Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.

The activation of protein kinase G (PKG) by cGMP has become of considerable interest as a novel molecular mechanism for the induction of apoptosis in cancer cells, because sulindac sulfone (exisulind, Aptosyn) and certain derivatives that inhibit cGMP-phosphodiesterases and thereby increase cellular levels of cGMP appear to induce apoptosis via this mechanism. However, other effects of these compounds have not been excluded, and the precise mechanism by which PKG activation induces apoptosis has not been elucidated in detail. To directly examine the effects of PKG on cell growth and apoptosis, we generated a series of mutants of PKG I␣: PKG I␣S65D, a constitutively activated point mutant; PKG I␣⌬, a constitutively activated N-terminal truncated mutant; and PKG I␣K390R, a dominant-negative point mutant. A similar series of mutants of PKG I␤ were also constructed (Deguchi et al., Mol. Cancer Ther., 1: 803-809, 2002). The present study demonstrates that when transiently expressed in SW480 colon cancer, the constitutively activated mutants of PKG I␤, and to a lesser extent PKG I␣, inhibit colony formation and induce apoptosis. We were not able to obtain derivatives of SW480 cells that stably expressed these constitutively activated mutants, presumably because of toxicity. However, derivatives that stably overexpressed wildtype PKG I␤ displayed growth inhibition, whereas derivatives that stably expressed the dominant-negative mutant (KR) of PKG I␤ grew more rapidly and were more resistant to Aptosyn-induced growth inhibition than vector control cells. Stable overexpression of PKG I␤ was associated with decreased cellular levels of ␤-catenin and cyclin D1 and increased levels of p21 CIP1 . Reporter assays indicated that activation of PKG I␤ inhibits the transcriptional activity of the cyclin D1 promoter. We also found that transient expression of the constitutively activated mutants of PKG I␤ inhibited cell migration. Taken together, these results indicate that activation of PKG I␤ is sufficient to inhibit growth and cell migration and induce apoptosis in human colon cancer cells and that these effects are associated with inhibition of the transcription of cyclin D1 and an increase in the expression of p21 CIP1 .

Pulmonary microvascular endothelial cells (PMVECs) form a more restrictive barrier to macromolecular flux than pulmonary arterial endothelial cells (PAECs); however, the mechanisms responsible for this intrinsic feature of PMVECs are unknown. Because cAMP improves endothelial barrier function, we hypothesized that differences in enzyme regulation of cAMP synthesis and/or degradation uniquely establish an elevated content in PMVECs. PMVECs possessed 20% higher basal cAMP concentrations than did PAECs; however, increased content was accompanied by 93% lower ATP-to-cAMP conversion rates. In PMVECs, responsiveness to β-adrenergic agonist (isoproterenol) or direct adenylyl cyclase (forskolin) activation was attenuated and responsiveness to phosphodiesterase inhibition (rolipram) was increased compared with those in PAECs. Although both types of endothelial cells express calcium-inhibited adenylyl cyclase, constitutive PMVEC cAMP accumulation was not inhibited by physiological rises in cytosolic calcium, whereas PAEC cAMP accumulation was inhibited 30% by calcium. Increasing either PMVEC calcium entry by maximal activation of store-operated calcium entry or ATP-to-cAMP conversion with rolipram unmasked calcium inhibition of adenylyl cyclase. These data indicate that suppressed calcium entry and low ATP-to-cAMP conversion intrinsically influence calcium sensitivity. Adenylyl cyclase-to-cAMP phosphodiesterase ratios regulate cAMP at elevated levels compared with PAECs, which likely contribute to enhanced microvascular barrier function.

Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitor yeast cells that are genetically engineered to display error-prone transcription. We discover that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease phenotypes. We further find that the error rate of transcription increases as cells age, suggesting that transcription errors affect proteostasis particularly in aging cells. Accordingly, transcription errors accelerate the aggregation of a peptide that is implicated in Alzheimer's disease, and shorten the lifespan of cells. These experiments reveal a previously unappreciated role for transcriptional fidelity in cellular health and aging.

The superfamily of cyclic nucleotide phosphodiesterases (PDEs) 1 is comprised of eleven known families of PDEs that vary in substrate specificity, regulatory properties, and tissue distribution (1, 2). The regulatory domains of five of the known PDE families (PDEs 2, 5, 6, 10, and 11) contain either one or two sequences known as GAF domains, and these are homologous among the five families. In three of these families (PDEs 2, 5, and 6), at least one of these sequences in each monomer forms an allosteric site for cGMP binding (3-10). These latter PDEs belong to a subgroup of PDE families known as cGMP binding PDEs. In PDE2, cGMP binding to the cGMP binding allosteric sites increases catalytic activity of the enzyme toward cAMP and cGMP by an unknown mechanism (11, 12). In PDE5, the effect of allosteric cGMP binding is still not clear, but cGMP binding to the PDE5 R domain controls phosphorylation of a specific serine that is near the amino terminus of the R domain (13,14). Phosphorylation at this serine activates both PDE5 catalytic and allosteric cGMP-binding activities (15). Phosphorylation of PDE5 occurs in intact vascular smooth muscle cells after cGMP elevation, and this phosphorylation is associated with increased catalytic activity of this enzyme (16 -18). Mechanism(s) by which cGMP binding and phosphorylation of the R domain of PDE5 alter enzyme properties are not understood.PDE5 exists in at least two conformational states. In absence of cGMP, the enzyme assumes an apparently more compact structure (19). Occupation of the PDE5 catalytic site by either cGMP or inhibitors such as 3-isobuty-1-methylxanthine, zaprinast, or sildenafil stimulates cGMP binding to the allosteric sites, and when both catalytic and binding sites are occupied PDE5 undergoes an apparent elongation (19). However, little is known about specific structural events that occur in PDEs upon interaction with regulatory agents or after post-translational modification by phosphorylation. Regulation of PDE5 involves cGMP interaction with the R domain, which is required for efficient post-translational modification through phosphorylation (13,15).Models of allosteric regulation of many enzymes include modulation of the equilibrium of distribution of proteins between two conformers, i.e. a relaxed (R) state that is active and has higher affinity for substrate and a taut (T) state that is less active and has lower affinity for substrate (20,21). Phosphorylase is an example of this model wherein either binding of an allosteric activator, 5ЈAMP, or phosphorylation by phosphorylase kinase alters equilibrium between the two states to produce a more active enzyme (21). PDE5 may conform to that model, because cGMP binding to allosteric binding sites induces an apparent conformational change in PDE5, perhaps converting it from a less active state to a more active state (15,19). However, phosphorylation of Ser 92 in bovine PDE5 and activation of PDE5 occur efficiently only when cGMP is elevated, and the enzyme is already in the cGMP-bound (R) conformat...