Anti-NO action of carvedilol in cell-free system and in vascular endothelial cells

Abstract

1. Carvedilol, an adrenoceptor blocker with antioxidant activity, was studied for its ability to interact with NO in a cell-free condition and in an endothelial cell line (ECV304). 2. In a cell-free system, carvedilol attenuated NO-dependent reduction of carboxy-2-phenyl-4,4, 5,5-tetramethyl-imidazoline-1-oxyl-3-oxide induced by a NO donor, 1-hydroxy-2-oxo-3-(aminopropyl)-3-isopropyl-1-triazene (NOC5), which was determined by electron paramagnetic resonance (EPR) spectrometry. The EPR study also showed that nitrosylhaemoglobin formation in rat red blood cells by the addition of NO-saturated solution was attenuated by prior incubation with 0.1 - 10 microM carvedilol. 3. NO-induced fluorescence in 4,5-diaminofluorescein-2 diacethyl (DAF-2DA)-loaded ECV304 cells was attenuated by carvedilol but not by labetalol. The IC(50) of carvedilol for NOC5 or sodium nitroprusside-induced fluorescence of DAF-2DA in ECV304 cells was 1. 0x10(-7) M, which was similar to the reported IC(50) of carvedilol for the antioxidant effect. 4. Cell toxicity induced by a NO donor determined by the number of viable cells after 24 h treatment with 2-2'(hydroxynitrosohydrazino)bis-ethanamine was significantly attenuated by pretreatment with 1 microM carvedilol. 5. Both free and cell-associated carvedilol quenched NO. Because NO mediates both physiological and pathophysiological processes, NO quenching by the drug may have diverse clinical implications depending upon specific functions of local NO in tissues where carvedilol is distributed.

Figure 1

Effect of carvedilol on NO donor-induced reduction of cPTIO. The interaction of carvedilol and NO was examined in a cell free system using an electron paramagnetic resonance (EPR). (a) The EPR spectrum of cPTIO. cPTIO (100 μM) in HBSS gave a 5-line EPR signal as previously reported (Iwamoto et al., 1996). (b) The EPR spectrum of cPTIO reacted with a NO donor. When 10 μM NOC5 was added to the cPTIO solution, NO-dependent reduction of cPTIO resulted in the appearance of a 9-line EPR signal of reduced cPTIO (i.e., cPTI). (c) The EPR spectrum of cPTIO reacted with the NO donor in the presence of carvedilol. When 10 μM carvedilol was present in the reaction mixture, 10 μM NOC5 produced little change in cPTIO signal, indicating that the NO-dependent reduction of cPTIO was attenuated by carvedilol. (D) In 100 μM carvedilol, NOC5-induced reduction of cPTIO was completely abolished. The signals at the both ends of the spectrum (MnO) is an internal standard (Mn) for the equipment and analysis conditions.

Figure 2

Attenuation of nitrosylhemoglobin formation by carvedilol in rat red blood cells (RBC) exposed to NO. The effect of cell-incorporated carvedilol on the formation of intracellular NO-haeme adduct was examined by EPR. (a–c) Rat RBC were suspended in PBS, pH 7.4. Six hundred μl of the RBC suspension was treated with the solvent (a mixture of 10% DMSO and 10% CH₃COOH, diluted 1000 times in PBS) (a), with the solvent followed by 6 μl of NO-saturated HBSS (b), and with 1 μM carvedilol followed by NO-saturated HBSS (c). EPR spectrometry was conducted in liquid nitrogen as previously described. The magnitude of the typical nitrosylhemoglobin signal at g=2.04 shown in (b) is markedly attenuated in (c). Although not shown, carvedilol (1–100 μM) did not affect the EPR signal of haemoglobin. (d) Rat RBC were treated with various concentrations of carvedilol followed by addition of NO-saturated HBSS as described above. The EPR signal of nitrosylhemoglobin was double integrated to calculate the concentration using CuSO₄ as a standard. Values are mean±s.d. of four determinations.

Figure 3

Fluorometry of DAF-2DA-loaded HUVEC treated with NOC5. ECV304 cells grown in a 48-well plate was incubated with 10 μM DAF-2DA in PBS, pH 7.4 for 60 min and washed twice with 100 mM PBS, pH 7.4. The cells were treated with given concentrations of NOC5 or hydrogen peroxide for 30 min and fluorometry was performed. The density of green fluorescence was measured by analysing the digital images using a computer as described in the Methods. Values are mean±s.d. of 16–20 determinations.

Figure 4

Fluorescence microscopy of the DAF-2DA-loaed ECV304 cells treated with NO donors and carvedilol. (a) ECV304 cells were loaded with DAF-2DA and fluorescent microscopy was performed. The unstimulated cells gave little fluorescence. (b–d) When DAF-2DA loaded cells were stimulated with 1×10⁻⁴ M NOC5 (b) or 1×10⁻⁴ M sodium nitroprusside (SNP) (c) for 30 min, the cells showed a marked increase in fluorescence, while 1×10⁻⁴ M hydrogen peroxide (d) showed no effect. (e,f) The ECV304 cells pretreated with 1 μM carvedilol showed marked attenuation in fluorescence induced by NOC5 (e) and SNP (f).

Figure 5

Fluorometric evaluation of NO production by ECV304 cells treated with NO donors, carvedilol and labetalol. ECV304 cells were incubated with different concentrations of carvedilol or with labetalol for 2 h, loaded with DAF-2DA and fluorescent images were obtained 30 min after treatment with 1×10⁻⁴ M NOC5 (a) or 1×10⁻⁴ M SNP (b). The intensity of green fluorescence was determined and the relative intensity to that of DAF-2DA-loaded cells treated with 1×10⁻⁴ M NOC5 (a) or of 1×10⁻⁴ M SNP (b) was calculated. Values are mean±s.d. of 20 determinations from three separate experiments.

Figure 6

Effect of carvedilol on the number of viable ECV304 cells after exposure to NO. ECV304 cells plated on a 96-well plate were incubated with 1 μM carvedilol or vehicle for 2 h followed by treatment with given concentrations of NOC18. The numbers of viable cells were estimated 24 h later by the oxidation of alamar blue determined by the difference of absorbance between 575 and 595 nm. The number of cells incubated for an identical period without any treatment was used as a reference to calculate the relative number of treated cells to untreated control cells. n=8 ea. ^*Denotes P<0.05 between groups.