Characterization and effects of methyl-2- (4-aminophenyl)-1, 2-dihydro-1-oxo-7- (2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel potent inhibitor of cGMP-binding cGMP-specific phosphodiesterase (PDE5)

An isoquinolone derivative, methyl-2-(4-aminophenyl)-1, 2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), was found to be a novel potent inhibitor of cyclic GMP (cGMP)-binding cGMP-specific phosphodiesterase (PDE5). We investigated the inhibitory effects of T-1032 on six PDE isozymes isolated from canine tissues. T-1032 specifically inhibited the hydrolysis of cGMP by PDE5 partially purified from canine lung, at a low concentration (IC(50) = 1.0 nM, K(i) = 1.2 nM), in a competitive manner. In contrast, the IC(50) values of T-1032 for PDE1, PDE2, PDE3, and PDE4 were more than 1 microM. T-1032 also inhibited PDE6 from canine retina with an IC(50) of 28 nM, which is of the same order of magnitude as the IC(50) of sildenafil. cGMP hydrolytic activities of two alternative splice variants of canine PDE5 expressed in COS-7 cells were inhibited by this compound to a similar extent. T-1032 increased the intracellular concentration of cGMP in cultured rat vascular smooth muscle cells in the presence and absence of C-type natriuretic peptide, an activator of membrane-bound guanylate cyclase, whereas the compound did not change cyclic AMP levels. These data indicated that T-1032, which belongs to a new structural class of PDE5 inhibitors, is a potent and selective PDE5 inhibitor. This compound may be useful in pharmacological studies to examine the role of a cGMP/PDE5 pathway in tissues.     

Inhibition by HS-142-1, a novel nonpeptide atrial natriuretic peptide antagonist of microbial origin, of atrial natriuretic peptide-induced relaxation of isolated rabbit aorta through the blockade of guanylyl cyclase-linked receptors.

HS-142-1, a specific nonpeptide antagonist for the atrial natriuretic peptide (ANP) receptor, equally blocked rat ANP (rANP)-, porcine brain natriuretic peptide-, or porcine C-type natriuretic peptide-stimulated GMP production in cultured bovine aortic smooth muscle (BASM) and bovine aortic endothelial (BAE) cells in a concentration-dependent fashion, at concentrations of 1-300 micrograms/ml. But, even at 300 micrograms/ml, HS-142-1 only weakly inhibited the specific binding of 125I-rANP to the BASM and BAE cells, where only a small portion of the binding sites are linked to guanylyl cyclase. Further, with BAE cell membranes, HS-142-1 recognized only the 135-kDa ANP receptor, which is thought from 125I-rANP affinity cross-linking studies to be the guanylyl cyclase-linked receptor. HS-142-1 also, if anything, inhibited the labeling of 135-kDa ANP receptors in the affinity cross-linking studies with BASM membranes, suggesting that a major portion of the 135-kDa ANP receptors are HS-142-1 insensitive and only a small portion of the 135-kDa ANP receptors are responsible for the blockade by HS-142-1 of GMP production in BASM cells. At a concentration of 100 micrograms/ml, HS-142-1 reversibly prevented ANP-induced relaxation of the isolated rabbit thoracic aorta induced to contract with 3 x 10(-7) M phenylephrine, but not the relaxation induced by sodium nitroprusside, isoproterenol, or papaverine. These results suggest that HS-142-1 specifically inhibits natriuretic peptide-induced vasorelaxation through the blockade of guanylyl cyclase-linked natriuretic peptide receptors. HS-142-1 thus will be a powerful tool for understanding the physiological roles, in vasculature, of natriuretic peptides, which contribute to the homeostasis of blood pressure and intravascular volume.     

