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(Stroke. 1997;28:181-185.)
© 1997 American Heart Association, Inc.

Articles

Mechanisms of Adrenomedullin-Induced Dilatation of Cerebral Arterioles

Markus G. Lang, PhD; Roberto Paterno, MD; Frank M. Faraci, PhD Donald D. Heistad, MD

the Departments of Internal Medicine and Pharmacology, Cardiovascular Center and Center on Aging, University of Iowa College of Medicine (Iowa City).

Correspondence to Frank M. Faraci, PhD, Associate Professor of Internal Medicine and Pharmacology, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242.

	Abstract

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Background and Purpose Adrenomedullin is a recently discovered vasoactive peptide that is structurally related to calcitonin gene–related peptide (CGRP). Adrenomedullin is produced by vascular endothelium and smooth muscle and is present in the brain. The goals of this study were to determine (1) whether adrenomedullin produces dilatation of cerebral arterioles and whether this effect is mediated by activation of CGRP receptors and (2) whether vasodilatation to adrenomedullin was mediated by K⁺ channels.

Methods Diameter of cerebral arterioles (mean±SE baseline, 46±1 µm) was measured using a closed cranial window in anesthetized rats.

Results Application of rat adrenomedullin (10^-7 and 10^-6 mol/L) increased vessel diameter by 16±3% and 45±8% (n=5), respectively. Vasodilator responses to repeated application of adrenomedullin were reproducible. Pretreatment of cerebral arterioles with the specific CGRP₁ receptor antagonist CGRP-(8-37) (5x10^-7 mol/L) selectively inhibited the vasodilator responses to adrenomedullin without inhibiting responses to ADP (10^-5 to 10^-3 mol/L). Responses to adrenomedullin (10^-7 and 10^-6 mol/L) were 14±1% and 40±3% before and 2±2% and 6±1% after CGRP-(8-37), respectively (P<.01). Glibenclamide (10^-6 mol/L), an inhibitor of ATP-sensitive K⁺ channels, reduced the responses to adrenomedullin without attenuating responses to ADP. Responses to adrenomedullin were 19±4% and 35±6% before and 6±3% and 19±5% after glibenclamide, respectively (P<.05). Iberiotoxin (10^-7 mol/L), an inhibitor of calcium-dependent K⁺ channels, also significantly attenuated responses to adrenomedullin and did not inhibit vasodilatation to papaverine. Responses to adrenomedullin were 16±2% and 55±8% before and 12±4% and 26±3% after iberiotoxin, respectively (P<.01 for 10^-6 mol/L adrenomedullin).

Conclusions Adrenomedullin produces substantial dilatation of cerebral arterioles in vivo, and the response is mediated in large part by activation of CGRP₁ receptors. Cerebral vasodilatation to adrenomedullin appears to be dependent on activation of K⁺ channels.

Key Words: cerebral circulation • peptides • potassium channels • vasodilation • rats

	Introduction

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Adrenomedullin is a recently discovered vasoactive peptide that was first isolated from human pheochromocytomas.¹ Adrenomedullin is structurally related to calcitonin gene–related peptide (CGRP) and is regarded as a member of the CGRP superfamily. This family of peptides elicits potent hypotensive and vasorelaxant effects in vivo and in vitro.^{1 2 3 4 5} Adrenomedullin is present in a variety of tissues, including the cerebral cortex, and can be produced by endothelium and vascular muscle.^{6 7 8 9} The latter finding suggests a possible role of adrenomedullin in autocrine or paracrine regulation of vascular tone and function.

Plasma and tissue concentrations of adrenomedullin appear to be altered in several disease states, including hypertension, renal failure, congestive heart failure, endotoxin shock, and brain ischemia.^{10 11 12 13 14 15} Increased expression of adrenomedullin mRNA has been observed in response to focal cerebral ischemia.¹⁵ Furthermore, adrenomedullin inhibits endothelin production, and endothelin has been suggested to contribute to brain injury after ischemia.^{16 17 18 19 20 21}

Effects and mechanisms of action of adrenomedullin in cerebral vessels are poorly defined. A recent study suggests that CGRP receptors may mediate responses of the vertebral artery in vivo to adrenomedullin. We²² and others^{23 24} have provided evidence that vasodilator responses of cerebral blood vessels to CGRP are mediated by activation of ATP-sensitive K⁺ channels.

