Adrenomedullin 2 Research Articles

Upregulated expression of intermedin and its receptor in the myocardium and aorta in spontaneously hypertensive rats

Intermedin (IMD), also called adrenomedullin 2 (ADM2), is a 47-amino acid peptide belonging to the calcitonin/calcitonin gene-related peptide (CGRP) family. IMD has similar or more potent vasodilatory and hypotensive actions compared with adrenomedullin (ADM) and CGRP. This study was designed to explore the role of IMD and its receptor in the pathogenesis of spontaneous hypertension and cardiac hypertrophy. Radioimmunoassay was employed to determine plasma immunoreactive IMD concentration and tissue immunoreactive IMD levels in the myocardium and aorta as well as cAMP concentration in the cardiovascular tissues in 13-week-old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). The mRNA expression of IMD, its receptor, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP)) were determined by semi-quantitative RT-PCR. Protein levels of CRLR and RAMPs were assayed by Western blotting. Our results showed that immunoreactive IMD concentration was enhanced in the SHR myocardium, aortas and plasma. Both the mRNA and protein levels of IMD, as well as those of CRLR and RAMP 1–3 were upregulated in SHRs. IMD affected cAMP generation in the myocardium and aorta, which were not attenuated by prior addition of either CGRP 8–37 or ADM 22–52 alone. These results indicate that the elevation of IMD and its receptor in the cardiovascular tissue may play an important role in the pathogenesis of spontaneous hypertension.

Adrenomedullin 2 microinjection into the nucleus tractus solitarius elevates arterial pressure and heart rate in rats

Adrenomedullin 2 (AM2), a novel member of the calcitonin gene-related peptide family, has emerged as a multifunctional peptide controlling endocrine and cardiovascular functions and physiological behaviors. Both central and peripheral administration of AM2 can have profound effects on systemic and/or pulmonary circulation in mammals. However, the target nuclei of AM2 and the role of central AM2 in cardiovascular regulation remain unknown. In the present study, we microinjected AM2 into the rat nucleus tractus solitarius (NTS), the central termination site of baroreceptor afferents. Consistent with previous reports showing the hypertensive effect of intracerebroventricular administration of AM2, the direct microinjection of the peptide into the NTS increased arterial pressure as well as heart rate in rats. Importantly, this effect of AM2 on cardiovascular regulation was significantly attenuated by an antagonist of receptor components for AM2 that were abundant in the rat NTS. Our results indicate that AM2 may play an important role in the regulation of the cardiovascular system at the NTS level.

Hemodynamic, Hormonal, and Renal Actions of Adrenomedullin 2 in Experimental Heart Failure

Adrenomedullin 2 (AM2) is a novel member of the calcitonin gene-related peptide family that is thought to play a regulatory role in circulatory homeostasis under normal physiological conditions. The effects of AM2 in heart failure have not been investigated previously. Two incremental doses of human AM2 (10 and 100 ng[kg.min] for 90 minutes each) were given by intravenous infusion to 8 sheep with pacing-induced heart failure. Compared with time-matched control infusions, AM2 produced dose-dependent increases in left ventricular dP/dt(max) (control 1168+/-138 mm Hg/s versus AM2 high-dose 1402+/-130 mm Hg/s; P<0.01) and cardiac output (2.09+/-0.66 L/min versus 3.81+/-0.30 L/min; P<0.001) and reductions in calculated total peripheral resistance (40+/-6 mm Hg(L.min) versus 21+/-4 mm Hg(L.min); P<0.001), mean arterial pressure (74.4+/-2.4 mm Hg versus 66.2+/-2.5 mm Hg; P<0.001), and left atrial pressure (23.3+/-1.0 mm Hg versus 18.8+/-1.3 mm Hg; P<0.001). AM2 administration also induced significant elevations in plasma cAMP (P<0.01) in association with rises in atrial (P<0.05) and brain (P<0.01) natriuretic peptides and plasma renin activity (P<0.01). Despite the increase in renin activity, plasma aldosterone levels were not significantly altered, whereas the aldosterone/plasma renin activity ratio was reduced (P=0.08). Plasma vasopressin, endothelin-1, and catecholamines levels were also unchanged by AM2. Renal effects of AM2 included increased excretion of sodium (P<0.05), cAMP (P<0.01), and creatinine (P<0.05), with augmented creatinine clearance (P<0.05), and a trend for urine output to rise (P=0.068). These results indicate that AM2 administration has favorable effects on cardiovascular, endocrine, and renal indexes in heart failure and identify the peptide as a potential therapeutic target in this disease.

