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A method for the estimation of dopamine beta-hydroxylase activity in human serum is described, based on a thin layer chromatographic separation of the substrate ([14C]tyramine) from the reaction product ([14C]octopamine). The basic properties of the human serum enzyme, investigated by this method are described.
Potato tuber disks, when treated with laminarin, a beta-1,3-glucooligosaccharide from Laminaria digitata, accumulate a hydroxycinnamoyl amide compound, N-p-coumaroyloctopamine (p-CO). The biosynthesis of p-CO was investigated by feeding experiments, in order to show that the precursors of N-p-coumaroyl and octopamine moieties of p-CO are L-phenylalanine and L-tyrosine, respectively. The treatment of potato tuber tissue with laminarin resulted in elevated activities of four enzymes which are putatively involved in p-CO biosynthesis: phenylalanine ammonia lyase (PAL; EC 4.3.1.5), 4-hydroxycinnamic acid:CoA ligase (4CL; EC 6.2.1.12), hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25). Among these, the response of TyrDC was specific to laminarin treatment, thus indicating that the regulation of TyrDC activity is critical for the accumulation of p-CO in potato tuber tissue.
In this article we describe recent advances in functional studies on the role of octopamine released in the periphery by efferent dorsal or ventral unpaired median neurons. In addition to the previously described modulatory effects on the neuromuscular junction, we describe a metabolic regulatory role for these neurons. Due to their activity glycolytic rates in target tissues, such as muscles, are increased. In flight muscles that use carbohydrate catabolism only at take-off but have to switch to lipid oxidation during prolonged flight, these neurons are only active at rest but are inhibited as soon as flight motor patterns are selected.
The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected as a band of 75-80 kDa in membranes from recombinant baculovirus-infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [(3)H-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca(2+)](i)) in fura 2-AM loaded sf21 cells, and promoted [(35)S]-GTP(gamma)S binding to membranes. The effects of bradykinin were dose dependent (with an EC(50) of 50 nmol/L for calcium mobilization) and were inhibited by N-alpha-adamantaneacetyl-D-Arg-[Hyp(3),Thi(5,8),D-phe(7)]-Bk, a specific B2 receptor antagonist. When the B2 antagonist was applied at the top of the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor occupation. No calcium mobilization was elicited by 1 micromol/L (Des-Arg(9))-Bk, a B1 receptor agonist that did not inhibit the subsequent action of 100 nmol/L bradykinin. No effect of bradykinin was detected in uninfected cells or cells infected with the wild-type baculovirus. Bradykinin-induced [Ca(2+)](i) mobilization was increased by genistein and tyrphostin A51. These tyrosine kinase inhibitors did not modify basal levels of [Ca(2+)](i). Homologous desensitization of the B2 receptor was observed after repeated applications of bradykinin, which resulted in attenuated changes in intracellular calcium. In addition, genistein promoted an increased response to a third exposure to the agonist when applied after washing the cells that had been previously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous octopamine G protein-coupled receptor or by thapsigargin. The B2 receptor, detected by confocal microscopy in unpermeabilized cells, remained constant at the surface of cells stimulated with bradykinin for 10 min, in the presence or absence of genistein. Agonist-promoted phosphorylation of the B2 receptor was markedly accentuated by genistein treatment. Phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by bradykinin, could act in a step previous to receptor phosphorylation. Interestingly, genistein prevented agonist-induced G protein uncoupling from B2 receptors, determined by in vitro bradykinin-stimulated [(35)S]-GTP(gamma)S binding, in membranes from bradykinin pretreated cells. Our results suggest that tyrosine kinase(s) regulate the activity of the human B2 receptor in sf21 cells by affecting its coupling to G proteins and its phosphorylation.
The effect of total colectomy and "intestinal sterilisation" following chronic portocaval shunt was investigated in 96 Sprague-Dawley rats: while both therapeutic procedures are of no significant impact on serum ammonia concentrations there is a significant reduction of brain octopamine, a known false neuro-chemical transmitter, and a clear response on aromatic amino acid levels in brain and plasma. The interference with central and peripheral neurotransmitters is suggested as an alternative mechanism in experimental encephalopathy.
