Particulate Guanylyl Cyclase Research Articles

The role of particulate guanylyl cyclase B (GC-B) in auditory function in adult mice

Background cGMP signaling triggered by the binding of C-type natriuretic peptide (CNP) to its receptor guanylyl cyclase B (GC-B; NPR2; NPRB) has been linked by genetic evidence to a remarkable variety of physiological functions like skeletal bone growth, female fertility, cardiac growth, fat metabolism and gastrointestinal function. For the nervous system it has been recently demonstrated that the CNP/GC-B/cGMP/cGMP-dependent protein kinase type I (cGKI) signaling pathway is essential for sensory axon branching at the dorsal root entry zone of the spinal cord and at the rhombomeres of the hindbrain during embryonic development [1]. Also in the auditory system, distinct auditory nerve fiber (ANF) types that differ in their discharge rate and sound sensitivity bifurcate in the cochlear nucleus (CN), sending collaterals to the anteroventral, posteroventral, and dorsal subdivisions. The lack of GC-B has been shown to lead to a central phenotype [2].

Structure, regulation and functions of particulate guanylyl cyclase type A

Guanylyl cyclase type A (GC-A) belongs to the particulate guanylyl cyclases (pGC), which, like the soluble guanylyl cyclases (sGC), catalyze the synthesis of a common secondary messenger, namely cyclic GMP (cGMP), involved in many cellular processes. Although both forms of guanylyl cyclases produce the same secondary messenger, activation of each of them triggers different signaling pathways leading to different cellular effects. This indicates that the final effect of cGMP depends on the site of its synthesis in the cell (cytosol or cell membrane). Particulate guanylyl cyclase type A is a homodimeric protein activated by natriuretic peptides (ANP - atrial natriuretic peptide and BNP - brain natriuretic peptide) binding in the extracellular domain of the enzyme. The widespread expression of GC-A in different cell types and tissues suggests that this protein may regulate many cellular processes. Besides the role of GC-A in the cardiovascular system, which is the most thoroughly documented in the literature, it was observed that this protein is also involved in carcinogenesis and regulation of inflammatory reactions. This review describes important information about the structure, functions and regulation of GC-A catalytic activity, and the regulation of GC-A gene expression.

B-type natriuretic peptide expression and cardioprotection is regulated by Akt dependent signaling at early reperfusion

Exogenously administered B-type natriuretic peptide (BNP) has been shown to offer cardioprotection through activation of particulate guanylyl cyclase (pGC), protein kinase G (PKG) and KATP channel opening. The current study explores if cardioprotection afforded by short intermittent BNP administration involves PI3K/Akt/p70s6k dependent signaling, and whether this signaling pathway may participate in regulation of BNP mRNA expression at early reperfusion. Isolated Langendorff perfused rat hearts were subjected to 30min of regional ischemia and 120min of reperfusion (IR). Applying intermittent 3×30s infusion of BNP peptide in a postconditioning like manner (BNPPost) reduced infarct size by >50% compared to controls (BNPPost 17±2% vs. control 42±4%, p<0.001). Co-treatment with inhibitors of the PI3K/Akt/p70s6k pathway (wortmannin, SH-6 and rapamycin) completely abolished the infarct-limiting effect of BNP postconditioning (BNPPost+Wi 36±5%, BNPPost+SH-6 41±4%, BNPPost+Rap 37±6% vs. BNPPost 17±2%, p<0.001). Inhibition of natriuretic peptide receptors (NPR) by isatin also abrogated BNPPost cardioprotection (BNPPost+isatin 46±2% vs. BNPPost 17±2%, p<0.001). BNPPost also significantly phosphorylated Akt and p70s6k at early reperfusion, and Akt phosphorylation was inhibited by SH-6 and isatin. Myocardial BNP mRNA levels in the area at risk (AA) were significantly elevated at early reperfusion as compared to the non-ischemic area (ANA) (Ctr(AA) 2.7±0.5 vs. Ctr(ANA) 1.2±0.2, p<0.05) and the ischemic control tissue (Ctr(AA) 2.7±0.5 vs. ischemia 1.0±0.1, p<0.05). Additional experiments also revealed a significant higher BNP mRNA level in ischemic postconditioned (IPost) hearts as compared to ischemic controls (IPost 6.7±1.3 vs. ischemia 1.0±0.2, p<0.05), but showed no difference from controls run in parallel (Ctr 5.4±0.8). Akt inhibition by SH-6 completely abrogated this elevation (IPost 6.7±1.3 vs. IPost+SH-6 1.8±0.7, p<0.05) (Ctr 5.4±0.8 vs. SH-6 1.5±0.9, p<0.05). In conclusion, Akt dependent signaling is involved in mediating the cardioprotection afforded by intermittent BNP infusion at early reperfusion, and may also participate in regulation of reperfusion induced BNP expression.

