Mechanisms of Toxicity and Cellular Resistance to 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and 1-Methyl-4-Phenylpyridinium in Adrenomedullary Chromaffin Cell Cultures

Bovine adrenomedullary chromaffin (BAMC) cells, cultured in a defined medium, were used to study the mechanisms of toxicity and cellular resistance to the catecholamine neuron toxicants 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+). The viability of the cells was assessed biochemically [cellular catecholamine content and the catalytic activities of tyrosine hydroxylase (TH) and lactate dehydrogenase (LDH)] and anatomically (by electron microscopy). When cultures of BAMC cells were exposed to MPTP or MPP+ for 3 days, a marked loss of cellular catecholamines and TH activity was observed. The addition of an inhibitor of monoamine oxidase (MAO) B (Ro 19-6327), but not MAO A (clorgyline), prevented the toxicity of MPTP but not that of MPP+. In addition, the cellular toxicity of MPP+, but not MPTP, was antagonized by desmethylimipramine, an inhibitor of cellular catecholamine uptake. The toxicity of MPP+ was time dependent, with losses of TH and the release of cellular LDH occurring after 48 h in culture. Catecholamine depletion occurred somewhat sooner, being evident after 24 h of exposure to MPP+. The cellular toxicity of MPP+ was concentration dependent and significantly enhanced by inhibitors of catecholamine vesicular uptake (reserpine, tetrabenazine, or Ro 4-1284). Electron microscopic examination of cells treated with either MPP+, tetrabenazine, or their combination revealed that MPP+ damaged BAMC cells and that this damage was markedly potentiated by the inhibition of vesicular uptake by tetrabenazine. The concentration of glucose in the culture media of untreated cells slowly decreased as a function of time. The rate of glucose consumption was markedly accelerated by MPP+ treatment and the losses in cell TH and the release of LDH into the media were preceded by a 99% depletion of glucose from the media. In cultures not treated with MPP+, lactate accumulated in the media as a function of time. Addition of MPP+ to the media increased the formation of lactate, in a concentration-dependent manner. Reserpine pretreatment further enhanced the production of lactate in response to MPP+. Culturing cells in glucose-free medium greatly potentiated the effects of MPP+ on cellular TH and catecholamines. The toxicity observed after 3 days' exposure of BAMC cells to MPP+ could be prevented when the medium was replaced with fresh medium every 24 h. The effects of glucose deprivation and reserpine were observed to be additive. The ability of MPP+ to affect mitochondrial function is determined by the capacity of the storage vesicle to sequester the pyridinium, acting as a cytosolic "buffer."(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the Characterization of the Inhibitory Mechanism of 4'-Alkylated 1-Methyl-4-Phenylpyridinium and Phenylpyridine Analogues in Mitochondria and Electron Transport Particles

Abstract: 1-Methyl-4-phenylpyridinium (MPP⁺), the toxic agent in MPTP-induced dopaminergic neurotoxicity, is thought to act by inhibiting mitochondrial electron transport at complex I. This study examined this latter action further with a series of 4′-alkylated analogues of MPP⁺. These derivatives had IC₅₀ values that ranged from 0.5 to 110 µM and from 1.6 to 3,300 µM in mitochondria and electron transport particles (ETPs), respectively. The IC₅₀ values of corresponding 4′-alkylated phenylpyridine derivatives to inhibit NADH-linked oxidation ranged from 10 to 205 µM in mitochondria and from 1.7 to 142 µM in ETPs. The potencies of both classes of inhibitors directly correlated with their ability to partition between 1-octanol and water. In mitochondria, increased hydrophobicity resulted in greater inhibition of NADH dehydrogenase but a smaller dependence on the transmembrane electrochemical gradient for accumulation of the pyridiniums as evidenced by an ～600-fold, versus only a 36-fold, increase in the IC₅₀ of MPP⁺ versus 4′-pentyl-MPP⁺, respectively, in the presence of uncoupler. In ETPs, the analogous increase in potencies of the more hydrophobic analogues was also consistent with an inhibitory mechanism that relied on differential partitioning into the lipid environment surrounding NADH dehydrogenase. However, the pyridinium charge must play a major role in explaining the inhibitory mechanism of the pyridiniums because their potencies are much greater than would be predicted based solely on hydrophobicity. For example, in ETPs, 4′-decyl-MPP⁺ was nearly 80-fold more potent than phenylpyridine although the latter compound partitions twice as much into 1-octanol. In addition, the lipophilic anion TPB^? was a more effective potentiator of inhibition by pyridiniums possessing greater hydrophilicity (0–5 carbons), consistent with facilitation of accumulation of these analogues within the membrane phase of complex I, probably via ion pairing. These studies delineate further the mechanisms by which this class of compounds is able to accumulate in mitochondria, inhibit complex I activity, and thereby, effect neurotoxicity.     