The heat-shock response was studied in Drosophila virilis strains with normal and impaired neurohormonal stress reaction. Flies from the latter strain were shown to have the impaired heat-shock response. In this strain, transcription of the heat shock gene hsp83 is reduced and synthesis of all heat shock proteins is suppressed. The neurohormonal stress reaction (status of dopamine, octopamine, and juvenile hormone metabolic systems) was examined in D. melanogaster strains having normal and impaired heat-shock response. The impairment of this response did not prevent the development of the stress reaction: in flies of both strains, the stress exposure resulted in an increase in the dopamine content and in a decrease in the activity of tyrosine decarboxylase (the first enzyme of dopamine synthesis) and in the level of juvenile hormone degradation. However, stress reactivity in mutant individuals differed from that in flies that did not carry stress-related mutations.
Insect flight is one of the most intense and energy-demanding physiological activities. High carbohydrate oxidation rates are necessary for take-off, but, to spare the limited carbohydrate reserves, long-distance flyers, such as locusts, soon switch to lipid as the main fuel. We demonstrate that before a flight, locust muscles are metabolically poised for take-off by the release of octopamine from central modulatory dorsal unpaired median (DUM) neurons, which increases the levels of the potent glycolytic activator fructose 2,6-bisphosphate in flight muscle. Because DUM neurons innervating the flight muscles are active during rest but selectively inhibited during flight, they stimulate carbohydrate catabolism during take-off but tend to decrease muscle glycolysis during prolonged flight. cAMP-dependent protein kinase A is necessary but not sufficient for signal transduction, suggesting parallel control via a calcium-dependent pathway. Locust flight is the first reported instance of a direct and specific involvement of neuronal activity in the control of muscle glycolysis in working muscle during exercise.
Pregnant rats were kept on an ethanol-containing (6.8% v/v) liquid diet either from the 13th or 18th day of gestation. In pups exposed to ethanol from the 13th day of gestation, synaptosomal uptake of 3H-tyramine and its conversion to 3H-octopamine were increased initially and exposure to ethanol from the 18th day of gestation produced increases in synaptosomal uptake and conversion for longer periods. These changes were not observed in adult rats administered ethanol. Brain tyrosin hydroxylase activity in the developing rats was unaltered by continuous exposure to ethanol. Withdrawal from ethanol either at 0, 3 or 5 days of age enhanced the tendancy toward increased synaptosomal uptake and conversion, but the magnitude of change depended upon the time of exposure and the age at which withdrawal was initiated. These results suggest that maternal ethanol ingestion can affect synaptic function in the developing central noradrenergic system, with consequent alterations in neurotransmitter uptake and storage.
The membranes of isolated bovine adrenal chromaffin granules take up catecholamines by an ATP-dependent process which fulfills many criteria of a carriermediated energy-requiring transport. This transport together with an intragranular mechanism probably explains the high storage capacity of the chromaffin granules for catecholamines. 5.2 Other amines (e.g. 5-hydroxytryptamine (5HT), octopamine) are also taken up by the membranes, but to a different degree. This probably explains why amines normally not present in storage organelles, if available in increased amounts, may accumulate within these organelles and function as false neurohumoral transmitters. 5.3. Two mechanisms of amine uptake seem to exist at the level of the granule membranes, one reserpine-sensitive (transporting, for instance, catecholamines and 5HT), and one reserpine-resistant (transporting tryptamine and partly metaraminol). Both mechanisms depend on ATP. 5.4. Chlorpromazine interferes with the reserpine-sensitive as well as the reserpine-resistant uptake of noradrenaline (NA) by granule membranes to a similar degree. Consequently the mechanism of action of neuroleptics and reserpine at the granule membrane level are probably different. 5.5. There is only an incomplete parallelism between the inhibition of NA-uptake by granule membranes in vitro and the degree of enhancement of neuronal DA turnover in vivo caused by various neuroleptics. Therefore, factors other than interference with the amine transport through the granule membranes must be co-determinant in the neuroleptic induced enhancement of DA turnover in vivo.
