J. S. Horng

Kinetics of serotonin accumulation into synaptosomes of rat brain-effects of amphetamine and chloroamphetamines

Abstract Synaptosomes from whole brain of rats accumulated serotonin by means of a high affinity process and a low affinity process, distinguishable kinetically. The high affinity process was stronly inhibited by 4-chloroamphetamine. 3-Chloroamphetamine, 2-chloroamphetamine and amphetamine were progressively weaker inhibitors of that process. 4-Chloroamphetamine was a less potent inhibitor of the low affinity process (about equal to amphetamine). When 4-chloroamphetamine was administered to rats and synaptosomes were isolated from whole brain, the high affinity uptake of serotonin was inhibited for as long as 22·5 hr after drug administration. In vitro, 4-chloroamphetamine but not chloroimipramine at 5 μM caused a release of serotonin from synaptosomes preincubated with radioactive serotonin. The ability of 4-chloroamphetamine to inhibit the high affinity uptake of serotonin by synaptosomes and to release serotonin from synaptosomes is probably related to the prolonged lowering of brain serotonin levels in rats treated with 4-chloroamphetamine.

Effects of antibiotic lonophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria

Abstract A23187, a new antibiotic with ionophore properties, uncoupled oxidative phosphorylation in mitochondria which oxidized either malate plus glutamate or succinate. Ca 2+ , but not Mg 2+ , enhanced the uncoupling effect. Fluorescence of ANS 1 was increased by A23187 suggesting the mitochondrial membranes were de-energized. This de-energization was presumably by activation of the energy-dependent uptake of Ca 2+ . The steady-state measurements of murexide-divalent cation complexes showed that A23187 caused mitochondria to release the accumulated Ca 2+ to the medium. This reduced the transmembrane Ca 2+ gradient even though normal active Ca 2+ uptake could take place. A23187 inhibited activity of ATPase induced by 2,4-dinitrophenol, valinomycin, and Ca 2+ . The addition of Mg 2+ could prevent this inhibition presumably by maintaining the endogenous Mg 2+ concentration. The above metabolic events could be explained by the fact that molecules of A23187 function in the mitochondrial inner membrane as mobile carriers for divalent cations.

Properties of 8,9-dichloro-2,3,4,5-tetrahydro-1H- 2-benzazepine, an inhibitor of norepinephrine N-methyltransferase

Abstract LY134046, 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride, was a potent inhibitor of norepinephrine N -methyltransferase (NMT) from rat brain or rabbit adrenal glands in vitro . The inhibition was competitive with respect to the methyl-accepting substrate, (-)-norepinephrine, the K i for LY134046 being 2.4 × 10 −8 M. LY134046 inhibited the NMT activity in rat brain stem and hypothalamus in vivo at doses of 10–40 mg/kg, i.p., and lowered the epinephrine (but not norepinephrine or dopamine) concentration in these brain regions. The epinephrine reduction produced by a single 40 mg/kg, i.p. dose of LY134046 persisted at 24 hr and daily injections of 10–40 mg/kg doses for 5 days produced cumulative reductions in epinephrine concentration. LY134046 was similar to SK&F 64139 (7,8-dichloro-1,2,3,4-tetrahydroisoquinoline hydrochloride), a structurally related compound, as an inhibitor of NMT in vitro and in vivo , but the two compounds differed in their relative abilities to block α 2 receptors. SK&F 64139 was 20-to 50-fold more potent than LY134046 in antagonizing [ 3 H]clonidine binding to rat brain membranes and phenylephrine-induced contractions of rat aortic strips, but it was only about twice as potent as LY134046 in inhibiting NMT activity. LY134046 seems to be more selective than other currently known inhibitors of NMT and may be useful for pharmacologic intervention in the function of epinephrine-forming neurons in brain.

Interactions of three inotropic agents, ASL-7022, dobutamine and dopamine, with α- and β-adrenoceptors in vitro

