Plasma levels of the proprotein convertase furin and incidence of diabetes and mortality.

BACKGROUND: Diabetes mellitus is linked to premature mortality of virtually all causes. Furin is a proprotein convertase broadly involved in the maintenance of cellular homeostasis; however, little is known about its role in the development of diabetes mellitus and risk of premature mortality. OBJECTIVES: To test if fasting plasma concentration of furin is associated with the development of diabetes mellitus and mortality. METHODS: Overnight fasted plasma furin levels were measured at baseline examination in 4678 individuals from the population-based prospective Malmö Diet and Cancer Study. We studied the relation of plasma furin levels with metabolic and hemodynamic traits. We used multivariable Cox proportional hazards models to investigate the association between baseline plasma furin levels and incidence of diabetes mellitus and mortality during 21.3-21.7 years follow-up. RESULTS: An association was observed between quartiles of furin concentration at baseline and body mass index, blood pressure and plasma concentration of glucose, insulin, LDL and HDL cholesterol (|0.11| ≤ ß ≤ |0.31|, P < 0.001). Plasma furin (hazard ratio [HR] per one standard deviation increment of furin) was predictive of future diabetes mellitus (727 events; HR = 1.24, CI = 1.14-1.36, P < 0.001) after adjustment for age, sex, body mass index, systolic and diastolic blood pressure, use of antihypertensive treatment, alcohol intake and fasting plasma level of glucose, insulin and lipoproteins cholesterol. Furin was also independently related to the risk of all-cause mortality (1229 events; HR = 1.12, CI = 1.05-1.19, P = 0.001) after full multivariable adjustment. CONCLUSION: Individuals with high plasma furin concentration have a pronounced dysmetabolic phenotype and elevated risk of diabetes mellitus and premature mortality.

Prognostic value of N-terminal natriuretic peptides in systemic sclerosis: a single centre study.

OBJECTIVES: Cardiac involvement is an important determinant of prognosis in systemic sclerosis (SSc). The identification of patients with high risk is of great importance. Our aim was to investigate the diagnostic and prognostic value of circulating concentrations of N-terminal fragments of A- and B-type natriuretic peptides (NT-proANP and NT-proBNP) in patients with SSc. METHODS: We prospectively studied 144 patients with SSc and followed them up for five years. Blood was collected for natriuretic peptide measurement at the time of the yearly scheduled cardiological check-up. The occurrence of clinically significant cardiac disease was measured as the composite of pulmonary arterial hypertension, cardiac revascularisation, development of left ventricular dysfunction or death. RESULTS: Patients diagnosed with heart involvement during the study had significantly higher levels of NT-proANP and NT-proBNP (791.4 ± 379.9 pmol/l vs. 608.0 ± 375.8 pmol/l, p<0.05 and 183.1 ± 162.6 vs. 125.7 ± 117.5 pmol/l, p<0.05, respectively). Receiver-operator-characteristic analysis identified <822.5 pmol/l as the best NT-proANP and <154.5 pmol/l as the best NT-proBNP threshold (sensitivity 56.3%, specificity 79.5%, negative predictive value: 86.4% and sensitivity 50.0%, specificity 76.8%, negative predictive value: 83.7%, respectively). During the follow-up, lower NT-proANP levels were significantly associated with a longer event-free survival (p<0.05), similar but a non-significant trend regarding NT-proBNP levels was also shown (p=0.052). CONCLUSIONS: In our cohort, NT-proANP had a supplementary prognostic value for cardiac involvement in systemic sclerosis. In addition, the high negative predictive value of natriuretic peptides supports the more extensive use in identifying SSc patients with high risk of future cardiac involvement.

Transcriptional cofactor dyxin mediates hypertrophic response in the heart during angiotensin II-induced hypertension.

Dyxin is a LIM-domain containing transcriptional regulator protein shown to play a role in a hypertrophic response in the heart. Here, the effect of adenoviral dyxin overexpression was studied on cardiac function and gene expression in the normal heart and in angiotensin II (Ang II)-induced hypertension in rats. The adenovirus-mediated intramyocardial gene transfer of dyxin (1.5x109 infectious units/animal) was performed into the left ventricle (LV) of Sprague-Dawley rats with and without the Ang II (33 µg/kg/h) infusion, administered via osmotic minipumps for 1 and 2 weeks. Echocardiography was used to assess the structural and functional changes. Dyxin expression and localization in the heart was analyzed with quantitative RT-PCR and immunohistochemistry, respectively. In the normal rat heart, the adenoviral overexpression of dyxin did not alter LV function in normal hearts as assessed by echocardiography. Dyxin was found to be localized in the cardiomyocytes as shown by the immunohistochemical staining. In Ang II-induced hypertrophy, echocardiographic data revealed a significant increase in the posterior wall diameter both in systole (21%, P<0.05) and diastole (21%, P<0.01) as well as in the diameter of the interventricular septum in systole (19%, P<0.05) in the dyxin-injected group compared with the LacZ-injected animals after two weeks of Ang II infusion. Interestingly, a significant decrease in the levels of both atrial natriuretic peptide (ANP) mRNA (55%, P<0.01) and B-type natriuretic peptide (BNP) mRNA (68%, P<0.05) was observed in the dyxin-injected group compared with the LacZ control group after one week of Ang II infusion. These results indicate that dyxin overexpression was deteriorative against pressure overload by inducing structural changes in the LV in rats. Interestingly, simultaneous adenoviral overexpression of dyxin suppressed the Ang II-induced changes of ANP and BNP genes suggesting that dyxin might have a role as a regulator of the cardiac hypertrophic gene program.