Toxicity of 1-Methyl-4-Phenylpyridinium for Rat Dopaminergic Neurons in Culture: Selectivity and Irreversibility

Cultures of dissociated embryonic rat mesencephalic cells were exposed to 10 microM 1-methyl-4-phenylpyridinium (MPP+), a concentration shown earlier to result in loss of greater than 85% of tyrosine hydroxylase (TH)-positive neurons without affecting the total number of cells observed by phase-contrast microscopy. To characterize better the selectivity of the toxic action of MPP+, other parameters were measured reflecting survival and function of dopaminergic or nondopaminergic neurons. Exposure of cultures to 10 microM MPP+ for 48 h reduced TH activity to 11% of control values without reducing protein levels. [3H]Dopamine uptake was reduced to less than 4% of control values, whereas the uptake of gamma-[3H]aminobutyric acid ([3H]GABA) was not affected in these cultures. This same treatment failed to reduce the number of cholinergic cells visualized in septal cultures and did not affect either choline acetyltransferase activity or high-affinity choline uptake. To assess for possible recovery of dopaminergic neurons, cultures were exposed to 10, 1.0, or 0.1 microM MPP+ for 48 h and then kept for up to 6 days in MPP(+)-free medium. After exposure to 10 microM MPP+, the number of TH-positive neurons, their neurite density, TH activity, and [3H]dopamine uptake remained at constant, reduced levels throughout the period of observation after termination of exposure, whereas GABA uptake remained normal. Treatment with lower concentrations of MPP+, i.e., 1.0 and 0.1 microM, induced less pronounced dopaminergic toxic effects. However, no recovery was seen after posttreatment incubation in toxin-free medium. These findings provide evidence that MPP+ treatment results in highly selective and irreversible toxicity for cultured dopaminergic neurons.     

1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Metabolism and l-Methyl-4-Phenylpyridinium Uptake in Dissociated Cell Cultures from the Embryonic Mesencephalon

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant found in a synthetic illicit drug, can elicit in humans and monkeys a severe extrapyramidal syndrome similar to Parkinson's disease. It also induces alterations of the dopamine (DA) pathways in rodents. MPTP neurotoxicity requires its enzymatic transformation into 1-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase followed by its concentration into target cells, the DA neurons. Here, we show that mesencephalic glial cells from the mouse embryo can take up MPTP in vitro, transform it into MPP+, and release it into the culture medium. MPTP is not taken up by neurons from either the mesencephalon or the striatum in vitro (8 days in serum-free conditions). However, mesencephalic neurons in culture revealed a high-affinity uptake mechanism for the metabolite MPP+, similar to that for DA. The affinity (Km) for DA uptake is fivefold higher than that for MPP+ (0.2 and 1.1 microM, respectively), whereas the number of uptake sites for MPP+ is double (Vmax = 25 and 55 pmol/mg of protein/min for DA and MPP+, respectively). Mazindol, a DA uptake inhibitor, blocks the uptake of DA and MPP+ equally well under these conditions. Moreover, by competition experiments, the two molecules appear to use the same carrier(s) to enter DA neurons. Small concentrations of MPP+ are also taken up by striatal neurons in vitro. The amount taken up represented less than 10% of the MPP+ uptake in mesencephalic neurons. Depolarization induced by veratridine released comparable proportions of labeled DA and MPP+ from mesencephalic cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Irreversible Inhibition of Mitochondrial Complex I by 1-Methyl-4-Phenylpyridinium: Evidence for Free Radical Involvement

