The control of nitric oxide dynamics and interaction with substituted zinc-phthalocyanines.

Phthalocyanines are artificial macrocycles that can harbour a central metal atom with four symmetric coordinations. Similar to metal-porphyrins, metal-phthalocyanines (M-PCs) may bind small molecules, especially diatomic gases such as NO and O2. Furthermore, various chemical chains can be grafted at the periphery of the M-PC macrocycle, which can change its properties, including the interaction with diatomic gases. In this study, we synthesized Zn-PCs with two different substituents and investigated their effects on the interaction and dynamics of nitric oxide (NO). Time-resolved absorption spectroscopy from picosecond to millisecond revealed that NO dynamics dramatically depends on the nature of the groups grafted to the Zn-PC macrocycle. These experimental results were rationalized by DFT calculations, which demonstrate that electrostatic interactions between NO and the quinoleinoxy substituent modify the potential energy surface and decrease the energy barrier for NO recombination, thus controlling its affinity.

The H-NOX protein structure adapts to different mechanisms in sensors interacting with nitric oxide.

Some classes of bacteria within phyla possess protein sensors identified as homologous to the heme domain of soluble guanylate cyclase, the mammalian NO-receptor. Named H-NOX domain (Heme-Nitric Oxide or OXygen-binding), their heme binds nitric oxide (NO) and O2 for some of them. The signaling pathways where these proteins act as NO or O2 sensors appear various and are fully established for only some species. Here, we investigated the reactivity of H-NOX from bacterial species toward NO with a mechanistic point of view using time-resolved spectroscopy. The present data show that H-NOXs modulate the dynamics of NO as a function of temperature, but in different ranges, changing its affinity by changing the probability of NO rebinding after dissociation in the picosecond time scale. This fundamental mechanism provides a means to adapt the heme structural response to the environment. In one particular H-NOX sensor the heme distortion induced by NO binding is relaxed in an ultrafast manner (∼15 ps) after NO dissociation, contrarily to other H-NOX proteins, providing another sensing mechanism through the H-NOX domain. Overall, our study links molecular dynamics with functional mechanism and adaptation.

Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy.

Mapping red blood cells (RBCs) flow and oxygenation is of key importance for analyzing brain and tissue physiology. Current microscopy methods are limited either in sensitivity or in spatio-temporal resolution. In this work, we introduce a novel approach based on label-free third-order sum-frequency generation (TSFG) and third-harmonic generation (THG) contrasts. First, we propose a novel experimental scheme for color TSFG microscopy, which provides simultaneous measurements at several wavelengths encompassing the Soret absorption band of hemoglobin. We show that there is a strong three-photon (3P) resonance related to the Soret band of hemoglobin in THG and TSFG signals from zebrafish and human RBCs, and that this resonance is sensitive to RBC oxygenation state. We demonstrate that our color TSFG implementation enables specific detection of flowing RBCs in zebrafish embryos and is sensitive to RBC oxygenation dynamics with single-cell resolution and microsecond pixel times. Moreover, it can be implemented on a 3P microscope and provides label-free RBC-specific contrast at depths exceeding 600 µm in live adult zebrafish brain. Our results establish a new multiphoton contrast extending the palette of deep-tissue microscopy.

Ultrafast dynamics of heme distortion in the O2-sensor of a thermophilic anaerobe bacterium.

Heme-Nitric oxide and Oxygen binding protein domains (H-NOX) are found in signaling pathways of both prokaryotes and eukaryotes and share sequence homology with soluble guanylate cyclase, the mammalian NO receptor. In bacteria, H-NOX is associated with kinase or methyl accepting chemotaxis domains. In the O2-sensor of the strict anaerobe Caldanaerobacter tengcongensis (Ct H-NOX) the heme appears highly distorted after O2 binding, but the role of heme distortion in allosteric transitions was not yet evidenced. Here, we measure the dynamics of the heme distortion triggered by the dissociation of diatomics from Ct H-NOX using transient electronic absorption spectroscopy in the picosecond to millisecond time range. We obtained a spectroscopic signature of the heme flattening upon O2 dissociation. The heme distortion is immediately (<1 ps) released after O2 dissociation to produce a relaxed state. This heme conformational change occurs with different proportions depending on diatomics as follows: CO < NO < O2. Our time-resolved data demonstrate that the primary structural event of allostery is the heme distortion in the Ct H-NOX sensor, contrastingly with hemoglobin and the human NO receptor, in which the primary structural events are respectively the motion of the proximal histidine and the rupture of the iron-histidine bond.

