Erik Pileblad

Reduction of Brain Glutathione by l-Buthionine Sulfoximine Potentiates the Dopamine-Depleting Action of 6-Hydroxydopamine in Rat Striatum

Abstract: Sprague‐Dawley rats were anesthetized with chloral hydrate, and plastic cannulae were permanently implanted into the lateral ventricles. The animals then were allowed to recover for 1–2 days. L‐Buthionine sulfoximine (l‐BSO), a selective inhibitor of glutathione (GSH) synthesis, and 6‐hydroxydopamine (6‐OH‐DA), a selective catecholaminergic neurotoxin, were administered intracerebroventricularly. The striatal concentrations of GSH and monoamines were determined by HPLC with electrochemical detection. Two injections of l‐BSO (3.2 mg, at a 48‐h interval) resulted in a 70% reduction of striatal GSH. 6‐OH‐DA (150 or 300 μg) reduced the concentrations of striatal dopamine and noradrenaline 7 days after the administration, but left the concentrations of 5‐hydroxytryptamine unaltered. L‐BSO treatment did not produce any changes in the levels of monoamines per se but it potentiated the catecholamine‐depleting effect of 6‐OH‐DA in the striatum. Thus, GSH appears to suppress the toxicity of 6‐OH‐DA, probably by scavenging the toxic species formed during 6‐OH‐DA oxidation. In view of these results one may suggest an important role for GSH in catecholaminergic neurons: protecting against the oxidation of endogenous catechols.

An electrophysiological analysis of the actions of the 3-PPP enantiomers on the nigrostriatal dopamine system.

SummaryExtracellular single unit recording and micro-iontophoretic studies were carried out in chloral hydrate-anesthetized and gallamine-paralyzed rats to investigate the actions of the enantiomers of the dopamine (DA) analogue 3-(3-hydroxyphenyl)-N-n-propylpiperidine, 3-PPP, on the nigrostriatal DA system. Intravenously administered (+)-or (−)-3-PPP consistently inhibited nigral DA neuronal activity; these actions were readily antagonized by haloperidol but were not affected by a pretreatment of reserpine plus alpha-methyltyrosine. In contrast to (+)-3-PPP, the (−)-enantiomer produced only partial inhibition of the majority of cells studied and was also capable of partially reversing the inhibitory action of apomorphine. A prior hemitransection of the brain did not alter the inhibitory action of either enantiomer. Whereas iontophoretically ejected (+)-3-PPP consistently reduced DA cell firing rate, similarly applied (−)-3-PPP reduced the activity of only some DA cells, while the majority were not influenced. In addition, iontophoresis of (−)-3-PPP could reduce the inhibitory effect of similarly applied DA or (+)-3-PPP. The (+)-enantiomer reduced caudate neuronal activity both after intravenous administration and iontophoresis. Intravenously administered (−)-3-PPP failed to influence or increased the activity of these neurons and reversed the inhibitory action of apomorphine. However, iontophoretically ejected drug reduced caudate cell activity and did not influence the inhibitory action of DA. The activity of non-DA zona reticulata neurons was inconsistently influenced by the 3-PPP enantiomers. It is concluded that (+)-3-PPP is a directly acting DA agonist, stimulating both DA autoreceptors and postsynaptic DA receptors. In contrast, (−)-3-PPP appears to be a partial agonist at nigral DA autoreceptors, whereas the action of the drug at putative postsynaptic DA receptors in the caudate remains to clarified.

