[Show abstract][Hide abstract] ABSTRACT: AimsCardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism.Methods and resultsTo circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to β-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca(2+)](i)-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser(16), the specific target site for cGKI, resulting in altered myocyte Ca(2+)(i) homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca(2+)(i)-handling, and contractility via cGKI.ConclusionThese results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca(2+)(i) handling and contractility.
European Heart Journal 12/2011; · 14.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A, to exert its diverse functions. This process involves a cGMP-dependent signaling pathway preventing pathological [Ca(2+)](i) increases in myocytes. In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization. Here we show that, in this situation, ANP binding to GC-A stimulates a unique cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca(2+) levels. This pathway involves the activation of Ca(2+)-permeable transient receptor potential canonical 3/6 (TRPC3/C6) cation channels by GC-A, which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting cation influx activates voltage-dependent L-type Ca(2+) channels and ultimately increases myocyte Ca(2)(+)(i) levels. These observations reveal a dual role of the ANP/GC-A-signaling pathway in the regulation of cardiac myocyte Ca(2+)(i) homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca(2+)(i)-enhancing action of hypertrophic factors such as angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca(2+)](i) might increase the propensity to cardiac hypertrophy and arrhythmias.
Proceedings of the National Academy of Sciences 11/2011; 108(45):18500-5. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiac atrial natriuretic peptide (ANP) locally counteracts cardiac hypertrophy via the guanylyl cyclase-A (GC-A) receptor and cGMP production, but the downstream signalling pathways are unknown. Here, we examined the influence of ANP on beta-adrenergic versus Angiotensin II (Ang II)-dependent (G(s) vs. G(alphaq) mediated) modulation of Ca(2+) (i)-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca(2+) currents and Ca(2+) (i) transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca(2+) currents and transients, but had no effect on isoproterenol stimulation. Ang II suppression by ANP was abolished in cardiomyocytes of mice deficient in GC-A, in cyclic GMP-dependent protein kinase I (PKG I) or in the regulator of G protein signalling (RGS) 2, a target of PKG I. Cardiac hypertrophy in response to exogenous Ang II was significantly exacerbated in mice with conditional, cardiomyocyte-restricted GC-A deletion (CM GC-A KO). This was concomitant to increased activation of the Ca(2+)/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, beta-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca(2+)-handling were absent in myocytes of mice deficient in TRPC3/TRPC6, the effects of isoproterenol were unchanged. Our data demonstrate a direct myocardial role for ANP/GC-A/cGMP to antagonize the Ca(2+) (i)-dependent hypertrophic growth response to Ang II, but not to beta-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT(1) signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for beta-adrenergic Ca(2+) (i)-stimulation in adult myocytes.
Archiv für Kreislaufforschung 03/2010; 105(5):583-95. · 7.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiac atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) modulate blood pressure and volume by activation of the receptor guanylyl cyclase-A (GC-A) and subsequent intracellular cGMP formation. Here we report what we believe to be a novel function of these peptides as paracrine regulators of vascular regeneration. In mice with systemic deletion of the GC-A gene, vascular regeneration in response to critical hind limb ischemia was severely impaired. Similar attenuation of ischemic angiogenesis was observed in mice with conditional, endothelial cell-restricted GC-A deletion (here termed EC GC-A KO mice). In contrast, smooth muscle cell-restricted GC-A ablation did not affect ischemic neovascularization. Immunohistochemistry and RT-PCR revealed BNP expression in activated satellite cells within the ischemic muscle, suggesting that local BNP elicits protective endothelial effects. Since within the heart, BNP is mainly induced in cardiomyocytes by mechanical load, we investigated whether the natriuretic peptide/GC-A system also regulates angiogenesis accompanying load-induced cardiac hypertrophy. EC GC-A KO hearts showed diminished angiogenesis, mild fibrosis, and diastolic dysfunction. In vitro BNP/GC-A stimulated proliferation and migration of cultured microvascular endothelia by activating cGMP-dependent protein kinase I and phosphorylating vasodilator-stimulated phosphoprotein and p38 MAPK. We therefore conclude that BNP, produced by activated satellite cells within ischemic skeletal muscle or by cardiomyocytes in response to pressure load, regulates the regeneration of neighboring endothelia via GC-A. This paracrine communication might be critically involved in coordinating muscle regeneration/hypertrophy and angiogenesis.