Soluble Guanylate Cyclase Inhibitors Discovered among Natural Compounds.

Soluble guanylate cyclase (sGC) is the human receptor of nitric oxide (NO) in numerous kinds of cells and produces the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) upon NO binding to its heme. sGC is involved in many cell signaling pathways both under healthy conditions and under pathological conditions, such as angiogenesis associated with tumor growth. Addressing the selective inhibition of the NO/cGMP pathway is a strategy worthwhile to be investigated for slowing down tumoral angiogenesis or for curing vasoplegia. However, sGC inhibitors are lacking investigation. We have explored a chemical library of various natural compounds and have discovered inhibitors of sGC. The selected compounds were evaluated for their inhibition of purified sGC in vitro and sGC in endothelial cells. Six natural compounds, from various organisms, have IC50 in the range 0.2-1.5 µM for inhibiting the NO-activated synthesis of cGMP by sGC, and selected compounds exhibit a quantified antiangiogenic activity using an endothelial cell line. These sGC inhibitors can be used directly as tools to investigate angiogenesis and cell signaling or as templates for drug design.

Role of surface defects in colloidal cadmium selenide (CdSe) nanocrystals in the specificity of fluorescence quenching by metal cations.

This study aimed to answer the question as whether crystal defects at the surface of soluble capped CdSe nanocrystals (or quantum dots, QDs) in water colloidal suspension are involved in the mechanism of fluorescence quenching induced by metal cations. Nanocrystals of CdSe were synthesized by an aqueous protocol, varying the ratio between the CdSe precursors and the grafted ligand mercaptosuccinic acid (MSA). Changing the MSA/CdSe ratio during synthesis impacts the crystal nucleation growth, which plays an important role in surface construction of CdSe QDs and changes the surface state. In this way, we could modulate the crystal surface defects of CdSe, as verified by analysis of the individual bands which constitute the emission spectra and are associated with different relaxation processes. We found that the various tested metal cations, which interact in solution with the MSA ligand grafted on the QDs, quench their fluorescence differently, depending on the MSA/CdSe ratio used in synthesis. The crystal defects modulate the excitonic relaxation in CdSe and we demonstrated here that the surface defects intervene in the quenching of QDs induced by the binding of cations.

Iron transitions during activation of allosteric heme proteins in cell signaling.

Allosteric heme proteins can fulfill a very large number of different functions thanks to the remarkable chemical versatility of heme through the entire living kingdom. Their efficacy resides in the ability of heme to transmit both iron coordination changes and iron redox state changes to the protein structure. Besides the properties of iron, proteins may impose a particular heme geometry leading to distortion, which allows selection or modulation of the electronic properties of heme. This review focusses on the mechanisms of allosteric protein activation triggered by heme coordination changes following diatomic binding to proteins as diverse as the human NO-receptor, cytochromes, NO-transporters and sensors, and a heme-activated potassium channel. It describes at the molecular level the chemical capabilities of heme to achieve very different tasks and emphasizes how the properties of heme are determined by the protein structure. Particularly, this reviews aims at giving an overview of the exquisite adaptability of heme, from bacteria to mammals.

Use of MPA-capped CdS quantum dots for sensitive detection and quantification of Co2+ ions in aqueous solution.