Strong relationship between NT-proXNP levels and cardiac output following cardiac surgery in neonates and infants.

BACKGROUND: NT-proXNP, a new natriuretic peptide analyte, incorporates information about the concentrations of both N-terminal pro-atrial and pro-brain natriuretic peptides (NT-proANP, NT-proBNP). We aimed to investigate whether NT-proXNP is a reliable indicator of the cardiac index (CI) and the hemodynamic state in neonates and infants undergoing an open heart surgery. METHODS: We enrolled 26 children under the age of 1 year into this prospective study. All patients underwent an elective cardiac operation with cardiopulmonary bypass (CPB) to achieve complete biventricular repair. Peri-operative hemodynamic parameters were assessed by transpulmonary thermodilution and natriuretic peptide levels were recorded. RESULTS: The NT-proXNP level correlated significantly with the simultaneously measured NT-proANP level (r=0.60, P<0.001), but more strongly with the NT-proBNP level (r=0.89, P<0.001) and the arithmetic sum of both (r=0.88, P<0.001). NT-proXNP had a strong correlation with CI (r=-0.85, P<0.001), the stroke volume index (r=-0.80, P<0.001) and the global ejection fraction (r=-0.67, P<0.009) throughout the post-operative period. Conventionally measured parameters such as heart rate, mean arterial pressure and pulse-pressure product exhibited weaker correlations with CI than NT-proXNP. Among laboratory values, creatinine levels correlated significantly with CI (r=-0.77, P<0.001) and NT-proXNP (r=0.76, P<0.001) during the post-operative period. A post-operative NT-proXNP level of 3079 pmol/l was diagnostic for CI <3 l/min/m(2) with 89% sensitivity and 90% specificity (area under the curve: 0.91 +/- 0.05). CONCLUSION: NT-proXNP is a good marker of cardiac output following pediatric cardiac surgery and might be a useful tool in the recognition of a low output state.

Protein remodeling of extracellular matrix in rat myocardium during four-day hypoxia: the effect of concurrent hypercapnia.

The combination of long-term hypercapnia and hypoxia decreases pulmonary vascular remodeling and attenuation of right ventricular (RV) hypertrophy. However, there is limited information in the literature regarding the first stages of acclimatization to hypercapnia/hypoxia. The purpose of this study was to investigate the effect of four-day hypoxia (10% O2) and hypoxia/hypercapnia (10% O2 + 4.4% CO2) on the protein composition of rat myocardium. Expression of the cardiac collagen types and activities of matrix metalloproteinases (MMPs) and of their tissue inhibitor TIMP-1 were followed. The four-day hypoxia changed protein composition of the right ventricle only in the hypercapnic condition; remodeling was observed in the extracellular matrix (ECM) compartments. While the concentrations of pepsin-soluble collagenous proteins in the RV were elevated, the concentrations of pepsin-insoluble proteins were decreased. Furthermore, the four-day hypoxia/hypercapnia increased the synthesis of cardiac collagen due to newly synthesized forms; the amount of cross-linked particles was not affected. This type of hypoxia increased cardiac collagen type III mRNA, while cardiac collagen type I mRNA was decreased. MMP-2 activity was detected on the zymographic gel through appearance of two bands; no differences were observed in either group. mRNA levels for MMP-2 in the RV were significantly lower in both the hypoxic and hypoxic/hypercapnic animals. mRNA levels for TIMP-1 were reduced in the RV of both the hypoxic and hypoxic/hypercapnic animals. Hypoxia with hypercapnia increased the level of mRNA (6.5 times) for the atrial natriuretic peptide (ANP) predominantly in the RV. The role of this peptide in remodeling of cardiac ECM is discussed.

Pressure overload increases GATA4 binding activity via endothelin-1.

BACKGROUND: The signaling cascades responsible for the activation of transcription factors in the hypertrophic growth of cardiac myocytes during hemodynamic overload are largely unknown. Several of the genes upregulated in the hypertrophied heart, including B-type natriuretic peptide (BNP) gene, are controlled by the cardiac-restricted zinc finger transcription factor GATA4. METHODS AND RESULTS: An in vivo model of intravenous administration of arginine(8)-vasopressin (AVP) for up to 4 hours in conscious normotensive rats was used to study the signaling mechanisms for GATA activation in response to pressure overload. Gel mobility shift assays were used to analyze the trans-acting factors that interact with the GATA motifs of the BNP promoter. AVP-induced increase in mean arterial pressure was followed by a significant increase in the BNP and c-fos mRNA levels in both the endocardial and epicardial layers of the left ventricle, whereas GATA4 and GATA6 mRNA levels remained unchanged. Pressure overload within 15 to 60 minutes produced an increase in left ventricular BNP GATA4 but not GATA5 and GATA6 binding activity, and at 30 minutes a 2.2-fold increase (P:<0.001) in GATA4 binding was noted. The mixed endothelin-1 ET(A)/ET(B) receptor antagonist bosentan but not the angiotensin II type 1 receptor antagonist losartan completely inhibited the pressure overload-induced increase in left ventricular BNP GATA4 binding activity. Bosentan alone had no statistically significant effect on GATA4 binding activity of the left ventricle in conscious animals. CONCLUSIONS: ET-1 is a signaling molecule that rapidly upregulates GATA4 DNA binding activity in response to pressure overload in vivo.

