William W. Youngblood

Clinical Studies of Methylphenidate Serum Levels in Children and Adults

Abstract Although methylphenidate (MPH) is used widely in the treatment of hyperactive children, and although it seems to be a safe and effective drug, very little is known about the pharmacology, pharmacokinetics, and metabolism of the drug, and the relationship between serum levels and subtle side effects. We report on the progress in applying a reliable and sensitive gas chromatography-mass spectrometric assay for MPH in serum to clinical studies of hyperactive children and adults. Although drug half-life in all subjects is remarkably constant (2 to 4 hours), there is a wide range of inter-individual variation in serum levels and, more disturbing, a wide range of intra-individual variations in serum levels. The clinical importance of this work is discussed.

Regional Distribution of Neurotensin in Human Brain

Abstract: Neurotensin (NT) is an endogenous neuropeptide that is active in many preclinical screening tests for neuroleptic drugs. Using a radioimmunoassay, we have studied the regional distribution of NT in postmortem human brain and in cerebrospinal fluid. Highest levels were present in the hypothalamus, substantia nigra, and limbic areas, whereas much lower amounts were found in the cortex and striatum. The chromatographic properties of hypothalamic immunoreactivity on ion‐exchange and high pressure liquid chromatography were similar to those of the synthetic tridecapeptide. We conclude that NT is present in human brain with a distribution resembling that seen in other species, such as rat and monkey.

The effect of castration, thyroidectomy and haloperidol upon the turnover rates of dopamine and norepinephrine and the kinetic properties of tyrosine hydroxylase in discrete hypothalamic nuclei of the male rat

Adult male rats were either castrated, thyroidectomized, or treated with haloperidol and the rates of turnover of dopamine (DA) and norepinephrine (NE) in the median eminence (ME), the arcuate and dorsomedial nuclei of the hypothalamus were estimated from the rate of decay of DA and NE concentrations as determined by radioenzymatic assay following blockade of catecholamine synthesis by alpha-methyl-p-tyrosine. The ME of animals similarly prepared was also examined for changes in the total activity and kinetic properties of tyrosine hydroxylase (TH). Four days following the administration of haloperidol (400 microgram/kg) or 10 days after castration, there was a significant increase in the rate of turnover of DA but not NE in the ME accompanied by an increase in the Vmax but not Km for the substrate or cofactor of TH. Furthermore, the administration of haloperidol to hypophysectomized rats also significantly increased the TH activity in the ME, indicating that such changes may occur independently of any changes in serum prolactin levels. Ten days after thyroidectomy, or three weeks after treatment with prophylthiouracil, there was a significant increase in the turnover rate of DA in both the ME and dorsomedial nucleus but not in the arcuate nucleus. No changes in the turnover rates of NE in any of the three areas were observed following thyroidectomy. In the ME, the increase in turnover of DA was accompanied by an increase in the total TH activity (Vmax) as welll as a decrease in Km for tetrahydrobiopterin but not tyrosine. From these results 4 conclusions were drawn: (1) following halperidol, castration, and thyroidectomy there are increases in the activity of dopaminergic terminals within the ME; (2) castration, haloperidol and thyroidectomy may influence the activity of dopaminergic terminals within the ME by different mechanisms; (3) changes in tyrosine hydroxylase and turnover of catecholamines within the ME may occur independently of changes in prolactin levels; and (4) local recurrent afferent circuits may exist in the arcuate nucleus region of the hypothalamus.

Prolactin in cerebrospinal fluid: A probable site of prolactin autoregulation

The purpose of this study was to examine the thesis that increasing concentrations of prolactin within the cerebrospinal fluid (CSF) increase the activity of dopaminergic terminals within the median eminence and that this increased dopaminergic activity is temporally associated with a suppression of endogenous prolactin secretion. To avoid difficulties encountered in performing catecholamine turnovers in the undisturbed rat, the measurement of tyrosine hydroxylase was validated as an index of dopaminergic activity within the median eminence. In the median eminence, but not the medial preoptic area, parallel increases in the activity of tyrosine hydroxylase and the turnover of dopamine (but not norepinephrine) occurred following hyperprolactinemia. Twenty-six hours but not 2.5 h after the subcutaneous administration of ovine prolactin, the activity of tyrosine hydroxylase was increased in the median eminence, and endogenous prolactin secretion was inhibited. During a 26 h continuous intracerebroventricular (icv) infusion (88 ng/h) of rat prolactin, there was a complete suppression of endogenous prolactin secretion. Twenty-six but not 2.5 h after the initiation of the icv infusion of prolactin, there was an increase in tyrosine hydroxylase activity in the median eminence. The results of these studies suggest that: (1) measurement of tyrosine hydroxylase activity within the median eminence is a useful index of the activity of dopaminergic terminals; (2) increasing concentrations of prolactin within the CSF suppressed prolactin secretion by the anterior pituitary; (3) this suppression of prolactin is accompanied by an increased activity of dopaminergic terminals within the median eminence; (4) those neural structures concerned with the regulation of prolactin secretion respond directly to prolactin itself; (5) the autoregulation by prolactin of its own secretion manifests a certain latency more characteristic of a tonic rather than a phasic inhibitory control; and finally, (6) dopaminergic terminals in the median eminence but not the preoptic area appear uniquely sensitive to prolactin.

