Chronic exposure to manganese decreases striatal dopamine turnover in human alpha-synuclein transgenic mice.

Interaction of genetic and environmental factors is likely involved in Parkinson's disease (PD). Mutations and multiplications of alpha-synuclein (α-syn) cause familial PD, and chronic manganese (Mn) exposure can produce an encephalopathy with signs of parkinsonism. We exposed male transgenic C57BL/6J mice expressing human α-syn or the A53T/A30P doubly mutated human α-syn under the tyrosine hydroxylase promoter and non-transgenic littermates to MnCl2-enriched (1%) or control food, starting at the age of 4 months. Locomotor activity was increased by Mn without significant effect of the transgenes. Mice were sacrificed at the age of 7 or 20 months. Striatal Mn was significantly increased about three-fold in those exposed to MnCl2. The number of tyrosine hydroxylase positive substantia nigra compacta neurons was significantly reduced in 20 months old mice (-10%), but Mn or transgenes were ineffective (three-way ANOVA with the factors gene, Mn and age). In 7 months old mice, striatal homovanillic acid (HVA)/dopamine (DA) ratios and aspartate levels were significantly increased in control mice with human α-syn as compared to non-transgenic controls (+17 and +11%, respectively); after Mn exposure both parameters were significantly reduced (-16 and -13%, respectively) in human α-syn mice, but unchanged in non-transgenic animals and mice with mutated α-syn (two-way ANOVA with factors gene and Mn). None of the parameters were changed in the 20 months old mice. Single HVA/DA ratios and single aspartate levels significantly correlated across all treatment groups suggesting a causal relationship between the rate of striatal DA metabolism and aspartate release. In conclusion, under our experimental conditions, Mn and human α-syn, wild-type and doubly mutated, did not interact to induce PD-like neurodegenerative changes. However, Mn significantly and selectively interacted with human wild-type α-syn on indices of striatal DA neurotransmission, the neurotransmitter most relevant to PD.

Carrier-mediated release, transport rates, and charge transfer induced by amphetamine, tyramine, and dopamine in mammalian cells transfected with the human dopamine transporter.

Amphetamine and related substances induce dopamine release. According to a traditional explanation, this dopamine release occurs in exchange for amphetamine by means of the dopamine transporter (DAT). We tested this hypothesis in human embryonic kidney 293 cells stably transfected with the human DAT by measuring the uptake of dopamine, tyramine, and D- and L-amphetamine as well as substrate-induced release of preloaded N-methyl-4-[3H]phenylpyridinium ([3H]MPP+). The uptake of substrates was sodium-dependent and was inhibited by ouabain and cocaine, which also prevented substrate-induced release of MPP+. Patch-clamp recordings revealed that all four substrates elicited voltage-dependent inward currents (on top of constitutive leak currents) that were prevented by cocaine. Whereas individual substrates had similar affinities in release, uptake, and patch-clamp experiments, maximal effects displayed remarkable differences. Hence, maximal effects in release and current induction were approximately 25% higher for D-amphetamine as compared with the other substrates. By contrast, dopamine was the most efficacious substrate in uptake experiments, with its maximal initial uptake rate exceeding those of amphetamine and tyramine by factors of 20 and 4, respectively. Our experiments indicate a poor correlation between substrate-induced release and the transport of substrates, whereas the ability of substrates to induce currents correlates well with their releasing action.

Induction by low Na+ or Cl- of cocaine sensitive carrier-mediated efflux of amines from cells transfected with the cloned human catecholamine transporters.

1. COS-7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [3H]-dopamine or [3H]-noradrenaline and superfused with buffers of different ionic composition. 2. In DAT cells lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux. Cocaine (10 microM) or mazindol (0.3 microM) blocked the efflux at low Na+, but not at 0 Na+. Lowering the Cl- concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 microM) was without effect in all the conditions tested. 3. In NAT cells, lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na+ than at 0 mM Na+. Efflux induced by 0, 5 or 10 mM Cl- was completely blocked by all three uptake inhibitors. 4. In cross-loading experiments, 5 mM Na(+)- or 0 Cl(-)-induced efflux was much lower from [3H]-noradrenaline-loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [3H]-dopamine-loaded NAT cells elicited by 5 mM Na+ or 0 Cl- was blocked by mazindol, as well as by desipramine. 5. Thus cloned catecholamine transporters display carrier-mediated efflux of amines if challenged by lowering the extracellular Na+ or Cl-, whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors.

Mechanism of the dopamine-releasing actions of amphetamine and cocaine: plasmalemmal dopamine transporter versus vesicular monoamine transporter.