On the basis of the structural homology of adrenomedullin and CGRP, it seemed likely that adrenomedullin might be a potent cerebral vasodilator. Thus, the first goal of this study was to determine effects of adrenomedullin on cerebral arterioles in vivo. The second goal of the study was to determine whether responses to adrenomedullin in vivo are mediated by CGRP receptors and whether activation of K⁺ channels contributes to the vascular responses.

	Materials and Methods

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Animal Preparation
All animal experiments were performed following the guidelines of the American Physiological Society. Adult male Sprague-Dawley rats (n=31, 300 to 400 g) were anesthetized with pentobarbital sodium (50 mg/kg IP). Anesthesia was regularly supplemented with pentobarbital at 20 mg/kg per hour IV. The trachea was cannulated, and the animal was mechanically ventilated with room air and supplemental oxygen. Skeletal muscle paralysis was produced with gallamine triethiodide (13 to 18 mg/kg). Depth of anesthesia was evaluated by applying pressure to a paw and observing changes in heart rate or blood pressure. If paw pressure produced a response, additional anesthetic was administered. Catheters were placed in the femoral artery to measure systemic arterial pressure and to obtain arterial blood samples and in the femoral vein for infusion of drugs. Arterial blood gases were monitored and maintained at physiological levels throughout the experiments (pH, 7.40±0.01; PCO₂, 42±1 mm Hg; and PO₂, 137±4 mm Hg [mean±SE]). Rectal temperature was maintained at 37±0.5°C with a heating pad.

A closed cranial window was prepared over the parietal cortex. The scalp, muscles, and periosteum overlying the skull were reflected, and bleeding was controlled with ferric chloride solution. A craniotomy was made in the parietal bone using an air-cooled drill. The dura and arachnoid overlying an arteriole were excised with microscissors. Two blunt 18-gauge needles were affixed to a dam of bone wax surrounding the cranial window, and a circular glass coverslip (12 mm) was fused to the wax. The preparation was reinforced with dental acrylic. An outlet tube was affixed to one needle and adjusted to maintain intracranial pressure at 10 cm H₂O. A stopcock was attached to the other needle, and the window was filled with artificial cerebrospinal fluid (CSF) warmed to 37°C and equilibrated with 90% N₂-5% O₂-5% CO₂ (pH, 7.43±0.01; PCO₂, 36±1 mm Hg; and PO₂, 61±2 mm Hg [mean±SE]). Cerebral arterioles were observed using a microscope (Olympus, BHMJ) equipped with a video camera (Panasonic, WV-1500), and images were recorded on videotape. Arteriolar diameter was measured with an image-shearing device (IPM, 908). The preparation was allowed to equilibrate for 1 hour, during which time the window was flushed with 2 mL artificial CSF every 30 minutes. Flushing the window with artificial CSF did not alter the diameter of cerebral arterioles.

After the equilibration period, arteriolar diameter was measured under basal conditions and in response to ADP (10^-5 to 10^-3 mol/L). Vasodilatation to ADP was examined to test the integrity of the endothelium and served as control. The window was then flushed with artificial CSF three times, and the preparation was allowed to recover for 30 minutes.

Experimental Protocol
Time Controls
We examined responses of cerebral arterioles to topical repeated application of adrenomedullin (10^-7 and 10^-6 mol/L). Agonists were mixed in artificial CSF and applied to the cranial window for at least 5 minutes. The diameter of cerebral arterioles was measured immediately before (baseline) and during the application of each concentration of adrenomedullin (beginning measurements after 1 minute).