Acute and chronic effects of T-1032, a novel selective phosphodiesterase type 5 inhibitor,on monocrotaline-induced pulmonary hypertension in rats

We examined the hemodynamic property of T-1032 (methyl 2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridylmethoxy)-4-(3,4,5-trimethoxy-phenyl)-3-isoquinoline carboxylate sulfate), a novel selective phosphodiesterase type 5 (PDE5) inhibitor, and evaluated the chronic effect of T-1032 on cardiac remodeling and its related death in monocrotaline (MCT)-induced pulmonary hypertensive rats. T-1032 (1, 10, 100 micro g/kg, i.v.) significantly reduced mean arterial pressure (MAP) and right ventricular systolic pressure (RVSP) without a change in heart rate. The change in RVSP was more potent than that in MAP with 1 micro g/kg T-1032 treatment (RVSP: -8.2+/-1.2%, mean arterial pressure: -5.7+/-1.2%), and reductions in RVSP and MAP reached a peak at doses of 1 and 10 micro g/kg, respectively. In contrast, nitroglycerin (0.1, 1, 10 micro g/kg, i.v.) and beraprost (0.1, 1 micro g/kg, i.v.) did not cause a selective reduction in RVSP at any dose. When T-1032 (300 ppm in diet) was chronically administered, it delayed the death, and significantly suppressed right ventricular remodeling (T-1032-treated: 0.318+/-0.021 g, control: 0.401+/-0.013 g, p<0.05). Our present results suggest that T-1032 selectively reduces RVSP, and resulting in the suppression of right ventricular remodeling with a delay of the death in MCT-induced pulmonary hypertensive rats.     

HS-142-1, a novel nonpeptide atrial natriuretic peptide (ANP) antagonist, blocks ANP-induced renal responses through a specific interaction with guanylyl cyclase-linked receptors.

HS-142-1, a novel microbial product, blocked 125I-labeled rat atrial natriuretic peptide (rANP) (= ANF(99-126)) binding to bovine adrenocortical membranes, where guanylyl cyclase-containing receptors are predominantly expressed. However, HS-142-1 only slightly inhibited [125I]rANP binding to bovine lung membranes where only a small portion of binding sites are coupled to guanylyl cyclase. Further, HS-142-1 only recognized the 135 kDa ANP receptor, which is considered to be the guanylyl cyclase-containing receptor based on the results obtained in affinity cross-linking studies with bovine adrenocortical and lung membranes. Under identical conditions, Atriopeptin I selectively recognized guanylyl cyclase-free receptors both in binding and affinity cross-linking experiments. When injected intravenously (1 mg/kg) to anesthetized rats, HS-142-1 abolished ANP-induced diuresis and natriuresis. These results suggest that HS-142-1 works in vivo through a specific interaction with the ANP functional receptor, and that HS-142-1 will be a powerful tool for understanding the physiological roles of ANP in distinction from its pharmacological effects.     

Effects of the neutral endopeptidase inhibitor thiorphan on cardiovascular and renal function in cirrhotic rats

1. Cirrhosis is associated with cardiovascular and renal dysfunction including sodium retention. Many vasoactive peptides such as atrial natriuretic peptide (ANP) and endothelin-1 (ET-1) are degraded by neutral endopeptidase 24.11 (NEP). We investigated the hemodynamic and renal effects of thiorphan, a NEP inhibitor, in a rat cirrhosis model. 2. Cirrhosis was induced by chronic bile duct ligation, and controls had sham operation. Systemic and renal hemodynamics in conscious, restrained animals were determined using radiolabeled microspheres, and glomerular filtration rate (GFR) was measured by (3)H-inulin clearance. Plasma ANP and ET-1, and renal cGMP and Na(+) - K(+) ATPase activity were assayed. These variables were measured at baseline and after intravenous infusion of thiorphan (0.5 mg kg(-1) loading dose followed by 0.1 mg kg(-1) min(-1) x 30 min). 3. Thiorphan significantly decreased cardiac output, and increased systemic vascular resistance in controls, whereas in cirrhotic rats these variables were unchanged. 4. Compared to the controls, cirrhotic rats showed a decreased baseline GFR and urine sodium excretion, and the latter was significantly increased by thiorphan. 5. Thiorphan increased plasma ET-1 levels in controls, but not cirrhotic rats. ANP levels were not significantly increased in either group by thiorphan. 6. Thiorphan significantly increased cGMP concentrations and decreased Na(+) - K(+) ATPase activity of renal medulla but not cortex in cirrhotic rats; no effect was observed in the control rats. 7. We conclude that thiorphan induces natriuresis in cirrhotic rats by a direct renal medullary mechanism via cGMP and Na(+) - K(+) ATPase, without affecting systemic hemodynamics. This may potentially be useful in patients with ascites.     

