News Physiol Sci 14: 255-259, 1999;
1548-9213/99 $5.00
News in Physiological Sciences, Vol. 14, No. 6, 255-259,
December 1999
© 1999 Int. Union Physiol. Sci./Am. Physiol. Soc.
Adrenomedullin: Is There Physiological Relevance in the Pathology and Pharmacology?
Willis K. Samson,
Zachary T. Resch,
Tonya C. Murphy,
Trini T. Vargas and
Debra A. Schell
W. K. Samson is in the Department of Pharmacological and Physiological Sciences, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104; Z. T. Resch, T. C. Murphy, and D. A. Schell are in the Department of Physiology, University of North Dakota School of Medicine and Health Sciences, 501 N. Columbia Rd., Grand Forks, ND 58202; and T. T. Vargas is in the Department of Pediatrics, University of Texas Medical Branch, Galveston, TX 77555-0359, USA.
 |
Abstract
|
|---|
The adrenomedullin gene encodes two potent hypotensive peptides, adrenomedullin and proadrenomedullin NH2-terminal 20 peptide. As with other vasoactive peptides, the most difficult challenge is to prove the physiological relevance of their recognized pathological and pharmacological actions and to establish, against that physiological background, their therapeutic potential.
|
Introduction
|
|---|
Over the past fifteen years, several families of vasoactive peptides have been identified and their multiple pharmacological actions have been characterized (for review, see Ref. 9). In addition, elevated levels of these peptide hormones have been characterized in numerous pathologies and roles for these peptides in the genesis of and compensation for these diseases have been hypothesized. For physiologists, the ultimate goal in the study of these pluripotent hormones is to establish their physiological relevance and in so doing determine the therapeutic value of either administration of the peptides or manipulation of endogenous production/secretion/action.
|
Adrenomedullin as an example of a multifaceted vasoactive hormone
|
|---|
Preproadrenomedullin (6) is posttranslationally modified to result in two vasoactive peptides (for review, see Ref. 10), the 52-amino acid adrenomedullin (AM) and the 20-amino acid proadrenomedullin NH2-terminal 20 peptide (PAMP). Unique mechanisms of action and receptors underlie the hypotensive actions of the two peptides. AM stimulates nitric oxide production, and PAMP exerts presynaptic inhibition of sympathetic fibers innervating the vasculature and acts in an autocrine fashion to antagonize cholinergic stimulation of catecholamine release in the adrenal medulla. Both peptides also act in the adrenal cortex to inhibit potassium and angiotensin II-stimulated aldosterone secretion and in the pituitary gland to inhibit adrenocorticotropic hormone release (for review, see Ref. 10). However, not all of the actions of AM are shared by PAMP (Table 1
), and some variability in bioactivity has been reported among species. AM, for example, is a potent renotropic agent, stimulating natriuresis and diuresis by a combination of effects on both renal perfusion pressure and tubular handling of sodium (5). Additionally, AM, but not PAMP, acts in brain to inhibit salt and water intakes (8, 11).
In general, the actions of the two adrenomedullin gene products appear focused on cardiovascular, endocrine, and renal mechanisms controlling fluid and electrolyte homeostasis. The multiple pharmacological effects of AM and PAMP are mirrored by elevations in circulating and tissue levels of the peptides in a variety of pathological states characterized by alterations in cardiovascular and renal function (3). Thus, in just five years since their initial discovery, much is known of the pharmacological effects of the peptides and their presence and production in human disease states and the appropriate animal models. However, relatively little is known of their physiological relevance. Are they necessary for the homeostatic regulation of fluid and electrolyte balance and does normal cardiovascular and renal function depend upon their presence? Alternatively, do the published pharmacological effects merely reflect supporting roles played by these peptides in the presence of more physiologically relevant factors? It is certainly more difficult to answer these questions than to simply characterize the multiple pharmacological actions of the hormones. This difficulty has handicapped our knowledge of the true function(s) of numerous biologically active peptides, and multiple strategies have been devised to overcome this knowledge gap. Fortunately, some of those strategies have worked in the study of the adrenomedullin gene products, and we have begun to understand the physiological relevance of these peptides.
