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Biomedical subjects

C R Creveling

Publications and source records attributed to C R Creveling.

At least 37 records · Page 2Linked to original sources
PubMed1990

Procaine isothiocyanate: an irreversible inhibitor of the specific binding of [3H]batrachotoxinin-A benzoate to sodium channels.

[3H]Batrachotoxinin-A benzoate ([3H]BTX-B) binds with high affinity to sites on voltage sensitive sodium channels in synaptoneurosomes from guinea pig cerebral cortex. Local anesthetics competitively antagonize the binding of [3H]BTX-B. An irreversible local anesthetic, procaine isothiocyanate (PRIT) and a tritiated derivative [( 3H]PRIT) have been prepared. PRIT inhibits the binding of [3H]BTX-B in a noncompetitive, irreversible manner (apparent Ki = 13 microM) whereas the parent compound, procaine, inhibits in a competitive, reversible manner (Ki = 40 microM). The dissociation rate of [3H]BTX-B from sites on the sodium channel is greatly accelerated in a concentration dependent manner in the presence of PRIT. A 50% increase in the dissociation rate of [3H]BTX-B is achieved in the presence of 0.98 microM PRIT. [3H]PRIT binds irreversibly to three proteins in synaptoneurosomes with apparent molecular weights of 20, 42, and 68 kDa. Protection studies with procaine and other local anesthetics suggest that only the 68 kDa species was related to local anesthetic binding.

Anesthetics, Local↗
PubMed1989

Isolation of the mRNA encoding rat liver catechol-O-methyltransferase.

A highly specific, well characterized rabbit antiserum to purified rat liver catechol-O-methyltransferase (COMT; EC 2.1.1.6) and the procedure of polysome immunoadsorption have been used to isolate a messenger RNA which encodes a single polypeptide when translated in vitro. Western blotting and immune fixation have shown multiple active forms of the enzyme to exist; a major, soluble one with MW of 23,000 and pI of 5.2 and another, membrane-bound one with MW of 26,000 and a pI of 6.2 (1). When translated in vitro, the purified message synthesizes a protein of molecular weight (MW) 23,000 and pI 5.2, values in agreement with those for purified enzyme reported by other investigators (2,3). Only the soluble form is seen after in vitro translation; the other immunoreactive proteins possibly arise due to post-translational modifications which do not occur in the lysate; or perhaps another mRNA exists. Cloning of the COMT cDNA will resolve this issue and should be feasible in light of our data indicating that the mRNA isolated here represents 0.46% of total rat liver polyadenylated message.

Animals↗
PubMed1988

Syntheses and adrenergic activities of ring-fluorinated epinephrines.

The study of chemical and biological effects of fluorine substitution on the aromatic ring of catecholamines has now been extended to epinephrine (Epi). 2- and 6-fluoroepinephrines (2-FEpi and 6-FEpi) have been synthesized. Fluorine substitution on the 2- or 6-carbon of the aromatic ring alters the selectivity of epinephrine toward alpha- and beta-adrenergic receptors, similar in manner to the change in selectivity seen with norepinephrine (NE). Thus, 2-FEpi is a relatively selective beta-adrenergic ligand, while 6-FEpi is a relatively selective alpha-adrenergic ligand. Fluorine substitution of Epi also can markedly increase potency at either alpha- or beta-adrenergic receptors.

Adenylyl Cyclase Inhibitors↗
PubMed1987

Effects of ring fluorination on the adrenergic properties of phenylephrine.

The adrenergic properties of 2-, 4- and 6-fluorophenylephrine (2-FPE, 4-FPE, 6-FPE) were compared to those of phenylephrine (PE). The order of affinities of these compounds for alpha 1-adrenoceptors as determined by displacement of [3H]prazosin and [3H]WB-4101 binding to brain membranes was the same as the order of potencies for eliciting two alpha 1-adrenergic metabolic responses in guinea-pig cerebral cortical synaptoneurosomes, namely the stimulation of phosphatidylinositol turnover and the potentiation of 2-chloroadenosine-induced accumulation of cyclic AMP. In all cases the order was 6-FPE greater than PE greater than 4-FPE greater than 2-FPE. The order of affinities for alpha 2-adrenoceptors as determined by displacement of binding of [3H]clonidine to brain membrane was 6-FPE greater than PE greater than or equal to 4-FPE = 2-FPE. In contrast, the order of potencies for inhibition of forskolin-stimulated adenylate cyclase activity in human platelet membranes via an alpha 2-adrenoceptor was 6-FPE approximately equal to PE greater than 4-FPE much greater than 2-FPE. The FPEs and PE were partial agonists compared to epinephrine in human platelets. The affinities of these compounds for beta-adrenoceptors as determined by displacement of binding of [3H]dihydroalprenolol to brain membranes are 2-FPE greater than PE greater than or equal to 4-FPE much greater than 6-FPE. The FPEs and PE had positive chronotropic and inotropic effects in isolated guinea-pig atria apparently through the activation of a beta-adrenoceptor, since pindolol blocked the response while prazosin did not. 6-FPE appeared less active than the other PEs in atria. In fat cell membranes, 2-FPE was more potent than PE in stimulating adenylate cyclase via a beta-adrenoceptor, while 4-FPE and 6-FPE were inactive. Both, 2-FPE and PE were partial agonists in fat cells compared to isoproterenol. Of the three FPEs, 6-FPE represents a more potent and more selective agonist for alpha-adrenoceptors compared to beta-adrenoceptors than PE, while 4-FPE and, in particular, 2-FPE are less potent and selective as alpha-agonists.