It's cold, mom! It's cyclic GMP.

Cyclic GMP is the signal transducer of a family of transmembrane, particulate guanylyl cyclase (GC) receptors with key roles in physiology and disease. GC-G, the last member of the membrane GCs identified in mammals, is an orphan receptor and its regulation and function have remained largely unknown. In this issue of The EMBO Journal, Chao et al (2015) show that the GC-G/cGMP pathway, which is expressed in a specific cluster of olfactory neurons of neonatal mice, functions as a cold-induced thermosensor, which triggers protective maternal care.

M‐Atrial Natriuretic Peptide and Nitroglycerin in a Canine Model of Experimental Acute Hypertensive Heart Failure: Differential Actions of 2 cGMP Activating Therapeutics

BackgroundSystemic hypertension is a common characteristic in acute heart failure (HF). This increasingly recognized phenotype is commonly associated with renal dysfunction and there is an unmet need for renal enhancing therapies. In a canine model of HF and acute vasoconstrictive hypertension we characterized and compared the cardiorenal actions of M‐atrial natriuretic peptide (M‐ANP), a novel particulate guanylyl cyclase (pGC) activator, and nitroglycerin, a soluble guanylyl cyclase (sGC) activator.Methods and ResultsHF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II infusion. We characterized the cardiorenal and humoral actions prior to, during, and following intravenous M‐ANP (n=7), nitroglycerin (n=7), and vehicle (n=7) infusion. Mean arterial pressure (MAP) was reduced by M‐ANP (139±4 to 118±3 mm Hg, P<0.05) and nitroglycerin (137±3 to 116±4 mm Hg, P<0.05); similar findings were recorded for pulmonary wedge pressure (PCWP) with M‐ANP (12±2 to 6±2 mm Hg, P<0.05) and nitroglycerin (12±1 to 6±1 mm Hg, P<0.05). M‐ANP enhanced renal function with significant increases (P<0.05) in glomerular filtration rate (38±4 to 53±5 mL/min), renal blood flow (132±18 to 236±23 mL/min), and natriuresis (11±4 to 689±37 mEq/min) and also inhibited aldosterone activation (32±3 to 23±2 ng/dL, P<0.05), whereas nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation.ConclusionsOur results advance the differential cardiorenal actions of pGC (M‐ANP) and sGC (nitroglycerin) mediated cGMP activation. These distinct renal and aldosterone modulating actions make M‐ANP an attractive therapeutic for HF with concomitant hypertension, where renal protection is a key therapeutic goal.

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC50 properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders.

M-atrial natriuretic peptide and nitroglycerin in a model of acute hypertensive heart failure: a comparison of two cGMP activating therapeutics

Purpose: Systemic hypertension is a common characteristic in acute heart failure (HF). This increasingly common phenotype is commonly associated with renal dysfunction and there is an unmet need for renal enhancing therapies. In a canine model of HF and acute vasoconstrictive hypertension we characterized and compared the cardiorenal actions of M-atrial natriuretic peptide (M-ANP), a novel particulate guanylyl cyclase activator (pGC), and nitroglycerin, a soluble guanylyl cyclase (sGC) activator. Methods: HF was induced by rapid right ventricular pacing (180 bpm) for 10 days. On day 11 hypertension was induced by continuous angiotensin II infusion. We characterized the cardiorenal and humoral actions prior to, during, and for 300 minutes following intravenous M-ANP (n=7), nitroglycerin (n=7), and vehicle (n=7) infusion. Results: Mean arterial pressure (MAP) was reduced by M-ANP (139±4 to 118±3mmHg, p 0.05) effects on these renal parameters or aldosterone activation. ![Figure][1] Conclusions: Our results advance the differential cardiorenal actions of pGC (M-ANP) and sGC (nitroglycerin) mediated cGMP activation. These distinct renal and aldosterone modulating actions make M-ANP an attractive therapeutic for HF with concomitant hypertension, where renal protection is a key therapeutic goal. [1]: pending:yes