|
Adrenomedullin and sodium homeostasis: what goes in must go out and vice versa
|
|---|
The natriuretic and diuretic actions of AM in several models of renal function are complemented by actions in brain to inhibit salt and water appetite. These effects of AM provide a potential physiological system in which to determine the peptide's physiological relevance. To prove physiological relevance one must demonstrate the abrogation of normal function in the absence of a given factor, or its functional blockade, and its restoration when the factor is replaced or the blocking strategy removed. These strategies generally fall into two categories, prevention of peptide production and prevention of peptide action. Embryonic or conditional gene "knockouts" have been employed to selectively remove a given peptide; however, both approaches have limitations that jeopardize interpretation. These approaches are best employed when the peptide targeted exerts limited biological action(s). Even conditional gene deletions have limitations because complete loss of a given factor may cause death or physiological compromise due to unpredicted actions of the peptide, not those being targeted. Even if not lethal, in time compensatory mechanisms can be recruited that "cover" the loss of one factor with the upregulation of another with a similar biological profile. Thus, to study the physiological relevance of a given peptide, it is best to examine a discrete action and to limit the abrogation to distinct tissue sites, when, as in the case of the AM gene, transcription occurs in multiple tissues throughout the body. This is not to say that both embryonic and conditional knockouts have limited value. Indeed, surprising results often open doors not previously recognized, and much that has been learned about the physiological relevance of the endothelins has been derived from such methodology (13).
Our strategy has been to attempt to examine the physiological relevance of a given peptide's action acutely, before the potential recruitment of redundant (compensatory) mechanisms, in a limited tissue site, and using reversible strategies. The antidipsogenic actions of AM provide such an opportunity. When injected into the lateral cerebroventricle of conscious rats driven to consume water by a variety of experimental manipulations, AM exerts dose-related inhibition of water drinking (8). Similarly, in animals stimulated to consume both water and salt, cerebroventricular injection of AM potently inhibits salt appetite (11). Both actions occur at physiological doses. Are these actions physiologically unique and relevant, and is AM the predominant factor determining salt appetite or thirst? AM is not alone in expressing antidipsogenic actions or inhibitory effects on salt appetite. Atrial natriuretic peptide (ANP) similarly inhibits both behaviors (for review, see Ref. 9), and abundant pharmacological and physiological evidence points to a role for oxytocin (OT) in the expression of salt appetite (1). Here lies the conundrum: If multiple factors can exert similar actions, which one is preeminent in normal physiology? Are there parallel elements in the biological mechanisms or can a series element be identified, establishing one factor then as the final, critical element? Only a combinatorial approach will lead to meaningful answers.
|
Is adrenomedullin a physiological regulator of sodium homeostasis?
|
|---|
Renal and adrenal production and actions of AM suggest a role for the peptide in the physiological regulation of sodium excretion. To date, physiological relevance in these models has been examined only in limited studies employing antagonists, and data from "knockout" models have not been forthcoming. As mentioned above, it is difficult to determine the physiological role of a peptide in a discrete function if the peptide has multiple biological actions and the methods chosen for abrogation of peptide production or action cannot be limited to single tissue sites. Thus it will be very difficult to establish the physiological relevance of the renal effects of AM on sodium excretion because the effects of antagonist or antisense administration would be difficult to limit to the kidney in vivo. We examine, on the other hand, the intake of sodium and thus can limit our tissue of interest to the central nervous system. Taking advantage of the blood-brain barrier, we have then the luxury of applying technologies within a limited distribution, avoiding the possible confusion of indirect effects on sodium appetite caused by alterations in the peripheral actions of the peptide.
When injected into the lateral cerebroventricle of conscious rats made isotonically hypovolemic by polyethylene glycol administration, AM dose-dependently inhibits saline drinking in a two-bottle preference test (11). This suggests that AM produced within the brain acts under normal conditions to limit sodium ingestion. If that pharmacological action of exogenous AM has physiological relevance, then several proofs must be obtained. First, levels of peptide production and/or release in brain should be affected by sodium status. At the present time, the available radioimmunoassays (RIAs) do not provide the necessary sensitivity to determine, in a quantitative sense, this possibility. Peptide production in discrete brain regions can be examined by Northern blot analysis or semiquantitative polymerase chain reaction analysis, and such studies are currently underway in the authors' laboratory. Regardless of the outcome of those studies, the results would be descriptive at best and would not establish physiological relevance. Instead, the role of AM in salt appetite can best be established with loss of function studies.
Because the peptide pharmacologically inhibits salt appetite, loss of peptide function should result in exaggerated saline drinking under these experimental conditions. Indeed, when animals made isotonically hypovolemic were treated with anti-AM antiserum (11) before access to the drinking tubes was provided, the amount of saline consumed over the first 5-h interval significantly exceeded that observed in normal controls or controls injected with normal rabbit serum (injection controls) (Fig. 1). This would suggest that AM produced in brain normally circumscribes salt appetite, at least under acute stimuli for saline drinking. The effect resolved within 24 h and thus was reversible. This was the first indication of a physiologically relevant role for AM, one that was possible because the technique employed could be limited to a single tissue site of peptide action. The interpretation of the results of these experiments was not without limitations, not the least of which was the possibility that the antiserum "sequestered" an unknown factor that also plays a role in this biological event. Although the antiserum employed displays excellent specificity when tested against other known peptides, there may be additional, yet to be identified peptides present in this tissue that can be "neutralized" by the immunoglobulins. Thus the data need corroboration with an alternative method.
