Alpha-tocopherol and beta-carotene do not protect marmosets against the dopaminergic neurotoxicity of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Idiopathic Parkinson's disease (PD) may possibly be caused by one or more unidentified neurotoxins present in the environment, or formed endogenously, which progressively damage dopaminergic nigrostriatal neurons. N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is an experimental neurotoxin which produces biochemical and neuropathological changes in humans, lower primates and mice that closely resemble those found in PD. Because the mechanisms of neuronal damage in both idiopathic PD and in the MPTP model of PD may involve free radical formation in the substantia nigra, antioxidants might protect dopaminergic neurons. Previously, we found that both alpha-tocopherol and beta-carotene partially protected mice against MPTP. However, in the experiments described in this paper, neither alpha-tocopherol nor beta-carotene, each administered in massive doses, had any demonstrable protective effect for dopaminergic nigrostriatal neurons in marmosets injected with low doses of MPTP. Without more knowledge about the identity of the neurotoxin(s) causing idiopathic PD, and their mechanism of action, it is not possible at this time to predict whether these 2 antioxidants might be clinically useful in preventing or ameliorating PD.

Inability to produce a model of dialysis encephalopathy in the rat by aluminum administration.

We attempted to produce a rat model of brain aluminum toxicity in order to explore whether or not aluminum accumulation produces the neurochemical changes observed in brains of patients who die with dialysis encephalopathy. Daily subcutaneous injection of Al(OH)3 caused marked elevation of serum aluminum concentrations, but did not increase brain aluminum contents, either in rats with normal renal function, or in rats with unilateral or 5/6 nephrectomies. LiCl pretreatment, which has been reported to cause irreversible renal failure, did not impair renal function nor aid in achieving elevated brain aluminum contents. No reductions in brain contents of gamma-aminobutyric acid (GABA) or in glutamic acid decarboxylase (GAD, E.C.4.1.1.15) and choline acetyltransferase (ChAT, E.C.2.3.1.6) activities were observed in aluminum-treated rats. We conclude that the rat is not a suitable laboratory animal to explore the role of aluminum toxicity in causing the GABA and ChAT deficits present in brains of hemodialyzed human patients.

Chronic organic manganese administration in the rat does not damage dopaminergic nigrostriatal neurons.

In an attempt to produce an animal model of Parkinson's disease, we injected rats repeatedly with high doses of methylcyclopentadienyl manganese tricarbonyl (MMT), a compound which has been reported to lower striatal dopamine content in mice. Chronic MMT administration for up to 5 months, even though it produced a substantial elevation in brain manganese content during the period of exposure, did not destroy dopaminergic nigrostriatal neurons. This was assessed by measurements of tyrosine hydroxylase activity and contents of dopamine and its metabolites in the striatum, and by histological examination of the substantia nigra. Our results differ from those of others who administered manganese chloride in drinking water to rats. This discrepancy is unlikely to be a consequence of differences in duration of exposure or route of administration. It could be due to our having used an organic rather than an inorganic manganese compound, or to a species difference in vulnerability to organic manganese between rats and mice.

Neurochemical abnormalities in brains of renal failure patients treated by repeated hemodialysis.

We examined autopsied brain from 10 patients with end-stage renal failure who had undergone repeated hemodialysis. Eight had classic symptoms, and two had suggestive symptoms of dialysis encephalopathy. Findings were compared with those in autopsied brain from control adults who had never been hemodialyzed. Mean gamma-aminobutyric acid (GABA) contents were significantly reduced in frontal and occipital cortex, cerebellar cortex, dentate nucleus, caudate nucleus, and medial-dorsal thalamus of the hemodialyzed patients, the reduction being greater than 40% in cerebral cortex and thalamus. Choline acetyltransferase activity was reduced by 25-35% in three cortical regions in the hemodialyzed patients. These two abnormalities were observed in the brain of each hemodialyzed patient, regardless of whether or not the patient died with unequivocal dialysis encephalopathy. Pyridoxal phosphate contents were substantially reduced in brains of the hemodialyzed patients, but metabolites of noradrenaline, 3,4-dihydroxyphenylethylamine (dopamine), and 5-hydroxytryptamine (serotonin) were present in normal amounts. Aluminum levels were abnormally high in frontal cortical gray matter in the hemodialyzed patients. Although this study does not clarify the role played by aluminum toxicity in the pathogenesis of dialysis encephalopathy, the abnormalities we found suggest the need for further neurochemical investigations in this disorder.

Retarded recovery of excitability and twitches of frog toe muscles after K-induced depolarization (slow inactivation) in the presence of alkyl amphipaths.

