Phenotypic changes in rat and guinea pig coronary microvascular endothelium after culture: loss of nitric oxide synthase activity

Objectives: Coronary microvascular endothelial cells (CMVEs) can modulate t he contractile performance of the adjacent myocardium by the release of age nts 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 Dun kin Hartley guinea pigs and then grown in culture for varying times. Fura-2 fluorescence was used to measure agonist-induced changes in CMVE intracell ular calcium levels. Agonist-induced changes in CMVE cGMP levels were measu red by commercial radioimmunoassay kit. Western blot analysis was used to m easure 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) CM VEs of the rat and guinea pig, bradykinin (BK) and the calcium ionophore A2 3187 (both 1 mu M) elicited significant (P<0.01) increases in the fura-2 34 0/380 fluorescence ratio. In cultured CMVEs, basal cGMP levels were unaffec ted by exposure to BK or A23187. Exposure to sodium nitroprusside (SNP) or atrial natriuretic peptide (ANP) (both 1 mu M) induced significant (P<0.01) increases in cGMP in guinea pig cells, whereas in rat cells only ANP produ ced a significant (P<0.01) response. By contrast, freshly isolated CMVEs of both species had higher basal cGMP levels than cultured cells, and on expo sure to BK and A23187, responded with significant (P<0.01) increases in cGM P. 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 analy sis demonstrated that ecNOS and sGC protein were lost from the rat CMVEs fo llowing culture. Furthermore, there was also a significant loss of ecNOS mR NA from the rat cells following culture. Conclusions: These data demonstrat e that freshly isolated rat and guinea pig CMVEs possess ecNOS activity, an d that this activity is downregulated following culture. At least for the r at, this effect would seem to lie at both the transcriptional and translati onal level. Furthermore, rat CMVEs have reduced activity of sGC following c ulture. (C) 1999 Elsevier Science B.V. All rights reserved.