Adrenomedullin 2/intermedin-like immunoreactivity in the hypothalamus and brainstem of rats

Adrenomedullin 2 (AM2) (identical to intermedin)-like immunoreactivity (LI) was examined in the rat brain by immunohistochemistry after intracerebroventricular administration of colchicine (100 µg/rat) and chronic salt loading (2% saline to drink) for 5 days. In both vehicle-treated and euhydrated rats, AM2-LI neurons were observed in the hypothalamus and brainstem, including in the organum vasculosum of the lamina terminalis, the median preoptic nucleus, the supraoptic nucleus (SON), the paraventricular nucleus (PVN), the ventromedial hypothalamic nucleus, the arcuate nucleus, the locus coeruleus, the nucleus of the tractus solitarius and the nucleus ambiguus. In colchicine-treated and salt loaded rats, AM2-LI neurons were visualized more strongly in the SON and the magnocellular part of the PVN than in those in each control. Some AM2-LI neurons appeared in the parvocellular part of the PVN in the colchicine-treated but not salt loaded rats. AM2-LI in the other areas of the hypothalamus and brainstem did not change after colchicine-treatment and chronic salt loading. These results suggest that AM2/intermedin in the hypothalamus and brainstem may play roles on neuroendocrine and autonomic functions, such as water/salt balance, in rats.

Adrenomedullin 2 (AM2)/intermedin is a more potent activator of hypothalamic oxytocin-secreting neurons than AM possibly through an unidentified receptor in rats

Central administration of either adrenomedullin 2 (AM2) or adrenomedullin (AM) activates hypothalamic oxytocin (OXT)-secreting neurons in rats. We compared AM2 with AM, given intracerebroventricularly (icv), across multiple measures: (1) plasma OXT levels in conscious rats; (2) blood pressure, heart rate and circulating catecholamine levels in urethane-anesthetized rats; and (3) the expression of the c- fos gene in the supraoptic (SON) and the paraventricular nuclei (PVN). We also tested the effects of the AM receptor antagonist, AM 22-52 and calcitonin gene-related peptide (CGRP) antagonist, CGRP 8-37 on these measures. Plasma OXT levels at 10 min after icv injection of AM (1 nmol/rat) were increased (compared with vehicle), but OXT levels after AM2 (1 nmol/rat) were nearly double the levels seen after AM injection. OXT levels remained elevated at 30 min. Pretreatment with AM 22-52 (27 nmol/rat) and CGRP 8-37 (3 nmol/rat), nearly abolished the increase in plasma OXT level after AM injection, but partially blocked OXT level changes due to AM2. Increases in blood pressure, heart rate and circulating catecholamines were all greater in response to central AM2 than to AM at the same dose. In situ hybridization histochemistry showed that both AM2 and AM induced expression of the c- fos gene in the SON and the PVN, but AM 22-52 + CGRP 8-37 could only nearly abolish the effects of centrally administered AM. These results suggest that the more potent central effects of AM2 and only partial blockade by AM/CGRP receptor antagonists may result from its action on an additional, as yet unidentified, specific receptor in the central nervous system.

Adrenomedullin-2, a Novel Calcitonin/Calcitonin-Gene-Related Peptide Family Peptide, Relaxes Rat Mesenteric Artery: Influence of Pregnancy

Adrenomedullin-2 (ADM2), a novel calcitonin/calcitonin-gene-related peptide family peptide, is reported to reduce blood pressure in both normal and hypertensive rats. This study demonstrates gestational regulation of circulatory ADM2 in rat plasma. ADM2 dose-dependently reduces the mean arterial pressure in rats, whereas the hypotensive effect of ADM2 is significantly higher during pregnancy. In addition, immunoreactive ADM2 protein is distributed in perivascular fibers of rat mesenteric artery, and levels of pre-pro-ADM2 are significantly (P<0.05) elevated in pregnant compared with nonpregnant rat mesenteric artery. Furthermore, incubation of endothelium intact arterial tissue from pregnant rats with ADM217-47, an ADM2 antagonist, shifted the dose-dependent relaxation curve to the right in wire myography. Inhibition of soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM) or endothelial nitric oxide synthase with N-nitro-L-arginine methyl ester (100 microM) reduced the relaxation of mesenteric artery induced by ADM2. Inhibition of adenylate cyclase with SQ22536 (10 microM) or protein kinase A with the Rp diastereomer of cyclic adenosine 3',5'-phosphorothioate (10 microM) also reduced the maximal relaxation responses induced by ADM2. Blockade of calcium-activated potassium channels with tetraethylammonium chloride (1 mM) inhibited the ADM2-induced relaxation, whereas blockade of ATP-sensitive potassium channels with glybenclamide (10 microM) did not affect the relaxation response. Hence the mechanism of ADM2-induced vasorelaxation is nitric oxide and receptor mediated and cGMP and cAMP dependent and occurs through activation of calcium-activated potassium channels. In conclusion, rat pregnancy is associated with increased levels of circulatory and vascular tissue ADM2 with concomitant increase in the in vivo hypotensive effect of ADM2 and vascular reactivity of mesenteric artery to ADM2, thus suggesting involvement of ADM2 in vascular adaptations during pregnancy.