Water soluble CdS quantum dots (QDs) were synthesized by a simple aqueous chemical route using mercaptopropionic acid (MPA) as a stabilizer. These QDs had a fluorescence emission band maximum at 540 nm with a FWHM ∼130 nm and a quantum yield of ∼12%. Transmission electronic microscopy images were used to determine the QD diameter of 8.9 ±â€¯0.4 nm. From this value we calculated the molecular mass M(QD) = 1.17 × 106 g mol-1 and the extinction coefficient at the band edge (450 nm) ε450 = 4.7 × 106 cm-1 M-1, which allowed to determine the true molar concentration of 17 nM for spectroscopic measurements in solution. The fluorescence intensity of MPA-CdS QDs was quenched only in the presence of Co2+ ions, but not in the presence of thirteen other metal cations. The fluorescence quenching of MPA-CdS QDs appeared proportional to the Co2+ concentration in the range 0.04-2 µM. Based on a fluorescence peak position and a lifetime both independent from Co2+ concentration, the quenching mechanism of MPA-CdS QDs appeared static. Because the strong electronic absorption of Co2+ overlaps the emission of QDs, our results can be explained by Förster energy transfer from QD to the bound Co2+ cations.

Density of Grafted Chains in Thioglycerol-Capped CdS Quantum Dots Determines Their Interaction with Aluminum(III) in Water.

We aimed to quantify the interaction of water-soluble-functionalized CdS quantum dots (QDs) with metal cations from their composition and physical properties. From the diameter of thioglycerol-capped nanoparticles (TG-CdS QDs) measured by electronic microscopy ( D = 12.3 ± 0.3 nm), we calculated the molecular mass of the individual particle MAQD = (3 ± 0.5) × 106 g·mol-1 and its molar absorption coefficient ε450 = 21 × 106 M-1·cm-1. We built a three-dimensional model of the TG-CdS QDs in agreement with the structural data, which allowed us to quantify the number of thioglycerol grafted chains to ∼2000 per QD. This value fully matches the saturation binding curve of Al3+ cations interacting with TG-CdS QDs. The reaction occurred with a slow association rate ( kon = 2.1 × 103 M-1·s-1), as expected for heavy QDs. The photophysical properties of the functionalized QDs were studied using an absolute QD concentration of 7 nM, which allowed us to investigate the interaction with 14 metallic cations in water. The fluorescence intensity of TG-CdS QDs could be quenched only in the presence of Al3+ ions in the range 0.2-10 µM but not with other cations and was not observed with other kinds of grafting chains.

Discovery of a Novel and Selective Indoleamine 2,3-Dioxygenase (IDO-1) Inhibitor 3-(5-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (EOS200271/PF-06840003) and Its Characterization as a Potential Clinical Candidate.

Tumors use tryptophan-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO-1) to induce an immunosuppressive environment. IDO-1 is induced in response to inflammatory stimuli and promotes immune tolerance through effector T-cell anergy and enhanced Treg function. As such, IDO-1 is a nexus for the induction of a key immunosuppressive mechanism and represents an important immunotherapeutic target in oncology. Starting from HTS hit 5, IDO-1 inhibitor 6 (EOS200271/PF-06840003) has been developed. The structure-activity relationship around 6 is described and rationalized using the X-ray crystal structure of 6 bound to human IDO-1, which shows that 6, differently from most of the IDO-1 inhibitors described so far, does not bind to the heme iron atom and has a novel binding mode. Clinical candidate 6 shows good potency in an IDO-1 human whole blood assay and also shows a very favorable ADME profile leading to favorable predicted human pharmacokinetic properties, including a predicted half-life of 16-19 h.

Structural changes and picosecond to second dynamics of cytochrome c in interaction with nitric oxide in ferrous and ferric redox states.

