Plasma levels of natriuretic peptides and year-by-year blood pressure variability: a population-based study.

Augmented blood pressure (BP) variability over various time periods has been recognized as a risk factor for cardiovascular diseases. Both atrial and B-type natriuretic peptides (ANP and BNP) are secreted in response to volume or pressure overload to the heart, exerting natriuretic and vasodilator actions. In this study, we examined the relationships between year-by-year BP variability and plasma levels of ANP and BNP in the general population. Study subjects were local residents receiving an annual heath checkup, who had an estimated glomerular filtration rate of >30 ml min-1 per 1.73 m2 and no history of heart disease. Of those, we selected 314 subjects that received checkups at least five times over the past 6 years. BP variability year-by-year was retrospectively evaluated by s.d., coefficient of variation, average real variability and variation independent of the mean of BP values of 6 or 7 time points. The four parameters of BP variability were each found to significantly correlate with logarithmically transformed ANP and BNP levels by simple regression. When classified by quartiles of s.d. of systolic BP, the highest quartile group showed significantly higher levels of the natriuretic peptides compared with other groups. Multivariate analyses revealed that BP variability was an independent determinant for the ANP and BNP levels. In conclusion, augmented year-by-year BP variability over the past 6 years was associated with elevation of plasma levels of ANP and BNP, suggesting a possible relationship between the BP variability and cardiac load, in the general population.

Angiotensin II stimulates cardiac adrenomedullin production and causes accumulation of mature adrenomedullin independently of hemodynamic stress in vivo.

Adrenomedullin is a potent hypotensive peptide that may act on myocytes to inhibit hypertrophy and on fibroblasts to inhibit growth in vitro induced by mechanical stretching and angiotensin II. Adrenomedullin is processed from the inactive intermediate adrenomedullin precursor with a glycine extension, which is subsequently converted to biologically active mature adrenomedullin by enzymatic amidation. Total adrenomedullin is the sum of intermediate and mature adrenomedullin. We examined the effect of a subpressor dose of angiotensin II on the production of left ventricular adrenomedullin and on protein levels of mature adrenomedullin in the left ventricle in vivo. We also investigated whether the effect is mediated by the angiotensin II type 1 receptor. Concentrations of total and mature adrenomedullin in the left ventricle and mature adrenomedullin-to-intermediate adrenomedullin ratio were significantly increased by angiotensin II infusion, regardless of pressure overload. Total and mature adrenomedullin concentrations significantly correlated with the weight of the left ventricle. Furthermore, increased adrenomedullin gene expression and protein levels were completely suppressed by a subdepressor dose of angiotensin II type 1 receptor blocker. In conclusion, angiotensin II stimulates the production of cardiac adrenomedullin and accumulates mature adrenomedullin in the left ventricle independently of hemodynamic stress. These processes are partially regulated through the angiotensin II type 1 receptor in vivo.

The seven amino acids of human RAMP2 (86) and RAMP3 (59) are critical for agonist binding to human adrenomedullin receptors.

When co-expressed with a receptor activity-modifying protein (RAMP) accessory protein, calcitonin receptor-like receptor (CRLR) can function as a calcitonin gene-related peptide receptor (CRLR-RAMP1) or an adrenomedullin (AM) receptor (CRLR-RAMP2/3). Here we report on the structural domain(s) involved in selective AM binding that were examined using various RAMP chimeras and deletion mutants. Co-expression of chimeric RAMPs and CRLR in HEK293 cells revealed that residues 77-101, situated in the extracellular N-terminal domain of human RAMP2 (hRAMP2), were crucial for selective AM-evoked cAMP production. More detailed analysis showed that deletion of hRAMP2 residues 86-92 significantly attenuated high-affinity (125)I-AM binding and AM-evoked cAMP production despite full cell surface expression of the receptor heterodimer and that deletion of hRAMP3 residues 59-65 had a similar effect. There is little sequence identity between hRAMP3 residues 59-65 and hRAMP2 residues 86-92; moreover, substituting alanine for Trp(86) (Ala(87)), Met(88), Ile(89), Ser(90), Arg(91), or Pro(92) of hRAMP2 had no effect on AM-evoked cAMP production. It thus seems unlikely that any one amino acid residue is responsible for determining selective AM binding or that AM binds directly to these peptide segments. Instead these findings suggest that the respective seven-amino acid sequences confer selectivity either by directly contributing to the structure of ligand binding pocket or by allosteric modulation of the conformation of CRLR.

