Linked Life Data

15769625

Source:http://linkedlifedata.com/resource/pubmed/id/15769625

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:dateCreated
2005-3-16
pubmed:abstractText
We have recently developed genetically encoded cGMP-indicators (cygnets) which have enabled us to study the spatial and temporal dynamics of intracellular cGMP in single cultured cells (1). However, primary mammalian cell types (dissociated cells or acute tissue samples) are often difficult to maintain undifferentiated in culture and the current established methods of introducing molecular reporters in single cells are laborious (micro-injection) and/or require cell culture techniques to accommodate the 1-2 day lag time of genetically mediated reporter expression. Here, we present an alternative, non-genetic method to rapidly introduce cGMP-indicators into cells and intact tissues using membrane permeable peptides (MPP). Five different 125 kDa MPP-cygnets were expressed and purified from insect SF9 cells. Three constructs showed high level cGMP-dependent FRET in vitro. One of the probes, Ant7-Cygnet, demonstrated emission ratio changes identical to the unmodified indicator. Ant7-Cygnet was rapidly (3 hours) and efficiently internalized in cultured smooth muscle cells and intact cerebral arteries. Furthermore, the internalized Ant7-Cygnet detected nitric oxide mediated elevations of intracellular cGMP in cultured smooth muscle cells and sensed increased levels of intracellular cGMP derived from C-type natriuretic peptide (CNP) induced guanylyl cyclase stimulation in intact arteries. These results demonstrate that MPP-cygnets provide a novel and potentially powerful technique to study intracellular cGMP in intact tissue.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
1093-4715
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
10
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1290-301
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Membrane-permeable cygnets: rapid cellular internalization of fluorescent cGMP-indicators.
pubmed:affiliation
Department of Pharmacology, College of Medicine, University of Vermont, Burlington 05405-0075, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural