Effects of sub-chronic in vivo chlorpyrifos exposure on muscarinic receptors and adenylate cyclase of rat striatum.

In this study dosing regimens were designed such that cholinesterase inhibition following exposure to chlorpyrifos was produced in one treatment group, but was absent in the other. The higher dosing regimen inhibited plasma and brain cholinesterase activities by 51 and 70%, respectively, and resulted in decreased [3H]cis-methyldioxolane ([3H]CD) binding, which was attributable to a decrease in Bmax. No concomitant loss of [3H]quinuclidinyl benzilate ([3H]QNB) binding sites was observed, indicating that the M2 muscarinic receptor subtype to which [3H]CD binds is particularly susceptible to alterations induced by chlorpyrifos treatment. As the M2 receptor subtype is surmised to be the muscarinic autoreceptor, decreases in this receptor may exacerbate poisoning by organophosphorus agents as a result of decreased ability to terminate synaptic acetylcholine release. The ability of carbachol to inhibit striatal adenylate cyclase, which is an effector molecule associated with the M2 receptor, was unaltered in chlorpyrifos-treated rats. Decreases in M2 receptors occurred with the higher dosing regimen, in the absence of any clinical manifestations. Thus, in the absence of overt clinical signs, perturbations of the muscarinic receptor system did occur as a result of sub-chronic chlorpyrifos exposure. Such alterations may contribute to neurological impairments that develop following chronic organophosphorus exposure.

Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes.

Dopamine transporters (DATs) are members of a family of Na+- and Cl--dependent neurotransmitter transporters responsible for the rapid clearance of dopamine from synaptic clefts. The predicted primary sequence of DAT contains numerous consensus phosphorylation sites. In this report we demonstrate that DATs undergo endogenous phosphorylation in striatal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [32P]orthophosphate, and solubilized homogenates were subjected to immunoprecipitation with an antiserum specific for DAT. Basal phosphorylation occurred in the absence of exogenous treatments, and the phosphorylation level was rapidly increased when synaptosomes were treated with the phosphatase inhibitors okadaic acid or calyculin. Treatment of synaptosomes with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also increased the level of phosphate incorporation. This occurred within 10 min and was dosedependent between 0.1 and 1 microM PMA. DAT phosphorylation was also significantly increased by two other protein kinase C activators, (-)-indolactam V and 1-oleoyl-2-acetyl-sn-glycerol. The inactive phorbol ester 4alpha-phorbol 12,13-didecanoate at 10 microM was without effect, and PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors staurosporine and bisindoylmaleimide. These results indicate that DATs undergo rapid in vivo phosphorylation in response to protein kinase C activation and that a robust mechanism exists in synaptosomes for DAT dephosphorylation. Dopamine transport activity in synaptosomes was reduced by all treatments that promoted DAT phosphorylation, with comparable dose, time, and inhibitor characteristics. The change in transport activity was produced by a reduction in Vmax with no significant effect on the Km for dopamine. These results suggest that synaptosomal dopamine transport activity is regulated by phosphorylation of DAT and present a potential mechanism for local neuronal control of synaptic neurotransmitter levels and consequent downstream neural activity.

Phorbol esters increase dopamine transporter phosphorylation and decrease transport Vmax.

Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport Vmax in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK1, cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport Vmax by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter Vmax suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.

In vitro effect of chlorpyrifos oxon on muscarinic receptors and adenylate cyclase.

Although the neurotoxicity of organophosphorus compounds is generally attributed to inhibition of acetylcholinesterase, recent reports have indicated that direct interactions with muscarinic receptors and signal transduction may be an additional mechanism of neurotoxicity. We have previously shown that the organophosphorus insecticide O,O-diethyl O-3,5,6-trichloro-2-pyridinyl phosphorothioate (chlorpyrifos) binds directly to muscarinic receptors and inhibits adenylate cyclase of rat striatum. We have further pursued those results in this study by investigating the effect of chlorpyrifos oxon in NG108-15 neuroblastoma-glioma cells and Chinese hamster ovary cells transfected with cDNA for human m2 or m4 muscarinic receptor subtypes. At millimolar concentrations, chlorpyrifos oxon inhibited [3H]QNB binding in all cell lines. Likewise, [3H]CD binding was inhibited in NG108-15 and CHO-Hm2 cells. When the effect of chlorpyrifos oxon on adenylate cyclase was examined, the oxon was found to inhibit adenylate cyclase at millimolar concentrations. Though this effect on cyclase required greater concentrations of oxon than the comparable effect in striatal cells, it displayed the common characteristic of being atropine-insensitive, suggesting that the effect on cyclase was not muscarinic receptor dependent. The inhibition of adenylate cyclase produced by chlorpyrifos oxon was not eliminated in pertussis toxin treated cells, lending further support to the idea that it is not a receptor-mediated event, and suggesting a potential direct interaction of chlorpyrifos oxon with the adenylate cyclase molecule.

