Kenneth G. Walton

Prolonged apomorphine-like behavioural effects of apomorphine esters☆

Abstract In vitro and in vivo activities of a series of O , O ′-diesters of apomorphine, including diacetyl-, dipropionyl-, diisobutyryl-, dipivaloyl-, and dibenzoylapomorphine, were evaluated in the rat. All esters provoked stereotyped gnawing behaviour in intact rats, as well as rotation in a direction contralateral to unilateral electrolytic lesions in the nigrostriatal pathway. The esters were somewhat less potent than apomorphine in inducing these behavioural responses, and had a delayed latency to maximal effect as well as a prolonged duration of action. Prolongation of the behavioural effects of the esters increased with the size of the ester substituent, the dibenzoyl ester being 5 or 6 times longer-acting than apomorphine at high doses. Acute intraperitoneal doses of all esters, up to 100 mg/kg, were well tolerated. The behavioural activity of the esters after intragastric administration was poor. None of these esters increased the synthesis of cyclic AMP in striatal homogenates exhibiting adenylate cyclase activity stimulated by apomorphine or dopamine, and the smaller esters appeared to inhibit stimulation by dopamine. We conclude that the desirable prolongation of action of labile esteric derivatives of apomorphine is probably due to their increased lipophilic properties or to slow hydrolysis of the larger esters that converts the depot “pro-drugs” to the probable active product, apomorphine. This characteristic of esteric derivatives of apomorphine may provide a strategy for the design of improved, clinically useful agents that interact with central dopamine receptors.

Inhibition of dopamine-stimulated adenylate cyclase activity by phenoxybenzamine☆

Phenoxybenzamine at a concentration of 2 X 10(-6 M) produced half-maximal inhibition of dopamine-stimulated adenylate cyclase activity in homogenates of rat striatum. Inhibition was sharply dependent on time and temperature of preincubation with the inhibitor. When included in the preincubation medium, dopamine was nearly 8 times more effective than norepinephrine at protecting against this inhibition, whereas neither isoproterenol nor methoxamine appeared to protect at all. These results suggest a direct, and possibly irreversible, interaction of phenoxybenzamine with the dopamine-binding component of the adenylate cyclase.

Effects of Mn2+ and other divalent cations on adenylate cyclase activity in rat brain.

Abstract— Mn2+ caused an 8‐to 16‐fold stimulation of adenylate cyclase activity in homogenates as well as synaptosomcs. isolated synaptic membranes, and slices prepared from rat brain. The stimulation occurred at low concentrations of Mn2+. with a doubling of activity at 50‐60μM. and was unaffected by a 60‐fold excess of Mg2+. Whether or not Mg2+ was added, inclusion of a low concentration of Mn2+ reduced, but did not prevent the stimulation of adenylate cyclase caused by dopaminc in homogenates of corpus striatum. In contrast, Ca2+. at a concentration that had little effect on basal cyclase activity, completely prevented the stimulation by dopamine. The increase of cyclase activity produced by Mn2+ in brain homogenates was potentiated by F−. Other ions, notably Hg2+. Pb2+. Cu2+ and Zn2+. in order of decreasing potency, inhibited both basal and Mn2‐‐stimulated cyclase activity. It is proposed that the effect of Mn2+ on adenylate cyclase activity may involve only the catalytic subunit of the enzyme, and that the mechanism is different from that by which either dopamine or F− stimulates the enzyme. These results suggest that the effects of low concentrations of Mn2+ and certain other divalent metal ions on adenylate cyclase activity may be involved in their neuropsychiatrie or other toxic effects, and that such ions may also participate in normal physiological mechanisms involving cyclic nucleotides.

Behavioral effects of apomorphine and diisobutyrylapomorphine in the mouse

Stereotyped climbing and clinging responses of the mouse to apomorphine or its ester prodrug, O,O′-diisobutyrylapomorphine were evaluated. Acute doses of the ester up to 0.3 mmoles/kg were tolerated without apparent ill effects. Both aporphines produced dose-dependent behavioral responses that were blocked by neuroleptics. The duration of action of the ester was much greater than that of apomorphine. When maximal initial behavior during the first hour was evaluated, low equimolar doses of apomorphine and the ester were similar in potency; in contrast, the total behavioral response to larger doses of the ester was greater than to apomorphine, evidently reflecting the greater duration of action of the ester. Behavioral responses to both agents during the first hour decreased at doses above 0.1 mmoles/kg. Oxidized or O-methylated apomorphine did not antagonize the behavioral effects of apomorphine. Systemic injection of apomorphine or diisobutyrylapomorphine led to detectable levels of free apomorphine as estimated by a sensitive and selective fluorimetric assay. The time-course and magnitude of the behavioral effects of both agents corresponded closely with brain levels of apomorphine. Apomorphine and dopamine (but not diisobutyrylapomorphine) stimulated adenylate cyclase activity in mouse striatal homogenates—an effect antagonized by neuroleptic drugs but not propranolol. Apomorphine exerted a biphasic effect on the cyclase in vitro and increased cyclic AMP levels in the striatum in vivo. The prolonged activity of apomorphine esters as depot prodrug agonists of putative dopaminergic mechanisms in the brain may provide clinically desirable characteristics.

Hydrolysis of diester prodrugs of apomorphine.

