Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes

We investigated the potential involvement of peroxynitrite (ONOO−) in the modulation of calcium current (ICa) in guinea pig ventricular myocytes with the whole‐cell patch clamp technique and with cyclic AMP (cAMP) measurements. Because of the short half‐life of ONOO− at physiological pH, we induced an increase in its intracellular levels by using donors of the precursors, nitric oxide (NO) and superoxide anion (O2−). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 μM) or SNAP (300 μM) increased basal ICa (50.3 ± 4.6%, n = 7 and 46.2 ± 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 μM) also stimulated basal ICa (44.6 ± 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 μM), although separately ineffective on ICa, enhanced the current if applied together (33.5 ± 0.7%, n = 7). The simultaneous donor of O2− and NO, SIN‐1 (500 μM), also stimulated basal ICa (22.8 ± 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 μM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 μM), ICa was still increased by SIN‐1 (32.0 ± 6.1%, n = 4 and 30.0 ± 5.4%, n = 8). Both Manganese(III)tetrakis(4‐benzoic acid) porphyrin chloride (MnTBAP, 100 μM) a ONOO− scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN‐1 on ICa (respectively −0.6 ± 4.1%, n = 4 and 3.6 ± 4.3%, n = 4). Intracellular cAMP level was unaltered by SIN‐1, while it was enhanced by blocking the NO–cGMP pathway with the NO synthase inhibitor L‐NMMA. These results suggest that peroxynitrite donors increase cardiac calcium current without the involvement of cAMP and cGMP. J. Cell. Physiol. 197: 284–296, 2003© 2003 Wiley‐Liss, Inc.