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1-Phenyl-3-(2-thiazolyl)-2-thiourea (PTTU) is a well-characterized dopamine β-hydroxylase inhibitor that prevents 6-hydroxydopamine-induced degenerative neuronal disease. However, the effect of PTTU on melanogenesis has not been reported. In this study, we examined the effect of PTTU on melanogenesis and studied its mechanism of action. We found that PTTU decreased melanin biosynthesis in a dose-dependent manner in normal human epidermal melanocytes (NHEMs). PTTU also inhibited tyrosinase catalytic activity in NHEMs. Moreover, PTTU treatment led to reduced protein levels of tyrosinase in NHEMs, while the protein levels of tyrosinase-related protein-1, tyrosinase-related protein-2, and microphthalmia-associated transcription factor were not affected. However, PTTU treatment did not affect the mRNA expression of tyrosinase. We found that PTTU-accelerated tyrosinase degradation via the ubiquitin-dependent proteasome pathway. In summary, we found that PTTU decreased melanin biosynthesis by decreasing the enzymatic activity and stability of tyrosinase. Our results indicate that PTTU could be used as a depigmentation agent for hyperpigmentation disorder.

Over 50 phenyl thiazolyl urea and carbamate derivatives were synthesized for evaluation as new inhibitors of bacterial cell-wall biosynthesis. Many of them demonstrated good activity against MurA and MurB and gram-positive bacteria including MRSA, VRE and PRSP. 3,4-Difluorophenyl 5-cyanothiazolylurea (3p) with clog P of 2.64 demonstrated antibacterial activity against both gram-positive and gram-negative bacteria.

The role of catecholamines in the expression of male courtship behavior in ring doves was examined using central administration of pharmacological agents. Males treated with 6-hydroxydopamine or U-14,624, which depleted norepinephrine (NE) levels in the preoptic-hypothalamic area, showed increased levels of bow-coo and nest-coo displays. Conversely, males treated with tyramine or desipramine, which elevated NE levels in the preoptic-hypothalamic area, showed decreased levels of bow-coo and nest-coo displays. Drug-induced changes in dopamine levels were not consistent with any changes in behavior. This suggests that in the male ring dove NE in the preoptic-hypothalamic area is important in the expression of courtship displays.

In this paper we describe our structure-based ligand design, synthetic strategy, and structure-activity relationship (SAR) studies that led to the identification of thiocarbamates (TCs), a novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), isosteres of phenethylthiazolylthiourea (PETT) derivatives. Assuming as a lead compound O-[2-(phthalimido)ethyl]phenylthiocarbamate 12, one of the precursors of the previously described acylthiocarbamates (Ranise, A.; et al. J. Med. Chem. 2003, 46, 768-781), two targeted solution-phase TC libraries were prepared by parallel synthesis. The lead optimization strategy led to para-substituted TCs 31, 33, 34, 39, 40, 41, 44, 45, and 50, which were active against wild-type HIV-1 in MT-4-based assays at nanomolar concentrations (EC50 range: 0.04-0.01 microM). The most potent congener 50 (EC50 = 0.01 microM) bears a methyl group at position 4 of the phthalimide moiety and a nitro group at the para position of the N-phenyl ring. Most of the TCs showed good selectivity indices, since no cytotoxic effect was detected at concentrations as high as 100 microM. TCs 31, 37, 39, 40, and 44 significantly reduced the multiplication of the Y181C mutant, but they were inactive against K103R and K103N + Y181C mutants. Nevertheless, the fold increase in resistance of 41 was not greater than that of efavirenz against the K103R mutant in enzyme assays. The docking model predictions were consistent with in vitro biological assays of the anti-HIV-1 activity of the TCs and related compounds synthesized.