Abstract: Incubation of 10 m M I-methyl-4-phenylpyridinium (MPP⁺) with sonicated beef heart mitochondria caused an irreversible time-dependent decrease in NADH-ubiquinone-l (CoQ₁) reductase activity (52% inhibition after 1 h). Inclusion of glutathione, ascorbate, or catalase in the incubation mixture protected the NADH-CoQ₁ reductase activity. These results suggest that the interaction of MPP⁺ with complex I induces free radical generation, which in turn leads to the irreversible inhibition of complex I activity. The generation of free radicals by neurotoxin-induced inhibition of complex I has important implications for our interpretation of the increased oxidative stress observed in Parkinson's disease substantia nigra and for our understanding of the cause(s) of dopaminergic cell death in this disorder.     

Tyrosine Hydroxylase mRNA Expression by Dopaminergic Neurons in Culture: Effect of 1 -Methyl-4-Phenylpyridinium Treatment

To enable us to study expression of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] as a measure of dopaminergic neuron function in future experiments, methods were developed to quantify TH mRNA levels in cultures of dopaminergic mesencephalic cells. The model of selective dopaminergic toxicity of 1-methyl-4-phenylpyridinium (MPP+) was used to verify the specificity of our methods. Fetal (embryonic day 15) rat ventral mesencephalic cell cultures were treated with 15 microM MPP+ for 48 h, conditions previously shown to reduce the number of TH-immunoreactive neurons, TH activity, and dopamine uptake to 5-10% of control values. This treatment decreased the number of neurons labeled by TH in situ hybridization to 9% of untreated controls and caused a strong reduction of the abundance of TH mRNA in Northern blots. Our findings establish TH mRNA expression as a parameter for future studies of toxic and trophic effects on cultured dopaminergic neurons, and they support the view that MPP+ destroys dopaminergic neurons.     

Uptake of 1-Methyl-4-Phenylpyridinium and Dopamine in the Mouse Brain Cell Nuclei

Abstract: The neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes, via its metabolite 1-methyl-4-phenylpyridinium (MPP⁺), parkinsonism in humans, monkeys, and mice but not in rats. When incubated with mouse brain homogenates, [³H]-MPP⁺ is recovered in relatively large concentrations in the brain cell nucleus. Although isolated cell nuclei from rat and mouse brain contain uptake systems for dopamine (DA), only brain cell nuclei from mice avidly take up [³H]MPP⁺. This nuclear uptake is ATP dependent and can be blocked by ouabain and Nethylmaleimide. It is not, however, affected by neuronal and vesicular blockers such as benztropine. mazindol, and reserpine. Selective uptake of MPP⁺ into brain cell nuclei may provide a new avenue for future investigation into the complex modes of action of the neurotoxin MPTP.     

EGb761 Pretreatment Reduces Monoamine Oxidase Activity in Mouse Corpus Striatum During 1-Methyl-4-Phenylpyridinium Neurotoxicity

EGb761 produces reversible inhibition of both monoamine oxidase (MAO) isoforms in the central nervous system. 1-Methyl-4-phenylpyridinium (MPP+) neurotoxicity is prevented by treatment with the MAO inhibitor pargyline. We investigated EGb761's effect on striatal MAO activity during MPP+ neurotoxicity. C-57 black mice were pretreated with EGb761 (10 mg/kg) daily for 17 days followed by administration of MPP+ (0.72 mg/kg). MPP+ enhanced striatal MAO (30%) activity at 6 h, and EGb761 prevented this effect. MAO-B activity in striatum was enhanced (70%) 6 h after MPP+ administration and was reduced to almost normal levels in EGb761 + MPP+ group compared to MPP+ group. Pretreatment with EGb761 partially prevented (32%) the striatal dopamine-depleting effect of MPP+ and prevented the reduction in striatal tyrosine hydroxylase activity (100%). Results suggest that EGb761 supplements may be effective in reducing MAO activity as well as enhancement in dopamine metabolism, thereby preventing MPP+-neurotoxicity.     

