George Gessner

Studies on the characterization and inhibition of rat brain phenylethanolamine N-methyltransferase

SummaryThe existence of phenylethanolamine N-methyltransferase (PNMT) activity in the rat brain and spinal cord was confirmed using both substrate specificity and selective inhibitors of the adrenal enzyme as biochemical tools. The enzyme was not generally localized throughout the central nervous system but was found primarily in the brain stem and spinal cord with lesser amounts occurring in the midbrain. No significant PNMT activity was found in the cerebellum or forebrain. The central enzyme was markedly inhibited both in vitro and in vivo by low concentrations (doses) of SK&F 64139, a potent inhibitor of the adrenal enzyme.

Binding of a novel dopaminergic agonist radioligand [3H]-fenoldopam1 (SKF 82526) to D-1 receptors in rat striatum

Fenoldopam (SKF 82526), a dopamine agonist which exhibits D-1 receptor subtype selectivity, was evaluated as a radioligand for this receptor subtype. In saturation studies in rat striatal membrane preparations, [3H]-fenoldopam appeared to label a single binding site with a KD of 2.3 +/- 0.1 nM and a Bmax of 590 +/- 40 fmoles/mg protein. In competition binding experiments, binding was shown to be stereoselective, and rank ordering of affinities of dopaminergic and non-dopaminergic compounds closely correlated with potencies of these compounds in stimulating or inhibiting dopamine-sensitive adenylate cyclase (D-1) and in binding to D-1 sites labelled with the antagonist [3H]-cis-flupenthixol. The most potent competitors were the recently identified D-1 selective antagonists, SCH 23390 and SKF R-83566. [3H]-Fenoldopam was also used to assess agonist/D-1 receptor interactions. The results suggest that [3H]-fenoldopam is a useful and selective agonist radioligand for the D-1 receptor.

Regulation of agonist and antagonist binding to striatal D-1 dopamine receptors: studies using the selective D-1 antagonist [3H]SK&F R-83566

The potent and D-1 versus D-2 selective dopamine receptor antagonist, SK&F R-83566, was radiolabelled with tritium and was used as a radioligand for examination of D-1 receptors in rat striatum. Binding of the radioligand was stereoselective, saturable and reversible. In homogenates of rat striatum, nonspecific binding of the radioligand was less than 5% of total binding, the KD was 1.1 +/- 0.2 nM and the Bmax was 1130 +/- 70 fmoles/mg protein. Results of competition binding analyses yielded a pharmacological profile that was characteristic of dopamine D-1 receptor interaction. Competition studies of dopamine agonists against the potent antagonist radioligand indicated multiple affinities of agonist binding to the D-1 receptor. Displacement was best fit to a two-site model of ligand binding and high and low affinities were subject to regulation by guanine, but not adenine, nucleotides. Antagonist binding was not complex and was unaffected by guanine nucleotides. The role of monovalent cations in regulating D-1 receptor binding was evaluated by comparing effects of Na+, Li+, and K+ on binding of the antagonist [3H]SK&F R-83566 and the agonist [3H]fenoldopam (SK&F 82526). Whereas agonist binding was reduced in a concentration dependent fashion by monovalent cations with a ranking of potency Li+ greater than Na+ greater than K+, antagonist binding was enhanced by the cation Na+ but little affected by Li+ or K+. This effect of relatively low concentrations of Na+ to decrease agonist binding and increase antagonist binding suggests similarities between the D-1 receptor which is positively-coupled to adenylate cyclase and other receptors, e.g. alpha 2 adrenergic receptors, which are negatively-coupled to adenylate cyclase.

The effects of an inhibitor of phenylethanolamine N-methyltransferase upon stimulated adrenal catecholamine release and excretion in the rat

SummarySK&F 64139, an inhibitor of adrenal phenylethanolamine N-methyltransferase (PNMT), was found to significantly decrease 2-deoxy-d-glucose (2-DG) induced epinephrine excretion in the conscious rat under conditions where the former agent was administered chronically at 50 and 200 mg/kg/day over a 12-day period and 2-DG was administered after 3, 7 and 11 days of treatment. The reduced epinephrine output caused by SK&F 64139 in response to 2-DG was accompanied by an increased norepinephrine excretion pattern at 200 mg/kg/day of the compound. The reductions in epinephrine excretion were also associated with significant decreases in adrenal epinephrine and increases in the norepinephrine content.

