Transnitrosylating nitric oxide species directly activate type I protein kinase A, providing a novel adenylate cyclase-independent cross-talk to beta-adrenergic-like signaling

Abstract

The transnitrosylating nitric oxide (NO) donor nitrocysteine (CysNO) induced a disulfide bond between the two regulatory RI subunits of protein kinase A (PKA). The conventional NO donor S-nitroso-N-acetylpenicillamine failed to do this, consistent with our observation that it also did not promote protein S-nitrosylation. This disulfide oxidation event activated PKA and induced vasorelaxation independently of the classical beta-adrenergic or NO signaling pathway. Activation of PKA had also been anticipated to exert a positive inotropic effect on the myocardium but did not. The lack of positive inotropy was explained by CysNO concomitantly activating protein kinase G (PKG) Ialpha. PKG was found to exert a partial negative inotropic influence regardless of whether PKA was activated by classical beta-receptor stimulation or by disulfide bond formation. This work demonstrates that NO molecules that can induce S-nitrosylation directly activate type I PKA, providing a novel cross-talk to beta-adrenergic-like signaling without receptor or adenylate cyclase stimulation. However, the expected positive inotropic consequences of PKA activation by this novel mechanism are countermanded by the simultaneous dual activation of PKGIalpha, which is also activated by CysNO.

FIGURE 1.

A, isolated perfused rat hearts were exposed to the NO donor SNAP for different durations or concentrations before being assayed for protein S-nitrosylation using the biotin-switch method. All attempts to induce protein S-nitrosylation by administering SNAP to intact hearts failed. However, if a cardiac homogenate was exposed to SNAP in vitro, S-nitrosylation was observed. B, although SNAP failed to induce protein S-nitrosylation in the perfused heart, it did modulate the phosphorylation status of phospholamban in a time- and dose-dependent manner, consistent with activation of the NO-cGMP-PKG pathway and demonstrating that the NO donor was indeed bioavailable to the tissue. C, in marked contrast to SNAP, when the NO donor CysNO was perfused into the heart, it induced a dose-dependent increase in protein S-nitrosylation. Mwt, molecular weight; P-Time, perfusion time.

FIGURE 2.

A, consistent with CysNO inducing widespread S-nitrosylation, this treatment also caused disulfide oxidation of the redox-modulated kinases PKGIα and PKA RI. B, CysNO also efficiently induced disulfide formation in recombinant N-terminal PKGIα, demonstrating that this oxidation state can result as a direct reaction of the oxidizing NO donor and the kinase. This supports the possibility that CysNO may directly S-nitrosylate the kinases in vivo prior to self-reduction and consequential disulfide bond formation. C, by themselves, SNAP and cysteine failed to induce PKGIα or PKA RI interprotein disulfide bond formation. However, together, they efficiently induced disulfides into both kinases, consistent with SNAP S-nitrosylating cysteine extracellularly before transport into the cell, where it modifies proteins.

FIGURE 3.

A, the LVDP of isolated rat hearts was rapidly depressed by 16. 6 ± 2.4% when exposed to CysNO. A positive inotropic response had been anticipated, as we equate disulfide PKA RI with activation of this kinase, which should increase cardiac work. We hypothesized that the negative inotropy was due to the simultaneous activation of PKG by CysNO, which activates this kinase classically by its NO donor capability and also by disulfide oxidation. B, to avoid the additional complexity of CysNO being an NO donor as well as a disulfide inducer, LVDP was monitored in hearts exposed to H₂O₂ with or without co-treatment with the PKG inhibitor KT5823. H₂O₂ induced disulfide bond activation of PKGIα and PKA RI without stimulating the NO-sGC pathway. By inhibiting PKG during the H₂O₂ treatment, an underlying activation of PKA by disulfide bond formation was unveiled, resulting in the observed positive inotropy. C, to examine the possibility that PKG dominates in the face of PKA activation and prevents positive inotropy, we exposed hearts to isoprenaline (ISO) with or without co-treatment with SNAP. Isoprenaline increased LVDP as expected, whereas the co-administration of SNAP rapidly exerted a negative inotropic effect.

FIGURE 4.

A, both CysNO and H₂O₂ rapidly lowered the perfusion pressure in rat coronary vessels, with the former exerting a more marked vasodilatory response. B, isoprenaline rapidly reduced coronary perfusion pressure. When SNAP was then administered simultaneously with isoprenaline (ISO), perfusion pressure remained depressed and similar to that observed with isoprenaline alone. This lack of effect of SNAP on perfusion pressure was in marked contrast to its negative influence on LVDP (Fig. 3C). C, aerobically perfused isolated hearts exposed to the PKG inhibitor KT5823 responded with a marginal time-dependent increase in perfusion pressure, which may be consistent with a limited contribution of this kinase to coronary tone under basal conditions. D, during the treatment of the aerobic heart with the KT5823 inhibitor, there was a small time-dependent depression in LVDP contractility.

FIGURE 5.

A, the vasorelaxive properties of SNAP and the thiol-oxidizing CysNO donor were compared in isolated vascular rings from rat thoracic aorta. SNAP elicited a classical dose-response vasorelaxation curve, which was significantly inhibited (rightward shift) by the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine (DDA). The SNAP-induced relaxations were entirely prevented by the sGC inhibitor ODQ. DMSO, dimethyl sulfoxide. B, CysNO also induced vasorelaxation, albeit this was only partially sensitive to ODQ. Thus, CysNO can induce vasorelaxation independently of the classical NO-sGC signaling paradigm, consistent with it directly activating PKA and PKG by disulfide oxidation. C, CysNO-induced relaxations of rat aorta were examined in the presence and absence of either a PKA or PKG inhibitor. The dose response to CysNO was significantly rightward-shifted by inhibitors of either kinase, but more so by the PKA antagonist (R_p)-8-bromo-cAMP. Overall, CysNO induced vasorelaxation independently of sGC, involving the activation of both PKA and PKG by interprotein disulfide bond formation.