T.R.A. Macedo

Toxic Effects of Opioid and Stimulant Drugs on Undifferentiated PC12 Cells

Abstract: Cell death and reactive oxygen species production have been suggested to be involved in neurodegeneration induced by the drugs of abuse. In this study we analyze the toxicity of the following drugs of abuse: heroin, morphine, d‐amphetamine, and cocaine in undifferentiated PC12 cells, used as dopaminergic neuronal models. Our data show that opioid drugs (heroin and morphine) are more toxic than stimulant drugs (d‐amphetamine and cocaine). Toxic effects induced by heroin are associated with a decrease in intracellular dopamine, an increase in DOPAC levels, and the formation of ROS, whereas toxic effects induced by amphetamine are associated with a decrease in intracellular dopamine and in ATP/ADP levels. In contrast with cocaine, both amphetamine and heroin induced features of apoptosis. The data suggest that the death of cultured PC12 cells induced by the drugs of abuse is correlated with a decrease in intracellular dopamine levels, which can be associated with an increased dopamine turnover and oxidative cell injury.

Electrochemical oxidation of mitoxantrone at a glassy carbon electrode

Mitoxantrone is an anthracycline used as an antitumour antibiotic for leukaemia and breast cancer treatment, due to its interaction with DNA. However, the molecular mechanism of the antitumour action is not completely understood. Using a glassy carbon electrode the electrochemical oxidation of mitoxantrone was shown to be a complex, pH-dependent, irreversible electrode process involving several metabolites. Comparison of the electrochemical oxidation behaviour of mitoxantrone, ametantrone and aminantrone enabled a deeper understanding of the mechanism and showed the relevance of electrochemical data for the understanding of the cytotoxicity of mitoxantrone. Since mitoxantrone and its oxidation products adsorb strongly on the electrode surface, causing severe problems of electrode fouling, reproducible electroanalytical determinations could only be done at very low concentrations and in an aqueous buffer supporting electrolyte containing 30% ethanol. The detection limit obtained was 10 ˇ7 M. # 1999 Elsevier Science B.V. All rights reserved.

Catecholamine and MHPG plasma levels, platelet MAO activity, and3H-imipramine binding in heroin and cocaine addicts

AbstractThis work evaluated in a population of heroin and heroin plus cocaine human addicts:1.Norepinephrine (NE), epinephrine (Epi), and 3-methoxy-4-hydroxyphenylglycol (MHPG) (the principal metabolite of brain NE) plasma levels;2.Monoamine oxidase (MAO) activity; and3.3H-imipramine specific binding to the amine carrier in platelets. NE plasma levels were significantly lower in the short-term heroin user groups (1–3 and 4–6 yr), a finding not observed in both the long-term heroin user (>6 yr) and heroin plus cocaine user (>6 yr) groups. Epi levels changed in a similar manner, except that a significant increase was noted in heroin plus cocaine abusers. Conversely, dopamine and MHPG plasma levels increased with the duration of heroin use, and even more with cocaine abuse. Platelet MAO activity increased in all groups. Specific3H-imipramine binding sites showed an increase after 3 yr of heroin abuse and in all heroin plus cocaine addicts. In conclusion, short-term use of heroin decreases NE or Epi release, but with prolonged use, a slow adpatation occurs. In contrast, cocaine inhibits the neuronal Epi uptake, even in a situation of long duration of abuse. Probably the amine levels additionally regulate the amine carrier, resulting in changes that show a different pattern from major depression. These drugs of abuse may also influence directly or indirectly related enzymatic systems.