Postischemic diazepam is neuroprotective in the gerbil hippocampus.

In this study, we address the hypothesis that enhancement of gamma-aminobutyric acid (GABA) neurotransmission following an ischemic episode is neuroprotective in the hippocampus. Mongolian gerbils were subjected to transient forebrain ischemia for 5 min by occlusion of the carotid arteries and then administered diazepam (10 mg/kg i.p.) 30 min or 30 and 90 min following ischemia. Diazepam produced a significant decrease in both rectal and brain temperature (4-6 degrees C) in the sham and ischemic gerbils. 1 day following the onset of reperfusion, diazepam substantially reduced the hyperactivity normally induced by the ischemic episode. 7 days later, neuronal viability in the hippocampus was assessed. The single dose of diazepam completely protected the CA1 pyramidal cells of the hippocampus in 62% of the gerbils and the double dose of diazepam completely protected CA1 pyramidal neurons in 67% of the gerbils. There was a significant correlation between the degree of pyramidal cell degeneration in the CA1 area of the hippocampus measured 7 days following ischemia and the degree of hyperactivity measured 1 day following ischemia. Diazepam also prevented the loss of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABA-gated chloride channels in the dendritic fields of the CA1 area of the hippocampus. Our findings support the hypothesis that enhancement of GABA neurotransmission following an ischemic event may offset neuronal excitability and prevent neuronal death in specific brain regions. We conclude that GABA-enhancing drugs, such as diazepam, are attractive candidates as neuroprotective agents following ischemic insults.

Chlorpyrifos oxon binds directly to muscarinic receptors and inhibits cAMP accumulation in rat striatum.

Although the acute effects of organophosphorus esters are generally ascribed to inhibition of acetylcholinesterase, work in this laboratory and others indicates that organophosphorus insecticides also interact directly with cholinergic receptors. The current study verifies that the insecticide O,O-diethyl O-3,5,6-trichloro-2-pyridinyl phosphorothionate (chlorpyrifos) and its oxon metabolite inhibits acetylcholinesterase (AChE). The metabolite inhibits rat brain AChE three orders of magnitude more rapidly than chlorpyrifos. In addition to their ability to inhibit AChE, these compounds were shown to interact directly with muscarinic receptors of rat striatum. The oxon metabolite bound at low concentrations to muscarinic receptors labeled by the muscarinic agonist [3H] cis-methyldioxolane; chlorpyrifos oxon bound with an IC50 value of 22.1 +/- 3.6 nM. The receptors bound by chlorpyrifos oxon account for approximately 30% of muscarinic receptors of the striatum and are of the m2 subtype. The binding of chlorpyrifos oxon to the m2 receptor results in a covalent modification of the receptor that does not interfere with the ability of the receptor to interact with the agonist carbachol. This receptor modification may be responsible for the inhibition of adenylate cyclase activity by chlorpyrifos oxon. The oxon inhibited adenylate cyclase with an IC50 of 155 +/- 78 nM. The inhibition of adenylate cyclase activity was not blocked by atropine and was additive to that produced by carbachol. The altering of postreceptor signal transduction by chlorpyrifos oxon may interfere with normal cellular signaling, thereby disturbing neurological function. Direct interaction of chlorpyrifos oxon with muscarinic receptors and associated signal transduction is a potential mechanism of neurotoxicity that is independent of AChE inhibition.

cis-Methyldioxolane specifically recognizes the m2 muscarinic receptor.