Bioactivity of natriuretic peptide coinfusions; no evidence for unique effects of BNP in conscious sheep

Few studies have addressed the possibility that brain natriuretic peptide (BNP) possesses a profile of bioactivity that is distinct from that of atrial natriuretic peptide (ANP). Accordingly, we assessed the biologic actions of BNP in the setting of maximal or near-maximal ANP-induced biologic activity. Background ANP infusions (7.5 pmol/kg/min) administered on all study days, increased plasma ANP (approximately 120 pM) and cyclic guanosine monophosphate (GMP) levels (approximately 40 nM), and induced significant decreases in arterial pressure and cardiac output associated with increased heart rate, hematocrit, diuresis, and natriuresis. Increasing the dose twofold after 1 h (experiment 1, n = 5) showed no enhancement of these actions despite a further twofold increase in plasma ANP and cyclic GMP (both p values <0.001). Addition of low-dose BNP (2 pmol/kg/min) after 1 h background infusion (experiment 2, n = 8), increased plasma BNP levels (30 pM, p < 0.001) but caused no significant effects on the hemodynamic, renal, or hormonal indices measured. In conclusion, in the setting of maximal hemodynamic, renal, and endocrine responses to high-dose background infusions of ANP, coinfusion of BNP exhibits no enhancement of, or additional, biologic activity. This study provides no evidence for unique short-term biologic actions of ANP and BNP.     

HS-142-1, a novel nonpeptide atrial natriuretic peptide (ANP) antagonist,blocks ANP-induced renal responses through a specific interaction with guanylyl cyclase-linked receptors

HS-142-1, a novel microbial product, blocked ¹²⁵I-labeled rat atrial natriuretic peptide (rANP) (= AN(99–126)) binding to bovine adrenocortical membranes, where guanylyl cyclase-containing receptors are predominantly expressed. However, HS-142-1 only slightly inhibited [¹²⁵I]rANP binding to bovine lung membranes where only a small portion of binding sites are coupled to guanylyl cyclase. Further, HS-142-1 only recognized the 135 kDa ANP receptor, which is considered to be the guanylyl cyclase-containing receptor based on the results obtained in affinity cross-linking studies with bovine adrenocortical and lung membranes. Under identical conditions. Atriopeptin I selectively recognized guanylyl cyclase-free receptors both in binding and affinity cross-linking experiments. When injected intravenously (1 mg/kg) to anesthetized rats, HS-142-1 abolished ANP-induced diuresis and natriuresis. These results suggest that HS-142-1 works in vivo through a specific interaction with the ANP functional receptor, and that HS-142-1 will be a powerful tool for understanding the physiological roles of ANP in distinction from its pharmacological effects.     

Phosphodiesterase-5A and neutral endopeptidase activities in human adipocytes do not control atrial natriuretic peptide-mediated lipolysis

BACKGROUND AND PURPOSE: Atrial natriuretic peptide (ANP) stimulates lipolysis in human adipocyte through a cGMP signalling pathway, the regulation of which is poorly known. Since phosphodiesterases (PDE) and neutral endopeptidase (NEP) play a major role in the regulation of the biological effects of natriuretic peptides in the cardiovascular and renal systems, we investigated whether these mechanisms could regulate cGMP signalling and ANP-mediated lipolysis in human adipocytes. EXPERIMENTAL APPROACH: The presence of cGMP-specific PDE and NEP in differentiated pre-adipocytes and in mature adipocytes was evaluated by real-time qPCR and Western blot. The effect of non-selective and selective inhibition of these enzymes on ANP-mediated cGMP signalling and lipolysis was determined in isolated mature adipocytes. KEY RESULTS: PDE-5A was expressed in both pre-adipocytes and adipocytes. PDE-5A mRNA and protein levels decreased as pre-adipocytes differentiated (10 days). PDE-5A is rapidly activated in response to ANP stimulation and lowers intracellular cGMP levels. Its selective inhibition by sildenafil partly prevented the decline in cGMP levels. However, no changes in baseline- and ANP-mediated lipolysis were observed under PDE-5 blockade using various inhibitors. In addition, NEP mRNA and protein levels gradually increased during the time-course of pre-adipocyte differentiation. Thiorphan, a selective NEP inhibitor, completely abolished NEP activity in human adipocyte membranes but did not modify ANP-mediated lipolysis. CONCLUSIONS AND IMPLICATIONS: Functional PDE-5A and NEP activities were present in human adipocytes, however these enzymes did not play a major role in the regulation of ANP-mediated lipolysis.     