The Journal of clinical investigation 07/2009; 119(7):2019-30. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atrial natriuretic peptide (ANP) regulates arterial blood pressure and volume. Its guanylyl cyclase-A (GC-A) receptor is expressed in vascular endothelium and mediates increases in cGMP, but the functional relevance is controversial. Notably, mice with endothelial-restricted GC-A deletion [EC GC-A knockout (KO) mice] exhibit significant chronic hypervolemic hypertension. The present study aimed to characterize the endothelial effects of ANP and their relevance for the acute regulation of intravascular fluid volume. We studied the effect of ANP on microvascular permeability to fluorescein isothiocyanate-labeled albumin (BSA) using intravital microscopy on mouse dorsal skinfold chambers. Local superfusion of ANP (100 nm) increased microvascular fluorescein isothiocyanate-BSA extravasation in control but not EC GC-A KO mice. Intravenous infusion of synthetic ANP (500 ng/kg x min) caused immediate increases in hematocrit in control mice, indicating intravascular volume contraction. In EC GC-A KO mice, the hematocrit responses were not only abolished but even reversed. Furthermore, acute vascular volume expansion, which caused release of endogenous cardiac ANP, did not affect resting central venous pressure of control mice but rapidly and significantly increased central venous pressure of EC GC-A KO mice. In cultured lung endothelial cells, ANP provoked cGMP-dependent protein kinase I-mediated phosphorylation of vasodilator-stimulated phosphoprotein. We conclude that ANP, via GC-A, enhances microvascular endothelial macromolecule permeability in vivo. This effect might be mediated by cGMP-dependent protein kinase I-dependent phosphorylation of vasodilator-stimulated phosphoprotein. Modulation of transcapillary protein and fluid transport may represent one of the most important hypovolemic actions of ANP.
[Show abstract][Hide abstract] ABSTRACT: D-Serine selectively causes necrosis of S(3) segments of proximal tubules in rats. This leads to aminoaciduria and glucosuria. Coinjection of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) prevents the tubulopathy. D-serine is selectively reabsorbed in S(3), thereby gaining access to peroxisomal D-amino acid oxidase (D-AAO). D-AAO-mediated metabolism produces reactive oxygen species. We determined the fractional excretion of amino acids and glucose in rats after intraperitoneal injection of d-serine alone or together with reduced glutathione (GSH) or AIB. Both compounds prevented the hyperaminoaciduria. We measured GSH concentrations in renal tissue before (control) and after D-serine injection and found that GSH levels decreased to approximately 30% of control. This decrease was prevented when equimolar GSH was coinjected with D-serine. To find out why AIB protected the tubule from D-serine toxicity, we microinfused D-[(14)C]serine or [(14)C]AIB (0.36 mmol/l) together with [(3)H]inulin in late proximal tubules in vivo and measured the radioactivity in the final urine. Fractional reabsorption of D-[(14)C]serine and [(14)C]AIB amounted to 55 and 70%, respectively, and 80 mmol/l of AIB or D-serine mutually prevented reabsorption to a great extent. D-AAO activity measured in vitro (using D-serine as substrate) was not influenced by a 10-fold higher AIB concentration. We conclude from these results that 1) D-AAO-mediated d-serine metabolism lowers renal GSH concentrations and thereby provokes tubular damage because reduction of reactive oxygen species by GSH is diminished and 2) AIB prevents d-serine-induced tubulopathy by inhibition of D-serine uptake in S(3) segments rather than by interfering with intracellular D-AAO-mediated D-serine metabolism.
American journal of physiology. Renal physiology 08/2007; 293(1):F382-90. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aldosterone plays a key role in cardiovascular and renal injury. The underlying mechanisms are not completely understood. Because the epidermal growth factor receptor (EGFR) is involved in the development of fibrosis and vascular dysfunction, upregulation of EGFR expression by aldosterone-bound mineralocorticoid receptor (MR) is an attractive hypothesis. We investigated the effect of aldosterone on EGFR expression in the aorta of adrenalectomized rats and in human aorta smooth muscle cells (HAoSMC) in primary culture. Aldosterone, but not dexamethasone, stimulated EGFR expression in vivo in the aorta as well as in HAoSMC. EGFR degradation was not affected. Aldosterone-induced EGFR expression in HAoSMC was dose dependent and prevented by spironolactone. Furthermore, incubation of HAoSMC with aldosterone led to enhanced EGF-induced ERK1/2 phosphorylation and an EGFR-dependent increase in media fibronectin. EGFR promoter reporter gene assay as well as chromatin immunoprecipitation data indicate that MR interacts with the EGFR promoter. With deletion constructs we gained evidence that this interaction takes place between the hMR and the EGFR promoter regions 316-163 (stronger activation site, EC50 approximately 1.0 nM) and 163-1 (weaker activation site, EC50 approximately 0.7 nM), which do not comprise canonical glucocorticoid response elements and are not activated by the human glucocorticoid receptor. The interactions require in part the NH2-terminal domains of MR. ELISA-based transcription factor DNA binding assay with in vitro synthesized hMR suggest direct binding to region 163-1. Our results indicate that aldosterone leads to enhanced EGFR expression via an interaction with the EGFR promoter, which is MR specific and could contribute to the aldosterone-induced increase in fibronectin abundance.
