Jane E. Harris

Uptake of [3H]-catecholamines by homogenates of rat corpus striatum and cerebral cortex: Effects of amphetamine analogues ☆

Abstract The effects of isomers and metabolites of amphetamine on the uptake of [3H]-catecholamines in synaptosomal preparations of the striatum and cortex of the rat were investigated. Dextroamphetamine was found to be four times more potent than the (−)-enantiomer against the uptake of [3H]-dopamine with striatal tissue. This difference may help to clarify the effects of the isomers on the development of stereotyped behaviour in the rat. The p-hydroxylation of amphetamine did not alter its potency, while β-hydroxylation of amphetamine or p-OH-amphetamine reduced the inhibitory potency markedly. With cerebral cortex, less than a 2-fold difference was found for the potencies of (+)- and (−)-amphetamine against the uptake of [3H]-norepinephrine. Although pretreatment with reserpine resulted in a marked inhibition of the initial uptake of norepinephrine, reserpine did not alter the relative potencies of the stereoisomers of amphetamine against the uptake of norepinephrine by cortex.

The uptake of (3H)dopamine by homogenates of rat corpus striatum: effects of cations.

Abstract The uptake of [ 3 H]dopamine was studied with a synaptosomal preparation of the corpus striatum. The accumulation of dopamine was found to be temperature-dependent and very rapid, but linear over time for at least 5 min. at 37°C with characteristics of saturable kinetics. The optimum concentrations for Na + and K + were 150–160 m M and 2.5–4.8 m M , respectively, while uptake was progressively inhibited at concentrations of K + greater than 5 m M . Rubidium was capable of substituting for potassium whereas cesium was a much less effective replacement. The uptake of DA was blocked by the antibiotics, valinomycin and gramicidin-D which bind K + or both Na + and K + , respectively, and thereby might interfere with the transport of cations across neuronal membranes. Similarly, ouabain which blocks the active transport of Na + markedly antagonized the accumulation of DA into striatal homogenates. In contrast, tetrodotoxin which does not prevent the active transport of Na + , had no effect. Uptake appeared not to require Ca ++ and it was not inhibited by increasing total osmolarity to 400 mos M . In general, the cationic requirements for DA-uptake in striatal tissue and its responses to several inhibition of ionic transport, do not appear to be greatly different from those reported for NE with synaptosomes prepared from whole brain.

Amphetamine-induced inhibition of tyrosine hydroxylation in homogenates of rat corpus striatum.

Tyrosine hydroxylase activity was measured in vitro by following the rate of production of 14CO2 from [14C]-DOPA newly formed from L-[1-14C]-tyrosine in a crude synaptosomal preparation of striatal homogenates which contained only endogenous pteridine cofactor. Administration of (+)-amphetamine in vivo (0·50–5·0 mg/kg, i.p.) led to a specific local inhibition of tyrosine hydroxylase activity, after a 15–30 min delay and persisted for at least 4 hr (50% inhibition at 2·5 mg/kg), in nerve endings prepared from the corpus striatum of the rat brain. (−)-Amphetamine was less potent (25% inhibition at 5 mg/kg). There was no change in the level of tyrosine hydroxylase in cell-free extracts and no inhibition of substrate-uptake into nerve endings or change in the level of endogenous tyrosine. When striatal nerve endings were exposed directly to amphetamine in vitro (10 μM), stimulation rather than inhibition of tyrosine hydroxylation occurred, although the hydroxylated metabolites p-hydroxyamphetamine and p-hydroxynorephedrine inhibited the reaction (at 0·1–10 μM). Nevertheless, the inhibitory effect of (+)-amphetamine in vivo persisted in the rat after blockade of microsomal oxidases with SKF-525A (40 mg/kg, i.p.) as well as in the guinea pig, a species in which amphetamine is weakly p-hydroxylated. Interruption of impulse flow in the nigro-striatal pathway by electrothermic lesions was found to decrease tyrosine hydroxylase activity in the striatum, possibly as a result of an increased intraneuronal accumulation of dopamine which could enhance the end-product feedback inhibition of tyrosine hydroxylase. Inhibitory effects of in vivo administration of amphetamine on tyrosine hydroxylase activity in striatal synaptosomes may similarly be mediated by a decreased impulse flow in striatal dopamine neurones. The inhibitory effect of (+)-amphetamine (5 mg/kg, i.p.) was interrupted by blockade of striatal dopamine-receptors with chlorpromazine (10 mg/kg, i.p.) and by blockers of γ-aminobutyric acid-receptors (bicuculline or picrotoxin, 2·5 mg/kg, i.p.). Local striatal receptors of dopamine may thus be involved in this action of amphetamine or descending γ-aminobutyric acid-neurones may mediate a neurophysilogical feedback regulation of activity in the nigro-striatal pathway.