Prolonged neutral endopeptidase inhibition in heart failure.

We studied the hormonal, renal and hemodynamic effects of prolonged treatment with SCH 39370, a new neutral endopeptidase (NEP) inhibitor, in experimental congestive heart failure (CHF). Coronary-ligated CHF rats and sham-operated controls received vehicle or SCH 39370 30 mg/kg s.c. twice daily for six days. In rats with heart failure, SCH 39370 elevated the high plasma atrial natriuretic peptide (ANP) and cyclic guanosine monophosphate (cGMP) levels 2-fold both initially and at the end of the experiment. Initially, water balance was more negative in SCH 39370-treated CHF rats than in those treated with vehicle. In all SCH 39370-treated rats, ANP, cGMP and electrolyte excretion and diuresis were pronounced for 6 h after injection but attenuated thereafter. Blood pressure and pulse remained unchanged. On reverse phase high performance liquid chromatography (HPLC), ANP-(99-126) appeared to be the only circulating form of ANP in rats with heart failure. Three forms have been discovered in patients with heart failure. HPLC revealed only intact ANP in plasma of rats with heart failure during SCH 39370 treatment. NEP inhibitors may provide a new tool for treating chronic heart failure.     

Sildenafil and T-1032, phosphodiesterase type 5 inhibitors, showed a different vasorelaxant property in the isolated rat aorta

The vasorelaxant effects of sildenafil and T-1032 [methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate], two phosphodiesterase type 5 inhibitors, were examined in the isolated rat aorta. Sildenafil and T-1032, both of which have almost the same potency and selectivity regarding phosphodiesterase type 5 inhibitory activity, produced a similar, moderate, relaxation at 10(-10) to 10(-7) M (sildenafil: 66.8 +/- 13.7%; T-1032: 77.9 +/- 10.8% at 10(-7) M). However, sildenafil, but not T-1032, produced further relaxation at the higher concentrations (sildenafil: 102.0 +/- 0.6%; T-1032: 81.0 +/- 7.2% at 10(-4) M, P < 0.05). Sildenafil also produced a more potent relaxation than did T-1032 at the high concentrations (10(-5) and 10(-4) M) in endothelium-denuded aortic rings and in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (3 x 10(-4) M). Moreover, the high concentrations of sildenafil, but not of T-1032, caused a rightward shift of the concentration-response curve for calcium chloride in K(+)-depolarized endothelium-denuded preparations. In the ligand binding assay for the L-type Ca(2+) channels, the affinities of sildenafil at 10(-5) M for binding sites of nitrendipine and (--)-desmethoxyverapamil [(--)- D888] (35.2 +/- 3.3% and 35.8 +/- 1.9%, respectively) were higher than those of T-1032 (11.8 +/- 4.0% and -13.1 +/- 1.3%, respectively, P < 0.05). Regarding cyclic nucleotide levels, both phosphodiesterase type 5 inhibitors increased cGMP levels at 10(-6) M. However, sildenafil, but not T-1032, further increased cGMP levels at the higher concentrations (sildenafil: 15.7 +/- 2.7 pmol/mg protein; T-1032: 5.6 +/- 0.6 pmol/mg protein at 10(-4) M, P < 0.05). These results suggested that high concentrations of sildenafil had additional vasorelaxant properties through mechanisms other than phosphodiesterase type 5 inhibition. Sildenafil-induced relaxation appears to be due to inhibition of the external Ca(2+)-dependent cascade for contraction and/or to an increase in cGMP levels. In contrast, T-1032 only showed a vasorelaxant property due to phosphodiesterase type 5 inhibition. In conclusion, T-1032 appears to be a specific phosphodiesterase type 5 inhibitor compared with sildenafil and a useful compound to examine the physiological function of phosphodiesterase type 5.     

Acute hemodynamic, hormonal, and renal effects of neutral endopeptidase inhibition in ovine heart failure.

