Adrenomedullin mRNA Levels Research Articles

Hypoxia Regulates the Expression of the Adrenomedullin and HIF-1 Genes in Cultured HL-1 Cardiomyocytes

Adrenomedullin (AM) is a hypotensive protein expressed in a variety of cells and tissues. We observed previously that the expression of the adrenomedullin gene increases substantially in the developing rat heart and in cultured adult rat ventricular cardiac myocytes in response to hypoxia as a function of time. An adrenomedullin promoter–luciferase reporter construct was used to show that this increase in adrenomedullin mRNA resulted from increased transcription in response to hypoxia. We report here additional evidence documenting that this hypoxia-induced transcription of the adrenomedullin gene is regulated by the hypoxia-inducible factor-1 (HIF-1) transcription factor. We used Northern blot analysis to show an increase in the levels of AM and HIF-1α mRNA but not HIF-1β mRNA in the HL-1 cardiac myocyte cell line in response to hypoxia. Furthermore, Western blot analysis revealed that the levels of both HIF-1α and HIF-1β protein increased under hypoxic conditions. Data from electrophoretic mobility shift assays indicate that the heterodimeric HIF-1 complex binds to the HIF-1-responsive elements. Combined data from these studies demonstrate that the AM gene is regulated by hypoxia-responsive elements localized in the AM promoter region.

Induction of adrenomedullin by hypoxia in cultured human coronary artery endothelial cells

To explore the role of adrenomedullin (ADM) in pathophysiology of ischemic heart disease, we investigated the effects of hypoxia on the production and secretion of ADM in cultured human coronary artery endothelial cells. Treatment with hypoxia (5% CO 2/94% N 2/1% O 2) for 6 and 12 h increased expression levels of ADM mRNA 2.2-fold and fivefold compared with the normoxia control, respectively. The levels of immunoreactive ADM in the media were increased by 12-h hypoxia about fivefold compared with the control (39.0 ± 1.1 fmol/10 5 cells per 12 h under hypoxia and 7.9 ± 0.4 fmol/10 5 cells per 12 h under normoxia; P < 0.01, n = 4, mean ± SEM). Reverse-phase high-performance liquid chromatography of the extracts of culture media under normoxia and hypoxia showed one major peak eluting in the position of human ADM standard. The production and secretion of ADM were increased in cultured human coronary artery endothelial cells under hypoxia. ADM may therefore play an important pathophysiological role in ischemic heart disease.

Hyperglycemia Increases Vascular Adrenomedullin Expression

We have reported that plasma adrenomedullin (AM) in hyperglycemic patients was significantly increased compared with normal volunteers. In this report we examined the effects of hyperglycemia on AM expression in the vasculature, the main site of AM production. AM mRNA level in the aorta was higher in the diabetic rats than in the control rats. AM mRNA level and protein kinase C (PKC) activity in cultured vascular smooth muscle cells (VSMC) increased as the glucose concentration in the medium changed from 100mg/dl to 450mg/dl. PKC inhibitors blocked this increase of AM mRNA. Similar osmotic change with mannitol had no effect on AM expression. We conclude that (1) hyperglycemia increases vascular AM expression through PKC-dependent pathway, and (2) the elevated plasma AM in hyperglycemic patients originates from the glucose induced vascular AM expression. We propose the possible role of AM in the pathogenesis of diabetic vascular complications.

Increased urinary levels of adrenomedullin in patients with cystitis.

In this study, we examined urinary levels of adrenomedullin (AM) in 18 healthy volunteers and 18 patients with cystitis. We also compared urinary levels of AM in 11 patients with cystitis before and after antibiotic treatment. Urinary AM concentrations were measured by a radioimmunoassay specific for human AM. Urinary AM levels in patients with cystitis were significantly elevated compared with those of healthy volunteers and correlated positively with the number of urine leukocytes. By antibiotic treatment, urinary AM levels significantly decreased as compared with before the treatment. By RNA blot analysis of AM transcript, we detected significant levels of AM mRNA in canine urinary bladder and ureter. Intravenous administration of lipopolysaccharide elevated the AM mRNA level in the urinary bladder. These data suggest that infection and inflammation stimulate AM production in the urinary tract, which results in increased urinary AM levels in patients with cystitis. Based on these results, it is deduced that AM participates in the pathophysiology of cystitis, and its urinary level could be used as an index of the degree of cystitis.

