Response of adrenomedullin system to cytokine in cardiac fibroblasts-role of adrenomedullin as an antifibrotic factor.
OBJECTIVE
The adrenomedullin system acts as an autocrine or paracrine factor (or both) in the development of cardiac hypertrophy and in the regulation of cardiac function. However, several aspects of the local action of adrenomedullin remain unclear. We studied the effects of interleukin 1-beta (IL-1beta) on the adrenomedullin system in cardiac fibroblasts and also examined the pathophysiological significance of such effects.


METHODS
We cultured rat neonatal cardiac fibroblasts with or without IL-1beta and measured (1) two molecular forms of adrenomedullin in culture medium by specific immunoradiometric assay; (2) gene expression of adrenomedullin, calcitonin receptor like receptor (CRLR), receptor activity modifying protein2 (RAMP2), and RAMP3, components of the adrenomedullin receptor, by Northern blot analysis or RT-PCR analysis; (3) intracellular cAMP levels in response to exogenously administered adrenomedullin; and (4) (3)H-proline incorporation with and without a specific adrenomedullin antisense oligodeoxynucleotide.


RESULTS
(1) IL-1beta time-dependently increased the levels of two molecular forms of adrenomedullin, adrenomedullin-mature and adrenomedullin-glycine (P<0.01). In contrast to known levels in plasma (about 10%), adrenomedullin-mature was a major molecular form in the culture medium of cardiac fibroblasts and myocytes (65-80%). (2) IL-1beta significantly increased gene expression of adrenomedullin and its receptor components (adrenomedullin: +46%, CRLR: +460%, RAMP2: +32%, RAMP3: +350%, all P<0.01). (3) Preincubated IL-1beta elevated the intracellular cAMP response to exogenous adrenomedullin administered at a concentration of 10(-7) M (+26%, P<0.05). (4) Adrenomedullin antisense oligodeoxynucleotide treatment significantly lowered adrenomedullin-mature levels in culture medium (-50%). Adrenomedullin nonsense oligodeoxynucleotide treatment did not change (3)H-proline incorporation or mRNA levels of collagen I and III, whereas adrenomedullin antisense oligodeoxynucleotide treatment significantly increased (3)H-proline incorporation and mRNA levels of collagen I and III in IL-1beta-treated cardiac fibroblasts.


CONCLUSIONS
These results provide evidence that the adrenomedullin system acts as an autocrine antifibrotic factor in the regulation of collagen synthesis in cardiac fibroblasts exposed to higher cytokine levels. This may beneficially modulate the pathophysiology of certain cardiac diseases.
Nishikimi
Toshio
T
Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan. nishikim@dokkyomed.ac.jp
Tadokoro
Kazuyoshi
K
Akimoto
Kazumi
K
Mori
Yosuke
Y
Ishikawa
Yayoi
Y
Ishimura
Kimihiko
K
Horio
Takeshi
T
Kangawa
Kenji
K
Matsuoka
Hiroaki
H
D053607:Adrenomedullin; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D058286:Calcitonin Receptor-Like Protein; D018929:Cell Culture Techniques; D024042:Collagen Type I / Q000378:metabolism; D024061:Collagen Type III / Q000378:metabolism; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism; D005342:Fibrinolysis / Q000502:physiology*; D015870:Gene Expression; D015592:Immunoradiometric Assay / Q000379:methods; D007375:Interleukin-1 / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D010455:Peptides / Q000235:genetics / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D058286:Calcitonin Receptor-Like Protein; C545580:Calcrl protein, rat; D024042:Collagen Type I; D024061:Collagen Type III; D007375:Interleukin-1; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D010455:Peptides; C545509:Ramp2 protein, rat; C545510:Ramp3 protein, rat; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D053607:Adrenomedullin; D000242:Cyclic AMP