David V. Maudsley

Ornithine Decarboxylase Stimulation in Rat Ovary by Luteinizing Hormone

In normal albino rats with a 4-day estrous cycle, the activity of ovarian ornithine decarboxylase undergoes a transitory rise on the evening of proestrus and only at that time. The response could be elicited by the administration of either luteinizing hormone or human chorionic gonadotrophin. When antiserum to luteinizing hormone was injected at 2 p.m. on the day of proestrus, the induction of ornithine decarboxylase was blocked, an indication that the enzyme is under luteinizing hormone control. The strategic positioning of the induction of ornithine decarboxylase between the normal release of luteinizing hormone and ovulation impties that putrescine is associated with the ovulatory process, and opens a new avenue of research on the control of ovulation.

Factors affecting the saturation assay of cyclic AMP in biological systems

Abstract Nonspecific (blank) effects interfering with the saturation analysis of cyclic AMP may be mediated either through the primary reaction between cyclic AMP and the binding protein or on the efficacy of the separation procedure. It is not normally permissible to substract blank values, measured at zero concentration of added nucleotide from the measurement of unknowns since the magnitude of the correction may vary with different nucleotide concentrations. Response curves should be set up in equivalent concentrations of the particular blank extract in every assay.

Release of diamine oxidase by heparin in the rat

Abstract Heparin produces a substantial rise in diamine oxidase activity in rat plasma. An increase in the plasma is evident within 10 min of an intravenous (i.v.) injection of heparin and a peak is observed between 30 and 60 min. The rise in the plasma can be accounted for by the release of diamine oxidase from the intestine, since the enzyme activity in this tissue is markedly reduced when the plasma level is at its peak. Only a slight increase in the plasma level is observed when heparin is given to eviscerated animals, suggesting that the contribution from tissues other than the intestine is small. As the rise in plasma activity is evident even with normal anticoagulant doses of heparin, care should be exercised in whole animal experiments when potential substrates for diamine oxidase, such as histamine and putrescine, are being studied.

A single-isotope enzyme assay for histamine

Abstract A simple, rapid, and sensitive radioenzymic method for histamine is described. The method utilizes a specific histamine enzyme, histamine N -methyltransferase, isolated from guinea pig brains and high specific activity tritiated S -adenosylmethionine. The sensitivity of the method as described, under best conditions, is 0.1 ng. All reagents are stable, readily prepared, and/or commercially available.

Induction of ornithine decarboxylase in rat ovary after administration of luteinizing hormone or human chorionic gonadotrophin

Abstract Ornithine decarboxylase in rat ovary is stimulated 5- to 10-fold by a subcutaneous injection of luteinizing hormone (LH) or human chorionic gonadotrophin (HCG). A peak is observed between 3 and 5 hr after which the enzyme activity declines rapidly. The pattern of enzyme response to hormonal administration is similar to that observed during late proestrus. S-adenosylmethionine decarboxylase is not altered during this period or after administration of LH or HCG. Cycloheximide administered in vivo markedly reduced ovarian ornithine decarboxylase within 15 min, suggesting that the turnover of this enzyme in the ovary is very rapid. When injected immediately prior to HCG, cycloheximide also prevented the rise in ornithine decarboxylase activity. High doses of actinomycin d were also effective in blocking the response to HCG. Ornithine decarboxylase responds to a single injection of HCG at all stages of the estrus cycle, but repeated injections do not prevent the decline in enzyme activity occurring between 5 and 7 hr. Anti-HCG injected immediately before the primary hormone completely inhbited the rise in ornithine decarboxylase, but when given 1 hr after the primary injection, it was ineffective. It is suggested that these characteristics of ornithine decarboxylase, i.e. high rate of turnover, rapid induction and the inability of repeated hormone injections to prevent the eventual decline of enzyme activity, ensure that the increase in the formation of putrescine during proestrus is restricted to a relatively narrow time period, thus supporting the view that putrescine may have a specific role in the regulation of protein and RNA synthesis involved in the early phase of LH action on the ovary.

Prostaglandin biosynthesis and stimulation of cyclic AMP in primary monolayer cultures of epithelial cells from mouse mammary gland

Cyclic AMP levels in primary monolayer cultures of epithelial cells prepared from mid-pregnant mice are stimulated by prostaglandin E1 and E2. Prostaglandin F1alpha and F2alpha have only a slight effect upon cyclic AMP levels. In the absence of phosphodiesterase inhibitors the rise in cyclic AMP produced by PGE1 is only transient and the levels return to normal within 30 minutes. High concentrations (16 mM) of theophylline are needed to prevent this decline, suggesting that the phosphodiesterase activity of epithelial cells in culture is high. However, theophylline alone produced only a small increase in basal cyclic AMP levels even over a 2-hour period indicating that basal cyclic AMP is turned over more slowly than cyclic AMP produced in response to stimulation with PGE1. Both PGE and PGF synthesis were monitored using radioimmunoassay procedures previously reported. The observed levels were found to decrease as cell density increased and were sensitive to the addition of agents such as collagen and naproxen.

Elevation of prostaglandin and cyclic AMP levels by arachidonic acid in primary epithelial cell cultures of C3H mouse mammary tumors.

Arachidonic acid causes a sharp transient increase in cyclic AMP levels in primary epithelial cell cultures obtained from C3H mouse mammary tumors. The effect is evident within two minutes and is enhanced by theophylline or 3-isobutyl-1-methylxanthine. Maximum increase in cyclic AMP levels are observed with a dose of 100 mug/ml of arachidonic acid (AA). At higher dose levels the increase in cyclic AMP levels is reduced. Naproxen, an inhibitor of prostaglandin synthesis in this system markedly reduces the stimulation of cyclic AMP by arachidonic acid but it does not affect the increase in cyclic AMP levels observed after the addition of prostaglandin Es, epinephrine or cholera enterotoxin. Arachidonic acid, under the same conditions, also causes a significant elevation of PGE and PGF media levels which is slower and more sustained than the cAMP response. The data strongly suggest that a metabolic of arachidonic acid is responsible for the cyclic rise, however, it is not certain whether this is due to PGE2 or some other product.

Effect of H2 receptor antagonists on histidine decarboxylase activity in rat gastric mucosa

Abstract Histidine decarboxylase activity in the gastric mucosa of the rat stomach is markedly increased by the H 2 receptor antagonists, burimamide and metiamide. The increase in enzyme activity is reduced by cycloheximide but not by actinomycin D. An inhibitor of histidine decarboxylase, 4-imidazolyl-3-amino-2-butanone (McN-A-1293), is also effective in reducing the enzyme activity stimulated by the H 2 receptor antagonists. The time course and the magnitude of the response of the enzyme with maximum doses of burimamide are similar to those obtained with pentagastrin. The histamine content of the mucosa is also reduced by burimamide and the data are discussed in relation to the hypothesis that H 2 receptor antagonists increase histidine decarboxylase activity through the release of endogenous gastrin.

Inhibition of histidine decarboxylase and diamine oxidase by 4-bromo-3-hydroxybenzyloxyamine

Abstract In freely fed rats, an intraperitoneal injection of 0.688 m-mole per kg of 4-bromo-3-hydroxybenzyloxyamine (NSD 1055) produces a transient drop in both the histidine decarboxylase activity of the glandular stomach and the diamine oxidase activity of the intestine. Both enzymes are inhibited by approximately 50 per cent within 30 min, but recovery to normal levels occurs within a few hours. The results obtained in vivo are in sharp contrast to the data in vitro , which show that the inhibitor is much more active against the decarboxylase. The transient effect of NSD 1055 against the two enzymes in vivo may be due primarily to the rapid clearance or metabolism of the drug, while the change in potency ratio from a situation in vitro to one in vivo may reflect the greater rate of turnover of histidine decarboxylase. The drug, however, is very effective in preventing the rise in histidine decarboxylase produced by insulin, pentagastrin or refeeding of starved animals. Presumably under these circumstances there is a greater opportunity for the drug to complex with the cofactor, pyridoxal phosphate.

Adrenocorticotropin stimulation of cyclic adenosine 3′,5′-monophosphate formation in isolated rat adrenal cells the role of membrane sialic acid

Abstract Neuraminidase treatment of isolated rat adrenal cells inhibited the cyclic AMP response to adrenocorticotropin stimulation. Greater percent inhibition was observed with the lower doses of adrenocorticotropin. Although the maximum amount of cyclic AMP produced by the neuraminidase-treated cells was similar to that obtained with untreated cells, the concentration of adrenocorticotropin required to produce it was greatly increased. The increase in adrenocorticotropin concentration to produce half-maximum amounts of cyclic AMP was 2-fold. Neuraminidase treatment caused a dose-related depletion of sialic acid from the cells with about 60% of the total sialic acid being released by 20 munits/ml of the enzyme. Stimulation of cyclic AMP formation by NaF in a particulate fraction obtained from homogenates of cells was unchanged after treatment of the cells with neuraminidase. The data implicate a role for sialic acid in the early events in the action of adrenocorticotropin on the cell membrane. Sialic acid may be involved in the interaction of the adrenocorticotropin molecule with the receptor or it may have a role in transmission of the signal arising from adrenocorticotropin-receptor interaction to the catalytic unit of adenyl cyclase.