Regulation of intracellular [Ca2+] and GABA release by presynaptic GABAB receptors in rat cerebrocortical synaptosomes

In this study we determined the changes in the intracellular free Ca2+ concentration, associated with the inhibitory modulation of the exocytotic release of GABA by GABAB receptor activation in rat cerebrocortical synaptosomes. We observed that SK&F 97541 and (-)baclofen both act as agonists of the presynaptic GABAB receptors in modulating GABA release and Ca2+ influx due to KCl (10 mM) depolarization, but SK&F 97541 is more potent than (-)baclofen in modulating both Ca2+ influx and GABA release. Thus, activation of GABAB receptors by either SK&F97541 (10 microM) or by (-)baclofen (100 microM) caused about 18% inhibition of the increase in [Ca2+]i, due to KCl depolarization, and inhibited the [3H]GABA release by about 30%. The pharmacological similarities of the GABAB receptor activation in producing inhibition of both calcium channel mediated influx of Ca2+ and transmitter release suggest that presynaptic inhibition of GABA release by GABAB receptor activation may result, at least in part, from inhibition of Ca2+ influx through P-type (or possibly Q-type) Ca2+ channels, sensitive to omega-Agatoxin IVA (200 nM). Furthermore, modulation of GABA release of GABAB receptors was abolished by preincubation with pertussis toxin, suggesting that a pertussis toxin sensitive G protein may be the coupling factor between GABAB receptors and the voltage-dependent Ca2+ channels associated with the exocytotic release of GABA in rat cerebrocortical nerve terminals.

Pharmacological characterization of the postsynaptic α-adrenoceptors in human uterine artery*

Prazosin and yohimbine were used to differentiate postjunctional α‐adrenoceptors in the human uterine artery in‐vitro. Two postjunctional α‐adrenoceptor subtypes were distinguished by the affinities of the receptor for yohimbine and prazosin. The pA2 for prazosin was 8.91 against phenylephrine with a slope not significantly different from unity (0.91), and the pA2 for yohimbine was 7.25 against naphazoline and 8.70 against clonidine, with slopes not significantly different from unity (1.11 and 1.18, respectively). Yohimbine was not very active against phenylephrine, while prazosin was very active against the mixed and selective α2‐adrenoceptor agonist noradrenaline and clonidine; the intercepts of the Schild plot were 8.80 and 8.82 but with slopes significantly less than unity (0.77 and 0.67, respectively). Prazosin competitively antagonized phenylephrine at the α1‐adrenoceptor, whereas yohimbine competitively antagonized naphazoline and clonidine at the α2‐adrenoceptor. It is concluded that both α1‐ and α2‐adrenoceptors are present in the human uterine artery.

Pharmacological Characterization of the Postsynaptic Serotonergic Receptor in the Human Uterine Artery

Serotonergic receptors are involved in many vascular functions, but only a few studies have been made in human vessels. Thus, this study aimed to characterize these receptors in the human uterine artery without endothelium. The pD2 value of serotonin (5-HT) was 5.96, but the intrinsic activity was 59% of that of noradrenaline. Spiperone and ketanserin shifted the concentration-response curves of 5-HT to the right (pA2 = 8.56 and 9.76; slopes = 0.98 and 0.83, respectively). Propranolol, yohimbine, prazosin and atropine did not significantly shift the concentration-response curves to 5-HT. Phentolamine inhibited the 5-HT response (pA2 = 6.69), and previous treatment of the vascular strips with 6-hydroxydopamine only partially reduced such an effect. The results demonstrate the existence, in the human uterine artery, of 5-HT2 receptors which are blocked by high concentrations of phentolamine. In this tissue, 5-HT does not release noradrenaline from perivascular nerves.

Effects of neuropeptides on the sumatriptan-disturbed circulation in the optic nerve head of rabbits.

