Phenotypic changes in rat and guinea pig coronary microvascular endothelium after culture: loss of nitric oxide synthase activity.
OBJECTIVE
Coronary microvascular endothelial cells (CMVEs) can modulate the contractile performance of the adjacent myocardium by the release of agents such as nitric oxide (NO). Most previous studies using CMVEs have been done in situ, in the intact organ. We set out to study possible differences in NO synthase (NOS) regulation between freshly isolated and cultured rat and guinea pig CMVEs.


METHODS
CMVEs were isolated from Wistar rats and Dunkin Hartley guinea pigs and then grown in culture for varying times. Fura-2 fluorescence was used to measure agonist-induced changes in CMVE intracellular calcium levels. Agonist-induced changes in CMVE cGMP levels were measured by commercial radioimmunoassay kit. Western blot analysis was used to measure endothelial, constitutive NOS (ecNOS) and soluble guanylate cyclase (sGC) protein levels. Reverse transcription, polymerase chain reactions and Southern blotting were used to measure ecNOS mRNA transcripts.


RESULTS
In both fresh (1 h post-isolation) and cultured (14 days with one passage) CMVEs of the rat and guinea pig, bradykinin (BK) and the calcium ionophore A23187 (both 1 microM) elicited significant (P < 0.01) increases in the fura-2 340/380 fluorescence ratio. In cultured CMVEs, basal cGMP levels were unaffected by exposure to BK or A23187. Exposure to sodium nitroprusside (SNP) or atrial natriuretic peptide (ANP) (both 1 microM) induced significant (P < 0.01) increases in cGMP in guinea pig cells, whereas in rat cells only ANP produced a significant (P < 0.01) response. By contrast, freshly isolated CMVEs of both species had higher basal cGMP levels than cultured cells, and on exposure to BK and A23187, responded with significant (P < 0.01) increases in cGMP. Moreover, exposure of both fresh rat and guinea pig CMVEs to SNP or ANP also resulted in significant (P < 0.01) increases in cGMP. Western blot analysis demonstrated that ecNOS and sGC protein were lost from the rat CMVEs following culture. Furthermore, there was also a significant loss of ecNOS mRNA from the rat cells following culture.


CONCLUSIONS
These data demonstrate that freshly isolated rat and guinea pig CMVEs possess ecNOS activity, and that this activity is downregulated following culture. At least for the rat, this effect would seem to lie at both the transcriptional and translational level. Furthermore, rat CMVEs have reduced activity of sGC following culture.
Lang
D
D
Department of Pharmacology, Therapeutics and Toxicology, University of Wales College of Medicine, Heath Park, Cardiff, UK. langd@cf.ac.uk
Bell
J P
JP
Bayraktutan
U
U
Small
G R
GR
Shah
A M
AM
Lewis
M J
MJ
D000704:Analysis of Variance; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000201:enzymology*; D006152:Cyclic GMP / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D006162:Guanylate Cyclase / Q000378:metabolism; D006168:Guinea Pigs; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D008833:Microcirculation; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D009599:Nitroprusside / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D007476:Ionophores; D012333:RNA, Messenger; D009599:Nitroprusside; D000001:Calcimycin; D009320:Atrial Natriuretic Factor; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D001920:Bradykinin