animals killed by rapid freczing than in animals killed by decapitation; a further increase in brain octopamine took place post-mortem. Brain octopamine was increased following treatment with MAO inhibitors, p-chlorophenylalanine, phenylalanine, tyrosine or phenylethylamine. The effects of tyrosine and phenylethylamine were greatly increased by pretreatment with a monoamine oxidase inhibitor. The antidepressants imipramine and iprindole gave rise to increased brain octopamine concentrations, possibly through an effect upon monoamine oxidase. Administration of chlordiazepoxide chlorpromazine, thyroxine, or reserpine had no effect upon brain octopamine.     

Development of an ionic liquid-based ultrasonic-assisted liquid-liquid microextraction method for sensitive determination of biogenic amines: application to the analysis of octopamine, tyramine and phenethylamine in beer samples

A simple and efficient method, ionic liquid-based ultrasound-assisted liquid-liquid microextraction, has been developed for the determination of three biogenic amines including octopamine (OCT), tyramine (TYR) and phenethylamine (PHE). Fluorescence probe 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester was applied for derivatization of biogenic amines and high-performance liquid chromatography coupled with fluorescence detection was used for the determination of the derivatives. The factors affecting the extraction efficiency, such as the type and volume of ionic liquid, ultrasonication time and centrifugation time have been investigated in detail. Under the optimum conditions, linearity of the method was observed in the range of 0.5-50 μgmL(-1) for OCT and TYR, and 0.025-2.5 μgmL(-1) for PHE, respectively, with correlation coefficients (γ)>0.996. The limits of detection ranged from 0.25-50 ngmL(-1) (S/N=3). The spiked recoveries of three target compounds in beer samples were in the range of 90.2-114%. As a result, this method has been successfully applied for the sensitive determination of OCT, TYR and PHE in beer samples.     

Trace amines differentially regulate adult locomotor activity, cocaine sensitivity, and female fertility in Drosophila melanogaster

The trace biogenic amines tyramine and octopamine are found in the nervous systems of animals ranging in complexity from nematodes to mammals. In insects such as Drosophila melanogaster, the trace amine octopamine is a well-established neuromodulator that mediates a diverse range of physiological processes, but an independent role for tyramine is less clear. Tyramine is synthesized from tyrosine by the enzyme tyrosine decarboxylase (TDC). We previously reported the identification of two Tdc genes in Drosophila: the peripherally-expressed Tdc1 and the neurally-expressed Tdc2. To further clarify the neural functions of the trace amines in Drosophila, we examined normal and cocaine-induced locomotor activity in flies that lack both neural tyramine and octopamine because of mutation in Tdc2 (Tdc2(RO54)). Tdc2(RO54) flies have dramatically reduced basal locomotor activity levels and are hypersensitive to an initial dose of cocaine. Tdc2-targeted expression of the constitutively active inward rectifying potassium channel Kir2.1 replicates these phenotypes, and Tdc2-driven expression of Tdc1 rescues the phenotypes. However, flies that contain no measurable neural octopamine and an excess of tyramine due to a null mutation in the tyramine beta-hydroxylase gene (TbetaH(nM18)) exhibit normal locomotor activity and cocaine responses in spite of showing female sterility due to loss of octopamine. The ability of elevated levels of neural tyramine in TbetaH(nM18) flies to supplant the role of octopamine in adult locomotor and cocaine-induced behaviors, but not in functions related to female fertility, indicates mechanistic differences in the roles of trace amines in these processes.     

Purification and properties of rat adrenal phenylethanolamine-N-methyl transferase

A procedure has been developed for the purification of phenylethanolamine-Nmethyl transferase (PNMT) (EC 2.1.1) from adrenal glands of rats. Ninety percent of the enzyme activity was in the 105,000g supernatant fraction. After chromatography on Sephadex G-150 and DEAE-cellulose, the PNMT showed two molecular species but the same specific activity on polyacrylamide gel electrophoresis. The final product was enriched nearly 100-fold. The methylation reaction is linear with increasing enzyme concentration, and the enzyme pH optimum was 8.0. The enzyme is relatively stable at 40 °C, but activity is partially destroyed by incubation at 60 °C. Several substrates were tested: octopamine, norepinephrine, tyramine, phenylethanolamine. Greatest affinity was for octopamine. All these substrates and the methyl group donor, S-adenosylmethionine, were inhibitory at high concentrations. Preincubation of the enzyme with norepinephrine accelerated the initial rate of the methylation reaction, while preincubation with S-adenosylmethionine had no such effect. A specific antibody against this purified enzyme was prepared. This antibody inhibited the enzyme activity and also precipitated it. Various immunological studies using this antibody are described.     