Alkyl amphipaths resemble conventional local anesthetics in their ability to retard the recovery of excitability and twitch tension after depolarization at high Ko, an effect that is attributed to slow inactivation of potential-dependent sodium channels. The similar effect of low temperature offers an explanation for its ability to enhance the frequency-dependent effects of these agents.

The influence of temperature and frequency of stimulation on the impairment of excitability of frog skeletal muscle by local anesthetics and alkyl amphipathic agents.

The potency of various types of alkyl amphipathic (cationic, anionic, and neutral) as well as tertiary amine local anesthetics in impairing the excitability of frog skeletal muscle was markedly enhanced by an increase in temperature from 20 to 30 degrees C. Enhancement of the local anesthetic effects of all types of agents was also produced by a decrease in temperature to 5 degrees C, but this effect was found to be frequency dependent. With abrupt increase or decrease in temperature, changes in excitability were rapid and unlikely to be the result of changes in the partition of the apolar portions of these molecules into the hydrophobic regions of the sarcolemma. These results are interpreted as indicating that both the presence of local anesthetics and alterations in temperature can influence the rates of potential-dependent changes in the conformation of membrane proteins that control the permeability of excitable sodium channels, possibly by modifying the fluidity of specific portions of their hydrophobic components or their immediate lipid environment. The accumulation of inactivation as the result of incomplete recovery from the effects of preceding depolarizations appears sufficient to explain the frequency-dependent effects produced by these agents.

Partial protection from the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by four different antioxidants in the mouse.

C57 black mice given a single injection of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 40 mg/kg, developed marked reduction of striatal dopamine content and loss of dopaminergic neurons in the zona compacta of the substantia nigra. However, pretreatment with any one of four different antioxidants, alpha-tocopherol, beta-carotene, ascorbic acid or N-acetylcysteine, significantly decreased MPTP-induced striatal dopamine loss, and alpha-tocopherol prevented neuronal loss in the substantia nigra. Four chemical analogues of MPTP (cinnamaldehyde, N,N-dimethylcinnamylamine, arecoline and 2-methyl-1,2,3,4-tetrahydro-6,7-isoquinolinediol) were all found to lack dopaminergic nigrostriatal neurotoxicity in the mouse.

Effects of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and its metabolite, N-methyl-4-phenylpyridinium ion, on dopaminergic nigrostriatal neurons in the mouse.

A single subcutaneous injection in the C57 black mouse of 40 mg/kg of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a 90% depletion of striatal dopamine, as well as loss of 33% of neuronal cell bodies in the substantia nigra, zona compacta. By 4.5 months, there appears to be partial recovery of striatal dopaminergic function. After injection into mice, the MPTP metabolite, N-methyl-4-phenylpyridinium ion (MPP+) enters the striatum, and through some unknown mechanism is even more toxic than MPTP. However, repeated injections of maximally tolerated amounts of MPP+ do not damage dopaminergic nigrostriatal neurons.

The influence of D2O, perchlorate, and variation in temperature on the potential-dependent contractile function of frog skeletal muscle.

D2O and perchlorate manifest opposing effects on the contractile function of skeletal muscle (amplitude of twitches and maximum K contractures, potential dependence of contraction activation and inactivation), and when combined the influence of one may effectively antagonize that of the other. The ratio of perchlorate concentrations required to produce effects of equal intensity (e.g., twitch enhancement and restoration of maximum K contractures in media lacking divalent cations or containing a depressant concentration of a cationic amphipath) in H2O and D2O solutions was generally rather constant. These findings are compatible with the view that both agents can influence contractile function by virtue of their effects on solvent structure. In the absence of divalent cations, the effects of reduced temperature resemble those of D2O whereas the effects of increased temperature resemble those of the chaotropic anion. However, in other media, variation in temperature was found to result in additional nonsolvent effects so that low temperature could oppose rather than enhance the effects of D2O. These observations are discussed in terms of a model postulates a role for solvent influences on the kinetics of two separate potential-dependent conformational transitions of membrane proteins which mediate the activation and inactivation of contraction in skeletal muscle.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) does not destroy nigrostriatal neurons in the scorbutic guinea pig.

Guinea pigs were injected subcutaneously with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in maximal tolerated doses (8 mg/kg, once daily) for 10 or 15 days. No neurological effects were noted, other than sedation and hypotonia lasting a few hours after each injection, either in animals maintained on normal diet or in animals fed an ascorbate-deficient diet and rendered severely scorbutic. Subsequent chemical analyses of the striatum showed no evidence of lasting damage to nigrostriatal dopaminergic neurons in MPTP treated guinea pigs on normal diet, and minimal evidence of permanent damage to these neurons in scorbutic animals. MPTP was undetectable in the urine of MPTP-treated animals, although a metabolite, presumably 1-methyl-4-phenylpyridinium ion (MPP+) was regularly present in urine. The relative lack of neurotoxicity of MPTP in the guinea pig remains unexplained. This species clearly is not a suitable small animal for MPTP-induced parkinsonism.

