Biomedical subjects
Publications and source records attributed to C R Creveling.
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19F nuclear magnetic resonance (NMR) has been used to examine the disposition of ring-fluorinated dopamine and norepinephrine in microsacs prepared from striata of guinea pig brains. Following incubation with a 10(-4)M initial concentration of 6-fluorodopamine (6F-DA), intact micmicrosacs at 4 degrees C gave a 19F NMR spectrum in which the 6F-DA present was sufficiently mobile to be visible. Intra-vesicular 6F-DA in striatal nerve terminals thus appears to exist in an environment resembling that in chromaffin vesicles but different from that prevailing inside the amine storage vesicles of platelets. Our data also suggest that the study of fluorinated compounds by 19F NMR can be used to expand our understanding of processes related to amine uptake, metabolism, and storage in nerves.
The sodium channel-specific agent batrachotoxin (BTX) has been shown to induce a time- and concentration-dependent depolarization of a vesicular preparation from guinea pig cerebral cortex. The K0.5 for depolarization by BTX was 0.011 microM at 30 min. Membrane potential was determined by the equilibrium distribution of [3H]triphenylmethylphosphonium ion. A series of seven local anesthetics was shown to inhibit BTX-induced depolarization competitively with Ki values ranging from 0.9 microM for dibucaine to 780 microM for lidocaine ethiodide. The specific binding of labeled batrachotoxinin-A 20 alpha-benzoate ([3H]BTX-B) to voltage-sensitive channels in vesicular preparations from mouse cerebral cortex in the presence of scorpion venom was measured and found to yield a range of Kd values from 25 to 30 nM and Bmax values of 0.5 and 1.0 pmole/mg of protein; the same preparation from guinea pig cerebral cortex was found to yield Kd values from 13 to 56 nM and Bmax values of 0.8-2.2 pmoles/mg of protein. A series of 14 local anesthetics was shown to inhibit the specific binding of [3H]BTX-B with Ki values ranging from 0.6 microM for dibucaine to 400 microM for benzocaine. The rank order of potency of the local anesthetics as antagonists of [3H]BTX-B binding was as follows: dibucaine greater than tetracaine greater than bupivacaine greater than diphenhydramine greater than piperocaine greater than cocaine greater than procaine greater than lidocaine greater than benzocaine. The quaternary local anesthetic dimethyl-di(phenylcarbamoylmethyl)ammonium chloride was comparable in potency to tetracaine. The rank order and relative potency of the local anesthetics tested in both paradigms were similar with the exception of lidocaine ethiodide, which was 18 times more potent as an inhibitor of binding of [3H]BTX-B than it was as an inhibitor of BTX-elicited depolarization.
The immunocytochemical localization of catechol methyltransferase was determined in normal and cancerous breast tissues of inbred female Swiss-Webster mice and in normal, lactating, and cancerous breast tissues of inbred Sprague-Dawley rats. The enzyme was found to be cytoplasmically localized in ductal epithelial cells of secretory tubules in both inactive and stimulated mammary glands, in endothelial cells lining blood vessels, in fibroblasts in the connective tissue matrix, and, especially, in tumor cells. Adipose cells were nonreactive. The intensity of the immunocytochemical reaction in tumor cells was stronger than that in lactating tissues, which, in turn, was more reactive than that in normal, unstimulated breast tissues.
1. The calcium-dependent K+-evoked release of [3H]norepinephrine from guinea pig cerebral cortical vesicular preparations is inhibited by norepinephrine, clonidine, and epinephrine. Isoproterenol has no effect and phentolamine prevents the inhibition by norepinephrine. The results indicate that an alpha-adrenergic receptor mediates an inhibitory input to the calcium-dependent release process. The inhibition by norepinephrine is prevented by high concentrations (3.0 mM) of calcium ions. 2. A cyclic AMP phosphodiesterase inhibitor, ZK 62771, slightly elevates [3H]cyclic AMP levels in the guinea pig cerebral cortical preparation and potentiates the marked elevation of [3H]cyclic AMP elicited by the adenylate cyclase activator, forskolin. 3. Neither ZK 62771 nor forskolin alone has significant effects on K+-evoked release of [3H]norepinephrine from the cerebral cortical vesicular preparation; however, a combination of ZK 62771 and forskolin inhibits K+-evoked release by as much as 60%. The inhibition is reversed by high concentrations (2.0 mM) of calcium ions. The results suggest that a marked accumulation of cyclic AMP elicited via both activation of adenylate cyclase and inhibition of phosphodiesterase can be inhibitory to neurotransmitter release from central synaptic terminals.
