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

Objective: Coronary microvascular endothelial cells (EC) may regulate the m yocardial contractile function by releasing cardioactive agents such as nit ric oxide (NO). However, understanding of these regulatory mechanisms is co mplicated by the fact that EC exhibit marked phenotypic changes, such as th e loss of endothelial NO synthase (eNOS), when they are placed into culture . Recently, it has been shown that eNOS gene expression is regulated by spe cific cell-cell interactions with mural cells depending on vascular beds. S ince EC and pericytes (PL) are closely associated in capillaries, we have e nzymatically isolated these cells from rat hearts to develop a primary cult ure of capillary cells favoring the re-establishment of cell interactions i n vitro. Methods: Expression of transcripts for both eNOS and the inducible isoform (iNOS), was evaluated by using reverse transcription, polymerase c hain reaction and Southern blot analysis. Expression of NOS proteins was de tected with specific rhodamine-labeled antibodies. Production of NO was ass essed (i) from nitrite measurements in culture supernatants by the Griess r eaction, 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. Results: In our heterotypic cultures, EC firstly proliferated to form spots of monolayers (i.e. first phase) before to be covered by PL on the followi ng 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, Duri ng the first phase of cell growth (5-8 days), cells released nitrite and a labile factor, clearly identified as NO, that inhibited the FIB proliferati on in reporter-cell bioassay. These effects, not observed during the second phase of cell growth (15-20 days), were prevented by hemoglobin (50 mu M) and by N omega-nitro-L-arginine methyl ester (L-NAME; 100 mu M). At the two periods of culture, EC increased rapidly their cytosolic Ca2+ concentratio n in response to bradykinin (10 nM). However, this calcium response was ass ociated with an increase in nitrite production only in older cultures. Conc lusions: Our data indicate that heterotypic cultures of native capillary ce lls 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 Ca2 +-mobilizing agonist, NO released by growing EC downregulates the prolifera tion of cardiac FIB, an effect which could be important in the cardiovascul ar plasticity. (C) 2000 Elsevier Science B.V. All rights reserved.