M-atrial natriuretic peptide and nitroglycerin in experimental acute hypertensive heart failure: differential actions of two cGMP activating therapeutics

Background Systemic hypertension is a common characteristic in acute heart failure (HF). This increasingly common phenotype is frequently associated with renal dysfunction and requires renal enhancing therapies. In a canine model of HF and acute hypertension we defined the cardiorenal actions of M-atrial natriuretic peptide (MANP), a novel particulate guanylyl cyclase A receptor activator (pGC-A) developed at the Mayo Clinic, and nitroglycerin (NTG), a soluble guanylyl cyclase (sGC) activator. Compared to native ANP, MANP has more sustained renal enhancing, blood pressure lowering and aldosterone inhibiting action. Based on the expression of the pGC-A receptor in the glomerulus, renal tubule and adrenal gland, compared to the more widespread expression of sGC in the renal vasculature, we hypothesized that MANP would have a more robust effect upon the glomerular filtration rate (GFR) and natriuretic actions and with greater aldosterone suppressing effects than sGC activation with NTG. To test these hypotheses, we used a canine model of combined HF and hypertension. Specifically, HF was induced by chronic (10 days) rapid right ventricular pacing (180 bmp), while hypertension was induced by continuous angiotensin II infusion (on day 11). We than characterized the cardiorenal and humoral actions of intravenous MANP (n=7), NTG (n=7), and vehicle (n=7) infusion. Results Mean arterial pressure (MAP) was reduced by both MANP (139±4 to 118±3 mmHg, p<0.05) and NTG (137 ±3 to 116±4 mmHg, p<0.05); similar findings were observed for reductions in pulmonary wedge pressure with MANP (12±2 to 6±2 mmHg, p<0.05) and NTG (12±1 to 6±1 mmHg, p<0.05). MANP enhanced renal function with significant increases in GFR (38±4 to 53±5 ml/min, p<0.05), renal blood flow (132±18 to 236±23 ml/ min, p<0.05), and natriuresis (11±4 to 689±37 mEq/min, p<0.05) with inhibition of aldosterone (32±3 to 23±2 ng/ dL, p<0.05), whereas NTG had no significant effect on these renal parameters or aldosterone activation.

Nucleotidyl Cyclase Activity of Particulate Guanylyl Cyclase A: Comparison with Particulate Guanylyl Cyclases E and F, Soluble Guanylyl Cyclase and Bacterial Adenylyl Cyclases Cyaa and Edema Factor

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.

Influence of phosphodiesterases and cGMP on cAMP generation and on phosphorylation of phospholamban and troponin I by 5-HT4 receptor activation in porcine left atrium

Our objective was to investigate the role of phosphodiesterase (PDE)3 and PDE4 and cGMP in the control of cAMP metabolism and of phosphorylation of troponin I (TnI) and phospholamban (PLB) when 5-HT4 receptors are activated in pig left atrium. Electrically paced porcine left atrial muscles, mounted in organ baths, received stimulators of particulate guanylyl cyclase (pGC) or soluble guanylyl cyclase (sGC) and/or specific PDE inhibitors followed by 5-HT or the 5-HT4 receptor agonist prucalopride. Muscles were freeze-clamped at different moments of exposure to measure phosphorylation of the cAMP/protein kinase A targets TnI and PLB by immunoblotting and cAMP levels by enzyme immunoassay. Corresponding with the functional results, 5-HT only transiently increased cAMP content, but caused a less quickly declining phosphorylation of PLB and did not significantly change TnI phosphorylation. Under combined PDE3 and PDE4 inhibition, the 5-HT-induced increase in cAMP levels and PLB phosphorylation was enhanced and sustained, and TnI phosphorylation was now also increased. Responses to prucalopride per se and the influence thereupon of PDE3 and PDE4 inhibition were similar except that responses were generally smaller. Stimulation of pGC together with PDE4 inhibition increased 5-HT-induced PLB phosphorylation compared to 5-HT alone, consistent with functional responses. sGC stimulation hastened the fade of inotropic responses to 5-HT, while cAMP levels were not altered. PDE3 and PDE4 control the cAMP response to 5-HT4 receptor activation, causing a dampening of downstream signalling. Stimulation of pGC is able to enhance inotropic responses to 5-HT by increasing cAMP levels, while sGC stimulation decreases contraction to 5-HT cAMP independently.

