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Serotonin-sensitive adenylate cyclase in neural tissue and its similarity to the serotonin receptor: a possible site of action of lysergic acid diethylamide.

An adenylate cyclase (EC 4.6.1.1) that is activated specifically by low concentrations of serotonin has been identified in homogenates of the thoracic ganglia of an insect nervous system. The activation of this enzyme by serotonin was selectively inhibited by extremely low concentrations of D-lysergic acid diethylamide (LSD), 2-bromo-LSD, and cyproheptadine, agents which are known to block certain serotonin receptors in vivo. The inhibition was competitive with respect to serotonin, and the calculated inhibitory constant of LSD for this serotonin-sensitive adenylate cyclase was 5 nM. The data are consistent with a model in which the serotonin receptor of neural tissue is intimately associated with a serotonin-sensitive adenylate cyclase which mediates serotonergic neurotransmission. The results are also compatible with the possibility that some of the physiological effects of LSD may be mediated through interaction with serotonin-sensitive adenylate cyclase.

Adenylyl Cyclases↗

Studies on the carbohydrate moiety of dopamine beta-hydroxylase: interaction of the enzyme with concanavalin A.

Dopamine beta-hydroxylase [EC 1.14.17.1; 3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating)], a glycoprotein, was found to interact strongly with the plant lectin, concanavalin A. This interaction did not appear to alter the catalytic properties of the enzyme and did not prevent interaction of the protein with specific antibodies. Dopamine-beta-hydroxylase is adsorbed to concanavalin A covalently bound to Sepharose and can be eluted with alpha-methylmannoside. The enzyme is catalytically active when it is immobilized on a concanavalin A-Sepharose column. Enzymic removal of four of seven mannose residues present in the monomer does not result in the loss of any catalytic activity. It is concluded that the active site is not near the point of carbohydrate attachment on the molecule and that this may be of significance in orienting the enzyme on the vesicular membrane.

Adrenal Glands↗

A Ca2+-linked increase in coupled cAMP synthesis and hydrolysis is an early event in cholinergic and beta-adrenergic stimulation of parotid secretion.

The dynamics and compartmental characteristics of cAMP metabolism were examined by 18O labeling of cellular adenine nucleotide alpha phosphoryls in rat parotid gland stimulated to secrete with beta-adrenergic and cholinergic agents. The secretory response occurred in association with a rapidly increased rate of cAMP hydrolysis apparently coordinated with an equivalent increase in the rate of cAMP synthesis, since the cellular concentration of cAMP remained unchanged. The magnitude of this metabolic response was equivalent to the metabolism of 10-75 times the cellular content of cAMP within the first minute of stimulation. This increased metabolic rate occurred only during the early (1-3 min) period of stimulation, in what appeared to be an exclusive cellular compartment distinguished by a unique distribution of 18O among adenine nucleotide alpha phosphoryls. This 18O distribution contrasted with that produced by forskolin, which increased cellular cAMP concentration and elicited only a delayed response missing the early secretory component. The early acceleration of cAMP metabolism appeared linked to a stimulus-induced increase in intracellular Ca2+ concentration, since the Ca2+ ionophore ionomycin produced the same metabolic response in association with secretion. These observations suggest that cAMP metabolism is involved in stimulus-secretion coupling by a Ca2+-linked mechanism different from that in which cAMP plays the role of a second messenger.

Adrenergic beta-Agonists↗

Serotonin and aggressive motivation in crustaceans: altering the decision to retreat.

In crustaceans, as in most animal species, the amine serotonin has been suggested to serve important roles in aggression. Here we show that injection of serotonin into the hemolymph of subordinate, freely moving animals results in a renewed willingness of these animals to engage the dominants in further agonistic encounters. By multivariate statistical analysis, we demonstrate that this reversal results principally from a reduction in the likelihood of retreat and an increase in the duration of fighting. Serotonin infusion does not alter other aspects of fighting behavior, including which animal initiates an encounter, how quickly fighting escalates, or which animal eventually retreats. Preliminary studies suggest that serotonin uptake plays an important role in this behavioral reversal.