The purpose of this work was to study ‘in vivo’ the vascular responses of retinal vessels of New Zealand white rabbits to substance P (SP), neurokinin A (NKA), neurokinin B (NKB), senktide, capsaicin (CAPS), and calcitonin gene related peptide (CGRP) before and after selective antagonist administration. We examined the effects of these neuropeptides on the normal circulation in the optic nerve head of the rabbit. Drugs were injected via pars plana through a micropipette system. Ten minutes before perivascular injection of 10 nmol/l sumatriptan (to contract the vessel), a selective antagonist or its solvent was administered. Then, cumulative injection of the agonist was performed. The other eye was used as control. Direct measurement of retinal arteriole diameters was performed using digital angiography. The quantification of the relaxing effect is expressed as percentage related to the precontracted vascular diameter. Microinjection of SP (NK1 receptor agonist) up to 10 nmol/l induced a dose-dependent arteriolar dilating effect [Emax (mean ± SEM) 21.3 ± 2.3%]. After the perivascular preinjection of 1 nmol/l L-668,169 or 1 nmol/l L-733,060 (NK1 receptor antagonists), the SP dose-response curve was shifted to the right. The same results were obtained with NKA (NK2 receptor agonist) which induced the most potent effect of all neuropeptides (Emax 53.3±2.5%). The NK2 receptor antagonists L-659,877 and GR 159897 (1 nmol/l) strongly inhibited this arteriolar vasodilation. As for CGRP, doses up to 10 nmol/l induced a marked vasodilation (Emax 41.1±0.4%) which decreased after microinjection of the selective antagonist CGRP8-37. The NK3 receptor agonists (senktide and NKB) showed a minor vasodilating effect (Emax 5.1±1.2 and 8.0±0.9%, respectively). On the contrary, CAPS showed a marked dose-dependent vasodilating effect (Emax 43.2±2.9%), antagonized by the tachykinin receptor antagonists and CGRP8-37. These results suggest, for the first time, the presence of NK1, NK2, and CGRP receptors on the retinal arteriolar wall of the rabbit.

In vitro study of the interaction of Tilia europeae L. aqueous extract with GABAA receptors in rat brain

The interaction of GABAA receptor‐complex in rat brain was investigated in vitro with aqueous extracts obtained from the inflorescences of Tilia europeae L., using the [3H]muscimol and [3H]flunitrazepam binding techniques to synaptic membranes and the uptake of 36Cl‐ to synaptoneurosomes from cortices. The extract inhibited [3H]muscimol binding, stimulated 36ClCl‐ uptake by synaptoneurosomes and displaced at high concentrations, the [3H]flunitrazepam bound to synaptic membranes. When analysed by HPLC, the aqueous extract of Tilia europeae L. contained several amino acids, including GABA (about 100 μM). This GABA content can justify the displacement of [3H]muscimol produced by the extract but it did not increase the binding of [3H]flunitrazepam, as expected. Probably the extract contains other benzodiazepine‐like substances which displace the [3H]flunitrazepam binding and counteract the expected GABA‐induced increase in [3H]flunitrazepam binding.

Monoamine oxidase activity in blood platelets of migraine patients

Biochemical changes in platelets of migraine patients during the attacks have been reported before, however there are some conflicting results. In an attempt to define the biochemical lesion in the platelets, we have carried out a survey of platelets monoamine oxidase in migraine patients with and without aura. Platelet MAO activity in platelets from migraine patients was significantly reduced when compared with normal platelets.

Electrical stimulation-induced release of dopamine and noradrenaline in human blood vessels

Abstract Noradrenaline (NA), dopamine (DA) and DA metabolites levels, as well as their ratios, have been compared in various human vessels. In the same tissue we have also studied the influence of electrical stimulation in the presence or absence of pargyline, or pargyline plus cocaine. Catecholamines and DA metabolites have been measured by HPLC with electrochemical detection. The highest levels of NA and DA were found in the splenic artery and the lowest in the pulmonary artery and saphenous vein. Appreciable concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) were found in splenic, gastric, mesenteric and uterine arteries, whereas homovanillic acid (HVA) was not measurable in any tissue. However, the vascular segments showed a different DA/NA ratio, which was significantly higher in the mesenteric artery. The electrical stimulation increased this ratio, especially in the mesenteric artery. The ratio increased still more after treatment with pargyline plus cocaine. After pargyline pretreatment and in the presence of cocaine, electrical stimulation significantly increased NA and DA release from all blood vessels, except pulmonary artery and saphenous vein. During a second period of electrical stimulation only the DA release remained high. The results suggest that DA is coreleased with NA from the nerve endings of the vascular wall, and after neuronal uptake it is metabolized to DOPAC. Moreover, deamination is more important than methylation. When the sympathetic nerve endings are depleted of NA, the synthesis of DA increases. The DA/NA ratio differs between the various vessels, suggesting different roles for DA. Since this ratio was higher in the mesenteric artery, DA probably works as a cotransmitter in this vessel.