In a recent experiment, it was found that the dopamine-beta-hydroxylase inhibitor, U-14,624, decreases the concentration of cytosol progestin receptors in guinea pig hypothalamus and causes an increase in the concentration of nuclear progestin receptors. In this series of experiments, the possibility that similar effects would be seen in the rat estrogen receptor system in mediobasal hypothalamus and pituitary was tested. U-14,624 caused a time-dependent decrease in the concentration of cytosol estrogen receptors and increase in the concentration of nuclear estrogen receptors in both mediobasal hypothalamus and anterior pituitary gland in ovariectomized rats, both in the absence and presence of low levels of estradiol, as well as in ovariectomized-adrenalectomized rats. The nuclear estrogen receptors that accumulate after U-14,624 injection do not require incubation at 25 degrees C to be assayed, suggesting that they are not occupied by an estradiol-like ligand. The nuclear estrogen receptors that accumulate after U-14,624 treatment are high affinity, with an apparent dissociation constant of approximately 0.1 nM. U-14,624 does not compete with (3H)estradiol, in vitro, suggesting that it does not directly interact with estrogen receptors. These results suggest that under some conditions, inhibition of dopamine-beta-hydroxylase causes a modification in unoccupied estrogen receptors so that they develop a higher affinity for cell nuclear components.

Bitemporal injections of puromycin that primarily affect the hippocampal-enthorhinal cortical areas suppress memory of maze-learning in mice for 3 days after training but are ineffective 6 or more days after training. At these later times, injections affecting widespread areas of the brain in addition to the hippocampal-entorhinal area are necessary for amnesia. These observations are interpreted to indicate that the locus of the memory trace has enlarged at 6 days to include other parts of the central nervous system in addition to the hippocampal-entorhinal area. To produce an imbalance of neurotransmitters and so to test their importance in enlargement of the memory trace's locus, we treated mice for 7 days after training with inhibitors of dopamine beta-hydroxylase. These mice, unlike untreated controls, developed amnesia after bitemporal injections of puromycin. In view of additional control experiments, we interpret these results to suggest that an imbalance of transmitters suppresses the normal enlargement of the locus of the memory trace.

Adult female Sprague-Dawley rats were ovariectomized and 2 weeks later were either hypophysectomized, sham hypophysectomized, or received no further surgical manipulation. Two weeks later, noradrenaline (NA) levels in the median eminence of hypophysectomized (HYPEX) rats were significantly elevated over those observed in sham-operated or control rats, while dopamine (DA) and epinephrine levels in this brain region of HYPEX rats were significantly lower than those observed in the other two groups. The concentration of NA in the remaining hypothalamus or in the telencephalon was not affected by hypophysectomy. The elevation of serum PRL by the implantation of two anterior pituitary homografts under the kidney capsule or the daily administration of 250 μg ovine PRL reduced the elevation of NA in the median eminence of HYPEX rats in the former case to a level significantly below that in controls and in the latter case to a level not significantly different from that observed in control rats. The greater effec...

The behavioral effects of low doses of the catecholamine (CA) synthesis inhibitor, alpha-methyl-p-tyrosine (alpha-MPT, 50 mg/kg i.p.), or the norepinephrine (NE) synthesis inhibitors (FLA-63, 15 mg/kg i.p., U-14624, 50 mg/kg i.p., or disulfiram 150 mg/kg i.p.) were studied in rats pretreated with reserpine (1 mg/kg i.p.) 24 h before. Rats were implanted either in the area ventralis tegmenti (AVT) or in the lateral hypothalamus (LH). The modifications of CA synthesis and endogenous CA levels were estimated in a parallel experiment. Reserpine treatment produced a slow decrease in self-stimulation (SS) rates during the first 12 h; SS rates were 85% of control values 24 h after reserpine treatment. Injection of alpha-MPT in reserpine-pretreated rats inhibited SS (85% decrease 3 h after administration either in AVT or LH rats), whereas dopamine beta-hydroxylase inhibition had no great effect on SS. The administration of very low doses of alpha-MPT (20 mg/kg i.p.) to rats treated with reserpine (24 h before) plus FLA-63 (1 h before) induced an important decrease in SS rates in AVT-implanted rats only. The major conclusion is that dopaminergic neurons seem to be involved in AVT and LH SS. The last experiment suggests the involvement of a balance between dopaminergic and noradrenergic neurons in AVT SS.

Bitemporal injections of puromycin that primarily affect the hippocampal-entorhinal areas consistently cause amnesia of maze-learning in mice for 3 days after training but become consistently ineffective if given 6 or more days after training. At these later times, additional puromycin injection sites covering widespread areas of the forebrain are necessary to induce amnesia. These observations are interpreted to indicate that the locus of the engram has become more widespread within the 6-day period. Treatment with inhibitors of dopamine β-hydroxylase for 3 days following training, retarded the spread of memory from a matter of days to a period of weeks. Repeated treatment with the inhibitors restricted engram spread for about 3 months; again spread was evident about a month after the last treatment. These observations imply that the mechanisms responsible for engram spread are capable of surviving for extraordinarily long periods of time.

