Jane Humm

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.

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.

A reliable method for the quantification of thyrotropin-releasing hormone (TRH) in tissue and biological fluids

A reliable technique for purification of crude tissue extracts and analysis for TRH by HPLC, TLC, radioimmunoassay and bioassay is presented. We conclude that authentic TRH is present throughout the mammalian brain and accounts for much of the TRH-like immunoreactivity in several peripheral organs, but that it is not present in urine, placenta, or pineal, and its concentration in blood is some 250-fold less than a value obtained from a direct radioimmunoassay of a sample of blood.

The effect of the presence of RNA upon diethyl pyrocarbonate inhibition of ribonuclease

Abstract The relationship of RNA, ribonuclease and diethylpyrocarbonate has been investigated using various concentrations of DEP, with and without the simultaneous addition of RNA. It has been found: 1. 1. The presence of any considerable amount of RNA interferes with the inhibition of ribonuclease by diethyl pyrocarbonate. 2. 2. The results do not appear to be explained by a direct interaction between diethyl pyrocarbonate and RNA. A question is raised as to the utility of diethyl pyrocarbonate as a ribonuclease inhibitor, if free RNA is present in the solution being extracted.

Assay of glutaminylpeptide cyclase.

Publisher Summary This chapter describes a simple assay for glutaminyl peptide cyclase applicable to a number of different tissues. The enzymes found in porcine pituitary are perhaps the best characterized. It is a glycoprotein exhibiting charge and size heterogeneity and is predominantly found in secretory vesicles. It also has a neutral pH optimum, is stimulated by high-salt concentrations, probably has catalytically important sulfhydryl, and requires no added cofactors. The substrate specificity of the enzyme is currently uncertain, but substrates with an aminoterminal glutamine followed by two or more amino acids may have the highest affinity for the catalytic site. The influence of the chemical characteristics of the amino acids following glutamine on affinity or reaction velocity is unknown. Interpretation of the results of the assay of crude tissue samples is complicated by the presence of pyroglutamyl peptide hydrolases (both soluble and particulate) and prolyl endopeptidase, which degrade the product, in the case of the former, and both substrate and product, in the case of the latter.

A rapid, sensitive assay for glycine-directed amidating enzymes

Current assays for glycine-directed, peptide-amidating enzymes have several shortcomings. In this report, we describe a rapid, sensitive microassay for amidating activity which overcomes these disadvantages. Tissue homogenates are incubated in the presence of D-Tyr-Val-Gly-OH and the product, D-Tyr-Val-NH2, is measured by radioimmunoassay using an antiserum with an affinity for the product, D-Tyr-Val-NH2 (Kaff 2 X 10(8) L/M), 4 orders of magnitude higher than for the substrate, D-Tyr-Val-Gly-OH (Kaff 4 X 10(4) L/M), and 3 orders of magnitude higher than for the deamidated product D-Tyr-Val-OH (Kaff 8 X 10(5) L/M). Addition of N-ethylmaleimide (0.5 mM) to the enzyme incubates prevents the degradation of D-Tyr-Val-NH2 and permits the measurement of enzymatic activity in crude homogenates. This assay is sensitive enough to permit the measurement of amidating activity in crude rat brain homogenates containing as little as 4-8 micrograms protein.

The absorption of fructose and methyl ethers of fructose from the intestine of the rat.

Abstract 1. 1. The absorption of fructose, 1-methylfructose, and 3-methylfructose was studied in unanesthetized rats. The relative absorption values (glucose: 100) were fructose: 53; 1-methylfructose: 47; 3-methylfructose: 32. 2. 2. 1-Methylfructose and 3-methylfructose are not demethylated by intestinal mucosa in vitro . 3. 3. 1-Methylfructose and 3-methylfructose are not absorbed against a concentration gradient.

AEROBIC AND ANAEROBIC CARBOHYDRATE UTILIZATION IN NORMAL AND MELANOTIC FISH PIGMENT CELLS

Since the first introduction of quantitative metabolic methods investigations have been carried out in which comparisons have been made between the metabolism of normal cells and their malignant or atypical counterparts in an attempt to establish some metabolic difference between normal and atypical growth. The early investigations of Warburgl and his school established the fact that the great majority of malignant cells as well as some embryonic tissues exhibited a pattern of metabolism that was different from most normal cells. Although the absolute level of oxygen consumption of the atypical cells remained well within normal limits the activity of the so-called glycolytic system leading to the production of lactic acid was very much more active in atypical growths than it was in normal cells. The over-all result of this change in metabolic emphasis was that the tumor cell, and some normal embryonic cells, were capable of producing lactic acid by glycolysis in the presence of air. Warburg2 has maintained then and since, that this change in the metabolic pattern resulting in aerobic glycolysis was due to a fundamental change in the oxidative capacities of the cancer cell and that this change led directly to the production of the atypical growth. Critical evaluation of this hypothesis requires the careful selection of biological material. The first criterion that would have to be met by such biological material would be that a strain of cells be available in which both normal and abnormal cells are present. The closer the genetic relationship of the normal cells to the tumor cells the fewer will be the imponderables involved in an analysis of the changes which have occurred in the transition from normal to malignant cells. Normal macromelanophores obtained from Xiphophorus maculatus (the platyfish) and the pigmented tumor cells arising from the interspecific cross between the platyfish and a closely related species Xiphophorus heZleri (the swordtail), fulfill the requirements as a test system. Pigment cells in the platyfish are normal in every respect. They never produce, in the wild condition, any type of atypical growth. The swordtail never exhibits any macromelanophore pigmentation. However, when the platyfish is bred to the swordtail the resulting hybrid is viable and fertile and may be crossed subsequently to either the swordtail or the platyfish. In the hybrid fish the expression of the X . maculatzls gene or genes for the occurrence of macromelanophores is modified in such a manner that the hybrid exhibits an overexpression of the macromelanophore pigmentation, the restricted areas of pigmentation typical of the normal fish are exceeded, and the cells cover large portions of the skin. I n time the hyperpigmented areas begin to show localized overgrowths that histologically have been shown by Grand et aL3 to be very similar to the melanotic tumors found in humans. Thus in these fish it is possible to obtain, in a