Biomedical subjects
Publications and source records attributed to D M Kuhn.
L-Tryptophan produces a significant antihypertensive effect in spontaneously hypertensive rats while D-tryptophan does not change blood pressure. Both isomers of tryptophan significantly increase brain serotonin to the same extent at a time when the antihypertensive effect of L-tryptophan is maximal. Thus, the antihypertensive effects of L-tryptophan do not appear to be mediated by brain serotonin.
The effects of L-tryptophan on blood pressure and brain serotonin have been investigated in normotensive and spontaneously hypertensive rats. Doses of L-tryptophan from 25 to 100 mg/kg increased the blood pressure of normotensive rats by 10 to 15 mm Hg. The lower doses of L-tryptophan also increased blood pressure in hypertensive rats but, in contrast, the highest dose (100 mg/kg) lowered blood pressure by 30 to 35 mm Hg. Blood pressure effects of L-tryptophan were evident within 30 min of treatment and reached maximal response by 60 min. L-Tryptophan-induced changes in serotonin neurochemistry were correlated on a temporal basis with changes in blood pressure. However, the dose-response relationship between blood pressure changes and increases in brain serotonin and 5-hydroxyindoleacetic acid content produced by L-tryptophan was not related. The effects of L-tryptophan on blood pressure in normotensive and spontaneously hypertensive rats cannot be explained entirely by its effects on brain serotonin.
Tyrosine hydroxylase [E.C. 1.14.16.2] is inactivated by incubation with its reduced pterin cofactors L-erythro-tetrahydrobiopterin, 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin. Each of the two diastereoisomers of L-erythro-tetrahydrobiopterin inactivates tyrosine hydroxylase but the natural (6R) form is much more potent than the unnatural (6S) form at equimolar concentrations. The pterin analog 6-methyl-5-deazatetrahydropterin, which has no cofactor activity, also inactivates the enzyme whereas the oxidized pterins 7,8 dihydrobiopterin and biopterin do not. The inactivation process is both temperature and time dependent and results in a reduction of the Vmax for both tetrahydrobiopterin and tyrosine. Neither tyrosine nor oxygen inactivates tyrosine hydroxylase.
The cardiovascular role of spinal serotonin (5-HT) neurones descending from 5-HT cells near the ventrolateral surface of the medulla oblongata was investigated by stimulating these cells in normal animals and in animals with selective chemical ablation of 5-HT nerves. These laterally placed 5-HT nerves fall within the B1 and B3 groups in the medulla and were identified using immunohistochemistry. 5,7-Dihydroxytryptamine (5,7-DHT) was injected into the lateral cerebral ventricle (i.c.v.) to produce a generalized destruction of central 5-HT pathways, with preliminary intraperitoneal administration of desipramine to prevent depletion of noradrenaline stores. In other experiments, 5,7-DHT was injected directly into the cervical spinal cord, after preliminary treatment with desipramine, to produce selective destruction of spinal 5-HT nerves, confirmed both biochemically and immunohistochemically. Electrical stimulation near the lateral 5-HT cells in the B1 and B3 cell groups elicited pressor responses in control (vehicle-injected) rats; the increase in mean arterial pressure was proportional to the intensity and to the frequency of stimulation. Microinjections of kainic acid or L-glutamate at the same sites also produced an increase in mean arterial pressure. Selective destruction of 5-HT nerves, whether produced by i.c.v. or intra-spinal administration of 5,7-DHT, reduced the magnitude of the pressor response to electrical stimulation by over 50%. These experiments suggest the activity of 5-HT nerve cells adjacent to the ventrolateral surface of the medulla oblongata and projecting to the intermediolateral cell column serves to elevate arterial pressure and maintain vasomotor tone.