Cardiac mechanotransduction: from sensing to disease and treatment.

In heart muscle a mechanical stimulus is sensed and transformed into adaptive changes in cardiac function by a process called mechanotransduction. Adaptation of heart muscle to mechanical load consists of neurohumoral activation and growth, both of which decrease the initial load. Under prolonged overload this process becomes maladaptive, leading to the development of left ventricular hypertrophy and ultimately to heart failure. Widespread synergism and crosstalk among a variety of molecules and signals involved in hypertrophic signaling pathways make the prevention or treatment of left ventricular hypertrophy and heart failure a challenging task. Therapeutic strategies should include either a complete and continuous reduction of load or normalization of left ventricular mass by interventions aimed at specific targets involved in mechanotransduction.

Mechanisms of mechanical load-induced atrial natriuretic peptide secretion: role of endothelin, nitric oxide, and angiotensin II.

There are three members in the natriuretic peptide hormone family, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP, brain natriuretic peptide), and C-type natriuretic peptide (CNP), that are involved in the regulation of blood pressure and fluid homeostasis. CNP is found principally in the central nervous system and vascular endothelial cells while ANP and BNP are cardiac hormones. ANP is synthesized mainly in the atria of the normal adult heart, while BNP is produced by both the atria and ventricles. The mechanisms controlling ANP release have been the subject of intense research, and are now fairly well understood. The major determinant of ANP secretion is myocyte stretch. Although much less is known about the factors regulating BNP release from the heart, myocyte stretch has also been reported to stimulate BNP release from both atria and ventricles. However, whether wall stretch acts directly or via factors such as endothelin- , nitric oxide, or angiotensin II liberated in response to distension has not been established. Recent studies show that by stimulating endothelin type A receptors endothelin plays an important physiological role as a mediator of acute-volume load-induced ANP secretion from atrial myocytes in conscious animals. In fact, endogenous paracrine/autocrine factors liberated in response to atrial wall stretch rather than direct stretch appears to be responsible for activation of ANP secretion in response to volume load, as evidenced by almost complete blockade of ANP secretion during combined inhibition of endothelin type A/B and angiotensin II receptors. Furthermore, under certain experimental conditions angiotensin II and nitric oxide may also exert a significant modulatory effect on stretch-activated ANP secretion. The molecular mechanisms by which endothelin-1, angiotensin II, and nitric oxide synergistically regulate stretch-activated ANP release are yet unclear.

Effects of long-term verapamil treatment on blood pressure, cardiac hypertrophy and collagen metabolism in spontaneously hypertensive rats.

The effects of long-term treatment with verapamil on blood pressure, cardiac hypertrophy and collagen content, collagen concentration and prolyl hydroxylase activity were studied in spontaneously hypertensive rats (SHR). Verapamil administration (0.75 mg . ml-1 in drinking water) was commenced: to pregnant SHR 3 to 5 days before delivery and continued to the mothers and offspring during the nursing period; or to SHR at 10 weeks of age. Both groups were maintained on verapamil treatment up to the age of 45 weeks. Verapamil treatment significantly decreased blood pressure, heart rate and the ratio of ventricular weight to body weight in treated SHR. Verapamil did not significantly change the cardiac collagen concentration and prolyl hydroxylase activity. Since, however, the cardiac muscle mass was diminished by verapamil administration, treatment actually slightly reduced the collagen content of the heart. In the aorta collagen concentration was increased by verapamil treatment. Contrary to these results, minoxidil treatment was observed to increase the cardiac collagen concentration, content and prolyl hydroxylase activity in SHR. These results suggest that the factors governing myocardial connective tissue proliferation and regression may be independent of those governing muscle fibre hypertrophy and that particular drug actions on myocardial collagen metabolism must be taken into account.

Comparison of the effects of supplementation with whey mineral and potassium on arterial tone in experimental hypertension.