Effects of Local Administration of L-, N-, and P/Q-Type Calcium Channel Blockers on Spontaneous Dopamine Release in the Striatum and the Substantia Nigra: A Microdialysis Study in Rat

Abstract: The pivotal role for voltage‐sensitive calcium channels in initiating synaptic transmitter release is undisputed, but it is only partly known to what extent the different subtypes contribute in vivo. Their importance for the dendritic release of dopamine has not been investigated in vivo previously. To evaluate comprehensively the relative importance of different voltage‐sensitive calcium channel subtypes for striatal dopamine release, and to further investigate the mechanism of dendritic dopamine release in the reticulate part of substantia nigra, dopamine was measured by in vivo microdialysis in the striatum or substantia nigra of awake rats. The calcium channel blockers nimodipine, ω‐conotoxin‐GVIA, ω‐agatoxin‐IVA, and neomycin were administered locally through the dialysis probes and compared with calcium‐free perfusion. Results indicate that dopamine release in the striatum is mainly dependent on N‐ and P/Q‐type channels, but the dendritic dopamine release in the substantia nigra is mediated mainly by some other calcium‐dependent mechanism, for example, calcium mobilization through T‐, O‐, or R‐type calcium channels. A portion of the dendritic release is calcium independent but can be inhibited partially by neomycin, which might suggest a role for inositol 4,5‐bisphosphate breakdown products.

Biochemical and functional evidence for a marked dopamine releasing action of N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (NMPTP) in mouse brain

N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (NMPTP), a recently identified neurotoxic compound, has been found to produce destruction of the nigrostriatal dopaminergic neurons in man and monkeys (Langston et al., 1983, Burns et al., 1983). Damage to both dopaminergic and noradrenergic neurons has been shown in the brains of mice, though in a higher dose range (Hallman, 1984). In order to further elucidate the action of NMPTP we have studied the acute effects of the agent in mice.

The effects of GBR 12909, a dopamine re-uptake inhibitor, on monoaminergic neurotransmission in rat striatum, limbic forebrain, cortical hemispheres and substantia nigra

SummaryIn order to investigate the physiological importance of the membrane pump in eliminating released dopamine (DA) we have studied the effects of the putative selective dopamine re-uptake inhibitor, GBR 12909, on synthesis and metabolism of monoamines in the rat striatum, limbic forebrain, cortical hemispheres and substantia nigra (SN). The effects of the drug on the firing rate of catecholamine containing neurons in the SN and locus coerulus (LC) were also investigated. For comparison we have investigated the effects of desipramine and maprotiline. As a measure of the synthesis of noradrenaline (NA), DA and 5-hydroxytryptamine (5-HT) we determined the 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) accumulation after inhibition of aromatic l-amino acid decarboxylase by 3-hydroxy-benzylhydrazine (NSD 1015). As indirect measurements of DA and NA release in vivo, we have assessed pargyline-induced 3-methoxytyramine (3-MT) and normetanephrine (NM) accumulation and disappearance rates of DA and NA after inhibition of their synthesis by α-methyl-p-tyrosine (α-MT). Administration of GBR 12909 (2.5, 5, 10, 20 or 40 mg/kg) decreased the NSD 1015-induced DOPA accumulation in the striatum and in the limbic forebrain. In contrast, only minor effects of the drug were seen on the DOPA accumulation in the cortical hemisphere and on the cerebral 5-HTP accumulation. GBR 12909 increased the 3-MT accumulation in the striatum, limbic forebrain and the cortical hemispheres, an effect that was even more pronounced in haloperidol-pretreated animals. However, GBR 12909 did not alter the 3-MT accumulation in the SN either when given alone or when given to haloperidol-pretreated rats. In haloperidol-pretreated rats GBR 12909 markedly enhanced the DA disappearance in the striatum and in the limbic forebrain, but not in the SN. Furthermore, GBR 12909 did not significantly affect the firing rate of dopaminergic neurons in the SN or that of noradrenergic neurons in the LC. Taken together, our results support the notion that GBR 12909 is a specific DA uptake inhibitor without a transmitter releasing action. In addition, our findings indicate that DA re-uptake is of physiological importance in the elimination of DA from the synaptic cleft in the striatum, limbic forebrain and cortical hemispheres, but not in the SN. Furthermore, a large part of the DA taken up by the dopaminergic terminals in the striatum and in the limbic forebrain seems to be re-incorporated into the storage vesicles.