The role of intra- and extravesicular ascorbate has been investigated in dopamine beta-monooxygenase (D beta M) turnover using adrenal medulla chromaffin granule ghosts. Resealing of vesicle ghosts with high levels of intravesicular ascorbate leads to viable vesicles, as evidenced from the high rates of the ATP-dependent accumulation of tyramine, Vmax = 14 +/- 1 nmol/min.mg and Km = 20 +/- 6 microM. However, the D beta M-catalyzed conversion of tyramine to octopamine occurs slowly, Vmax = 0.50 +/- 0.13 nmol/min.mg and Km = 29 +/- 18 mM. When ascorbate is present instead in the external buffer, the D beta M rate increases 3.6-fold for a final Vmax = 1.8 +/- 0.2 and Km = 1.2 +/- 0.3 mM. This relatively high rate of enzyme turnover is retained in ghosts resealed with a large excess of ascorbate oxidase, ruling out contamination by intravesicular ascorbate as the source of enzyme activity. The synergistic effect of intravesicular ascorbate was examined under conditions of 2 mM external ascorbate, showing that the enzymatic rate increases 2.7-fold, from 1.2 (0 internal ascorbate) to 3.2 +/- 0.4 nmol/min.mg (saturating internal ascorbate). This result confirms that high levels of internal ascorbate are not damaging to intravesicular D beta M. These studies demonstrate very clearly that external ascorbate is the preferred reductant for the membranous form of D beta M in chromaffin granule ghosts.
Graded chemical synaptic transmission is important for establishing the motor patterns produced by the pyloric central pattern generator (CPG) circuit of the lobster stomatogastric ganglion (Raper, 1979; Anderson and Barker, 1981; Graubard et al., 1983). We examined the modulatory effects of the amines dopamine (DA), serotonin (5-HT), and octopamine (Oct) on graded synaptic transmission at all the central chemical synapses made by the pyloric dilator (PD) neuron onto its follower cells, using synaptic input-output curves measured from cell somata. DA strongly reduced the graded synaptic strength at all the PD synapses. DA reduction of chemical synaptic strength from PD onto the inferior cardiac (IC) neuron could change the sign of synaptic interaction between these 2 cells from inhibitory to excitatory by uncovering a weak electrical connection. 5-HT had weaker and more variable effects, reducing graded synaptic strength from the PD onto the lateral pyloric and pyloric neurons and enhancing the weak synapse from the PD to the IC cell. Oct strongly enhanced the graded synaptic strength at all the PD central synapses. Oct enhancement of graded synaptic strength between the PD and IC cells could also change the sign of the interaction: weak, excitatory electrical coupling, which was sometimes dominant before Oct, was masked by the enhanced chemical inhibitory interaction during Oct application. Measurements of electrical coupling between 2 PD cells and between 2 postsynaptic cells suggest that Oct does not change the input resistance of these cells and may act directly at the PD synapses. The effects of DA and 5-HT are most easily explained by their general reductions in pre- and postsynaptic input resistance. DA, 5-HT, and Oct each produce a distinct pyloric motor pattern (Flamm and Harris-Warrick, 1986a). These amine-induced motor patterns may be explained by the unique actions of each amine on the intrinsic membrane properties of different pyloric CPG neurons (Flamm and Harris-Warrick, 1986b) and by modulation of graded synaptic transmission between the pyloric neurons.
Cholera toxin, injected into living fireflies, caused a delayed, non-hormone-dependent activation of adenylate cyclase, an elevation of cyclic AMP levels, and a brilliant persistent glow of the firefly light organ 8-20 hours following injection. During periods of spontaneous flashing, onset of toxin-induced glowing was abrupt and step-like in character, due to a markedly prolonged off-time of individual flashes. These observations provide a striking demonstration of the mechanism of cholera toxin action, and, together with other data, suggest that the initiation of the normal adult firefly flash is mediated through elevation of cyclic AMP levels secondary to the activation of an octopamine-sensitive adenylate cyclase.