Aggression↗

The reduction of membrane-bound dopamine beta-monooxygenase in resealed chromaffin granule ghosts. Is intragranular ascorbic acid a mediator for extragranular reducing equivalents?

The role of internal and external reductants in the dopamine beta-monooxygenase (D beta M)-catalyzed conversion of dopamine to norepinephrine has been investigated in resealed chromaffin granule ghosts. The rate of norepinephrine production was not affected by the exclusion of internal ascorbate. The omission of ascorbate from the external medium drastically reduced the norepinephrine production without affecting the net rate of dopamine uptake. In the presence of the external reductant, the internal ascorbate levels were constant throughout the incubation period. The rate of norepinephrine production was not affected when ghosts were resealed to contain the D beta M reduction site inhibitor, imino-D-glucoascorbate. Ghosts incubated with external imino-D-glucoascorbate reduced the norepinephrine production. The weak D beta M reductant, 6-amino-L-ascorbic acid, was found to be a good external reductant for granule ghosts. The outcome of the above experiments was not altered when dopamine was replaced with the reductively inactive D beta M substrate, tyramine. These results and the known topology of membrane-bound D beta M disfavor the direct reduction of the enzyme by the external reductant. Our observations are consistent with the hypothesis that external ascorbate is the sole source of reducing equivalents for D beta M monooxygenation and that internal soluble ascorbate (or dopamine) may not directly reduce or mediate the reduction of membrane-bound D beta M in resealed granule ghosts.

Adenosine Triphosphate↗

Metabolic flux analysis of the phenylpropanoid pathway in wound-healing potato tuber tissue using stable isotope-labeled tracer and LC-MS spectroscopy.

The metabolic flux of two phenylpropanoid metabolites, N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA), in the wound-healing potato tuber tissue was quantitatively analyzed by a newly developed method based upon the tracer experiment using stable isotope-labeled compounds and LC-MS. Tuber disks were treated with aqueous solution of L-phenyl-d(5)-alanine, and the change in the ratio of stable isotope-labeled compound to non-labeled (isotope abundance) was monitored for p-CO and CGA in the tissue extract by LC-MS. The time-dependent change in the isotope abundance of each metabolite was fitted to an equation that was derived from the formation and conversion kinetics of each compound. Good correlations were obtained between the observed and calculated isotope abundances for both p-CO and CGA. The rates of p-CO formation and conversion (i.e. fluxes) were 1.15 and 0.96 nmol (g FW)(-1) h(-1), respectively, and for CGA, the rates 4.63 and 0.42 nmol (g FW)(-1) h(-1), respectively. This analysis enabled a direct comparison of the biosynthetic activity between these two compounds.

Algorithms↗

Plasma amino acids as predictors of the severity and outcome of sepsis.

Sepsis is a major catabolic insult resulting in a peripheral energy deficit which is made up in part by increased breakdown of lean body mass and oxidation of amino acids, principally the branched chain amino acids. The prognosis in any given case of sepsis is difficult to predict, but should theoretically be related to the degree of disturbance in peripheral energy deficit, which may in turn, be related to plasma amino acid pattern. In order to study whether this hypothesis was correct, plasma amino acids and some of their metabolic byproducts, the beta-hydroxyphenylethanolamines, were studied in 25 septic patients, and were used as discriminant variables in a series of computer performed discriminant analyses and multiple regressions. The two functions tested were the degree of metabolic septic encephalopathy as a determinant of the severity of sepsis and the final outcome in the septic patient. Plasma amino acid patterns exhibited elevated levels of the aromatic and sulfur containing amino acids, phenylalanine, tryosine, tryptophan, methionine, cysteine, and taurine, normal concentrations of alanine, and low normal concentrations of the branched chain amino acids, valine, leucine and isoleucine. Arginine levels, as previously noted, were very low. Patients not surviving the septic episode exhibited higher concentrations of aromatic and sulfur containing amino acids, while patients surviving sepsis had higher concentrations of the branched chain amino acids and arginine. When the degree of encephalopathy as a determinant of the severity of sepsis and step wise discriminant analysis with multiple crescent techniques were used, the best discriminant function between patients with and without encephalopathy was found to result from the interaction of cysteine, methionine, phenylalanine, isoleucine, leucine, and valine. These amino acids gave a correct classification in 82% of patients with no encephalopathy, and 80% of patients with septic encephalopathy. When the same amino acids were used for the discriminant analysis for patients dying of sepsis and patients surviving, the best discriminant function was achieved by using plasma concentrations of alanine, cysteine, methionine, isoleucine, arginine, tyrosine and phenylalanine resulting in 91% of the nonsurvivors, and 79% of the survivors correctly classified. The results suggest a close and significant relationship between the deranged energy metabolism and muscle protein breakdown in sepsis, and the outcome. This further suggests a central role for certain amino acids in perhaps predicting the severity of sepsis and its outcome.