Adrenomedullin 2 Antagonist Infusion to Rats During Midgestation Causes Fetoplacental Growth Restriction Through Apoptosis1

Adrenomedullin 2 (ADM2) is a recently discovered member of the calcitonin/calcitonin gene-related peptide family with an exon-intron structure similar to that of ADM. The mRNA of ADM2 is expressed in several tissues, including uterus and ovary. The present study was designed to assess the effects of ADM2 antagonist (ADM2(17-47)) infusion to pregnant rats on fetal and placental growth. On Day 15 of gestation, rats were implanted s.c. with osmotic minipumps delivering 50 and 200 mug per rat per day of ADM2(17-47) and were killed on Gestational Day 18. In ADM2(17-47)-treated rats, placental weights were significantly inhibited in a dose-related manner, with an 11% reduction in the group of rats receiving 200 microg/day, whereas the fetal weights were reduced by 17% without significant differences between the two doses. 2 In ADM2(17-47)-infused rats, increased apoptosis was demonstrated in the labyrinth and junctional zones of rat placenta by the TUNEL method compared with the control animals. Western blot analysis demonstrated that in ADM2(17-47)-treated rats Bcl-2, mitochondrial cytochrome c, and active caspase-9 and caspase-3 were significantly increased compared with the controls. No significant treatment-associated changes were observed in Bax, Bid, p53, and caspase-8 and caspase-10 proteins in the treated placentas. In addition, infusion of ADM2(17-47) caused a significant decline in the transcripts of nitric oxide synthase 3 (NOS3) and NOS2. These findings show that ADM2(17-47) infusion in rats during midpregnancy cause fetoplacental growth restriction through the activation of mitochondrial apoptotic pathways. This study demonstrates for the first time (to our knowledge) a potential role for ADM2 in placental functions during pregnancy.

Hemodynamic, Hormonal, and Renal Actions of Adrenomedullin-2 in Normal Conscious Sheep

Although blood pressure and heart rate effects have been reported for adrenomedullin 2 (AM-2), a new member of the calcitonin gene-related peptide superfamily, little information is available regarding other biological actions of systemically administered AM-2. Accordingly, we report for the first time the integrated hemodynamic, hormonal, and renal actions of the AM-2 in normal conscious sheep. AM-2 induced significant reductions in mean arterial pressure (P < 0.001). This was associated with dose-dependent rises in heart rate (P < 0.001) and cardiac output (P < 0.001) and dose-dependent falls in calculated total peripheral resistance (P < 0.001). Right atrial pressure was increased post infusion (P = 0.026), whereas hematocrit fell post infusion (P = 0.001). AM-2 also induced significant hormonal changes, particularly in the renin-angiotensin-aldosterone system where plasma renin activity was significantly activated (P < 0.001) associated with a dose-dependent rise in plasma aldosterone (P < 0.001). Plasma cAMP also rose in response to AM-2 (P < 0.001), as did circulating levels of the natriuretic peptides, particularly post infusion. In conclusion, iv infusions of AM-2 administered to normal conscious sheep induced significant hemodynamic actions including reduced mean arterial pressure and calculated total peripheral resistance and increased heart rate and cardiac output. Concurrently, AM-2 activated plasma cAMP, plasma renin activity, aldosterone, and the natriuretic peptides. With the exception of actions on aldosterone, these actions are similar to those previously reported for AM. Thus, AM-2 may be another important regulator of volume and pressure homeostasis and may play a role in the pathophysiology of heart disease.

Roles of adrenomedullin 2 in regulating the cardiovascular and sympathetic nervous systems in conscious rats

Recently, a new member of the calcitonin gene-related peptide (CGRP) family, adrenomedullin 2 (AM2) or intermedin (IMD), was identified. AM2/IMD has been shown to have a vasodilator effect in mice and rats and an effect on urine formation in rats. In the present study, we investigated the effects of intravenously infused rat AM2 (rAM2) on blood pressure (BP), heart rate (HR), renal sympathetic nerve activity (RSNA), and renal blood flow (RBF) in conscious unrestrained rats relative to the effects of rat adrenomedullin (rAM) and proadrenomedullin NH2-terminal 20 peptide (rPAMP). Intravenous infusion of rAM2 (5 nmol/kg) significantly decreased BP and increased HR, RSNA, and RBF. These hypotensive and sympathoexcitatory effects diminished after 20 min, and HR returned to control levels 30 min after cessation of the infusion. In contrast, a significant increase in RBF was still evident 60 min after cessation of the peptide infusion. The duration of BP, HR, and RSNA responses was longer with rAM (5 nmol/kg) than with rAM2 infusion, whereas the increases in RBF induced by rAM2 and rAM were similar in their amplitude and duration. Infusion of rPAMP (200 nmol/kg) increased HR and RSNA but had no effect on RBF. Baroreceptor denervation suppressed, but did not diminish, the increases in HR and RSNA to rAM2. These findings indicate that the physiological roles of rAM2 and rAM are similar and that rAM2 also has a long-lasting vasodilator action on the renal vascular bed.

