Cyclic AMP‐dependent synthesis and release of adrenomedullin and proadrenomedullin N‐terminal 20 peptide in cultured bovine adrenal chromaffin cells

Abstract

Adrenomedullin and proadrenomedullin N-terminal 20 peptide are peptides with multiple physiological functions and are most abundant in adrenal medulla. We studied whether the cAMP-dependent pathway is involved in the regulation of synthesis and release of adrenomedullin and proadrenomedullin N-terminal 20 peptide in cultured bovine adrenal chromaffin cells. Exposure of the cells to dibutyryl cAMP (dbcAMP) increased a progressive accumulation of immunoreactive-adrenomedullin and immunoreactive-proadrenomedullin N-terminal 20 peptide in the extracellular medium, while reciprocally decreasing their cellular content in a time-dependent manner. The decrease of levels of both peptides in the cells was much greater in extent than the increase of the peptides in the medium. H89, an inhibitor of cAMP-dependent protein kinase attenuated these changes, induced by dbcAMP. The resulting changes by dbcAMP and H89 were similar to those of chromogranin B, a marker peptide of chromaffin granule. Northern blot analysis showed that the mRNA encoding these peptides, detected as a band of 1.6 kb, was decreased by the treatment with dbcAMP. The effect of dbcAMP on mRNA was attenuated by H89, and was reversible as the decreased mRNA level caused by dbcAMP could be returned to control levels by culturing cells after removal of dbcAMP. These results suggest that the cAMP-dependent protein kinase pathway stimulates the release of adrenomedullin and proadrenomedullin N-terminal 20 peptide, whereas it lowers synthesis of these peptides via the reduction of their transcript level. The AM gene also produces another biologically active peptide, proadrenomedullin N-terminal 20 peptide (PAMP) [10±13]. The C-terminal of PAMP is amidated similarly to AM, but its amino acid sequence differs from that of AM. Although PAMP has hypotensive action [12], the mechanism of action of PAMP is different from that of AM, and has not yet been well defined. Several mechanisms have been proposed for the hypotensive action of PAMP depending on vascular beds and species, including: reduction of vascular tone by suppressing norepinephrine release from sympathetic nerve in rat mesenteric arteries [14], dilatation of the hindlimb vascular bed by a direct cAMP-dependent mechanism in cat [15], and dilatation by a tone-dependent mechanism in rat hindlimb vascular bed [16].The immunoreactivity and mRNA of AM and PAMP are most abundant in the adrenal medulla [11,17±20]. However, the intracellular regulatory mechanisms of the synthesis and release of the both peptides are not known. cAMP is a second messenger involved in the regulation of a wide variety of cell functions. The cAMP level in adrenal chromaffin cells is regulated by neurotransmitters and hormones such as acetylcholine [21], dopamine [22], vasoactive intestinal polypeptide [23] and pituitary adenylate cyclase-activating peptide [24]. Therefore, in this paper, we studied whether the cAMP-dependent pathway was involved in the synthesis and release of AM and PAMP in bovine adrenal chromaffin cells.

Regulation of adrenomedullin expression and release

“…Bovine adrenal chromaffin cell was reported to secrete AM at a rate of 0.68 Ϯ 0.02 fmol/10 5 cells/hour, and dibutyl-cAMP increased the secretion rate 1.5-fold (Kobayashi et al, 1999). Cholinergic stimulation was reported to stimulate AM secretion from bovine chromaffin cell (Katoh et al, 1994).…”

Pathophysiological Function of Adrenomedullin and Proadrenomedullin N-Terminal Peptides in Adrenal Chromaffin Cells

“…ir-AM and ir-PAMP20 are also released gradually during culture of the bovine chromaffin cells (28). This gradual release of ir-AM and ir-PAMP was increased by a membrane permeable analog of cAMP, dibutyryl cyclic AMP (dbc-AMP).…”

Variations of Human Adrenomedullin Gene and Its Relation to Cardiovascular Diseases

“…On the other hand, AM gene expression has been shown to be decreased by cAMP (27,28). Although it has not yet been proven, the consensus sequence of the cAMP responsive element existing in intron 1 may transmit this negative feedback signal.…”