NGF promotes the survival and enhances the neurotransmitter phenotype of basal forebrain and striatal cholinergic neurons in brain of rat. The objective of the present study was to determine whether stimulations of the cholinergic neuronal markers ChAT and high-affinity choline uptake are reflected in enhanced synthesis and release of ACh. Enhancement of ACh release in brain of adult and aged rats could result in increased cholinergic neurotransmission, and altered animal behavior. NGF (1.2 micrograms/d) was administered intracerebroventricularly for 2 weeks by osmotic minipump to male Fischer-344 rats aged 4 and 24 months. Cholinergic neuronal functional parameters were measured in frontal cortex, hippocampus, and striatum. In young adult rats, increased ChAT and choline uptake activities were accompanied by enhanced ACh synthesis, basal and depolarization-induced release of both endogenous and newly synthesized transmitter, with the largest effect generally being observed in striatum. In aged animals, the responses to NGF were less uniform. Whereas the pattern for changes in ChAT activity was similar to that seen in younger animals, choline uptake activity was increased only in frontal cortex and striatum. Coincidentally, ACh synthesis was also increased only in these two brain regions. ACh content of synaptosomes was not affected by age or NGF treatment, and the ACh levels in microwaved samples of striatum and basal forebrain were not affected by NGF treatment. However, profound deficits in both basal and evoked release of newly synthesized ACh were observed in the aged rats. NGF treatment had no significant effect on the basal release of newly synthesized ACh in aged rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF‐mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen‐activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF‐mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)‐1/2 and p42/p44‐MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nω‐nitro‐L‐arginine methylester and s‐methylisothiourea and exposed to NGF for up to 24 h. NGF‐induced activation of MEK‐1/2 and p42/p44‐MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras‐Raf‐MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF‐mediated phosphorylation of MEK. Expression of constitutively active‐MEKK1 in cells led to phosphorylation of p42/p44‐MAP kinase and robust neurite outgrowth; constitutively active‐MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF‐mediated nuclear translocation of phospho‐MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor‐treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.
The high-affinity choline transporter (CHT1) is responsible for uptake of choline from the synaptic cleft and supplying choline for acetylcholine synthesis. CHT1 internalization by clathrin-coated vesicles is proposed to represent a mechanism by which high-affinity choline uptake can be modulated. We show here that internalized CHT1 is rapidly recycled back to the cell surface in both human embryonic kidney cells (HEK 293 cells) and SH-SY5Y neuroblastoma cells. This rapidly recycling pool of CHT1 comprises about 10% of total CHT1 protein. In the SH-SY5Y neuroblastoma cell line K(+)-depolarization promotes Ca(2+)-dependent increase in the rate of CHT1 recycling to the plasma membrane without affecting the rate of CHT1 internalization. K(+)-depolarization also increases the size of the pool of CHT1 protein that can be mobilized to the plasma membrane. Thus, the activity-dependent increase in plasma membrane CHT1 localization appears to be regulated by two mechanisms: (i) an increase in the rate of externalization of the intracellular CHT1 pool; and (ii) the recruitment of additional intracellular transporters to the recycling pool.
1 The ability of hexamethonium (C6) to reverse the neuromuscular blocking action of tubocurarine (Tc) has been reinvestigated at the voltage clamped endplate of the omohyoid muscle of rat. The possibility that a weak anticholinesterase action of C6 could contribute to the paradoxical potentiation of the peak amplitude of the endplate response has been examined. 5 When tested against responses to short ionophoretic pulses of agonists, C6 was less effective against ACh ((EC5o ca. 300 JiM) than against carbachol (CCh) (EC50 100 JIM). When cholinesterase was irreversibly inhibited, C6 blocked responses to both agonists equally (EC50 ca. 100 JiM). 6 The effectiveness of C6 in blocking the action of CCh was reduced 10 fold in the presence of 0.6 JIM Tc, implying that the two antagonists compete for the same binding site. 7 C6 in the presence of Tc (0.6 JM) increased the response to ionophoretically applied ACh but not that to CCh.8 C6 was equipotent in blocking m.e.p.cs and responses to ionophoretically applied ACh whereas Tc was more potent against the exogenously applied agonist.9 C6 was a weak inhibitor of acetylcholinesterase activity in rat muscle homogenates (EC50 1.5 mM).10 The results are discussed in terms of the kinetic hypothesis advanced by Ginsborg & Stephenson (1974) to account for the Tc reversal phenomenon. It is concluded that this theory can explain most of the effect on e.p.cs, but that the weak anticholinesterase action of C6 is also a factor, particularly in the reversal of Tc block of ionophoretic responses.
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