CAPS1 regulates catecholamine loading of large dense-core vesicles.

CAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs.

STC1 induction by PACAP is mediated through cAMP and ERK1/2 but not PKA in cultured cortical neurons.

The neuroprotective actions of PACAP (pituitary adenylate cyclase-activating polypeptide) in vitro and in vivo suggest that activation of its cognate G protein coupled receptor PAC1 or downstream signaling molecules,and thus activation of PACAP target genes, could be of therapeutic benefit. Here, we show that cultured rat cortical neurons predominantly expressed the PAC1hop and null variants. PACAP receptor activation resulted in the elevation of the two second messengers cAMP and Ca(2+) and expression of the putative neuroprotectant stanniocalcin 1(STC1). PACAP signaling to the STC1 gene proceeded through the extracellular signal-regulated kinases 1 and 2(ERK1/2), but not through the cAMP-dependent protein kinase (PKA), and was mimicked by the adenylate cyclase activator forskolin. PACAP- and forskolin-mediated activation of ERK1/2 occurred through cAMP, but not PKA.These results suggest that STC1 gene induction proceeds through cAMP and ERK1/2, independently of PKA, the canonical cAMP effector. In contrast, PACAP signaling to the BDNF gene proceeded through PKA, suggesting that two different neuroprotective cAMP pathways co-exist in differentiated cortical neurons. The selective activation of a potentially neuroprotective cAMP-dependent pathway different from the canonical cAMP pathway used in many physiological processes, such as memory storage, has implications for pharmacological activation of neuroprotection in vivo.

PAC1hop, null and hip receptors mediate differential signaling through cyclic AMP and calcium leading to splice variant-specific gene induction in neural cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated activation of its G protein-coupled receptor PAC1 results in activation of the two G proteins Gs and Gq to alter second messenger generation and gene transcription in the nervous system, important for homeostatic responses to stress and injury. Heterologous expression of the three major splice variants of the rat PAC1 receptor, PAC1hop, null and hip, in neural NG108-15 cells conferred PACAP-mediated intracellular cAMP generation, while elevation of [Ca(2+)](i) occurred only in PAC1hop-, and to a lesser extent in PAC1null-expressing cells. Induction of vasoactive intestinal polypeptide (VIP) and stanniocalcin 1 (STC1), two genes potentially involved in PACAP's homeostatic responses, was examined as a function of the expressed PAC1 variant. VIP induction was greatest in PAC1hop-expressing cells, suggesting that a maximal transcriptional response requires combinatorial signaling through both cAMP and Ca(2+). STC1 induction was similar for all three receptor splice variants and was mimicked by the adenylate cyclase activator forskolin, indicating that cAMP elevation is sufficient to induce STC1. The degree of activation of two different second messenger pathways appears to determine the transcriptional response, suggesting that cellular responses to stressors are fine-tuned through differential receptor isoform expression. Signaling to the VIP gene proceeded through cAMP and protein kinase A (PKA) in these cells, independently of the MAP kinase ERK1/2. STC1 gene induction by PACAP was dependent on cAMP and ERK1/2, independently of PKA. Differential gene induction via different cAMP dependent signaling pathways potentially provides further targets for the design of treatments for stress-associated disorders.

PACAP: a master regulator of neuroendocrine stress circuits and the cellular stress response.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is released from stress-transducing neurons. It exerts postsynaptic effects required to complete the hypothalamo-pituitary-adrenocortical (HPA) and hypothalamo-sympatho-adrenal (HSA) circuits activated by psychogenic and metabolic stressors. Upon activation of these circuits, PACAP-responsive (in cell culture models) and PACAP-dependent (in vivo) transcriptomic responses in the adrenal gland, hypothalamus, and pituitary have been identified. Gene products produced in response circuits during stress include additional neuropeptides, neurotransmitter biosynthetic enzymes, and neuroprotective factors. Major portions of HPA and HSA stress responses are abolished in PACAP-deficient mice. This deficit occurs at the level of both the hypothalamus (HPA axis) and the adrenal medulla (HSA axis). PACAP-dependent transcriptional stress responses are conveyed through noncanonical cyclic AMP- and calcium-initiated signaling pathways within the HSA circuit. PACAP transcriptional regulation of the HPA axis, in the hypothalamus, is likely to be mediated via canonical cyclic AMP signaling through protein kinase A.