Apart from its role in electron transfer, mitochondrial cytochrome c also plays a role in apoptosis and is subject to nitrosylation. The cleavage of the Fe-Met80 bond plays a role in several processes including the release of Cyt c from mitochondria or increase of its peroxidase activity. Nitrosylation of Cyt c precludes the reformation of the disrupted Fe-Met80 bond and was shown to occur during apoptosis. These physiological properties are associated with a conformational change of the heme center of Cyt c. Here, we demonstrate that NO binding induces pronounced heme conformational changes in the six-coordinate Cyt c-NO complex. Equilibrium and time-resolved Raman data reveal that the heme structural conformation depends both on the nature of the distal iron ligand (NO or Met80) and on the Fe2+ or Fe3+ heme redox state. Upon nitrosylation, the heme ruffling distortion is greatly enhanced for ferrous Cyt c. Contrastingly, the initial strong heme distortion in native ferric Cyt c almost disappears after NO binding. We measured the heme coordination dynamics in the picosecond to second time range and identified Met80 and NO rebinding phases using time-resolved Raman and absorption spectroscopies. Dissociation of NO instantly produces 5-coordinate heme with a domed structure which continues to rearrange within 15 ps, while the initial ruffling distortion disappears. The rates of Cyt c-NO complex formation measured by transient absorption are kon = 1.81 × 106 M-1 s-1 for ferric Cyt c and 83 M-1 s-1 for ferrous Cyt c. After NO dissociation and exit from the heme pocket, the rebinding of Met80 to the heme iron takes place 6 orders of magnitude more slowly (3-5 µs) than Met80 rebinding in the absence of NO (5 ps). Altogether, these data reveal the structural and dynamic properties of Cyt c in interaction with nitric oxide relevant for the molecular mechanism of apoptosis.

The Dynamics Behind the Affinity: Controlling Heme-Gas Affinity via Geminate Recombination and Heme Propionate Conformation in the NO Carrier Cytochrome c'.

Nitric oxide (NO) sensors are heme proteins which may also bind CO and O2. Control of heme-gas affinity and their discrimination are achieved by the structural properties and reactivity of the heme and its distal and proximal environments, leading to several energy barriers. In the bacterial NO sensor cytochrome c' from Alcaligenes xylosoxidans (AXCP), the single Leu16Ala distal mutation boosts the affinity for gas ligands by a remarkable 106-108-fold, transforming AXCP from one of the lowest affinity gas binding proteins to one of the highest. Here, we report the dynamics of diatomics after photodissociation from wild type and L16A-AXCP over 12 orders of magnitude in time. For the L16A variant, the picosecond geminate rebinding of both CO and NO appears with an unprecedented 100% yield, and no exit of these ligands from protein to solvent could be observed. Molecular dynamic simulations saliently demonstrate that dissociated CO stays within 4 Å from Fe2+, in contrast to wild-type AXCP. The L16A mutation confers a heme propionate conformation and docking site which traps the diatomics, maximizing the probability of recombination and directly explaining the ultrahigh affinities for CO, NO, and O2. Overall, our results point to a novel mechanism for modulating heme-gas affinities in proteins.

Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase.

We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τG1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τG2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 µs with [NO] = 200 µM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10(8) M(-1)⋅s(-1). In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ5C1 = 0.66 µs and τ5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 µs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed.

Reactivity and dynamics of H2S, NO, and O2 interacting with hemoglobins from Lucina pectinata.

Hemoglobin HbI from the clam Lucina pectinata is involved in H2S transport, whereas homologous heme protein HbII/III is involved in O2 metabolism. Despite similar tertiary structures, HbI and HbII/III exhibit very different reactivity toward heme ligands H2S, O2, and NO. To investigate this reactivity at the heme level, we measured the dynamics of ligand interaction by time-resolved absorption spectroscopy in the picosecond to nanosecond time range. We demonstrated that H2S can be photodissociated from both ferric and ferrous HbI. H2S geminately rebinds to ferric and ferrous out-of-plane iron with time constants (τgem) of 12 and 165 ps, respectively, with very different proportions of photodissociated H2S exiting the protein (24% in ferric and 80% in ferrous HbI). The Gln(E7)His mutation considerably changes H2S dynamics in ferric HbI, indicating the role of Gln(E7) in controling H2S reactivity. In ferric HbI, the rate of diffusion of H2S from the solvent into the heme pocket (kentry) is 0.30 µM(-1) s(-1). For the HbII/III-O2 complex, we observed mainly a six-coordinate vibrationally excited heme-O2 complex with O2 still bound to the iron. This explains the low yield of O2 photodissociation and low koff from HbII/III, compared with those of HbI and Mb. Both isoforms behave very differently with regard to NO and O2 dynamics. Whereas the amplitude of geminate rebinding of O2 to HbI (38.5%) is similar to that of myoglobin (34.5%) in spite of different distal heme sites, it appears to be much larger for HbII/III (77%). The distal Tyr(B10) side chain present in HbII/III increases the energy barrier for ligand escape and participates in the stabilization of bound O2 and NO.