AJP Endocrinology and Metabolism 07/2007; 292(6):E1790-800. · 4.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mice with genetic disruption of the guanylyl cyclase-A (GC-A) receptor for atrial natriuretic peptide (ANP), have chronic arterial hypertension and marked cardiac hypertrophy. Intriguingly, despite pronounced remodeling, cardiac contractile functions and cardiomyocyte Ca(2+)-handling are preserved and even enhanced. The present study aimed to characterize the specific molecular mechanisms preventing cardiac failure.
Contractile function and expression as well as phosphorylation of regulatory proteins were evaluated in isolated perfused working hearts from wild-type and GC-A KO mice under baseline conditions and during beta(1)-adrenergic stimulation. Ca(i)(2+)-transients were monitored in Indo-1 loaded isolated adult cardiomyocytes. Cardiac contractile, especially lusitropic responsiveness to beta-adrenergic stimulation was significantly increased in GC-A KO mice. This was concomitant to enhanced expression and activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), increased dual-site phosphorylation of phospholamban (PLB) at Ser(16) and Thr(17), enhanced amplitude of Ca(i)(2+) transients, and accelerated Ca(i)(2+) decay. In contrast, the expression of cardiac ryanodine receptors and phosphorylation at Ser(2809) and Ser(2815) was not altered. Pharmacological inhibition of CaMKII-but not of protein kinase A-mediated PLB phosphorylation totally abolished the increased effects of beta-adrenergic stimulation on cardiac contractility and Ca(i)(2+)-handling. Thus, acceleration of sarcoplasmic reticulum Ca(2+)-uptake and increased availability of Ca(2+) for contraction, both secondary to increased CaMKII-mediated PLB phosphorylation, seem to mediate the augmented responsiveness of GC-A KO hearts to catecholamines.
Our observations show that increased CaMKII activity enhances the contractile relaxation response of hypertrophic GC-A KO hearts to beta-adrenergic stimulation and emphasize the critical role of CaMKII-dependent pathways in beta(1)-adrenoreceptor modulation of myocardial Ca(2+)-homeostasis and contractility.
Cardiovascular Research 04/2007; 73(4):678-88. · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proximal tubular receptor-mediated endocytosis (RME) of filtered proteins prevents proteinuria. Pharmacological and genetic studies in cultured opossum kidney cells have shown that the apical Na(+)/H(+) exchanger isoform 3 (NHE3) supports RME by interference with endosomal pH homeostasis and endocytic fusion events. However, it is not known whether NHE3 also supports proximal tubular RME in vivo. We analyzed proximal tubular protein reabsorption by microinfusion experiments in rats and investigated renal protein excretion in NHE3 knockout (Nhe3 -/-) mice. Inhibition of NHE3 by EIPA or S-3226 reduced the fractional reabsorption of [(14)C]cytochrome c by approximately 50% during early proximal microinfusion. During early distal microinfusion, no protein reabsorption could be detected. Urinary protein excretion of Nhe3 -/- or heterozygous mutant mice was significantly higher compared with wild-type mice. SDS-PAGE analysis of urinary proteins revealed that Nhe3 -/- animals excreted proteins the size of albumin or smaller. Thus a reduction in NHE3 activity or abundance causes tubular proteinuria. These data show that NHE3 supports proximal tubular RME of filtered proteins in vivo.