Effects of 1-Methyl-4-Phenylpyridinium on Isolated Rat Brain Mitochondria: Evidence for a Primary Involvement of Energy Depletion

Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP⁺) on the oxygen consumption, ATP production, H₂O₂ production, and mitochondrial NADH-CoQ₁ reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP⁺ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP⁺ produced a concentration-dependent decrease in H₂O₂ production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP⁺ caused no loss of complex I activity after washing of MPP⁺ from the mitochondrial membranes. These data are consistent with MPP⁺ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H₂O₂ by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H₂O₂ in the presence of Cu²⁺ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP⁺ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.     

Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and 1-Methyl-4-Phenylpyridinium Ion on Activities of the Enzymes in the Electron Transport System in Mouse Brain

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) on activities of enzyme complexes in the electron transport system were studied using isolated mitochondrial preparations from C57BL/6J mouse brains. Both MPTP and MPP+ dose-dependently inhibited activity of NADH-ubiquinone oxidoreductase (EC 1.6.5.3). The inhibition was reversible. Preincubation of freeze-thawed mitochondria with MPTP or MPP+ had no effect on the inhibition; however, when nonfrozen mitochondria were used, NADH-ubiquinone oxidoreductase activity was reduced to 46% of that in the nonincubated sample after a 5-min preincubation with MPTP and to 77% of that in the nonincubated sample after a 5-min preincubation with MPP+. Kinetic analyses revealed that inhibition of MPTP was noncompetitive and that of MPP+ uncompetitive with respect to NADH. On the other hand, inhibition of MPTP was uncompetitive and that of MPP+ noncompetitive with respect to ubiquinone. Succinate-ubiquinone oxidoreductase (complex II), dihydroubiquinone-cytochrome c oxidoreductase (complex III), and ferrocytochrome c-oxygen oxidoreductase (EC 1.9.3.1) activities were either slightly inhibited or not inhibited by MPTP or MPP+. The significance of these findings is discussed in relation to the mechanism of MPTP-induced neuronal degeneration.     

Studies on the Neurotoxicity of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine: Inhibition of NAD-Linked Substrate Oxidation by Its Metabolite, 1-Methyl-4-Phenylpyridinium

Abstract: The effects of the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its 4-electron oxidation product 1-methyl-4-phenylpyridinium (MPP⁺) were studied in isolated mitochondria and in mouse brain striatal slices. ADP-stimulated oxidation of NAD-linked substrates was inhibited in a time-dependent manner by MPP⁺ (0.1–0.5 m M ), but not MPTP, in mitochondria prepared from rat brain, mouse brain, or rat liver. Under identical conditions, succinate oxidation was relatively unaffected. In neostriatal slices prepared from the mouse, a species susceptible to the dopaminergic neurotoxicity of MPTP, incubation with either MPP⁺ or MPTP caused metabolic changes consistent with inhibition of mitochondnial oxidation, i.e., an increase in the formation of lactate and accumulation of the amino acids glutamate and alanine with concomitant decreases in glutamine and aspartate levels. The changes resulting from incubation with MPTP were prevented by the monoamine oxidase inhibitor pargyline, which blocks formation of MPP⁺ from MPTP. The results suggest that compromise of mitochondrial function and its metabolic sequelae within dopaminergic neurons could be an important factor in the neurotoxicity observed after MPTP administration.     

Uptake, Release, and Subcellular Localization of l-Methyl-4-Phenylpyridinium in Blood Platelets