2-Hydroxyphenethylamine↗

Early multiple organ failure after recurrent endotoxemia in the presence of vasoconstrictor-masked hypovolemia.

OBJECTIVE: Critically ill patients who develop multiple organ failure during systemic inflammatory states are often predisposed to hypovolemia and vasoconstrictor therapy. Although numerous investigations have evaluated the sequelae of systemic inflammation, no data are available on the contribution of chronic vasoconstrictor-masked hypovolemia to organ dysfunction and morphology. DESIGN: Prospective, randomized laboratory investigation. SETTING: University research laboratory. SUBJECTS: Eighteen adult chronically instrumented sheep. INTERVENTIONS: The animals were randomly assigned to one of three groups. In the norfenefrine-masked hypovolemia plus endotoxemia (NMH+ENDO) group, mean arterial pressures of 80 mm Hg were maintained by using the alpha1-adrenergic catecholamine norfenefrine for 52 hrs during hypovolemia. Hypovolemia was induced by hemorrhage (about 23 mL x kg(-1)) until mean arterial pressures reached 40 mm Hg. Endotoxin (0.5 microg x k(-1)) was then injected after 4, 16, 28, and 40 hrs. The NMH group received norfenefrine-masked hypovolemia but no endotoxin. In the ENDO group, recurrent endotoxemia was induced during normovolemia. MEASUREMENTS AND MAIN RESULTS: Despite profound differences in fluid management, cardiovascular filling pressures were not statistically different between groups. Endotoxemia induced norfenefrine-refractory shock (p < .05 vs. the other groups) and contributed to renal dysfunction only during vasoconstrictor-masked hypovolemia. Norfenefrine-masked hypovolemia caused disseminated cardiac cell necrosis independent of endotoxemia (p < .05 vs. ENDO). CONCLUSIONS: Hypovolemia can be masked when volume status is monitored by filling pressures. In this new model of endotoxemia-associated multiple organ failure, chronic vasoconstrictor-masked hypovolemia turned systemic inflammation into a life-threatening condition with renal and cardiovascular failure. Cardiomyocyte necroses were caused by vasoconstrictor-masked hypovolemia but were unrelated to cardiovascular failure.

Adrenergic alpha-Agonists↗

Amino acid residues involved in interaction with tyramine in the Bombyx mori tyramine receptor.

To identify amino acid residues interacting with tyramine (TA) in the Bombyx mori TA (BmTA) receptor, several mutant receptors were expressed in HEK-293 cells and examined for their abilities to bind TA and to attenuate forskolin-stimulated cAMP production in response to TA. The D134A BmTA receptor showed no specific [3H]TA binding and no TA-attenuation of cAMP levels. Although the S218A and S222A BmTA receptors showed no specific [3H]TA binding, they still had the ability to mediate the attenuation of cAMP levels in response to the high concentration (100 microM) of TA. The double mutation of Ser218 and Ser222 to Ala, however, led to the loss of TA-attenuation of cAMP levels. The present study thus confirms that at least three amino acid residues play key roles in interaction with TA in the BmTA receptor.