Effects of endothelin on adrenomedullin secretion and expression of adrenomedullin receptors in rat cardiomyocytes.

Both endothelin (ET) and adrenomedullin (AM), produced by cardiac myocytes, are thought to be locally-acting hormones in the heart. Recently, calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) have been shown to function together to serve as AM receptors stimulating cAMP production. In the present study, we examined the effects of ET on AM secretion, intracellular cAMP response to AM, and gene expressions of CRLR and RAMPs in cultured cardiac myocytes. Synthetic ET-1 dose-dependently increased AM secretion from the cardiomyocytes. AM increased the intracellular cAMP level in a dose-dependent manner and the cAMP accumulation by AM was significantly amplified by 24 h preincubation with ET-1. 10 nmol/L ET-1 significantly increased the CRLR mRNA level without any effect on RAMP1 mRNA. 1 micromol/L ET-1 significantly reduced the RAMP2 mRNA level, but ET-1 dose-dependently increased the RAMP3 mRNA level in the cardiac myocytes. These findings suggest that ET-1 not only stimulates AM secretion, but also modulates intracellular cAMP responses to AM probably by altering the expressions of CRLR and RAMPs in rat cardiomyocytes.

Secretion of proadrenomedullin N-terminal 20 peptide from cultured neonatal rat cardiac cells.

Proadrenomedullin N-terminal 20 peptide (PAMP) is generated from post-transcriptional enzymatic processing of a 185-amino acid precursor for adrenomedullin (AM), a potent vasodilator peptide. We have reported that AM is secreted from cultured neonatal rat cardiac myocytes and fibroblasts, and that secreted AM modulates the growth of these cells; however, it is unknown whether or not the cardiac cells produce PAMP. In this study, we examined the production of PAMP in cultured neonatal rat cardiac myocytes and fibroblasts. Both the cardiac myocytes and fibroblasts cultured with serum-free media secreted PAMP time-dependently at rates of 5.7+/-0.9 fmol/10(5) cells/40 h and 8.4+/-0.7 fmol/5x10(4) cells/48 h (mean+/-SD), respectively. Reverse-phase high performance liquid chromatography showed that immunoreactive PAMP secreted from these cells was identical to PAMP[1-20], a whole active molecule. PAMP and AM secretions were significantly (P<0.01) stimulated by 10(-6) mol/L angiotensin II (Ang II) and 10% fetal bovine serum (FBS) in myocytes and fibroblasts, whereas the ratio of PAMP to AM secretion in the myocytes was smaller than that of the fibroblasts. These results suggest that PAMP is secreted along with AM from rat cardiac myocytes and fibroblasts, and the secretion is augmented by the growth-promoting stimuli of Ang II and FBS for these cells.

Selective inhibition of nicotinic cholinergic receptors by proadrenomedullin N-terminal 12 peptide in bovine adrenal chromaffin cells.