Most behavioral reactions that habituate can also be dishabituated by strong stimuli. In the best studied cases, dishabituation seems to be the result of an independent "sensitization" of the behavioral reaction that compensates for habituation without necessarily abolishing it. Crayfish lateral giant (LG) neuron-mediated escape reactions are one of the most fully analyzed behavioral reactions that are prone to habituation; however, sensitization/dishabituation of LG escape has not previously been reported. Here, the effect of strong AC shocks to head or abdomen on the ability of 0.1 msec "test" shocks to sensory roots innervating the tailfan to elicit an LG escape response was examined. Following single AC shocks, test shock threshold for eliciting LG escape reliably fell 5-80% and recovered over 15 min to 1 hr. When AC shocks and test shocks alternated at 90 sec intervals, test shock threshold rapidly dropped to an asymptote that was maintained as long as AC shocks were given (up to 2 hr); following such repeated AC shocks, recovery often required a number of hours but was complete within 24. Comparable sensitization is seen in the response of interneuron A, the largest of a set of sensory interneurons that links afferents to LGs. AC shocks (to either head or tail) no longer sensitize abdominal LG reflex circuitry if the nerve cord is severed between thorax and abdomen. Thus, sensitization appears to depend on a neurally conducted influence that arises in the rostral half of the animal. Pharmacological evidence suggests that octopamine may mediate the sensitization.
Sensitive and specific radioimmunoassays of metanephrine and normetanephrine were developed by use of 125I-labeled synephrine and specific metanephrine antibody, and 125I-labeled octopamine and specific normetanephrine antibody. Specific antibody for both metanephrine and normetanephrine was raised in rabbits by immunization with bovine serum albumin conjugated with the corresponding hapten, prepared by the method of Grota and Brown (Endocrinology 1976;98:615). The detection limits of the metanephrine and the normetanephrine radioimmunoassays were 2 and 6 pg/tube, respectively. Mean plasma metanephrine and normetanephrine values for 24 normal subjects were 62 (SD 14) and 100 (SD 40) ng/L, respectively. Mean urinary metanephrine and normetanephrine values for 22 normal subjects were 154 (SD 74) and 217 (SD 109) micrograms/day. For 14 pheochromocytoma patients, plasma metanephrine and normetanephrine values ranged from 29 to 683 and from 28 to 7850 ng/L, and urinary metanephrine and normetanephrine values were 606 to 6630 and 296 to 4800 micrograms/day, respectively. The present methods are simple and suitable for routine tests or for mass screening for pheochromocytoma.