Abstract O,O′-diesters of apomorphine exert behavioral effects identical to those of apomorphine, but prolonged in proportion to the bulk of the esters. This prolonged activity may reflect depot properties of the esters, and/or decreasing rates of hydrolysis to the presumably active metabolite, apomorphine. To test the latter hypothesis, mouse brain apomorphine was assayed by a fluorimetric method that can distinguish apomorphine from its monophenolic analogues and was confirmed by thin-layer chromatography. Apomorphine fluorescence was recovered after systemic injection of di-isobutyrylapomorphine as well as after apomorphine, but its half-life in brain was greatly prolonged after administration of the ester. Ester hydrolysis also occurred in vitro with minces or homogenates of rat tissue, yielding a product with the fluorescent and Chromatographic properties of free apomorphine which also stimulated the activity of a dopamine-sensitive adenylate cyclase in rat striatal homogenates, sensitive to apomorphine but not its esters. Most of the activity resided in the high-speed supernatant fraction of liver; high esterase activity was found in rat plasma and was present in most tissues. This reaction was dependent on temperature, tissue and substrate concentration, followed saturable kinetics, and was inhibited by triorthocresylphosphate, an esterase inhibitor. This apparent enzymatic reaction proceeded at rates varying inversely with the size of the ester substituent. These results support the suggestion that the rate of hydrolysis may be related to desirable prolongation of action of ester prodrugs of apomorphine.

Esters of apomorphine and N,N-dimethyldopamine as agonists of dopamine receptors in the rat brain in vivo.

Abstract Subcutaneous injections of O,O-diacetylapomorphine, at doses of 0.1 to 10.0 mg kg , produced stereotyped gnawing behaviour in the rat, indistinguishable from that induced by apomorphine. The dose-response relationship and time course of this effect were similar for the two drugs, although the ester appeared to be somewhat more potent and longer-lasting at higher doses. While apomorphine had no behavioural effects when given orally at doses up to 100 mg kg , diacetylapomorphine produced discernible stereotypy at oral doses as low as 10 mg kg . The injection of diacetylapomorphine into animals previously lesioned electrothermally in the left nigro-striatal tract provoked turning behaviour toward the side contralateral to the lesions, with an effectiveness similar to that of apomorphine. Apomorphine, but not diacetylapomorphine, stimulated the production of cyclic AMP when incubated with homogenates of corpus striatum. In analogous experiments, N,N-dimethyldopamine, but not its ester, O,O-diacetyl-N,N-dimethyldopamine (another apparent agonist of central dopamine receptors in vivo), also stimulated the production of cyclic AMP in homogenates, and a similar failure to stimulate the production of cyclic AMP was obtained with O,O-dibenzylapomorphine, which had prolonged behavioural effects in vivo. These results are compatible with the conclusion that catechol esters of certain structural analogues of dopamine can be hydrolysed in vivo to yield free catechols capable of stimulating central dopamine receptors.

Prenatal and early postnatal β-adrenergic receptor-mediated increase of cyclic AMP in slices of rat brain

The levels of cyclic AMP in slices of cerebral cortex and cerebellum from newborn rats were significantly, but transiently, increased by exposure to the beta-adrenergic agonist, isoproterenol. Isobutylmethyxanthine, an inhibitor of phosphodiesterase, enhanced this effect and permitted its detection in cerebral cortex obtained from the prenatal rat. These results are consistent with the possibilities that functional noradrenergic synapses are formed early in the ontogeny of the CNS, and that norepinephrine may exert cyclic AMP-mediated influences on brain development.

Effects of isoproterenol on the development of β-adrenergic receptors in brain cell aggregates

The development of binding sites for the beta-adrenergic receptor antagonist [125I]iodohydroxybenzylpindolol in reaggregate cultures of fetal rat brain has been investigated. beta-Adrenergic receptor density increased rapidly between 6 and 22 days in culture at which time maximal density was attained. When cultures were grown in the presence of the beta-adrenergic receptor agonist isoproterenol, the development of receptors was markedly inhibited. Cultures were also grown for 15 days in the presence of isoproterenol and then for 7-14 days in the absence of isoproterenol. Following the removal of the agonist, binding sites developed, but to only approximately 50% of the density seen in control cultures.

The noradrenergic system in cultured aggregates of fetal rat brain cells: morphology of the aggregates and pharmacological indices of noradrenergic neurons.

Spherical aggregates formed rapidly in culture by re-aggregation of trypsin-dissociated brain cells from the 17-day-old fetal rat. Over about 10 days in initially random distribution of cells evolved into a 3-layered arrangement; cells with characteristics of neurons were found largely in the intermediate layer. The survival of neuronal and glial cell types was evaluated histologically and verified by electron microscopy, which revealed synaptic and myelin structures that rapidly increased in number after 18 days in culture. Levels of norepinephrine (NE) and dopamine (DA) reached peaks of 9.5 and 4.4 ng/mg protein, respectively, at culture day 21. Uptake of [3H]NE paralleled these amine levels and was blocked by desipramine or pretreatment with either reserpine or 6-OH-DA. Autoradiographs of aggregates labeled with [3H]NE showed a high density of silver grains over cells, apparently neurons, with branching processes traced for 120 micrometer. Previously accumulated [3H]NE was released under depolarizing conditions (high [K+] or vertridine) only in the presence of Ca2+. Release was induced to a lesser extent by kainic greater than glutamic acid. Thus, such aggregates appear to contain catecholaminergic neurons capable of synthesis, uptake and release of NE. The time course of development of these functions supports suggestions that aggregate preparations might be useful in studying neurochemical or morphological aspects of brain development and function in vitro.

Effects of alternative transmitter amines on cyclic AMP formation in rat brain tissue.

Aromatic amines and related compounds, some of which are taken up and released from nerve terminals, might act at brain receptors ordinarily stimulated by traditional amine neurotransmitters. Several of these compounds were evaluated for their ability to stimulate or impede synthesis of cyclic AMP in rat striatal homogenates and cortical slices. In contrast to catecholamines, most had no effect, consistent with their possible role as false transmitters.