Kennard Grimes

Sildenafil citrate does not affect cardiac contractility in human or dog heart

SUMMARY Objective: This study evaluated whether sildenafil citrate, an oral treatment for erectile dysfunction and a selective inhibitor of phosphodiesterase type 5 (PDE5) with modest vasodilating properties, affects cardiac contractility in vitro. Research design and methods: Slices of freshly obtained human (n = 2) or dog (n = 3) atrial appendage were suspended in organ baths containing Krebs—Ringer bicarbonate buffer (pH 7.4, 37°C) bubbled continuously with 95% O2 and 5% CO2, and isometric tension was recorded using a Gould physiograph. Contractions were elicited by 1-Hz electric pacing. After 15min of equilibration, 1\M sildenafil was added to the bath, followed 15min later (human and dog) by 5|iM epinephrine, an inotropic agent, and 10min later (dog) by 88|iM 3-isobutyl-1 -methylxanthine (IBMX), a nonselective PDE inhibitor. In a separate experiment, cyclic guanosine monophosphate levels and PDE, protein kinase G, and protein kinase A activities were determined. Results: Addition of 1 |iM sildenafil to isolated dog or human atrial tissue had no significant effect on force of cardiac contraction, whereas epinephrine produced a robust increase in contractile force in the same muscle strip. The addition of IBMX produced a marked stimulation of contractile force in dog atrial tissue. Very low amounts of PDE5 were found in extracts of human heart, consistent with its known primary location in the smooth muscle of systemic vasculature. Conclusions: Sildenafil is unlikely to directly produce inotropic effects on cardiac muscle in patients being treated for erectile dysfunction.

Mechanisms associated with cGMP binding and activation of cGMP-dependent protein kinase

Using small-angle x-ray scattering, we have observed the cGMP-induced elongation of an active, cGMP-dependent, monomeric deletion mutant of cGMP-dependent protein kinase (Δ1–52PKG-Iβ). On saturation with cGMP, the radius of gyration of Δ1–52PKG-Iβ increases from 29.4 ± 0.1 Å to 40.1 ± 0.7 Å, and the maximum linear dimension increases from 90 Å ± 10% to 130 Å ± 10%. The elongation is due to a change in the interaction between structured regulatory (R) and catalytic (C) domains. A model of cGMP binding to Δ1–52PKG-Iβ indicates that elongation of Δ1–52PKG-Iβ requires binding of cGMP to the low-affinity binding site of the R domain. A comparison with cAMP-dependent protein kinase suggests that both elongation and activation require cGMP binding to both sites; cGMP binding to the low-affinity site therefore seems to be a necessary, but not sufficient, condition for both elongation and activation of Δ1–52PKG-Iβ. We also predict that there is little or no cooperativity in cGMP binding to the two sites of Δ1–52PKG-Iβ under the conditions used here. Results obtained by using the Δ1–52PKG-Iβ monomer indicate that a previously observed elongation of PKG-Iα is consistent with a pure change in the interaction between the R domain and the C domain, without alteration of the dimerization interaction. This study has revealed important features of molecular mechanisms in the biochemical network describing PKG-Iβ activation by cGMP, yielding new insight into ligand activation of cyclic nucleotide-dependent protein kinases, a class of regulatory proteins that is key to many cellular processes.

Activation by Cyclic GMP Binding Causes an Apparent Conformational Change in cGMP-dependent Protein Kinase*

Cyclic nucleotide binding activates cyclic nucleotide-dependent protein kinases, but the molecular mechanism is unknown. In the present studies, cGMP binding to type Iα or type Iβ cGMP-dependent protein kinase (PKG) caused (i) a large electronegative charge shift of each enzyme on ion exchange chromatography, (ii) an increase in the Stokes radius (>3 Å) of each enzyme, and (iii) a decreased mobility of type Iβ PKG on native gel electrophoresis. These physical changes were not detected in the monomeric form of type Iβ PKG upon activation by cGMP. However, the results of partial proteolysis of type Iα PKG revealed some degree of cGMP-induced conformational change within the PKG-monomer, since cGMP binding protects the PKG-monomer against chymotryptic cleavage. The altered sensitivity to proteolysis occurs at Met-200, which is located between the B and C α-helices in the high affinity site (site A), and implies that the cGMP-induced structural perturbations in this region may participate in activation of dimeric PKG. The cGMP-induced conformational effects observed using the physical separation methods are likely to reflect altered interactions within the dimeric PKG that are caused by structural alterations within the subunits.

