Effect of fluorine substitution on the agonist specificity of norepinephrine.

Substitution of fluorine for hydrogen in position 2, 5, or 6 of the aromatic ring of norepinephrine markedly alters the alpha- and beta-adrenergic agonist properties of norephinephrine. The 6-fluoro isomer is an beta-adrenergic agonist with virtually no beta agonist activity, while the 2-fluoro isomer is a beta-adrenergic agonist with little alpha activity. The 5-fluoro isomer is equipotent with norepinephrine as an alpha agonist and significantly more potent as a beta agonist. The possible physiochemical basis for these differences is discussed.

Immunohistochemical localization of catechol methyltransferase in normal and cancerous breast tissues of mice and rats.

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.

Calcium-dependent cyclic nucleotide phosphodiesterase. Inhibition of basal activity by heat-stable factors from rat cerebrum.

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca2+-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca2+-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in Km for cyclic GMP and a decrease in V. In the presence of calcium ions and purified activator protein, the Ca2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was minimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca2+-independent activation of Ca2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phoshodiesterase activity.

Electron microscopic identification of adrenergic nerve endings on thyroid epithelial cells.

Electron microscopic observations were made of the microinnervation of the thyroids of normal mice and of mice given false adrenergic neurotransmitters, and the effects of the drugs on thyroidal norepinephrine uptake were measured. In addition to adrenergic nerve terminals on thyroid blood vessels, structures resembling nerve endings were seen in close apposition to the bases of thyroid epithelial cells. Because they take up false adrenergic neurotransmitters, and undergo degenerative change after administration of cytotoxic false neurotransmitters (6-hydroxydopamine and 5,7-dihydroxytryptamine), which also reduce thyroid norepinephrine uptake, they probably represent adrenergic nerve terminals on thyroid follicles. If so, they afford a morphological basis for the direct effects of catecholamines on thyroid function, which have been reported.

Immunocytochemical localization of catechol-O-methyltransferase in the pregnant rat uterus.

Light microscopic immunocytochemical observations of catechol-O-methyl-transferase (COMT) localization in the pregnant rat uterus were made with a specific antibody to soluble rat liver COMT and the peroxidase-antiperoxidase technique. The changes in cellular localization of COMT were followed from the onset of pregnancy to 12 h after delivery of the last fetus. COMT first appeared on day 5 of pregnancy in endometrial epithelial cells of the uterus. On day 7, COMT appeared in the decidual cells on the antimesometrial side of the uterus close to the implanting blastocyst. Two morphologically distinct, COMT-positive, decidual cells were observed: polygonal cells and stellate cells. After parturition, COMT-positive decidual cells remained in the decidua basalis. The possible physiological roles of COMT in the pregnant uterus are discussed.

Norepinephrine uptake sites in cardiac tissue. Lack of affinity of 6-hydroxynorepinephrine and related compounds.

The effects of a series of phenethylamines and the corresponding phenethanolamines on (i) rate of uptake of radioactive norepinephrine into cardiac tissue in vivo and (ii) the rate of efflux of radioactive norepinephrine from prelabeled cardiac storage sites have been determined. The results indicate that m- and p-hydroxuphenethylamines and the corresponding phenethanolamines have high affinities for uptake into cytoplasm and storage vesicles of noradrenergic terminals in the heart. o-Hydroxyphenethylamines such as 2-hydroxyphenethylamine and 2,4,5-trihydroxyphenethylamine (6-hydroxydopamine) also have moderate to high activity as inhibitors of norepinephrine uptake and as releasing agents for norepinephrine, but o-hydroxyphenethanolamines such as 2-hydroxyphenethanolamine, 2,5-dihydroxyphenethanolamine, and 2,4,5-trihydroxyphenethanolamine (6-hyproxynorepinephrine) have little or no activity as inhibitors of uptake or as releasing agents. 2,6-Dihydroxyphenethylamines have little or no activity as inhibitors of uptake or as releasing agents. The results are consonant with significant binding of the gauche conformers of 2-hydroxyphenethylamines to uptake sites. Such conformers would be preferred because of stabilization by hydrogen bonding between nitrogen and phenolic oxygen. Apparently a hydrophobic region of the site prevents binding of such stabilized gauche conformers of 2-hydroxyphenethanolamines and 2,6-dihydroxyphenetylamines.