Adrenomedullin attenuates the hypertension in hypertensive pregnant rats induced by NG-nitro- l-arginine methyl ester

We examined the effect of adrenomedullin on the cardiovascular system of an animal model for preeclampsia. An inhibitor of nitric oxide synthase, N G-nitro- l-arginine methyl ester ( l-NAME), was infused subcutaneously into rats at a constant rate from day 14 of pregnancy to make an animal model for preeclampsia. Adrenomedullin was continuously infused intravenously at a dose of 3 or 10 pmol/h from day 17 of pregnancy. The basal systolic blood pressure was significantly higher in the l-NAME treated rats than in the control rats. The adrenomedullin administration at day 19 of pregnancy showed a significant decrease in the blood pressure in the l-NAME treated rats than in vehicle rats during infusion. The blood pressure of normal pregnant rats did not significantly decrease by adrenomedullin infusion. The adrenomedullin decreased pup mortality of the l-NAME treated rats. Adrenomedullin attenuated the l-NAME induced hypertension and pup mortality. On the other hand, adrenomedullin administration in both pregnant rats in early gestation (5–11 days of pregnancy) and in non-pregnant rats did not show any significant effect on l-NAME-induced hypertension. The adrenomedullin mRNA level was predominantly expressed at high levels in the ovary, uterus and placenta, but at low levels in other tissues in pregnant rats in late gestation. The adrenomedullin mRNA level of the l-NAME treated rats in placenta decreased more than in the normal pregnant rats in late gestation ( P<0.05). These findings suggest that the adrenomedullin might play an important role in the regulation of the cardiovascular system of the mother and fetoplacental unit in rats.

Expression and effect of adrenomedullin on rat granulosa cell.

Adrenomedullin (AM) is a potent hypotensive peptide recently discovered in extracts of human pheochromocytoma. To elucidate the regulation of AM production in the ovary, the effect of gonadotropin on the production of AM was studied in the cultured rat granulosa cells. A Northern blot analysis of the LH receptor and adrenomedullin yielded a major hybridizing band at 5.4 kb and 1.6 kb, respectively. In our culture system of rat granulosa cells, without any stimulus, the LH receptor mRNA was undetectable and the AM mRNA level was stably expressed for 6 days. FSH significantly induced LH receptor mRNA and suppressed AM mRNA for 4 days of culture and with the addition of hCG after 2 days of pretreatment with FSH, AM mRNA levels were markedly suppressed. FSH and 8-Br-cAMP significantly increase LH receptor mRNA and suppress AM mRNA in a dose-dependent manner. These data indicated that the differentiation of granulosa cells mediated by gonadotropins were associated by suppression in AM expression through a cAMP-dependent mechanism. On the other hand, AM stimulated a rapid rise in intracellular cAMP levels, which peaked within 15 min of addition, in a dose-dependent manner with a maximal response seen at 100 nM. Additionally, AM enhanced the effects of FSH, acting additionally to produce cAMP in the cells. AM may play a role in the process of granulosa cell differentiation as a local regulator through an autocrine/paracrine mechanism.

Adrenomedullin regulation by thyroid hormone in the rat

To investigate the effect of thyroid hormone on adrenomedullin (AM) changes during hyper- and hypothyroid states, we evaluated plasma AM concentrations and AM mRNA levels in lung tissue from hyper- and hypothyroid rats. AM peptide concentrations were significantly higher in plasma from hyperthyroid rats and lower in plasma from hypothyroid rats compared with control rats. AM mRNA transcripts were significantly increased in lung tissue from hyperthyroid rats and significantly decreased in lung tissue from hypothyroid rats compared with normal rats. These changes are paralleled alterations in AM production in cultured rat vascular smooth muscle cells, in which AM is regulated at the transcriptional level by thyroid hormone. Thus, thyroid hormone positively regulates AM production in vivo.

Induction of adrenomedullin mRNA and protein by lipopolysaccharide and paclitaxel (Taxol) in murine macrophages.

Lipopolysaccharide (LPS), a potent inflammatory stimulus derived from the outer membrane of gram-negative bacteria, has been implicated in septic shock. Plasma levels of adrenomedullin (AM), a potent vasorelaxant, are increased in septic shock and possibly contribute to the characteristic hypotension. As macrophages play a central role in the host response to LPS, we studied AM production by LPS-stimulated macrophages. When peritoneal exudate macrophages from C3H/OuJ mice were treated with protein-free LPS (100 ng/ml) or the LPS mimetic paclitaxel (Taxol; 35 microM), an approximately 10-fold increase in steady-state AM mRNA levels was observed, which peaked between 2 and 4 h. A three- to fourfold maximum increase in the levels of immunoreactive AM protein was detected after 6 to 8 h of stimulation. While LPS-hyporesponsive C3H/HeJ macrophages failed to respond to protein-free LPS with an increase in steady-state AM mRNA levels, increased levels were observed after stimulation of these cells with a protein-rich (butanol-extracted) LPS preparation. In addition, increased AM mRNA was observed following treatment of either C3H/OuJ or C3H/HeJ macrophages with soluble Toxoplasma gondii tachyzoite antigen or the synthetic flavone analog 5, 6-dimethylxanthenone-4-acetic acid. Gamma interferon also stimulated C3H/OuJ macrophages to express increased AM mRNA levels yet was inhibitory in the presence of LPS or paclitaxel. In vivo, mice challenged intraperitoneally with 25 microg of LPS exhibited increased AM mRNA levels in the lungs, liver, and spleen; the greatest increase (>50-fold) was observed in the liver and lungs. Thus, AM is produced, by murine macrophages, and furthermore, LPS induces AM mRNA in vivo in a number of tissues. These data support a possible role for AM in the pathophysiology of sepsis and septic shock.

Coronary pressure as a determinant of B-type natriuretic peptide gene expression in isolated perfused adult rat heart

The role of coronary flow in the regulation of ventricular B-type natriuretic peptide (BNP) gene expression was studied in isolated perfused rat heart preparation. The increase of coronary flow from 5 ml/min to 20 ml/min for 2 h resulted in a 132 ± 6 mm Hg increase in aortic perfusion pressure. The changes in BNP mRNA and immunoreactive BNP (IR-BNP) levels in response to hemodynamic stress were compared to those of c-fos and adrenomedullin (ADM) gene expression. The increase of coronary flow resulted in 1.5-fold increases in the left ventricular BNP mRNA ( P < 0.001) and IR-BNP ( P < 0.05) levels in 2-month old rats. There was also a 1.5-fold ( P < 0.05) increase in ventricular c-fos mRNA levels, whereas ADM mRNA levels decreased by 74% ( P < 0.001) in the left ventricle. In 18-month old rats, the increase in coronary flow decreased left and right ventricular BNP mRNA levels by 18% ( P < 0.05) and 39 % ( P < 0.001), respectively. There were no changes in IR-BNP peptide and c-fos mRNA levels, whereas ADM mRNA levels decreased by 46% ( P < 0.001) in the left ventricles. The results show that increased aortic perfusion pressure results in differential expression of cardiac genes including up-regulation of ventricular BNP and c-fos gene expression and down-regulation of ADM gene expression. Furthermore, aging seems to elevate the threshold at which hemodynamic stress of the heart results in a response at BNP gene level.

Adrenomedullin Gene Expression Is Developmentally Regulated and Induced by Hypoxia in Rat Ventricular Cardiac Myocytes

Adrenomedullin is a recently discovered hypotensive peptide that is expressed in a variety of cell and tissue types. Using the technique of differential display, the adrenomedullin gene was observed to be differentially expressed in developing rat heart. Reverse transcription-polymerase chain reaction analysis revealed that the level of adrenomedullin mRNA was significantly higher in adult ventricular cardiac muscle as compared with embryonic day 17 ventricular cardiac muscle. Adrenomedullin receptor mRNA was constitutively expressed throughout development of the ventricular heart. Two potential hypoxia-inducible factor-1 (HIF-1) consensus binding sites were identified in the mouse adrenomedullin promoter at -1095 and -770 nucleotides from the transcription start site. Exposure of cultured adult rat ventricular cardiac myocytes to hypoxia (1% O2) resulted in a significant, time-dependent increase in adrenomedullin mRNA levels. Transfection studies revealed that the 5'-flanking sequence of adrenomedullin was capable of mediating a hypoxia-inducible increase in transcription. Mutation of the putative HIF-1 consensus binding sites revealed that the major regulatory sequence that mediates the hypoxia-inducible transcriptional response is located at -1095. These data demonstrate that the adrenomedullin gene is developmentally regulated in ventricular cardiomyocytes, that adrenomedullin transcription can be induced by hypoxia, and that this response is primarily mediated by HIF-1 consensus sites in the adrenomedullin promoter.

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.

Differential Regulation of Ventricular Adrenomedullin and Atrial Natriuretic Peptide Gene Expression in Pressure and Volume Overload in the Rat

1. Adrenomedullin is a recently discovered vasodilating and natriuretic peptide whose physiological and pathophysiological roles remain to be established. Like atrial natiuretic peptide adrenomedullin is expressed in the left ventricle. Ventricular expression of atrial natriuretic peptide is known to be markedly increased by volume or pressure overload. In this study we investigated whether ventricular expression of adrenomedullin is similarly stimulated under such conditions. 2. Ventricular adrenomedullin and atrial natriuretic peptide mRNA levels as well as those of a loading control mRNA (glyceraldehyde-3-phosphate dehydrogenase) were quantified by Northern blot analysis in (a) rats with severe post-infarction heart failure induced by left coronary ligation at 30 days post-surgery and (b) in rats with pressure-related cardiac hypertrophy induced by aortic banding at several time points (0.5, 1 and 4 h, and 1, 4, 7 and 28 days) after surgery. Levels were compared with those in matched sham-operated controls. 3. The mRNA level of atrial natriuretic peptide was markedly increased (8-10-fold) in the left ventricle of animals with post-infarction heart failure. In contrast, there was only a modest (40%) increase in the level of adrenomedullin mRNA. In rats with pressure-induced cardiac hypertrophy the ventricular level of atrial natriuretic peptide mRNA was again markedly increased (maximum 10-fold). The increase was first noticeable at 24 h post-banding and persisted until 28 days. In contrast, there was no change in adrenomedullin mRNA level compared with sham-operated rats at any time point. 4. Despite having similar systemic effects, the expression of adrenomedullin and atrial natriuretic peptide in the left ventricle is differently regulated. The findings imply distinct roles for the two peptides. The results do not support an important role for ventricular adrenomedullin expression in the remodelling process that occurs during the development of cardiac hypertrophy but suggest that ventricular adrenomedullin participates in the local and/or systemic response to heart failure.

Glucocorticoid regulation of adrenomedullin in a rat model of endotoxic shock

Wistar rats were injected intravenously with bacterial lipopolysaccharide (LPS) and developed endotoxic shock with severe hypotension. This was accompanied by significantly elevated concentrations of adrenomedullin (AM) in the plasma and expression of high levels of AM mRNA in the lung. Pretreatment of the rats with dexamethasone (DEX) prevented hypotension caused by LPS administration, but plasma AM concentrations and AM mRNA levels in the lung remained elevated. Adrenalectomized (ADX) rats developed a more severe form of circulatory shock in response to a low-dose of LPS. This was accompanied by only a slight increase in circulating AM in the plasma. However, pretreatment of ADX rats with DEX caused substantial elevations of plasma AM concentrations and expression of AM mRNA in the lung. Our studies demonstrate that glucocorticoid upregulates the expression and secretion of AM in vivo, and endogenous glucocorticoid is required for increased AM secretion under certain conditions such as endotoxic Shock.

Induction of Adrenomedullin by Hypoxia and Cobalt Chloride in Human Colorectal Carcinoma Cells

Adrenomedullin (ADM) is a vasodilator peptide, first isolated from human pheochromocytoma. To explore the pathophysiological role of ADM in ischemic conditions, we investigated the effects of hypoxia on ADM production and ADM mRNA expression in a cultured human colorectal carcinoma cell line, DLD-1. Northern blot analysis and radioimmunoassay showed that hypoxia stimulated the accumulation of ADM mRNA in the DLD-1 cells and immunoreactive ADM (ir-ADM) in the cultured media. Exposure to hypoxia for 12 hours increased ADM mRNA levels about 6-fold and ir-ADM levels about 4-fold. Moreover, treatment of DLD-1 cells with cobalt chloride, which mimics hypoxic states, significantly increased ADM mRNA levels about 18-fold and ir-ADM levels about 4-fold. These results suggest that ADM plays an important role in the pathophysiology of ischemic states.

Decrease in circulating and urine adrenomedullin concentrations in stroke-prone spontaneously hypertensive rats.

Adrenomedullin (AM) is a peptide with potent vasodilatory and hypotensive properties. Plasma AM levels in rats with experimentally induced hypertension, such as Dahl salt-sensitive rats and two-kidney, one-clip hypertensive rats, are higher than those in normotensive rats. We previously noted, however, that plasma AM levels in spontaneously hypertensive rats (SHR) are similar to those in Wistar-Kyoto rats. To define the role of AM in rats with severe hypertension, we investigated changes in circulating and tissue AM levels in stroke-prone spontaneously hypertensive rats (SHRSP/Izm). The immunoreactive rat AM levels in plasma, urine, and tissue measured with a sensitive radioimmunoassay, and the AM mRNA levels in various tissues in 15-wk-old SHRSP/Izm were compared with those in age-matched Wistar-Kyoto rats (WKY/Izm). The plasma and urinary AM levels in SHRSP/Izm were significantly lower than those in WKY/Izm [plasma AM, 2.14+/-0.06 (SE) vs. 3.24+/-0.16 fmol/ml, p< 0.001; urinary AM, 16.36+/-3.21 vs. 36.12+/-6.09 fmol/ml, p< 0.01]. A negative correlation was found between the plasma AM level and the systolic blood pressure in both SHRSP/Izm and WKY/Izm. Reverse-phase high-performance liquid chromatography showed that the molecular components of plasma immunoreactive AM in SHRSP/Izm were similar to those in WKY/Izm. Furthermore, tissue AM levels in various organs in SHRSP/Izm were not lower than those in WKY/Izm. In conclusion, low levels of circulating AM may contribute to the maintenance of high blood pressure in 15-wk-old SHRSP/Izm. These low plasma AM levels may be caused by accelerated metabolism of circulating AM in SHRSP/Izm.

Oxidative stress increases adrenomedullin mRNA levels in cultured rat vascular smooth muscle cells.

We investigated the effect of oxidative stress on the expression of adrenomedullin (AM) mRNA in cultured rat vascular smooth muscle cells (VSMCs), using diethyldithiocarbamate (DDC), which is known to inhibit endogenous Cu, Zn superoxide dismutase (SOD) and to increase superoxide (O2-). DDC (10 mM) increased O2- levels produced from rat VSMCs in a time-dependent fashion. Concomitantly, DDC increased the expression of AM mRNA for up to 24 h, although it did not affect the expression of beta-actin mRNA. Thus, oxidative stress enhanced AM production in rat VSMCs, which may compensate for oxidative-stress-induced vasoconstriction.

Dexamethasone-induced Up-regulation of Adrenomedullin and Atrial Natriuretic Peptide Genes in Cultured Rat Ventricular Myocytes

To determine whether adrenomedullin (AM), a novel 51-residue vasodilator peptide originally isolated from human pheochromocytoma, is expressed by the heart, and whether the expression of cardiac AM gene is regulated by glucocorticoids, the effect of dexamethasone (DEX) on the expression of protein and mRNA of AM and atrial natriuretic peptide (ANP) was tested in cultured ventricular myocytes prepared from the neonatal rats. Northern blot analysis of rat ventricular myocytes with cDNAs for rat AM and ANP as probes revealed distinct bands corresponding to the sizes of rat AM mRNA (1.6 kb) and rat ANP mRNA (1 kb), respectively. Dexamethasone increased steady-state levels of both AM and ANP mRNA as well as secretion of both AM and ANP immunoreactivity in a time- and dose-dependent manner. The stimulatory effects of dexamethasone were completely abolished by a glucocorticoid antagonist (RU38486) and a RNA synthesis inhibitor (actinomycin D). The approximate half-lives of basal and dexamethasone-induced AM mRNA were about 4 h, suggesting that dexamethasone-induced up-regulation of AM mRNA was unlikely due to its decreased degradation. These data suggest that glucocorticoids directly stimulate gene expression of AM as well as ANP in rat ventricular myocytes.

Adrenomedullin gene expression in the rat heart is stimulated by acute pressure overload: blunted effect in experimental hypertension.

The levels of adrenomedullin (ADM), a newly discovered vasodilating and natriuretic peptide, are elevated in plasma and ventricular myocardium in human congestive heart failure suggesting that cardiac synthesis may contribute to the plasma concentrations of ADM. To examine the time course of induction and mechanisms regulating cardiac ADM gene expression, we determined the effect of acute and short-term cardiac overload on ventricular ADM mRNA and immunoreactive ADM (ir-ADM) levels in conscious rats. Acute pressure overload was produced by infusion of arginine8-vasopressin (AVP, 0.05 microg/kg/min, i.v.) for 2 h into 12-week-old hypertensive TGR(mREN-2)27 rats and normotensive Sprague-Dawley (SD) rats. Hypertension and marked left ventricular hypertrophy were associated with 2.2-times higher ir-ADM levels in the left ventricular epicardial layer (178 +/- 36 vs. 81 +/- 23 fmol/g, P<0.05) and 2.6-times higher ir-ADM levels in the left ventricular endocardial layer (213 +/- 23 vs. 83 +/- 22 fmol/g, P<0.01). The infusion of AVP for 2 h in normotensive rats produced rapid increases in the levels of left ventricular ADM mRNA (epicardial layer: 1.6-fold, P<0.05) and ir-ADM (endocardial layer: from 83 +/- 22 to 140 +/- 12 fmol/g, P<0.05), whereas ventricular ADM mRNA and ir-ADM levels did not change significantly in hypertensive rats. Short-term cardiac overload, induced by administration of angiotensin II (33.3 microg/kg/h, s.c., osmotic minipumps) for two weeks in normotensive SD rats resulted in left ventricular hypertrophy (3.05 +/- 0.17 vs. 2.75 +/- 0.3 mg/g, P<0.05) and a 1.5-fold increase (P<0.05) in ventricular ADM mRNA levels. In conclusion, the present results show that pressure overload acutely stimulated ventricular ADM gene expression in conscious normotensive rats suggesting a potential beneficial role for endogenous ADM production in the heart against cardiac overload. Since pressure overload-induced increase in ADM synthesis was attenuated in hypertensive rats, alterations in the ADM system may contribute to the pathogenesis of hypertension in the TGR(mREN-2)27 rat.

Production and secretion of adrenomedullin by cultured choroid plexus carcinoma cells.

Adrenomedullin is a potent vasodilator peptide that was originally isolated from pheochromocytoma. The production and secretion of adrenomedullin by cultured choroid plexus carcinoma cells were studied by radioimmunoassay and northern blot hybridization. Choroid plexus carcinoma is a rare malignant tumor derived from the epithelium of the choroid plexus. Immunoreactive adrenomedullin was detected in the conditioned medium of choroid plexus carcinoma cells (40.8 +/- 7.5 fmol/10(5) cells/24 h; mean +/- SEM, n = 5). Reverse-phase HPLC of the conditioned medium showed one major peak of the immunoreactive peptide eluting in the position of synthetic human adrenomedullin and two smaller peaks eluting earlier. Addition of interleukin-1 beta (10 ng/ml) alone or in combination with three cytokines, interferon-gamma (100 U/ml), tumor necrosis factor-alpha (20 ng/ml), and interleukin-1 beta (10 ng/ml), caused significant increases in the immunoreactive adrenomedullin concentrations in the medium (approximately 175 and 293% of the control level, respectively). Northern blot analysis showed the expression of 1.6-kb adrenomedullin mRNA in the total RNA sample prepared from cultured choroid plexus carcinoma cells. Treatment with either interleukin-1 beta or the combination of three cytokines caused significant increases in levels of adrenomedullin mRNA in parallel with those in immunoreactive adrenomedullin concentrations in the conditioned medium. These findings raise a possibility that adrenomedullin is secreted from the choroid plexus and has physiological roles in the CNS via the CSF. In addition, adrenomedullin secreted from choroid plexus carcinoma may be related to the pathophysiology of the tumor.

Production and Secretion of Adrenomedullin From Glial Cell Tumors and its Effects on cAMP Production

Takahashi, K., F. Satoh, E. Hara, M. Sone, O. Murakami, T. Kayama, T. Yoshimoto and S. Shibahara. Production and secretion of adrenomedullin from glial cell tumors and its effects on cAMP production. Peptides 18(8)1117–1124, 1997.—The expression of adrenomedullin (ADM) and its mRNA was studied in human glial cell tumors and cultured glioblastoma cell lines, T98G and A172. Northern blot analysis showed that ADM mRNA was expressed in all brain tumors examined (three anaplastic astrocytomas and two glioblastomas multiforme) and in the glioblastoma cell lines. Immunoreactive (IR-) ADM was detectable in these brain tumors by radioimmunoassay (0.31–2.0 pmol/g wet weight), except for one anaplastic astrocytoma. Reverse phase high performance liquid chromatography of the tumor extracts showed a single peak eluting in the position of ADM-(1–52). IR-ADM concentrations in the cultured media of T98G cells were 205.5 ± 8.4 fmol/10 5 cells/24 h (mean ± SEM, n = 5). Treatment of T98G cells with interferon γ or interleukin 1β increased the expression levels of ADM mRNA and the IR-ADM concentrations in the cultured media, whereas tumor necrosis factor α decreased them in a dose-dependent manner. Treatment with synthetic ADM-(1–52) (10 −8 or 10 −7 mol/l) increased the cAMP concentrations in the cultured media of T98G cells. These findings suggest that ADM is secreted from glial cell tumors and is related to the pathophysiology of these tumors.