Centrally administered adrenomedullin 2 activates hypothalamic oxytocin-secreting neurons, causing elevated plasma oxytocin level in rats

We examined the effects of intracerebroventricular (i.c.v.) administration of adrenomedullin 2 (AM2) on plasma oxytocin (OXT) and arginine vasopressin (AVP) levels in conscious rats. Plasma OXT levels were markedly increased 5 min after i.c.v. administration of AM2 (1 nmol/rat) compared with vehicle and remained elevated in samples taken at 10, 15, 30, and 60 min. By contrast, plasma AVP levels were not significantly elevated in samples taken between 5 and 180 min after i.c.v. administration of AM2 except at the 30-min time point. Fos-like immunoreactivity (Fos-LI) was observed in various brain areas, including the paraventricular (PVN) and the supraoptic nuclei (SON) after i.c.v. administration of AM2 (2 nmol/rat) in conscious rats (measured at 90 min post-AM2 infusion). Dual immunostaining for OXT/Fos and AVP/Fos showed that OXT-LI neurons predominantly exhibited nuclear Fos-LI compared with AVP-LI neurons in the PVN and the SON. In situ hybridization histochemistry showed that i.c.v. administration of AM2 (0.2, 1, and 2 nmol/rat) caused marked induction of the expression of the c-fos gene in the PVN and the SON. This induction was significantly reduced by pretreatment with both the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37) (3 nmol/rat) and the AM receptor antagonist AM-(22-52) (27 nmol/rat). These results suggest that centrally administered AM2 mainly activates OXT-secreting neurons in the PVN and the SON, at least in part through the CGRP and/or AM receptors with marked elevation of plasma OXT levels in conscious rats.

A comparison study of adrenomedullin (AM) and adrenomedullin 2 (AM2) in distribution of rat tissue

A comparison study of adrenomedullin (AM) and adrenomedullin 2 (AM2) in distribution of rat tissue

Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake

Intermedin (IMD)/adrenomedullin-2 (AM2) is a novel peptide that was independently discovered by two groups. The 47-amino acid peptide is homologous to adrenomedullin (AM) and can activate both the AM and calcitonin gene-related peptide (CGRP) receptors. IMD should therefore have actions similar to those of AM and CGRP. Indeed, like AM and CGRP, intravenous administration of IMD decreased blood pressure in rats and mice. We demonstrate here that immunoreactive IMD is present in plasma as well as heart, lung, stomach, kidney, pituitary, and brain. Because IMD is present in brain and both AM and CGRP have potent central nervous system (CNS) effects, we examined the ability of IMD within brain to regulate blood pressure and ingestive behaviors. Administration of IMD into the lateral cerebroventricle of rats caused significant, long-lasting elevations in mean arterial pressure and heart rate. These elevations are similar to the effects of CGRP and significantly greater than the effects of AM. IMD-induced elevations in mean arterial pressure were inhibited by intravenous administration of phentolamine, indicating that IMD activates the sympathetic nervous system. Intracerebroventricular administration of IMD also inhibited food and water intake in sated and in food- and water-restricted animals. The effects on feeding are likely related to activation of the CGRP receptor and are independent of the effects on water intake, which are likely through the AM receptor. Our data indicate that IMD has potent actions within the CNS that may be a result of the combined activation of both AM and CGRP receptors.

CORONARY VASODILATORY RESPONSE TO A NOVEL PEPTIDE, ADRENOMEDULLIN 2

1. Adrenomedullin-2 (AM2) is a novel peptide originally found in a fish and it is structurally related to mammalian AM or fish AM1. Cloning of AM2 cDNA in the mouse, rat and human has been successful. In the present study, the vasodilatory effect of synthetic human AM2 was analysed in isolated artery ring preparations of porcine. 2. Vasodilatory effect of AM2 was the most potent in the coronary artery, followed by the carotid and supramesenteric arteries, but absent in the femoral, pulmonary and renal arteries. 3. The effect of AM2 was equipotent with that of human AM/AM1 in these arteries. 4. AM2-induced relaxation was inhibited by human CGRP (8-37), but not by human AM/AM1 (22-52), suggesting that the vasodilatory response to AM2 is mediated through the CGRP1 receptor. 5. AM2 may share the same receptor (complex) with AM/AM1 for the vasorelaxation. This is the first report of the vasodilatory responses to AM2 in isolated vasculature.