Summary. Injection of the dopamine hydroxylase inhibitor U-14,624, to ovariectomized rats resulted in the reduction of hypothalamic norepinephrine concentration over a 48-h period and the elevation of hypothalamic dopamine concentration over a 24-h period. Plasma LH and FSH levels were markedly suppressed up to 48 h after drug treatment. These results suggest that U-14,624 is a much more potent inhibitor of gonadotrophin secretion than has been previously reported.

1. The effects of the dopamine-beta-hydroxylase inhibitors bupicamide, fusaric acid, FLA-63 and U-14,624 on blood pressure and heart rate of spontaneously hypertensive rats were examined. 2. Bupicamide and fusaric acid caused marked tachycardia whereas FLA-63 and U-14,624 caused modest bradycardia; all drugs decreased blood pressure. 3. In normotensive rats, fusaric acid caused the same degree of tachycardia as in spontaneously hypertensive rats, but blood pressure was only slightly reduced. 4. Tachycardia after fusaric acid was not due to increased sympathetic activity or decreased parasympathetic activity but required intact catecholamine stores. 5. It is concluded that fusaric acid causes tachycardia by releasing catecholamines indirectly and that a metabolite of fusaric acid is also involved.

Involvement of noradrenergic and/or dopaminergic processes of the brain in self-administration behavior toward ethanol was assessed in rats allowed to lever-press for 25 mg/kg intragastric doses on a CRF schedule. Initial access to infusions of saline for establishing an operant baseline was followed by one 10-hr session on acquisition contingencies for ethanol and then one extinction session on saline. Prior to a reacquisition session, rats were treated with either (a) saline, (b) alpha-methyl-p-tyrosine (AMT; 225 mg/kg), (c) 1-phenyl-3(2-thiazolyl)-2-thiourea (U-14,624; 600 mg/kg or 300 mg/kg), or (d) haloperidol (3.5 mg/kg). Only the saline- pretreated control group and the haloperidol-treated rats reacquired lever-press behavior. Groups treated in like fashion, but pressing for a sweet milk reinforcer, all showed reacquisition. Thus, the effects of AMT and U-14,624 are attributed to an interference with the reinforcing effects of ethanolinfusions. Brain levels of norepinephrine were depeleted by both compounds, dopamine was depleted only by AMT, and serotonin was elevated by 600 mg/kg of U-14,624 but unaffected by 300 mg/kg. These results suggest that a cerebral noradrenergic system plays an important role in the reinforcing effect of ethanol without an involvement of dopaminergic systems

We have recently shown that the dopamine-beta-hydroxylase inhibitor, U-14,624, decreases the concentration of cytosol estrogen receptors in the mediobasal hypothalamus (MBH) and anterior pituitary gland (AP) in ovariectomized rats, but that it also causes cell nuclear accumulation of estrogen receptors. We tried to determine if this is the mechanism by which other catecholaminergic inhibitors decrease the concentration of cytosol estrogen receptors in either the MBH or AP. The previously reported decrease in the concentration of cytosol estrogen receptors in AP by the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine was confirmed. Also, the decrease in the concentration of cytosol estrogen receptors in MBH after treatment with the dopamine-beta-hydroxylase inhibitors, diethyldithiocarbamate and FLA 63 was demonstrated. In no case was an increase in the concentration of nuclear estrogen receptor accumulation detected after treatment with the drugs. Results of assays of norepinephrine and dopamine levels in MBH after the various treatments suggest that, at the dosage used, U-14,624 has a greater effect on norepinephrine and dopamine levels that the other dopamine-beta-hydroxylase inhibitors. The results of these experiments suggest that inhibitors of dopamine-beta-hydroxylase and tyrosine hydroxylase cause decreases in the concentration of cytosol estrogen receptors in either the MBH or AP that are not referable to increased cell nuclear accumulation of estrogen receptors.