Animals↗
PubMed1987

Immunocytochemical evidence for the coexistence of catecholestrogen and catechol-O-methyltransferase in the rat parotid gland.

Catechol-O-methyltransferase (COMT) (EC 2.1.1.6) and catecholestrogen were localized in the parotid gland of the rat by immunocytochemical methods. Specific immunoreactive deposits for COMT and catecholestrogen were found in the cytoplasm of duct cells, but only those for COMT in myo-epithelial cells. The pattern of localization of COMT and catecholestrogen in the parotid gland suggests a functional relationship between COMT and catecholestrogen.

Animals↗
PubMed1986

The presence of catechol-o-methyltransferase activity in separately cultured cerebromicrovascular endothelial and smooth muscle cells.

The activity of catechol-o-methyltransferase (COMT) was investigated in cultured and propagated cerebromicrovascular endothelial and smooth muscle cells using high performance liquid chromatography and immunocytochemistry. The existence of COMT was detected in both cell types. The demonstration of this enzyme activity in the cerebromicrovascular smooth muscle cells, in addition to the endothelium, indicates that the enzymatic barrier to catecholamine is not limited to capillaries, the main constituents of the blood-brain barrier.

Animals↗
PubMed1986

Regioselectivity of catechol O-methyltransferase. The effect of pH on the site of O-methylation of fluorinated norepinephrines.

Selectivity of catechol O-methyltransferase has been examined for the three ring-fluorinated norepinephrines to elucidate the role of acidity of the phenolic groups in their methylation. Substitution of fluorine at the 5-position of norepinephrine reverses the selectivity of catechol O-methyltransferase so that p-O-methylation predominates. The 5-fluoro substituent also causes the pKa of the p-hydroxyl group to decrease substantially. In contrast, 2- and 6-fluoronorepinephrines are methylated predominantly at the m-hydroxyl group. These results suggest that acidity of a phenolic group can play an important role in its ability to be methylated by catechol O-methyltransferase. Percentages of p-O-methylation of norepinephrine and its fluorinated derivatives increase with pH. This relative increase in p-O-methylation appears to accompany ionization of a group with pKa of 8.6, 7.7, 7.9, and 8.4 for norepinephrine and its 2-, 5-, and 6-fluoro derivative, respectively. These pKa values are the same as or similar to the pKa values of a phenolic hydroxyl group of these substrates. 3,4-Dihydroxybenzyl alcohol and its 5-fluoro derivative are O-methylated by catechol O-methyltransferase to form p- and m-O-methyl products in approximately 1:1 and 4:1 ratios, respectively, at all pH values. Based on the above results, a catechol-binding site model for catechol O-methyltransferase is proposed in which the two phenolic hydroxyl groups of catechol substrates are postulated to be approximately equally spaced from the methyl group of the cosubstrate S-adenosylmethionine.

Binding Sites↗
PubMed1986

Immunocytochemical localization of catechol-O-methyltransferase in the oviduct and in macrophages in corpora lutea of rat.

Catechol-O-methyltransferase (COMT) (EC 2.1.1.6) was localized in rat ovary, oviduct, and uterus using immunocytochemical methods. Immunoreactive deposits were found in the cytoplasm of macrophages in the ovary, epithelial cells of the oviduct, and glandular epithelial cells of the non-pregnant uterus. The pattern of localization observed in the extraneuronal elements suggest that enzyme may function in extraneuronal inactivation of catechols in the ovary, oviduct, and uterus.

Animals↗
PubMed1986

Anticonvulsant activity of piperidinol and (dialkylamino)alkanol esters.