American journal of physiology. Renal physiology 10/2004; 287(3):F469-73. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Filtered myo-inositol, an important renal intracellular organic osmolyte, is almost completely reabsorbed. To examine tubule sites and specificity and, thus possible mechanism of this reabsorption, we microinfused myo-[(3)H]inositol or D-[(3)H]glucose into early proximal (EP), late proximal (LP), or early distal tubule sections of superficial nephrons and into long loops of Henle (LLH) of juxtamedullary nephrons and papillary vasa recta in rats in vivo et situ and determined urinary fractional recovery of the (3)H label compared with comicroinfused [(14)C]inulin. To determine the extent to which the proximal convoluted tubule (PCT) alone contributes to myo-inositol reabsorption, we also microperfused this tubule segment between EP and LP puncture sites. We examined specificity of reabsorptive carrier(s) by adding high concentrations of other polyols and monosaccharides to the infusate. The results show that >60% of the physiological glomerular load of myo-inositol can be reabsorbed in the PCT and >90% in the short loop of Henle (SLH) by a saturable, phloridzin-sensitive process. myo-Inositol can also be reabsorbed in the ascending limb of LLH and can move from papillary vasa recta blood into ipsilateral tubular structures. Essentially no reabsorption occurred in nephron segments beyond the SLH or in collecting ducts. Specificity studies indicate that reabsorption probably occurs via a luminal Na(+)-myo-inositol cotransporter.
American journal of physiology. Renal physiology 06/2003; 284(6):F1181-9. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study was designed 1) to localize and 2) to characterize betaine reabsorption from the tubular lumen in rat kidney in vivo, and 3) to test whether reabsorption is modulated by the diuretic state. [(14)C]betaine (+ [(3)H]inulin) was microperfused through the proximal convoluted tubule (PCT) and microinfused into late proximal (LP) and early distal (ED) tubules, long loops of Henle (LLH), and vasa recta of the rat in vivo et situ, and the fractional recovery of the (14)C label was determined end proximally (PCT) and in the final urine, respectively. [(14)C]betaine was not reabsorbed during ED microinfusion, whereas fractional reabsorption during LP microinfusion was 82% at 0.06 mM betaine and decreased gradually to 4.8% at 60 mM. L-Proline had lower Michaelis-Menten constant (K(m)) and sarcosine a higher K(m) than betaine. Chronic, but not acute, diuresis inhibited betaine reabsorption in Henle's loops. Fractional [(14)C]betaine reabsorption in PCT was much smaller than that during LP microinfusion. [(14)C]betaine (7.28 mM) microinfused 1) into LLH was reabsorbed to 30% and 2) into vasa recta appeared in the ipsilateral urine to a much higher extent than contralaterally. In both cases, no saturation was detected at 70 mM. We conclude that betaine is reabsorbed by mediated transport from descending limbs of short Henle's loops by a proline-preferring carrier in a diuresis-modulated manner. In the deep medulla, bidirectional blood/urine betaine transport exists.
American journal of physiology. Renal physiology 04/2000; 278(3):F434-9. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amino acids are apparently recycled between loops of Henle and vasa recta in rat papilla in vivo. To examine this process in the absence of metabolism, we performed continuous microinfusions of rat renal papillary ascending thin limbs (ATLs) and vasa recta in vivo, and microperflusions of isolated rat renal papillary descending thin limbs (DTLs) and ATLs in vitro using the nonmetabolizable, synthetic, neutral amino acid cycloleucine. Like naturally occurring amino acids, approximately = 25% of radiolabeled cycloleucine microinfused into ATLs in vivo was reabsorbed by a process that was not saturable or inhibitable. Also, like naturally occurring amino acids, approximately = 47% (relative to inulin) of radiolabeled cycloleucine microinfused into ascending vasa recta in vivo was transferred directly into ipsilateral tubular structures (probably DTLs) by a saturable and inhibitable process. In DTLs perfused in vitro, unidirectional bath-to-lumen fluxes (Jbl) tended to exceed unidirectional lumen-to-bath fluxes (Jlb), whereas in ATLs perfused in vitro Jlb tended to exceed Jbl, but the differences were not statistically significant. Moreover, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from ATLs in vivo but incompatible with apparent movement from vasa recta to DTLs in vivo. These in vitro observations are like those made previously for the naturally occurring neutral amino acid L-alanine. The lack of saturation and inhibition, like the previous data on L-alanine, suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route and that regulation of amino acid movement in vivo may involve vasa recta, not DTLs. They also suggest that cycloleucine is a good nonmetabolizable surrogate for the study of neutral amino acid transport in the kidney.
Pflügers Archiv - European Journal of Physiology 04/2000; 439(5):517-23. · 4.87 Impact Factor