OBJECTIVE: The aim of this work was to compare the effects of supplementation of rat chow diet with potassium (K+) and whey mineral concentrate (Whey), a diet rich in milk minerals, on blood pressure and arterial responses in vitro in spontaneously hypertensive rats (SHR). METHODS: Thirty young SHR and twenty Wistar-Kyoto rats (WKY) were allocated into five groups: SHR, Whey-SHR, K(+)-SHR, WKY and Whey-WKY. Whey-supplementation was performed by adding 25% whey mineral concentrate to the chow, which in particular increased the intake of potassium (from 1.0% to 3.6%) and also that of calcium (from 1.0% to 1.3%) and magnesium (from 0.2% to 0.3%) in the rats. The K(+)-SHR were given extra potassium chloride (KCl) so that the final potassium content in the chow was 3.6%. Blood pressures were measured indirectly by the tail-cuff method. Responses of mesenteric arterial rings were examined in standard organ chambers after 12 study weeks. RESULTS: During the 12-week study systolic blood pressures in control SHR increased steadily from 160 to about 230 mmHg, while supplementation with either Whey or potassium had a clear antihypertensive effect of about 50 mmHg in the hypertensive rats. Blood pressures in the WKY and Whey-WKY groups remained comparable during the whole study. In noradrenaline-precontracted arterial rings, endothelium-dependent relaxation to acetylcholine (ACh), as well as endothelium-independent relaxations to nitroprusside and isoprenaline were attenuated in untreated SHR, while all these dilatory responses were similarly improved by Whey and potassium supplementation. The cyclooxygenase inhibitor diclofenac, which reduces the synthesis of dilatory and constricting prostanoids, clearly enhanced the relaxation to ACh in untreated SHR, but was without effect in the other groups. In the presence of the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester the relaxation to ACh was markedly reduced in all SHR groups, whereas in the two WKY groups, distinct relaxations to ACh were still present. The remaining responses were partially prevented by tetraethylammonium, an inhibitor of calcium-activated potassium channels, and the difference between untreated and potassium-supplemented SHR was abolished. When endothelium-mediated hyperpolarization of smooth muscle was prevented by precontracting the preparations with 50 mM KCl, only marginal differences were observed in relaxations to ACh between untreated SHR and the other groups. Interestingly, the impaired endothelium-independent relaxations to cromakalim, a hyperpolarizing vasodilator acting via ATP-sensitive potassium channels, were normalized by Whey mineral and potassium diets. CONCLUSION: Supplementation with Whey mineral and a comparable dose of potassium similarly opposed the development of experimental genetic hypertension, an effect which was associated with improved arterial dilatory properties. Both supplements augmented the hyperpolarization-related component of arterial relaxation, increased the sensitivity of smooth muscle to nitric oxide, and decreased the production of vasoconstrictor prostanoids. Therefore, the beneficial effects of the Whey diet could be attributed to increased intake of potassium in SHR.

Intrapericardial infusion of endothelin-1 induces ventricular arrhythmias in dogs.

OBJECTIVES: Recently, extremely high levels of endothelin-1 (ET-1) were detected in the pericardial fluid of patients with heart disease; however, the pathophysiological importance of this finding is not known. The present study was designed to characterize ET-1 levels in canine pericardial fluid and to investigate the effects of local high concentrations of exogenous ET-1 in vivo. METHODS: In anesthetized, open-chest dogs ET-1 (Groups 1 and 2: 11 and 33 pmol.kg-1.min-1; n = 6 and 6, respectively) or physiological saline (Group 3, n = 5) were infused into the closed pericardial sac for 40 min. In serial pericardial fluid and aortic blood plasma samples, ET-1 levels were measured by radioimmunoassay, and analysed by high-performance liquid chromatography (HPLC). Systemic arterial blood pressure, heart rate, cardiac output (CO), standard ECG and right ventricular endocardial monophasic action potentials (MAPs) were recorded. RESULTS: Basal pericardial fluid ET-1 levels were significantly higher than respective plasma levels (342 +/- 210 vs. 8.0 +/- 5.2 pmol.l-1, n = 14, P < 0.001. In HPLC analysis pericardial fluid ET-1 was indistinguishable from ET-1(1-21). Infusion of exogenous ET-1 into the pericardial space induced ventricular arrhythmias in all instances, which were associated with 9.7-fold increase in pericardial fluid ET-1 levels. Ventricular tachycardias developed in 9 of 12 animals. The arrhythmogenic effect of ET-1 was more apparent in dogs with the larger dose. Before the onset of arrhythmias, intrapericardial infusion of ET-1 increased QT time (Group 1: 207 +/- 18 to 230 +/- 23 ms, P < 0.01; Group 2: 220 +/- 12 to 277 +/- 17 ms, P < 0.01) and MAP duration at 90% repolarization (at 300 ms cycle length) (Group 1: 192 +/- 9 to 216 +/- 9 ms, P < 0.01; Group 2: 205 +/- 9 to 255 +/- 9 ms, P < 0.001). Hemodynamic variables did not change significantly prior to the onset of ventricular tachyarrhythmias. In Group 3, arrhythmias were not observed and all electrophysiological and hemodynamic parameters remained unchanged. CONCLUSIONS: Administration of exogenous ET-1 into the pericardial space induces ventricular arrhythmias associated with prolongation of QT time and MAP duration. Whether pericardial fluid ET-1 under pathophysiological conditions can ever reach sufficiently high levels to induce ventricular arrhythmias remains to be elucidated.

Basal and volume expansion-stimulated plasma atrial natriuretic peptide concentrations and hemodynamics in conscious rats: effects of SCH 39.370, an endopeptidase inhibitor, and C-ANF-(4-23), a clearance receptor ligand.