We studied whether a novel proadrenomedullin derived peptide was present and what was its physiological function in cultured bovine adrenal chromaffin cells. We found a high level of proadrenomedullin N-terminal 12 peptide (PAMP-12) which consists of a peptide from 9th amino acid to 20th amino acid of proadrenomedullin N-terminal 20 peptide (PAMP-20). PAMP-12 was released from the cells along with catecholamine upon stimulation of nicotinic cholinergic receptors. When PAMP-12 was added in the incubation medium, this peptide inhibited nicotinic receptor-mediated catecholamine release and influx of Na(+) and Ca(2+) into the cells. PAMP-12 did not affect catecholamine release evoked by histamine or by depolarization by high concentration of potassium. PAMP-12 also inhibited synthesis of catecholamines as well as the activation of tyrosine hydroxylase by nicotinic stimulation. Thus, PAMP-12 is an endogenous peptide that regulates release and synthesis of catecholamines by acting on nicotinic cholinergic receptors in an autocrine manner in adrenal chromaffin cells.

Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system.

Previously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi et al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta-D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure-function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.

Visualization of the calcitonin receptor-like receptor and its receptor activity-modifying proteins during internalization and recycling.

Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3). A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without co-transfection of RAMPs. CRLR-GFP failed to generate responses to CGRP or AM without RAMPs. Furthermore, CRLR-GFP was not found in the plasma membrane and its localization was unchanged after agonist exposure. When stably coexpressed with RAMPs, CRLR-GFP appeared on the cell surface and was fully active in intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs. This internalization was strongly inhibited by hypertonic medium (0.45 m sucrose) and paralleled localization of rhodamine-labeled transferrin, suggesting that CRLR endocytosis occurred predominantly through a clathrin-dependent pathway. A significant proportion of CRLR was targeted to lysosomes upon binding of the ligands, and recycling of the internalized CRLR was not efficient. In HEK 293 cells stably expressing CRLR-GFP and Myc-RAMPs, these rhodamine-labeled RAMPs were co-localized with CRLR-GFP in the presence and absence of the ligands. Thus, the CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway.

Increased plasma proadrenomedullin N-terminal 20 peptide in patients with essential hypertension.

The novel hypotensive peptide, proadrenomedullin N-terminal 20 peptide (PAMP), is processed from the adrenomedullin precursor. Recently, we identified PAMP-12 [PAMP(9-20)] from the porcine adrenal medulla as a major endogenous and biologically active peptide. Using a new, sensitive radioimmunoassay which recognizes the C-terminal region of PAMP-20 [PAMP(1-20)], we investigated the role of PAMP in patients with essential hypertension who had normal renal function, and whether PAMP-12 is present in humans. The mean PAMP plasma concentration, like that of adrenomedullin, was significantly higher in hypertensive [1.51 fmol/mL, standard error of the mean (SEM) 0.09 fmol/mL] than normotensive participants (1.08 fmol/mL, SEM 0.05). The increase in plasma PAMP concentration in patients with organ damage accompanied by hypertension was significantly higher than that in patients without organ damage. The PAMP concentration had a significant positive correlation with mean blood pressure and adrenomedullin concentration. The immunoreactive PAMP in human tissue and plasma was characterized by reverse-phase high-performance liquid chromatography. PAMP-12, as well as PAMP-20, was abundant in the phaeochromocytoma tissue. These findings suggest that PAMP plays some pathophysiological role against the development of essential hypertension.

The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.

Adrenomedullin, a potently hypotensive peptide isolated from human pheochromocytoma, is known to elicit a rise in cAMP levels within mammalian endothelial and smooth muscle cells. Until now, however, little has been known about the adrenomedullin receptor. Recently, a group called receptor activity-modifying proteins that complex with the calcitonin receptor-like receptor, and thereby regulate its transport and ligand specificity, were identified. Here we show that mRNA for both the calcitonin receptor-like receptor and the receptor activity-modifying protein 2, but not the receptor activity-modifying protein 1 or receptor activity-modifying protein 3, are expressed in human endothelial and vascular smooth muscle cells. We also found that adrenomedullin increased cAMP levels in HeLa EBNA and 293 EBNA cells, expressing both the receptor activity-modifying protein 2 and calcitonin receptor-like receptor proteins. Thus, the receptor activity-modifying protein 2/calcitonin receptor-like receptor complex apparently serves as a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.

