C-type natriuretic peptide is synthesized and secreted from leukemia cell lines, peripheral blood cells, and peritoneal macrophages.

OBJECTIVE: C-type natriuretic peptide (CNP) is the third member of the natriuretic peptide family. Cultured endothelial cells secrete CNP, and its secretion rate from the endothelial cells is augmented by lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha, which participate in the pathophysiology of inflammation. In this study, we investigated the regulation of CNP secretion from monocytes and macrophages to estimate its contribution to the progression of inflammation. MATERIALS AND METHODS: CNP secretion rates from two human leukemia cell lines (THP-1 and HL-60), human peripheral blood lymphocytes, granulocytes, monocytes, monocyte-derived macrophages, and mouse peritoneal macrophages were measured under conditions with or without stimulation. Immunoreactive CNP levels in the culture media of these cells were measured by a specific radioimmunoassay. RESULTS: The secretion rates of CNP from THP-1 and HL-60 cells were augmented according to the degree of their differentiation into macrophage-like cells under the stimulation with phorbol ester. Peripheral blood monocytes also increased the CNP secretion rate after their differentiation into macrophages. Retinoic acid elicited synergistic effects on the CNP secretion rate from HL-60 cells when administered with lipopolysaccharide, interferon-gamma, interleukin-1beta, tumor necrosis factor-alpha, or phorbol ester. In contrast, the phorbol ester-stimulated CNP secretion rate from THP-1 cells was suppressed with dexamethasone, which inhibits monocyte differentiation into macrophage. CONCLUSIONS: The secretion rate of CNP from monocytes was shown to be regulated based on the degree of their differentiation. This study provides evidence that the monocyte/macrophage system is one of the sources of CNP, especially under inflammatory conditions.

Cardiac fibroblasts are major production and target cells of adrenomedullin in the heart in vitro.

OBJECTIVE: Adrenomedullin (AM) is a potent vasodilator peptide. Plasma AM concentration is increased in patients with various heart diseases, and both myocytes (MCs) and non-myocytes (NMCs) secrete AM and express its receptors. These facts suggest that cardiac cells possess an autocrine/paracrine capability mediated by AM. METHODS: MCs and NMCs were prepared from cardiac ventricles of neonatal rats. AM and endothelin-1 concentrations were measured by radioimmunoassays, and interleukin-6 level by a specific bioassay. Total nitrite/nitrate contents were measured with a fluorescence assay kit. RESULTS: A basal secretion rate of AM from NMCs was 2.8-fold higher than that from MCs. Interleukin-1beta, tumor necrosis factor-alpha and lipopolysaccharide stimulated AM secretion from NMCs but not from MCs. AM stimulated interleukin-6 production in the presence of these cytokines or lipopolysaccharide, which was more prominent in NMCs. In the presence of interleukin-1beta, AM augmented nitric oxide synthesis 2.7-fold in NMCs, but slightly in MCs. NMCs secreted endothelin-1 at a rate nine times higher than MCs, and AM inhibited endothelin-1 secretion from NMCs. CONCLUSION: This in vitro study suggests that AM in the heart is mainly produced in NMCs and exerts its effects through NMCs, especially under inflammatory conditions.

Interaction between monocytes and vascular endothelial cells induces adrenomedullin production.

Adrenomedullin (AM), a potent vasodilator peptide, has natriuretic effects, and its plasma concentration is elevated in cardiovascular diseases. In the present study, we investigated the induction of AM expression due to interactions between THP-1 cells (human monocytic cell line) and human umbilical cord vein endothelial cells (HUVECs). AM levels in the culture medium were measured by radioimmunoassay. The luciferase vector containing the 5'-flanking region of the human AM gene was transfected into either HUVECs or THP-1 cells. Addition of THP-1 cells to HUVECs for 48 h induced marked increases in AM levels, which were 16-fold higher than those of HUVECs alone. Luciferase vectors containing the 5'-flanking region of human AM gene (pLCF-1534) were transferred into THP-1 cells or HUVECs. Addition of THP-1 cells to pLCF-1534-transfected HUVECs induced an increase in luciferase activity in cell lysates, which was 5-fold higher than that of the transfected HUVECs alone. In contrast, the luciferase activity of lysates from pLCF-1534-transfected THP-1 cells was not affected by coculture with HUVECs. A separate coculture experiment revealed that direct contact of THP-1 cells and HUVECs contributed to enhanced AM production in the cocoulture. Co-incubation of the cell membrane fraction from THP-1 cells augmented AM production by HUVECs. Both anti-interleukin (IL)-1alpha antibody and IL-1 receptor antagonist significantly inhibited AM production in the cocultures. The cell-to-cell interaction between monocytes and HUVECs induces AM production by HUVECs, which may play an important role in the pathogenesis of vascular disorders.

Regulation of endothelin-1 production in cultured rat vascular smooth muscle cells.

Endothelin-1 (ET-1) is secreted from all rat vascular smooth muscle cells (VSMCs) examined, in addition to endothelial cells (ECs). An average secretion rate of ET-1 from rat VSMCs was determined to be 10% that excreted from ECs. We examined the effects of 22 substances on ET-1 secretion from VSMCs and compared them with those from ECs. Transforming growth factor-beta1 (TGF-beta), acidic and basic fibroblast growth factors, epidermal growth factor, angiotensin II, and adrenaline stimulated ET-1 secretion from VSMCs, whereas forskolin, thrombin, and platelet-derived growth factor-BB reduced it. Only TGF-beta and phorbol ester elicited consistent effects on ET-1 secretion from VSMCs and ECs. Regulation of ET-1 and adrenomedullin secretion from VSMCs was distinctly different. These data suggest that ET- 1 production in VSMCs is regulated by a mechanism separate from that in ECs and from adrenomedullin production in VSMCs. Chromatographic analysis showed immunoreactive ET-1 secreted from VSMCs was mainly composed of big ET- 1, whereas approximately 90% of that from ECs was ET-1. By TGF-beta stimulation of VSMCs, the ratio of big ET-1 to ET-1 was further increased. Because big ET-1 is converted into ET-1 only on the surface of the ECs in the culture system, big ET-1 secreted from the VSMCs may function as a mediator transmitting a signal from VSMCs to ECs in vivo.

Regulation of adrenomedullin secretion from cultured cells.

Characterization of immunoreactive adrenomedullin (AM) secreted from cultured human vascular smooth muscle cells and 7 other cells indicates that AM is synthesized and secreted from all cultured cells we surveyed. The secretion rate of AM measured ranges from 0.001-6.83 fmol/10(5) cells/h, and endothelial cells, vascular smooth muscle cells and fibroblasts generally secrete AM at high rates. Based on the results of regulation of AM secretion from vascular wall cells, fibroblasts, macrophages and other cells measured in this and previous studies, AM secretion is found to be generally stimulated by inflammatory cytokines, lipopolysaccharide (LPS) and hormones. Especially, vascular smooth muscle cells and fibroblasts elicited uniform and strong stimulatory responses of AM secretion to tumor necrosis factor (TNF), interleukin-1 (IL-1), LPS and glucocorticoid, but endothelial cells did not elicit such prominent responses. AM secretion of monocyte-macrophage was mainly regulated by the degree of differentiation into macrophage and activation by LPS and inflammatory cytokines including interferon-gamma. The other examined cells showed weaker responses to LPS and IL-1. Although cultured cells may have been transformed as compared with those in the tissue, these data indicate that AM is widely synthesized and secreted from most of the cells in the body and functions as a local factor regulating inflammation and related reactions in addition to as a potent vasodilator. The responses of AM secretion to LPS and inflammatory cytokines suggest that fibroblasts, vascular smooth muscle cells and macrophage are the major sources of AM in the septic shock.

Adrenomedullin suppresses interleukin-1beta-induced tumor necrosis factor-alpha production in Swiss 3T3 cells.

We demonstrated that adrenomedullin (AM) inhibited interleukin-1beta-induced tumor necrosis factor-alpha (TNF-alpha) secretion and gene transcription in Swiss 3T3 fibroblasts maximally to 23% and 18% of control, while the other peptides elevating intracellular cAMP levels elicited much weaker effects. AM rapidly reduced the gene transcript level of TNF-alpha, inducing a maximal effect within 1 h. The inhibitory effect of AM was restored with an AM receptor antagonist as well as a cAMP-dependent protein kinase inhibitor. These findings indicate that AM is a potent and quick suppressor of TNF-alpha production in Swiss 3T3 cells acting through the cAMP protein kinase A pathway. As TNF-alpha is a major inflammatory cytokine and stimulates AM production in fibroblasts, AM is deduced to be an autocrine or paracrine factor suppressing inflammation through the inhibition of TNF-alpha production.

Effects of thiopental, ketamine, etomidate, propofol and midazolam on the production of adrenomedullin and endothelin-1 in vascular smooth muscle cells.