The effects of a neutral endopeptidase (NEP) inhibitor, SCH 39.370, and a clearance receptor ligand, C-atrial natriuretic factor-(4-23) [C-ANF-(4-23)] on the plasma concentration of atrial natriuretic peptide (ANP) and hemodynamics under basal conditions and during increased circulating ANP levels produced by acute volume loading in conscious rats were studied. Measurements of plasma immunoreactive N-terminal fragment of pro-ANP (IR-NT-ANP) concentrations were used to characterize the endogenous secretion of the biologically active peptide in response to drug infusions and volume expansion. Infusion of SCH 39.370 increased plasma IR-ANP levels dose-dependently in conscious normotensive Wistar rats; maximal increases of 17% (P less than 0.02) after the dose of 3 mg/kg, iv, and 67% (P less than 0.002) after the dose of 10 mg/kg, iv, SCH 39.370 were noted. Similarly, infusion of C-ANF-(4-23) alone (30 micrograms/kg, iv bolus, followed by infusion of 3 micrograms/kg.min for 30 min) increased plasma IR-ANP levels by 37% (P less than 0.001). Given in combination, SCH 39.370 and C-ANF-(4-23) produced a greater increase in plasma IR-ANP concentration (83%; P less than 0.001) than when either substance was infused alone. Neither SCH 39.370 nor C-ANF-(4-23), alone or in combination, had any effect on basal plasma IR-NT-ANP concentrations. The combination reduced mean arterial pressure (8 +/- 2 mm Hg; P less than 0.01) and right atrial pressure (0.67 +/- 0.20 mm Hg; P less than 0.01), while administration of SCH 39.370 or C-ANF-(4-23) alone had no effect on mean arterial pressure, heart rate, or right atrial pressure in conscious rats. Acute volume expansion with 0.9% saline (1.1 ml/100 g BW) resulted in an increase in right atrial pressure (2.7 +/- 0.2 mm Hg; P less than 0.001) as well as in plasma IR-ANP (55%; P less than 0.001) and IR-NT-ANP concentrations (24%; P less than 0.03). Volume expansion in rats pretreated with SCH 39.370 resulted in a greater increase in plasma IR-ANP concentrations than in control animals; the relative ANP increases corresponding to the 2.5-mm Hg increase in right atrial pressure were 1.48-, 1.69-, and 2.28-fold in control, 3 mg/kg SCH 39.370-treated, and 10 mg/kg SCH 39.370-treated groups, respectively. When the relation between changes from control in plasma IR-ANP and right atrial pressure in response to acute volume expansion was analyzed in the presence of C-ANF-(4-23), no difference was noted between control and treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Endothelin stimulates basal and stretch-induced atrial natriuretic peptide secretion from the perfused rat heart.

To examine the role of intracellular signals in the regulation of atrial natriuretic peptide (ANP) release, the effects of endothelin (ET), a putative endogenous agonist for voltage-dependent Ca2+ channels on basal and atrial stretch-stimulated ANP release as well as on hemodynamic parameters (perfusion pressure, heart rate, contractile force) in isolated perfused rat hearts were studied. Infusion of ET (0.9 x 10(-9)-2.3 x 10(-9) M) alone for 30 min caused a dose-dependent sustained increase in the perfusate immunoreactive ANP (IR-ANP) concentration and coronary vasoconstriction. An initial inotropic response with a later decrease in the contractile force in response to ET was observed, while heart rate remained unchanged. A phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase-C activity, at a dose of 4.6 x 10(-8) M caused a gradual, slowly progressive increase in perfusate IR-ANP levels and a more rapid increase in perfusion pressure. ET, when infused in combination with TPA, enhanced IR-ANP secretion induced by the phorbol ester. When hearts from spontaneously hypertensive rats (SHR) were examined, the vasoconstrictor response to infusion of ET was greater than that in the normotensive Wistar-Kyoto (WKY) rats. Infusion of eguipressor doses of ET increased the release of IR-ANP in WKY rats, but had no effect on perfusate IR-ANP levels in SHR. To examine effects of ET on stretch-stimulated ANP release, the modified perfused rat heart preparation that enabled the stepwise distension of the right atrium was used. The increase in right atrial pressure (2.65 +/- 0.13 mm Hg) was accompanied by an increase in the perfusate IR-ANP concentration (from 8.3 +/- 1.1 to 13.9 +/- 2.0 ng/5 min; P less than 0.05; n = 15). The increase in right atrial pressure during the ET infusions resulted in a significantly greater increase in the perfusate IR-ANP concentration than vehicle alone. The calculated ANP increase corresponding to the 2-mm Hg increase in the right atrial pressure was 1.52-fold in the control group and 1.74-fold when 1.9 x 10(-9) M ET was infused (P less than 0.05). This study shows that ET stimulates both basal and atrial stretch-stimulated ANP secretion from the isolated perfused heart and suggests that ET is involved in the regulation of stretch-induced ANP release. The results further confirm the potent vasoconstrictor and cardiac effects of ET.

Mechanisms of atrial and brain natriuretic peptide release from rat ventricular myocardium: effect of stretching.