Aromatic and heterocyclic esters of 1-methyl-4-piperidinol and 1,4-dimethyl-4-piperidinol and aromatic esters of (dialkylamino)alkanols were prepared and evaluated for antiepileptic activity by the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole seizure threshold (scMet) assays and for minimal central neurotoxicity by the rotorod ataxia test. The most potent compound, namely the 2-phenylbenzoate (57) of 3-(diethylamino)propanol, was slightly more potent than diphenylhydantoin in the MES assay, while the 2-phenylbenzoate (24) of 1-methyl-4-piperidinol and the 2-phenylbenzoate (56) of (diethylamino)ethanol displayed activity comparable to that of diphenylhydantoin. The 2-phenethylbenzoate ester (6) of 1-methyl-4-piperidinol exhibited one-third the activity of diphenylhydantoin. The 2,4,5-trimethylbenzoate 40 and 2,4,6-trimethylbenzoate 41 of 1-methyl-4-pieridinol were even less potent, but did display activity in the phenobarbital-methsuximide range. Certain compounds interact with sites associated with the GABA receptor-chloride channel complex, but their potencies as anticonvulsant agents do not correlate with interaction at sites on the channel complex. Certain analogues antagonize binding of a batrachotoxin analogue to sodium channel sites, a property indicative of local anesthetic activity. There are structural similarities between 2-phenylbenzoates 57, 56, and 24 and diphenylhydantoin, and the latter anticonvulsant also antagonizes binding of the batrachotoxin analogue.

Amino Alcohols↗
PubMed1986

Synthesis and adrenergic activity of ring-fluorinated phenylephrines.

2-Fluoro-, 4-fluoro-, and 6-fluorophenylephrine (6-FPE) were synthesized from the corresponding fluorinated 3-hydroxybenzaldehydes. New routes to 2-fluoro- and 6-fluoro-3-hydroxybenzaldehydes were developed based on regioselective lithiation of 2- and 4-[(dimethyl-tert-butylsilyl)oxy]fluorobenzene ortho to fluorine. As with norepinephrine and isoproterenol analogues, the adrenergic properties of phenylephrine were markedly altered by ring fluorination. The order of potency of the fluoro analogues as alpha 1-adrenergic agonists in the stimulation of contraction of aortic strips and of phosphatidylinositol turnover and potentiation of cyclic AMP accumulation in guinea pig synaptoneurosomes was 6-FPE greater than PE greater than 4-FPE greater than 2-FPE. The same pattern was observed for the displacement of radioligands specific for alpha 1- and alpha 2-adrenergic receptors on brain membranes. The order of potency for the displacement of [3H]dihydroalprenolol, a beta-specific adrenergic ligand from brain membranes, was 2-FPE greater than 4-FPE = PE much greater than 6-FPE. 6-FPE was much more selective for alpha-adrenergic receptors compared to beta-receptors than was phenylephrine. A rationale for the observed fluorine-induced alterations in potency and selectivity of the FPEs for alpha- and beta-adrenergic systems is presented based on fluorine-induced conformations due to electrostatic repulsion of fluorine and the benzyl hydroxyl group.

Animals↗
PubMed1985

The effect of ring-fluorination on the rate of O-methylation of dihydroxyphenylalanine (DOPA) by catechol-O-methyltransferase: significance in the development of 18F-PETT scanning agents.

The three ring-fluorinated analogs of dihydroxyphenylalanine (DOPA) were synthesized and the kinetics of their O-methylation catalyzed by catechol-O-methyltransferase compared to those of DOPA. The affinities (Km) of 2-FDOPA, 5-FDOPA, DOPA, and 6-FDOPA were 20, 23, 55, and 552 microM, respectively, whilst the Vmax values were 4.0, 4.4, 5.0 and 5.4 nmol per min per mg protein. The importance of the low affinity and decreased rate constant of 6-FDOPA for COMT are discussed with regard to the use of 6-FDOPA as an 18-fluorine labeled probe for positron emission transaxial tomography (PETT).

Animals↗
PubMed1985

Local anesthetic properties of opioids and phencyclidines: interaction with the voltage-dependent, batrachotoxin binding site in sodium channels.

[3H]Batrachotoxinin-A 20-alpha-benzoate ([3H]BTX-B) binds specifically and with high affinity (Kd = 30 nM) to a site on voltage-dependent Na+ channels. Compounds with local anesthetic activity inhibit the binding of [3H]BTX-B by a mutually exclusive, allosteric mechanism. The potential local anesthetic potency of a series of 23 opioids and phencyclidine-like compounds has been estimated by their inhibition of [3H]BTX-B binding to Na+ channels in a preparation of synaptoneurosomes from guinea pig cerebral cortex. The potency of these compounds were also tested as inhibitors of the specific binding of [3H]phencyclidine ([3H]PCP) to a high affinity site on rat brain membranes. Opioids such as morphine and codeine show little affinity for the [3H]BTX-B binding site or for the [3H]PCP binding site. Other analgesics, many of the PCP-like compounds and dioxadrol derivatives are potent versus [3H]BTX-B binding and display both stereospecificity and high affinity towards the PCP-binding site. However, there was no correlation between local anesthetic potency assessed as antagonism of [3H]BTX-B binding and affinity towards the PCP site. Five classical local anesthetics had no affinity for the PCP-site, but did displace [3H]BTX-B from its binding site.

