Donald M. Kuhn

Review of the role of the central serotonergic neuronal system in blood pressure regulation.

SUMMARY Alterations in the dynamics of brain serotonin biosynthesis can lead to changes in cardiovascular function. It appears that the activation of cerebral serotonin receptors produces a pressor effect in normotensive rats but produces a depressor effect in nonnotensive cats or dogs. On the other hand, reductions in the levels of serotonin can prevent the onset of hypertension in some experimental hypertensive models and lower the blood pressure of organisms with established hypertension. The ability of brain serotonin to modulate arterial blood pressure may be mediated by the influences of the serotonergic neuronal systems on efferent sympathetic activity. Finally, the reduction in sympathetic outflow produced by increasing brain serotonin levels in dogs protects the heart against ventricular fibrillation and may, therefore, constitute a reasonable adjunct in the management of high-risk, cardiac-arrest patients.

Gene expression profile of the nucleus accumbens of human cocaine abusers: evidence for dysregulation of myelin

Chronic cocaine abuse induces long‐term neural adaptations as a consequence of alterations in gene expression. This study was undertaken to identify those transcripts differentially regulated in the nucleus accumbens of human cocaine abusers. Affymetrix microarrays were used to measure transcript abundance in 10 cocaine abusers and 10 control subjects matched for age, race, sex, and brain pH. As expected, gene expression of cocaine‐ and amphetamine‐regulated transcript (CART) was increased in the nucleus accumbens of cocaine abusers. The most robust and consistent finding, however, was a decrease in the expression of a number of myelin‐related genes, including myelin basic protein (MBP), proteolipid protein (PLP), and myelin‐associated oligodendrocyte basic protein (MOBP). The differential expression seen by microarray for CART as well as MBP, MOBP, and PLP was verified by RT–PCR. In addition, immunohistochemical experiments revealed a decrease in the number of MBP‐immunoreactive oligodendrocytes present in the nucleus accumbens and surrounding white matter of cocaine abusers. These findings suggest a dysregulation of myelin in human cocaine abusers.

Serotonin neurochemistry revisited: A new look at some old axioms

A number of principles have long guided research into serotonin neurochemistry and some of them are presently reconsidered with a slightly different outlook. An increasing amount of experimental data does not always support these older axioms and as more empirical observations are made, some of these may require modification. For example, increases in 5-HT produced by injections of l-tryptophan do not necessarily indicate that tryptophan hydroxylase is less than saturated with its substrate in vivo; injections of l-tryptophan increase 5-HT and its turnover but do not appear to increase its release; 5-HT release can apparently occur from the cytoplasm and need not involve exocytosis; presynaptic autoreceptors do not appear to modulate 5-HT release; and, 5-HIAA most accurately reflects monoamine oxidase activity, not release of 5-HT or activity of 5-HT neurons.

Does brain 5-HIAA indicate serotonin release or monoamine oxidase activity?

The question of whether serotonin is deaminated by MAO before it can be released or after release has occurred was investigated by studying the 5-HT behavioral syndrome in acutely reserpinized rats. The release of serotonin from vesicles by reserpine does not produce the serotonin behavioral syndrome which is an in vivo index of serotonin release and receptor activation. However, if rats are first pretreated with a nonselective monoamine oxidase inhibitor (e.g., tranylcypromine), the injection of reserpine is followed by symptoms which are characteristic of the behavioral syndrome including forepaw treading, hindlimb abduction and head weaving. Neither selective MAO-A or -B inhibition with clorgyline or deprenyl, respectively, nor inhibition of serotonin reuptake with fluoxetine prior to reserpine produced the serotonin behavioral syndrome. However, the combination of clorgyline and deprenyl followed by reserpine does so. These behavioral data along with neurochemical analyses of serotonin and 5-hydroxyindoleacetic acid levels lead to the conclusion that serotonin does not have to be released before it is metabolized to 5-hydroxyindoleacetic acid. Consequently, the levels of 5-hydroxyindoleacetic acid in brain reflect MAO activity and not serotonin release or utilization.

The regional distribution of hydroxylase cofactor in rat brain

TETRAHYDROBIOPTERIN (BH,) is thought to be the natural (in ciuo) cofactor for both tyrosine hydroxylase and tryptophan hydroxylase (KAUFMAN, 1974), the initial and rate limiting enzymes in the biosynthesis of the katecholamines (UDENFRIEND, 1966) and serotonin (JEQUIER et al., 1967), respectively. It has been suggested that the concentration of BH, in tissue may play a regulatory role in determining the rates of hydroxylase enzyme activity (KAUFMAN, 1973; LOVENBERG & VICTOR, 1974; LOVENBERG & KUHN, 1978). However, only recently have attempts been made to quantify BH, in brain and to correlate the distribution of BH, with the activity of these hydroxylase enzymes (GAL et al., 1976; BULLARD et al., 1978). The results of this communication confirm the presence of high concentrations of hydroxylase cofactor (presumably BH4) in brain areas known to contain large amounts of tyrosine (striatum) and tryptophan (mesencephalic tegmentum) hydroxylase activity. In addition, our results demonstrate the existence of large quantities of hydroxylase cofactor in other areas known to be involved in neuroendocrine function, but having relatively little hydroxylase activity.