Natriuretic peptides induce weak VASP phosphorylation at Serine 239 in platelets

Cyclic guanosine-3′,5′-monophoshate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides (NP; e.g. atrial NP [ANP]), which activate soluble and particulate guanylyl cyclases, respectively. The role of NO in regulating cGMP and platelet function is well documented, whereas there is little evidence supporting a role for NPs in regulating platelet reactivity. By studying platelet aggregation and secretion in response to a PAR-1 peptide, collagen and ADP, and phosphorylation of the cGMP-dependent protein kinase (PKG) substrate vasodilator-stimulated phosphoprotein (VASP) at serine 239, we evaluated the effects of NPs in the absence or presence of the non-selective cGMP and cAMP phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Our results show that NPs, possibly through the clearance receptor (natriuretic peptide receptor-C) expressed on platelet membranes, increase VASP phosphorylation but only following PDE inhibition, indicating a small, localised cGMP synthesis. As platelet aggregation and secretion measured under the same conditions were not affected, we conclude that the magnitude of PKG activation achieved by NPs in platelets per se is not sufficient to exert functional inhibition of platelet involvement in haemostasis.

Molecular Mechanisms of Gonadotropin-Releasing Hormone Signaling: Integrating Cyclic Nucleotides into the Network

Gonadotropin-releasing hormone (GnRH) is the primary regulator of mammalian reproductive function in both males and females. It acts via G-protein coupled receptors on gonadotropes to stimulate synthesis and secretion of the gonadotropin hormones luteinizing hormone and follicle-stimulating hormone. These receptors couple primarily via G-proteins of the Gq/ll family, driving activation of phospholipases C and mediating GnRH effects on gonadotropin synthesis and secretion. There is also good evidence that GnRH causes activation of other heterotrimeric G-proteins (Gs and Gi) with consequent effects on cyclic AMP production, as well as for effects on the soluble and particulate guanylyl cyclases that generate cGMP. Here we provide an overview of these pathways. We emphasize mechanisms underpinning pulsatile hormone signaling and the possible interplay of GnRH and autocrine or paracrine regulatory mechanisms in control of cyclic nucleotide signaling.

Pharmacological characterization of receptor guanylyl cyclase reporter cell lines

Receptor guanylyl cyclases are implicated in a growing number of pathophysiologies and, therefore, represent an important target class for drug development. We report here the generation and pharmacological characterization of three particulate guanylyl cyclase (pGC) reporter cell lines. Plasmid constructs encoding the natriuretic peptide receptors GC-A and GC-B, and the heat-stable enterotoxin receptor GC-C, were stably transfected in a parental reporter cell line expressing a cyclic nucleotide-gated (CNG) cation channel, acting as the biosensor for intracellular cGMP. In our reporter cell lines pGC activity can be monitored in living cells in real-time . By using different natural as well as synthetic receptor ligands of the natriuretic and guanylin peptide families, we show that our reporter assay monitors pGC activity with very high sensitivity. In contrast to previous findings, we could detect significant stimulation of GC-A and GC-B by each of the natriuretic peptides ANP, BNP and CNP. In addition, the clearance receptor ligand Cys-ANF(4–18) and the ANP receptor antagonist Arg-ANF(6–18) were characterized as partial GC-A agonists. The results imply that our novel pGC reporter cell lines are well suited for the characterization of receptor pharmacology and may be used for natural ligand characterization of guanylyl cyclase orphan receptors.

Phosphodiesterase-5 and Retargeting of Subcellular cGMP Signaling During Pathological Hypertrophy