Increase in rat brain glutathione following intracerebroventricular administration of γ-glutamylcysteine

The effects of intracerebroventricularly (i.c.v.) administered gamma-glutamylcysteine (gamma-GC) and glutathione (GSH) monoethyl ester, subcutaneously (s.c.) injected L-2-oxo-4-thiazolidinecarboxylic acid (OTC) and intraperitoneally (i.p.) administered cysteine on the concentration of GSH in rat brain were investigated. The brain content of GSH, cysteine and gamma-GC was determined by HPLC with electrochemical detection (gold/mercury electrode) using N-acetylcysteine as internal standard. A dose-dependent increase in the GSH concentration (145-170% of controls) was found in the substantia nigra (SN) and in the rest of the brain stem after injection of gamma-GC, whereas no significant alterations in GSH were observed in the striatum and in the cerebral cortex. High levels of gamma-GC could be detected in the brain tissue after the administration, and the concentration of cysteine did also increase markedly after gamma-GC injection in all brain regions assessed. I.c.v. administration of L-buthionine sulfoximine (L-BSO) reduced the brain concentration of GSH by 50-70% within 24 hr. Injection of gamma-GC 24 hr after L-BSO resulted in an increase in GSH up to control values within 1-3 hr in the SN and the rest of the brain stem, whereas only a slight increase in GSH was observed in the striatum and the cerebral cortex. The concentration of GSH in the striatum and SN did not change after i.p. injection of cysteine, but a slight increase in the GSH concentration in the limbic region was observed. GSH monoethyl ester (i.c.v.) and OTC (s.c.) did not produce any significant increase in the GSH concentration in the brain. When the GSH concentration had been reduced by administration of L-BSO (i.c.v.; 24 hr) subsequent injection of GSH monoethyl ester led to a slight increase in the striatal and limbic GSH levels. These data show that, of the drugs studied, gamma-GC was the most effective in increasing brain GSH. It could thus serve as a valuable tool in future studies regarding metabolism and function of GSH in the brain. The observed difference in the effects of gamma-GC in different brain regions indicate that the brain tissue is not homogeneous with regard to GSH synthesizing capacity.

Reference range of prostate-specific antigen after transurethral resection of the prostate

OBJECTIVES The aim of the present study was to investigate how transurethral resection of the prostate (TURP) affected the serum levels of prostate-specific antigen (PSA) and to establish reference ranges of PSA in patients who have undergone TURP. METHODS PSA was determined preoperatively and 3 months postoperatively in 190 patients who underwent TURP because of benign prostatic hyperplasia (BPH). RESULTS Mean PSA levels were reduced by 70%, from 6.0 to 1.9 ng/mL. Prostate volume was reduced by 58% from 63.3 to 26.5 cc, which is close to the reported normal volume in men without BPH. Ninety percent of the patients had a postoperative PSA value of less than 4 ng/mL and 98% less than 10 ng/mL. CONCLUSIONS After a complete TURP with a benign histopathologic specimen, PSA should be expected to be within the normal reference range, that is, less than 4 ng/mL.

Intracerebroventricular Administration of l‐Buthionine Sulfoximine: A Method for Depleting Brain Glutathione