Arginine 75 in the Pseudosubstrate Sequence of Type Iβ cGMPdependent Protein Kinase Is Critical for Autoinhibition, Although Autophosphorylated Serine 63 Is Outside This Sequence

Autoinhibitory domains in many protein kinases include either a phosphorylatable substrate-like sequence or a pseudosubstrate sequence. This study shows that Iβ cGMP-dependent protein kinase (cGK) autophosphorylates Ser-63, which is in an atypical cGK substrate sequence (-59AQKQSAS-) that is amino-terminal to the pseudosubstrate motif (-74KRQAI-). cGMP increases the rate of autophosphorylation (∼0.8 phosphate/cGK monomer) ∼3-fold. Autophosphorylation is an intramolecular process since it is independent of cGK concentration. cGMP activation of cGK enhances proteolysis within and near the pseudosubstrate site; treatment of dimeric cGK with three proteases produces three cGK monomers (∼67-70 kDa each). Their amino-terminal sequences are 75RQAISAEPT-, 76QAISAEPTAF-, and 86DIQDLSXV-, respectively. cGMP stimulates these kinases by 10-, 2.5-, and 1.4-fold, respectively, compared with a 10-fold effect on intact cGK. Increased basal activity accounts for the diminished stimulation. Thus, the primary autophosphorylation site of Iβ cGK is well outside the pseudosubstrate site, but Arg-75 in the pseudosubstrate site is critical for autoinhibition. Autoinhibition also involves elements that are carboxyl-terminal to Arg-75.

cGMP-dependent protein kinase protects cGMP from hydrolysis by phosphodiesterase-5.

The physiological effects of cGMP are largely determined by the activities of intracellular receptors, including cGMP-dependent protein kinase (PKG) and cGMP-binding cyclic nucleotide phosphodiesterases (PDEs), and the distribution of cGMP among these receptors dictates activity of the signalling pathway. In the present study, the effects of PKG-Ialpha or PKG-Ibeta on the rate of cGMP hydrolysis by the isolated PDE5 catalytic domain were examined. PKG-Ialpha strongly inhibited cGMP hydrolysis with an IC(50) value of 217 nM, which is similar to the physiological concentration of PKG in pig coronary artery reported previously. By contrast, PKG-Ibeta, which has lower affinity for cGMP than does PKG-Ialpha, inhibited cGMP hydrolysis with an IC(50) of approx. 1 microM. Inhibition by PKG-Ialpha was more effective than that by PKG-Ibeta, consistent with their relative affinities for cGMP. Autophosphorylation of PKGs increased their cGMP-binding affinities and their inhibitory effects on PDE5 hydrolysis of cGMP. Autophosphorylation of PKG-Ibeta increased its inhibitory potency on PDE5 hydrolysis of cGMP by 10-fold compared with a 2-fold increase upon autophosphorylation of PKG-Ialpha. The results indicate that cGMP bound to allosteric cGMP-binding sites of PKG is protected from hydrolysis by PDE5 and that persistent protection of cGMP by either non-phosphorylated or autophosphorylated PKGs may be a positive-feedback control to sustain cGMP signalling.

Distinguishing the Roles of the Two Different cGMP-binding Sites for Modulating Phosphorylation of Exogenous Substrate (Heterophosphorylation) and Autophosphorylation of cGMP-dependent Protein Kinase

The role of each of the two different cGMP-binding sites (referred to as slow and fast sites) of type I cGMP-dependent protein kinase (PKG) in altering the rate of catalysis of phosphorylation of exogenous substrates (heterophosphorylation) or the rate of autophosphorylation has not been resolved. In the present study, the cGMP concentration required for half-maximal activation (A50) of wild-type PKG type Iβ (WT) was 5-fold higher for heterophosphorylation than for autophosphorylation. cGMP occupation of the slow site was associated with an increase in the autophosphorylation rate, whereas occupation of the fast and slow site together was associated with a decrease in the autophosphorylation rate compared with the rate observed with occupation of the slow site alone. The contributions of each cGMP-binding site were investigated using PKG mutants containing substitutions of an invariant threonine residue that is critical for high affinity cGMP-binding in each site. Site-directed mutagenesis of Thr-317 of the fast site (T317A) increased the cGMP A50 for heterophosphorylation 4-fold at 30 °C, with nominal effect on cGMP A50 for autophosphorylation compared with WT. The analogous slow site mutation (T193A) increased the cGMP A50 for heterophosphorylation and autophosphorylation 32- and 64-fold, respectively. Compared with WT, the cGMP A50 of the double mutant (T193A/T317A) for heterophosphorylation was increased 300-fold, whereas the cGMP A50 for autophosphorylation was similar to that of T193A. Thus, occupation of both cGMP-binding sites of PKG is required for maximal stimulation of heterophosphorylation, whereas occupation of the slow site alone is sufficient for stimulation of the rate of autophosphorylation, and additional occupation of the fast site reduces this rate.