The present study was performed to examine the role of brain norepinephrine in the control of copulation and the post-ejaculatory refractory period in the male rat. Disruption of central noradrenergic systems was achieved by (1) selective electrolytic lesion of noradrenergic cell bodies in the locus coeruleus or (2) administration of specific inhibitors of norepinephrine synthesis, sodium diethyldithiocarbamate (DDC) or 1-phenyl-3-(2-thiazolyl)-2 thiourea (U-14, 624). Electrolytic lesions of the locus coeruleus produced a significant increase in the duration of the post-ejaculatory refractory period and its concomitant 22-kHz ultrasonic vocalization. Administration of norepinephrine synthesis inhibitors significantly increased both mount and intromission latencies and caused a dramatic increase in the length of the post-ejaculatory refractory period. These findings support the hypothesis that norepinephrine-containing neural pathways are involved in the control of sexual arousal and suggest that a functional noradrenergic system is essential to the integrity of normal masculine copulatory behavior.

1-Phenyl-3-(2-thiazolyl)-2-thiourea (PTTU) prevented the neurotoxic actions of 6-hydroxydopamine on adrenergic nerves in the mouse atrium and iris. This is the first reported 6-hydroxydopamine antagonist that does not act by blocking uptake of catecholamines into nerve terminals. PTTU also prevented the diabetogenic action of alloxan.

The effect of adrenalectomy and corticosterone treatment on dopamine beta-hydroxylase (DBH) activity, catecholamine content and norepinephrine formation and metabolism were studied in the hypothalamus and other parts of the brain of male rats. Two days after adrenalectomy, there was a decrease in DBH activity in the hypothalamus and the brain stem but no change in norepinephrine or dopamine content. Conversion of intraventricularly administered tritiated dopamine to tritiated norepinephrine was slightly increased and norepinephrine was metabolized at a more rapid rate than normal. Corticosterone in a dose of 100 mg/kg increased DBH activity but decreased hypothalamic norepinephrine and copamine content. In adrenalectomized rats, smaller, more physiological doses of corticosterone did not change DBH activity or catecholamine content. The fact that norepinephrine formation and metabolism were increased at the same time that DBH activity in vitro was decreased suggests that DBH is not rate-limiting in adrenergic neurons in the hypothalamus, or that a change in the in vitro activity of the enzyme was not accompanied by a parallel change in its activity in vivo.

The relationship between the medial basal hypothalamus (MBH) LH-RH activity and LH release was studied following progesterone (P) treatment of oestrogen-primed ovariectomized rats (day 0). Following P administration at 10.00 h (day 2) serum LH levels increased rapidly after 13.00 h to peak levels attained at 15.00 h and maintained until 18.00 h. Coincident with the onset of augmented release and peak serum LH concentrations at 15.00 h there was a significant enhancement in the MBH LH-RH activity. Thereafter, the MBH LH-RH stores promptly fell and remained at morning low levels through the rest of the LH surge period. P treatment also stimulated release of FSH and prolactin in the afternoon. Administration of norepinephrine (NE) synthesis inhibitors, diethyldithiocarbamate (DDC) and U-14,624 before P blocked the afternoon increments in serum gonadotrophins and the MBH LH-RH levels; prolactin release was also suppressed in DDC treated rats. In contrast, lergotrile mesylate (dopamine agonist) treatment prior to P administration suppressed only the afternoon increase in prolactin release. These studies show that (1) P can stimulate MBH LH-RH activity in oestrogen-primed rats and these effects are transmitted to the LH-RH peptidergic neurons via NE synapses in the preoptic area and (2) a common central NE system may mediate the stimulatory feedback effects of P on gonadotrophin and prolactin release.

The inhibition of pituitary luteinizing hormone (LH) release by the norepinephrine depletor U-14624 was investigated in ovariectomized rats. Ovariectomized rats were injected at 1100 hours with either U-14624 (200 mg/kg) or vehicle alone. The serum from trunk blood was analyzed for LH and LH-RH (releasing hormone by radioimmunoassay in the 1st experiment and estimated after extraction with methanol in the 2nd experiment. In both experiments LH-RH was significantly reduced. Serum LH was also significantly depressed. It is suggested that a disruption of norepinephrine systems in the hypothalamus leads to impaired release of LH-RH.

