Intracellular protons (H+ ions) are major modulators of cardiac myocyte function. The sodium-hydrogen exchanger (NHE1) plays an important role in the control of intracellular pH by being a main mechanism for H+ ion extrusion from cardiac myocytes. Due to the stoichiometric coupling with Na+ ions, NHE1 has been implicated in the modulation of Ca2+ handling which physiologically may result in positive inotropy, but aberrant NHE1 function is associated with hypertrophy and heart failure1. Another major modulator of cardiac function is nitric oxide (NO)2, however, its interaction with H+ signalling remains poorly understood. We sought to investigate the effect of NO-evoked signalling on the modulation of intracellular pH via effects on NHE in adult rat myocytes.Myocytes were isolated enzymatically from adult male Sprague-Dawley rat ventricles (Langendorff perfusion). Isolated myocytes were loaded with a pH-sensitive dye, carboxy-SNARF-1 (10µM), and imaged using an epifluorescence microscope. Cells were superfused with HEPES-buffered Tyrode’s solution to eliminate HCO3–dependent acid-extrusion. H+-extrusion by NHE was activated by acid-loading cells (20 mM ammonium pre-pulse solution manoeuvre). Data are presented as mean±SEM, Student’s t-tests, 2-way RM ANOVA were used to test for statistical significance where P<0.05.NO release from the exogenous donor NOC-12 (5µM) attenuated NHE1 H+-efflux (10.2±4.3 mM.min-1 vs. 4.8±1.8 mM.min-1 at pH 6.82, n=10-16, P<0.01), in agreement with a previous report3. This inhibition was prevented by pre-treatment with the guanylate cyclase (GC) inhibitor ODQ (5µM) suggesting that inhibition by exogenous NO is via the NO-GC-cGMP pathway. In the absence of NO donors, exposure to AAAN (5µM), a specific inhibitor of neuronal NOS (nNOS), reduced NHE1 activity (9.0±3.6 mM.min-1 vs. 5.31±1.9 mM.min-1 at pH 6.82 n=17-19, P<0.001). This finding argues that basal NO production has an excitatory effect on NHE1 activity, i.e. opposite to the effect of exogenously delivered NO. Inhibition of GC with ODQ (5µM) under basal conditions also reduced NHE1 flux (12.73±1.6 mM.min-1 vs. 6.38±3.5 mM.min-1 at pH 6.82 n=5-6, P<0.001), arguing that the basal levels and distribution of cGMP exerts an excitatory effect on NHE1. This may be the downstream pathway triggered by basal nNOS-derived NO release. To test further the hypothesis that nNOS activity stimulates NHE1, adenoviral nNOS gene delivery and subsequent up-regulation of nNOS4 enhanced H+ flux via NHE1 relative to sham-controls (21.9±0.3 mM.min-1 vs. 16.5±0.6 mM.min-1 at pH 6.92 n=3, P<0.05). Our findings show that NO can modulate NHE1 activity, and hence pH-control either via a biphasic dose-dependence or by operating over different spatial domains possibly as a result of compartmentalized cGMP-signalling.