Ventricular hypertrophy is characterized by augmentation of the synthesis and storage of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). To evaluate in vitro the cellular mechanisms of immunoreactive ANP (IR-ANP) and BNP (IR-BNP) release from ventricular cardiocytes, we measured the secretory response to graded passive myocardial stretch in isolated atrialectomized perfused hypertrophied hearts of 14- to 18-month-old spontaneously hypertensive rats. At this age, the ventricular levels of both IR-ANP and IR-BNP were markedly higher in spontaneously hypertensive (182 +/- 27 and 32 +/- 3 pmol/ventricle, respectively) than in age-matched normotensive Wistar-Kyoto rats (35 +/- 4 and 12 +/- 1 pmol/ventricle, respectively; P < 0.001), whereas the differences between the strains in atrial levels of these peptides were small. The release of natriuretic peptides from ventricles in response to stretch was examined by increasing the volume of the intraventricular balloon for 10 min. Stretching of the hypertrophied ventricles produced a rapid transient (from 1-5 min) increase in both IR-ANP and IR-BNP secretion. As left ventricular pressure rose from 0 to 26 +/- 1 mm Hg, IR-ANP and IR-BNP release into the perfusion fluid increased 1.8 +/- 0.4- and 2.5 +/- 0.2-fold, respectively. Infusion of staurosporine, known to inhibit protein kinase-C activity in heart cells, blocked the stretch-induced increase in IR-ANP release (F = 3.10; P < 0.001, by analysis of variance), but had no effect on basal ventricular IR-ANP secretion (F = 0.87; P = NS). An L-type calcium channel antagonist, diltiazem, had no significant effect on basal (F = 1.20; P = NS) or stretch-stimulated (F = 1.47; P = NS) IR-ANP release from hypertrophied rat myocardium. Chromatographical analysis revealed that the ventricles primarily release the active processed 28- and 45- amino acid ANP- and BNP-like peptides, respectively, both before and during stretch. This study indicates that stretch stimulates both ANP and BNP secretion from hypertropic ventricular myocytes. The results further suggest that protein kinase-C may be involved in stretch-induced ventricular ANP release, whereas the influx of extracellular calcium may not be necessary.

Phorbol esters enhance stretch-induced atrial natriuretic peptide secretion.

Stretching of atrial myocytes stimulates atrial natriuretic peptide (ANP) secretion, but the cellular processes linking mechanical distention to ANP release are unknown. We studied whether or not protein kinase C activation by phorbol ester affects atrial stretch-induced ANP secretion using the modified perfused rat heart preparation that enabled stepwise distention of the right atrium as an experimental model for stretch-stimulated ANP release. The increase in right atrial pressure (2.65 +/- 0.13 mm Hg) was accompanied by an increase in the perfusate immunoreactive ANP (IR-ANP) concentration (from 8.3 +/- 1.1 ng/5 min to 13.9 +/- 2.0 ng/5 min, P less than 0.05, n = 14). During stretch, a slight inotropic response was observed, while heart rate and perfusion pressure remained unchanged. Increase in right atrial pressure in the presence of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase C activity in heart cells, resulted in a significantly greater increase in the perfusate IR-ANP concentration than after vehicle infusion. The calculated ANP increase corresponding to the 2 mm Hg increase in the right atrial pressure was 1.52-fold in the control group and 1.84-fold when 10 nM TPA was infused (P less than 0.05). Infusion of TPA at a dose of 24 nM further increased the stretch-induced ANP release by causing 2.22-fold (P less than 0.01) increase in IR-ANP secretion. As judged by gel filtration chromatography, abnormal release of the large mol wt stored ANP could not account for the secretory response to phorbol ester. Additionally, a phorbol ester analog, 4 alpha-phorbol 12,13-didecanoate, which is incapable of binding to and activating protein kinase C, was inactive as an ANP secretagogue. In contrast, drugs known to increase the concentration of intracellular Ca2+ in myocytes, Bay K8644 (3 and 6 microns) and forskolin (0.14 microM), significantly inhibited the stretch-stimulated ANP release. This study shows that phorbol ester enhances atrial stretch-stimulated ANP secretion from the isolated perfused heart, suggesting that protein kinase C activity is positively coupled to the stretch-induced ANP release. The results further demonstrate the negative effect of increase in intracellular Ca2+ on stretch-induced ANP release.

Atrial natriuretic peptide (ANP) inhibits its own secretion via ANP(A) receptors: altered effect in experimental hypertension.