Comparison of the effects of SK & F 29661 and 64139 upon adrenal and cardiac catecholamines

After 7 day dosing with SK & F 64139, an inhibitor of adrenal and CNS PNMT, a stoichiometric relationship is observed between epinephrine decrease and norepinephrine increase in adrenal tissue. However, this relationship is not found with the adrenal PNMT inhibitor, SK & F 29661. SK & F 64139 was found to possess significant adrenal MAO inhibitory activity, but SK & F 29661 does not. Three factors are presented which argue against MAO inhibition as the cause of the catecholamine profile difference. We then performed various studies with SK & F 29661 and 64139 to examine whether enzymes in the norepinephrine biosynthetic pathway are differentially affected by these drugs and found that they were not. However. when rats were dosed with either SK & F 29661 or 64139 over 24 h and the adrenal catecholamine stores were then radioactively labeled with a tracer dose of 3H-norepinephrine, the rate of 3H-norepinephrine disappearance was reduced in the SK & F 64139-treated animals as compared with those given SK & F 29661. A similar result was observed in the heart. These results suggest that SK & F 64139 may reduce norepinephrine turnover in the adrenal gland and heart via an effect not related to peripheral PNMT inhibition. These findings also demonstrate quite clearly that in intact animals all PNMT inhibitors do not produce identical endogenous catecholamine profiles.

Effect of triamterene on folic reductase activity and reproduction in the rat

Abstract Triamterene has been found to inhibit the activity of folic acid reductase from murine leukemic cells by 50% at approximately 8 × 10−7 M. The inhibition due to triamterene was competitive with folic acid at low inhibitor concentrations. At all concentrations, the inhibition obtained with triamterene was either partially or completely reversed by increasing the folic acid concentration. At folic acid concentrations above 10−4 M, addition of substrate inhibited folic acid reductase activity. Competition of 9-methylfolic acid and folic acid for folic reductase could not be demonstrated, since 9-methylfolic acid itself is a substrate for murine leukemic folic reductase. Under the experimental conditions employed for the study of reproduction, 9-methylfolic acid administered to rats at varying doses on days 8 and 9 after conception resulted in resorptions (67%), a reduction in live-birth index, and the birth of young with congenital defects including short tail, hydrocephalus, gastroschises with ectopia of the abdominal wall, unilateral and bilateral microphthalmia, and anophthalmia. In contrast, triamterene caused no significant changes in any of the parameters monitored. No physical abnormalities were noted in the progeny, clearly demonstrating that triamterene has no significant antifolic activity in vivo even under experimental conditions sensitive to this liability.

Studies on lung N-methyltransferases, a pharmacological approach.

SummaryPhenylethanolamine N-methyltransferase (PNMT) was identified as the primary, high affinity N-methylating enzyme for phenylethanolamine (PEA) in rat, dog and Rhesus monkey lung. Human and rabbit lung, however, do not contain this enzyme but possess a more non-specific or general N-methyltransferase with a relatively low affinity for PEA and for which β-phenethylamine (β-PE) is also a substrate. The former but not the latter enzyme is markedly inhibited by micromolar concentrations of the PNMT antagonist, SK & F 64139. This evidence indicates that certain species differences exist for the enzyme system(s) available for the N-methylation of phenethylamine-type compounds in pulmonary tissue.

The long term effects of an inhibitor of phenylethanolamine N-methyltransferase upon adrenal epinephrine biosynthesis

SummaryChronic administration of SK & F 64139, an inhibitor of phenylethanolamine N-methyltransferase (PNMT), initially resulted in a lowered adrenal epinephrine content in both the rat and squirrel monkey adrenal gland. With continued dosing, however, these levels returned toward control. The latter changes were accompanied by increased adrenal levels of norepinephrine and PNMT, but not by decreased plasma drug levels. These results suggest that long-term pharmacological PNMT inhibition may evoke compensatory mechanisms to maintain adrenal epinephrine biosynthesis under basal laboratory conditions.

Studies on the long term effects of SK&F 29661 upon adrenal catecholamines.

SummarySK&F 29661 (1,2,3,4-tetrahydro-7-isoquinoline-sulfonamide) is a potent and selective in vitro and in vivo inhibitor of adrenal phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28). Its Ki value for in vitro inhibition of rat adrenal PNMT was 133 nM. In vivo, the adrenal conversion of 3H-norepinephrine to 3H-epinephrine was maximally inhibited by a single oral dose of 100 mg/kg. In long term chronic studies in rats, adrenal epinephrine was reduced by SK&F 29661 in a dose dependent fashion to greater than 90%, without substantial increases in norepinephrine. Urinary excretion of epinephrine was significantly reduced by the drug both basally and following 2-deoxy-d-glucose stimulation. No drug related changes were found in plasma corticosterone values and only small effects were observed on adrenal tyrosine hydroxylase and PNMT enzyme levels. The cardiac norepinephrine pool and its turnover time were both significantly reduced; its turnover rate, however, was only slightly increased. Our studies indicate that SK&F 29661 is a highly effective, non-toxic and novel pharmacological tool which is useful in depleting adrenal epinephrine stores via inhibition of its biosynthesis.