L-Tryptophan, 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid (5-HIAA) from discrete regions of brain can be resolved by isocratic elution on a C-18 reversed-phase column and quantitated by native fluorescence measurement. This method is rapid and simple and can reproduceably detect 5-hydroxytryptamine, tryptophan and 5-HIAA with picogram sensitivity. The detection of 5-HTP in brain extracts from untreated animals is difficult because of the extremely low endogenous levels of 5-HTP. Quantitation of 5-HTP presents no problem in animals injected with 5-HTP or with an inhibitor of L-aromatic amino acid decarboxylase. Brain tissue requires minimal treatment for assay and the hydroxyindoles are stable enough to allow automated processing.
Calmodulin was removed from brain cytosol by DEAE-52 chromatography or by affinity chromatography employing fluphenazine-Sepharose. The substrates phosphorylated by endogenous protein kinase after chromatography differed depending on the method used, and both chromatographic methods altered the phosphorylation pattern as compared to untreated cytosol. Cytosol, chromatographed on fluphenazine-Sepharose, retained most of the characteristics of untreated cytosol. Both calmodulin and phospholipids increased the phosphorylation of specific but separate brain cytosol proteins in a Ca2+-dependent manner. The effects of phospholipids could be mimicked by the detergent, sodium dodecyl sulfate, and the hydrophobic probe, 8-anilino-1-naphthalenesulfonate. Furthermore, the calmodulin-induced increase in phosphorylation, but not that produced by phospholipids, was blocked by 8-anilino-1-naphthalenesulfonate. These results suggest that the effects of phospholipids may not be due to the presence of a specific phospholipid-sensitive protein kinase in cytosol, but rather to a general interaction of hydrophobic probes with either specific substrate proteins or with the Ca2+-calmodulin-dependent protein kinase itself.
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Tryptophan hydroxylase can be activated 2.0- to 2.5-fold in vitro by ATPa dn Mg2+. This apparent phosphorylation effect is not dependent on cyclic nucleotides but is dependent on the presence of calcium. The activation of tryptophan hydroxylase by ATP-Mg2+ reduces the apparent Km of the enzyme for its cofactor, 6-methyltetrahydropterin, from 0.21 to 0.09 mM. The addition of certain antipsychotic drugs known to bind to calmodulin in a phosphorylation reaction mixture prevents the activation to tryptophan hydroxylase by ATP-Mg2+ in the concentration-dependent fashion. External addition of purified calmodulin protects the enzyme from the drug-induced effects. Preparation of calmodulin-free tryptophan hydroxylase by affinity chromatography on fluphenazine-Sepharose 4B yields an enzyme that is no longer activated by ATP-Mg2+, whereas the readdition of calmodulin to a calmodulin-free enzyme restores the responsiveness of tryptophan hydroxylase to ATP-Mg2+. This restoration is dependent on Ca2+. Taken together, these results indicate that the activation of tryptophan hydroxylase by phosphorylating conditions is dependent on both calcium and calmodulin.
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Electrical stimulation of the dorsal raphe nucleus or direct microinjection of serotonin into the preoptic region of the anterior hypothalamus produces a transient rise in arterial blood pressure in both spontaneously hypertensive rats (SRH) and Wistar--Kyoto (WKY) controls. SRHs are more responsive to raphe stimulation but are somewhat less responsive to serotonin injections when compared to WKYs. The serotonin antagonist metergoline blocks the pressor response to serotonin in both strains. These results suggest that the central serotonergic neuronal system play a similar, but not identical, role in blood pressure modulation in hypertensive and normotensive rats.
The effects of serotonin applied directly to the region of the medulla oblongata which contains the nucleus tractus solitarii, nucleus intercalatus, nucleus originis dorsalis vagi and the nucleus originis nervi hypoglossi were investigated in anesthetized rats. A dose dependent increase in blood pressure with variable changes in heart rate was observed after unilateral application of serotonin into the nucleus tractus solitarius. The serotonergic antagonists, 2-bromolysergic acid diethylamide (locally applied) and metergoline (systemically administered) significantly attenuated the serotonin-induced pressor response. Fluoxetine, a serotonin uptake inhibitor, significantly enhanced the magnitude of the pressor response, but did not prolong it. The present data suggest that enhancement of serotonergic activity in the region of the nucleus tractus solitarius produces a neurogenic pressor response.