The initial aim of the present studies was to examine the influence of blockade of dopamine (DA) receptors with pimozide or inhibition of norepinephrine (NE) synthesis with U-14,624 on acute, ovariectomy (OVX)-induced changes in pulsatile LH release. Either treatment instituted at the time of OVX suppressed or inhibited the rapid increase in LH pulse amplitude and frequency normally occurring within 24 hr following ovarian removal on diestrus 1. While administration of pimozide at either 24 hr or 48 hr following OVX suppressed pulsatile LH release by selectively reducing LH pulse frequency, by 8 days following OVX pimozide failed to exert any effect on LH pulse frequency and therefore on pulsatile LH secretion. To determine if there was a transient critical period following OVX of at least 2 days but less than 8 when endogenous DA was excitatory to pulsatile LH release, piribedil (a DA receptor agonist) was given 24 hr following OVX. Rather than increase LH secretion, piribedil markedly suppressed pulsatile LH release indicating that DA does not stimulate LH secretion in acutely ovariectomized rats. These experiments indicate that 1. (1) NE is involved in stimulating the acute, OVX-induced increase that occurs in pulsatile LH release; 2. (2) DA receptorblockade by pimozide has a differential effect on pulsatile LH secretion which depends on the time following OVX when the compound is administered; 3. (3) this differential effect cannot be explained by a transient critical period of a few days duration following OVX during which DA is excitatory to pulsatile LH release.

The role of brain noradrenergic neurons in mediating the reinforcing properties of small intravenous doses of morphine and d-amphetamine was investigated by pretreatment of rats with the norepinephrine-depleting agents diethyldithiocarbamate and U-14,624, inhibitors of dopamine-β-hydroxylase (DBH). Such treatment prevented the reacquisition of a self-administration response (bar-press) for morphine (32 μg/kg/injection) or d-amphetamine (15 μg/kg/injection) made available on a CRF schedule. Pretreatment with a DBH inhibitor also prevented the development of a secondary (conditioned) reinforcer based on primary reinforcement associated with either drug. Observations indicating that the orienting reflex was intact are taken as evidence that depressant effects of the DBH inhibitors were not severe enough to disrupt the associative process. Therefore, any effect on learning does not seem sufficient to explain the present results. Thus, it is inferred that the mechanisms mediating reinforcement for both morphine and amphetamine were disrupted by the inhibition of central noradrenergic functions.

In Experiment 1, the dose-response effects of three dopamine-β-hydroxylase (DBH) inhibitors (diethyldithiocarbamate, FLA-63 and U-14, 624) on the endogenous levels of norepinephrine and dopamine in pons-medulla of rat brain were determined. In Experiment 2, the effect of low doses of diethylithiocarbamate (2.5 to 120 mg/kg) on the level of norepinephrine- 3 H produced from dopamine-H 3 was determined. The data obtained by extrapolation of the curves in both experiments provided an estimation of the in vivo level of DBH activity and suggested that it was not present in excess. Finally, in Experiment 3, the three DBH inhibitors reduced self-stimulation (a behavior dependent upon catecholamines) in a dose-related manner and intraventricular injections of 1-norepinephrine reinstated normal rates of self-stimulation. The results from the three experiments are consistent with the idea that DBH is involved in the regulation of norepinephrine biosynthesis. The relationship of this finding to our earlier report of a deficit of DBH in post-mortem brains of schizophrenics is discussed.

Phenol oxidase may be involved in the formation of the eggshell in Schistosoma mansoni. 3H-Labeled female S. mansoni proteins were polymerized in vitro following incubation with S. mansoni phenol oxidase and excess l -tyrosine. Peroxidase inhibitors, autoxidation inhibitors, inhibitors of lipid peroxidation, and inactive analogs of phenol oxidase inhibitors did not inhibit eggshell formation. Fluorescent substances found in eggshell hydrolysates were similar to those formed from the reaction of phenol oxidase-generated quinones with protein-bound lysine. These observations support the classical concept of phenol oxidase-catalyzed protein hardening. However, fluorescent globules of egg material were still formed after the administration of 200 mg/kg of the phenol oxidase inhibitor diethyldithiocarbamate. These globules could not be destroyed by inhibitors of autoxidation and lipid peroxidation.

