Coincident Elevation of cAMP and Calcium Influx by PACAP-27 Synergistically Regulates Vasoactive Intestinal Polypeptide Gene Transcription through a Novel PKA-Independent Signaling Pathway

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) causes calcium influx, intracellular calcium release, and elevation of cAMP in chromaffin cells. Calcium influx is required for PACAP-stimulated secretion of catecholamines and neuropeptides. The role of cAMP elevation in the action of PACAP at either sympathetic or adrenomedullary synapses, however, is unknown. Here, we show that PACAP-27-induced calcium influx through voltage-sensitive calcium channels (VSCCs), together with elevation of intracellular cAMP, was sufficient to stimulate vasoactive intestinal polypeptide (VIP) biosynthesis at least 40-fold. Combined treatment of chromaffin cells with 40 mm KCl, which elevates intracellular calcium, and 25 micrometer forskolin, which elevates intracellular cAMP, caused an increase in VIP peptide and mRNA much greater than that elicited by either agent alone, and comparable to the increase caused by 10-100 nm PACAP-27. Elevation of VIP mRNA by either KCl plus forskolin, or PACAP, (1) was independent of new protein synthesis, (2) was blocked by inhibition of calcium influx through voltage-sensitive calcium channels, (3) was calcineurin dependent, and (4) was dependent on MAP kinase activation but not activation of protein kinase A. The degree of activation of two different second-messenger pathways, calcium influx and cAMP elevation, appears to determine the magnitude of transcriptional activation of the VIP gene in chromaffin cells. Maximal stimulation of VIP biosynthesis by PACAP appears to require the coincident activation of both of these pathways.

A New Site and Mechanism of Action for the Widely Used Adenylate Cyclase Inhibitor SQ22,536

“…Whether this is attributable to differential cell penetration of these compounds into neurons, compared with neuroendocrine cell lines, has not been determined. Thus, AC inhibitors with greater potency for all AC isoforms, but preservation of specificity for AC, compared with other ATP-and cAMP-binding proteins, are needed tools for use in studies of cAMP-mediated signaling, particularly in neuroendocrine cells with multiple defined cAMP signaling pathways causing diverse cellular responses (Emery and Eiden, 2012;Hamelink et al, 2002;Vaudry et al, 2002;Ravni et al, 2008) The present studies indicate that SQ22,536 is a more potent AC inhibitor than is ddAd but has an additional downstream target that is related to the intracellular actions of cAMP. Specifically, SQ22,536 blocks the action of two distinct targets involved in cAMP-related signaling: AC, as previously reported, and the neuritogenic cAMP sensor or NCS, a signaling component linking cAMP elevation to activation of ERK (Emery and Eiden, 2012).…”

“…Working in neuroendocrine chromaffin, PC12, and neuroscreen-1 (NS-1) cells, we identified a cAMP-dependent signaling pathway for cellular differentiation, transcription of neuroendocrine-specific genes, and neurite extension (Emery and Eiden, 2012;Hamelink et al, 2002;Ravni et al, 2008). This signaling pathway requires sequential activation of a neuritogenic cAMP sensor (NCS) and the MAP kinase ERK, and it is wholly independent of both PKA and Epac.…”

Nerve Growth Factor Regulates Adrenergic Expression

“…To isolate nuclear protein and total RNA, cells were grown in 100-mm culture dishes to a density of 5 ϫ 10 5 to 1 ϫ 10 7 cells/dish before drug treatment. NGF (0-100 ng/ml), 10 nM PACAP, and the signaling pathway drugs H89 (30 M), a selective protein kinase A (PKA) inhibitor (Chijiwa et al, 1990;; forskolin (10 M), an adenylate cyclase activator; GF109203X (100 nM), a protein kinase C (PKC) inhibitor (Tsuji et al, 2001;; phorbol 12-myristate 13-acetate (PMA; 80 nM), a PKC activator at low concentrations (Morita et al, 1995); U0126 (10 M), an ERK1/2 MAPK inhibitor (Harada et al, 2001;Hamelink et al, 2002;Hou et al, 2003); SB203580 (50 M), a p38 MEK inhibitor (Cheng et al, 2000); wortmannin and LY294002 (10-50 M), phosphatidylinositol 3-kinase inhibitors (Tsuji et al, 2001;Chang et al, 2003;Ha et al, 2003); and U-73122 (50 M), a phospholipase C inhibitor (Hamelink et al, 2002) were obtained from Sigma-Aldrich. Initial treatment concentrations were based on literature values, and doseresponse curves and time courses were executed to optimize treatment conditions (data not shown).…”

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

“…PACAP also stimulates the release of brain natriuretic peptide and enkephalins, two regulatory peptides that are cosequestered with catecholamines in chromaffin granules (Babinski et al, 1996;Hahm et al, 1998) as well as a 15-fold increase in VIP mRNA expression (Lee et al, 1999a). The effect of PACAP on VIP biosynthesis and catecholamine secretion requires the coincident elevation of calcium and cAMP (Fukushima et al, 2001b;Hamelink et al, 2002a;Morita et al, 2002). It is noteworthy that the induction of VIP in septic shock is blocked in PACAP deficient mice (Ait-Ali et al, 2009).…”