The neurotoxic compound 1-[methyl-3H]-4-phenylpyridinium ([3H]MPP+) was actively taken up by human, rabbit, and guinea pig platelets incubated in plasma. In human platelets, the apparent Km of this uptake (22.6 microM) was 50 times higher than that for serotonin [5-hydroxytryptamine (5-HT]). The uptake of [3H]MPP+ by human platelets was inhibited by selective 5-HT uptake blockers [cianopramine, (-)-paroxetine, and clomipramine], by metabolic inhibitors (KCN and ouabain), and by drugs that interfere with amine storage in the 5-HT organelles (reserpine, mepacrine, and Ro 4-1284). Impairment of the transmembrane proton gradient by ionophores (monensin and nigericin) induced a marked release of radioactivity from platelets preincubated with [3H]MPP+. Fractionation of homogenates of rabbit platelets preincubated with [3H]MPP+ showed that the drug was concentrated to a great extent in the 5-HT organelle fraction. MPP+ competitively inhibited [14C]5-HT uptake by human platelets and reduced the endogenous 5-HT content of human, rabbit, and guinea pig platelets. These investigations show that MPP+ is transported into the platelets via the 5-HT carrier and is accumulated predominantly in the subcellular organelles that store 5-HT and other monoamines. It is suggested that an accumulation of MPP+ in amine storage vesicles of neurons may be involved in the effects of the drug in the CNS, e.g., by protecting other subcellular compartments from exposure to high concentrations of MPP+, by sustaining a gradual release of the toxin, or both.     

1, 2, 3, 4-Tetrahydro-2-Methyl-4, 6, 7-Isoquinolinetriol Depletes Catecholamines in Rat Brain

1, 2, 3, 4-Tetrahydro-2-methyl-4, 6, 7-isoquinolinetriol (TMIQ) was synthesised and tested for activity as a dopamine-depleting agent in rat brain. After intracerebroventricular infusion, TMIQ caused reductions in dopamine concentrations in substantia nigra, striatum, hypothalamus, and dorsal raphe, and reduction in noradrenaline concentrations in locus coeruleus. TMIQ also reduced 5-hydroxytryptamine concentrations in dorsal raphe and substantia nigra, although with a lower potency. Comparisons between TMIQ and MPTP showed that they were approximately equipotent in depleting dopamine in the substantia nigra, hypothalamus, and dorsal raphe. Pretreatment of animals with a combination of monoamine oxidase A and B inhibitors completely prevented the TMIQ-induced reductions in dopamine concentrations in substantia nigra and hypothalamus. Direct unilateral intrastriatal injections of TMIQ produced marked ipsilateral reductions in striatal dopamine, correlating with a behavioural response consisting of turning towards the side of injection. The results suggest that TMIQ should be evaluated further as a possible MPTP-like compound, which may derive from endogenous β-hydroxylated catecholamines.     

Diethyldithiocarbamate Potentiates the Neurotoxicity of In Vivo l-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine and of In Vitro 1-Methyl-4-Phenylpyridinium

Diethyldithiocarbamic acid (DDC) potentiates in vivo neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in vitro neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Male C57B1/6 mice were given two or five injections of MPTP (30 mg/kg i.p.) preceded 0.5 h by DDC (400 mg/kg i.p.). The mice were tested for catalepsy, akinesia, or motor activity during and after the period of dosing. Striatal and hippocampal tissues were obtained at 2 and 7 days following the last injection and evaluated for dopamine and norepinephrine levels, respectively. These same tissues were also analyzed for the levels of glial fibrillary acidic protein (GFAP), an astrocyte-localized protein known to increase in response to neural injury. Pretreatment with DDC potentiated the effect of MPTP in striatum and resulted in substantially greater dopamine depletion, as well as a more pronounced elevation in GFAP. In hippocampus, the levels of norepinephrine and GFAP were not different from controls in mice receiving only MPTP, but pretreatment with DDC resulted in a sustained depletion of norepinephrine and an elevation of GFAP, suggesting that damage was extended to this brain area by the combined treatment. Mice receiving MPTP preceded by DDC also demonstrated a more profound, but reversible, catalepsy and akinesia compared to those receiving MPTP alone. Systemically administered MPP+ decreased heart norepinephrine, but did not alter the striatal levels of dopamine or GFAP, and pretreatment with DDC did not alter these effects, but did increase lethality. DDC is known to increase brain levels of MPP+ after MPTP, but our data indicate that this is not due to a movement of peripherally generated MPP+ into CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Involvement of Mitochondrial Permeability Transition in Cytotoxicity of 1-Methyl-4-Phenylpyridinium and 6-Hydroxydopamine

