Regioselective O-methylation of tetrahydropapaveroline and tetrahydroxyberbine in vivo in rat brain.

Enzymatic O-methylation is a primary pathway for the metabolism of catecholamines in mammals and of isoquinoline alkaloids in plants. This report describes the differential O-methylation patterns of the racemates and enantiomers of two catecholamine-derived alkaloids, tetrahydropapaveroline (THP) and 2,3,10,11-tetrahydroxyberbine (THB), in the brain of the rat. One hour after intracerebroventricular administration of a specific isomeric form of each alkaloid, the O-methylated metabolites were isolated from the rat brain and subsequently quantified using high performance liquid chromatography. The isomeric form of THP or THB which was administered markedly influenced the pattern of O-methylation. The racemate and R-(+)-enantiomer of THP were mono-O-methylated predominantly at the 7 and 3' positions, while the S-(-)-enantiomer of THP was mono-O-methylated to an essentially equal degree at the 6, 7 and 3' positions. Minimal mono-O-methylation at the 4' position was detectable only with the racemate and (-)-enantiomer of THP. The racemate and enantiomers of THB were mono-O-methylated predominantly at the 2 and 11 positions and to a lesser extent at the 3 and 10 positions. Although minimal with the R-(+)-enantiomer, the 3 and the 10-O-methylation pathways were enhanced significantly with the S-(-)-enantiomer of THB. These results demonstrate that both enantiomers of THP and THB are O-methylated in vivo in rat brain and that the chiral centers of these alkaloids influence the position of O-methylation, thereby dictating the relative amounts of specific products formed.

Interaction of catecholamine-derived alkaloids with central neurotransmitter receptors.

Catecholamine-derived alkaloids of the simple tetrahydroisoquinoline, 1-benzyl-tetrahydroisoquinoline and tetrahydroprotoberberine classes have been tested for their ability to inhibit the binding of seven different radioligands to neurotransmitter receptors of brain synaptic membranes. Alkaloids of all three classes were active in inhibiting 3H-clonidine binding to alpha 2-adrenergic receptors. Stereoselectivity of tetrahydropapaveroline in binding to alpha 2-adrenergic receptors was evidenced by the marked activity of the S-(--) isomer (IC50 = 0.65 microM) in comparison to the R-(+) enantiomer (IC50 = 50 microM). The simple tetrahydroisoquinolines (3,4-dihydroxytetrahydroisoquinoline and salsolinol), the four isomeric mono-O-methyl derivatives of 2,3,10,11-tetrahydroxyberbine and tetrahydropapaveroline were the most potent inhibitors of 3H-apomorphine binding to dopaminergic receptor agonist sites. The tetrahydroprotoberberines, as a class, were the most potent inhibitors of 3H-spiroperidol binding to dopaminergic receptor antagonist sites and of 3H-WB-4101 binding to alpha 1-adrenergic receptors. The 1-benzyl-tetrahydroisoquinolines exhibited varying degrees of interaction with beta 1-adrenergic receptors. Tetrahydropapaveroline (IC50 = 0.3 microM) was the most active of the 24 alkaloids tested in inhibiting binding of 3H-dihydroalprenolol to beta 1-adrenergic receptors. None of the alkaloids significantly affected 3H-QNB binding to muscarinic-cholinergic receptors, and selected alkaloids from each class interacted only moderately with serotonergic receptors.

Pumiliotoxin-C and synthetic analogues. A new class of nicotinic antagonists.