Phosphorimetric assay for dopamine beta-hydroxylase in rat plasma

A sensitive phosphorimetric method for the assay of dopamine β-hydroxylase in rat plasma is described. Octopamine, formed enzymatically from the substrate tyramine, is separated by Dowex 50W-X4 column chromatography and oxidized with periodate to p-hydroxybenz-aldehyde. The aldehyde is extracted with ether and then determined phosphorimetrically in a mixture of ether and ethanolic potassium hydroxide. The assay requires as little as 40 μl of rat plasma for the test and the blank with the lower limit of detection for octopamine at 60 pmol.     

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.     

effect of stress on levels of octopamine,dopamine and serotonin in the American cockroach (Periplaneta americana L.)

1. Various biogenic amines including octopamine, dopamine and serotonin, and their precursors and metabolites in haemolymph and the central nervous system from American cockroaches (Periplaneta americana L.) were measured using electrochemical detection.2. Octopamine was found in similar high relative abundances in haemolymph and the central nervous system.3. The amount of octopamine was much higher than that of tyramine and synephrine in haemolymph and thoracic nerve cord, whereas tyramine was at the highest level followed by octopamine and synephrine in the brain.4. Insects were stressed by vibrating at 100 or 1000 Hz, visually by flashing light at 4 Hz for 15 min or by immersing the insect in water at 60°C for 30 sec, which resulted in the elevation of octopamine, tyramine, synephrine and tyrosine levels in thoracic nerve cord.     

Assay for dopamine β-hydroxylase in rat serum and adrenal medulla by high-performance liquid chromatography with fluorescence detection

A highly sensitive method for the assay of dopamine β-hydroxylase in rat serum and in sample solution prepared from rat adrenal medulla is described which employs high-performance liquid chromatography with fluorescence detection. Octopamine, formed enzymatically from the substrate tyramine, is separated by Dowex 50W-X4 column chromatography and oxidized with periodate to p-hydroxybenzaldehyde, which is then converted into a fluorescent compound with 2,2′-dithiobis(1-aminonaphthalene). The derivative, after extraction with n-hexane—chloroform, is separated by normal-phase chromatography on Alox T. The limit of detection for octopamine formed enzymatically is 10 pmol. This method requires as little as 5 μl of rat serum.     

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.     

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.     

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.     

DEVELOPMENTAL CHARACTERISTICS OF PHENYLETHANOLAMINE AND OCTOPAMINE IN THE RAT BRAIN

Abstract— Phenylethanolamine and octopamine have been detected in the developing rat brain. Maximum concentration of these amines occurs early in development (16-17 days of gestation). At this developmental stage, the brain concentration of these amines is higher than that of norepinephrine. There is a sharp decline in the phenylethanolamine and octopamine concentrations on day 18 of gestation to approximately those of the adult. This decrease coincides with an increase in-monoamine oxidase activity of fetal brain, with an increase in the activities of tyrosine hydroxylase and dopamine-β-hydroxylase, and with the appearance of a saturable active uptake mechanism for norepinephrine. The administration of iproniazid, a monoamine oxidase inhibitor, to pregnant rats produced an increase in phenylethanolamine, octopamine and norepinephrine concentrations in the fetal rat brain at 16 days of gestation. p -Chlorophenylalanine, an inhibitor of phenylalanine hydroxylase, decreased fetal brain norepinephrine; this drug increased brain levels of phenylethanolamine and octopamine. The combined administration of iproniazid, p -chlorophenylalanine and phenylalanine to pregnant rats resulted in increased concentrations of octopamine and in a several-fold increase of phenylethanolamine levels; norepinephrine concentrations were sharply reduced. The possible significance of these findings in relation to pathological conditions such as phenylketonuria is discussed.     