Effects of amphetamines on the metabolism of catecholamines in the rat brain

THE BIZARRE, stereotyped behavior observed after the administration of amphetamines to several mammahan species (RANDRUP and MUNKVAD, 1967r, 1970”) has been attributed to an increase of dopamine (DA) at its receptors, due to an increased release or an inhibition of reuptake of DA in dopaminergic terminals of the basal ganglia and portions of the limbic system (CARLSSON et aZ.,3 COYLE and SNYDER~). The dextrorotatory isomer appears to be at least 2 (TAYLOR and SNYDER~) and possibly four-to-six-times (SCHEEL-Kx~_?GER~) more potent than levoamphetamine in vivo with respect to the development of stereotyped behavior in the rat and this difference in potency remains unexplained. When we studied the uptake of [3H]DA by a synaptosomal preparation of the rat corpus striatum (HARRIS and BALDESSARINI’), the inhibitory concentration (IC,,,) required to produce a 50 per cent reduction of the accumulation of 0.1 PM rH]DA for the (+)-isomer of amphetamine was about four-times less than for the ( -)-isomer in inhibiting the net accumulation of DA (Table 1). Similarly, in kinetic experiments, the apparent inhibitory constants (&) were found to be O-1 and 0.4 PM for (+)- and (-)-amphetamine, respectively. Furthermore, the (+)-isomer of the para-hydroxylated product of amphetamine was about equipotent against DA-uptake with the corresponding (+)-isomer of amphetamine, whereas racemic mixtures of the beta-hydroxylated products of amphetamine and p-OHamphetamine (norephedrine and p-OH-norephedrine, respectively) had much less inhibitory potency than (-J-)-p-OH-amphetamine or (-)-amphetamine (Table 1). In addition, (-)amphetamine, cocaine and (-)-metaraminol were very similar in their abilities to inhibit DA-accumulation. In contrast to the results with striatal tissues, with cortical homogenates, the dextrorotatory isomer of amphetamine was only slightly more potent in inhibiting the uptake of NE (0.5 pM) and DA (0.2 ,uM) than the (-)-enantiomer. By varying the concentration of either the catecholamine or amphetamine, kinetic analysis by two methods indicated that the &-value for levoamphetamine was less than twice that of dextroamphetamine.

β-Adrenergic receptor-sensitive adenosine cyclic 3′,5′-monophosphate accumulation in homogenates of the rat corpus striatum. A comparison with the dopamine receptor-coupled adenylate cyclase☆

Abstract The conversion of newly formed [ 3 H]adenosine triphosphate (ATP) to [ 3 H]adenosine cyclic 3′,5′-monophosphate (cAMP) was studied in osmotically shocked, crude synaptosomal fractions of the rat corpus striatum. Of the β-hydroxylated catecholamines tested, the potency of isoproterenol (Ec 50 about 0.01 μM) was greater than that of norepinephrine (Ec 50 about 1.0 μM). Stereoselectivity was displayed with the (−)isomer of isoproterenol being more potent that its (+)isomer. Of the non-β-hydroxylated catecholamines studied, N -isopropyldopamine demonstrated greater potency than dopamine, whereas apomorphine was inactive. No “additive stimulatory effect” was observed when dopamine was combined with a maximum effective concentration of isoproterenol. The β-adrenergic antagonist propranolol, completely blocked both the dopamine- and norepinephrine-induced increases in cAMP formation. Properties characteristic of a β-type system were, likewise, exhibited by homogenates of both the cerebral cortex and hindbrain. Furthermore, the conversion to [ 3 H]cAMP was increased only slightly (about 16 per cent) by exposure to sodium fluoride, and the stimulatory response to isoproterenol was not altered significantly by high concentrations of ATP but was lost upon sonication of the tissues. In contrast, by assaying adenylate cyclase activity with a high saturating concentration of exogenous [ 3 H]ATP, striatal and cerebral cortical homogenates exhibited responses characteristic of a specific dopamine receptor-coupled adenylate cyclase. Sonication of tissues did not alter the stimulatory effect of dopamine, and sodium fluoride produced about a 2-fold stimulation of the adenylate cyclase activity. Thus, findings in osmotically shocked, crude synaptosomal fractions of the corpus striatum suggest that the paniculate component of adenylate cyclase, which utilizes exogenous ATP as substrate, exhibits properties characteristic of a dopamine receptor-coupled adenylate cyclase. In contrast, the membrane-enclosed, particulate component of adenylate cyclase, which utilizes endogenously synthesized ATP as substrate, conforms to criteria identified with a β-adrenoreceptor-linked adenylate cyclase.

EFFECTS OF AMPHETAMINES ON THE METABOLISM OF CATECHOLAMINES IN THE RAT BRAIN

Publisher Summary The bizarre, stereotyped behavior observed after the administration of amphetamines to several mammalian species has been attributed to an increase of dopamine (DA) at its receptors due to an increased release or an inhibition of reuptake of DA in dopaminergic terminals of the basal ganglia and portions of the limbic system. The dextrorotatory isomer appears to be at least two and possibly four-to-six-times more potent than levoamphetamine in vivo with respect to the development of stereotyped behavior in the rat and this difference in potency remains unexplained. The chapter discusses a study to examine the effects of amphetamines on the metabolism of catecholamines in the rat brain.