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Article: The dynamics of cGMP metabolism in neuroblastoma N1E-115 cells determined by 18O labeling of guanine nucleotide α-phosphoryls

TitleThe dynamics of cGMP metabolism in neuroblastoma N1E-115 cells determined by 18O labeling of guanine nucleotide α-phosphoryls
Authors
KeywordsChemicals And Cas Registry Numbers
Issue Date1987
PublisherSpringer New York LLC. The Journal's web site is located at http://springerlink.metapress.com/openurl.asp?genre=journal&issn=0364-3190
Citation
Neurochemical Research, 1987, v. 12 n. 6, p. 551-560 How to Cite?
AbstractThe rates of phosphodiesterase-promoted hydrolysis of cGMP and cAMP have been measured in intact neuroblastoma N1E-115 cells by determining of 18O incorporation from 18O-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 this 18O-labeling rate in conjunction with 28- and 50-fold increases in cellular cGMP concentration at 3 and 6 min, respectively. This 18O-labeling rate (795 pmol·mg protein-1·min-1) corresponded with the sum of the low (1.7 μM) and high (34 μM) K(m) phosphodiesterase activities assayable in cell lysates which exhibited a combined maximum velocity of 808 pmol·mg protein-1·min-1 to which the high K(m) species contributed 84%. This information and the characteristics of the profile of 18O-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 of 18O 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 of 18O 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 of 18O 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.
Persistent Identifierhttp://hdl.handle.net/10722/132592
ISSN
2015 Impact Factor: 2.472
2015 SCImago Journal Rankings: 1.102
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorGraeff, RMen_HK
dc.contributor.authorWalseth, TFen_HK
dc.contributor.authorGoldberg, NDen_HK
dc.date.accessioned2011-03-28T09:26:35Z-
dc.date.available2011-03-28T09:26:35Z-
dc.date.issued1987en_HK
dc.identifier.citationNeurochemical Research, 1987, v. 12 n. 6, p. 551-560en_HK
dc.identifier.issn0364-3190en_HK
dc.identifier.urihttp://hdl.handle.net/10722/132592-
dc.description.abstractThe rates of phosphodiesterase-promoted hydrolysis of cGMP and cAMP have been measured in intact neuroblastoma N1E-115 cells by determining of 18O incorporation from 18O-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 this 18O-labeling rate in conjunction with 28- and 50-fold increases in cellular cGMP concentration at 3 and 6 min, respectively. This 18O-labeling rate (795 pmol·mg protein-1·min-1) corresponded with the sum of the low (1.7 μM) and high (34 μM) K(m) phosphodiesterase activities assayable in cell lysates which exhibited a combined maximum velocity of 808 pmol·mg protein-1·min-1 to which the high K(m) species contributed 84%. This information and the characteristics of the profile of 18O-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 of 18O 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 of 18O 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 of 18O 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.en_HK
dc.languageengen_US
dc.publisherSpringer New York LLC. The Journal's web site is located at http://springerlink.metapress.com/openurl.asp?genre=journal&issn=0364-3190en_HK
dc.relation.ispartofNeurochemical Researchen_HK
dc.subjectChemicals And Cas Registry Numbersen_US
dc.titleThe dynamics of cGMP metabolism in neuroblastoma N1E-115 cells determined by 18O labeling of guanine nucleotide α-phosphorylsen_HK
dc.typeArticleen_HK
dc.identifier.emailGraeff, RM: graeffr@hku.hken_HK
dc.identifier.authorityGraeff, RM=rp01464en_HK
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.pmid2439934-
dc.identifier.scopuseid_2-s2.0-0023254202en_HK
dc.identifier.volume12en_HK
dc.identifier.issue6en_HK
dc.identifier.spage551en_HK
dc.identifier.epage560en_HK
dc.identifier.isiWOS:A1987H384000010-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridGraeff, RM=7003614053en_HK
dc.identifier.scopusauthoridWalseth, TF=7005424273en_HK
dc.identifier.scopusauthoridGoldberg, ND=23085828700en_HK

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