We quantified the effects amphetamine (AMPH), phenylethylamine (PEA), tyramine (TYR), octopamine (OCT) and DA, on initial rates of DA uptake (striatal minces), binding of [3H]mazindol to the neuronal uptake (NU) site (striatal membranes) and on DA and dihydroxyphenylacetic acid (DOPAC) efflux (striatal slices). In general, the order of potency for the three paradigms was: AMPH less than DA = TYR = PEA less than OCT. The Km values for uptake were positively correlated with the Ki values for inhibition of mazindol binding (r = 0.91; P less than .01) and with the potencies to induce DA efflux (r = 0.96; P less than .005). Potencies for inhibition of mazindol binding and for eliciting DA efflux also were highly correlated (r = 0.92; P less than .01). Correlations were lost if data for nomifensine (NOM), a NU inhibitor, were included in the analysis. Despite the significant correlations, AMPH, TYR, PEA and OCT Despite the significant correlations, AMPH, TYR, PEA, and OCT were 10 to 20 times more potent in inhibiting NU than in eliciting efflux or inhibiting mazindol binding. Conversely, the potency of NOM to inhibit mazindol binding was 10 and 800 times greater than that required to inhibit NU or to elicit DA release, respectively. NOM inhibited competitively AMPH-induced DA release. These results suggest that: 1) AMPH-like drugs bind to and are likely to be transported by the NU carrier, and 2) inward transport of these agents appears to have multiple effects (e.g., an increase in intracellular Na+ and Cl-) that act cooperatively to increase Vmax and reduce Km for the outward, carrier-mediated DA transport. AMPH, TYR, PEA and OCT had qualitatively similar effects on endogenous DA and [3H]DA release. Monoamine oxidase inhibition potentiated these effects. All these agents released more endogenous DA from control than from reserpine-treated slices. In control slices, TYR and OCT increased DA and DOPAC efflux; whereas AMPH and PEA increased DA and reduced DOPAC efflux, except at high concentrations in which DOPAC efflux also was increased. After reserpine, these agents reduced DOPAC efflux in proportion to the increased DA efflux. In summary, we propose that AMPH-like drugs increase DA efflux from a single cytoplasmic pool maintained by DA synthesis and spontaneous and drug-induced efflux of DA from storage vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)
The pathogenesis of hepatic encephalopathy has been investigated in a two-stage devascularization model in the rat with portavacal shunt and hepatic artery ligation. There is a significant increase in brain octopamine and phenylethanolamine and a decrease in brain norepinephrine (NE) 6 to 9 hours after hepatic artery ligation. The depletion of NE seems the sequel of diminished synthesis in the presence of an unaltered turnover rate, due to a blockade of tyrosine hydroxylase either by accumulation of false neurochemical transmitters or by phenylalanine. It is most marked in the cortex and midbrain. The high-energy phosphate compounds, ATP, phosphocreatine and glucose-6-phosphate are not diminished in hepatic coma, nor is glucose, indicating that other mechanism are involved in the pathogenesis of metabolic state by the increased ammonia level. "intestinal sterilization" and total colectomy have no significant effect on the ammonia level, but cause a decrease in the level or aromatic precursor amino acids in the plasma and brain, with normalization of the level of cerebral transmitters. These results permit the formulation of a unified concept of the hepatic coma syndrome and its clinical manifestations such as flapping tremor, the hyperdynamic cardiovascular state and the hepatorenal syndrome. Moreover, they form the basis for the introduction of a new therapeutic principle in the management of hepatic encephalopathy by L-dopa or modified amino acid solutions, which act by altering the central and peripheral neurotransmitters.
Four different adsorbents (activated charcoal, XAD-4, a strong base anion and a strong acid cation-exchange resin) were tested in vitro for their capacity to remove substances that may be important in the development of hepatic encephalopathy. Separate columns packed with one of these adsorbents were perfused for three hours with a reconstituted plasma solution containing simultaneously high concentrations of amino-acids, ammoniumchloride, short-chain fatty acids, octopamine and bile salts. Effective removal of all these substances was only obtained when either activated charcoal, or XAD-4, were combined with the cation-exchange resin. Possible implications for the treatment of hepatic coma are discussed.
In fourteen subjects with various stages of hepatic encephalopathy (HE), Visual Evoked Potential (VEP) recording and blood ammonia (NH3), octopamine (OCT) and phenylethanolamine (PEA) determinations were performed before and during a four-day treatment with branched chain amino acid (BCAA) i.v. infusion. All the subjects with HE showed significant basal VEP alterations, namely an increased latency and a lowered amplitude of the P100 wave, in comparison with a control group of 26 normal subjects. A significant improvement in P100 wave amplitude occurred just about 60' after the beginning of BCAA infusion, while P100 latency was still unaffected, as were OCT, PEA and NH3 levels. After the fourth day of BCAA treatment, both P100 wave amplitudes and latencies were strongly improved, together with OCT, PEA and NH3 levels. VEP improvements seemed to be well correlated with HE clinical evolution, and were able to detect modifications of central nervous system reactivity earlier than serum parameters.