Serotonin synthesis and release in brain slices: Independence of tryptophan

The purpose of the present study was to examine the role of substrate availability in the regulation of the release and synthesis of serotonin by brain slices. Electrical field depolarization of the brain slices stimulated the synthesis and release of serotonin in the absence of changes in intracellular tryptophan concentration, in the absence of tryptophan in the incubation bath, and in the absence of changes in total tryptophan uptake. Furthermore, electrical stimulation decreased the apparent Km for tryptophan required for synthesis of serotonin by the slices. Several conclusions were drawn. (1) Rates of serotonin release and synthesis in brain slices may increase independently of the tissue tryptophan concentration or tryptophan uptake. (2) There is little difference in the synthetic rate of serotonin in tissues exposed to pargyline or not at all. (3) Blockade of monoamine oxidase by pargyline appears to give estimates of the synthetic rates of serotonin which are comparable to estimates derived from [3H]-tryptophan incorporation. (4) Newly synthesized serotinin is preferentially released. (5) Only in tissue pretreated with pargyline do increasing concentrations of tryptophan increase the releasable pool of 5-HT. (6) Electrical depolarization may allosterically activate tryptophan hydroxylase; an effect which may be mimicked by homogenization of the tissue.

Biochemical and histochemical evidence for the existence of a tuberoinfundibular cholinergic pathway in the rat

Although considerable biochemical, physiological and pharmacological data indicate that cholinergic pathways exist in the hypothalamus 9,1°,13, the exact location of these circuits has yet to be determined. Choline acetyltransferase (ChAc; EC 2.3.1.6), the enzyme responsible for the synthesis of acetylcholine (ACh), is believed to be present exclusively in cholinergic neurons 10. In fact, levels of this enzyme correlate well with the amount of ACh in different brain regions 1. The enzyme is present in several diencephalic nuclei and its highest activity in the hypothalamus is in the median eminence (ME) 1. It seems reasonable to assume that the ChAc in the ME is present in nerve terminals, since this region is devoid of neuronal perikarya 8 and also because ChAc has not yet been identified in glial cells la. Previous studies have demonstrated that after Halasz deafferentation (hypothalamic islands composed primarily of the isolated arcuate nucleus and the ME), ChAc activity in the ME is not significantly reduced 19. These data suggested that an intrinsic cholinergic ventrobasal pathway exists in the rat hypothalamus. We have utilized chemical lesioning and microdissection techniques coupled with microchemical and histochemical methods to demonstrate the existence of a tuberoinfundibular ACh pathway. The administration of high doses of monosodium L-glutamate (MSG) to neonatal rodents or primates results in a central nervous system lesion, largely restricted to the arcuate nucleus of the hypothalamus16,17. Since this lesion destroys 80-90 ° o of arcuate perikarya 17, but apparently spares axons in passage, examination of the arcuate nucleus and ME of MSG-treated rats provides a convenient means to examine the arcuate contribution to cholinergic terminals in the ME. Timed-pregnant Sprague-Dawley rats (Holtzman) were housed in rooms with controlled lighting (14 h light; 10 h dark) and fed laboratory chow and water ad libitum. Their offspring were injected intraperitoneally with MSG (4 mg/g BW) on alternate days during the first ten days of life as previously described 16. Control animals received

TRH-like immunoreactivity in rat pancreas and eye, bovine and sheep pineals, and human placenta: Non-identity with synthetic Pyroglu-His-Pro-NH2 (TRH)

Extracts from albino rat eyes and pancreas, bovine and sheep pineals and human placenta containing TRH-like immunoreactivity were chromatographed on silica-gel plates. Comparison of elution profiles for TRH-like immunoreactivity with that of TRH revealed the presence of substances other than TRH in these samples. Chromatography of TRH-like immunoreactivity obtained from Rana pipiens skin eluted in two solvent systems produced elution profiles identical with that of synthetic Pyroglu-His-Pro-NH2 consistent with reports that frog skin contains large quantities of TRH. Implications of these findings are discussed.

