Abstract
AimsWhen activated, Na+/H+ exchanger-1 (NHE1) produces some of the largest ionic fluxes in the heart. NHE1-dependent H+ extrusion and Na+ entry strongly modulate cardiac physiology through the direct effects of pH on proteins and by influencing intracellular Ca2+ handling. To attain an appropriate level of activation, cardiac NHE1 must respond to myocyte-derived cues. Among physiologically important cues is nitric oxide (NO), which regulates a myriad of cardiac functions, but its actions on NHE1 are unclear.Methods and resultsNHE1 activity was measured using pH-sensitive cSNARF1 fluorescence after acid-loading adult ventricular myocytes by an ammonium prepulse solution manoeuvre. NO signalling was manipulated by knockout of its major constitutive synthase nNOS, adenoviral nNOS gene delivery, nNOS inhibition, and application of NO-donors. NHE1 flux was found to be activated by low [NO], but inhibited at high [NO]. These responses involved cGMP-dependent signalling, rather than S-nitros(yl)ation. Stronger cGMP signals, that can inhibit phosphodiesterase enzymes, allowed [cAMP] to rise, as demonstrated by a FRET-based sensor. Inferring from the actions of membrane-permeant analogues, cGMP was determined to activate NHE1, whereas cAMP was inhibitory, which explains the biphasic regulation by NO. Activation of NHE1-dependent Na+ influx by low [NO] also increased the frequency of spontaneous Ca2+ waves, whereas high [NO] suppressed these aberrant forms of Ca2+ signalling.ConclusionsPhysiological levels of NO stimulation increase NHE1 activity, which boosts pH control during acid-disturbances and results in Na+-driven cellular Ca2+ loading. These responses are positively inotropic but also increase the likelihood of aberrant Ca2+ signals, and hence arrhythmia. Stronger NO signals inhibit NHE1, leading to a reversal of the aforementioned effects, ostensibly as a potential cardioprotective intervention to curtail NHE1 overdrive.
Highlights
Sarcolemmal Naþ/Hþ exchanger-1 (NHE1) is the most powerful regulator of intracellular pH in the heart, capable of correcting acid–base disturbances within minutes.[1,2,3] When fully activated, NHE1 can generate acid-extrusion fluxes of the order of tens of mM/min, coupled to a matching influx of Naþ ions.[1,2,3] Such high acid-extrusion fluxes are conducive for cardiomyocyte pHi homeostasis, in periods of elevated metabolic acid production, driven by a heightened demand for cardiac work
Using manipulations that include nNOS knockout, adenoviral nNOS gene delivery, nNOS inhibition, and pharmacological nitric oxide (NO) titrations, we demonstrate that low levels of NO activate NHE1, whereas higher doses switch to a net inhibitory effect
The biphasic effect of NO on NHE1 was not observed in neonatal myocytes (Supplementary material online, Figure S6A and B) or MDA-MB-468 cells (Supplementary material online, Figure S6C and D). These findings argue that NO signals modulate NHE1 in a highly context-sensitive manner, involving elements that are present in adult cardiac myocytes, but not necessarily in other cell types
Summary
Sarcolemmal Naþ/Hþ exchanger-1 (NHE1) is the most powerful regulator of intracellular pH (pHi) in the heart, capable of correcting acid–base disturbances within minutes.[1,2,3] When fully activated, NHE1 can generate acid-extrusion fluxes of the order of tens of mM/min, coupled to a matching influx of Naþ ions.[1,2,3] Such high acid-extrusion fluxes are conducive for cardiomyocyte pHi homeostasis, in periods of elevated metabolic acid production, driven by a heightened demand for cardiac work. This balancing act is exercised by appropriately controlling NHE1 activity
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