CGRP-(8-37)
Responses to adrenomedullin and ADP were obtained under control conditions. The specific CGRP₁ receptor antagonist CGRP-(8-37) (5x10^-7 mol/L)²⁵ was then applied 10 minutes before and during the application of adrenomedullin (10^-7 and 10^-6 mol/L) or ADP (10^-5 to 10^-3 mol/L).

Glibenclamide
Responses to adrenomedullin and ADP were obtained under control conditions. Glibenclamide (10^-6 mol/L), a selective blocker of ATP-sensitive K⁺ channels,²² was then applied 10 minutes before and during the application of adrenomedullin (10^-7 and 10^-6 mol/L) or ADP (10^-5 to 10^-3 mol/L).

Iberiotoxin
Responses to adrenomedullin and papaverine were obtained under control conditions. Thereafter, iberiotoxin (10^-7 mol/L), which is reported to be a specific inhibitor of calcium-dependent K⁺ channels at the concentration used in this study,²⁶ was applied 10 minutes before and during the application of adrenomedullin (10^-7 and 10^-6 mol/L) or papaverine (5x10^-5 and 2x10^-4 mol/L). Papaverine instead of ADP or sodium nitroprusside was used as control in these experiments because recent studies suggest that agonists that increase cyclic GMP produce iberiotoxin-sensitive dilatation of cerebral arterioles.²⁷

In some instances, the protocols of both control responses (ie, ADP or papaverine) and responses to adrenomedullin were performed in the same animals.

Drugs
The following drugs were used: ADP, glibenclamide, and papaverine from Sigma Chemical Co; rat adrenomedullin and human CGRP-(8-37) from Peptides International Inc; and iberiotoxin from Research Biochemicals International. All concentrations of the drugs are expressed as final molar concentration in the cranial window.

Adrenomedullin was dissolved in distilled water to furnish aliquots of 10^-4 mol/L and was immediately stored at -70°C. Further dilutions were made using artificial CSF. Because aliquots of adrenomedullin seemed to lose their potency even when stored at -70°C for more than 3 weeks, we used aliquots within about 3 weeks.

Calculations and Statistical Analysis
Relaxation is expressed as percentage of baseline values, which were measured immediately before applying the agonists. Data are given as mean±SE; n refers to the number of animals used. Maximal responses of agonists applied under control conditions and after intervention were compared with paired Student's t test. A two-tailed value of P<.05 was considered statistically significant.

	Results

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Baseline Values
Under control conditions, average diameter of cerebral arterioles was similar in all animals and averaged 46±1 µm (n=31). Mean arterial pressure was 114±3 mm Hg and did not change detectably during topical application of the drugs.

Responses to Adrenomedullin
Topical application of adrenomedullin (10^-7 and 10^-6 mol/L) produced concentration-dependent dilatation of cerebral arterioles. Repeated responses to adrenomedullin were reproducible (Fig 1).

View larger version (15K): [in this window] [in a new window]   Figure 1. Dilator responses of cerebral arterioles to two applications (Appl) of adrenomedullin. Baseline arteriolar diameter was 46±1 µm. Values are mean±SE in 5 rats.

CGRP-(8-37) (5x10^-7 mol/L), a selective blocker of CGRP₁ receptors, had no effect on baseline diameter (change in diameter, 2±1%). CGRP-(8-37) almost abolished responses to adrenomedullin but not to ADP (Fig 2). Responses to adrenomedullin (10^-7 and 10^-6 mol/L) were inhibited by 86% and 85%, respectively (P<.01). Responses to ADP (10^-5, 10^-4, and 10^-3 mol/L) were reduced by 27% and 11% and increased by 3%, respectively (NS).

View larger version (14K): [in this window] [in a new window]   Figure 2. Effects of human calcitonin gene-related peptide (hCGRP)-(8-37) (5x10-7 mol/L) on responses to adrenomedullin (n=5) and ADP (n=3). hCGRP-(8-37) reduced responses to adrenomedullin (*P<.01) but not ADP. Values are mean±SE.