Influence of fluorine substitution on the site of enzymatic O-methylation of fluorinated norepinephrines.

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.

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.

N-(2,4,5-Trihydroxyphenehtyl)normetazocine, a potential irreversible inhibitor of the narcotic receptor.

The reaction of N-2,4,5-tribenzyloxyphenyl)ethyl methanesulfonate, prepared in a seven-step sequence, with normetazocine followed by hydrogenolysis of the benzyloxy-protecting groups, gave N-(2,4,5-trihydroxyphenethyl)normetazocine. This compound was prepared to study the effect of a narcotic analgesic containing a functional group which could be activated in situ to a moiety potentially capable of reacting irreversibly with the narcotic receptor. This 6-hydroxydopamin derivative of normetazocine did not prove to be a useful affinity label. Its low toxicity could indicate the necessity for the formation of an aminochrome system for the expression of toxicity by 6-hydroxydopamine.

Syntheses and adrenergic agonist properties of ring-fluorinated isoproterenols.

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.

[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.

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.

Synthesis and biological properties of 2-, 5-, and 6-fluoronorepinephrines.

2-Fluoro-, 5-fluoro- and 6-fluorodimethoxybenzaldehydes were prepared by photochemical decomposition of the corresponding diazonium fluoroborates. The aldehydes were converted to the cyanohydrin trimethylsilyl ethers, which, in turn, were reduced to the dimethoxyphenethanolamines. Boron tribromide demethylation afforded the racemic ring-fluorinated norepinephrines. An alternate route, using the dibenzyloxyfluoroaldehyde, was also used to prepare 6-fluoronorepinephrine. The fluorine substituent markedly increases the phenolic acidities of these analogues. The biological properties conferred upon norepinephrine by the fluorine substituents in peripheral and central adrenergically responsive systems clearly demonstrate that 2-fluoronorepinephrine is a nearly a pure beta-adrenergic agonist, while 6-fluoronorepinephrine is an alpha-adrenergic agonist. 5-Fluoronorepinephrine retains both beta- and alpha-adrenergic agonist properties. Receptor-binding studies with specific radiolabeled ligands indicate that the specificity conferred by the site of fluorine substituents results from a change in the affinity of these analogues for the alpha- and beta-adrenergic receptors.

Syntheses of 2,5- and 2,6-difluoronorepinephrine, 2,5-difluoroepinephrine, and 2,6-difluorophenylephrine: effect of disubstitution with fluorine on adrenergic activity.

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.

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.

Effect of fluorine substitution on the adrenergic properties of 3-(tert-butylamino)-1-(3,4-dihydroxyphenoxy)-2-propanol.

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.

Induction of catechol-O-methyltransferase in the luminal epithelium of rat uterus by progesterone.

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.

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.

Pumiliotoxin alkaloids: a new class of sodium channel agents.

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.

Studies on catechol-O-methyltransferase in rat brain using two-dimensional gel electrophoresis.

The presence of catechol-O-methyltransferase (COMT) in the rat brain was studied using a combination of two-dimensional gel electrophoresis (2-DE), protein blotting and a specific antiserum. Two major immunoreactive proteins were identified-one with mol. wt 23 kdalton and an isoelectric point of 5.2, the other of mol. wt 25 kdalton and an isoelectric point of 5.1. In addition, multiple lower molecular weight immunoreactive proteins, possibly corresponding to breakdown products of the enzyme, were also detected. The 23 kdalton form of COMT, which is probably the soluble form of the enzyme, is a major protein visible on silver-stained 2-D gels of rat brain. In contrast, the other proteins recognized by the antiserum were not detected by the silver stain. These results demonstrate, using 2-DE, that at least two distinct forms of catechol-O-methyltransferase are present in rat brain. In addition, since one of these proteins is stained by silver, these results also serve to identify another protein visible on 2-D electrophoretograms of rat brain.