A time-resolved assay of dopamine β-hydroxylase activity utilizing high-pressure liquid chromatography

A time-resolved assay of dopamine β-hydroxylase (EC 1.14.17.1) activity utilizing high-pressure liquid chromatography is described. The conversion of tyramine to octopamine by the enzyme was used as a standard reaction. The analytical separation of the assay substrate and product employed a reversed-phase ion-pair chromatographic system, with ultraviolet absorbance detection of eluents at 280 nm. Aliquots of the assay solution were injected directly onto the high-pressure liquid chromatography column and were separated in 6 min total elapsed time, thus permitting time-resolved determination of the produet. Quantities of octopamine as small as 20 pmol could be measured. This facile method is more straightforward, convenient, and sensitive than previously published physical and spectroscopic methods of determining dopamine β-hydroxylase activity.     

Influence of the biogenic amine tyramine on ethanol-induced behaviors in Drosophila

The biogenic amine tyramine has been implicated in drug-induced behavior. The Drosophila inactive mutant is characterized by reduced tyramine and octopamine levels and is defective in cocaine sensitization. To test whether there is an overlap in the use of the amine neurotransmitter system in ethanol- and cocaine-induced behaviors, mutant analyses were extended to the phenotypic characterization of inactive and other mutants effecting the tyramine and octopamine neurotransmitter system. The inactive mutant displays increased ethanol sensitivity and is impaired in the initial startle response upon ethanol application. Furthermore, this mutant fails to regulate its alcohol-induced hyperactivity properly. In contrast to the defects seen after cocaine application, inactive mutants develop normal ethanol tolerance and sensitize to the locomotor activating effect of ethanol. The tyramine-beta-hydroxylase mutant (TbetaH) with increased tyramine and depleted octopamine levels displays normal ethanol sensitivity, a startle repression, and hyperactivates more in response to ethanol. In addition, TbetaH mutants fail to develop a tolerance to the hyperactivating effect of ethanol. Ethanol-induced sensitization does not seem to be impaired in either mutant, suggesting that tyramine is not required for this process. The comparative analysis of the phenotypes associated with inactive and TbetaH mutants suggests that the fine tuning of ethanol-induced hyperactivity can be correlated with different tyramine levels. Defects in other aspects of ethanol-induced behaviors might be due to different molecules or mechanisms.     

Tyramine and octopamine have opposite effects on the locomotion of Drosophila larvae

Biogenic amines are believed to play important roles in producing behaviors. Although some biogenic amines have been extensively studied in both vertebrates and invertebrates, little is known about the effects of trace amines like tyramine and octopamine. We investigated how trace amines affect behaviors using quantitative morphometric methods on Drosophila Tbetah(nM18) and iav(N) mutants that have altered levels of tyramine and octopamine. Locomotion of wild-type and mutant third instar larvae was analyzed using Dynamic Image Analysis System (DIAS) software. We found that Tbetah(nM18) mutants, with elevated tyramine levels and reduced octopamine levels, had a severe locomotion phenotype. Mutant larvae spent much more time in pausing episodes than wild-type larvae and displayed a reduction in speed and linear translocation. The locomotion phenotype was partially rescued by feeding Tbetah(nM18) larvae octopamine, an effect that could be nullified with simultaneous feeding of tyramine. Feeding Tbetah(nM18) larvae yohimbine, an agent that inhibits the activity of Drosophila tyramine receptors, also improved some locomotion parameters. Feeding both octopamine and yohimbine further improved rescue efficiency. Simultaneously reducing the octopamine and tyramine levels as in iav(N) larvae, in contrast, led to a less severe behavioral phenotype than that of Tbetah(nM18) mutants. Feeding iav(N) larvae either tyramine or octopamine exerted only a minor improvement in locomotion. These results suggest that tyramine and octopamine have opposite effects on Drosophila larval locomotion regulation and that a balance between the two is important in producing normal behavior.     

Effects of biogenic amines and related agonists and antagonists on the isolated heart of the common cuttlefish Sepia officinalis L

1. Effects of noradrenaline and the related compounds adrenaline, dopamine, octopamine, tyramine, clonidine and isoprenaline were studied in isolated heart preparations from the cuttlefish Sepia officinalis L. 2. All analogues produced a positive inotropic affect, with noradrenaline being the most potent substance. The chronotropic effects of the tested compounds differed widely. 3. The action of substances of the phenylethanolamine group were not antagonized by propranolol but were partly antagonized by phentolamine. 4. Serotonin and its analogues also produced cardio-excitation. These effects were blocked by cyproheptadine but not by methysergide. 5. These results indicate the presence of two different receptors in the Sepia myocardium: one type reacting with noradrenaline most effectively and a second type being stimulated by serotonin.