Biomedical subjects
Publications and source records attributed to C R Creveling.
Recent genetic epidemiological studies implicate a low activity form of catechol-O-methyltransferase (COMT) with increased risk factor for breast cancer. Taking into account, the role of COMT in the metabolism of otherwise carcinogenic catecholestrogens, it is reasonable to propose that environmental or dietary products that inhibit COMT pose a risk for breast cancer.
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In the present study serotonin (5-hydroxytryptamine, 5-HT) and monoamine oxidase (MAO) were both found localized in the blood vessel walls of human dental pulp. Our discovery of MAO activity in human dental pulp suggests a functional relationship between serotonin and MAO in this region.
Catechol-O-methyltransferase (COMT) plays both a regulatory and protective role in catechol homeostasis. It contributes to the regulation of tissue levels of catecholamines and catecholestrogens (CEs) and, by blocking oxidative metabolism of catechols, prevents endogenous and exogenous catechols from becoming a source of potentially mutagenic electrophiles. Evidence implicating CEs in carcinogenesis, in particular in the hamster kidney model of estrogen-induced cancer, has focused attention on the protective role of COMT in estrogen target tissues. We have previously reported that treating hamsters with estrogens causes translocation of COMT to nuclei of epithelial cells in the renal cortex, the site of CE biosynthesis and where the cancers arise. This finding suggested that nuclear COMT may be a marker of a threat to the genome by catechols, including CEs. It is postulated that CEs play a role in the genesis of breast cancer by contributing to a state of chronic oxidative stress that is presumed to underlie the high incidence of this disease in the United States. Therefore, here we used immunocytochemistry to re-examine human breast parenchyma for nuclear COMT. In addition to confirming previous reports of cytoplasmic COMT in mammary epithelial cells, we identified nuclear COMT in foci of mammary epithelial cells in histologically normal breast tissue of virtually all control (macromastia) and cancer patients and in breast cancer cells. There was no correlation between tissue histology and the numbers of cells with nuclear COMT, the size of foci containing such cells, or intensity of nuclear COMT immunostaining. The focal nature of the phenomenon suggests that nuclear COMT does not serve a housekeeping function but that it reflects a protective response to an increased local catechol load, presumably of CEs and, as such, that it may be a characteristic of the population of women studied who share the same major risk factor for developing breast cancer, that of living in the industrialized West.
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Catecholestrogens are postulated to contribute to carcinogenesis by causing DNA damage mediated by reactive oxygen species generated during redox cycling between catechol and quinone estrogens, and by quinone estrogens that can form depurinating adducts. The above hypothesis is based principally on studies of the cancers that develop in renal cortex of hamsters treated with primary estrogens: Hamster kidney can catalyze 2- and 4-hydroxylation of estrogens and support their redox cycling, and the kidneys of estradiol-treated hamsters show evidence of oxidative cellular and DNA damage. Here we used immunocytochemisty to test the postulate that catechol-O-methyltransferase (COMT), the enzyme that can prevent oxidation of catecholestrogens to their quinone derivatives, would be induced in renal cortex of hamsters treated with estradiol or ethinyl estradiol. In kidneys of control hamsters, COMT was localized in cytoplasm of epithelial cells of proximal convoluted tubules, predominantly in the juxtamedullary region where the estrogen-induced cancers arise. After 2- or 4-weeks of treatment with either estrogen, COMT was seen in epithelial cells of proximal convoluted tubules throughout the cortex, and many cells also showed intense nuclear COMT immunoreactivity. Estradiol-induced renal cancers were negative for COMT, but were surrounded by tubules with intense cytoplasmic and nuclear immunostaining. The nucleus-associated COMT was shown by immunoblot analysis to be the soluble form of the enzyme. Using reverse transcription-polymerase chain reaction amplification, hamster kidney COMT was shown to lack the putative nuclear localization signal sequence present in human COMT. A second phase II enzyme, CuZn-superoxide dismutase (CuZnSOD), was shown by immunocytochemistry to remain extranuclear in proximal convoluted tubules of estrogen-treated hamsters, which indicates entry of COMT into the nucleus to be selective. The findings are consistent with the catechol/quinone estrogen hypothesis of estrogen-induced cancer, while the translocation of the enzyme to the nucleus following estrogen treatment suggests a response to a threat to the genome by electrophilic products of catechols.
Relative large amounts of DOPA as compared with the concentrations of norepinephrine are found in human dental pulp. AADC and COMT are localized in blood vessel walls of human dental pulp. This localization suggests a functional relationship between COMT and AADC with regard to the metabolism of DOPA.
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dopa, norepinephrine, and traces of dopamine, epinephrine were present in in rat dental pulp. L-dopa was localized in nerve fibers in dental pulp. The results suggest that L-dopa-positive nerve fibers are present in dental pulp as well as classical adrenergic fibers.
Synthetic routes to difluorinated analogs of the adrenergic agonists, norepinephrine (NE), epinephrine (E), and phenylephrine (PE) have been developed. The syntheses were based on elaboration of the ethanolamine side chains from the appropriately polyfunctionalized benzaldehydes. The benzaldehydes were prepared from precursor difluorinated benzenes by sequential regioselective lithiations and reaction with electrophiles to introduce hydroxyl and carboxaldehyde functionalities. Binding and functional assay data demonstrate that the 2,6-difluorinated analogs are relatively inactive at both alpha- and beta-adrenergic receptors. These results are consistent with earlier observations that 2-fluoro substitution of adrenergic agonists decreases alpha-adrenergic activity whereas 6-fluoro substitution decreases beta-adrenergic activity.
The tissue distribution and time course of incorporation into acid insoluble (bound) and acid soluble (free) fractions of [3H]2-fluorohistidine is compared to that of U[14C]Histidine in mouse tissues in vivo. The cycloheximide-sensitive incorporation of 2-FHis is between 9 and 17 percent of that of His. Unlike [14C]His a major fraction, approximately 90% at 72 hrs, of isotope derived from [3H]2-FHis remains in tissues for a prolonged period in an acid soluble form. The excretion of isotope derived from [14C]His (T1/2 = 5 hr) is more rapid than from [3H]2-FHis (T1/2 = 11.4 hrs). 2-FHis, at doses from 100 to 250 mg/kg produce a reversible inhibition of growth in mice.
The alkaloid pumiliotoxin B (PTX-B) "activates" voltage-dependent sodium channels in synaptoneurosomes and neuroblastoma cells. It appears that PTX-B activates sodium channels by interacting with a site that is allosterically coupled to other sites on the sodium channel, namely two scorpion toxin sites and the brevetoxin site. In guinea pig cortical synaptoneurosomes, alpha-scorpion toxin, beta-scorpion toxin, and brevetoxin induce a dose-dependent potentiation of PTX-B-induced 22Na+ influx. The synergism with beta-scorpion toxin differentiates PTX-B from the alkaloid veratridine, which induces an activation of sodium channels that is not affected by beta-scorpion toxin. PTX-B does not inhibit [3H]batrachotoxinin-A benzoate ([3H]BTX-B) binding to the alkaloid site on sodium channels. On the other hand, aconitine, which activates sodium channels and inhibits [3H]BTX-B binding, induces a 22Na+ influx that, like PTX-B-induced 22Na+ influx, is potentiated by alpha-scorpion toxin, beta-scorpion toxin, and brevetoxin. Inhibition of [3H]BTX-B binding by aconitine is reduced in the presence of PTX-B. Both a type I pyrethroid (allethrin) and a type II pyrethroid (fenvalerate) inhibit PTX-B- and PTX-B/alpha-scorpion toxin-mediated 22Na+ influx. Allethrin and fenvalerate also inhibit aconitine-mediated 22Na+ flux but not BTX-mediated 22Na+ influx. It is proposed that on the sodium channel there is an "alkaloid-binding domain" at which alkaloids exert stimulatory actions. However, depending on the region on the domain to which the binding occurs, different allosteric interactions with other sites can be observed. PTX-B is proposed to interact with a part of the alkaloid-binding domain that is shared by aconitine but not by batrachotoxin or veratridine, whereas aconitine interacts with a part of the domain shared by PTX-B and by batrachotoxin/veratridine.
The 2- and 6-fluoro derivatives of the potent beta-adrenergic agonist 3-(tert-butylamino)-1-(3,4-dihydroxyphenoxy)-2-propanol were prepared and their adrenergic properties examined. The order of potency was as follows: beta-adrenergic activity (simulation of cyclic AMP formation in C6 glioma cells), 2-F = parent much greater than 6-F; beta 1-activity (rate of contraction, guinea pig atria), parent greater than 2-F much greater than 6-F; beta 2-activity (relaxation of guinea pig tracheal strip), 2-F greater than parent much greater than 6-F. The affinity of the 2-fluoro analogue for beta 1-adrenergic receptors (inhibition of the specific binding of [3H]dihydroalprenolol, rat cerebral cortical membranes) was 2 times greater, while the 6-fluoro analogue was 1450 times less than the parent. These results suggest that the aromatic rings of phenoxypropanolamine adrenergic agonists and phenylethanolamine adrenergic agonists bind in similar fashion to the adrenergic receptor, and that if interactions between fluorine and the side-chain hydroxyl group are critical in defining beta-adrenergic selectivity, the interactions are similar in both phenoxypropanolamines and phenylethanolamines.
Immunocytochemical observations by light and electron microscopy of catechol-O-methyltransferase (COMT) were conducted in dental pulp by use of a specific antibody to soluble rat-liver COMT and the peroxidase-antiperoxidase technique. Immunoreactive deposits were found in macrophages. The pattern of localization suggests that COMT may function in extraneuronal inactivation of catecholamines in dental pulp.