Picosecond binding of the His ligand to four-coordinate heme in cytochrome c': a one-way gate for releasing proximal NO.

We provide a direct demonstration of a "kinetic trap" mechanism in the proximal 5-coordinate heme-nitrosyl complex (5c-NO) of cytochrome c' from Alcaligenes xylosoxidans (AXCP) in which picosecond rebinding of the endogenous His ligand following heme-NO dissociation acts as a one-way gate for the release of proximal NO into solution. This demonstration is based upon picosecond transient absorption changes following NO photodissociation of the proximal 5c-NO AXCP complex. We have determined the absolute transient absorption spectrum of 4-coordinate ferrous heme to which NO rebinds with a time constant τ(NO) = 7 ps (k(NO) = 1.4 × 10(11) s(-1)) and shown that rebinding of the proximal histidine to the 4-coordinate heme takes place with a time constant τ(His) = 100 ± 10 ps (k(His) = 10(10) s(-1)) after the release of NO from the proximal heme pocket. This rapid His reattachment acts as a one-way gate for releasing proximal NO by precluding direct proximal NO rebinding once it has left the proximal heme pocket and requiring NO rebinding from solution to proceed via the distal heme face.

Picosecond to second dynamics reveals a structural transition in Clostridium botulinum NO-sensor triggered by the activator BAY-41-2272.

Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor that synthesizes cGMP upon NO activation. In synergy with the artificial allosteric effector BAY 41-2272 (a lead compound for drug design in cardiovascular treatment), sGC can also be activated by carbon monoxide (CO), but the structural basis for this synergistic effect are unknown. We recorded in the unusually broad time range from 1 ps to 1 s the dynamics of the interaction of CO binding to full length sGC, to the isolated sGC heme domain ß(1)(200) and to the homologous bacterial NO-sensor from Clostridium botulinum. By identifying all phases of CO binding in this full time range and characterizing how these phases are modified by BAY 41-2272, we show that this activator induces the same structural changes in both proteins. This result demonstrates that the BAY 41-2272 binding site resides in the ß(1)(200) sGC heme domain and is the same in sGC and in the NO-sensor from Clostridium botulinum.

Absorption band III kinetics probe the picosecond heme iron motion triggered by nitric oxide binding to hemoglobin and myoglobin.

To study the ultrafast movement of the heme iron induced by nitric oxide (NO) binding to hemoglobin (Hb) and myoglobin (Mb), we probed the picosecond spectral evolution of absorption band III (∼760 nm) and vibrational modes (iron-histidine stretching, ν(4) and ν(7) in-plane modes) in time-resolved resonance Raman spectra. The time constants of band III intensity kinetics induced by NO rebinding (25 ps for hemoglobin and 40 ps for myoglobin) are larger than in Soret bands and Q-bands. Band III intensity kinetics is retarded with respect to NO rebinding to Hb and to Mb. Similarly, the ν((Fe-His)) stretching intensity kinetics are retarded with respect to the ν(4) and ν(7) heme modes and to Soret absorption. In contrast, band III spectral shift kinetics do not coincide with band III intensity kinetics but follows Soret kinetics. We concluded that, namely, the band III intensity depends on the heme iron out-of-plane position, as theoretically predicted ( Stavrov , S. S. Biopolymers 2004 , 74 , 37 - 40 ).

