Compartment-specific nitric oxide signaling in mouse cardiac endothelial cells: Intra- and extracellular measurements and the impact of cryopreservation

Nitric oxide (NO) produced by endothelial cells plays a central role in regulating vascular tone, blood flow, and inflammatory responses. Although NO freely diffuses across cell membranes, intracellular and extracellular NO-related signals are often measured interchangeably, complicating interpretation of NO production, signaling, and bioavailability. Moreover, how cryopreservation and experimental context influence these two measurement compartments remains poorly defined. Here, we systematically compared intracellular and extracellular NO-related fluorescence in mouse cardiac endothelial cells (MCECs) under defined stimulatory, inhibitory, and culture conditions. Using DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) and DAF-FM-based assays, we examined (i) the impact of cryopreservation on intra- and extracellular NO-related signals following calcium ionophore A23187 stimulation, and (ii) the effects of physiological stimulation (bradykinin), enzymatic inhibition (Nᴳ-Nitro-l-arginine methyl ester (l-NAME)), and NO scavenging (carboxy-PTIO potassium salt (cPTIO)) in non-cryopreserved cells. Calcium ionophore A23187 increased intracellular NO-related fluorescence across non-cryopreserved and post-thaw groups, whereas extracellular responses were smaller and more variable, with increases observed primarily in glycerol-cryopreserved cells. The removal of phenol red from the medium enhanced extracellular NO-related fluorescence (reflecting improved detection sensitivity) but reduced intracellular signals, indicating a strong influence of medium composition on NO-related measurements. Bradykinin produced dose-dependent increases in extracellular signals accompanied by reductions in intracellular signals, consistent with rapid diffusion and context-dependent intracellular detection. l-NAME reduced intracellular fluorescence and suppressed extracellular signals only under simplified buffer conditions, while cPTIO produced modest reductions in extracellular fluorescence with substantial variability. Together, these findings indicate that intracellular and extracellular NO-related fluorescence are differentially regulated by stimulation, membrane properties, oxidative environment, and assay conditions. The results underscore the importance of compartment-specific, method-aware interpretation when using fluorescence-based approaches to assess NO signaling in endothelial cell models, particularly in the context of cryopreservation and experimental reproducibility.