cis-Methyldioxolane (CD) is a muscarinic receptor agonist. [3H]CD has been used to label a subpopulation of muscarinic receptors described as exhibiting high agonist affinity. Pharmacological evidence suggests that the population of receptors labeled by [3H]CD consists of m2 and/or m4 subtypes; however, no studies have directly addressed the subtype selectivity of [3H]CD. The present study characterizes binding of this ligand to individual human receptor subtypes expressed in transfected Chinese hamster ovary cells. Results indicate that [3H]CD binds with high affinity only to Hm2 receptors but not to all Hm2 receptors. Twenty-eight percent of Hm2 receptors bound [3H]CD with a KD of 3.5 +/- 0.5 nM. Binding was eliminated in the presence of guanosine 5'-O-(3-thiotriphosphate), indicating that the Hm2 receptors labeled by [3H]CD are those that are associated with GDP-bound G protein. Binding of [3H]CD by only a subpopulation of Hm2 receptors is in agreement with data generated from studies of [3H]CD binding in mammalian brain. Because muscarinic receptors have been implicated to play a role in the pathogenesis of both Alzheimer's and Parkinson's disease, as well as the neurotoxicity of organophosphorus compounds, knowledge of the binding specificity of the muscarinic agonist [3H]CD should aid research in these areas.

Application of alkaline unwinding to analysis of strand breaks induced by bleomycin in hamster lung DNA in vivo.

Strand breaks in hamster lung DNA were analyzed following in vivo exposure to bleomycin. An alkaline unwinding procedure involving separation of single-strand from double-strand DNA by hydroxylapatite chromatography followed by fluorescence detection of DNA with bisbenzimide was adapted for these studies. Procedures were developed that allowed preparation of DNA from lungs of control animals with a relatively low amount of single-strand DNA relative to double-strand DNA. Time dependence of unwinding was demonstrated using samples that were damaged deliberately by brief probe sonication. To verify that residual bleomycin remaining in lungs at the time of sacrifice did not cause strand breaks during sample preparation, bleomycin was added to minced lung in vitro. Under these conditions, the ratio of single-strand to double-strand DNA was not increased significantly. Substantial strand breaks were produced in vivo at 15 min and 1 h following intratracheal instillation of bleomycin into hamsters, as evidenced by a 5-6-fold increase in the ratio of single-strand to double-strand DNA relative to controls. The DNA damage appeared to be repaired within 1 day following exposure.

Glucocorticoids regulate the ontogenetic transition of adrenergic receptor subtypes in rat liver.

During neonatal development, adrenergic control of hepatic glucose metabolism undergoes a transition from beta-receptor to alpha 1-receptor-mediated dominance coincident with the onset of function of the hypothalamus-pituitary-adrenocortical axis at the conclusion of the third to fourth week postpartum. To determine whether glucocorticoids contribute to this switch, neonatal rats were given 1 mg/kg of dexamethasone on postnatal days 13, 14 and 15 and the adrenergic receptor population examined by radioligand binding techniques. Dexamethasone accelerated the maturational replacement of beta-receptors with the alpha 1-subtype; the loss of beta-receptors was not reversible upon discontinuing treatment. When the glucocorticoid was given earlier, on days 7, 8 and 9, similar effects were obtained, but the suppression of the beta-subtype was only temporary; treatment before parturition (gestational days 17, 18 and 19) failed to suppress beta-receptor binding. These results suggest that, during a critical period, adrenocorticosteroids provide an important signal for the transition of adrenergic control of hepatic function.

Mutagenicity and DNA-damaging activity for several pesticides tested with Bacillus subtilis mutants.

The active pure compounds of 4 pesticides were tested for DNA-damaging and mutagenic activity in Bacillus subtilis and Salmonella typhimurium tester strains. Included were zinc ethylenebisdithiocarbamate (dithane), 1,2-dihydropyridazine-3,6-dione (maleic hydrazide), O,O-dimethylphosphorodithioate (malathion), and 1,2-dibromoethane (fumazone). These agents gave either weak or negative mutagenic responses with the Salmonella/microsome tests for mutagenicity, but were all positive when the tester was B. subtilis strain TKJ6321. Of the 4 chemicals, only fumazone required metabolic activation with rat-liver S9 mix. Upon activation, it produced a volatile mutagenic product. Dithane, maleic hydrazide, and malathion were all mutagenic and did not require metabolic activation. Among these agents, dithane was strongly mutagenic while fumazone, maleic hydrazide and malathion were moderately mutagenic. Only dithane gave significant DNA-damaging activity when applied to a battery of repair-deficient B. subtilis mutants. For the chemicals reported, it is concluded that B. subtilis is superior to S. typhimurium in the detection of mutagenic activity. We strongly recommend its use for prescreening procedures in combination with the S. typhimurium testers.