Quaternary structure controls ligand dynamics in soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor. The mechanisms of activation and deactivation of this heterodimeric enzyme are unknown. For deciphering them, functional domains can be overexpressed. We have probed the dynamics of the diatomic ligands NO and CO within the isolated heme domain ß(1)(190) of human sGC by piconanosecond absorption spectroscopy. After photo-excitation of nitrosylated sGC, only NO geminate rebinding occurs in 7.5 ps. In ß(1)(190), both photo-dissociation of 5c-NO and photo-oxidation occur, contrary to sGC, followed by NO rebinding (7 ps) and back-reduction (230 ps and 2 ns). In full-length sGC, CO geminate rebinding to the heme does not occur. In contrast, CO geminately rebinds to ß(1)(190) with fast multiphasic process (35, 171, and 18 ns). We measured the bimolecular association rates k(on) = 0.075 ± 0.01 × 10(6) M(-1) · S(-1) for sGC and 0.83 ± 0.1 × 10(6) M(-1) · S(-1) for ß(1)(190). These different dynamics reflect conformational changes and less proximal constraints in the isolated heme domain with respect to the dimeric native sGC. We concluded that the α-subunit and the ß(1)(191-619) domain exert structural strains on the heme domain. These strains are likely involved in the transmission of the energy and relaxation toward the activated state after Fe(2+)-His bond breaking. This also reveals the heme domain plasticity modulated by the associated domains and subunit.

Picosecond primary structural transition of the heme is retarded after nitric oxide binding to heme proteins.

We investigated the ultrafast structural transitions of the heme induced by nitric oxide (NO) binding for several heme proteins by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We probed the heme iron motion by the evolution of the iron-histidine Raman band intensity after NO photolysis. Unexpectedly, we found that the heme response and iron motion do not follow the kinetics of NO rebinding. Whereas NO dissociation induces quasi-instantaneous iron motion and heme doming (<0.6 ps), the reverse process results in a much slower picosecond movement of the iron toward the planar heme configuration after NO binding. The time constant for this primary domed-to-planar heme transition varies among proteins (approximately 30 ps for myoglobin and its H64V mutant, approximately 15 ps for hemoglobin, approximately 7 ps for dehaloperoxidase, and approximately 6 ps for cytochrome c) and depends upon constraints exerted by the protein structure on the heme cofactor. This observed phenomenon constitutes the primary structural transition in heme proteins induced by NO binding.

Hypericin activates L-type Ca2+ channels in cardiac myocytes.

The effects and the mode of action of hypericin (1) were studied, in the dark, on the action potential (AP) and the L-type Ca2+ channel of frog atrial heart muscle, using intracellular microelectrode and patch-clamp techniques, respectively. In the presence of Ca2+ in Ringer solution, hypericin (1 to 4 microM) did not markedly modify the AP. Total replacement of Ca2+ by Sr2+ in the solution (Ringer Sr2+) revealed that hypericin (4 microM) prolonged the AP duration (APD). Hypericin dose-dependently increased the magnitude of the Sr2+current, which develops through L-type Ca2+ channels in the Ringer solution containing tetrodotoxin (0.7 microM) and tetraethylammonium (10 mM), but did not modify the kinetics of activation and inactivation. This revealed that hypericin increased L-type Ca2+ channel conductance, which accounted for the APD lengthening. The hypericin-induced APD lengthening recorded in the Ringer Sr2+ was not prevented by (i) a blockade of alpha- and beta-adrenoceptors by yohimbine (1 microM), urapidil (1 microM), and propanolol (50 microM), respectively, and (ii) PKC blockade by staurosporine (1 microM). The hypericin-induced APD lengthening recorded in the Ringer Sr2+ was prevented by blocking soluble guanylate cyclase (sGC) activity by 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (13 microM), which mimicked the effects of hypericin. Hypericin decreased the cellular cGMP level by 69% in atrial myocytes. The compound also decreased the cellular cGMP level by inhibiting sGC, thus cancelling the nucleotide inhibitory effect on the cardiac L-type Ca2+ channel.