Regulation of stimulus-dependent hippocampal acetylcholine release by okadaic acid-sensitive phosphoprotein phosphatases.

Isolated nerve endings (synaptosomes) from rat hippocampus were used to characterize the influence by serine/threonine-specific phosphoprotein phosphatase (PP) inhibitors on acetylcholine release. Brief exposure to low concentrations of selective PP inhibitors (okadaic acid and calyculin A) caused a concentration-dependent attenuation of stimulus-dependent (calcium-evoked or potassium-evoked) [3H]acetylcholine ([3H]ACh) release, while having no effect on the rate of basal transmitter efflux. In view of the observed potencies for okadaic acid and calyculin A (pseudo-IC50 values near 3 nM), these data indicate that Type 1 (PP1) or Type 2A (PP2A) enzymes play a permissive role in exocytotic [3H]ACh release. In contrast, the absence of any measurable effect by sodium orthovanadate argues against a similar influence by tyrosine-specific phosphoprotein phosphatases. While the neuronal substrate(s) responsible for PP regulation of [3H]ACh release are unknown, the underlying mechanism clearly differs from that through which muscarinic autoreceptors act since inhibition by okadaic acid and oxotremorine (an autoreceptor agonist) are additive and the former is not blocked by the muscarinic receptor antagonist atropine. Based upon these results, we conclude that dephosphorylation steps catalyzed by okadaic acid-sensitive PP represent an important regulatory mechanism for stimulus-dependent transmitter release in septo-hippocampal cholinergic neurons.

Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: evidence for feedback autoregulation.

Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N omega-nitro-L-arginine methyl ester-sensitive conversion of L-[3H]arginine into L-[3H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC50 approximately 100 microM) and prevented by the NO scavenger oxyhemoglobin. L-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells.

Irreversible blockade of high-affinity choline uptake in rat brain by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).

N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an agent that causes irreversible covalent modification of protein carboxyl residues, has been used previously to produce irreversible occlusion of neurotransmitter receptors as well as other cellular proteins. The present investigation was undertaken to ascertain the mechanism by which EEDQ inhibits stimulus-dependent acetylcholine (ACh) release from rat brain hippocampal synaptosomes. Brief pretreatment with EEDQ (up to 100 microM) eliminated completely calcium-evoked [3H]acetylcholine ([3H]ACh) release and reduced de novo synthesis of transmitter by greater than 90%. Studies revealed that pretreatment with EEDQ in vitro caused a time- and concentration-dependent inhibition of high-affinity [3H]choline uptake (HACU) by synaptosomes. EEDQ-induced inhibition of HACU was not reversed by repeated tissue washing; however, co-incubation with hemicholinium-3, a highly specific and reversible inhibitor of HACU, protected against EEDQ-induced inhibition of HACU, as well as the loss of stimulus-dependent [3H]-ACh release. In vivo administration of EEDQ (20 mg/kg, s.c.) to rats caused marked reductions (46-65%) in synaptosomal HACU as well as the number of membrane binding sites for the muscarinic cholinergic antagonist L-[benzilic-4,4'-3H]quinuclidinyl benzilate ([3H]QNB) in the hippocampus and striatum. Treatment with atropine (100 mg/kg) prevented the reduction in [3H]QNB binding but did not influence EEDQ-induced inhibition of HACU. Taken together, these results indicate that EEDQ causes a direct and irreversible inhibition of high-affinity choline transporters on CNS cholinergic nerve terminals and, therefore, may be a useful investigational tool for characterization of the turnover and regulation of this transporter protein in vivo.

Absence of receptor reserve at hippocampal muscarinic autoreceptors which inhibit stimulus-dependent acetylcholine release.

The extent of reserve among inhibitory muscarinic autoreceptors on hippocampal cholinergic nerve terminals was examined in superfused calcium-naive synaptosomes. The tissues were treated with the irreversible muscarinic cholinergic receptor antagonist propylbenzilycholine mustard (PrBCM) and then used to assess the functional status of autoreceptors through acetylcholine (ACh)-induced inhibition of calcium-evoked [3H]ACh release. PrBCM treatment caused a marked reduction in the density of high-affinity [3H]quinuclidinyl benzilate binding sites (46%, 72% and 90% reductions after 3, 6 or 10 nM PrBCM, respectively) but had no apparent influence on the binding affinities or relative proportions of high- and low-affinity binding sites for the M1-selective antagonist pirenzepine or the agonist ACh. In vehicle-treated tissues, ACh was a potent (EC50 = 240 nM) and efficacious (maximal inhibition of stimulated [3H]ACh release = 65%) agonist at muscarinic autoreceptors. However, after PrBCM treatment, the maximal inhibition for ACh was greatly attenuated (35% and 17% for 3 and 6 nM PrBCM, respectively) with no concurrent changes in the EC50 or slope factor. Comparisons of equieffective agonist concentrations before and after receptor occlusion revealed a direct linear relationship between autoreceptor occupancy and inhibition of [3H]ACh release with close agreement between the calculated agonist dissociation constant (KA = 220 nM) and the EC50 for ACh. Pretreatment with 100 nM atropine methylbromide completely prevented PrBCM-induced reductions in muscarinic cholinergic receptor binding and autoreceptor function. These results support the conclusion that muscarinic autoreceptors on hippocampal nerve endings exhibit little or no reserve for inhibition of ACh release by the endogenous neurotransmitter.