Both cAMP and cGMP are critical intracellular second messengers regulating fundamental physiological processes in the myocardium, from acute contraction/relaxation to chronic gene expression, cell growth and apoptosis, and cardiac structural remodeling. cAMP is synthesized by adenylate cyclases on activation of G-protein–coupled receptors. cGMP is generated from the cytosolic purine nucleotide GTP by guanylyl cyclases (GCs) with Mg2+ or Mn2+ as cofactors. Two isoforms of GCs exist in vertebrate cells and tissues: a nitric oxide (NO)–sensitive cytosolic or soluble GC (sGC) and atrial natriuretic peptides (NP)–activated, plasma membrane–bound, particulate GC (pGC).1 Once produced, the effects of cGMP occur through 3 main groups of cellular target molecules: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and phosphodiesterases (PDEs). PDEs are metallohydrolases that catalyze the breakdown of cAMP or cGMP into the inactive 5′-AMP, thus modulating the duration and intensity of their intracellular response. PDEs have 11 families (PDE1–PDE11) that are encoded by 21 different genes. More than 80 enzyme variants are generated from multiple promoters and as a consequence of alternative splicing.2 PDE1 through PDE3, PDE10, and PDE11 are dual-specificity esterases because they hydrolyze both cAMP and cGMP; PDE4, PDE7, and PDE8 specifically degrade cAMP; and PDE5, PDE6, and PDE9 hydrolyze cGMP.2 The NH2-terminal portion of the PDE enzyme may undergo phosphorylation/dephosphorylation events, binding of Ca2+/calmodulin, and allosteric binding of cGMP and can mediate interactions with other proteins. PDE1, PDE3, PDE4, and PDE5 contain phosphorylation sites for various kinases. PDE1 also contains Ca2+-calmodulin binding sites, and stimuli that increase or decrease intracellular Ca2+ hereby profoundly affecting its activity. PDE2, PDE6, and PDE9 contain allosteric binding sites for cGMP called GAF. The binding of cGMP to GAFB in PDE2 activates the enzyme, whereas the binding of cGMP to GAFA in PDE5 favors …

Study of the regulation of the inotropic response to 5-HT4 receptor activation via phosphodiesterases and its cross-talk with C-type natriuretic peptide in porcine left atrium

We studied how 5-HT(4) receptor-mediated inotropic responses are regulated at the level of cAMP in porcine left atrium. We used selective phosphodiesterase (PDE) inhibitors to assess which PDE subtypes are responsible for the fade with time of inotropic responses to 5-HT(4) receptor activation with 5-HT and the 5-HT(4) receptor agonist prucalopride. A possible cross-talk via PDEs between cGMP and 5-HT(4) receptor-induced cAMP signalling was evaluated. Electrically paced left atrial pectinate muscles from young male pigs (15-25kg) were studied in vitro. Simultaneous inhibition of PDE3 plus PDE4 subtypes was necessary to increase the amplitude and completely prevent the fade of the inotropic response to 5-HT and prucalopride. When responses to 5-HT or prucalopride had faded 1h after addition, the nonspecific PDE-inhibitor IBMX still fully recovered inotropic responses. Stimulation of particulate guanylyl cyclase, together with PDE2 and PDE4 inhibition, delayed the fade of the response to 5-HT, while stimulation of soluble guanylyl cyclase independently of PDEs accelerated the fade of the response to 5-HT. In conclusion, both PDE3 and PDE4 subtypes are responsible for the suppression and the fade of the inotropic response to 5-HT and prucalopride. Signalling through the 5-HT(4) receptor remains fully active for at least 90min with PDEs continuously regulating the response. cGMP levels, elevated by activation of particulate guanylyl cyclase under PDE2 inhibition, can indirectly enhance 5-HT(4) receptor-mediated signalling, at least when also PDE4 is inhibited, presumably through inhibition of PDE3. Elevation of cGMP generated by soluble guanylyl cyclase attenuates responses to 5-HT independently of PDEs.

Cyclic GMP-Dependent Signaling in Cardiac Myocytes

Cyclic GMP (cGMP) and its effector kinase PKG regulate diverse cellular functions. In cardiac myocytes, cGMP is produced by soluble and particulate guanylyl cyclases (GCs), the former stimulated by nitric oxide and the latter by natriuretic peptides, and is hydrolyzed to inactive 5'-GMP by cGMP-phosphodiesterases (PDEs). cGMP-PKG modulates cardiac contractility, hypertrophy and remodeling, and exerts cardioprotection. Although early research efforts have mostly focused on cGMP synthetic pathways, recent studies have revealed that cGMP degradation controlled by PDEs plays a critical role in the physiological action of cGMP. Among several cGMP-PDEs, cGMP-specific PDE5 has been intensively investigated. Studies in experimental animal models and humans consistently demonstrate benefits from PDE5 inhibitors in various cardiac pathologies. Several clinical trials are ongoing or planned to test the efficacy of PDE5 inhibitors in human heart disease, including a large multicenter clinical trial (RELAX) led by the NIH evaluating sildenafil efficacy in heart failure with preserved ejection fraction. This review underscores the current understanding of cGMP-PKG signal regulation and its pathophysiological role in the heart, focusing on cardiac myocytes.