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2-Fluoro-, 5-fluoro-, and 6-fluoroisoproterenol were synthesized by reduction of the Schiff base formed between the corresponding ring-fluorinated 3,4-bis(benzyloxy)phenethanolamine and acetone, followed by reductive debenzylation in the presence of oxalic acid to yield crystalline neutral oxalates. The apparent beta-adrenergic potencies were determined in the isolated guinea pig atria. 2-Fluoro- and 5-fluoroisoproterenol were equipotent with (+/-)-isoproterenol, while 6-fluoroisoprotenol was virtually inactive. No alpha-adrenergic agonist activity (guinea pig aorta) was shown by any of the fluoroisoproterenols. Displacement of alpha- and beta-specific radioligands from isolated membrane preparations from rat brain by the fluoroisoproterenols were in agreement with the responses of the organ preparations. Thus, the apparent fluorine-induced specificity is due to specificity at the receptor binding site. The effects of fluorine substitution are discussed with regard to the apparent negative influence of the 6-fluoro substituent on the beta-agonist properties of isoproterenol, the lack of any increase in potency due to the 2-fluoro substituent, and the possibility of fluorine-induced changes in the electron density of the aromatic ring as a possible rational for the fluorine-induced specificity of both the fluoroisoproterenols and the fluoronoirepinephrines.
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Catechol-O-methyltransferase (COMT) was localized in the organum vasculosum of the lamina terminals, subfornical organ, subcommissural organ and area postrema of rat brain using an indirect immunofluorescence technique. COMT immunofluorescence was apparent in neuroglia within the organum vasculosum and was most intense in the ependyma between this structure and the optic recess of the third ventricle. In both the subfornical organ and the area postrema, COMT was localized in a neuroglial network, but was noticeably absent in the ependymal layer. COMT immunofluorescence in the ependyma of the subcommissural organ was continuous with the more intense immunofluorescence of the cuboidal ependyma of the third ventricle. Each of the circumventricular organs studied, with the exception of the subcommissural organ, lies outside the blood-brain barrier. However, the unique pattern of COMT immunofluorescence in the area postrema and the subfornical organ suggests that these two structures, of all circumventricular organs, are most likely to permit the entry of peripherally circulating catechols to the cerebrospinal fluid.
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Catechol-O-methyltransferase (COMT) was localized in cells of the pia-arachnoid, and in epithelial cells of the choroid plexus, using an indirect immunofluorescence technique. The specific activity of COMT derived from these tissues was determined by radioenzymatic assay, and in the case of the choroid plexus was found to be 9-fold greater than that measured in whole rat brain. The level of COMT specific activity in pia-arachnoid was twice as high as that in whole brain. Indirect immunofluorescence studies also revealed an intensity of COMT immunofluorescence in the ciliary epithelium at the blood-aqueous barrier in the rat eye, similar to that visualized in the epithelium of the choroid plexus at the blood-cerebrospinal fluid barrier. The localization of COMT in the leptomeninges, choroid plexus, and ciliary epithelium is consistent with a role for this enzyme in the separation of catechol compounds synthesized in the central nervous system, from those of peripheral origin. Thus, catecholamines derived from the peripheral sympathetic system may be prevented from entering the brain parenchyma, which is innervated by the functionally distinct central catecholaminergic systems.
The extent of meta- and para-O-methylation by catechol O-methyltransferase of 2-fluoro-, 5-fluoro-, and 6-fluoronorepinephrine (FNE) at pH 7 and 9 was determined. The rank order of preference for para-O-methylation is 5FNE much greater than NE greater than 6FNE greater than 2FNE. In all cases, increasing the pH to 9 results in an increase in para-O-methylation. Results with 2F- and 5FNE demonstrate the importance of ionization in the methyltransferase reaction when fluorine is situated ortho to one of the phenolic groups. To establish unequivocally the identities of the products, the isomeric, monofluorinated vanillins and isovanillins were synthesized and directly related to the products formed enzymatically from the monofluorinated norepinephrines.
Catecholamine, histamine, and adenosine-mediated accumulations of radioactive cyclic AMP were assessed in adenine-labeled slices from eight rat brain regions. 2-Fluoronorepinephrine, a selective beta-adrenergic agonist, elicited an an accumulation of cyclic AMP in cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, thalamus, hypothalamus, and medulla-pons. In cerebral cortex and most other brain regions, the beta-adrenergic-mediated response appeared to involve primarily beta 1-adrenergic receptors, while in cerebellum, there was a significant involvement of beta 2-adrenergic receptors. 6-Fluoronorepinephrine, a selective alpha-adrenergic agonist, elicited accumulations of cyclic AMP in all regions except cerebellum. Combinations of the two fluoro derivatives afforded in all brain regions an accumulation of cyclic AMP identical with that elicited by norepinephrine. In hypothalamus, the alpha- and beta-adrenergic responses were significantly greater than additive. In cerebral cortex, the alpha-adrenergic receptor-mediated response appeared to involve alpha 1-adrenergic receptors and to be nearly completely dependent on adenosine, while in other brain regions, the dependence of the alpha-adrenergic response on adenosine was less or absent. Combinations of 6-fluoronorepinephrine and histamine had greater than additive effects in cortex and hippocampus. The results indicate that the interactive control of cyclic AMP-generating systems by alpha-adrenergic, beta-adrenergic, adenosine, and histamine receptors differs significantly among rat brain regions.