Endothelin-1 (ET-1) and adrenomedullin (AM) are potent vasoconstrictive and vasodilative peptides. Vascular smooth muscle cells are one of the major producing tissues of these peptides. We investigated effect of thiopental, ketamine, etomidate, midazolam and propofol on the production of ET-1 and AM in cultured rat aorta vascular smooth muscle cells. Rat cultured vascular smooth muscle cells are used and the dose-dependent effect (10(-8)-10(-4) M) of thiopental, ketamine, etomidate, midazolam, and propofol on the production of ET-1 and AM was examined. The concentration of these peptides in the culture medium were measured by radioimmunoassay. Ketamine, etomidate, propofol and midazolam, but not thiopental, decreased the production of ET-1 at a concentration of 10(-4) M and the effect of midazolam is the most potent. Thiopental and etomidate produced small but significant increases in the production of AM at a concentration of 10(-4) M. Ketamine did not affect the production, but propofol and midazolam decreased the production at 10(-4) M and 10(-5) M, respectively. These results suggest that thiopental, ketamine, etomidate, propofol and midazolam differentially affect the production of ET-1 and AM in rat vascular smooth muscle cells.

Production of adrenomedullin in macrophage cell line and peritoneal macrophage.

We demonstrate that adrenomedullin (AM) is produced and secreted from cultured murine monocyte/macrophage cell line (RAW 264.7) as well as mouse peritoneal macrophage. Immunoreactive (IR) AM secreted from RAW 264.7 cells was chromatographically identified to be native AM. To elucidate the regulation mechanism of AM production in macrophage, we examined the effects of various substances inducing differentiation or activation of monocyte/macrophage. Phorbol ester (TPA), retinoic acid (RA), lipopolysaccharide (LPS), and interferon-gamma (IFN-gamma) increased AM production 1.5-7-fold in RAW 264.7 cells in a dose- as well as time-dependent manner. By LPS stimulation, the AM mRNA level in RAW 264.7 cells was augmented up to 7-fold after 14 h incubation. RA exerted a synergistic effect when administered with TPA, LPS, or IFN-gamma, whereas IFN-gamma completely suppressed AM production in RAW 264.7 cells stimulated with LPS. Dexamethasone, hydrocortisone, estradiol, and transforming growth factor-beta dose-dependently suppressed AM production in RAW 264.7 cells. AM production was also investigated in mouse peritoneal macrophage. Primary mouse macrophage secreted IR-AM at a rate similar to that of RAW 264.7 cells, and its production was enhanced 9-fold by LPS stimulation. AM was found to increase basal secretion of tumor necrosis factor alpha (TNF-alpha) from RAW 264.7 cells, whereas AM suppressed the secretion of TNF-alpha and interleukin-6 from that stimulated with LPS. Thus, macrophage should be recognized as one of the major sources of AM circulating in the blood. Especially in cases of sepsis and inflammation, AM production in macrophage is augmented, and the secreted AM is deduced to function as a modulator of cytokine production.

Development of guanine analyzer to measure activity of guanylate cyclase.

A previous analyzer of adenine compounds by high-performance liquid chromatography was converted for the determination of guanine, its nucleoside and nucleotides by a post-column fluorescence derivatization with phenylglyoxal (PGO) in place of bromoacetoaldehyde. The gel filtration column (Asahipak GS-320H) was used for separation by a mobile phase consisting of 25 mM sodium citrate buffered (pH 4.0)-150 mM NaCl solution and CH3CN (85:15, v/v) containing 15 mM PGO. The separated analytes reacted with flow through PGO in a reaction coil at 90 degrees C into fluorescent derivatives. Those derivatives were detected fluorimetrically, highly selective and quantitatively. The activity of soluble guanylate cyclase (sGC) in the neuroblastoma N1E-115 cell was measured by tracing the peak height of cGMP synthesized from substrate GTP using this guanine analyzer. The sensitivity of the present method was lower than the radioisotope method. However, our modified method was simpler, safer and quicker than the radioisotope method. Furthermore, this method could trace other guanine compounds simultaneously, allowing measurement of guanine metabolizing enzymatic activity. Therefore, it will be useful for screening of effectors on sGC.

Adrenomedullin production is correlated with differentiation in human leukemia cell lines and peripheral blood monocytes.

We demonstrated that adrenomedullin (AM) is produced and secreted from human leukemia cell lines (THP-1 and HL-60) as well as peripheral blood granulocytes, lymphocytes, monocytes and monocyte-derived macrophages. Immunoreactive AM accumulated in the culture media of THP-1 and HL-60 cells increased according to their differentiation into macrophage-like cells. Retinoic acid exerted synergistic effects on AM secretion from THP-1 and HL-60 cells when administered with tumor necrosis factor-alpha, lipopolysaccharide or 12-O-tetradecanoyl phorbol-13-acetate. AM was shown to increase the scavenger receptor activity on THP-1 cells. Thus, monocytes/macrophages should be recognized as sources of AM, and the secreted AM may modulate the function of macrophages.