Altered regulation of nitric oxide and natriuretic peptide system in cisplatin-induced nephropathy

Cisplatin is a chemotherapeutic agent used for treating solid tumors. However, nephrotoxicity is the dose-limiting factor in its clinical use. The present study was aimed to determine whether altered regulation of the local nitric oxide (NO) and natriuretic peptide (NP) systems is involved in the pathogenesis of cisplatin-induced nephropathy. Cisplatin (6mg/kg) was injected intraperitoneally into male Sprague–Dawley rats. The control group was not treated with cisplatin. Expression levels of nitric oxide synthase (NOS), nitrotyrosine, soluble guanylyl cyclase and neutral endopeptidase (NEP) in the kidneys were determined 4days after treatment by semiquantitative immunoblotting. mRNA expression of NPs and natriuretic peptide receptors (NPRs) was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclase were determined by measuring the amount of cyclic 3′,5′-guanosine monophosphate (cGMP) generated in responses to sodium nitroprusside and atrial natriuretic peptide (ANP), respectively. In the test rats, creatinine clearance was decreased, while sodium and water excretion were increased. The expression of inducible NOS (iNOS) and nitrotyrosine was increased in the cortex/outer stripe of outer medullar and inner medullar, while that of endothelial and neuronal NOS was decreased in the inner medullar. Excretion of NO metabolites was increased in these rats. The catalytic activity of soluble guanyly cyclase was blunted in the papilla after cisplatin was administered. The mRNA expression of ANP, brain natriuretic peptide, and C-type natriuretic peptide was increased, while that of NPR-A and NPR-C were decreased in the test rats. The catalytic activity of soluble and particulate guanylyl cyclase in the papilla was blunted after cisplatin was administered. In conclusion, increased production of NO by iNOS may contribute to cytotoxic injury, resulting in cisplatin-induced nephropathy, while the up-regulation of renal natriuretic peptide synthesis together with the down-regulation of NEP and NPR-C may contribute to the natriuresis and diuresis seen in cisplatin-induced nephropathy.

CATALYTIC RECEPTORS

CATALYTIC RECEPTORS

Increase of nitric oxide production by C-type natriuretic peptide in beating rabbit atria

Objective: C-type natriuretic peptide (CNP) is a specific activator for the particulate (p) guanylyl cyclase (GC)-coupled natriuretic peptide receptor-B (NPR-B) in the heart. Nitric oxide (NO) is an endogenous activator for sGC activity. However, the relationship between CNP and NO in the regulation of atrial function has not yet been clearly defined. The aim of the present study was to define whether CNP regulates NO production in beating rabbit atria. Methods: Experiments were performed in perfused beating rabbit left atria.

Agents increasing cyclic GMP amplify 5-HT4-elicited positive inotropic response in failing rat cardiac ventricle

Activation of 5-HT4 receptors in failing ventricles elicits a cAMP-dependent positive inotropic response which is mainly limited by the cGMP-inhibitable phosphodiesterase (PDE) 3. However, PDE4 plays an additional role which is demasked by PDE3 inhibition. The objective of this study was to evaluate the effect of cGMP generated by particulate and soluble guanylyl cyclase (GC) on the 5-HT4-mediated inotropic response. Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats, exhibiting heart failure 6 weeks after surgery. Contractility was measured in left ventricular preparations. Cyclic GMP was measured by EIA. In ventricular preparations, ANP or BNP displayed no impact on 5-HT4-mediated inotropic response. However, CNP increased the 5-HT4-mediated inotropic response as well as the β1-adrenoceptor (β1-AR)-mediated response to a similar extent as PDE3 inhibition by cilostamide. Pretreatment with cilostamide eliminated the effect of CNP. Inhibition of nitric oxide (NO) synthase and soluble GC by l-NAME and ODQ, respectively, attenuated the 5-HT4-mediated inotropic response, whereas the NO donor Sin-1 increased this response. The effects were absent during PDE3 inhibition, suggesting cGMP-dependent inhibition of PDE3. However, in contrast to the effects on the 5-HT4 response, Sin-1 inhibited whereas l-NAME and ODQ enhanced the β1-AR-mediated inotropic response. cGMP generated both by particulate (NPR-B) and soluble GC increases the 5-HT4-mediated inotropic response in failing hearts, probably through inhibition of PDE3. β1-AR and 5-HT4 receptor signalling are subject to opposite regulatory control by cGMP generated by soluble GC in failing hearts. Thus, cGMP from different sources is functionally compartmented, giving differential regulation of different Gs-coupled receptors.