SummaryThree inotropic agents, ASL-7022, dobutamine and dopamine, were evaluated for their α-and β-adrenoceptor mediated effects in vitro in a variety of isolated organs and in radioligand binding studies. All compounds were α1-adrenoreceptor agonists in rat and guinea pig aortae, but the rank orders of potency were exactly opposite in these two tissues. Only the rank potency order of dobutamine>ASL-7022>dopamine obtained in rat aorta was consistent with the results obtained in radioligand binding studies to α1-adrenoreceptors in rat cerebral cortex and to previous results obtained in vivo in the pithed rat. The results obtained in guinea pig aorta did not parallel the radioligand binding studies in rat brain or our previous results in pithed rat, and suggests that species differences exist between postsynaptic vascular α1-adrenoreceptors in rat and guinea pig aorta, consistent with previous conclusions. ASL-7022 was found to be a potent α2-adrenoreceptor agonist in field-stimulated guinea pig ileum, and was approximately 10-fold more potent than dobutamine in this respects, which was also confirmed by radioligand binding studies to α2-adrenoreceptors in rat cerebral cortex. The β1-adrenoreceptor mediated effects of these compounds were evaluated in guinea pig atria, where the rank order of potency was dobutamine>ASL-7022>dopamine. An identical rank order of affinity was established for these compounds by displacement of 3H-dihydroalprenolol from β1-adrenoreceptors in rat cerebral cortex. The β1-adrenoreceptor mediated effects of dobutamine and ASL-7022 in guinea pig atria were completely direct in nature and not secondary to the release of endogenous catecholamines. In contrast, a major component of the β1-adrenoreceptor mediated tachycardia produced by dopamine in guinea pig atria was indirect in nature as evidenced by the marked attenuation in potency that occurred following catecholamine depletion with reserpine. All three compounds elicited β2-adrenoreceptor mediated inhibition of tone in rat uterus, with the rank order of potency being ASL-7022>dobutamine>dopamine. Again, this rank order of β2-adrenoreceptor potency was also reflected in β2-adrenoreceptor affinity as assessed by displacement of 3H-dihydroalprenolol from β2-adrenoreceptors in rat cerebellum. Based on these results, it may be concluded that for α-adrenoceptors, dobutamine is a selective β2-adrenoreceptor agonist, ASL-7022 is a selective α2-adrenoreceptor agonist, and dopamine is a nonselective α-adrenoceptor agonist. For β-adrenoceptor mediated effect, ASL-7022 is a selective α2 agonist, while dobutamine and dopamine are nonselective β-adrenoceptor agonists. It is likely that the complex inotropic and hemodynamic activities of ASL-7022, dobutamine and dopamine result from the sum of their individual effects at the α-and β-adrenoceptor subtypes.

Accumulation of amphetamine and p‐chloroamphetamine into synaptosomes of rat brain

p-Chloroamphetamine, but not amphetamine, lowers brain 5-hydroxytryptamine concentrations (Pletscher, Bartholini & others, 1964; Fuller, Hines & Mills, 1965) by either inhibition of its synthesis (Sanders-Bush & Sulser, 1970) or by releasing bound 5-HT (Fuller, 1966; Pletscher, Da Prada & Burkard, 1970). On the other hand, both p-chloroamphetamine and amphetamine interfere with noradrenaline uptake into brain nerve endings (Strada, Sanders-Bush & Sulser, 1970). Perhaps, the distinct difference between the two drugs is their binding ability to the particulate matter of rat brain (Fuller & Hines, 1967). The present report is an extension of our observation in the localization of p-chloroamphetamine and amphetamine in subcellular particles of the rat brain. Groups of four Sprague-Dawley rats (150-180 g) were separately treated with equimolar doses of amphetamine (1 8 mg/kg) and p-chloroamphetamine (24 mg/kg) and were decapitated at 1 h. A crude mitochondria1 preparation was obtained from a 30 % brain homogenate in 0 . 3 2 ~ sucrose after stepwise differential centrifugation of 1085 g (10 min) and 17 300 g (20 min). It was further fractionated by centrifugation in a discontinuous Ficoll gradient (Kurokawa, Sakamoto & Kato, 1965). A microsomal fraction was isolated from the post-mitochondria1 supernatant fraction after centrifugation at 100 000 g (60 min). The subcellular distribution of the two drugs is shown in Table 1. Values in the first two columns show the relative amounts in percentage of drug in the 1085g supernatant fraction. Most of the amphetamine (77%) was found in the postmitochondrial supernatant fraction; 62.5 % of the amphetamine was associated with the high-speed supernatant fraction. After the crude mitochondrial fraction was subjected to centrifugation in a discontinuous Ficoll gradient, four sub-fractions resulted including a supernatant fraction, myelin, synaptosomes and mitochondria. The particulate fractions, except mitochondria, contained 6 to 8 % amphetamine. The particles sedimented further away from the soluble fraction contained less drug per mg protein. A total of 31.5 % (7.9 % + 23.6 %) of the amphetamine was particle-bound.

α2 andrenoreceptor affinity of some inhibitors of norepinephrine N-methyltransferase

Because certain inhibitors of norepinephrine N-methyltransferase (NMT, the epinephrine-forming enzyme) are α2 adrenoreceptor blockers, we compared the ability of various compounds to inhibit rat brain NMT activity and the binding of tritiated clonidine to rat brain membranes in vitro. There was no correlation between potency of NMT inhibition and potency of α2 receptor antagonism (as measured by inhibition of tritiated clonidine binding) among NMT inhibitors representing several chemical classes or among members of a single class of compounds, l-aminoindans. In addition, several potent α2 blocking drugs were essentially inactive as NMT inhibitors. These findings indicate that NMT inhibition and α2 blockade are dissociable activities. Future development of NMT inhibitors should include this dissociation as a goal to increase the usefulness of NMT inhibitors as pharmacologic tools.