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Tryptophan hydroxylase (EC 1.14.16.4) from rat mid-brain is inactivated upon exposure to oxygen. The degree of inactivation is dependent both on the temperature and partial pressure of oxygen to which the enzyme is exposed. Furthermore, molecular oxygen, and not an oxygen or hydroxyl radical, is responsible for the inactivation. Sulfhydryl compounds and reductants partially protect the hydroxylase from inactivation by oxygen. Enzyme inhibited by oxygen can be reconstituted by anaerobic incubation in the presence of dithiothreitol and Fe2+ at 25 degrees C and in some experiments the inclusion of inorganic sulfide, in addition to dithiothreitol and Fe2+, led to even greater recoveries of activity. Preincubation of tryptophan hydroxylase with various sulfhydryl reagents or disulfide compounds also produces inactivation which can be rapidly reversed by dithiothreitol. The substrate tryptophan protects the enzyme from inactivation by sulfhydryl reagents and disulfides but not from inactivation by oxygen. Finally, the enzyme was inhibited by a variety of iron chelators. These results suggest that the catalytic activity of tryptophan hydroxylase is dependent on the oxidation-reduction status of--SH groups and iron sites, which are probably located at the catalytic (substrate binding) site of the enzyme.
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Tryptophan hydroxylase [tryptophan 5-monooxygenase, L-tryptophan,tetrahydropterin:oxygen oxidoreductase (5-hydroxylating), EC 1.14.16.4] is activated by phosphorylating conditions (ATP-Mg2+) in a calcium-dependent, cyclic AMP-independent manner. Addition to the phosphorylation reaction of certain antipsychotic drugs that bind to calmodulin, the heat-stable calcium-binding protein, prevents the activation of tryptophan hydroxylase by ATP-Mg2+ in a concentration-dependent fashion. External addition of purified calmodulin protects the enzyme from the drug-induced effects. Calmodulin-free tryptophan hydroxylase prepared by affinity chromatography on fluphenazine-Sepharose is not activated by ATP-Mg2+ whereas addition of calmodulin to calmodulin-free enzyme restores the responsiveness of the hydroxylase to ATP-MG2+ only in the presence of Ca2+. These results indicate that the activation of tryptophan hydroxylase by phosphorylating conditions is dependent on both calcium and calmodulin.
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Electrical stimulation (1--50 Hz, 100--500 muA, 0.3 msec pulse duration) of the dorsal or median raphe nucleus in anesthetized rats produces a transient increase in arterial blood pressure. Heart rate and respiratory rate do not appear to change systematically after stimulation. Transections rostral or caudal to the raphe nuclei or electrolytic lesions in the area of the nucleus linearis, pars caudalis abolishes the pressor response to stimulation. Depletion of brain serotonin with p-chlorophenylalanine significantly attenuates the pressor effect of both dorsal and median raphe stimulation. The p-chlorophenylalanine effect can be partially reversed by Ro4-4602, a peripherally acting decarboxylase inhibitor, plus 5-hydroxytryptophan. Fluoxetine, a specific serotonin uptake inhibitor, prolongs the duration of the raphe pressor response and slightly increases its magnitude. Injections of 2-bromolysergic acid diethylamide (BOL), a putative serotonin antagonist, into the anterior hypothalamus/preoptic area significantly attenuates the dorsal raphe pressor effect, whereas treatment of rats with the sympathetic postganglionic blocking agent bretylium prevents the stimulation-induced pressor effect. Taken together, these data suggest that stimulation of the ascending serotonergic neuronal system produces a phasic pressor effect which is mediated, at least in part, by synaptic serotonin.
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