View larger version (17K):
[in this window]
[in a new window]
|
FIGURE 1. Combinatorial approach for establishing physiological relevance of actions of adrenomedullin (AM) on salt appetite. Top: pharmacological evidence. Central administration of AM dose dependently inhibits salt intake (see Ref. 11). Middle: initial physiological evidence. Blockade of action of endogenous AM with anti-AM antiserum results in exaggerated salt intake compared to normal rabbit serum (NRS)-injected controls (see Ref. 11). Bottom: predicted results from antisense studies. Abrogation of endogenous AM production would result in exaggerated salt intake. [Note added in proof: The predicted exaggeration of salt appetite after adrenomedullin antisense oligonucleotide treatment, and the accompanying selective diminution of brain AM content, have recently been observed (Samson et al., Brain Res. 818: 164–167, 1999).]
|
|
One such method would be the abrogation of peptide production using antisense oligonucleotides. If it was possible to block AM production in brain with such technology, then it could be predicted (Fig. 1
) that those animals, in addition to having significant diminutions in AM immunoreactivity in selected brain sites, would respond to the hypovolemic challenge much the same as those that were treated with the anti-AM antiserum. Furthermore, it would be predicted that the effect of the antisense oligonucleotide would be selective (i.e., levels of colocalized peptides such as OT would be unaffected) and reversible. The case for a physiological role for AM in central mechanisms regulating sodium homeostasis recently has been strengthened using this second approach. In our hands, abrogation of peptide production yielded data (Samson et al., Brain Res. 818: 164–167, 1999) that corroborated the findings of the blockade of peptide function studies (11). Indeed, treatment of rats with an antisense oligonucleotide directed against the adrenomedullin gene product compromised adrenomedullin content, but not that of colocalized OT, and resulted in a significant exaggeration of sodium appetite.
It could be argued that the reversibility of the effect of passive immunoneutralization or abrogation of peptide production would not reflect the return of normal function of AM but instead compensation by other peptides that act in a similar fashion. The results of the antiserum study (11) cannot refute this potential argument, and a combinatorial approach employing methodologies designed to abrogate function of multiple peptides, demonstrated by themselves to have potential relevance, must be attempted. Again, this could be demonstrated in the saline drinking paradigm. Abrogation of the action of OT by either immunosequestration or selective compromise of OT-receptive elements (e.g., neurons) in brain also results in exaggerated saline drinking (1). Similarly, compromise of ANP-receptive elements in brain results in exaggerated saline drinking (1). Are these three peptides linked functionally in their biological action? We do know that the saline inhibition exerted by OT differs qualitatively from that of ANP, in terms of selectivity of the pure sodium versus general osmolar stimuli (1); however, we do not know the endogenous circuitry that employs these three, and perhaps more to be discovered, peptides. Would antisense-mediated abrogation of AM production block the salt appetite suppression caused by exogenous OT or ANP? If so, then one could argue that AM is further downstream in the neural network controlling sodium intake. This would also argue against those peptides acting as compensatory agents in the absence of AM.
|
Is this the only physiologically relevant action of AM?
|
|---|
There are several clinical conditions in which the elevated circulating or tissue levels of AM suggest quite strongly physiologically relevant actions of the peptide (3). The difficulty lies in obtaining proof in whole animal models. Most promising are the renotropic actions of AM, as well as the peptide's antimitogenic effects in the vasculature and the heart. Can the natriuresis that accompanies sepsis in humans, or that observed in animal models after lipopolysaccharide (LPS) administration, be blocked with AM antagonists? Clearly, plasma AM levels are elevated in sepsis and LPS activates AM gene transcription in a variety of tissues and secretion into the general circulation.
Plasma levels of AM are elevated in numerous syndromes characterized by elevated blood pressure and are significantly depressed in stroke-prone spontaneously hypertensive rats (4). There may be physiological relevance in these observations because AM gene delivery lowers blood pressure in spontaneously hypertensive rats (2). The antimitogenic effects of AM may be physiologically relevant because immunosequestration of endogenously produced AM with a selective monoclonal antibody enhanced DNA synthesis in cultured endothelial and mesangial cells in vitro (7). Similarly, protein synthesis in cultured cardiomyocytes appears to be inhibited by endogenously produced AM because addition of a specific anti-AM monoclonal antibody to the cultures resulted in significantly increased phenylalanine incorporation into these cells (12).
