Alfreda Beasley

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.

[7] Regulatory subunits of bovine heart and rabbit skeletal muscle cAMP-dependent protein kinase isozymes

Publisher Summary This chapter explains regulatory subunits of bovine heart and rabbit skeletal muscle cAMP-dependent protein kinase isozymes. Cyclic AMP-dependent protein kinase exists in two major isozymic forms, types I and II, which are present in varying relative concentrations, depending upon the tissue and species studied. Regulatory subunits from both isozymes can be isolated from a single tissue source when both types are present in approximately equal amounts, such as in porcine skeletal muscle. The choice of tissue and method of elution ultimately depends upon the quantity of R I or R II subunits needed and the nature of the studies for which they are to be used. The procedures described in the chapter for purification of R I and R II are based on relatively large amounts of starting material. If scaling down is appropriate, the final volume of cAMP-binding material which is eluted from diethylaminoethyl (DEAE)-cellulose may be small enough that concentration by (NH 4 ) 2 SO 4 before applying it to the affinity matrix is unnecessary. It is advisable to centrifuge all material to prevent blockage of the column. Omitting the (NH 4 ) 2 SO 4 precipitation step saves at least 6 hours and makes it possible to go from intact tissue to the cAMP-affinity column in

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.

Conversion of Phosphodiesterase-5 (PDE5) Catalytic Site to Higher Affinity by PDE5 Inhibitors

Phosphodiesterase-5 (PDE5) specifically hydrolyzes cGMP, thereby contributing to modulation of intracellular levels of this nucleotide. In the present study, preincubation with cGMP increased PDE5 catalytic activity for cGMP degradation, and it converted the PDE5 catalytic site to a form that was more potently inhibited by each of the three PDE5 catalytic site-specific inhibitors: sildenafil, vardenafil, and tadalafil. These results implied that elevated cGMP initiates a physiological negative feedback on the cGMP pathway by increasing the affinity of the PDE5 catalytic site for cGMP. This increase in catalytic site activity or affinity for inhibitors could be caused by binding of cGMP to either the PDE5 allosteric sites, catalytic site, or both. Whether occupation of the catalytic site alone could mediate the effect was examined using radiolabeled PDE5 inhibitors in the absence of cGMP. Exchange-dissociation of [3H]sildenafil (Viagra), [3H]vardenafil (Levitra), or [3H]tadalafil (Cialis) from full-length PDE5 or isolated catalytic domain revealed two kinetic components (slow and fast). Extended preincubation of full-length PDE5, but not isolated catalytic domain, with 3H inhibitors converted the biphasic pattern to a single slow (high-affinity) component. Studies of amino-terminally truncated PDE5 established that full-length mammalian GAF-B (cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlA) subdomain conjoined with the catalytic domain was sufficient for this conversion. In conclusion, binding of substrate or substrate analogs such as PDE5 inhibitors to the catalytic site converts a fast (low-affinity) inhibitor dissociation component of the PDE5 catalytic site to a slow (high-affinity) inhibitor dissociation component. This effect is predicted to improve the substrate affinity or inhibitory potencies of these compounds in intact cells.

A photoaffinity probe covalently modifies the catalytic site of the cGMP-binding cGMP-specific phosphodiesterase (PDE-5).

The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors or the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photo-affinity labeling was progressively blocked by cGMP at concentrations higher than 10 μM, whereas cAMP or 5′-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, clMP, and β-phenyl-1,N2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 μM, which compared favorably with an IC50 of 5 μM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.