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The Journal of Neuroscience, October 15, 2002, 22(20):8819-8826

Patch Cramming Reveals the Mechanism of Long-Term Suppression of Cyclic Nucleotides in Intact Neurons

Bhavya Trivedi¹ and Richard H. Kramer^{1, 2}

¹ Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101, and ² Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720

To understand cyclic nucleotide dynamics in intact cells, we used the patch-cramming method with cyclic nucleotide-gated channels as real-time biosensors for cGMP. In neuroblastoma and sympathetic neurons, both muscarinic agonists and nitric oxide (NO) rapidly elevate cGMP. However, muscarinic agonists also elicit a long-term (2 hr) suppression (LTS) of subsequent cGMP responses. Muscarinic agonists elevate cGMP by triggering Ca²⁺ mobilization, which activates NO synthase to produce NO, leading to the activation of soluble guanylate cyclase (sGC). Here we examine the mechanism of LTS. Experiments using direct intracellular cGMP injection demonstrate that enhancement of phosphodiesterase (PDE) activity, rather than depression of sGC activity, is responsible for LTS. Biochemical measurements show that both cGMP and cAMP content is suppressed, consistent with the involvement of a nonselective PDE. Application of pharmacological agents that alter Ca²⁺ mobilization from intracellular stores and experiments involving injection of the Ca²⁺ chelator BAPTA show that Ca²⁺ mobilization is necessary and sufficient for LTS induction but also show that LTS maintenance is Ca²⁺-independent. Protein phosphatase injection reverses LTS, and specific inhibitors of Ca²⁺/calmodulin kinase II (CaMKII) prevent induction and inhibit maintenance. The switch between the Ca²⁺ dependence of LTS induction to the Ca²⁺ independence of LTS maintenance is consistent with CaMKII autophosphorylation, similar to proposed mechanisms of hippocampal long-term potentiation. Because the molecular machinery underlying LTS is common to many cells, LTS may be a widespread mechanism for long-term silencing of cyclic nucleotide signaling.

Key words: sympathetic neuron; neuroblastoma; cGMP; nitric oxide; CaMKII; cyclic nucleotide-gated channel; synaptic plasticity

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Copyright © 2002 Society for Neuroscience  0270-6474/02/22208819-08$05.00/0

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