Thus not only the central effect of AM on sodium appetite but also renal, vascular, and cardiac effects of the peptide appear to have potential physiological relevance. The challenge facing both basic and clinical scientists now is twofold. Can the apparent physiological relevance in brain, kidney, blood vessel, and heart be established in human models, and is AM, or for that matter PAMP, a potentially therapeutic agent that will demonstrate efficacy in a clinical setting? The challenge can be addressed with a combination of available methodologies only if the focus on the whole animal is maintained.
|
Acknowledgments
|
|---|
Our work on adrenomedullin is supported by the Max Baer Heart Fund and the Fraternal Order of Eagles.
The authors acknowledge the multiple, important contributions of numerous investigators in the field and apologize for being unable to cite those contributions within the guidelines for this publication. For an extensive reference list, which acknowledges those contributions, please consult Ref. 10.
|
References
|
|---|
-
Blackburn, R. E., W. K. Samson, R. J. Fulton, E. M. Stricker, and J. G. Verbalis. Central oxytocin and ANP mediate osmotic inhibition of salt appetite in rats. Am. J. Physiol. 269 (Regulatory Integrative Comp. Physiol. 38): R245–R251, 1995.[Abstract/Free Full Text]
-
Chao, J., L. Jin, K. F. Lin, and L. Chao. Adrenomedullin gene delivery reduces blood pressure in spontaneously hypertensive rats. Hypertens. Res. 20: 269–277, 1997.[Medline]
-
Cheung, L., and R. Leung. Elevated plasma levels of human adrenomedullin in cardiovascular, respiratory, hepatic and renal disorders. Clin. Sci. 92: 59–62, 1997.[Medline]
-
Hirano, S., Y. Ishiyama, T. Matsuo, T. Imamura, J. Sakata, K. Kitamura, Y. Koiwaya, and T. Eto. Decrease in circulating and urine adrenomedullin concentrations in stroke-prone spontaneously hypertensive rats. Hypertens. Res. 21: 23–28, 1998.[Medline]
-
Jougasaki, M., C. M. Wei, L. L. Aarhus, D. M. Heublein, S. M. Sandberg, and J. C. Burnett. Renal localization and actions of adrenomedullin: a natriuretic peptide. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F657–F663, 1995.[Abstract/Free Full Text]
-
Kitamura, K., K. Kangawa, M. Kawamoto, Y. Ichiki, S. Nakamura, H. Matsuo, and T. Eto. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun. 192: 553–560, 1993.[Medline]
-
Michibata, H., M. Mukoyama, I. Tanaka, S. Suga, M. Nakagawa, R. Ishibashi, M. Goto, K. Akaji, Y. Fujiwara, Y. Kiso, and K. Nakao. Autocrine/paracrine role of adrenomedullin in cultured endothelial and mesangial cells. Kidney Int. 53: 979–985, 1998.[Medline]
-
Murphy, T. C., and W. K. Samson. The novel vasoactive hormone, adrenomedullin, inhibits water drinking in the rat. Endocrinology 136: 2459–2463, 1995.[Abstract]
-
Samson, W. K. Cardiovascular Hormones. In: Endocrinology: Basic and Clinical Principles, edited by P. M. Conn and S. Melmed. Totowa, NJ: Humana, 1997, p. 361–376.
-
Samson, W. K. Proadrenomedullin-derived peptides. Front. Neuroendocrinol. 19: 100–127, 1998.[Medline]
-
Samson, W. K., and T. C. Murphy. Adrenomedullin inhibits salt appetite. Endocrinology 138: 613–616, 1996.[Abstract/Free Full Text]
-
Tsuruda, T, J. Kato, K. Kitamura, K. Kuwasako, T. Imamura, Y. Koiwaya, T. Tsuji, K. Kangawa, and T. Eto. Adrenomedullin: a possible autocrine or paracrine inhibitor of hypertrophy of cardiomyocytes. Hypertension 31: 505–510, 1998.[Abstract/Free Full Text]
-
Yanagisawa , H., R. E. Hammer, J. A. Richardson, S. C. Williams, D. E. Clouthier, and M. Yanagisawa. Role of endothelin-1/endothelin-A receptor-mediated signaling pathway in the aortic arch patterning in mice. J. Clin. Invest. 102: 22–33, 1998.[Medline]
This article has been cited by other articles:
|
|
|
|
 
I. Haulica, W. Bild, C. Mihaila, D. N Serban, L. Serban, D. Boisteanu, T. Ionita, and O. Radasanu
Comparative study of the inhibitory effects of adrenomedullin on angiotensin II contraction in rat conductance and resistance arteries
Journal of Renin-Angiotensin-Aldosterone System,
June 1, 2004;
5(2):
79 - 83.
[Abstract]
[PDF]
|
|
|
Top
Introduction
Adrenomedullin as an example...