Synthesis and release of serotonin by brain slices: Effect of ionic manipulations and cationic ionophores

The effect of various ionic manipulations and cationic ionophores on the rates of release and synthesis of serotonin were investigated in brain slices prepared from adult male rats. Two depolarizing, univalent cationic ionophores, gramacidin (10 μg/ml) and valinomycin (10 μ/ml), and the non-depolarizing, calcium-specific ionophore, A23187 (190 μM), stimulated both the release and synthesis of serotonin in this tissue preparation. Electrical field depolarization of the brain slices also stimulated both the release and synthesis of serotonin, effects which were completely blocked by a calcium-free media and 1 mM EGTA or by 10 mM Mg2+. Lithium partially blocked the release of serotonin by the stimulated brain slices, but markedly augmented the rates of serotonin biosynthesis. Although electrical stimulation significantly increased the rate of [3H]tryptophan uptake by the slices, the increased rates of release and synthesis of serotonin following treatment of the tissues with the ionophores and lithium were not associated with increased rates of tryptophan uptake. Furthermore, despite the inhibition of the release and synthesis of serotonin by stimulated slices incubated in the presence of magnesium or in the absence of calcium, rates of tryptophan uptake by the stimulated brain slice remained increased. These results taken together with the results of other studies suggest that release and synthesis of serotonin by the serotonergic neuron is tightly coupled to the ionic events attending depolarization and further argue than transcellular calcium fluxes may be of special importance in this regulation. These results do not support the postulate that regulation of serotonin release and synthesis is dependent upon substrate availability as a major variable.

Studies of the neural mechanisms by which hypothyroidism decreases prolactin secretion in the rat

These studies report that chronic hypothyroidism in the rat is accompanied by decreased serum prolactin. An investigation of those mechanisms by which hypothyroidism decreases prolactin secretion revealed the following findings: (1) activation of tyrosine hydroxylase in the median eminence (ME) and increase in the turnover-rate of dopamine (DA) in the ME; (2) a decreased content and in vitro release of prolactin by pituitaries from hypothyroid rats; (3) decreased [3H]spiroperidol binding to pituitary homogenates obtained from hypothyroid rats, and (4) normal increase of serum prolactin following administration of haloperidol. Comparison of the effects of hypothyroidism on dopaminergic terminals of the striatum with those of the ME was made and the following tentative conclusions proposed. Hypothyroidism presumably increases the release of DA into the pituitary portal system. A deficiency of thyroid hormone desensitizes the pituitary lactrotrope to inhibition by DA, decreases the total pituitary prolactin content and presumable also reduces the peripheral catabolism of prolactin. The overall net effect of hypothyroidism is, therefore, a decrease in serum prolactin levels which can increase normally following haloperidol. The mechanism by which a deficiency of thyroid hormone alters the function of the tuberoinfundibular and striatal dopaminergic systems is unknown.

Studies of substrate requirements, kinetic properties, and competive inhibitors of the enzymes catabolizing TRH in rat brain

The enzymes catalyzing the breakdown of TRH are soluble and dependent upon active sulfhydryl groups. One of these enzymes is a pyroglutamyl aminopeptidase (EC 3.4.11.8) (apparent molecular weight 60,000 daltons) with a specificity restricted to pyroglutamyl peptide bonds. The second enzyme is a prolyl endopeptidase (apparent molecular weight 70,000 daltons) with a specificity restricted to proline peptide bonds wherein the proline is preceded by an alpha adjacent amino acid possessing a blocked N-terminus. Substrate requirements for this latter enzyme are identical to those reported previously for the TRH deamidase of rat brain, the prolyl endopeptidase of rabbit brain and the post proline cleaving enzyme of rat brain. We conclude that this enzymatic activity variously described as TRH deamidase, post proline cleaving enzyme, and prolyl endopeptidase is that of a single enzyme best denoted as a prolyl endopeptidase (EC 3.4.22.16, proposed). The pyroglutamyl aminopeptidase has a Km for TRH of 45 micro M as compared to a Km for TRH of 2400 micro M for the prolyl endopeptidase. Total enzymatic activity of the prolyl endopeptidase, however, is approximately 3500 nmol/h/rat brain with the total enzymatic activity of the pyroglutamyl aminopeptidase being approximately 600 nmol/h/rat brain. The 5- to 6-fold higher affinity of the pyroglutamyl aminopeptidase for TRH suggests that of these two catabolic pathways, removal of the N-terminal pyroglutamyl moiety is likely to be the more important pathway for the initial catabolism of TRH in rat brain. Analysis of substrate specificity permitted the synthesis of several effective competitive inhibitors of the two enzymes. Of these, the most effective inhibitors of the pyroglutamyl aminopeptidase were p-glu-NH2 (Ki 185 micro M) and p-glu-his-OCH3 (Ki 16.5 micro M). The most effective inhibitors of the prolyl endopeptidase were Ac-gly-pro-NH2 (Ki 1143 micro M) and Z-gly-pro-NH2 (Ki 442 micro M).