Background-Adrenomedullin (ADM), a new vasorelaxing and natriuretic peptide, may function as an endogenous regulator of cardiac function, because ADM and its binding sites have been found in the heart. We characterize herein the cardiac effects of ADM as well as the underlying signaling pathways in vitro. Methods and Results-In isolated perfused, paced rat heart preparation, infusion of ADM at concentrations of 0.1 to 1 nmol/L for 30 minutes induced a dose-dependent, gradual increase in developed tension, whereas proadrenomedullin N-20 (PAMP; 10 to 100 nmol/L), a peptide derived from the same gene as ADM, had no effect. The ADM-induced positive inotropic effect was not altered by a calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP 8 -37 , or H-89, a cAMP-dependent protein kinase inhibitor. ADM also failed to stimulate ventricular cAMP content of the perfused hearts. Ryanodine (3 nmol/L), a sarcoplasmic reticulum Ca 2ϩ release channel opener, suppressed the overall ADM-induced positive inotropic effect. Pretreatment with thapsigargin (30 nmol/L), which inhibits sarcoplasmic reticulum Ca 2ϩ ATPase and depletes intracellular Ca 2ϩ stores, attenuated the early increase in developed tension produced by ADM. In addition, inhibition of protein kinase C by staurosporine (10 nmol/L) and blockade of L-type Ca 2ϩ channels by diltiazem (1 mol/L) significantly decreased the sustained phase of ADM-induced increase in developed tension. Superfusion of atrial myocytes with ADM (1 nmol/L) in isolated left atrial preparations resulted in a marked prolongation of action potential duration between 10 and Ϫ50 mV transmembrane voltage, consistent with an increase in L-type Ca 2ϩ channel current during the plateau. Conclusions-Our results show that ADM enhances cardiac contractility via cAMP-independent mechanisms including Ca 2ϩ release from intracellular ryanodine-and thapsigargin-sensitive Ca 2ϩ stores, activation of protein kinase C, and Ca 2ϩ influx through L-type Ca 2ϩ channels. (Circulation. 1998;97:1062-1070.)Key Words: adrenomedullin Ⅲ contractility Ⅲ calcium Ⅲ peptides Ⅲ signal transduction A drenomedullin is a newly discovered, potent, vasorelaxing and natriuretic peptide that was originally isolated from human pheochromocytoma.1 The peptide, consisting of 52 amino acids in humans and 50 amino acids in the rat, is classified in the CGRP family.2,3 ADM may function as a paracrine and/or autocrine factor in the regulation of cardiac function, because high mRNA expression, 4 a considerable amount of ADM-like immunoreactivity, 5-7 and a high level of 125 I-ADM binding 8 have been found in the heart. In agreement with this hypothesis, ADM has been reported to increase cardiac output and left ventricular contractility in vivo 9,10 and exert a direct inotropic effect in vitro.11 Recently, the plasma concentration of circulating ADM has been shown to be increased in patients with congestive heart failure.5,12-14 Moreover, Jougasaki et al 5 reported that immunohistochemical staining for ADM is significantly increased...

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.

IntroductionGenomewide analysis of diagnostic bone marrow samples from patients for whom therapy fails may provide insight into particular profiles of gene expression that determine outcome. Understanding the relationships of known biologic markers to outcome using genomic profiles should help develop rational targeted therapies.In acute myeloid leukemia (AML), mutations of the Fms-like tyrosine kinase 3 (FLT3) receptor are markers of poor outcomes. Several studies in children and adults suggested that FLT3 internal tandem duplication (FLT3-ITD) confers poor prognosis in AML. [1][2][3][4][5][6] The FLT3 receptor is a member of the class III receptor tyrosine kinase (RTK) family. 7,8 High FLT3 receptor expression has been reported in more than 60% of precursor B-cell acute lymphocytic leukemia and AML cases, 9-11 and it is the most frequently mutated RTK 2,4,6 in adult and childhood de novo AML. Most FLT3 mutations result in constitutive activation and autophosphorylation of the FLT3 receptor. This results in downstream activation of the signal transducer and activator of transcription 5 (STAT5), phosphatidylinositol 3 (PI-3) kinase, and Ras pathways. 6,[12][13][14][15][16][17][18][19][20][21][22][23] Activation of these pathways leads to enhanced proliferation and resistance to apoptosis. 24,25 There are 2 types of FLT3 receptor mutations that have been described: internal tandem duplications (ITDs) in the juxtamembrane region [26][27][28][29][30][31] and activation loop mutations (ALMs) in the tyrosine kinase domain. [32][33][34] The incidence of FLT3-ITD is 11% to 16% in children 3,5 and 20% or more in adults 2,35 ; whereas, the incidence of point mutations is 7% to 8% in adults and children. 6,35 In studies by Whitman et al 4 and Liang et al, 5 a higher ratio of ITD to wild-type (WT) allele had a strong correlation with poor outcome, whereas a lower ratio correlated with an outcome similar to a FLT3-WT receptor. Meshinchi et al 3 reported that FLT3-ITD correlated highly with refractoriness to induction therapy in the Children's Cancer Group study 2891. Zwaan et al 36 reported similar results after analyzing a larger set of samples from several Berlin-Frankfurt-Müenster and Dutch Childhood Leukemia Group studies. As with most biologic markers, FLT3-ITD and FLT3-ALM convey a high but not absolute risk of poor outcome.In this study, we used DNA microarray analysis to identify a pattern of gene expression associated with a high-risk of treatment failure in specimens with FLT3 mutations (FLT3-MUs) in childhoodAML patients. There are 2 genes included in this expression profile that are also predictive of outcome when analyzed separately and provide insight into a possible mechanism by which FLT3-MUs confer a poor prognosis. Patients, materials, and methods Arrays Childhood de novo acute myeloid leukemia specimensThe specimens used in this study were archived at the Children's Oncology Group AML cell bank from Pediatric Oncology Group (POG) study 9421. Institutional review board (IRB) approval was obtained for gen...