Anesthetics, Local↗
PubMed1985

[3H]Batrachotoxinin A 20 alpha-benzoate binding to voltage-sensitive sodium channels: a rapid and quantitative assay for local anesthetic activity in a variety of drugs.

[3H]Batrachotoxinin A benzoate ( [3H]BTX-B) binds with high affinity to sites on voltage-dependent sodium channels in a vesicular preparation from guinea pig cerebral cortex. In this preparation, local anesthetics competitively antagonize the binding of [3H]BTX-B. The potencies of some 40 classical local anesthetics and a variety of catecholamine, histamine, serotonin, adenosine, GABA, glycine, acetylcholine, and calcium antagonists, tranquilizers, antidepressants, barbiturates, anticonvulsants, steroids, vasodilators, antiinflammatories, anticoagulants, analgesics, and other agents have been determined. An excellent correlation with the known local anesthetic activity of many of these agents indicate that antagonism of binding of [3H]BTX-B binding provides a rapid, quantitative, and facile method for the screening and investigation of local anesthetic activity.

Adrenergic alpha-Antagonists↗
PubMed1985

Soluble and particulate forms of rat catechol-O-methyltransferase distinguished by gel electrophoresis and immune fixation.

Catechol-O-methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of all tissue homogenates examined revealed three major immune-specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 X g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an "aggregate" of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two-dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pI of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1-5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is "soluble" with an apparent Mr of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.

Animals↗
PubMed1985

Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3':5'-monophosphate-generating systems, receptors, and enzymes.

A particulate preparation was obtained by low speed centrifugation of guinea pig cerebral cortical homogenates prepared with a Krebs-Henseleit buffer. Light microscopic examination, using a reflected light differential interference contrast system, reveals the presence of intact neurons, axonal fragments, glial cells, and erythrocytes along with an abundance of small spherical entities (diameter about 1.1 micron) and snowman-shaped entities (diameter of larger sphere about 1.1 micron, diameter of attached smaller sphere about 0.6 micron). Many unattached smaller spherical entities are also present (diameter about 0.6 micron). Pressure filtration through 5- or 10-micron Millipore filters, followed by low speed centrifugation and resuspension, removes most of the larger entities to afford a suspension composed mainly of the small spherical and snowman-shaped entities. Electron microscopic examination reveals the presence of many synaptosomes with attached resealed postsynaptic entities. It is proposed that these correspond to the snowman-shaped entities to be termed synaptoneurosomes. Accumulations of cyclic AMP elicited by 2-chloroadenosine and histamine, and by combinations of 2-chloroadenosine, histamine, norepinephrine, and forskolin, are lower in filtered than in unfiltered preparations, whereas accumulations elicited by forskolin are unchanged. Levels of adenylate cyclase are reduced by filtration, whereas levels of phosphodiesterase are unchanged. Filtration reduces levels of markers for whole cells and endothelial cells, whereas neuronal markers such as acetylcholinesterase activity and norepinephrine uptake are increased. Levels of S-100 protein, a marker for glial cells, are not significantly decreased. There is no apparent change in the density of many receptors or ion channels. Levels of A1-adenosine and H1-histamine receptors are increased, whereas levels of so-called peripheral benzodiazepine-binding sites are decreased.

2-Chloroadenosine↗
PubMed1984

Alpha-adrenergic potentiation of beta-adrenergic stimulation of rat pineal N-acetyltransferase. Studies using cirazoline and fluorine analogs of norepinephrine.

Recent evidence indicates that melatonin production is controlled by norepinephrine acting via alpha 1-and beta 1-adrenoceptors on pinealocytes; activation of alpha 1-adrenoceptors appears to potentiate the effects of beta 1-adrenoceptor activation. However, alpha-adrenergic potentiation of beta 1-adrenergic activation has been demonstrated with only one alpha-adrenergic agonist. For this reason, this issue was reinvestigated using two other alpha-adrenergic agonists, 6-fluoronorepinephrine and cirazoline. Both compounds, which were found to have a high affinity for pineal alpha 1-adrenoceptors, potentiated the stimulatory effects of isoproterenol on pineal N-acetyltransferase. 6-Fluoronorepinephrine also potentiated the stimulation of N-acetyltransferase activity produced by another beta-adrenergic agonist, 2-fluoronorepinephrine. These findings support the hypothesis that pineal N-acetyltransferase activity is regulated by norepinephrine acting through both alpha 1- and beta 1-adrenoceptors.

Acetyltransferases↗