The effects of neutral endopeptidase inhibition (NEP-I) were studied in 6 conscious sheep with heart failure (HF) induced by rapid ventricular pacing for 7 days. Measurements were performed 1 h before and for 6 h after intravenous (i.v.) bolus administration of vehicle and SCH 39370 (1.25 and 5 mg/kg) on separate days. After the higher dose, an index of serum NEP activity decreased from 0.83 +/- 0.05 to 0.13 +/- 0.07 nmol/ml/min (p less than 0.001) at 1 h and then returned to control levels at 6 h. Plasma atrial natriuretic peptide (ANP) and cyclic GMP rose from 328 +/- 28 and 20.2 +/- 4.3 to a peak of 570 +/- 65 pmol/L (p less than 0.001) and 28.7 +/- 6.3 nmol/L (p less than 0.05) respectively. Natriuresis and diuresis were significant and left atrial pressure (LAP) decreased from 21.9 +/- 1.1 to 20.1 +/- 0.8 mm Hg (p less than 0.05). Despite high endogenous ANP levels in HF, NEP-I further increases both ANP and its "second messenger." Its natriuretic and hemodynamic effects are consistent with enhanced ANP activity in renal and vascular tissues, suggesting that NEP-I may be useful for treating HF.     

Pulmonary hypertension: inhaled nitric oxide, sildenafil and natriuretic peptides

Phosphodiesterase-5 (PDE5) inhibitors and other agents that modulate intracellular cGMP are now emerging as promising, safe, and easy to administer therapies for pulmonary hypertension, with relatively few side effects. Recent studies have shown that PDE5 inhibitors are potent acute pulmonary vasodilators in experimental models that partially reverse established pulmonary arterial hypertension and blunt chronic pulmonary hypertension. In addition, studies on animals reveal that PDE5 inhibitors work in concert with nitric oxide and/or natriuretic peptide levels by enhancing intracellular cGMP and cGMP-mediated vasodilator effects. Further, the combination of PDE5 inhibitors and agents that increase cGMP or cAMP also yields additive beneficial effects on pulmonary hemodynamics in patients with pulmonary arterial hypertension.     

Relaxation and potentiation of cGMP-mediated response by ibudilast in bovine tracheal smooth muscle

The effects of ibudilast, an inhibitor of phosphodiesterases (PDEs), on tension, levels of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) were investigated in bovine tracheal smooth muscle. We especially examined the combined effect of ibudilast with the cGMP-elevating agents on these parameters. Ibudilast was equipotent to attenuate the precontractions induced by both 0.3 microM methacholine and 40 mM K(+). By contrast, the relaxant effects of sodium nitroprusside and salbutamol on 40 mM K(+)-contracted preparations were smaller than those on 0.3 microM methacholine-contracted ones. Neither N(omega)-nitro-L-arginine (100 microM), an inhibitor of nitric oxide synthase, nor ODQ (1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 5 microM), an inhibitor of soluble guanylyl cyclase, affected the ibudilast-induced relaxation. The relaxations induced by ibudilast and diltiazem on 40 mM K(+)-contracted preparations were significantly attenuated when extracellular CaCl(2) was increased from 2.54 mM to 10 mM. Ibudilast (10 microM), which caused only minor effect by itself, significantly ( P<0.05) shifted the concentration-response curves for the relaxant responses to sodium nitroprusside (SNP), atrial natriuretic peptide (ANP) and salbutamol to the left. On the other hand, ibudilast did not change the relaxant responses to diltiazem. Unlike ibudilast, diltiazem (3 microM) failed to affect the SNP- and salbutamol-induced relaxations. Ibudilast significantly ( P<0.05) increased basal levels of cGMP and cAMP. Furthermore, ibudilast enhanced SNP (0.3 microM)- and ANP (0.3 microM)-induced cGMP accumulation and salbutamol (10 nM)-induced cAMP accumulation. Zaprinast (10 microM), a type 5 PDE inhibitor, enhanced both relaxation and cGMP accumulation induced by SNP and ANP without changing salbutamol-induced responses. These findings suggest that blockade of voltage-gated Ca(2+) channels is involved in the relaxing action of ibudilast in bovine tracheal smooth muscle. However, ibudilast potentiates relaxation responses to ANP and SNP by inhibition of PDE 5, not by blockade of Ca(2+) channels. The enhancement of cGMP-mediated response may contribute to the therapeutic effects of ibudilast.     