A series of experiments was performed to study the possible behavioral relevance of the apparent regulation of hypothalamic cytosol progestin receptors by noradrenergic transmission in female guinea pigs. In the first experiment, ovariectomized guinea pigs were injected with estradiol benzoate followed 36 h later by the dopamine-beta-hydroxylase inhibitor, U-14,624 or vehicle. Twelve h later, they were injected with progesterone and tested hourly for sexual behavior. Six h after the progesterone injection, a time at which inhibition of sexual behavior by the U-14,624 was confirmed, they were killed, and cytosol and nuclear progestin receptors were assayed in the hypothalamus and cerebral cortex. No difference was seen in the concentration of progestin receptors after drug treatment in these animals that also received a progesterone injection. In subsequent experiments, it was found that the U-14,624-inhibition of progesterone-facilitated sexual behavior is not accompanied by an inhibition of nuclear progestin receptor accumulation. Furthermore, it was found that the decreased cytosol progestin receptor level caused by U-14,624 prior to progesterone injection was accompanied by an increase in the concentration of nuclear progestin receptors. The increase in nuclear progestin receptors was also seen after treatment with the alpha-adrenergic antagonist, prazosin, U-14,624 does not compete with [3H]R 5020 for binding to the progestin receptor, suggesting that it does not directly cause translocation of progestin receptors. The results of these experiments suggest that the decrease in the concentration of cytosol progestin receptors caused by noradrenergic inhibitors is not due entirely to an interference with the formation of cytosol progestin receptors. Rather, it seems that these drugs, in some way, also cause the accumulation of progestin receptors in cell nuclei. Furthermore, they suggest that the mechanism by which these drugs inhibit sexual behavior may not be by interference with the progestin receptor system.

1-Phenyl-3-(2-thiazolyl)-2-thiourea (PTTU) administered i.p. to mice prevented the neurodegenerative actions of subsequently injected (1 hour later) 6-hydroxydopamine (6-OHDA) or 6-aminodopamine on peripheral adrenergic nerve terminals. Destruction of nerve terminals was studied in vitro in the left atrium by measuring the accumulation of 3H-norepinephrine (3H-HE), and in the iris by both 3H-NE accumulation and fluorescence microscopy methods. Strong protection was observed at 4, 24 and 72 hours after 6-OHDA. The degree of protection was dose-dependent and showed step-wise decrements for concentrations of PTTU below 200 mg/kg (viz., 100, 50 and 20 mg/kg) or concentrations of 6-OHDA-HBr above 7.5 mg/kg (viz., 10, 20 and 50 mg/kg). Protection also fell off at time intervals greater than 1 hour after administration of PTTU (viz., 3 and 5 hours). The appearance (fluorescence microscopy) of the nerve plexus of fully protected mice and the remaining plexus in partially protected mice was essentially normal at 24 hours, except for infrequent large swellings. PTTU proved to be a very effective scavenger of hydroxyl radicals; the formation and scavenging of these radicals was studied by gas chromatography in a system in which the hydroxyl radicals (which were generated during the autoxidation of 6-aminodopamine) gave rise to ethylene, a hydrocarbon gas. Other hydroxyl radical scavengers, namely diethyldithiocarbamate and methimazole, exhibited a protective action on sympathetic nerves in the left atrium; PTTU, diethyldithiocarbamate and methimazole are also recognized as copper chelating compounds. Ethanol, n-butanol and cysteamine, which are well known hydroxyl radical scavengers, also exhibited some degree of protection against 6-OHDA. Blockade of transport of 6-OHDA into sympathetic nerves was ruled out as a protective mechanism by the observation that none of the protective compounds inhibited the accumulation of tritium by the left atrium when 3H-NE was injected in place of 6-OHDA. The mechanism of action for these protective agents has not been definitively established, but scavenging of cytotoxic hydroxyl radicals within neurons may play a significant role.

