cGMP-Dependent Protein Kinase I Mediates the Negative Inotropic Effect of cGMP in the Murine Myocardium

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133

115

To explore the functional significance of cGMP-dependent protein kinase type I (cGKI) in the regulation of erythrocyte survival, gene-targeted mice lacking cGKI were compared with their control littermates. By the age of 10 weeks, cGKI-deficient mice exhibited pronounced anemia and splenomegaly. Compared with control mice, the cGKI mutants had significantly lower red blood cell count, packed cell volume, and hemoglobin concentration. Anemia was associated with a higher reticulocyte number and an increase of plasma erythropoietin concentration. The spleens of cGKI mutant mice were massively enlarged and contained a higher fraction of Ter119 ؉ erythroid cells, whereas the relative proportion of leukocyte subpopulations was not changed. The Ter119 ؉ cGKI-deficient splenocytes showed a marked increase in annexin V binding, pointing to phosphatidylserine (PS) exposure at the outer membrane leaflet, a hallmark of suicidal erythrocyte death or eryptosis. Compared with control erythrocytes, cGKI-deficient erythrocytes exhibited in vitro a higher cytosolic Ca 2؉ concentration, a known trigger of eryptosis, and showed increased PS exposure, which was paralleled by a faster clearance in vivo. Together, these results identify a role of cGKI as mediator of erythrocyte survival and extend the emerging concept that cGMP/cGKI signaling has an antiapoptotic/prosurvival function in a number of cell types in vivo.apoptosis ͉ Ca2ϩ channels ͉ phosphatidylserine ͉ spleen N itric oxide (NO) has been shown to be a powerful regulator of cell survival (1, 2). Depending on the source or concentration of NO and the influence of additional regulators, NO may stimulate or inhibit apoptosis (3). NO exerts its effects in part through S-nitrosylation of target proteins. However, the effect of NO donors on Ca 2ϩ -induced phosphatidylserine (PS) exposure, a hallmark of apoptosis, could be mimicked by cGMP analogs (4), suggesting the involvement of soluble guanylyl cyclase, cGMP, and cGMP-dependent protein kinase type I (cGKI), a well known signaling cascade downstream of NO (5, 6).Recent studies indicated that erythroid cells possess a functional NO/cGMP pathway (7-9), which may be involved in the regulation of eryptosis, the suicidal death of erythrocytes (10). Erythrocyte cGMP production might also be stimulated by NO generated in the endothelium. The cGKI can also be activated independent of cGMP in response to oxidative stress (11). Eryptosis may follow osmotic shock, energy depletion, and oxidative stress (10), which activate Ca 2ϩ -permeable cation channels (12). Subsequent Ca 2ϩ entry leads to activation of Ca 2ϩ -sensitive K ϩ channels, exit of KCl with osmotically obliged water, and thus cell shrinkage (13). In addition, Ca 2ϩ entry triggers Ca 2ϩ -sensitive scrambling of the cell membrane (14, 15) with subsequent exposure of PS at the erythrocyte surface (12). Cell membrane scrambling may further be triggered by ceramide (16) and activation of protein kinase C (17). PS-exposing erythrocytes bind to PS receptors (18) and are recognized, ...

Section: Resultsmentioning

confidence: 94%

Anemia and splenomegaly in cGKI-deficient mice

Föller

Feil²,

Ghoreschi³

et al. 2008

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133

115

“…The integrated reduction in myofibril contractility, induced by cGMP in response to metabolic stress, decreases tissue indices of energy demand, including force of contraction (31), tension (32), fractional shortening (33), and maximum rate of shortening (34).…”

Section: Resultsmentioning

confidence: 99%

Guanylyl cyclase is an ATP sensor coupling nitric oxide signaling to cell metabolism