Adrenomedullin production in fibroblasts: its possible function as a growth regulator of Swiss 3T3 cells.

In addition to endothelial cells and vascular smooth muscle cells, we demonstrated that adrenomedullin (AM) is synthesized and secreted from fibroblasts, Swiss 3T3, Hs68, and NHLF cells, in a native and biologically active form. Synthesis and secretion of AM from these fibroblasts was regulated by inflammatory cytokines, such as tumor necrosis factor and interleukin-1, lipopolysaccharide, growth and differentiation factors, and hormones in a manner similar to that of vascular smooth muscle cells and endothelial cells. Tumor necrosis factor-alpha, interleukin-1beta, and dexamethasone elevated AM secretion, whereas transforming growth factor-beta1 and interferon-gamma suppressed it in these three fibroblasts. Swiss 3T3 cells were shown to express receptors specific for AM by both cAMP production and receptor binding assay, and AM was found to stimulate DNA synthesis of quiescent cells through the cAMP-mediated pathway. AM secreted from Swiss 3T3 cells was also confirmed to augment cAMP production and DNA synthesis in the cells themselves. These effects were inhibited by a neutralizing monoclonal antibody against AM. These findings raise the possibility that AM functions as a growth regulator in the case of Swiss 3T3 cells. As AM receptors are widely distributed, AM secreted from fibroblast may play a role as a local regulator in mesenchymal cells of inflammatory or wounded regions.

Regulation of adrenomedullin production in rat endothelial cells.

Adrenomedullin (AM) is a potent vasorelaxant peptide recently identified in extracts of pheochromocytoma. We have found that AM is actively secreted from endothelial cell (EC) and vascular smooth muscle cell (VSMC). To elucidate the function of AM secreted from EC, the effects of 43 substances on secretion of AM from cultured rat EC were examined in this study. We first confirmed that synthesized AM was not stored but constitutively secreted from EC, indicating that the amount secreted could be used as an index of AM synthesis in EC. EC secreted AM at a rate 5.8 times higher than VSMC, and AM gene transcription in EC significantly contributed to the total aortic AM messenger RNA. Tumor necrosis factor, interleukin-1, and lipopolysaccharide augmented AM secretion from EC, showing cooperative effects, which suggests that AM secreted from EC participates in the induction of hypotension in septic shock. Transforming growth factor beta1 and FCS suppressed AM secretion but stimulated endothelin-1 (ET-1) secretion. Thrombin potently stimulated AM secretion from EC but suppressed it from VSMC. Thyroid hormone and phorbol ester increased AM and ET-1 secretion but to a lesser extent. Interferon-gamma inhibited AM secretion from EC, whereas oxidized LDL stimulated it. Regulation of AM production in EC is found to be similar to that of VSMC with several exceptions, but AM and ET-1 production in EC are deduced to be controlled independently and by different mechanisms. AM stimulates cAMP production in EC, though receptors expressed on cultured rat EC are not specific to AM but to calcitonin gene-related peptide. Based on these findings, AM production in EC is thought to be regulated by a variety of substances coming from blood and neighboring cells, and the secreted AM is deduced to dilate blood vessels as an endothelium-derived relaxing factor competing with ET-1.

Adrenomedullin stimulates interleukin-6 production in Swiss 3T3 cells.

Adrenomedullin (AM) has very recently been demonstrated to be produced and secreted from fibroblasts. The production of AM in the fibroblasts is augmented by inflammation-related substances, and Swiss 3T3 fibroblast cells express AM specific receptors coupled with adenylate cyclase. To assess the functions of AM secreted from fibroblasts, we measured the effect of AM on production in Swiss 3T3 cells of interleukin-6 (IL-6), a typical cytokine involved in the general inflammatory reactions. AM stimulated basal secretion of IL-6 5.5-fold, while other peptides elicited much weaker stimulatory effects. The effect of AM was inhibited with an AM receptor antagonist and a cAMP-dependent protein kinase (PKA) inhibitor. Furthermore, AM remarkably potentiated stimulatory effects of tumor necrosis factor-alpha, IL-1 beta and lipopolysaccharide on IL-6 production. This stimulatory effect of AM was induced through activation of gene transcription, which reached maximum within 30 min. These findings verify that AM is a rapid and extraordinarily potent regulator of IL-6 production in Swiss 3T3 cells acting through the cAMP-PKA pathway. The data thus obtained suggest that AM is a peptidergic regulator of inflammation.