The effects of amphetamine and cocaine were studied in [3H]-dopamine-loaded and superfused COS-7 cells transfected with either the cDNA of the plasmalemmal dopamine transporter ("DAT cells") or the cDNA of the vesicular amine transporter ("VAT cells"), or with both transporters ("DAT/VAT cells"). Amphetamine (0.01-100 microM, added for 4 min of superfusion) led to a concentration-dependent increase in dopamine release in DAT cells, as well as in DAT/VAT cells. The EC50 of the effect of amphetamine on DAT cells was 1.1 +/- 0.6 microM; the effect on DAT/VAT cells did not reach a plateau in the concentration range tested. With longer exposure to amphetamine, dopamine efflux from DAT cells reached a peak and quickly returned to baseline, in spite of the continued presence of the drug, whereas in DAT/VAT cells and in VAT cells the effect was sustained. Cocaine (up to 100 microM) did not exert any effect of its own in DAT cells or VAT cells but inhibited the amphetamine-induced release of dopamine from DAT cells in a competitive manner. In DAT/VAT cells cocaine and its analogue (-)-2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane caused an efflux of dopamine resembling that caused by amphetamine but quantitatively much smaller. The rank order of potency was the same as in uptake experiments [(-)-2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane > cocaine]. The effect of cocaine was mimicked by the reduction of chloride. The results indicate that there is a plasmalemmal component and a vesicular component in the dopamine-releasing action of amphetamine. The releasing action of cocaine is dependent on the existence of a vesicular pool of the neurotransmitter and seems to be linked to inhibition of the plasmalemmal dopamine transporter.

Functional sensitization of striatal dopamine D1 receptors in the 6-hydroxydopamine-lesioned rat.

Dopamine-stimulated adenylyl cyclase activity was measured in striatal homogenates of rats in which the nigrostriatal pathway was lesioned by 6-hydroxydopamine 20-24 months before the experiments. In the intact (contralateral) striatum the potency and the efficacy of dopamine in stimulating adenylyl cyclase was lower in the presence of high NaCl concentrations (120 mM) compared with the effects of dopamine in an NaCl-poor assay medium (20 mM). The same effect of NaCl was observed in the striatum on the side of a weak, behaviourally ineffective 6-hydroxydopamine lesion resulting in a loss of 57% of striatal dopamine. This effect of NaCl was absent in the strongly denervated striatum, i.e. in rats having a 99.8% dopamine loss and rotating when challenged with a low dose of apomorphine. Thus, in denervated vs intact striatum, in the presence of a physiological concentration of NaCl, dopamine-stimulated adenylyl cyclase showed a sensitization which was absent in assays with 20 mM NaCl. The inhibition of adenylyl cyclase by dopamine via D2 receptors, which was seen in the presence of 120 mM NaCl and the D1 antagonist SCH 23390, was not affected by denervation. We suggest that chronic dopaminergic denervation of the striatum results in a stabilized, i.e. NaCl-insensitive, high affinity state of D1 receptors. This may be the basis for a sensitization of the coupling mechanism of the denervated D1 receptors to adenylyl cyclase.

Uptake mechanism of technetium-99m-d, 1-HMPAO in cell cultures of the dissociated postnatal rat cerebellum.

The accumulation and retention mechanisms of 99mTc-d, 1-hexamethylpropyleneamine oxime (99mTc-d, 1-HMPAO) were investigated in cultures of the dissociated rat cerebellum. Our experiments indicate a linear dependency of the uptake on incubation time and on the concentration of the radioligand. Upon chloroform extraction and distribution between the lipophilic and the hydrophilic phases, we located 69.1% of the retained radioactivity in the hydrophilic phase, 24.1% in a bound state and 6.8% in the lipophilic phase. The water-soluble, unbound radioactive contents of the cultures were identified as 99mTcO4- by HPLC analysis. Treatment of cultures with diethyl maleate (DEM) inhibited the accumulation of radioactivity along with a reduction of the GSH contents of the cultures. However, even in the absence of GSH, significant amounts of radioactivity were accumulated. DEM reduced the radioactive contents of cultures predominantly by diminishing the aqueous phase of the chloroform-extracted material. By contrast, the metabolic state, manipulated by treating the cultures with oligomycin B or 2,4-dinitrophenol, had no significant effect on the accumulation of radioactivity. Our experiments suggest two major mechanisms for the retention of radioactivity following the exposure of neuronal tissue to 99mTc-d, 1-HMPAO: Conversion of the lipophilic complex to the hydrophilic product, 99mTcO4-, and binding to non-diffusible cell components.

Ornipressin in the treatment of functional renal failure in decompensated liver cirrhosis. Effects on renal hemodynamics and atrial natriuretic factor.

