Phosphorylation of vasodilator-stimulated phosphoprotein: a consequence of nitric oxide- and cGMP-mediated signal transduction in brain capillary endothelial cells and astrocytes.
There is contradictory information on the relevance of nitric oxide (NO) and cGMP for the function of brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). Therefore, NO/cGMP-mediated signal transduction was investigated in cell cultures of BCEC and of astrocytes (AC) inducing BBB properties in BCEC. Constitutive, Ca2+-activated isoforms of NO synthase (NOS) were found in BCEC (endothelial NOS: eNOS) and in AC (neuronal NOS: nNOS), leading to increased NO release after incubation with the Ca2+-ionophore A23187. Both cell types expressed inducible NOS (iNOS) after incubation with cytokines. Soluble guanylate cyclase (sGC) was detected in both cell types. NO-dependent cGMP formation were observed in BCEC and, less pronounced, in AC. Furthermore, both cell types formed cGMP independently of NO via stimulation of particulate guanylate cyclase (pGC). cGMP-dependent protein kinase (PKG) type Ibeta, but not type II, was expressed in BCEC and AC. In BCEC, vasodilator-stimulated phosphoprotein (VASP) was detected, an established substrate of PKG and associated with microfilaments and cell-cell contacts. Phosphorylation of VASP was intensified by increased intracellular cGMP concentrations. The results indicate that BCEC and, to a smaller degree, AC can form NO and cGMP in response to different stimuli. In BCEC, NO/cGMP-dependent phosphorylation of VASP is demonstrated, thus providing a possibility of influencing cell-cell contacts.
Sporbert
A
A
Forschungsinstitut für Molekulare Pharmakologie, Alfred-Kowalke Str. 4, 10315, Berlin, Germany.
Mertsch
K
K
Smolenski
A
A
Haseloff
R F
RF
Schönfelder
G
G
Paul
M
M
Ruth
P
P
Walter
U
U
Blasig
I E
IE
D000818:Animals; D001253:Astrocytes / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D001798:Blood Proteins / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000502:physiology*; D002196:Capillaries / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000378:metabolism*; D002450:Cell Communication / Q000502:physiology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000032:analysis / Q000378:metabolism*; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins; D009566:Nitrates / Q000032:analysis; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D000098662:Vasodilator-Stimulated Phosphoprotein
D001798:Blood Proteins; D015815:Cell Adhesion Molecules; D008840:Microfilament Proteins; D009566:Nitrates; D009573:Nitrites; D010750:Phosphoproteins; D012333:RNA, Messenger; D000098662:Vasodilator-Stimulated Phosphoprotein; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D006152:Cyclic GMP