An autocrine or a paracrine role of adrenomedullin in modulating cardiac fibroblast growth.

OBJECTIVE: The aim of the present study was to determine the role of adrenomedullin (AM) in cardiac fibroblasts. METHODS: The production and secretion of AM were examined in cultured neonatal rat cardiac fibroblasts, and the effects of AM on proliferation and protein synthesis of these cells were assessed by [3H]thymidine and [3H]phenylalanine incorporation, respectively. RESULTS: Cultured cardiac fibroblasts secreted AM into the medium time-dependently at a rate of 20.3 +/- 3.0 fmol/5 x 10(4) cells/48 h, mean +/- S.D. Northern blot analysis showed expression of preproAM mRNA of 1.6 kb in these cells. In addition, 10(-6) mol/l of angiotensin II (Ang II) and endothelin-1 (ET-1) significantly increased the AM secretion by 55 and 48%, respectively. Synthetic AM significantly reduced 10(-6) mol/l Ang II- or 10(-7) mol/l ET-1-stimulated [3H]thymidine and [3H]phenylalanine incorporation in a dose-dependent manner, and these effects were attenuated by a calcitonin gene-related peptide (CGRP) type 1 receptor antagonist, CGRP(8-37). Synthetic AM also had a dose-dependent stimulatory effect on cAMP accumulation in these cells, which was significantly attenuated by CGRP(8-37). A cAMP analogue, 8-bromo-cAMP, mimicked the AM effects, inhibiting the Ang II-stimulated [3H]thymidine and [3H]phenylalanine incorporation. Blockage of the effect of endogenous AM by anti-AM monoclonal antibody not only significantly reduced the basal level of intracellular cAMP, but also enhanced the [3H]thymidine and [3H]phenylalanine incorporation into the cells. CONCLUSIONS: Cultured neonatal rat cardiac fibroblasts produce and secrete AM, and the secreted AM may inhibit proliferation and protein synthesis of these cells. AM may exert these inhibitory effects partly by elevating intracellular cAMP. It is suggested that AM has an important role in modulating the growth of cardiac fibroblasts in an autocrine or a paracrine manner.

Adrenomedullin: a possible autocrine or paracrine inhibitor of hypertrophy of cardiomyocytes.

Adrenomedullin (AM), a potent vasodilator peptide, exists in the cardiac ventricle; however, the role of AM in the ventricular tissue remains unknown. In the present study, we investigated the production and secretion of AM in cultured neonatal rat cardiomyocytes, and we examined the effect of AM on de novo protein synthesis in these cells by measuring [14C]phenylalanine incorporation. The cardiomyocytes cultured with serum-free media secreted AM into the media in a time-dependent manner at the rate of 12.2+/-0.5 fmol/10(5) cells/48 hours (mean+/-SEM). Angiotensin II (1 micromol/L) or 10% fetal bovine serum significantly (P<.01) increased the AM secretion by 115% and 305%, respectively. In addition, Northern blot analysis of total RNA extracted from the myocytes disclosed the expression of prepro-AM mRNA of 1.6 kb. Synthetic AM at 1 micromol/L significantly reduced the 10(-6) mol/L angiotensin II- and 10% fetal bovine serum-stimulated [14C]phenylalanine incorporation into the cells, by 16% (P<.05) and 20% (P<.01), respectively. The inhibitory effect of AM on the angiotensin II-stimulated [14C]phenylalanine incorporation was abolished dose-dependently by a calcitonin gene-related peptide receptor antagonist, CGRP(8-37). Furthermore, blockade of the action of endogenous AM by either 10(-6) mol/L CGRP(8-37) or anti-AM monoclonal antibody significantly enhanced the basal and 10(-6) mol/L angiotensin II-stimulated [14C]phenylalanine incorporation. In summary, cultured neonatal rat cardiomyocytes produce and secrete AM, and the secreted AM inhibits the protein synthesis of these cells. Thus, AM may act on cardiomyocytes as an autocrine or a paracrine factor modulating the cardiac growth.