Effects of Glibenclamide and Iberiotoxin
Glibenclamide (10^-6 mol/L), an inhibitor of ATP-sensitive K⁺ channels, did not cause any changes in baseline diameter (change in diameter, 0±1%). Glibenclamide reduced responses to adrenomedullin but not to ADP (Fig 3). Responses to adrenomedullin were reduced by 68% and 46%, respectively (P<.05). Responses to ADP (10^-5, 10^-4, and 10^-3 mol/L) were diminished by 39%, 0%, and 10%, respectively (NS).

View larger version (14K): [in this window] [in a new window]   Figure 3. Effects of glibenclamide (10-6 mol/L) on responses to adrenomedullin (n=6) and ADP (n=6). Glibenclamide inhibited responses to adrenomedullin but not to ADP (*P<.05). Values are mean±SE.

Iberiotoxin (10^-7 mol/L), a selective blocker of calcium-dependent K⁺ channels, did not affect baseline diameter (change in diameter, 2±1%). Iberiotoxin inhibited responses to 10^-6 mol/L adrenomedullin but not to papaverine (Fig 4). Responses to adrenomedullin were inhibited by 25% and 53%, respectively (P<.01 for 10^-6 mol/L). Responses to papaverine (5x10^-5 and 2x10^-4 mol/L) were unchanged in the presence of iberiotoxin.

View larger version (12K): [in this window] [in a new window]   Figure 4. Effects of iberiotoxin (10-7 mol/L) on responses to adrenomedullin (n=6) and papaverine (n=5). Iberiotoxin inhibited dilatation to adrenomedullin 10-6 mol/L but not papaverine (*P<.01). Values are mean±SE.

	Discussion

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This study demonstrates that adrenomedullin produces substantial dilatation of cerebral arterioles in vivo. Vasodilator responses to adrenomedullin were inhibited markedly by CGRP-(8-37), which suggests that the response was mediated in large part by activation of CGRP₁ receptors. Furthermore, the findings with glibenclamide and iberiotoxin suggest that K⁺ channels, perhaps both ATP-sensitive and calcium-dependent K⁺ channels, are involved in dilatation of cerebral arterioles to adrenomedullin.

Effect of Adrenomedullin
Adrenomedullin, a recently discovered vasoactive peptide, is synthesized by vascular endothelium and smooth muscle and is present in the brain.^{6 7 8 9} In previous studies, adrenomedullin was shown to be a potent hypotensive agent when injected intravenously.^{1 2} Furthermore, adrenomedullin exhibited profound vasodilator effects on dog vertebral and basilar arteries in vivo.⁴ Studies of vascular rings in vitro suggest heterogeneous sensitivity to adrenomedullin, with greater responses in canine basilar and mesenteric arteries than in renal, coronary, and femoral arteries.⁵ In contrast, another study reported only modest relaxation to adrenomedullin in isolated dog basilar and middle cerebral arteries.⁴

In this study, adrenomedullin produced substantial dilatation of cerebral arterioles at concentrations of 1 µmol/L. This finding is in line with results obtained in the vertebral circulation in vivo.⁴ Moreover, we found that repeated responses of the peptide were reproducible, without evidence of tachyphylaxis.

The concentrations of adrenomedullin may appear to be somewhat high for a peptide, especially in comparison with CGRP. However, adrenomedullin was applied extraluminally, which may limit access of adrenomedullin to the vascular smooth muscle. Under physiological conditions, adrenomedullin is mainly released by the endothelium and adrenal medulla, which does not require passing of the adventitia. Indeed, Baskaya et al⁴ reported a similar potency for adrenomedullin and CGRP when the drugs were administered intraluminally in the vertebral artery. Thus, the potency of adrenomedullin may be even greater than that observed in this study.

Role of CGRP Receptors
In the absence of cloning of CGRP receptor subtypes, the classification of CGRP receptors has been the subject of controversy. On the basis of pharmacological evidence, two subtypes of CGRP receptors have been proposed (CGRP₁ and CGRP₂). The receptor-antagonist CGRP-(8-37) has higher affinity for CGRP₁ receptors than CGRP₂ receptors.²⁸

Previous studies provided evidence that effects of adrenomedullin, such as relaxation of isolated mesenteric arteries or stimulation of cyclic AMP in vascular muscle, could be blocked by CGRP-(8-37).^{29 30} These findings suggested that adrenomedullin and CGRP share the same CGRP₁ receptor subtype, at least in some tissues. Not all responses to adrenomedullin, however, can be blocked by CGRP-(8-37). For example, effects of adrenomedullin on rat kidney in vivo such as renal vasodilatation, increase in glomerular filtration rate, diuresis, and natriuresis were not blocked by CGRP-(8-37). Similarly, CGRP-(8-37) did not affect cyclic AMP accumulation by adrenomedullin in human endothelial tissue culture, suggesting the presence of a more specific adrenomedullin receptor.^{31 32} CGRP receptors and adrenomedullin receptors are also thought to be coupled to G-proteins and adenylate cyclase.^{28 32} Indeed, CGRP, as well as adrenomedullin, produces an increase in cyclic AMP levels, an effect that at least in the case of CGRP can be completely blocked by CGRP-(8-37).^{30 32 33}

The role of endothelium in CGRP-induced vasorelaxation varies among different species and vascular beds.^{34 35 36 37} Responses of cerebral vessels to CGRP generally appear to be endothelium independent.^{22 37} Endothelial removal only minimally reduced responses to adrenomedullin in five different vascular beds,⁵ suggesting that adrenomedullin acts in large part by direct effects on vascular muscle.

In this study, pretreatment of vessels with CGRP-(8-37), a selective inhibitor of CGRP₁ receptors, nearly abolished responses to adrenomedullin but did not inhibit responses to ADP. Thus, responses to adrenomedullin appear to be mediated in large part by activation of CGRP₁ receptors. A similar role of CGRP₁ receptors in responses to adrenomedullin has been shown previously in rat mesenteric and dog vertebral arteries.^{4 29} Furthermore, these findings indicate that vascular responses that are inhibited by CGRP-(8-37) may not necessarily be mediated by CGRP.

Role of K⁺ Channels
Potassium channels are important regulators of cerebral vascular tone under physiological and pathophysiological conditions.³⁸ Differences in basal activity of K⁺ channels between large cerebral arteries and cerebral arterioles have been reported previously. For example, inhibition of calcium-dependent K⁺ channels produces contraction of large cerebral arteries but not cerebral arterioles.^{24 26 27 39} The latter observation in cerebral arterioles is consistent with the findings in the present study. In contrast, glibenclamide, a selective inhibitor of ATP-sensitive K⁺ channels, does not affect tone of either large cerebral arteries or cerebral microvessels.^{24 39} This finding also was confirmed in the present study. Together, these findings suggest that in cerebral microvessels, both ATP-sensitive and calcium-dependent K⁺ channels are not activated under basal conditions.

Pretreatment of vessels with either glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, or iberiotoxin, an inhibitor of calcium-dependent K⁺ channels, reduced responses to adrenomedullin but not to ADP or papaverine, respectively. These results suggest a role of both ATP-sensitive and calcium-dependent K⁺ channels in dilator responses to adrenomedullin. The fact that responses to adrenomedullin (10^-7 mol/L) were inhibited only by glibenclamide but not iberiotoxin may be explained by the relatively large current passing through calcium-dependent K⁺ channels, thereby limiting the effectiveness of iberiotoxin. A similar role of both ATP-sensitive and calcium-dependent K⁺ channels in dilatation of cerebral arterioles has been reported recently for CGRP.²⁴ Taken together, these findings suggest that adrenomedullin and CGRP share similar mechanisms of action, at least in cerebral arterioles.