Ruiz-Stewart

Tiyyagura

Lin

et al. 2003

Defending cellular integrity against disturbances in intracellular concentrations of ATP ([ATP]M etabolically active cells with high-energy requirements are particularly susceptible to structural and functional impairment when deprived of oxygen and substrates obligatory for generating ATP (1, 2). Maintenance of homeostasis requires these cells to respond to rapidly fluctuating energy demands in the context of varying supplies of metabolic substrates. Tight coordination between energy supply and demand is predicated on efficient communication and integration between metabolism, the steady-state and local availability of high-energy phosphates, and signaling mechanisms regulating cell functions. Although specific components and their interactions that transduce metabolic and energetic signaling have been described (1), molecular mechanisms mediating their coordination with cellular signaling remain incompletely understood.Production and release of NO represents one paracrine͞ autocrine mechanism coordinating energy supply and demand in tissues. Metabolic stress, such as ischemia, stimulates NO synthases to produce NO from arginine (3). In turn, NO regulates the supply of energy by improving the delivery of substrates and oxygen to deprived tissues (4-6), regulating the intracellular delivery of metabolic substrates (7-10), selection of substrates that support metabolism (6, 10-14), oxidation of metabolic substrates (15), generation of ATP by improving metabolic efficiency (16), and biogenesis of mitochondria (17). Conversely, NO reduces the demand for ATP by decreasing energyconsuming cell functions, for example, by reducing the activity of the contractile apparatus in muscle cells (ref. 18 and references therein). Moreover, in some models of severe hypoxic stress, NO induces complete and reversible metabolic stasis required to survive the insult (19). The importance of NO in defending against ischemia is underscored by its regulation of the transcription factor hypoxia inducible factor 1, a master regulator of cellular homeostasis in the context of oxygen insufficiency (20).Coordination of energy supply and demand is mediated, in part, by NO activation of soluble guanylyl cyclase (sGC) and the associated accumulation of intracellular cGMP concentration ([cGMP] i ) (9,10,12,15,16,18), yet the mechanisms coordinating cGMP-dependent and metabolic signaling, beyond the production of NO, remain undefined. Recently, a novel mechanism was identified by which adenine nucleotides inhibit GCs (21,22) that is analogous to P site inhibition of adenylyl cyclases (23). Indeed, the two-substituted adenine nucleotides 2-methylthio-ATP and 2-chloro-ATP inhibit NO-dependent cGMP production by directly binding to sGC (22). Synthetic two-substituted adenine nucleotides presumably exploit allosteric mechanisms regulated by endogenous cell products. The present study revealed that ATP is the native ligand mediating adenine nucleotide-dependent inhibition of sGC by binding directly to an allosteric purinergic site on the enzyme. More...

“…The generation of mice carrying a conditional loxP-flanked cGKI allele (L2) or a recombined cGKI-null allele (LϪ) and the detection of the cGKI WT(ϩ), L2, and LϪ alleles by PCR have been described (14). Mice carrying the SM-CreER T2 knock-in allele (Cre) were genotyped as described (15).…”

Section: Methodsmentioning

confidence: 99%

A proatherogenic role for cGMP-dependent protein kinase in vascular smooth muscle cells

Wolfsgruber¹,

Feil²,

Brummer³

et al. 2003

Self Cite

Nitric oxide (NO) exerts both antiatherogenic and proatherogenic effects, but the cellular and molecular mechanisms that contribute to modulation of atherosclerosis by NO are not understood completely. The cGMP-dependent protein kinase I (cGKI) is a potential mediator of NO signaling in vascular smooth muscle cells (SMCs). Postnatal ablation of cGKI selectively in the SMCs of mice reduced atherosclerotic lesion area, demonstrating that smooth muscle cGKI promotes atherogenesis. Cell-fate mapping indicated that cGKI is involved in the development of SMC-derived plaque cells. Activation of endogenous cGKI in primary aortic SMCs resulted in cells with increased levels of proliferation; increased levels of vascular cell adhesion molecule-1, peroxisome proliferator-activated receptor ␥, and phosphatidylinositol 3-kinase͞Akt signaling; and decreased plasminogen activator inhibitor 1 mRNA, which all are potentially proatherogenic properties. Taken together, these results highlight the pathophysiologic significance of vascular SMCs in atherogenesis and identify a key role for cGKI in the development of atherogenic SMCs in vitro and in vivo. We suggest that activation of smooth muscle cGKI contributes to the proatherogenic effect of NO and that inhibition of cGKI might be a therapeutic option for treating atherosclerosis in humans.A therosclerosis causes heart attack and stroke, the major causes of death in industrial nations. The pathophysiology of atherogenesis is complex. It is considered to be a chronic inflammatory condition that results from the interaction between modified lipoproteins and various cell types, including leukocytes, platelets, and cells of the vessel wall (1). The development of an atherosclerotic plaque involves, in addition to inflammation, the phenotypic modulation of vascular smooth muscle cells (SMCs) to proliferating and dedifferentiated cells. However, the mechanisms that contribute to the development of plaque SMCs and their pathophysiologic significance are not well understood (2).The signaling molecule nitric oxide (NO) has critical roles in the pathogenesis of atherosclerosis (3). Analysis of transgenic mice that lack or overexpress NO synthases indicated that NO exerts both protective (4, 5) and atherogenic (6-9) effects. The double role of NO might explain why NO-generating drugs (e.g., glyceryl trinitrate) have not been reported to limit the progression of atherosclerosis in humans. The opposing actions of NO on atherogenesis might depend on the spatiotemporal profile of its production and are likely mediated by different cellular and molecular mechanisms. Signaling pathways in SMCs that contribute to NO modulation of atherogenesis have not been identified. In vascular SMCs, NO is thought to exert many of its effects by activation of soluble guanylyl cyclase, synthesis of the second messenger cGMP, and activation of cGMP-dependent protein kinase I (cGKI) (10). The analysis of the functional significance of cGKI in NO͞cGMP signaling is complicated by the existence of multiple receptors ...