Ana Kilic

Enhanced activity of the myocardial Na+/H+ exchanger NHE-1 contributes to cardiac remodeling in atrial natriuretic peptide receptor-deficient mice.

Background— Atrial natriuretic peptide (ANP), through its guanylyl cyclase-A (GC-A) receptor, not only is critically involved in the endocrine regulation of arterial blood pressure but also locally moderates cardiomyocyte growth. The mechanisms underlying the antihypertrophic effects of ANP remain largely uncharacterized. We examined the contribution of the Na+/H+ exchanger NHE-1 to cardiac remodeling in GC-A–deficient (GC-A−/−) mice. Methods and Results— Fluorometric measurements in isolated adult cardiomyocytes demonstrated that cardiac hypertrophy in GC-A−/− mice was associated with enhanced NHE-1 activity, alkalinization of intracellular pH, and increased Ca2+ levels. Chronic treatment of GC-A−/− mice with the NHE-1 inhibitor cariporide normalized cardiomyocyte pH and Ca2+ levels and regressed cardiac hypertrophy and fibrosis, despite persistent arterial hypertension. To characterize the molecular pathways driving cardiac hypertrophy in GC-A−/− mice, we evaluated the activity of 4 prohypertrophic signaling pathways: the mitogen-activated protein kinases (MAPK), the serine-threonine kinase Akt, calcineurin, and Ca2+/calmodulin-dependent kinase II (CaMKII). The results demonstrate that all 4 pathways were activated in GC-A−/− mice, but only CaMKII and Akt activity regressed during reversal of the hypertrophic phenotype by cariporide treatment. In contrast, the MAPK and calcineurin/NFAT signaling pathways remained activated during regression of hypertrophy. Conclusions— On the basis of these results, we conclude that the ANP/GC-A system moderates the cardiac growth response to pressure overload by preventing excessive activation of NHE-1 and subsequent increases in cardiomyocyte intracellular pH, Ca2+, and CaMKII as well as Akt activity.

Increased effects of C-type natriuretic peptide on contractility and calcium regulation in murine hearts overexpressing cyclic GMP-dependent protein kinase I

C‐type natriuretic peptide (CNP) and its receptor guanylyl cyclase (GC‐B) are expressed in the heart and modulate cardiac contractility in a cGMP‐dependent manner. Since the distal cellular signalling pathways remain unclear, we evaluated the peptide effects on cardiac function and calcium regulation in wild‐type (WT) and transgenic mice with cardiac overexpression of cGMP‐dependent protein kinase I (PKG ITG). In isolated, perfused working WT hearts, CNP (10 nM) provoked an immediate increase in the maximal rates of contraction and relaxation, a small increase in the left ventricular systolic pressure and a decrease in the time of relaxation. These changes in cardiac function were accompanied by a marked increase in the levels of Ser16‐phosphorylated phospholamban (PLB). In PKG ITG hearts, the effects of CNP on cardiac contractility and relaxation as well as on PLB phosphorylation were markedly enhanced. CNP increased cell shortening and systolic Cai2+ levels, and accelerated Cai2+ decay in isolated, Indo‐1/AM‐loaded WT cardiomyocytes, and these effects were enhanced in PKG I‐overexpressing cardiomyocytes. 8‐pCPT‐cGMP, a membrane‐permeable PKG activator, mimicked the contractile and molecular actions of CNP, the effects again being more pronounced in PKG ITG hearts. In contrast, the cardiac reponses to β‐adrenergic stimulation were not different between genotypes. Taken together, our data indicate that PKG I is a downstream target activated by the CNP/GC‐B/cGMP‐signalling pathway in cardiac myocytes. cGMP/PKG I‐stimulated phosphorylation of PLB and subsequent activation of the sarcoplasmic reticulum Ca2+ pump appear to mediate the positive inotropic and lusitropic responses to CNP.

Identification of an orphan guanylate cyclase receptor selectively expressed in mouse testis.

We have identified a novel membrane form of guanylate cyclase (GC) from a mouse testis cDNA library and termed it mGC-G (mouse GC-G) based on its high sequence homology to rat GC-G. It encodes a potential type I transmembrane receptor, with the characteristic domain structure common to all members of the family of membrane GCs, including an extracellular, putative ligand-binding domain, a single membrane-spanning segment and cytoplasmic protein kinase-like and cyclase catalytic domains. Real-time quantitative reverse transcriptase--PCR and Northern-blot analyses showed that mGC-G is highly and selectively expressed in mouse testis. Phylogenetic analysis based on the extracellular protein sequence revealed that mGC-G is closely related to members of the subfamily of natriuretic peptide receptor GCs. When overexpressed in HEK-293T cells (human embryonic kidney 293T cells) or COS-7 cells, mGC-G manifests as a membrane-bound glycoprotein, which can form either homomeric or heteromeric complexes with the natriuretic peptide receptor GC-A. It exhibits marked cGMP-generating GC activity; however, notably, all ligands known to activate other receptor GCs failed to stimulate enzymic activity. The unique testis-enriched expression of mGC-G, which is completely different from the broader tissue distribution of rat GC-G, suggests the existence of as-yet-unidentified ligands and unappreciated species-specific physiological functions mediated through mGC-G/cGMP signalling in the testis.