Cardiac capillary cells release biologically active nitric oxide at an early stage of in vitro development

Coronary microvascular endothelial cells (EC) may regulate the myocardial contractile function by releasing cardioactive agents such as nitric oxide (NO). However, understanding of these regulatory mechanisms is complicated by the fact that EC exhibit marked phenotypic changes, such as the loss of endothelial NO synthase (eNOS), when they are placed into culture. Recently, it has been shown that eNOS gene expression is regulated by specific cell-cell interactions with mural cells depending on vascular beds. Since EC and pericytes (PL) are closely associated in capillaries, we have enzymatically isolated these cells from rat hearts to develop a primary culture of capillary cells favoring the re-establishment of cell interactions in vitro.

Expression of transcripts for both eNOS and the inducible isoform (iNOS), was evaluated by using reverse transcription, polymerase chain reaction and Southern blot analysis. Expression of NOS proteins was detected with specific rhodamine-labeled antibodies. Production of NO was assessed (i) from nitrite measurements in culture supernatants by the Griess reaction, and (ii) from its antiproliferative action on cardiac fibroblasts (FIB) in non-contacted cocultures (reporter-cell bioassay) compared to that of sodium nitroprusside in homotypic FIB cultures. Fura-2 fluorescence was used to measure agonist-induced changes in cytosolic free calcium levels.

In our heterotypic cultures, EC firstly proliferated to form spots of monolayers (i.e. first phase) before to be covered by PL on the following days (i.e. second phase). The data from RT-PCR analysis demonstrate the presence of mRNAs of both eNOS and iNOS at all developmental stages of the culture. However, eNOS protein was only detected and restricted to EC. During the first phase of cell growth (5-8 days), cells released nitrite and a labile factor, clearly identified as NO, that inhibited the FIB proliferation in reporter-cell bioassay. These effects, not observed during the second phase of cell growth (15-20 days), were prevented by hemoglobin (50 microM) and by N omega-nitro-L-arginine methyl ester (L-NAME; 100 microM). At the two periods of culture, EC increased rapidly their cytosolic Ca(2+) concentration in response to bradykinin (10 nM). However, this calcium response was associated with an increase in nitrite production only in older cultures.

Our data indicate that heterotypic cultures of native capillary cells preserve the eNOS expression by EC. This enzyme is basally active at an early stage of in vitro development, and then becomes activatable by a Ca(2+)-mobilizing agonist. NO released by growing EC downregulates the proliferation of cardiac FIB, an effect which could be important in the cardiovascular plasticity.