Interactions of the enantiomers of 3-O-methyldobutamine with α- and β-adrenoceptors in vitro

SummaryThe enantiomers of 3-O-methyldobutamine, a metabolite of dobutamine, were evaluated for their α- and β-adrenoceptor mediated effects in vitro in a variety of isolated organs and in radioligand binding studies. Neither enantiomer of 3-O-methyldobutamine possessed α1-adrenoceptor agonist activity in isolated guinea pig aorta. However, both enantiomers of 3-O-methyldobutamine were competitive α1-adrenoceptor antagonists, with the (+)-enantiomer being approximately 10-fold more potent than the (-)-enantiomer as assessed either in guinea pig aorta or by displacement of 3H-prazosin binding from α1-adrenoceptors in rat cerebral cortex. The α1-adrenoceptor blocking activity of (+)-3-O-methyldobutamine was relatively potent and corresponded to a pA2 of 7.33 in guinea pig aorta and a-log Ki of 7.72 in radioligand binding studies. Neither enantiomer of 3-O-methyldobutamine possessed α2-adrenoceptor agonist activity in field-stimulated guinea pig ileum. Although (+)-3-O-methyldobutamine weakly inhibited the twitch response in field-stimulated guinea pig ileum, the response was not blocked by the selective α2-adrenoceptor antagonist, yohimbine, and was found to result from weak anticholinergic activity (pA2=5.06). Neither enantiomer of 3-O-methyldobutamine possessed β1-adrenoceptor agonist activity in guinea pig atria, however the (+)-enantiomer was a weak noncompetitive antagonist at β1-adrenoceptors. In contrast, both enantiomers of 3-O-methyldobutamine were weak β2-adrenoceptor agonists in rat uterus, however these weak effects were not highly stereoselective, which was also confirmed in radioligand binding studies. The results of the present study indicate that 3-O-methyldobutamine is a potent and highly selective α1-adrenoceptor antagonist, with minimal activity at α2-, β1- and β2-adrenoceptors. It is hypothesized that the potent α1-adrenoceptor antagonist activity of 3-O-methyldobutamine, which resides predominantly in the (+)-enantiomer, may play a role in the hemodynamic effects of dobutamine, by contributing, in part, to the decrease in total peripheral vascular resistance observed following administration of dobutamine.

Demonstration of two mitochondrial populations by rate zonal centrifugation

Abstract A bimodal distribution of rat liver mitochondria was observed after rate zonal centrifugation in a Ficoll gradient. Addition of succinate and ATP to the gradient increased the sedimentation coefficient of both mitochondrial populations but did not affect the sedimentation of lysosomes. An uncoupler of oxidative phophorylation, carbonyl cyanide m-chlorophenylhydrazone, reversed the effect of succinate.

Robenzidene, an inhibitor of oxidative phosphorylation.

Robenzidene at 20 μM, inhibits the ADP stimulated respiration of intact rat liver mitochondria and induces respiratory control in submitochondrial fragments. At concentrations of the order of 16 nmole per mg protein, it also inhibits the Cl-CCP induced ATPase of mitochondria and ATPase of mitochondrial fragments. These properties of robenzidene resemble those of other inhibitors of oxidative phosphorylation such as oligomycin and DCCD.

Alpha-2-Adrenoceptor-Mediated Effects of Pergolide

The alpha-adrenoceptor-mediated effects of pergolide were investigated in a number of biochemical and pharmacological test systems. In radioligand binding studies, pergolide more effectively competed for alpha 2-adrenoceptors than for alpha 1-adrenoceptors in membranes prepared from rat cerebral cortex. Consistent with this finding was the observation that pergolide was several orders of magnitude more effective in activating presynaptic alpha 2-adrenoceptors in rat vas deferens to inhibit stimulation-evoked [3H]-norepinephrine release than in activating postsynaptic alpha 1-adrenoceptors in the same tissue to produce a contractile response. Pergolide elicited a potent vasopressor response in pithed rats that was highly sensitive to antagonism by the selective alpha 2-adrenoceptor antagonist, yohimbine, and resistant to blockade by the selective alpha 1-adrenoceptor antagonist, prazosin, suggesting that pergolide selectively activates postsynaptic vascular alpha 2-adrenoceptors. The results of the present study indicate that the alpha-adrenoceptor-mediated effects commonly associated with pergolide result from selective stimulation of alpha 2-adrenoceptors.