Three atrial natriuretic peptide (ANP) receptors, ANP(A), ANP(B), and ANP(C), have been identified in the heart, suggesting that natriuretic peptides may have direct effects on cardiac function. To characterize the possible role of atrial natriuretic peptide (ANP) in the regulation of its own secretion, we studied here the effects of ANP (greater affinity for ANP(A) than for ANP(B) receptors) and C-type natriuretic peptide (CNP), a potent activator of ANP(B) receptors, on the release of atrial peptides under basal conditions and during acute volume expansion in conscious normotensive Sprague-Dawley rats. The effects of HS-142-1, a nonpeptide ANP(A) and ANP(B) receptor antagonist, on volume load-induced atrial peptide release in 1-yr-old conscious normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were also studied. As an index of secretion of atrial peptides from the heart, plasma levels of N-terminal fragment of pro-ANP (NT-ANP) were measured. In Sprague-Dawley rats, i.v. infusion of ANP for 30 min in doses of 0.3 and 1.0 microg/kg x min blocked the plasma immunoreactive NT-ANP (IR-NT-ANP) response to volume load (P < 0.001), whereas CNP had no significant effect. Neither ANP nor CNP infusion had any effect on plasma IR-NT-ANP levels under basal conditions. Bolus administration of HS-142-1 increased baseline plasma IR-ANP concentrations in both WKY and SHR strains (WKY: 3 mg/kg, 46 +/- 8 pmol/liter, P < 0.001; SHR: 1 mg/kg, 26 +/- 9 pmol/liter, P < 0.01; SHR: 3 mg/kg, 40 +/- 12 pmol/liter, P < 0.01). The corresponding increases in plasma IR-NT-ANP concentrations in the SHR in response to administration of HS-142-1 were 0.17 +/- 0.06 nmol/liter (P < 0.01) and 0.40 +/- 0.14 nmol/liter (P < 0.01). Moreover, HS-142-1 (3 mg/kg) augmented plasma IR-ANP and IR-NT-ANP responses to acute volume load in WKY rats. In contrast, HS-142-1 did not enhance the plasma IR-ANP response to acute volume load in SHR and resulted in a smaller increase in the plasma IR-NT-ANP concentration in SHR than in WKY rats. In conclusion, the findings that ANP, but not CNP, inhibited volume expansion-stimulated NT-ANP release and that HS-142-1, an antagonist of guanylate cyclase-linked natriuretic peptide receptors, increased plasma ANP and NT-ANP concentrations show that endogenous ANP directly modulates its own release via ANP(A) receptors in vivo. Furthermore, this modulation of acute volume expansion-induced atrial peptide release appears to be altered in experimental hypertension.

Atrial stretch induces rapid increase in brain natriuretic peptide but not in atrial natriuretic peptide gene expression in vitro.

Pressure and volume overload in vivo is characterized by induction of the expression of two cardiac hormones, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), but whether stretch directly or other pathophysiological factors associated with cardiac overload cause the activation of these genes is not known. In the present study we examined the effect of short-term (from 30 min to 2 h) direct myocardial stretch on atrial ANP and BNP synthesis and release in modified perfused rat heart preparation that enabled the stepwise distension of the right atrium by pressures approximating those found in vivo. The increase in right atrial pressure by 3.6 mm Hg for 2 h resulted in a 3.3- (p < 0.001) and 1.7-fold (p < 0.02) increase in the rate of IR-ANP and IR-BNP release, respectively, into the perfusate. The maximal increase in both ANP and BNP release was seen after 20 min distension. Thereafter the perfusate IR-ANP and IR-BNP concentration gradually decreased, reaching control values within 2 hours. Chromatographic analysis showed that the hearts primarily release the active, processed 28- and 45-amino acid ANP- and BNP-like peptides, respectively, both before and during atrial stretch. Atrial stretch induced rapid stimulation of BNP gene expression: 1.9- (p < 0.001) and 4.5-fold (p < 0.001) increase in right auricular BNP mRNA levels after 1.0 and 2.0 hours' stretching, respectively, was found on Northern blot analysis, while no change was seen after 30 min distension. In contrast, stretching for up to 2 h did not change auricular ANP mRNA, IR-ANP or IR-BNP levels. Our results show for the first time that atrial stretch induces rapid stimulation of both synthesis and secretion of BNP. The induction of BNP gene expression in the very early stages of cardiac overload mimics the induction of protooncogenes and occurred without involvement of humoral or neural factors. The lack of response of atrial ANP mRNA levels demonstrates that the regulation of BNP gene expression differs from that of ANP.

Role of nitric oxide on cardiac hormone secretion: effect of NG-nitro-L-arginine methyl ester on atrial natriuretic peptide and brain natriuretic peptide release.

Endocardial cells, like endothelial cells, release nitric oxide (NO) and may play a role in modulating the contractility of cardiac muscle. We have studied the effects of NG-nitro-L-arginine methyl ester (L-NAME), a selective NO synthase inhibitor, on basal and volume expansion-induced secretion of two cardiac hormones, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), in vivo. In conscious chronically cannulated rats, bolus injection of L-NAME at doses of 1, 3, and 10 mg/kg, iv, caused a dose-dependent increase in mean arterial pressure and sustained bradycardia, whereas right atrial pressure remained unchanged. The hemodynamic effects of L-NAME were reversed by simultaneous administration of L-arginine, a precursor of NO. Administration of 3 and 10 mg/kg L-NAME alone increased plasma levels of immunoreactive ANP (IR-ANP) from 30 +/- 5 to 52 +/- 9 pmol/liter and from 38 +/- 6 to 91 +/- 16 pmol/liter (P < 0.01), respectively, but had no effect on plasma levels of immunoreactive BNP (IR-BNP). The increase in plasma IR-ANP concentration in response to L-NAME infusions showed a positive linear correlation (P < 0.01) with the increase in mean arterial pressure and a negative correlation (P < 0.01) with the changes in heart rate. Acute volume expansion with 0.9% saline in conscious animals resulted in a 3.2-fold increase in plasma IR-ANP levels (from 35 +/- 7 to 113 +/- 15 pmol/liter; P < 0.01), but plasma IR-BNP levels did not change. In the rats pretreated with L-NAME, the relation between the changes from control in plasma IR-ANP and right atrial pressure shifted to the left; the absolute increases corresponding to the 3 mm Hg increase in right atrial pressure were 66 +/- 13, 76 +/- 15, 135 +/- 33, and 148 +/- 24 pmol/liter in the control and 1 mg/kg L-NAME, 3 mg/kg L-NAME-, and 10 mg/kg L-NAME-treated groups, respectively. The combined infusion of L-NAME and L-arginine attenuated the L-NAME-induced increase in ANP release. Our results show that L-NAME increases basal plasma IR-ANP levels and enhances stretch-induced ANP release, suggesting that secretion of ANP in response to volume load may be modulated by the locally released nitric oxide from the endothelium. Further, acute regulation of BNP secretion in response to inhibition of nitric oxide synthase and volume load differs from that of ANP.