Calcium-dependent activation of tryptophan hydroxylase by ATP and magnesium

Abstract Tryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)] in rat brainstem extracts is activated 2 to 2.5-fold by ATP and Mg++ in the presence of subsaturating concentrations of the cofactor 6-methyltetrahydropterin (6MPH4). The activation of tryptophan hydroxylase under these conditions results from a reduction in the apparent Km for 6MPH4 from 0.21 mM to 0.09 mM. The activation requires Mg++ and ATP but is not dependent on either cAMP or cGMP. The effect of ATP and Mg++ on enzyme activity was enhanced by μM concentrations of Ca++ and totally blocked by EGTA. These data suggest that tryptophan hydroxylase can be activated by a cyclic nucleotide independent protein kinase which requires low calcium concentrations for the expression of its activity.

Uptake and release of tryptophan and serotonin: An HPLC method to study the flux of endogenous 5-hydroxyindoles through synaptosomes

Abstract: An HPLC assay with fluorometric detection has been developed that is sensitive enough to measure simultaneously endogenous levels of tryptophan, serotonin (5‐hydroxytryptamine, or 5‐HT), and 5‐hydroxyindoleacetic acid (5‐HIAA) inside synaptosomes as well as that released into the incubation medium. Using this assay, we have observed that tryptophan is rapidly taken up by synaptosomes and turned over to 5‐HIAA without a concurrent release of 5‐HT. Exogenous 5‐HT is also rapidly taken up, and, within 20–30 min, 80% of the 5‐HT is deaminated. Veratridine induces release of both tryptophan and 5‐HT from synaptosomes. Changes in the disposition of exogenous tryptophan or 5‐HT can be completely accounted for by uptake or by stoichiometric changes in metabolites. This assay method should be valuable in the study of 5‐HT pools and in the determination of from which pool 5‐HT release occurs.

Comparisons of tryptophan hydroxylase from a malignant murine mast cell tumor and rat mesencephalic tegmentum

Abstract A comparative study of the effects of a wide variety of substances on tryptophan hydroxylase from a transplantable murine mast cell tumor and rat brain (mesencephalic tegmentum) was made. Heparin, calcium, limited tryptic proteolysis, sodium dodecyl sulfate, selected phospholipids, and protein phosphorylation each produced activations of tryptophan hydroxylase from rat brain varying from two- to fivefold in magnitude. In contrast to these results, most of these same activators either had no effect (trypsin, phosphorylation) or inhibited the activity of the mast cell hydroxylase (sodium dodecyl sulfate, calcium, phospholipids, phosphorylation). Only heparin activated the mast cell enzyme. The present data taken together with previous results from our laboratory (8) suggest that the tryptophan hydroxylating enzymes from the malignant murine mast cell tumor and rat tegmentum have different molecular, functional, and regulatory properties.

Tyrosine hydroxylase: purification from PC-12 cells, characterization and production of antibodies

Tyrosine hydroxylase has been purified to homogeneity from cultured PC-12 cells. The protein migrates as a single band with a molecular weight of 60,000 on sodium dodecyl sulfate polyacrylamide electrophoresis. Two-dimensional electrophoresis of the pure enzyme resolves three spots (each with molecular weights of 60,000) with isoelectric points of 5.4, 5.8 and 5.9. This charge heterogeneity cannot be explained by the presence of sugar or lipid moieties on the enzyme. Amino acid analysis indicates a relatively high content of hydrophobic amino acids and a lower serine content than other preparations of tyrosine hydroxylase. The enzyme hydroxylates tryptophan at approximately 1% of its rate of tyrosine hydroxylation but will not catalyze the hydroxylation of phenylalanine. Polyclonal antibodies were produced in rabbits against pure tyrosine hydroxylase and were judged to be monospecific by Western blot analysis. The IgG fraction was isolated from serum, and when coupled to cyanogen bromide activated Sepharose, could be used to purify tyrosine hydroxylase from crude extracts in a single step. The antiserum proved to be very useful in immunoprecipitation and immunocytochemical experiments with tyrosine hydroxylase.

Pressor effects of dorsal raphe stimulation and intrahypothalamic application of serotonin in the spontaneously hypertensive rat

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.