In 11 patients with decompensated cirrhosis and deteriorating renal function, the effect of the vasoconstrictor substance 8-ornithin vasopressin (ornipressin; POR 8; Sandoz, Basel, Switzerland) on renal function, hemodynamic parameters, and humoral mediators was studied. Ornipressin was infused at a dose of 6 IU/h over a period of 4 hours. During ornipressin infusion an improvement of renal function was achieved as indicated by significant increases in inulin clearance (+65%), paraaminohippuric acid clearance (+49%), urine volume (+45%), sodium excretion (+259%), and fractional elimination of sodium (+130%). The hyperdynamic circulation was reversed to a nearly normal circulatory state. The increase in systemic vascular resistance (+60%) coincided with a decrease of a previously elevated renal vascular resistance (-27%) and increase in renal blood flow (+44%). The renal fraction of the cardiac output increased from 2.3% to 4.7% (P less than 0.05). A decline of the elevated plasma levels of noradrenaline (2.08-1.13 ng/mL; P less than 0.01) and renin activity (27.6-14.2 ng.mL-1.h-1; P less than 0.01) was achieved. The plasma concentration of the atrial natriuretic factor increased in most of the patients, but slightly decreased in 3 patients. The decrease of renal vascular resistance and the increase of renal blood flow and of the renal fraction of cardiac output play a key role in the beneficial effect of ornipressin on renal failure. These changes develop by an increase in mean arterial pressure, the reduction of the sympathetic activity, and probably of an extenuation of the splanchnic vasodilation. A significant contribution of atrial natriuretic factor is less likely. The present findings implicate that treatment with ornipressin represents an alternative approach to the management of functional renal failure in advanced liver cirrhosis.

Lower efficacy of the dopamine D1 agonist, SKF 38393, to stimulate adenylyl cyclase activity in primate than in rodent striatum.

The selective D1 agonist, SKF 38393, stimulated adenylyl cyclase by about 40% of basal activity in rat striatum but by only about 10% in the striatum of rhesus monkeys. In contrast, dopamine stimulated striatal adenylyl cyclase in both species with equal efficiency (70-80%). SKF 38393 30 microM inhibited the effect of 30 microM dopamine by about 45% in rat and by about 75% in primate tissue. This difference may be due to a lower D1 receptor reserve in primate than in rodent tissue and suggests that only selective D1 agonists with full efficacy at D1 receptors can be expected to have beneficial effects in patients with Parkinson's disease.

Cholinergic deficit induced by ethylcholine aziridinium (AF64A) in rat hippocampus: effect on glutamatergic systems.

The reaction of the glutamatergic systems in the rat hippocampus to the withdrawal of cholinergic function after cholinergic degeneration induced by ethylcholine aziridinium (AF64A) was investigated. Furthermore, the question whether blockade of N-methyl-D-aspartate (NMDA) receptors by MK-801 has an impact on the extent of the cholinergic lesion was addressed. After bilateral intracerebroventricular injection of AF64A (2 nmol/ventricle) the activity of choline acetyltransferase (ChAT) started to decline in the hippocampus within 24 h. The reduction of ChAT activity reached its maximum within 4 days (65%) and persisted during the observation period of 65 days. The loss of ChAT activity was accompanied by a transient decline in the level of glutamate, which was most pronounced 1 to 2 days after AF64A (25% reduction). Within 65 days the glutamate level returned to normal. A detailed subdissection of the hippocampus revealed that the cholinergic system was most affected in the ventral part of the hippocampus and the CA3 subfield. On the other hand, the transient reduction in glutamate was restricted to the CA1 and CA3 area. In the dentate gyrus the marked loss of cholinergic function was not accompanied by any reduction in glutamate level. Treatment of the AF64A-injected rats with the muscarinic agonist pilocarpine prevented the decline in glutamate levels. The transient nature of the decline in glutamate as well as its reversal by treatment with pilocarpine are suggestive of an increased release of glutamate in response to the withdrawal of cholinergic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Manufacture and use of fused silica cannulas for intracerebral injections in freely moving rats.

This report describes in detail the assembly and use of miniature cannulas for intracerebral injections in freely moving rats. Guide and injection cannulas were prepared from polyimid-coated fused silica capillaries as routinely in use for gas chromatography, in this way avoiding the permanent contact of stainless-steel with brain tissue. The outer diameter of the guide capillary measured 300 microns, that of the injection capillary 170 microns. In spite of their fragile appearance, the capillaries were robust and easy to handle. In the same animal, up to 20 consecutive injections were possible. In the course of 2 years 204 cannulas directed to the amygdala or the lateral ventricle were implanted, and only in 8 rats (4%) the inner capillary broke and plugged the guide cannula, making injections impossible. Histological examination of brains implanted for up to 2 months demonstrated only blunt scar formation along the cannula's track. No changes in transmitter levels (biogenic amines, amino acids) could be detected in chronically implanted rat brains except for a slight increase in serotonergic activity in the contralateral piriform cortex. Intra-amygdaloid injection of 0.32 microliters 0.9% saline resulted in a mild and transient reduction of GABA levels in both piriform cortices, without any detectable persistent histopathological consequences.

Evidence for neuronal localization of histamine-N-methyltransferase in rat brain.

There is evidence that histamine may be a neurotransmitter in mammalian brain. Histamine in neurons of the central nervous system is easily released and rapidly turned over. The cellular localization of histamine-N-methyltransferase, the proposed histamine-inactivating enzyme, was investigated by measuring its activity in rat striatum after applying neurochemical or electrolytic lesions. The results indicate a major neuronal localization of the enzyme in this area.