Nitric oxide modulates cardiomyocyte pH control through a biphasic effect on sodium/hydrogen exchanger-1

Figures

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  Figure 1. Phosphorylation of the NHE1 C-terminus by PKA and PKG. (A) Various combinations of kinase (PKA or PKG; 8 ng/µL) and protein (His182; 20 ng/µL) were reacted in vitro and run on a Phos-tag gel to seek evidence for differences in phosphorylation pattern. (B) His182 (100 ng/µL) was reacted in vitro with PKA (100 ng/µL) or PKG (50 ng/µL) and analysed by LC-MS/MS mass spectrometry for phosphorylation sites. Results indicated a mean phosphorylation stoichiometry of five and two phosphate groups introduced by PKA and PKG, respectively. (C) Residues identified as being phosphorylated by PKA and PKG. (D) Time course of reaction between His182 (20 ng/µL; plus 500 µM ATP) and either PKG (8 ng/µL; left) or PKA (8 ng/µL; right). Western blots were performed using an antibody raised against phosphorylated serine residues. (E) Western blot performed using antibody against the phosphorylated 14-3-3 binding motif (detecting Ser703 in NHE1). (F) Western blot performed using antibody against phosphorylated Akt substrate (Ser648 in NHE1). Note: blots for PKA- and PKG-reacted His182 were prepared with the same amount of substrate and developed simultaneously with identical exposure time. See…

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  Figure 2. nNOS-derived NO activates NHE1 activity. (A) Histogram of resting [H+]i in ventricular myocytes isolated from nNOS knockout mice (N = 267 from five mice) or their wild-type littermates (N = 244 from five mice). (B) Time course of ammonium prepulse (mean ± SEM; N = 64 from five nNOS knockout mice and N = 54 from five wild-type littermates). Hepes-buffered superfusates, 37°C. Solution manoeuvre acid-loaded myocytes to interrogate acid-extrusion fluxes during recovery phase (blocked by 30 µM dimethylamiloride, DMA). (C) Flux calculated from the rate of pHi change and buffering capacity (Supplementary material online, Figure S2). NHE1-dependent fluxes were slower in nNOS knockout mice (P < 0.0001, two-way ANOVA). (D) NHE1 flux measured in wild-type rat ventricular myocytes. Hepes-buffered superfusates, 37°C. Treatment with selective nNOS inhibitor AAAN (5 µM; N = 16 from five rats) reduced activity relative to control (N = 18 from five rats) (P < 0.0001, two-way ANOVA). (E) Ammonium prepulse performed on adenovirally infected rat ventricular myocytes cultured overnight. Hepes-buffered superfusates, 37°C. Infected cells were identified by eGFP fluorescence (inset). (F) NHE1 activity…

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  Figure 3. NO delivered from donors produces a concentration-dependent biphasic effect on NHE1 activity. (A) Wild-type rat myocytes. Hepes-buffered superfusates, 37°C. The short half-life NO donor, sodium nitroprusside (SNP), was added to solutions 4–6 min before activating NHE1. With 1 µM SNP, NO is expected to peak at 600 nM during pHi recovery (control: N = 35 cells from five animals; SNP: N = 31 cells from five animals). (B) Experiments repeated on myocytes that had been pre-treated with 6 µM ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one), an inhibitor of guanylyl cyclase (GC; ODQ: N = 24 cells from four animals; ODQ+SNP: N = 26 cells from four animals). (C) CO2/HCO3−-buffered superfusates, 37°C; 30 µM dimethylamiloride (DMA) included to block NHE isoforms. pHi recovery mediated by HCO3− dependent transporters is unaffected by NO (control: N = 15 cells from three animals; SNP: N = 15 cells from three animals). (D) Wild-type rat myocytes. Hepes-buffered superfusates, 37°C. The long half-life NO donor NOC12 was added to solutions 10 min before activating NHE1. At a concentration of 5 µM, NOC12 is expected to maintain NO above 300 nM for the duration of pHi recovery. (E) NHE1 fluxes.…