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the influence of the mitochondrial membrane permeability transition inhibition against the toxicity of 1-methyl-4-phenylpyridinium (MPP⁺) and 6-hydroxydopamine (6-OHDA) in relation to the mitochondria-mediated cell death process and role of oxidative stress. Both MPP⁺ and 6-OHDA induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Cyclosporin A (CsA), trifluoperazine and aristolochic acid, inhibitors of mitochondrial permeability transition, significantly attenuated the MPP⁺-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. In contrast to MPP⁺, the cytotoxicity of 6-OHDA was not reduced by the addition of the mitochondrial permeability transition inhibitors. The results show that the cytotoxicity of MPP⁺ may be mediated by the mitochondrial permeability transition formation, which is associated with formation of reactive oxygen species and the depletion of GSH. In contrast, the 6-OHDA-induced cell injury appears to be mediated by increased oxidative stress without intervention of the mitochondrial membrane permeability transition.     

Intracarotid 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Administration: Biochemical and Behavioral Observations in a Primate Model of Hemiparkinsonism

Cynomolgus monkeys received intracarotid injections of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to produce a chronic unilateral model of parkinsonism. Extensive dopamine (DA) depletion was observed in the caudate nucleus and putamen on the side ipsilateral to the injection and this was associated with contralateral tremor, rigidity, and bradykinesia. A dose of 1.25 mg of MPTP caused ipsilateral DA loss of 99.4% in the caudate nucleus, 99.8% in the putamen, and 74.2% in the nucleus accumbens. A dose of 2.5 mg caused ipsilateral DA depletion of 99.3% in the caudate nucleus, 99.5% in putamen, and 90.1% in the nucleus accumbens. The unilateral aspect of the lesion was dose sensitive, with the 2.5-mg dose causing bilateral asymmetric DA depletion. Tissue concentrations of serotonin were not affected by the toxin. These findings confirm that intracarotid injection of MPTP may produce a useful primate model of hemiparkinsonism that can be associated with selective unilateral DA depletion when the appropriate dose of toxin is used.     

Membrane Potential and Catecholamine Secretion by Bovine Adrenal Chromaffin Cells: Use of Tetraphenylphosphonium Distribution and Carbocyanine Dye Fluorescence

Changes in plasma membrane potential of isolated bovine adrenal chromaffin cells were measured independently by two chemical probe methods and related to corresponding effects on catecholamine secretion. The lipophilic cation tetraphenylphosphonium (TPP+) and the carbocyanine dye 3,3'-dipropylthiadicarbocyanine [DiS-C3-(5)] were used. The necessity of evaluating the subcellular distribution of TPP+ among cytoplasmic, mitochondrial, secretory granule, and bound compartments was demonstrated and the resting plasma membrane potential determined to be -55 mV. The relationship between membrane potential and catecholamine secretion was determined in response to variations in extracellular K+ and to the presence of several secretagogues including cholinergic receptor ligands, veratridine, and ionophores for Na+ and K+. The dependence of potential on K+ concentration fit the Goldman constant field equation with a Na/K permeability ratio of 0.1. The dependence of both K+- and veratridine-evoked catecholamine secretion on membrane potential exhibited a potential threshold of about -40 mV before a significant rise in secretion occurred. This is likely related to the threshold for opening of voltage-sensitive Ca2+ channels. Acetylcholine and nicotine evoked a large secretory response without a sufficiently sustained depolarization to be detectable by the relatively slow potential sensitive chemical probes. Decamethonium induced a detectable depolarization of the chromaffin cells. Veratridine and gramicidin evoked both membrane depolarization and catecholamine release. By contrast the K ionophore valinomycin evoked significant levels of secretion without any depolarization. This is consistent with its utilization of an intracellular source of Ca2+ and the independence of its measured secretory response on extracellular Ca2+.     

Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin Levels and the Role of the Cofactor in Tyrosine Hydroxylation in Primary Cultures of Adrenomedullary Chromaffin Cells

Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N-acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N-acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three-day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl- and 8-bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP-cyclohydrolase, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8-bromo cyclic AMP. The increase in GTP-cyclohydrolase and tetrahydrobiopterin induced by 8-bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP-cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP-cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.