Patch cramming reveals the mechanism of long-term suppression of cyclic nucleotides in intact neurons.
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.
Trivedi
Bhavya
B
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Kramer
Richard H
RH
D000818:Animals; D015374:Biosensing Techniques; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase; D007124:Immunoenzyme Techniques; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008845:Microinjections; D018721:Muscarinic Agonists / Q000494:pharmacology; D009433:Neural Inhibition / Q000502:physiology*; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism*; D009473:Neuronal Plasticity / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D009712:Nucleotides, Cyclic / Q000032:analysis / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods*; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase; D013564:Sympathetic Nervous System / Q000166:cytology; D013995:Time; D014981:Xenopus
D054815:Cyclic Nucleotide-Gated Cation Channels; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007473:Ion Channels; D018721:Muscarinic Agonists; D009712:Nucleotides, Cyclic; D018160:Receptors, Cytoplasmic and Nuclear; D009569:Nitric Oxide; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP