0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society
Vol. 82, No. 1 Printed in U.S.A.
Circulating Adrenomedullin Does Not Regulate Systemic Blood Pressure but Increases Plasma Prolactin after Intravenous Infusion in Humans: A Pharmacokinetic Study* KARIM MEERAN†, DONAL O’SHEA‡, PAUL D. UPTON, CAROLINE J. SMALL§, MOHAMMAD A. GHATEI, PETER H. BYFIELD, AND STEPHEN R. BLOOM Division of Endocrinology and Metabolism, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom W12 0NN ABSTRACT Adrenomedullin has been proposed to be a circulating hormone regulating systemic and pulmonary blood pressure. A potential therapeutic role in the management of pulmonary hypertension has been suggested based on animal studies, but the pharmacokinetics and pharmacodynamics in human subjects have not been studied. We have infused adrenomedullin into volunteers at 3.2 pmol/kgzmin, which more than quadrupled (52 pmol/L) normal circulating concentrations. At this dose no change in heart rate or blood pressure was noted. When infused at 13.4 pmol/kgzmin to achieve a concentration over 40 times normal circulating levels (448 pmol/L), there was a significant fall in diastolic blood pressure from 69 6 2 to 53 6 2 mm Hg and a significant increase in pulse rate from 57 6 3 to 95 6 4 beats/min. Circulating PRL concentrations rose from 197 6 46 to 372 6 64 IU/L (mean 6 SEM; P , 0.01). No effect was seen on ACTH, TSH, FSH, LH, or cortisol. When the infusion was discontinued,
baseline pulse and blood pressure were reestablished after 20 min. Adrenomedullin has a MCR of 27.4 6 3.6 mL/kgzmin, with a circulating half life of 22 6 1.6 min and an apparent volume of distribution of 880 6 150 mL/kg. Column chromatography of plasma taken during infusion and decay of adrenomedullin showed no evidence of the production of additional molecular forms. These results are consistent with a peptide that is markedly tissue bound. Plasma adrenomedullin concentrations were increased in patients with renal impairment (14.1 6 0.9 pmol/L) compared to those in healthy volunteers (8.1 6 0.7 pmol/L), with a good correlation (r 5 0.86) between circulating adrenomedullin and plasma creatinine. The circulating concentration of adrenomedullin necessary to affect blood pressure greatly exceeds that observed in healthy volunteers and in patients with a range of pathological conditions. Thus, adrenomedullin may be a paracrine regulator of vascular smooth muscle in humans. (J Clin Endocrinol Metab 82: 95–100, 1997)
A
pituitary based on studies in dispersed rat anterior pituitary cells (5). In animal studies, it has been shown to have pulmonary (6, 7) and systemic (8, 9) hypotensive properties. We have previously demonstrated that the recently cloned adrenomedullin receptor (10) is expressed in several rat tissues, with particularly high levels in the lung (11). In animals with pharmacologically induced pulmonary hypertension, adrenomedullin dramatically reduces pulmonary pressures (6, 7), and a possible role for adrenomedullin in this disease in humans has been suggested. Ishimitsu et al. (12) found increased circulating plasma concentrations of adrenomedullin in patients with essential hypertension and proposed that it acts to protect the cardiovascular system from the effects of hypertension. The effects of adrenomedullin in human subjects have not been investigated to date. The present study was designed to elucidate the pharmacokinetics of adrenomedullin and to investigate whether adrenomedullin has a role in the regulation of blood pressure. The gene expression of adrenomedullin is higher in endothelial cells than in other tissues, including the adrenal (2), and for this reason we also studied several patients with diseases known to cause endothelial damage, such as diabetic retinopathy or the vasculitides such as systemic lupus erythematosus (SLE) or Wegener’s granulomatosis.
DRENOMEDULLIN is a 52-amino acid peptide originally isolated from human pheochromocytomas (1). It is a member of the calcitonin family of peptides, with sequence homology to calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide. Its physiological role is unknown. Adrenomedullin appears to be actively synthesized and secreted by vascular endothelial cells into both the bloodstream and the space between endothelial cells and vascular smooth muscle cells (2). The major source of circulating adrenomedullin is probably the vascular endothelium (3) rather than the adrenal gland (4). Adrenomedullin may act in a paracrine fashion on vascular smooth muscle cells by regulating vascular tone, rather than as a circulating hormone. Adrenomedullin has also been proposed to be a paracrine regulator of the Received June 10, 1996. Revision received August 21, 1996. Accepted August 23, 1996. Address all correspondence and requests for reprints to: Prof. S. R. Bloom, Division of Endocrinology and Metabolism, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, United Kingdom W12 0NN. E-mail:
[email protected]. * This work was supported by the United Kingdom Medical Research Council. † United Kingdom Medical Research Council Research Fellow. ‡ Wellcome Trust Research Fellow. § Wellcome Trust Prize student.
95
96
JCE & M • 1997 Vol 82 • No 1
MEERAN ET AL.
Materials and Methods Synthesis of human adrenomedullin The 52-residue peptide amide was synthesized in an Applied Biosystems 431A peptide synthesizer (Foster City, CA). The product comprised one major peak that was purified to homogeneity by reverse phase high performance liquid chromatography. The peptide was fully active when given intracerebroventricularly to rats (13).
Human infusions of adrenomedullin Eight healthy male volunteers (aged 24 –33 yr) were studied. Informed consent was obtained from each subject, and all experiments had prior approval from the Royal Postgraduate Medical School ethical committee. After an overnight fast, subjects were infused with either adrenomedullin or saline in random order into the antecubital vein of the left arm. There was an interval of at least 7 days between each infusion. Infusions were commenced with a 30-min period when only saline was infused. To minimize peptide adsorption to the infusion system (14), adrenomedullin was dissolved in 5 mL of the volunteer’s own plasma, which was then diluted to 50 mL with 0.9% saline. The infusion rate, initially 3.2 pmol/kgzmin, was increased every 10 min to a maximum of 13.4 pmol/kgzmin, which was maintained for 40 min. Subjects were supine throughout the study, except for measurement of standing blood pressure during the period of maximum adrenomedullin infusion. An iv cannula was inserted into the antecubital vein of the right arm for blood sampling. Pulse and blood pressure were recorded every 5 min. Blood was collected in tubes containing heparin and aprotinin from the cannula in the antecubital vein of the right arm and centrifuged immediately. Preliminary experiments (results not shown) indicated that assay of plasma after storage at 220 C is fully comparable with assay of the fresh plasma. Plasma was thus frozen on dry ice and stored at 220 C for assay of adrenomedullin, cortisol, glucose, and the pituitary hormones PRL, LH, FSH, ACTH, and TSH. Pharmacokinetics were studied in seven other volunteers, aged 24 –29 yr. A calculated loading dose of 20 pmol/kgzmin adrenomedullin was infused over 10 min, and an infusion of 6.5 pmol/kgzmin was maintained for 60 min to achieve a steady state. Blood was collected every 10 min for adrenomedullin assay to confirm that a steady state had been achieved. At the end of this period, the infusion was discontinued, and frequent samples were taken. The mean basal concentration of adrenomedullin was subtracted from the mean plateau concentration and from each subsequent sample. The postinfusion values were normalized by expressing them as a percentage of the previous steady state concentration. These values were plotted for each volunteer, and the half-life was derived from the resulting decay curve. The concentration of adrenomedullin was measured in a sample of each volunteer’s infusate to confirm the infusion rate. The MCR of adrenomedullin was calculated for each volunteer from the steady state concentration (CSS) and infusion rate at which this concentration was stable, where MCR 5 infusion rate/CSS. The apparent volume of distribution (VD) was calculated from the half-life and the steady state clearance, where VD 5 MCR 3 half-life 3 1.44
Circulating adrenomedullin concentrations in various diseases The study population consisted of 11 healthy volunteers, 17 patients with background diabetic retinopathy, 8 patients suffering from endstage renal failure on continuous ambulatory peritoneal dialysis, 6 patients with SLE, 12 patients with Wegener’s granulomatosis, 5 subjects with chronic stable asthma, and 4 subjects with rheumatoid arthritis. All patients were attending the out-patient department at Hammersmith Hospital and were being venesected for routine plasma electrolyte and creatinine determinations. Blood was also collected from several other patients attending the out-patients clinic who were having their creatinine levels measured. Additional patient details are given in Table 2. Blood (10 mL) was collected in lithium heparin tubes containing aprotinin (2000 IU) and centrifuged immediately at 4 C. Plasma was frozen on dry ice and stored at 220 C until extraction for assay of adrenomedullin. Plasma from patient samples was extracted using SepPak cartridges, as previously described (15), and submitted directly to assay.
Samples from volunteers receiving adrenomedullin infusions were assayed directly without extraction because plasma concentrations during the infusion were well above the detection limit for the assay. Aliquots of plasma from both patients and volunteers during infusion were analyzed by gel permeation chromatography to characterize immunoreactive adrenomedullin. The samples were applied to a Sephadex G-50 column (60 3 0.9 cm) and eluted with 0.06 mol/L sodium phosphate buffer containing 0.3% (wt/vol) BSA and 0.2 mol/L sodium chloride. Fractions (0.6 mL) were collected and submitted for direct assay. The elution coefficient (Kav) for each fraction was calculated relative to the elution positions of the markers dextran blue and Na125I according to the method of Laurent and Killander (16) (Figs. 2 and 5).
Adrenomedullin assay Adrenomedullin antiserum was raised in a rabbit against synthetic human adrenomedullin conjugated to BSA using carbodiimide (17) and used in the assay at a final dilution of 1:10,000. The detection limit was 2 fmol/tube at 95% confidence limits, and the assay did not cross-react with synthetic CGRP, islet amyloid polypeptide, or calcitonin. The intraand interassay coefficients of variation were 8% and 12.5%, respectively. Synthetic human adrenomedullin was used as the standard, and radiolabeled ligand was prepared using the adrenomedullin-(22–52) fragment by the Iodogen method (18) and purified by reverse phase high performance liquid chromatography. The specific activity of the tracer was 22.1 becquerels/fmol. Assays were performed in a final volume of 700 mL using 100 mL plasma with 0.06 mol/L sodium phosphate (pH 7.2) containing 0.3% (wt/vol) BSA, 10 mmol/L ethylenediamine tetraacetate, and 7 mmol/L sodium azide and incubated at 4 C for 3 days. Bound and free tracers were separated using dextran-coated charcoal.
Other assays Glucose was measured by the glucose oxidase method using a Yellow Springs YSI 2300 glucometer (Yellow Springs, OH). Plasma LH, FSH, TSH, PRL, and cortisol were measured using an automated analyzer. Plasma ACTH was determined using a two-site immunoradiometric assay kit (19) (Euro-Diagnostica, Europath Ltd., Cornwall, UK). Samples and standards were incubated overnight at 4 C with 100 ml each of N-terminally directed 125I-labeled sheep anti-ACTH antibody and Cterminally directed rabbit anti-ACTH antibody. The bound radioactive complex was separated from the free radioactive antibody by the addition of sheep antirabbit IgG and centrifugation. The pellet was counted using a g-counter for 60 s. The ACTH concentration in the samples was determined by comparison with a standard curve. The sensitivity of the ACTH immunoradiometric assay was 0.8 ng/L.
Statistical analysis Blood pressure and plasma levels of pituitary hormones during infusion of either saline or adrenomedullin were expressed as the mean 6 sem and compared by ANOVA with post-hoc Tukey’s test using the Systat computer package (Systat, Evanston, IL). ANOVA was also used to compare plasma adrenomedullin concentrations in patients with different conditions.
Results Adrenomedullin infusions
Cardiovascular effects. There was no change in pulse or blood pressure when adrenomedullin was infused at 3.2 pmol/ kgzmin, achieving a circulating concentration of 52 pmol/L. Higher dose adrenomedullin infusions (13.4 6 0.5 pmol/ kgzmin) were well tolerated with no adverse effects apart from facial flushing. At this infusion rate, the plasma adrenomedullin concentration was 448 6 58 pmol/kg. Diastolic blood pressure was significantly reduced, accompanied by tachycardia, although there was no change in systolic blood pressure. The fall in diastolic blood pressure was not associated with any postural hypotension. Standing diastolic
PHARMACOKINETICS OF ADRENOMEDULLIN IN MAN
FIG. 1. Blood pressure during a stepwise increasing adrenomedullin infusion in eight healthy volunteers. Thick stripes, Baseline during saline infusion; closed box, 3.2 pmol/kgzmin adrenomedullin infusion; open box, 6.4 pmol/kgzmin adrenomedullin infusion; mesh box, 13.4 pmol/kgzmin (peak) adrenomedullin infusion; thin stripes, standing during peak infusion.
blood pressure at the maximum infusion rate was significantly higher than supine diastolic blood pressure (Fig. 1). Gel filtration chromatography confirmed that the majority of the immunoreactivity eluted in the position of synthetic adrenomedullin standard from samples taken during both high dose infusion and recovery (Fig. 2). Endocrine effects. Plasma PRL was significantly increased (372 6 64 IU/L) when adrenomedullin was infused at 13.4 pmol/kgzmin compared to levels during baseline (197 6 46 IU/L) and control (204 6 30 IU/L) infusions (Table 1). There was no change in plasma concentrations of ACTH, LH, FSH, or TSH during either infusion. There was no difference between adrenomedullin and saline (data not shown) in the fall in cortisol seen during the course of infusion. Pharmacokinetics. During the second set of infusions, a plasma concentration of circulating adrenomedullin of 258 6 33.8 pmol/L was achieved within 10 min (Fig. 3) with a steady state infusion rate of 6.45 6 0.36 pmol/kgzmin. A decline in the plasma concentration of adrenomedullin was observed when the infusion was discontinued. From this rate of decline, the plasma half-life of adrenomedullin in humans was calculated to be 22 6 1.6 min. The mean MCR was 27.4 6 3.6 mL/kgzmin, and the apparent distribution space was 880 6 150 mL/kg. Basal circulating concentrations of adrenomedullin
The mean circulating concentration of adrenomedullin was significantly elevated in patients with any cause of renal failure (Table 2 and Fig. 4). Adrenomedullin was not increased in patients with normal renal function. Patients with conditions known to cause endothelial damage, such as the vasculitides Wegener’s granulomatosis and SLE, also have normal plasma adrenomedullin levels, provided that renal function is normal.
97
FIG. 2. Representative Sephadex G-50 column chromatography of plasma from a volunteer during high dose infusion (solid line) and 15 min (dashed line) and 30 min (dotted line) after cessation of the infusion. The immunoreactive peak seen at the void volume is thought to be due to high mol wt interference factors and is unchanged before (intermittent line) and after infusion. The two subsequent immunoreactive peaks decline as adrenomedullin is metabolized, confirming that the apparent long half-life is not due to the generation of new immunoreactive fragments. The arrow shows the elution position of the synthetic human adrenomedullin standard.
Discussion
Adrenomedullin has not previously been infused into humans and was well tolerated in our volunteers. Although our first infusion was not a dose-response study, we found that more than quadrupling the circulating concentration had no effect on blood pressure. Even during high dose adrenomedullin infusion, a plasma concentration of 448 pmol/L was not associated with any change in systolic blood pressure and with only minor changes in diastolic blood pressure. All biological effects of adrenomedullin disappeared within one half-life of the infusion being discontinued, confirming that high plasma levels (10-fold higher than normal) have no effect on systemic blood pressure. We have previously shown that this adrenomedullin is fully biologically active (13). These results strongly suggest that circulating adrenomedullin plays no role in the control of systemic blood pressure in humans. The proposal by Ishimitsu et al. (12) that circulating adrenomedullin is involved in a defense mechanism, preserving the integrity of the cardiovascular system in hypertension, is not supported by our findings. Adrenomedullin is more likely to have a paracrine role in the control of vascular tone. Endothelial cells actively secrete adrenomedullin (2), which can directly stimulate vascular smooth muscle cells through specific adrenomedullin receptors (20 –22). The proximity of vascular smooth muscle cells to endothelial cells means that the concentration of adrenomedullin around these cells is likely to be much higher than the basal concentrations that we have found in plasma and may be similar to those achieved by our maximal infusion rate. Adrenomedullin has been shown to inhibit endothelin production (23) and to be an antimigration factor (24)
98
JCE & M • 1997 Vol 82 • No 1
MEERAN ET AL.
TABLE 1. Endocrine effects of adrenomedullin infusion
Baseline (time zero) 6.7 pmol (130) 13.4 pmol/kg/min (170; peak infusion rate) Recovery (1110)
Adrenomedullin (pmol/L)
PRL (IU/L)
ACTH (ng/L)
Cortisol (nmol/L)
8.1 6 0.7 116 6 28.3 448 6 57.6
197 6 46 190 6 38 372 6 64a
7.2 6 1.2 5.4 6 1.1 7.6 6 1.7
150 6 21.0
275 6 38a
6.0 6 1.1
LH (IU/L)
FSH (IU/L)
TSH (IU/L)
Glucose (mmol/L)
330 6 56 217 6 32 233 6 56
2.6 6 0.4 1.9 6 0.3 2.3 6 0.7
3.0 6 0.3 2.7 6 0.3 2.7 6 0.3
1.4 6 0.2 1.2 6 0.2 1.1 6 0.2
4.5 6 0.1 4.5 6 0.1 4.8 6 0.2
223 6 108
2.0 6 0.6
2.6 6 0.4
1.0 6 0.1
4.8 6 0.1
P , 0.01 compared to values during saline infusions in the same individuals (data not shown). A similar decline in cortisol occurred with time during the saline infusion, presumably as the volunteers progressively relaxed. There was no rise in plasma PRL during the saline infusion. All values are the mean 6 SEM. a
FIG. 3. Infusion of adrenomedullin in seven additional volunteers to achieve a steady state.
in vascular smooth muscle cells. Cultured vascular smooth muscle cells express adrenomedullin messenger ribonucleic acid at a 3- to 4-fold higher concentration than that in adrenal gland as demonstrated by Northern blot analysis (3). In addition, adrenomedullin messenger ribonucleic acid appears to be under hormonal regulation in endothelial and vascular smooth muscle cells (25, 26), suggesting that adrenomedullin has an important function in these cell types. Plasma samples were prepared by solid phase extraction of medium and low mol wt proteins as previously described (15). This technique is commonly used in RIAs to remove the nonspecific high mol wt antigen-antibody uncoupling factors (interference factors) (27). In the case of our adrenomedullin infusion studies, we have shown the chromatographic profile obtained in unextracted plasma to illustrate that exogenous adrenomedullin does not alter the high mol wt interference factor component in a particular individual. Thus, Sep-Pak-ing is not required for the infusion studies where comparison is made of a volunteer’s plasma at different times after adrenomedullin infusion. The interference factor remains unchanged. Sep-Pak-ing of plasma completely removes this interference factor and, hence, removes this variable, which is essential when comparing basal adrenomedullin levels between individuals. The profile in Fig. 5 shows an unextracted profile. The high mol wt peak is completely removed by Sep-Pak-ing. The half-life and apparent volume of distribution of adrenomedullin are larger than those of other related peptides.
Rat CGRP has a plasma half-life of 6.9 min and a MCR of 11.3 mL/kgzmin in humans (28). Human calcitonin has a plasma half-life of 10.1 min and a MCR of 8.4 mL/kgzmin (29). Amylin has a half-life of 11.8 min, a MCR of 5.7 mL/kgzmin, and an apparent distribution space of 94 mL/kg (30). The very high apparent distribution space for adrenomedullin suggests that adrenomedullin is extensively tissue bound, possibly to the receptors found both on the endothelium (8, 31) and in vascular smooth muscle cells (20, 21). Plasma adrenomedullin was reported to be raised by 26% in patients with hypertension without organ damage (12) and by 78 –214% in patients with renal impairment. We found a similar increase in patients with renal impairment and a significant correlation between plasma adrenomedullin and degree of renal impairment as previously described (32). Circulating adrenomedullin concentrations were normal in our patients with normal renal function, even when they had diseases known to affect the endothelium, including Wegener’s granulomatosis, systemic lupus erythematosus, or background diabetic retinopathy. It is likely that adrenomedullin is metabolized or cleared at least in part by the kidney, and that this metabolism is impaired in patients with renal failure. The small increase in plasma adrenomedullin reported in patients with hypertension (12) may indicate early renal impairment. Creatinine clearance was not checked in their study (12). Adrenomedullin has been proposed to act through CGRP1 receptors in the isolated perfused mesenteric vascular bed of rats (33), although the effect on blood pressure in the intact animal is known not to act through these receptors (8, 9). The change in diastolic blood pressure during our human infusions did not have a postural component and was associated with facial cutaneous flushing, suggesting that the effect may have occurred distal to the arterioles at the capillary level. Intravenous CGRP caused similar facial flushing when infused at between 0.96 –1.92 pmol/kgzmin (achieved plasma concentration of 184 6 9 pmol/L) in humans (28). Samson et al. proposed a paracrine role for adrenomedullin in the pituitary based on their studies demonstrating that adrenomedullin inhibits ACTH release from dispersed rat anterior pituitary cells (5). We found that high dose adrenomedullin infusion was associated with increased release of PRL, but no change in other pituitary hormones, including ACTH. This would suggest a possible role for adrenomedullin as a regulator of lactotroph function. A paracrine rather than an endocrine effect is likely because the high dose iv adrenomedullin infusion may achieve local concentrations around the pituitary cells, consistent with those of a para-
PHARMACOKINETICS OF ADRENOMEDULLIN IN MAN
99
TABLE 2. Demographic data on the patients and volunteers and mean circulating adrenomedullin concentrations Patient group
n (male)
Age (range)
Adrenomedullin (pmol/L)
% of control
Controls Asthma Rheumatoid arthritis Retinopathy (creatinine ,100) Retinopathy (creatinine .150) CAPD patients SLE (creatinine ,100) SLE (creatinine .150) Wegeners (creatinine ,100) Wegeners (creatinine .150) All renal failure
11 (2) 5 (4) 4 (0) 13 (10) 4 (2) 8 (3) 2 (0) 4 (0) 7 (1) 5 (3) 21 (8)
40 (25–58) 53 (34 – 62) 63 (32– 86) 54 (26 – 80) 67 (51– 83) 52 (34 – 68) 45 (41– 49) 51 (37– 61) 52 (20 –70) 62 (54 –71) 57 (34 – 83)
8.1 6 0.7 9.3 6 0.3 8.2 6 0.2 8.1 6 0.3 13.3 6 1.1 13.9 6 1.5 6.5 6 0.4 12.9 6 3.4 10.7 6 0.7 16.0 6 1.4 14.1 6 0.9
100 115 101 100 164a 172b 80 159 132 198b 174b
Age is expressed as a mean (range). The adrenomedullin concentration is expressed as the mean 6 SEM and also as a percentage compared to the control value (100%). a P , 0.05. b P , 0.01.
FIG. 4. Correlation of basal circulating adrenomedullin with plasma creatinine in patients.
crine peptide. Adrenomedullin is known to be synthesized in the anterior pituitary (34). A possible role for adrenomedullin in the management of primary pulmonary hypertension has been suggested after studies of its effects in animal models of this disease (6, 7). Under conditions of resting (low) pulmonary vasomotor tone, intralobar arterial injection of adrenomedullin had little effect on baseline lobar arterial or systemic blood pressure. In contrast, when pulmonary vasomotor tone was actively increased by intralobar arterial infusion of the thromboxane A2 mimic U-46619, adrenomedullin decreased lobar arterial pressure in a dose-dependent manner, with minimal effects on systemic blood pressure. There is at present no pulmonary vasodilator available that does not cause systemic hypotension. Angiotensin-converting enzyme inhibitors, calcium antagonists (35), CGRP (36), prostacyclin (37, 38), nitric oxide (35), and oxygen have been used with minimal success. We have shown that high dose adrenomedullin is well tolerated in human volunteers, and that adrenomedullin has a minimal effect on systemic blood pressure. Some patients with elevated pulmonary pressure might respond to infusion of adrenomedullin with improved pulmonary pressure and consequent improve-
FIG. 5. Representative Sephadex G-50 gel filtration profile of adrenomedullin-like immunoreactivity in human plasma before SepPak-ing from a patient with renal impairment. Similar profiles were obtained for plasma samples from patients with other disorders. The second peak probably reflects an immunoreactive fragment. This profile is similar to that described previously (39). The arrow shows the elution position of synthetic human adrenomedullin standard. The interference factor in this individual is smaller than in the volunteer from Fig. 2.
ment in ventilation-perfusion matching. Adrenomedullin is known to be metabolized or cleared in the lung (4), as concentrations are higher in the pulmonary artery than in the aorta. This may be because the lung has a large number of adrenomedullin receptors. In conclusion, we found that adrenomedullin could lower blood pressure in humans, as predicted from animal and tissue experiments (8). The circulating concentrations required to achieve this effect, however, were well above those found in plasma taken from patients with a variety of conditions. Thus, we conclude that adrenomedullin is likely to influence vascular tone mainly through paracrine mechanisms. References 1. Kitamura K, Kangawa K, Kawamoto M, et al. 1993 Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun. 192:553–560.
100
MEERAN ET AL.
2. Sugo S, Minamino N, Kangawa K, et al. 1994 Endothelial cells actively synthesize and secrete adrenomedullin. Biochem Biophys Res Commun. 201:1160 –1166. 3. Sugo S, Minamino N, Shoji H, et al. 1994 Production and secretion of adrenomedullin from vascular smooth muscle cells: augmented production by tumor necrosis factor-alpha. Biochem Biophys Res Commun. 203:719 –726. 4. Nishikimi T, Kitamura K, Saito Y, et al. 1994 Clinical studies on the sites of production and clearance of circulating adrenomedullin in human subjects. Hypertension. 24:600 – 604. 5. Samson WK, Murphy T, Schell DA. 1995 A novel vasoactive peptide, adrenomedullin, inhibits pituitary adrenocorticotropin release. Endocrinology. 136:2349 –2352. 6. Dewitt BJ, Cheng DY, Caminiti GN, et al. 1994 Comparison of responses to adrenomedullin and calcitonin gene-related peptide in the pulmonary vascular bed of the cat. Eur J Pharmacol. 257:303–306. 7. Lippton H, Chang JK, Hao Q, Summer W, Hyman AL. 1994 Adrenomedullin dilates the pulmonary vascular bed in vivo. J Appl Physiol. 76:2154 –2156. 8. Nandha KA, Taylor GM, Smith DM, et al. 1996 Specific adrenomedullin binding sites and hypotension in the rat systemic vascular bed. Regul Pept. 62:145–151. 9. Gardiner SM, Kemp PA, March JE, Bennett T. 1995 Regional haemodynamic effects of human and rat adrenomedullin in conscious rats. Br J Pharmacol. 114:584 –591. 10. Kapas S, Catt KJ, Clark AJL. 1995 Cloning and expression of cDNA encoding a rat adrenomedullin receptor. J Biol Chem. 270:25344 –25347. 11. Owji AA, Smith DM, Coppock HA, et al. 1995 An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat. Endocrinology. 136:2127–2134. 12. Ishimitsu T, Nishikimi T, Saito Y, et al. 1994 Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J Clin Invest. 94:2158 –2161. 13. Taylor GM, Meeran K, O’Shea D, Smith DM, Ghatei MA, Bloom SR. 1996 Adrenomedullin inhibits feeding in the rat by a mechanism involving calcitonin gene-related peptide receptors. Endocrinology. 137:3260 –3264. 14. Kraegen EW, Lazarus L, Meler H, Campbell L, Chia YO. 1975 Carrier solutions for low-level intravenous insulin infusion. Br Med J. 3:464 – 466. 15. Kitamura K, Ichiki Y, Tanaka M, et al. 1994 Immunoreactive adrenomedullin in human plasma. FEBS Lett. 341:288 –290. 16. Laurent TC, Killander J. 1964 A theory of gel filtration and its experimental verification. J Chromatogr. 14:317–330. 17. O’Shaughnessy DJ. 1982 Antibodies. In: Bloom SR, Long WB, eds. Radioimmunoassay of gut regulatory peptides. London: Saunders; 11–20. 18. Fraker PJ, Speck JC. 1978 Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril Biochem Biophys Res Commun. 80:849 – 857. 19. Hodgkinson SC, Allolio B, Landon J, Lowry PJ. 1984 Development of a non-extracted ‘two-site’ immunoradiometric assay for corticotropin utilizing extreme amino- and carboxy-terminally directed antibodies. Biochem J. 218:703–711. 20. Eguchi S, Hirata Y, Iwasaki H, et al. 1994 Structure-activity relationship of adrenomedullin, a novel vasodilatory peptide, in cultured rat vascular smooth muscle cells. Endocrinology. 135:2454 –2458. 21. Eguchi S, Hirata Y, Kano H, et al. 1994 Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells. FEBS Lett. 340:226 –230.
JCE & M • 1997 Vol 82 • No 1
22. Ishizaka Y, Tanaka M, Kitamura K, et al. 1994 Adrenomedullin stimulates cyclic AMP formation in rat vascular smooth muscle cells. Biochem Biophys Res Commun. 200:642– 646. 23. Kohno M, Kano H, Horio T, Yokokawa K, Yasunari K, Takeda T. 1995 Inhibition of endothelin production by adrenomedullin in vascular smooth muscle cells. Hypertension. 25:1185–1190. 24. Horio T, Kohno M, Kano H, et al. 1995 Adrenomedullin as a novel antimigration factor of vascular smooth muscle cells. Circ. Res. 77:660 – 664. 25. Imai T, Hirata Y, Iwashina M, Marumo F. 1995 Hormonal regulation of rat adrenomedullin gene in vasculature. Endocrinology. 136:1544 –1548. 26. Minamino N, Shoji H, Sugo S, Kangawa K, Matsuo H. 1995 Adrenocortical steroids, thyroid hormones and retinoic acid augment the production of adrenomedullin in vascular smooth muscle cells. Biochem Biophys Res Commun. 211:686 – 693. 27. Adrian TE. 1982 Measurement in plasma. In: Bloom SR, Long WB, eds. Radioimmunoassay of gut regulatory peptides. London: Saunders; 28 –35. 28. Kraenzlin ME, Ch’ng JL, Mulderry PK, Ghatei MA, Bloom SR. 1985 Infusion of a novel peptide, calcitonin gene-related peptide (CGRP) in man. Pharmacokinetics and effects on gastric acid secretion and on gastrointestinal hormones. Regul Pept. 10:189 –197. 29. Huwyler R, Born W, Ohnhaus EE, Fischer JA. 1979 Plasma kinetics and urinary excretion of exogenous human and salmon calcitonin in man. Am J Physiol. 236:E15–E19. 30. Bretherton Watt D, Gilbey SG, Ghatei MA, Beacham J, Bloom SR. 1990 Failure to establish islet amyloid polypeptide (amylin) as a circulating beta cell inhibiting hormone in man. Diabetologia. 33:115–117. 31. Shimekake Y, Nagata K, Ohta S, et al. 1995 Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca21 mobilization, in bovine aortic endothelial cells. J Biol Chem. 270:4412– 4417. 32. Kohno M, Hanehira T, Kano H, et al. 1996 Plasma adrenomedullin concentrations in essential hypertension. Hypertension. 27:102–107. 33. Nuki C, Kawasaki H, Kitamura K, et al. 1993 Vasodilator effect of adrenomedullin and calcitonin gene-related peptide receptors in rat mesenteric vascular beds. Biochem Biophys Res Commun. 196:245–251. 34. Washimine H, Asada Y, Kitamura K, et al. 1995 Immunohistochemical identification of adrenomedullin in human, rat, and porcine tissue. Histochem Cell Biol. 103:251–254. 35. Rubin LJ. 1995 Pathology and pathophysiology of primary pulmonary hypertension. Am J Cardiol. 75:51A–54A. 36. Uren NG, Ludman PF, Crake T, Oakley CM. 1992 Response of the pulmonary circulation to acetylcholine, calcitonin gene-related peptide, substance P and oral nicardipine in patients with primary pulmonary hypertension. J Am Coll Cardiol. 19:835– 841. 37. Higenbottam TW, Spiegelhalter D, Scott JP, et al. 1993 Prostacyclin (epoprostenol) and heart-lung transplantation as treatments for severe pulmonary hypertension. Br Heart J. 70:366 –370. 38. Barst RJ, Rubin LJ, Long WA, et al. 1996 A comparison of continuous intravenous epoprostenol (prostacyclin) with conventional therapy for primary pulmonary hypertension. N Engl J Med. 334:296 –301. 39. Ichiki Y, Kitamura K, Kangawa K, Kawamoto M, Matsuo H, Eto T. 1994 Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett. 338:6 –10.