We performed light microscopic immunocytochemical observations of the localization of catechol-O-methyltransferase (COMT) in rat uterus, using a rabbit anti-rat serum specific for the soluble form of rat liver COMT, biotinylated goat anti-rabbit immunoglobulin, and peroxidase conjugated with streptavidin. In the non-pregnant rat, COMT was minimal but detectable in the uterine luminal and glandular epithelium, with greater amounts present in uteri from rats in estrus than those in diestrus. In early pregnancy a robust accumulation of COMT was observed in the luminal epithelium. To more precisely define both the timing and the factors contributing to the appearance of COMT, uteri were examined on Days 1-5 in pregnant and pseudopregnant rats. Accumulation of COMT in the luminal epithelium was observed by Day 3 in uteri from pregnant and pseudopregnant rats and by Day 4 in lactating post-partum rats. No immunostaining of COMT was observed in uteri from non-lactating post-partum rats. Ovariectomy on Day 0 or 1 but not on Day 2 of pregnancy prevented the appearance of COMT on Day 4. Progesterone treatment immediately after ovariectomy on Day 0 or 1 of pregnancy restored the COMT.
Pumiliotoxin B (PTX-B) and a variety of congeneric alkaloids and synthetic analogs stimulated sodium flux and phosphoinositide breakdown in guinea pig cerebral cortical synaptoneurosomes. The effects of PTX-B and active congeners and analogs on sodium flux in synaptoneurosomes were potentiated markedly by scorpion venom (Leiurus quinquestriatus). In neuroblastoma cells, PTX-B and active congeners had no effect on sodium flux unless synergized by alpha-scorpion toxin or scorpion venom. Certain inactive congeners, lacking hydroxyl groups in the 6-alkylidene side chain, inhibited sodium flux elicited by PTX-B, scorpion venom, or the sodium channel activator batrachotoxin. Such inhibition appeared different from inhibition by local anesthetics, since pumiliotoxins, unlike local anesthetics, had little or no effect on binding of [3H]batrachotoxinin A benzoate to sodium channels. Thus, it appears likely that some "inactive" congeners bind to the PTX-B binding site, but do not activate sodium channels. In the absence of scorpion venom the stimulation of phosphoinositide breakdown in synaptoneurosomes was consonant with the stimulatory effects of these compounds on sodium flux through voltage-dependent sodium channels.
22Na+ influx and binding of [3H]saxitoxin ([3H]STX) and [3H]batrachotoxin-A 20-alpha-benzoate ([3H]BTX-B) were studied in guinea pig cerebral synaptoneurosomes. STX and tetrodotoxin (TTX) completely blocked the stimulation of sodium influx induced by 1 microM BTX. The IC50 values for STX and TTX closely matched the Ki values for inhibition of [3H]STX binding, suggesting that the sites labelled by [3H]STX are associated with a population of BTX-sensitive channels. BTX induced a dose-dependent stimulation of sodium influx in synaptoneurosomes (EC50 280 nM). The potency of BTX for stimulation of sodium influx was increased (EC50 24 nM) in the presence of 0.6 microgram/ml scorpion venom without any change in maximal influx. In contrast, specific binding of [3H]BTX-B to synaptoneurosomes was minimal in the absence of scorpion venom, but it was increased several fold in the presence of 60 micrograms/ml scorpion venom. With proparacaine isothiocyanate (PROPRIT), an irreversible local anesthetic, the inhibition of [3H]BTX-B binding by PROPRIT did not occur in parallel with an inhibition of sodium influx induced by BTX. Preincubation of synaptoneurosomes with 10 microM PROPRIT for 10 min resulted in approximately 70% inhibition of [3H]BTX-B binding in the presence of scorpion venom. Such preincubation did not alter BTX-induced sodium uptake in synaptoneurosomes. Preincubations of synaptoneurosomes with 100 microM PROPRIT for 10 min completely inhibited [3H]BTX-B binding, and under these conditions BTX-induced sodium influx was reduced only by 50%. The results indicate that virtual elimination of binding sites labeled by [3H]BTX-B in the presence of scorpion venom by PROPRIT has little effect on sodium influx induced by BTX.(ABSTRACT TRUNCATED AT 250 WORDS)
PC12 cells are a nerve growth factor-responsive clone derived from a rat pheochromocytoma. The cells contain catecholamines and secrete them in response to depolarizing stimuli and cholinergic agonists. Treatment of the cells with nerve growth factor produces a number of very rapid changes, including the structural rearrangement of the cell membrane, the generation of a number of different second messengers, and the phosphorylation of several proteins. The present studies show that nerve growth factor treatment increases the release of dopamine and norepinephrine from the cells within a few minutes and does so independently of its effect on their metabolism. The experiments indicate that this effect on nerve growth factor is dependent on the presence of extracellular calcium and can be blocked by calcium channel antagonists. K-252a, an alkaloid-like material, usually found to inhibit the actions of nerve growth factor on PC12 cells, also increases the release of catecholamines under these conditions.