The uptake-accumulation and binding of radioactivity in mouse heart after administration of the catecholamine neurotoxin [3H]6-hydroxydopamine (6-OH-DA, 1 or 3 mg/kg, i.v.) has been investigated. It was confirmed that a substantial portion (8--20%) of the radioactivity taken up and retained by the heart could not be extracted with perchloric acid, in all probability representing covalently bound oxidation products of 6-OH-DA to tissue proteins. Pharmacological analysis showed that a large part of this fraction was associated with the adrenergic nerves. The time-course of the perchloric acid resistant binding to the adrenergic nerves was found to parallel that of the neurotoxic action of 6-hydroxydopamine as evaluated by monitoring the change in [3H]noradrenaline uptake. Calculation of the intranelronal 6-hydroxydopamine concentration (average) needed to induce degeneration showed it to be in the order of 50 mM. The binding ratio for tritium deriving from [3H]6-OH-DA between the intraneuronal and extraneuronal compartments was found to be 10,000 to 30,000, pointing to a very high neuronal specificity for 6-hydroxydopamine. The 'covalent' binding of oxidation products of [3H]6-OH-DA was considerably reduced after desipramine or 1-phenyl-3(2-thiazolyl)-2-thiourea administration, treatments both known to protect the adrenergic nerves from undergoing degeneration. Conversely it was found that the binding increased during conditions known to potentiate the neurotoxic action of 6-hydroxydopamine, e.g., after monoamine oxidase inhibition with nialamide. Subcellular fractionation studies indicated that the predominant site of interaction between 6-hydroxydopamine oxidation products and neuronal proteins is the cytoplasm and the axonal membrane. Analysis of the effect in vivo administration of 6-hydroxydopamine on the field-stimulated induced release of [3H]noradrenaline previously taken up in the adrenergic nerves showed a 6-hydroxydopamine indiced reduction in [3H]noradrenaline release which was approximately proportional to the reduction in the number of nerve terminals. These findings further support the view that 6-hydroxydopamine acts largely in an "all-or-none' fashion with respect to the neurodegenerative action. Administration of [3H]dopamine also resulted in a fraction which was not extractable with perchloric acid, although this fraction was very small compared to that found after an equal dose of [3H]6-hydroxydopamine. These data may indicate that oxidation products of dopamine can interact with tissue proteins. From the present results it can be concluded that there is a close relationship between 'covalent' binding of 6-hydroxydopamine oxidation products to neuronal elements and the cytotoxic action of 6-hydroxydopamine, indicating that this binding may play an important role in the neurodegenerative action of 6-hydroxydopamine on catecholamine neurons.

A bipolar electrode was stereotaxically implanted in or near the medial forebrain bundle at the level of the posterior lateral hypothalamus of male albino Wistar-Imamichi rats. 1) Two electrode sites implanted in a rat both in the lateral hypothalamus and in the dorsal noradrenaline bundle supported self-stimulation (SS) behavior. 2) Methamphetamine facilitated the SS dose-dependently in the "threshold"-intensity reinforcement. The pretreatment of imipramine enhanced the effects of methamphetamine, but that of chlorpromazine inhibited those effects. Methamphetamine facilitated the SS also in the moderate-intensity reinforcement. Imipramine had no significant effects. On the other hand, chlorpromazine inhibited markedly the SS behavior. 3) FLA63 and U-14,624, dopamine beta-hydroxylase inhibitors, suppressed the SS behavior. Both drugs significantly reduced brain norepinephrine levels. FLA63 significantly increased the levels of dopamine, but the increase with U-14,624 was not significant. 4) Apomorphine, stimulant of dopamine receptor, did not facilitate, but rather suppressed the SS in the "threshold"-intensity reinforcement. 5) l-Norepinephrine injected into the lateral ventricle facilitated the rate of SS, dose-dependently. On the other hand, dopamine had no detectable effects. 7) These results suggest that the noradrenergic system in the brain plays a more important role in the positive reinforcement of the lateral hypothalamic SS behavior than does the dopaminergic system.

A bipolar electrode was stereotaxically implanted in the substantia nigra of male albino Wistar-Imamichi rats. 1) Rats lifted and turned their heads and contralateral forelimb towards the contralateral side to the electrode during the substantia nigral self-stimulation (SS) behavior. 2) Methamphetamine facilitated the SS dose-dependently in the "threshold"-intensity reinforcement. The pretreatment of imipramine facilitated the effects of methamphetamine. 3) FLA63 and U-14,624, dopamine beta-hydroxylase inhibitors, suppressed the SS in the moderate-intensity reinforcement. 4) Apomorphine, stimulant of dopamine receptor, did not facilitate, but rather inhibited the SS dose-dependently in the "threshold"-intensity reinforcement. 5) l-Norepinephrine injected into the lateral ventricle facilitated the SS dose-dependently. On the other hand, dopamine had no marked effects. 6) These results suggest that the noradrenergic system in the brain plays a more important role in the positive reinforcement of the substantia nigral SS behavior than does the dopaminergicsystem.

1-Phenyl-3-(2-thiazolyl)-2-thiourea (200 mg/kg, 1 h) protected the adrenergic nerve plexus in the mouse atrium against the destructive action of i.v. 5,7-dihydroxytryptamine. Protection was also observed with ethanol (4 g/kg, 1 h) and with nialamide (50 mg/kg, 2 h).