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  Figure 4. NO regulates NHE1 activity via a diffusible messenger and not via S-nitros(yl)ation. (A) Hearts were Langendorff-perfused with normal Tyrode, or Tyrode with high (5 µM NOC12) or low (300 µM NOC12 + 100 µM CPTIO) [NO]. S-nitros(yl)ation assay was performed on membrane fractions of heart lysates. NHE1 staining visible as bands near 90 kDa. Re-probed for TMT as readout of protein S-nitros(yl)ation: no increase in nitros(yl)ation was detectable in the presence of NO donors. (B) Direct NHE1 S-nitros(yl)ation (SNO) was evaluated by an independent method that uses organo-mercury enrichment. (C) No difference in the extent of SNO-enriched NHE1 was observed between control and high NO-treated hearts. N = 7 per group; not significant (P = 0.9); Mann–Witney U-test. (D) Dual microperfusion of isolated rat myocyte. Left microstream produced a NO microdomain from slow-releasing NOC12 (5 µM); NHE1 was inactivated locally by dimethylamiloride (30 µM). Right microstream contained CPTIO to scavenge any spill-over of NO from the left microstream. (E) Measurement of NO microdomain using fluorescent dye DAR-4M, showing gradient of [NO] along length of cell (N = 9 cells from three animals). (F) NHE1…

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  Figure 5. Biphasic regulation of NHE1 by NO is achieved by balancing cGMP-cAMP crosstalk. (A) Rat myocyte infected with H187, a FRET-based sensor of cAMP (inset). NOC12 (5 µM) produced a significant increase in signal. As a positive control, F/I (forskolin and IBMX) raises cAMP to levels that saturate the sensor. Isoprenaline (ISO) is a cAMP-mobilizing β-agonist. NO release from NOC12 produces a rise in [cAMP] equivalent to the effect of ∼1 nM Isoprenaline (Iso). (B) Proposed mechanism through which cGMP increases [cAMP]. (C) Effect of 0.5 nM Iso on NHE1 flux (N = 10 Iso-treated cells from three animals; N = 19 controls from three animals), showing significant reduction in flux (P < 0.0001; two-way ANOVA). (D) Effect of 20 µM 8Br-cAMP on NHE1 flux (N = 15 treated cells from four animals; N = 15 control cells from four animals), showing significant reduction in flux (P < 0.0001; two-way ANOVA). (E) Effect of 5 µM NOC12 when adenylyl cyclase activity is inactivated with membrane-permeable inhibitor dideoxyadenosine (ddA; N = 40 myocytes from five animals). NHE1 flux was significantly greater if cAMP production was ablated (P < 0.0001; two-way ANOVA). (F) Effect of two different…

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  Figure 6. Biphasic regulation of NHE1 by NO affects the propensity of aberrant forms of Ca2+ signalling evoked by mild acidosis. (A) Schematic model of mechanism linking mild acidosis (attained with 30 mM acetate) to sarcoplasmic reticulum (SR) overload. (B) Rat myocytes (loaded with pH-sensitive cSNARF1) under superfusion with Hepes-buffered normal Tyrode with [Ca2+] raised to 5 mM to produce Ca2+ overload and increase the incidence of waves. After a 6 min period in control solution (NT, normal Tyrode), cells were exposed to 30 mM acetate (Ac) which acidifies the myocyte and activates NHE1 (13 cells from two animals). (C) Predicted Na+ influx on NHE1, based on pH-sensitivity of flux determined in Figure 2-2, mapped onto the pHi time course. (D) Rat myocyte loaded with Ca2+ dye Fluo3, imaged in line scan mode. Exemplar line scans are shown for a cell in control conditions, and after 6 min of superfusion with 30 mM acetate. Line scan measurements repeated on cells stimulated with either high or low [NO] released from 5 µM NOC12 (producing [NO]>300 nM) or with 300 µM NOC12 plus 100 µM CPTIO (producing [NO]<30 nM), respectively. Results presented as Ca2+ frequency normalized to baseline level…

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  Figure 7. Model of cardiac NHE1 regulation by NO signalling. (A) The various experimental manoeuvres performed in this study are ranked by strength of NO signal. Higher NO stimulations will evoke greater cGMP production. At higher [cGMP], inhibition of PDE3 permits a steady rise in [cAMP]. The balance between cGMP and cAMP signals sets the level of NHE1 activity. Based on literature values for [NO] measured in vivo, NO-evoked NHE1 activation is expected to be within physiological signalling, whereas NO-evoked NHE1 inhibition would occur over the pathophysiological range of elevated [NO]. (B) Schematic of the signalling pathways, evoked by NO, that regulate NHE1 activity by means of a differential activation of PKG-and PKA-dependent cascades.