Left ventricular hypertrophy is characterized by stimulation of ventricular synthesis of atrial natriuretic peptide (ANP). This study was designed to test the hypothesis that the increased ventricular ANP levels participate in the release of ANP into the circulation. Swimming was used as a physiologic model to induce ANP release from the heart, and atrial and ventricular levels of immunoreactive ANP (IR-ANP) and ANP messenger RNA (mRNA) were measured simultaneously in the spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats at rest and after swimming. IR-ANP concentration in the left ventricle of l-year-old SHR with severe left ventricular hypertrophy was increased in association with the augmentation of ANP mRNA levels, whereas right ventricular levels of ANP were reduced in SHR compared with normotensive controls. A 30-minute exercise in hypertensive and in normotensive rats resulted in marked increases in mean arterial pressure, heart rate, plasma catecholamine levels, blood lactate levels, and plasma IR-ANP concentration. The increased ANP secretion was associated with a decrease in left (34-39%) and right (24%) ventricular concentration of IR-ANP; transmurally, this depletion of ventricular IR-ANP was greatest (28%) in the endocardial layer of the left ventricle of SHR. No significant differences were noted in total atrial and left or right auricular IR-ANP concentration between SHR and WKY rats or between the resting and swimming rats. When studied in vitro with an isolated, perfused heart preparation, the hypertrophic ventricular tissue after atrialectomy secreted more ANP into the perfusate than did control hearts; in SHR, ventricles contributed 28% of the total ANP release to perfusate, and in normotensive control rats, ventricles contributed 8%. These studies show that stimulated release of ANP is associated with depletion of endocardial left ventricular stores. The amount of ANP released in vitro and in vivo correlated with the degree of hypertrophy of the ventricle. Finally, the phorbol ester, known to increase ANP secretion from intact perfused hearts, had only a limited effect on ANP release after atrialectomy, suggesting that the secretion of ANP from ventricular cells may be mainly of the constitutive type. (Circulation 1989;80:390-400) A trial natriuretic peptide (ANP), a potent natriuretic, diuretic, and vasorelaxant cardiac hormone that inhibits renin, aldosterone, and vasopressin secretion, is synthesized and secreted predominantly by the atria in healthy adult mammals.1-5 Atrial wall stretch is a predom- The aim of this study was to determine whether or not ventricular ANP levels participate in the release of ANP into the circulation. Physical exercise in the normotensive and genetically hypertensive rats was used as an experimental model to stimulate release of ANP from the heart, and ventricular levels of immunoreactive ANP (IR-ANP) in normal and hypertrophic ventricles were measured at rest and after exercise. In addition, the biosynthesis of ANP in the atria and ventricles, it...

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The FK506-binding protein, FKBP12, is a putative target of type I receptors for transforming growth factor-beta (TbetaR-I). As the FK506 motif that competes with TbetaR-I for FKBP12 resembles an invariant Leu-Pro dipeptide in TbetaR-I, we replaced Leu193 and Pro194 with Ala, along with mutations across the Gly/Ser box. L193A, P194A, and L193A/P194A do not alter TbetaR-I function; T204D partially activates, independent of ligand; L193A/P194A/T204D was an even more potent constitutive mutation. Association with FKBP12 in a yeast two-hybrid assay was disrupted by P194A, L193A/P194A, and L193A/P194A/T204D, but not L193A or T204D alone. Thus, FKBP12 recognition is dispensable for TGFbeta signaling.

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