Short-term modulation of the renin-angiotensin system does not alter plasma adrenomedullin concentration in humans.

OBJECTIVE: Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are novel hypotensive peptides produced from the same precursor. A relationship between AM and the renin-angiotensin-aldosterone (RAA) axis was reported in several studies, but the response of the above two peptides to short-term modulation of the RAA axis in humans is not yet clear. Here, we assessed the responses of AM and PAMP in patients with varying RAA system status, including renovascular hypertension (RVH) and primary aldosteronism (PA). DESIGN AND METHODS: Essential hypertension (EHT), RVH and PA patients were hospitalized and maintained on a standard diet (NaCl 10 g/day). The patients underwent a captopril (25 mg) loading test. A renin-secretion stimulating test (furosemide 1 mg/kg, i.v. +2 h of walking) and an ACTH loading test were performed for the PA patients. The plasma renin activity (PRA), plasma aldosterone concentration (PAC), and plasma AM and PAMP levels were monitored before and after the loadings. RESULTS: In the basal state, significantly higher concentrations of AM and PAMP were shown in the RVH patients compared to the other groups. AM and PAMP were significantly correlated with PRA but not PAC in all patients. The AM and PAMP levels were not affected by the captopril loading with or without a hypotensive reaction. The AM and PAMP levels were increased only slightly despite the large increase in PAC induced in the PA patients by the renin-secretion stimulating and ACTH loading tests. CONCLUSION: The responses of plasma AM and PAMP to a short-term modulation of the RAA system were relatively small, despite the correlations observed between PRA and AM or PAMP.

Purification and characterization of PAMP-12 (PAMP[9-20]) in porcine adrenal medulla as a major endogenous biologically active peptide.

Proadrenomedullin N-terminal 20 peptide (PAMP-20) is a potent hypotensive peptide processed from the adrenomedullin (AM) precursor. We developed a specific radioimmunoassay which recognizes the C-terminal region of PAMP-20. Using this radioimmunoassay, the distribution of immunoreactive (ir-) PAMP was determined in porcine tissues. High concentrations of ir-PAMP were observed in the adrenal medulla and in the atrium, and these values were comparable to the corresponding concentrations of ir-AM. The concentration of ir-PAMP was almost the same as that of ir-AM in the kidney, while ir-PAMP was significantly lower than ir-AM in the ventricle, lung, and aorta. Reversed-phase high performance liquid chromatography in each porcine tissue sample revealed that two major peaks of ir-PAMP existed: one emerged at a position identical to that of authentic porcine PAMP-20; the other unknown peak was eluted earlier. The unknown peptide was purified to homogeneity from porcine adrenal medulla, and its complete amino acid sequence was determined. This peptide was found to be PAMP[9-20] with a C-terminal amide structure, and was named PAMP-12. Intravenous injections of PAMP-12 in anesthetized rats showed a significant hypotensive effect in a dose-dependent fashion, and the effect was comparable to that of PAMP-20. These data indicate that PAMP-12, a major component of ir-PAMP, is processed from the AM precursor, as is PAMP-20, and may participate in cardiovascular control.

Plasma adrenomedullin in cerebrovascular disease: a possible indicator of endothelial injury.