Amino Acid Sequence↗

Two new beta 2-adrenoceptor agonists, D-2343 and QH 25, studied in asthmatic patients.

Two new beta 2-adrenoceptor agonists, D-2343 and QH 25, have been studied in asthmatic patients. Animal experiments indicated less effect on skeletal muscle than on bronchial muscle. D-2343 (four i.v. cumulatively increasing doses, 0.21--6.3 micrograms kg-1 min-1) and i.v. terbutaline (four cumulative doses 0.07--2.1 micrograms kg-1 min-1) were single-blindly compared in a randomized crossover study in eight asthmatics. It was shown that D-2343 is an effective beta 2-adrenoceptor agonist with 5--6 times lower potency than terbutaline. The tremor-inducing effect of D-2343 was the same as that of terbutaline, when the same degree of bronchodilation was obtained. The effect of QH 25 (0.25, 0.75 and 2.25 mg cumulatively) administered orally, was single-blindly compared with the effect of oral salbutamol (2, 6 and 18 mg) in a randomized crossover study in eight asthmatics. QH 25 was shown to be an effective beta 2-adrenoceptor stimulant about 12 times more potent than salbutamol. The tremor-inducing capacity was the same as for salbutamol when equipotent bronchodilating doses were studied.

Adrenergic beta-Agonists↗

A new beta 2-adrenoceptor agonist--D 2343--with long duration. Inhalation comparison with terbutaline in asthmatics.

A new beta 2-adrenoceptor agonist D2343 has been shown in a human i.v. study to be less potent than terbutaline. Animal experiments showed an increased affinity to lung tissue and a longer bronchodilating effect than terbutaline when inhaled. In a randomized single-blind, crossover comparison, 2.5 and 5 mg D2343 and 5 mg terbutaline were inhaled by eight asthma patients. We found that 5 mg of D2343 was equipotent to 5 mg terbutaline in bronchodilatory effect measured with FEV1. The duration was longer with an increased bronchodilation (P less than 0.01) 6 h after D2343 inhalation compared with terbutaline. The mean fall in FEV1 3-6 h after inhalation was significantly greater with terbutaline than with D2343 (P less than 0.05). Thus, this study shows that inhaled D2343 has a longer bronchodilating effect than terbutaline.

Asthma↗

Ion currents and spiking properties of identified subtypes of locust octopaminergic dorsal unpaired median neurons.

Efferent dorsal unpaired median (DUM) neurons are key elements of an insect neuromodulatory system. In locusts, subpopulations of DUM neurons mediate octopaminergic modulation at specific targets depending on their activity during different behaviours. This study investigates whether in addition to synaptic inputs, activity in DUM neurons depends on intrinsic membrane properties. Intracellular in situ recordings and whole-cell patch-clamp recordings from freshly isolated somata characterize somatic voltage signals and the underlying ion currents of individual subtypes of DUM neurons identified beforehand by a vital retrograde tracing technique. Na(+), Ca(2+), K(+) currents and a hyperpolarization-activated (I(h)) current are described in detail for their (in-)activation properties and subtype-specific current densities. In addition, a Ca(2+)-dependent K(+) current is demonstrated by its sensitivity to cadmium and charybdotoxin. This complex current composition determines somatic excitability similar in all subtypes of DUM neurons. Both Na(+) and Ca(2+) currents generate overshooting somatic action potentials. Repolarizing K(+) currents, in particular transient, subthreshold-activating A-currents, regulate the firing frequency and cause delayed excitation by shunting depolarizing input. An opposing hyperpolarization-activated (I(h)) current contributes to the resting membrane potential and induces rebound activity after prolonged inhibition phases. A quantitative analysis reveals subtype-specific differences in current densities with more inhibitory I(K) but less depolarizing I(Na) and I(h) - at least in DUM3 neurons promoting a reliable suppression of their activity as observed during behaviour. In contrast, DUM neurons that are easily activated during behaviour (DUM3,4,5 and DUMETi) express less I(K) and a pronounced depolarizing I(h) promoting excitability.

Action Potentials↗