T-1032, a cyclic GMP phosphodiesterase-5 inhibitor, acutely blocks physiologic insulin-mediated muscle haemodynamic effects and glucose uptake in vivo

1. Cyclic GMP phosphodiesterase-5 inhibitors have been shown to alter blood flow in specific tissues by potentiating local NO-dependent vasodilatory mechanisms. Since the haemodynamic effects of physiologic insulin, particularly capillary recruitment, may be critical for muscle glucose uptake in vivo and are blocked by inhibitors of nitric oxide synthase, we have explored the acute effects of the specific cGMP phosphodiesterase-5 inhibitor T-1032 on physiologic insulin action in anaesthetized healthy rats in vivo. 2. Whole-body glucose infusion (GIR), femoral blood flow (FBF), hind leg vascular resistance (VR), hind leg glucose uptake (HGU), 2-deoxyglucose uptake into muscles of the lower leg (R'g), hind leg metabolism of infused 1-methylxanthine (1-MX), a measure of capillary recruitment, and muscle cGMP were determined. The experimental groups were T-1032 (10 microg min-1 kg-1) infused for 1 h before and during a euglycaemic insulin clamp (3 mU min-1 kg-1 x 2 h), T-1032 infused for 3 h with saline, T-1032 during a 2 h clamp, T-1032 with saline for 2 h, and a 2 h saline control. 3. Insulin increased GIR from zero to 13 mg min-1 kg-1, HGU from 0.1+/-0.01 to 0.43+/-0.05 micromol min-1, R'g and 1-MX, marginally increased FBF, and had no effect on blood pressure or heart rate. T-1032 alone had no effect on blood pressure, heart rate, FBF, VR, HGU, R'g or 1-MX, but increased muscle cGMP. T-1032 1 h before and during insulin completely blocked GIR (1 h), HGU (2 h), R'g (2 h), and 1-MX (2 h). T-1032 commenced with insulin had only partial blocking activity against insulin. 4. We conclude that T-1032 is a potent acutely acting inhibitor of the muscle effects of physiologic insulin on capillary recruitment and glucose uptake in vivo. These, together with inhibition of whole-body glucose infusion during insulin, may caution against the use of isoenzyme-5-specific cyclic GMP phosphodiesterase inhibitors as therapeutic agents.     

cGMP: transition from bench to bedside: a report of the 6th International Conference on cGMP Generators,Effectors and Therapeutic Implications

Essential physiological homeostatic processes such as vascular tone, fluid balance, cardiorenal function, and sensory processes are regulated by the second messenger cyclic guanosine 3′, 5′-monophosphate (cGMP). Dysregulation of cGMP-dependent pathways plays an important role in cardiovascular diseases such as hypertension, pulmonary hypertension, heart failure, or erectile dysfunction. Thus, the cGMP pathway consisting of the cGMP-generating guanylyl cyclases, protein kinases, and phosphodiesterases (PDE) has evolved to an important drug target and is the focus of a wide variety of research fields ranging from unraveling mechanisms on the molecular level to understanding the regulation of physiological and pathophysiological processes by cGMP. Based on the results from basic and preclinical research, therapeutic drugs have been developed which modulate the cGMP pathway and are investigated in clinical trials. Riociguat, a nitric oxide (NO)-independent soluble guanylyl cyclase stimulator; recombinant B-type natriuretic peptide (BNP); or recombinant atrial natriuretic peptide (ANP) and PDE5 inhibitors are cGMP-modulating drugs that are already available for the treatment of pulmonary hypertension, acute heart failure, and erectile dysfunction, respectively. The latest results from basic to clinical research on cGMP were presented on the 6th International Conference on cGMP in Erfurt, Germany, and are summarized in this article.     

Effects of intrapericardially administered endothelin-1 on pericardial natriuretic peptide concentrations of the in situ dog heart

It has recently been proposed that endothelin-1 (ET-1) is an important activator of natriuretic peptide [atrial natriuretic peptide (ANP) and the brain natriuretic peptide (BNP)] release in the heart and may mediate ANP and BNP deliberation to myocardial stretch and ischemia. The close inter-relationship between ET-1 and natriuretic peptide release was indicated mainly by the results of in vitro studies. In vivo, the local alterations of ANP and BNP levels in the myocardial interstitium can be well characterized by the changes of their pericardial fluid concentrations. The effect of the intrapericardially administered ET-1 on natriuretic peptide concentrations was studied on the in situ dog heart (n = 8). Control and three consecutive infusate samples were removed from the pericardial sac following ET-1 administration (150 pmol/kg) and parallel peripheral blood samples were taken to determine the ANP and BNP concentrations (enzyme-linked immunosorbent assay). Standard hemodynamic parameters were recorded continuously. In our results the intrapericardial ET-1 increased pericardial ANP but not BNP concentrations significantly (control versus ANPmax, 37 +/- 5 ng/mL versus 94 +/- 12 ng/mL; P < 0.02). ET-1 elicited significant ST elevations without changes of the hemodynamic values and the natriuretic peptide levels in the arterial plasma samples. In conclusion, intrapericardial ET-1 effectively stimulated the myocardial ANP release, which was reflected as elevation in the pericardial ANP level. The results also support the hypothesis that important regulatory mechanisms might be activated from the pericardium.     

