DAF-FM (4-Amino-5-methylamino-2′,7′-difluorofluorescein) Diacetate Detects Impairment of Agonist-Stimulated Nitric Oxide Synthesis by Elevated Glucose in Human Vascular Endothelial Cells: Reversal by Vitamin C and l-Sepiapterin

Abstract

Elevated plasma glucose, as commonly seen in types I and II diabetes mellitus, is known to result in endothelial dysfunction, a condition characterized by a loss of nitric oxide (NO)-dependent regulation of vascular tone. In the present study, we have utilized a recently developed NO-sensitive fluorescent dye, DAF-FM (4-amino-5-methylamino-2Ј,7Ј-difluorofluorescein) diacetate to directly examine the consequences of elevated glucose on agonist-evoked NO synthesis in cultured human vascular endothelial cells. Exposure of cells for 5 to 7 days to high (20 mM) external glucose markedly reduced NO production in response to ATP, histamine, or the calcium ionophore calcimycin A23187 compared with 5 and 10 mM glucose concentrations. However, high glucose did not affect agonist-evoked elevations in cytosolic-free calcium, as monitored by Fluo-3.The addition of vitamin C (150 M) and L-sepiapterin (20 M) for ϳ24 h to 20 mM glucose-treated cells improved stimulusevoked NO synthesis but had no effect on cells exposed to either 5 or 10 mM glucose. Likewise, impaired NO production in high glucose-treated cells was largely reversed by exposure (ϳ3 h) to superoxide dismutase. Cellular levels of endothelial nitric-oxide synthase protein were unaltered by elevated glucose treatment, and no further change was observed after the addition of vitamin C and L-sepiapterin. Taken together, the results of our study serve to directly explain at the cellular level how glucose-impaired NO production in human endothelial cells may be reversed by agents that are reported clinically to improve endothelium-dependent vasorelaxation in patients.The vascular endothelium exerts precise control over mean systemic blood pressure through the synthesis and release of vasoactive factors (e.g., nitric oxide, prostaglandins, endothelin, arachadonic acid metabolites, etc.) that act on the surrounding smooth muscle layer(s) (Mombouli and Vanhoutte, 1999). In diabetes mellitus, an early consequence of elevated plasma glucose is the development of endothelial dysfunction, which is characterized by an impairment of nitric oxidedependent vasorelaxation that can lead to retinopathy, end stage renal failure, peripheral neuropathy, atherosclerosis, and heart disease . It is now well recognized that a major consequence of the glucose-mediated metabolic alterations in vascular endothelium is the disruption of normal NO signal transduction (Cosentino and Lü scher, 1998;Creager et al., 2003), which may represent the primary cellular event that initiates the pathogenesis of more severe cardiovascular complications. The increased generation of superoxide radicals, as a result of glucosemediated metabolic disruption, has been identified as a causative event contributing to endothelial cell dysfunction and the loss of NO synthesis/bioavailability (Brownlee, 2001). For example, impaired endothelium-dependent vasorelaxation in diabetic patients and rodent models of diabetes is improved upon administration of either an antioxidant (e.g., vitamin C)

Endogenous Nitric Oxide Is a Key Promoting Factor for Initiation of Seizure-Like Events in Hippocampal and Entorhinal Cortex Slices

“…It is noteworthy that DAF-fm fluorescence recovered to baseline after SNAP application. This recovery is not a true reversal, but it rather indicates increased susceptibility of the fluorescent NO adduct to photobleaching (Sheng et al, 2005) (see Fig. 2 A).…”

Inhibitory motor neurons of the esophageal myenteric plexus are mechanosensitive

“…The change in F360 is an indicator of cell stretch induced by changes in the osmolality of extracellular solution. [Ca 2ϩ ]cyt in the neurons was measured using fura 2-AM at 340-and 380-nm excitation and 510-nm emission, as described previously (2,9 cyt) production in the neurons was measured as described previously (5,38). Briefly, after neurons were cultured on 10-mm glass coverslips for 2 wk, they were loaded with 1 M 4-amino-5-methylamino-2=,7=-difluorofluorescein (DAF-FM) diacetate in PSS at room temperature (22°C) for 30 min and washed in PSS for Ն20 min.…”

Glucose enhances expression of TRPC1 and calcium entry in endothelial cells

“…Although there is a strong association between endothelial dysfunction and the development of cardiovascular disease, the cellular mechanism(s) remain poorly defined (13,20,40). The effects of hyperglycemia on endothelial nitric oxide (NO) synthase (eNOS) activity and NO generation are variable with suppressed basal NO levels reported for cultured endothelial cells with either no change or a decrease or an increase of eNOS expression and activity (11,17,18,19,49), whereas in streptozocin-induced diabetes in rats and mouse, an increase in eNOS expression has been described (16,24).…”

“…Endothelial cells do not express voltage-dependent Ca 2ϩ channels (53), and agonist-stimulated Ca 2ϩ influx depends on the Ca 2ϩ driving force and the opening of store-operated cation channels (SOCs) by a process referred to as capacitative Ca 2ϩ entry or store-operated Ca 2ϩ entry (SOCE) (31,36,41,42). Although there is a lack of a clear understanding concerning how hyperglycemia affects eNOS function, Sheng et al (49) reported that 5-7 days exposure of human vascular endothelial cells to 20 mmol/l glucose significantly impaired agonist-induced NO generation but was without effect on agonist-induced changes in Ca 2ϩ . In contrast, a 96-h exposure of human umbilical vein endothelial cells to 30 mM glucose induced apoptosis that was linked to an enhanced Ca 2ϩ entry via SOCs (50).…”