Phosphorylation of phosphodiesterase-5 is promoted by a conformational change induced by sildenafil, vardenafil, or tadalafil.

Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, vardenafil, or tadalafil) or phosphorylation by cyclic nucleotide-dependent protein kinase causes an apparent conformational change in PDE5, as indicated by a shift in migration on non-denaturing PAGE gels and an altered pattern of tryptic digestion. Combination of cGMP and a PDE5 inhibitor or phosphorylation does not cause a further gel shift or change in tryptic digest. Phosphorylation of PDE5 is stimulated by inhibitors, and combination of cGMP and inhibitor does not cause further phosphorylation. Dephosphorylation of PDE5 by either purified phosphoprotein phosphatase-1 or -2A catalytic subunit or by a crude phosphatase mixture is not affected by cGMP or inhibitors, suggesting that phosphorylation itself maintains conformational exposure of the phosphorylation site. The combined results imply that cGMP binding to the catalytic site initiates negative feedback control of many cellular cGMP signaling pathways by directly stimulating phosphorylation and activation of PDE5; by exploiting this molecular mechanism, PDE5 inhibitors stimulate their own potencies.

Phosphorylation Increases Affinity of the Phosphodiesterase-5 Catalytic Site for Tadalafil

Phosphodiesterase-5 (PDE5) is phosphorylated at a single serine residue by cyclic nucleotide-dependent protein kinases. To test for a direct effect of phosphorylation on the PDE5 catalytic site, independent of cGMP binding to the allosteric sites of the enzyme, binding of the catalytic site-specific substrate analog [3H]tadalafil to PDE5 was measured. Phosphorylation increased [3H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [3H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [3H]tadalafil exchange-dissociation rate from PDE5 more than 6-fold. Finally, phosphorylation increased affinity for hydrolysis of a catalytic site-specific cGMP analog, 2′-O-anthraniloyl-cGMP, by ∼3-fold. The combined results showed that phosphorylation activates PDE5 catalytic site independently of cGMP binding to the allosteric sites. The results suggested that phosphorylation acts in concert with allosteric cGMP binding to stimulate the PDE5 catalytic site, which should promote negative feedback regulation of the cGMP pathway in intact cells. By increasing the affinity of the catalytic site, phosphorylation should also consequently increase the potency and duration of PDE5 inhibitor action.

Allosteric sites of phosphodiesterase-5 sequester cyclic GMP.

Phosphodiesterase-5 (PDE5) and cGMP-dependent protein kinase (PKG) play key roles in cGMP signaling. PDE5 has a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that binds cGMP at allosteric sites. We recently demonstrated that in corpus cavernosum, PDE5 concentration exceeds basal cGMP by ~5-fold making it possible that its allosteric sites could bind a significant fraction of the total cellular cGMP. It is hypothesized that the allosteric sites regulate cGMP signaling by sequestering cGMP. At 60 nM cGMP in vitro, which approaches a stimulated concentration of cGMP in rabbit corpus cavernosum, isolated R domain inhibits both cGMP hydrolysis by C domain and activation of PKG (IC50 values of 388 and 100 nM, respectively). Prior phosphorylation of R domain by cyclic nucleotide-dependent protein kinases, which increases its cGMP-binding affinity, also increases its potency for inhibiting both cGMP hydrolysis by C domain and cGMP activation of PKG (IC50 values of 58 and 38 nM, respectively). In rabbit corpus cavernosum, PDE5 concentration (94 nM) exceeds these values. These findings support our hypothesis that physiological concentrations of R domain regulate cGMP signaling by sequestering this nucleotide and that phosphorylation of R domain modulates this effect. This could provide for negative feedback control of cGMP-signaling.