Vascular natriuretic peptide receptor-linked particulate guanylate cyclases are modulated by nitric oxide-cyclic GMP signalling

(1) The sensitivity of the particulate guanylate cyclase-cyclic guanosine-3',5'-monophosphate (cGMP) system to atrial (ANP) and C-type (CNP) natriuretic peptides was investigated in aortae and mesenteric small arteries from wild-type (WT) and endothelial nitric oxide synthase (eNOS) knockout (KO) mice. (2) ANP and CNP produced concentration-dependent relaxations of mouse aorta that were significantly attenuated by the natriuretic peptide receptor (NPR)-A/B antagonist HS-142-1 (10(-5) M). Both ANP and CNP were more potent in aortae from eNOS KO mice compared to WT. (3) The potency of ANP and CNP in aortae from WT animals was increased in the presence of the NOS inhibitor, N(G)-nitro-L-arginine (3 x 10(-4) M) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolol[4,3,a]quinoxalin-1-one (5 x 10(-6) M). (4) In contrast, the potency of ANP and CNP in aortae from eNOS KO animals was reduced following pretreatment of tissues with supramaximal concentrations of the NO-donor, glyceryl trinitrate (3 x 10(-5) M, 30 min) or ANP (10(-7) M, 30 min). (5) Responses to acetylcholine in aortae from WT mice (dependent on the release of endothelium-derived NO) were significantly reduced following pretreatment of tissues with GTN (3 x 10(-5) M, 30 min) and ANP (10(-7) M, 30 min). (6) CNP and the NO-donor, spermine-NONOate caused concentration-dependent relaxations of mesenteric small arteries from WT animals that were significantly increased in eNOS KO mice compared to WT. ANP was unable to significantly relax mesenteric arteries from WT or eNOS KO animals. (7) In conclusion, both NPR-A- and NPR-B-linked pGC pathways are modulated by NO-cGMP in murine aorta and mesenteric small arteries and crossdesensitisation occurs between NPR subtypes. The biological activity of endothelium-derived NO is also influenced by the ambient concentration of NO and natriuretic peptides. Such an autoregulatory pathway may represent an important physiological homeostatic mechanism and link the paracrine activity of NO and CNP with the endocrine functions of ANP and BNP in the regulation of vascular tone and blood pressure.     

RELATIONSHIP BETWEEN THE SUPPRESSIVE ACTIONS ON INTESTINAL ABSORPTION AND ON cGMP PRODUCTION FOR THE NATRIURETIC PEPTIDE FAMILY IN DOGS

1. The aim of this study was to investigate whether the suppressive effects of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) on net intestinal absorption were related to guanosine 3′,5′-cyclic monophosphate (cGMP) production in the intestine. 2. We measured the plasma cGMP concentration of the arterial, jejunal and ileal venous blood after intravenous infusions of natriuretic peptides (97 pmol/ kg per min for 30 min) in anaesthetized dogs. 3. The infusion of ANP increased cGMP concentration of the aortic blood by 49.9 ± 9.0 (pmol/mL), BNP by 71.8 ± 12.3 and CNP by 5.5 ± 1.3. The increases in cGMP after ANP and BNP were larger than after CNP. The infusion of ANP increased jejunal arteriovenous differences in cGMP concentration by 69.9 ± 3.5 (pmol/mL) and ileal arteriovenous differences by 8.7 ± 3.2. In BNP infusion, the jejunal and ileal arteriovenous differences in cGMP concentration tended to increase by 15.6 ± 5.8 (pmol/mL) and by 14.8 ± 6.6 but neither were significant. CNP infusion did not change the jejunal and ileal arteriovenous differences in cGMP concentration. 4. These results suggest that, while the actions of ANP on intestinal absorption may be mediated by cGMP, those of BNP and CNP are not.     