Thus, adrenomedullin produces substantial dilatation of cerebral arterioles, and responses are mediated by activation of CGRP₁ receptors. Activation of both ATP-sensitive and calcium-dependent K⁺ channels appears to be important in mediating microvascular responses to adrenomedullin. The potency of this novel peptide, as well as recent reports describing increased expression after cerebral ischemia,¹⁵ implies the potential for an important role of adrenomedullin in cerebrovascular physiology or pathophysiology.

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL-16066, NS-24621, HL-14388, AG-10269, and HL-38901 and by a Grant-in-Aid from the American Heart Association (95014510). Dr Lang is a recipient of a National Research Service Award (AG00214-04), a Michael J. Brody Fellowship, and a stipend of the Janggen-Pohn-Foundation (Switzerland). Dr Faraci is an Established Investigator of the American Heart Association.

Received July 19, 1996; revision received September 20, 1996; accepted September 26, 1996.

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Editorial Comment

Jeffrey R. Kirsch, MD, Guest Editor

Department of Anesthesiology and Critical Care MedicineJohns Hopkins Medical InstitutionsBaltimore, Md

	Introduction

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The accompanying article evaluates the mechanism of vasodilation produced by adrenomedullin, a compound with structural homology to CGRP. In addition, many of the cerebrovascular effects of adrenomedullin appear to be similar to those of CGRP. For example, in a previous study, Baskaya et al^1R demonstrated that the vasodilator effects of adrenomedullin on conducting arteries of the cerebral circulation were inhibited by the CGRP receptor antagonist CGRP-(8-37) and that prior administration of CGRP prevented subsequent adrenomedullin-induced vasodilation. This previous study also ruled out the possibility that adrenomedullin produced cerebral vasodilation by a mechanism that involved production of nitric oxide or vasodilator prostanoids. Because intracisternal administration of adrenomedullin caused an increase in cyclic AMP concentration in CSF, the authors speculated that adrenomedullin-induced cerebral vasorelaxation was due to a mechanism involving stimulation of adenylate cyclase.

The present investigation demonstrates that adrenomedullin-induced dilation of pial arterioles is not subject to tachyphylaxis. In addition, like CGRP, adrenomedullin-induced cerebral vasodilation involves opening of both ATP-sensitive and Ca-dependent K⁺ channels. The precise physiological role of adrenomedullin in the cerebral circulation remains uncertain because of the lack of specific pharmacological probes that separate the effects of adrenomedullin from CGRP. Indeed, some of the physiological effects attributed to CGRP could be due to adrenomedullin because the CGRP antagonist CGRP-(8-37), was found to inhibit adrenomedullin-induced vasodilation in this and a past study.^1R

The clinical significance of adrenomedullin may be related to its role in the mechanism of injury from focal cerebral ischemia. During focal ischemia, there is an increased expression of adrenomedullin rRNA.^2R As a cerebral vasodilator, it would be expected that excess production may lead to improved postischemic neurological outcome. On the contrary, exogenous administration of adrenomedullin is associated with an increase in infarction size.^2R Additional studies will be required to determine why adrenomedullin increases infarction size whereas CGRP administration is associated with cerebral vasodilation and a reduction in infarction size.^3R

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 

1. Baskaya MK, Suzuki Y, Anzai M, Seki Y, Saito K, Takayasu M, Shibuya M, Sugita K. Effects of adrenomedullin, calcitonin gene-related peptide, and amylin on cerebral circulation in dogs. J Cereb Blood Flow Metab.. 1995;15:827-834.
2. Wang X, Yue T, Barone FC, White RF, Clark RK, Willette RN, Sulpizio AC, Aiyar NV, Ruffolo RR Jr, Feuerstein GZ. Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage. Proc Natl Acad Sci U S A.. 1995;92:11480-11484.
3. Holland JP, Sydserff SGC, Taylor WAS, Bell BA. Calcitonin gene-related peptide reduces brain injury in a rat model of focal cerebral ischemia. Stroke.. 1994;25:2055-2059.[Abstract]

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