Endothelin-induced atrial natriuretic peptide release from cultured neonatal cardiac myocytes: the role of extracellular calcium and protein kinase-C.

Regulation of atrial natriuretic peptide (ANP) secretion from neonatal rat myocytes cultured on microcarriers was studied using endothelin-1 (ET-1) as a secretagogue. Myocytes were cultured for 3 days on microcarriers, packed in a chromatography column, and perifused with Krebs-Henseleit bicarbonate buffer. ANP secretion was measured by RIA, and the cytosolic free calcium concentration ([Ca2+]f) was measured continuously during secretion by the fluorescent calcium indicator fura-2. In perifused atrial and ventricular cells, basal values for [Ca2+]f were 146 and 167 nM, and immunoreactive ANP (IR-ANP) secretion rates were 61 and 65 pg/min.mg protein, respectively. ET-1 at concentrations of 1, 10, and 100 nM caused a concentration-dependent increases in [Ca2+]f and IR-ANP secretion in atrial myocytes. The maximal increases in [Ca2+]f and IR-ANP secretion were 30% and 100%, respectively. Diltiazem (1 microM), an inhibitor of voltage-sensitive Ca2+ channels, inhibited [Ca2+]f increments, but had no effect on ET-induced IR-ANP secretion. Staurosporine (10 nM), a protein kinase-C inhibitor, augmented [Ca2+]f changes, but inhibited the sustained phase of ET-induced IR-ANP secretion (P less than 0.05). Diltiazem abolished the stimulatory effect of staurosporine on [Ca2+]f and its inhibitory effect on IR-ANP secretion. ET-1 caused increases in [Ca2+]f and IR-ANP secretion in ventricular myocytes similar to those in atrial myocytes. Peptides corresponding in size to pro-ANP and ANP-(1-28) were detected in the original cell culture medium and perifusion effluent, and ET-1 did not change their concentration ratio in the eluate. Lactate dehydrogenase was not detected in the effluents before or during ET infusion, showing that the increase in IR-ANP secretion was not due to cell damage. This study shows that ET stimulates atrial and ventricular ANP secretion. The results also suggest that sustained ET-induced atrial ANP secretion is dependent on protein kinase-C, but does not require the influx of extracellular calcium.

Brain natriuretic peptide in plasma, atria, and ventricles of vasopressin- and phenylephrine-infused conscious rats.

To evaluate the mechanisms of brain natriuretic peptide (BNP) gene expression, we determined the effect of acute cardiac overload (from 30 min to 4 h) on atrial and ventricular BNP mRNA levels in normal and hypertrophied myocardium. Arginine8 vasopressin (AVP; 0.05 microgram/kg.min) and l-phenylephrine (PHE; 20 micrograms/kg.min) were infused iv to increase cardiac workload in conscious spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. At the age of 10-22 months, during the established phase of ventricular hypertrophy, baseline BNP synthesis was increased in the hypertrophic ventricular cells of SHR, as reflected by about 2-fold (P < 0.05-0.001) elevation of levels of immunoreactive BNP (IR-BNP) and BNP mRNA. Intravenous infusions of AVP and PHE increased mean arterial pressure, plasma IR-BNP levels, and ventricular BNP mRNA levels within 1 h of pressure overload; peak levels of BNP mRNA were reached at 4 h. The increase in BNP mRNA levels was slightly greater in the epicardial (2.0- to 2.6-fold; P < 0.01) than in the endocardial layer (1.9- to 2.0-fold; P < 0.01) of the left ventricle. The rapid stimulation of ventricular BNP mRNA synthesis induced by AVP and PHE was accompanied by the simultaneous activation of left atrial BNP gene expression. Left atrial BNP mRNA levels were increased significantly in response to 1-h infusions, and values peaked in both the AVP- and PHE-infused SHR at 2 h, i.e. a 3.6-fold increase in BNP mRNA levels in left atria in AVP-infused SHR, and a 2.5-fold increase in PHE-infused SHR. Right atrial BNP mRNA levels remained unchanged during drug infusion, except for a transient increase in the WKY after 30 min of infusion. The induction of BNP synthesis was also reflected by increased ventricular IR-BNP levels, whereas AVP and PHE did not affect atrial IR-BNP concentrations or contents. In conclusion, the present study shows that pressure overload rapidly stimulates BNP gene expression in the hearts of normal and hypertensive rats. Thus, locally generated BNP in the heart muscle may play a significant role in cardiac adaptation to acute changes in mechanical load.