T-1032, a novel phosphodiesterase type 5 inhibitor,increases the survival of cardiomyopathic hamsters

To evaluate the influence of T-1032 (methyl2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate), a potent and relatively selective phosphodiesterase 5 inhibitor, on chronic heart failure, we examined the acute hemodynamic profile of T-1032 and its chronic effect on the survival of Bio 14.6 cardiomyopathic hamsters. In the acute study, T-1032 (1, 10, 100 microg/kg) was administered intravenously by means of a dose-escalating procedure in 55-week-old hamsters. T-1032 significantly reduced both the right and left ventricular end-diastolic pressure in a dose-dependent manner. T-1032 modestly reduced the systemic arterial pressure at the highest dose (100 microg/kg i.v.). T-1032 did not change the heart rate or left ventricular dp/dt(max). In the survival study, chronic administration of T-1032 (50 and 500 ppm in a diet) increased survival, and the survival rate was 24.2%, 45.4% and 48.5% in the control, 50 and 500 ppm-treated groups, respectively. The median survival was 55, 58 and 58 weeks in control, 50 and 500 ppm-treated groups, respectively. Analysis of the survival curves revealed that T-1032 (500 ppm) significantly increased the survival of these hamsters (P<0.05 vs. control). It was concluded that T-1032 had beneficial hemodynamic effects on heart failure in Bio 14.6 cardiomyopathic hamsters, and the favorable hemodynamic changes induced by T-1032 were partly related to the increase in the survival of these hamsters. Phosphodiesterase type 5 inhibitors may have therapeutic potential for the treatment of chronic heart failure.     

Renal effects of prolonged intrarenal infusions of angiotensin II and atrial natriuretic peptide in sheep.

Angiotensin II (AngII) and atrial natriuretic peptide (ANP) are two hormones that have antagonistic effects on volume and pressure regulation. Plasma levels of both hormones are elevated in sheep pregnancy. However, during pregnancy, volume expansion occurs despite elevated plasma ANP, implying an overriding role of AngII. In addition to counteracting the effects of ANP on the physiological level, AngII also may act on the receptor level. Therefore this study was designed to investigate the hemodynamic and renal effects of ANP and AngII separately and to define their selective effects on the renal natriuretic peptide receptor types in the various segments of the nephron. Eight unilaterally nephrectomized nonpregnant sheep received separately for 10 days, low doses of AngII (1 ng/kg/min) and ANP (0.5 ng/kg/min) directly infused into the renal arteries to avoid systemic effects. Intrarenal AngII infusion decreased sodium excretion (UNaV) from 111+/-11 to 36 +/-8 and 45+/-6 mmol/day (p<0.05) on days 3 and 8-10, respectively. Mean arterial pressure (MAP) increased from 94 +/-6 mm Hg to a maximum of 107+/-8 mm Hg on day 5 of infusion and stabilized at 101+/-7 mm Hg on days 8-10 (p<0.05). Intrarenal ANP infusion significantly increased UNaV on day 1 from 93+/-9 to 188+/-20 mmol/day (p<0.05), followed by sodium retention on days 4-6 (average, 60+/-13 mmol/day; p<0.05). UNaV again increased above control levels on days 8-10 to an average level of 111+/-15 mmol/day. MAP decreased from 99+/-4 to 90+/-5 mm Hg (p<0.05) on days 1-3, and remained lower than control throughout the infusion period. The kidneys were collected at control nephrectomy and at the end of infusion. The natriuretic peptide receptors were characterized by competitive-binding radioreceptor assays on glomerular, outer medullary, and inner medullary membranes. AngII infusion increased the dissociation constant (Kd) of inner medullary natriuretic peptide receptors from 186 +/-11 to 267+/-22 pM (p<0.05), and ANP infusion decreased maximal binding capacity (Bmax) of inner medullary receptors from 134+/-10 to 89+/-15 fmol/mg protein (p<0.05). Glomerular and outer medullary natriuretic peptide receptors were not affected by either AngII or ANP infusion. In conclusion, AngII stimulates antinatriuresis and counteracts the hemodynamic and renal effects of ANP in part by downregulating the renal inner medullary natriuretic peptide receptors.