BACKGROUND: Cultured vascular endothelial and smooth muscle cells actively produce adrenomedullin, a novel vasodilator peptide discovered in human pheochromocytoma tissue. This present study was designed to determine whether the plasma level of adrenomedullin is a useful indicator for estimating the degree of endothelial injury in patients with atherosclerotic disease. METHODS: We used a radioimmunoassay to measure plasma adrenomedullin concentrations in 51 patients with chronic cerebrovascular disease (34 infarctions and 17 haemorrhages) and in 10 subjects without symptomatic cerebrovascular disease. We also measured the plasma concentrations of thrombomodulin and endothelin as markers of endothelial injury. The patients were divided into three groups (A, B, and C) on the basis of the number of risk factors for atherosclerosis: hypertension, hyperlipidemia, smoking, low HDL-cholesterol, diabetes mellitus, and hyperuricaemia. Group A (68.7+/-2.7 years) consisted of patients with 0 or 1 risk factors; B (68.3+/-4.2 years) those with 2 risk factors; and C (69.2+/-3.6 years) those with 3 or more risk factors. RESULTS: The plasma concentration of adrenomedullin in these patients showed a significant positive correlation with age (r=0.33, p<0.05), as well as with the plasma concentrations of thrombomodulin (r=0.54, p<0.001) and endothelin (r=0.53, p<0.001). Moreover, the plasma concentrations of adrenomedullin and thrombomodulin (p<0.005 and p<0.02, respectively) tended to be higher in Group B and to be significantly higher in Group C as compared to Group A. Plasma adrenomedullin concentrations did not, however, significantly differ between the infarction and haemorrhage patients. CONCLUSIONS: These findings suggest that the plasma adrenomedullin concentrations reflect the degree of endothelial injury in patients with atherosclerotic disease.

Plasma adrenomedullin in patients with primary aldosteronism.

Adrenomedullin (AM) is a novel hypotensive peptide originally isolated from the pheochromocytoma tissue of humans. To examine the pathophysiological role of AM in primary aldosteronism (PA), the plasma concentration of AM in patients with PA was measured with a specific radioimmunoassay and compared to that in age- and sex-matched healthy normotensive subjects. In addition, the concentrations of AM as well as catecholamines in the plasma from both the adrenal vein and the inferior vena cava (IVC) were measured to determine whether or not the circulating AM in these PA patients is supplied from the adrenal medulla, which contains a much higher concentration of AM than any other human tissue does. The plasma concentration of AM in the PA patients (4.57 +/- 0.32 fmol/mL, n = 6) was significantly (P < .01) higher than that in the healthy subjects (3.06 +/- 0.20 fmol/mL, n = 12). A significant positive correlation (r = 0.62, P < .01) was observed between the mean blood pressure and the plasma AM level. The AM concentration in plasma from the adrenal vein was almost the same level as that from the IVC although the concentrations of both epinephrine and norepinephrine in the adrenal vein were much higher than those in the IVC. Therefore, it seems unlikely that the plasma AM in the PA patients is mainly supplied from the adrenal medulla. Judging from the potent hypotensive activity of AM, the present findings suggest that AM participates in defense mechanisms acting against the elevation of blood pressure in the patients with PA.

Human proadrenomedullin N-terminal 20 peptide in pheochromocytoma and normal adrenal medulla.

Proadrenomedullin N-terminal 20 peptide (PAMP) is a novel hypotensive peptide found in adrenomedullin precursor. Using a radioimmunoassay for human PAMP, we purified immunoreactive PAMP (ir-PAMP) from human pheochromocytoma and determined its complete amino acid sequence. The major component of PAMP-like immunoreactivity was found to be PAMP [1-20] NH2 with an amino acid sequence identical to that of the deduced amino acid sequence by cDNA analysis. Both ir-PAMP and ir-adrenomedullin were found to be abundant in normal adrenal medulla as well as pheochromocytoma tissue arising from adrenal medulla, and there was a significantly (p < 0.05) positive correlation between ir-adrenomedullin and ir-PAMP concentrations in these tissues. However, the PAMP/adrenomedullin ratio in pheochromocytoma tissues (0.197 +/- 0.013) was significantly (p < 0.005) lower than that in adrenal medullae (0.384 +/- 0.041). The present data indicate that PAMP is biosynthesized from adrenomedullin precursor, but the biosynthesis or metabolism of PAMP in pheochromocytoma may be different from that of normal adrenal medulla.