Abstract-␤-Adrenergic agonists stimulate cardiac contractility and simultaneously blunt this response by coactivating NO synthase (NOS3) to enhance cGMP synthesis and activate protein kinase G (PKG-1). cGMP is also catabolically regulated by phosphodiesterase 5A (PDE5A). PDE5A inhibition by sildenafil (Viagra) increases cGMP and is used widely to treat erectile dysfunction; however, its role in the heart and its interaction with ␤-adrenergic and NOS3/cGMP stimulation is largely unknown. In nontransgenic (control) murine in vivo hearts and isolated myocytes, PDE5A inhibition (sildenafil) minimally altered rest function. However, when the hearts or isolated myocytes were stimulated with isoproterenol, PDE5A inhibition was associated with a suppression of contractility that was coupled to elevated cGMP and increased PKG-1 activity. In contrast, NOS3-null hearts or controls with NOS inhibited by N G -nitro-Larginine methyl ester, or soluble guanylate cyclase (sGC) inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one, showed no effect of PDE5A inhibition on ␤-stimulated contractility or PKG-1 activation. This lack of response was not attributable to altered PDE5A gene or protein expression or in vitro PDE5A activity, but rather to an absence of sGC-generated cGMP specifically targeted to PDE5A catabolism and to a loss of PDE5A localization to z-bands. Re-expression of active NOS3 in NOS3-null hearts by adenoviral gene transfer restored PDE5A z-band localization and the antiadrenergic efficacy of PDE5A inhibition. These data support a novel regulatory role of PDE5A in hearts under adrenergic stimulation and highlight specific coupling of PDE5A catabolic regulation with NOS3-derived cGMP attributable to protein subcellular localization and targeted synthetic/catabolic coupling. Key Words: PDE5 Ⅲ phosphodiesterase Ⅲ sildenafil Ⅲ nitric oxide synthase Ⅲ contractility Ⅲ z-band B eta-adrenergic regulation of cardiac contraction is coupled to elevations in adenosine (cAMP) and guanosine (cGMP) cyclic nucleotides. Increased cAMP enhances contractility 1,2 by activating protein kinase A (PKA), whereas concomitant stimulation of cGMP opposes this in part by activating protein kinase G (PKG-1). 3,4 The latter response is thought to be attributable to stimulation of soluble guanylate cyclase (sGC) by NO. 3-9 Cyclic GMP is also synthesized by receptor GC (rGC) coupled to natriuretic peptide stimulation, and both sources can modulate cardiac function and structure, particularly in hearts stimulated by neurohormones or mechanical stress. 3-5,10 -13 cGMP is also regulated by catabolic phosphodiesterases such as phosphodiesterase 5A (PDE5A), and PDE5A inhibition by sildenafil (Viagra; SIL) and similar compounds augments cGMP in vascular tissue and is the primary therapy for erectile dysfunction. 14,15 However, the role for PDE5A in regulating cardiac function has remained unclear. 16 -18 Such clarification has become increasingly important because PDE5A inhibitors are poised to become chronic treatments for diseases such as pul...

The signaling molecule nitric oxide (NO), first described as endothelium-derived relaxing factor (EDRF), acts as physiological activator of NO-sensitive guanylyl cyclase (NO-GC) in the cardiovascular, gastrointestinal, and nervous systems. Besides NO-GC, other NO targets have been proposed; however, their particular contribution still remains unclear. Here, we generated mice deficient for the ␤1 subunit of NO-GC, which resulted in complete loss of the enzyme. GC-KO mice have a life span of 3-4 weeks but then die because of intestinal dysmotility; however, they can be rescued by feeding them a fiber-free diet. Apparently, NO-GC is absolutely vital for the maintenance of normal peristalsis of the gut. GC-KO mice show a pronounced increase in blood pressure, underlining the importance of NO in the regulation of smooth muscle tone in vivo. The lack of an NO effect on aortic relaxation and platelet aggregation confirms NO-GC as the only NO target regulating these two functions, excluding cGMP-independent mechanisms. Our knockout model completely disrupts the NO/cGMP signaling cascade and provides evidence for the unique role of NO-GC as NO receptor.cardiovascular ͉ knockout mice ͉ cGMP ͉ platelet aggregation ͉ smooth muscle relaxation T he nitric oxide (NO)/cGMP signaling cascade regulates a plethora of physiological functions in the cardiovascular, neuronal, and gastrointestinal systems (1-3). In the vascular system, NO, first recognized as endothelium-derived relaxing factor (EDRF; ref. 4), has been shown to mediate smooth muscle relaxation and inhibition of platelet aggregation. NO is synthesized by the family of NO synthases which exist in endothelial, neuronal, and inducible forms. The prominent receptor known to date is the enzyme NO-sensitive guanylyl cyclase (NO-GC). Stimulation of NO-GC by NO results in the production of the second messenger, cGMP, which exerts its effects via cGMPdependent kinases, channels, or phosphodiesterases (5-8). Besides these cGMP-mediated effects, NO is thought to mediate a variety of effects via cGMP-independent mechanisms in the cardiovascular system (for a review, see ref. 9).To gain further insight into the NO/cGMP signaling cascade, mice deficient in NO synthases (NOS) have been generated (10)(11)(12)(13)(14). Although these mouse lines have tremendously helped to understand NO/cGMP signaling, it is still not known which of NO's effects are mediated via NO-GC and thus cGMP, or alternatively, via pathways not involving cGMP.To address this point and to further investigate the physiological role of the enzyme and of the NO/cGMP signaling cascade in vivo, we generated an NO-GC-deficient mouse line. NO-GC is a heterodimer made up of two subunits, ␣ and ␤. Two isoforms are known to exist (␣ 1 ␤ 1 and ␣ 2 ␤ 1 ; ref. 15) in which the ␤ 1 subunit acts as the dimerizing partner for either ␣ subunit. ␣ subunits in the absence of the ␤ 1 subunit do not form dimers and are not catalytically active. Thus, deletion of the ␤ 1 subunit should completely eliminate NO-GC and yield a mouse line ...