Pumiliotoxin-C (PTX-CI), a cis-decahydroquinoline alkaloid, and synthetic analogues inhibited directly and indirectly elicited twitches in rat phrenic nerve diaphragm preparations. At 40 microM the most potent analogue, 2,5-di-n-propyl-cis-decahydroquinoline (PTX-CII), reversibly blocked the indirectly elicited twitch and markedly depressed the amplitude of end-plate potentials and miniature end-plate potentials without affecting muscle membrane potential. PTX-CII did not depress the directly elicited twitch in diaphragm muscle treated with alpha-bungarotoxin. At 40 microM, the compound blocked extrajunctional acetylcholine (ACh) sensitivity in chronically denervated soleus muscle of the rat. The peak amplitude of the end-plate current in frog sartorius muscle was depressed by PTX-CII at both positive and negative membrane potentials. Nonlinearity in the current-voltage relationship appeared at membrane potentials greater than -100 mV with concentrations of PTX-CII from 2.5 to 7.5 microM. The decay phase of the end-plate current was shifted to lower values such that it decayed faster at all membrane potentials but retained voltage sensitivity. PTX-CII, the most potent of the analogues, inhibited binding of [3H]perhydrohistrionicotoxin to Torpedo electroplax membranes with an IC50 value of 1.5 microM in the absence of carbamylcholine (Carb) and 0.1 microM in the presence of Carb. The other analogues were less potent, but all showed higher affinities in the presence of Carb. PTX-CI, PTX-CII, and PTX-CIII had no effect on the binding of [125I]alpha-bungarotoxin to ACh receptors in Torpedo membranes while slightly enhancing the binding of [3H]ACh, whereas PTX-CIV gave slight inhibition of both receptor ligands at concentrations greater than 10 microM. The pumiliotoxin-C class of alkaloids therefore appears to block neuromuscular transmission primarily via interactions with sites associated with the ion channel controlled by the ACh receptor.

Binding of 2',5'-dideoxyadenosine to brain membranes. Comparison to P-site inhibition of adenylate cyclase.

Membranes from rat cerebral cortex and striatum contain a relatively large number of high-affinity binding sites for [3H]2',5'-dideoxyadenosine, [3H]adenine arabinoside, and [3H]adenosine. The binding of [3H]2',5'-dideoxyadenosine and [3H]adenine arabinoside was virtually unaffected by relatively specific agonists and antagonists for adenosine receptors, such as 2-chloroadenosine, N6-phenylisopropyladenosine or theophylline. Binding of [3H]adenosine was partially blocked by such receptor ligands. The specific binding of all three ligands was antagonized by a variety of adenosine analogs which inhibit adenylate cyclase by interaction with the so-called P-site associated with this enzyme. However, potencies of adenosine analogs as P-site inhibitors of adenylate cyclase and as antagonists of binding do not correlate well. 5'-Methylthioadenosine had high potency and efficacy versus binding of [3H]2',5'-dideoxyadenosine but had virtually no effect on activity of adenylate cyclase. 2-Fluoroadenosine was less potent than adenosine as an antagonist of specific binding of [3H]2',5'-dideoxyadenosine, while 2-fluoroderivatives of adenosine, adenine arabinoside and adenine xylofuranoside were more potent than the parent compounds as P-site inhibitors. The significance of the binding sites for [3H]2',5'-dideoxyadenosine remains unclear, but their presence complicates the use of [3H]adenosine and certain analogs as ligands for adenosine membrane sites associated with adenylate cyclase.

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.

Adenosine and cyclic AMP in rat cerebral cortical slices: effects of adenosine uptake inhibitors and adenosine deaminase inhibitors.

The endogenous levels of adenosine functionally linked to cyclic AMP systems in rat cerebral cortical slices are regulated by both adenosine deaminase and adenosine uptake systems. 2'-Deoxycoformycin (2'-DCF), an adenosine deaminase inhibitor, slightly increased basal, adenosine, and norepinephrine-elicited accumulations of cyclic AMP, whereas dipyridamole, an uptake inhibitor, had an even greater effect on cyclic AMP accumulations under the same conditions. Combinations of 2'-DCF and dipyridamole elicited a greater effect than either compound alone. Neither 2'-DCF nor dipyridamole significantly augmented accumulations of cyclic AP elicited by a depolarizing agent, veratridine, suggesting that the adenosine "released" during neuronal depolarization of brain slices is not as subject to inactivation by uptake or deamination as endogenous adenosine in control brain slices. The accumulation of cyclic AMP elicited by a combination of norepinephrine and veratridine was greater than additive. The response to a pure beta-adrenergic agonist, isoproterenol, was not potentiated by 2'-DCF, dipyridamole, or veratridine, consonant with minimal interaction of endogenous adenosine with beta-adrenergic systems.