Abstract: Sprague‐Dawley rats (200‐260 g) were anesthetized with chJoral hydrate (400 mg/kg) and polyethylene cannulae were permanently implanted into the lateral ventricles. One or two days later, l‐buthionine‐[S,R]‐sulfoximine (L‐BSO), an apparently selective inhibitor of γ‐glutamylcysteine synthetase, was administered intracerebroventricularly through the cannulae. The brain content of glutathione (GSH) was determined by HPLC with electrochemical detection (gold/ mercury electrode) using N‐acetylcysteine as internal standard. A time‐course study of the changes in the striatum following a single dose of l‐BSO (3.2 mg) revealed a maximal depletion of GSH (‐60%) approximately 48 h after the administration. The effects of various doses of l‐BSO on GSH in the striatum, in the limbic region, and in the cortex were assessed at 24 h and 48 h after the administration. l‐BSO (0.02‐3.2 mg) produced dose‐dependent reductions of GSH in all brain regions studied at both time intervals. In a long‐term experiment l‐BSO (3.2 mg) was administered every second day. After 4 days, i.e., after two injections, striatal GSH was reduced by approximately 70%. No further depletion of GSH was obtained by additional injections of l‐BSO, but GSH was maintained at this low level for the 12 days studied. These results suggest that l‐BSO, administered intracerebroventricularly, would serve as a useful tool for evaluation of the biological role of GSH in the CNS.

Outcome of Laterally Directed Sextant Biopsies of the Prostate in Screened Males Aged 50–66 Years

Objective: Prostate cancer has its most frequent location in the posterior–lateral part of the gland. The aim of this study was to evaluate the cancer detection rate of six systemic prostate biopsies with mid lobar biopsies taken far laterally in the prostate. Patients and Methods: A total of 692 patients (aged 50–66 years) enrolled in a screening study underwent prostate biopsies because of an elevated serum prostate–specific antigen (PSA; ≧3ng/ml) level. The outcome of the biopsies was related to findings at digital rectal examination (DRE) and transrectal ultrasound (TRUS) and to the location within the prostate. Results: Prostate cancer was detected in 164 patients. DRE and TRUS were suspicious of malignancy in 66 cases (40%) and 84 cases (51%), respectively. The two biopsies taken far laterally midlobar in the prostate detected as many as 83% of the cancers and when combined with two apical biopsies, 96% of all cancers were detected. Conclusion: At PSA screening in this age–group, only 57% of the prostate cancers detectable by sextant biopsies were palpable or visible at TRUS. Most of the cancers (96%) were detectable by only four systematic, carefully directed biopsies. In men with normal DRE, the two lateral midlobar biopsies should be taken first during the biopsy procedure.

Effective depletion of glutathione in rat striatum and substantia nigra by L-buthionine sulfoximine in combination with 2-cyclohexene-1-one

The effects of L-buthionine sulfoximine (L-BSO), 2-cyclohexene-1-one and diethylmaleate (DEM) on the concentration of rat brain glutathione (GSH) were investigated. Both DEM and 2-cyclohexene-1-one, administered subcutaneously, produced marked and rapid reduction of brain GSH, but 2-cyclohexene-1-one appeared less toxic than DEM. Six hours after 2-cyclohexene-1-one (100 microliters/kg) the striatal GSH concentration was 35% of control values, whereas the level was 55% of controls at 24 h and 80% of controls at 48 h. Similar results were obtained with DEM (800 microliters/kg). L-BSO (3.2 mg), administered intracerebroventricularly, produced a slower depletion of brain GSH. A 55% reduction of striatal GSH was obtained 24 h after the administration, and the level was approximately 50% of control at 48 h. Thus, the effect of 2-cyclohexene-1-one and DEM is rapid in onset but relatively short lasting, whereas the disappearance of brain GSH after L-BSO is slower but the effect is more long-lasting. By combining L-BSO with either 2-cyclohexene-1-one or DEM both a rapid and long-lasting GSH depletion was obtained that was more profound than after any of the drugs alone. The combination of L-BSO and 2-cyclohexene-1-one was well tolerated, but the combination of L-BSO and DEM led to death in half of the rats the second day after injection. The disappearance rate of GSH after L-BSO alone gives an estimate of the turn-over of GSH. We found the turn-over of GSH to be higher in the substantia nigra pars compacta than in the striatum. The present work suggest that L-BSO and 2-cyclohexene-1-one would be very useful for evaluation of the biological role of GSH in the central nervous system.