Dynamics of cyclic GMP signaling in neuronal cells

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)



First Committee Member

Richard L. Rotundo - Committee Chair


Cyclic GMP (cGMP) is a crucial intracellular messenger used by nearly all eukaryotic cells. In neurons, cGMP is particularly important in mediating intracellular responses to transmitters such as acetylcholine, bradykinin, and natriuretic peptides. In addition, most of the effects of nitric oxide (NO) have been attributed to activation of soluble guanylate cyclase (sGC) and production of cGMP. While there is a growing awareness about the physiological importance and biochemistry of signaling cascades, very little is known about the intracellular dynamics of cGMP. This study used the patch-cram detection method to obtain the first real-time measurements of cGMP in intact, living N1E-115 neuroblastoma cells. This method involves impaling a cell with an electrode holding an excised, inside-out membrane patch containing cyclic nucleotide gated (CNG) ion channels, used as a detector for cGMP. Detector patches were obtained from Xenopus oocytes expressing a chimeric CNG channel that exhibits high specificity and sensitivity for cGMP (K 1/2 = 4 muM). The conductance of the detector patch was precalibrated with known concentrations of cGMP. After cramming the detector patch into a recipient cell, changes in patch conductance indicated changes in free cytoplasmic cGMP.Patch-cramming experiments on neuroblastoma cells show that both muscarinic agonists and NO rapidly elevate cGMP. NO elicits cGMP responses repeatedly without decrement, whereas responses to muscarinic agonists exhibit a profound and prolonged desensitization. Remarkably, muscarinic agonists also cause long-term (up to 2 hrs) suppression (LTS) of cGMP responses to G-protein coupled receptor activation and NO or other NO-independent sGC activators. Biochemical measurements demonstrate that rat sympathetic neurons exhibit a similar prolonged suppression of cGMP, suggesting that LTS may also occur in primary neurons.The mechanism of LTS was examined using the patch cramming method for detection of cGMP, and pharmacological agents that affect the enzymes involved in cGMP metabolism. Direct injection of cGMP into recipient cells demonstrates that enhancement of phosphodiesterase (PDE) activity, rather than depression of sGC, is responsible for LTS. Biochemical measurements show that both cGMP and cAMP are suppressed, suggesting that a non-selective PDE is responsible. Ca2+ mobilization is necessary and sufficient for LTS induction, but LTS maintenance is Ca2+-independent. Protein phosphatase injection reverses LTS, suggesting that a phosphorylation event is required for LTS maintenance. Specific inhibitors of Ca2+/calmodulin kinase II (CaMKII) but not protein kinase C prevent induction and inhibit maintenance of LTS. The Ca2+-independence of LTS maintenance is consistent with CaMKII autophosphorylation, similar to proposed mechanisms of some hippocampal long-term potentiation. Since the molecular machinery underlying LTS is common to many cells, LTS may be a widespread new mechanism for long-term silencing of cyclic nucleotide signaling.


Biology, Neuroscience

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