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An antiserum to rat liver catechol-O-methyltransferase (COMT) was utilized in the immunological characterization of COMT from rat kidney, brain, and choroid plexuses, in addition to rat liver. The presence of anti-COMT activity was confirmed by the direct inhibition of the activity of the enzyme from rat liver by small quantities of the antiserum and by the inhibition of the activity of the enzyme from rat brain. The specificity of the antiserum was demonstrated both by immunoelectrophoresis of rat liver COMT, and by a partial purification of rat liver COMT in which changes in COMT specific activity were correlated with the appearance of a precipitin line in double-immunodiffusion experiments. The antigenic similarity of the enzyme derived from rat liver, kidney, brain, and choroid plexuses was demonstrated by the formation of a precipitin line of identity when preparations from these four tissues were diffused against the antiserum.
Catechol-O-methyltransferase (COMT) activity in the liver and kidneys of adult Fischer-344 (F-344) rats is only half of that in the same organs of Wistar-Furth (W-F) rats. The trait of low COMT activity in these animals is inherited in an autosomal recessive fashion. A comprehensive study of patterns of change in COMT activity during growth and development was performed to determine whether "temporal gene" effects might play a role in the inherited differences in enzyme activity present in adult animals. The COMT activity expressed per mg protein in liver and kidneys of newborn F-344 rats is only 50-60% of that in the same organs of W-F animals. The liver and the kidneys of newborn rats of both strains have COMT activity an order of magnitude higher than those in brain, heart, or blood. In addition, in both strains there are much larger increases in liver and kidney COMT activities during growth and development (5-10 fold) than in blood, brain, or heart (one- to twofold). Immunotitration with antibodies against rat COMT demonstrates that differences in immunoreactive COMT parallel differences in COMT activity, both between strains and within strains during growth and development. However, when the temporal patterns of change in enzyme activities in the liver and the kidneys of the two strains of rat are compared at multiple times during growth and development, no differences in the patterns are present. These results make it unlikely that temporal gene effects can explain the inherited differences in COMT activity in liver and kidneys of F-344 and W-F rats.
Norepinephrine, histamine, adenosine, glutamate, and depolarizing agents elicit accumulations of radioactive cyclic AMP from adenine-labeled nucleotides in particulate fractions from Krebs-Ringer homogenates of guinea pig cerebral cortex. The particulate fractions contain sac-like entities, which apparently are associated with a significant portion of the membranal adenylate cyclase. Particulate fractions from sucrose homogenates are a less effective source of such responsive entities. Activation of the adenine-labeled cyclic AMP-generating systems by norepinephrine is by means of alpha-adrenergic receptors, while activation by histamine is through H1- and H2-histaminergic receptors. Adenosine responses are potentiated by the amines and are antagonized by alkylxanthines. Glutamate and depolarizing agents appear to elicit accumulations of cyclic AMP via "release" of endogenous adenosine. It is proposed, based on the virtual absence of an alpha-adrenergic or H1-histaminergic response in the presence of a combination of potent adenosine and H2-histaminergic antagonists, that alpha-adrenergic and H1-histaminergic receptor mechanisms do not activate adenylate cyclase directly in brain slices or Krebs-Ringer particulate fractions, but merely facilitate activation by beta-adrenergic, H2-histaminergic, or adenosine receptors.
The accumulations of radioactive cyclic AMP elicited by adenosine, norepinephrine, and histamine in adenine-labeled vesicular entities of a particulate fraction from guinea pig cerebral cortex are greatly reduced as a result of prolonged preincubation. The presence of adenosine deaminase during preincubations largely prevents the loss of adenosine, norepinephrine and histamine responses. Adenosine deaminase was inactivated by deoxycoformycin prior to stimulation of cyclic AMP accumulation by adenosine or amines. If adenosine deaminase is not inactivated, responses to norepinephrine are not significant and histamine responses are reduced by 50%. Adenosine deaminase cannot restore responsiveness of the cyclic AMP-generating systems. It is proposed that, in particulate fractions of guinea pig cerebral cortex, low levels of adenosine cause a slow loss of receptors and/or coupling of receptors to cyclic AMP-generating systems.