The dynamics of cGMP metabolism in neuroblastoma N1E-115 cells determined by18O labeling of guanine nucleotide α-phosphoryls

Richard M. Graeff, Timothy F. Walseth, Nelson D. Goldberg

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3 Scopus citations

Abstract

The rates of phosphodiesterase-promoted hydrolysis of cGMP and cAMP have been measuted in intact neuroblastoma N1E-115 cells by determining rates of18O incorporation from18O-water into the α-phosphoryls of guanine and adenine nucleotides. The basal rate of guanine nucleotide α-phosphoryl labeling ranged from 180 to 244 pmol·mg protein-1·min-1. Sodium nitroprusside (SNP) caused a sustained 3,4-fold increase in this18O-labeling rate in conjunction with 28- and 50-fold increases in cellular cGMP concentration at 3 and 6 min, respectively. This18O-labeling rate (795 pmol·mg protein-1·min-1) corresponded with the sum of the low (1.7 μM) and high (34 μM) Km phosphodiesterase activities assayable in cell lysates which exhibited a combined maximum velocity of 808 pmol·mg protein-1·min-1 to which the high Km species contributed 84%. This information and the characteristics of the profile of18O-labeled molecular species indicate that cGMP metabolism was restricted to a very discrete cellular compartment(s) of approximately 12% of the cell volume. Carbachol (1 mM) produced a transient increase (6-fold) in cellular cGMP concentration and a transient increase (90%) in the rate of18O labeling of α-GTP during the first minute of treatment which translates into 30 additional cellular pools of cGMP hydrolyzed in this period. IBMX (1 mM) produced a relatively rapid increase in cellular cGMP (3- to 5-fold) and cAMP (2-fold) concentrations and a delayed inhibition of18O labeling of guanine and adenine nucleotide α-phosphoryls without further elevation of cyclic nucleotide levels. These results indicate that besides inhibiting cyclic nucleotide hydrolysis, IBMX also imparts a time-dependent inhibitory influence on the generation of cyclic nucleotides. The data obtained show that measurement of18O labeling of guanine and adenine nucleotide α-phosphoryls combined with measurements of cyclic nucleotide steady state levels provides a means to assess the rates of cyclic nucleotide synthesis and hydrolysis within intact cells and to identify the site(s) of action of agents that alter cellular cyclic nucleotide metabolism.

Original languageEnglish (US)
Pages (from-to)551-560
Number of pages10
JournalNeurochemical Research
Volume12
Issue number6
DOIs
StatePublished - Jun 1 1987

Keywords

  • O
  • cAMP metabolism
  • cGMP metabolism
  • neuroblastoma

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