Is a circulating neurotoxin involved in the pathogenesis of Huntington's chorea?

We tested the hypothesis that the premature neuronal death which occurs in Huntington's chorea (HC) might be the result of a genetically-determined enzymatic failure in the degradation of a circulating neurotoxin of either endogenous of exogenous origin. Infant rats were given daily subcutaneous injections of large quantities of whole serum (for 24 days), or of a concentrated serum ultrafiltrate (for 37 days), obtained from HC patients or control subjects. Animals were killed 4 months after the end of injections, and their striata were examined neurochemically. There was a significant but small (16%) reduction in the mean striatal content of gamma-aminobutyric acid (GABA) in rats treated with whole serum from HC patients, but no striatal GABA deficiency was observed in rats treated with ultrafiltrates of serum from HC patients. Nor did these rats have any reduction in their striatal choline acetyltransferase activity. We conclude that if a circulating neurotoxin does contribute to the pathogenesis of HC, it must either be a small molecule which is tightly bound to serum proteins, or less likely a large compound with a molecular weight greater than 10 000.

A comparison of the effects of cationic, anionic, and neutral amphipathic agents on the contractile behaviour of frog skeletal muscle. I. Twitches and potential threshold for contraction.

Cationic, anionic, and neutral amphipathic agents displayed striking differences as well as similarities in their effects on the contractile function of frog skeletal muscle. Slowed repolarization during the action potential appeared to account for twitch potentiation by low concentrations of alkyl trimethylammonium and by small n-alkanols (propanol, butanol). Small n-alkanols also caused a decrease in the potential threshold for K contractures and slower relaxation of submaximum K contractures as well as enhancement of chloride withdrawal and caffeine contractures, but these effects were not observed with larger alkanols. For the ionic amphipathic agents, the direction of the changes in the relation between Ko and K-contracture tension could be accounted for on the basis of the expected changes in surface charge, but the effects of these two types of agents on the rate of relaxation of submaximum K contractures were disproportionate and with the cationic series were opposite in direction to those produced by inorganic divalent cations. The reductions in the amplitude of chloride-withdrawal contractures by cationic as well as anionic amphipaths indicated that both types of agents can impair excitation-contraction coupling. Similar depressant effects on caffeine contractures demonstrate that these responses also can be influenced by events restricted to the external lamina of the sarcolemma. It is concluded that opposite effects can be produced by similar perturbations in different regions of the sarcolemma and that electrostatic as well as hydrophobic interactions can make an important contribution to the effects of amphipathic agents on twitches and contractures in skeletal muscle.

A comparison of the effects of cationic, anionic, and neutral amphipathic agents on the contractile behaviour of frog skeletal muscle. II. Amplitude of depolarization and repolarization-induced contractures.

Sufficiently high concentrations of cationic (n-alkyl trimethylammonium) or neutral (n-alkanols) amphipathic agents reduced the amplitude of maximum K contractures of frog toe muscles, an effect which was antagonized by reduced temperature, by the presence of perchlorate anions, or (to a lesser extent) by an increased concentration of divalent cations. Enhancement of the similar effect of tetracaine was prominent only with alkyl trimethylammonium compounds. Enhancement of the depressant effect of acidity (pH 5.0) was observed with octyl trimethylammonium and octanol but not with octanesulfonate or butanol. Partial restoration of potassium contractures in media lacking divalent cations was produced by octane- or nonane-sulfonate and by propanol but not by octyl trimethylammonium or octanol. The alkyl sulfonates differed from the other agents studied in producing tonic contractures at concentration which did not reduce maximum K-contracture tension. The alkyl sulfonates also differed from other amphipaths of similar size in their ability to elicit small repolarization-induced contractures in the absence of perchlorate, although this property also was shared by small alkanols. Sufficient concentrations of all amphipaths reduced the amplitude of repolarization-induced contractures in the presence of perchlorate. The intensity of the effects of these agents on contractile function usually was proportional to the size of their apolar group, and with ionic apmphipaths such effects were apparent only with compounds having hydrocarbon chains containing eight or more carbon atoms. These experiments indicate that hydrophobic interactions in the external lamina of the sarcolemma can influence the potential-dependent control of contractile function in skeletal muscle, presumably by effects on the conformational transitions of integral membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Nigrostriatal dopaminergic neurons remain undamaged in rats given high doses of L-DOPA and carbidopa chronically.

Rats were fed maximally tolerated doses of L-3,4-Dihydroxyphenylalanine (L-DOPA) and carbidopa daily for 120 days in order to achieve a sustained elevation in brain dopamine levels. Some animals were also given buthionine sulfoximine, a gamma-glutamylcysteine synthetase inhibitor, in an unsuccessful effort to reduce brain glutathione contents. L-DOPA- and carbidopa-treated animals displayed no behavioral changes suggestive of nigrostriatal dopaminergic neuronal loss. When sacrificed 60 days after L-DOPA treatment ended, all rats had normal tyrosine hydroxylase activities and dopamine contents in their striata, and cell counts were normal in the substantia nigra. It therefore seems unlikely that a model of Parkinson's disease, suitable for exploring the etiological importance of glutathione deficiency, can be produced in rats merely by administering the largest tolerable doses of L-DOPA.

A comparison of the local anaesthetic effects of cationic, anionic, and neutral amphipathic agents in frog skeletal muscle.

Impermeant alkyl amphipathic agents reduced the excitability of frog twitch muscle fibers, indicating that local anaesthesia can be produced by perturbations within the external lamina of the sarcolemma. Cationic (n-alkyl trimethylammonium) and anionic (n-alkyl sulfonate) as well as permeant neutral (n-alkanol) agents have been compared with regard to their local anaesthetic potency. Small impermeant agents (fewer than six carbon atoms) alone were ineffective. Within each series the concentration required to reduce excitability was proportional to the length (hydrophobicity) of the hydrocarbon chain attached to the polar group. However, corresponding members of these three series of compounds differed considerably in their local anaethetic potency. Hence, charged groups as well as apolar moieties can influence local anaesthetic efficacy. The supra-additive character of the local anaesthesia produced by combining impermeant alkyl anions and cations indicates that these two types of amphipaths may produce their similar effects by perturbations at different membrane sites.

The role of electrostatic interactions in modulating the local anaesthetic effects of amphipathic agents in frog skeletal muscle.

The local anaesthetic effect of cationic, anionic, and neutral alkyl amphipathic agents was similarly enhanced in an apparently nonspecific way by circumstances which modulate electrostatic interactions (acidity, modification of charged groups at the sarcolemmal surface by group-specific reagents, or changes in the calcium concentration), presumably as the result of secondary effects on the conformation of membrane proteins. However, the selective enhancement of the local anaesthetic effect of alkyl trimethylammonium compounds by perchlorate implies a more specific interaction which may influence the penetration of hydrophobic groups into the membrane interior.

Effect of an impermeant arginine-modifying reagent on the responses of rabbit platelets to agonists.

The importance of surface arginyl residues in platelet aggregation was investigated by studying the effects of an impermeant arginine-modifying reagent, p-sulfonylphenylglyoxal (PSPG), on platelet responses to various agonists. Pretreatment of resuspended rabbit platelets with 2-15 mM PSPG resulted in complete inhibition of aggregation responses to ADP and 5-HT, and a concentration-dependent inhibition of the preceding shape change. Aggregation responses to thrombin also were inhibited in a concentration-dependent manner. The protective effects of antagonists of these three agonists (beta, gamma-methylene ATP for ADP, hirudin for thrombin and phentolamine for 5-HT) during pretreatment of platelets with PSPG indicated that intact arginine residues form part of the receptor sites for ADP and for thrombin. Arginine residues are not part of the 5-HT receptor site itself, but seem to be important for the maintenance of the functional integrity of this site.

The effect of local anaesthetics and antiarrhythmic agents on the responses of rabbit platelete to ADP and thrombin.

Local anaesthetics and antiarrhythmic agents produced changes in responses of rabbit platelets to ADP and thrombin that varied with the agent and its concentration. In high concentrations all local anaesthetics decreased aggregation rates. At lower concentrations several local anaesthetics increased aggregation rates. Depending on the agonist and local anaesthetic, increases were produced at all or only at low or high agonist concentrations. Local anaesthetics failed to influence shape change except at concentrations much greater than those which inhibited aggregation. Inhibition of aggregation by procaine was potentiated by small organic anions suggesting effects at different sites on the platelet membrane. The inhibitory effect of local anaesthetics was decreased by increasing Ca. Kinetic analysis indicated different mechanisms for this Ca effect, i.e., predominantly competitive for procaine or lidocaine and predominantly non-competitive for tetracaine or ethyl lidocaine. Local anaesthetics may affect aggregation by modifying the participation of Ca in this process.