“Soluble” adenylyl cyclase‐generated cyclic adenosine monophosphate promotes fast migration in PC12 cells

Abstract

In a model for neuronal movement, PC12 cells undergo fast migration in response to nerve growth factor (NGF) and phorbol ester (PMA). We previously showed that NGF increases intracellular cAMP via activation of soluble adenylyl cyclase (sAC). In this report, we demonstrate that sAC activation is an essential component of NGF-+PMA-induced fast migration in PC12 cells. Interestingly, PMA also raises intracellular cAMP but does so by stimulating transmembrane adenylyl cyclases (tmAC); however, this tmAC-generated cAMP does not contribute to fast migration. Therefore, cells must possess independent pools of cAMP capable of modulating distinct functions.Keywords adenylyl cyclase; nerve growth factor; neuron; compartmentalization; protein kinase C Neuronal migration and differentiation constitute key processes in the development of the central nervous system. The rat pheochromocytoma cell line PC12 has been extensively used as a model for the morphological changes that accompany both processes. When treated with nerve growth factor (NGF) for long periods (48-72 hr), PC12 cells extend neurites in a process analogous to differentiation of sympathetic neurons (Levi-Montalcini and Angeletti, 1968;Greene and Tischler, 1982;Vaudry et al., 2002). Conversely, when PC12 cells, plated on laminin-coated surfaces, are simultaneously stimulated with NGF and phorbol 12-myristate-13-acetate (PMA), the cell body recedes away from points of attachment to the plate surface, and the cells assume a crescent shape (Glowacka and Wagner, 1990). The latter phenomenon is rapid (observable within 1 hr of NGF + PMA addition) and has been considered a model for "fast neuronal migration."The signaling cascades that mediate the fast migration process have not been adequately described. Specifically, it is known that PMA exerts its effects via the activation of protein kinase C (PKC), which was independently shown to be essential for cell motility in primary (Choe et al., 2003). NGF has been shown to promote the survival and differentiation of multiple neuronal populations within the central nervous system (LeviMontalcini and Angeletti, 1968), but the signaling mechanisms involved in NGF-induced neuritogenesis remain controversial (Vaudry et al., 2002).We recently identified "soluble" adenylyl cyclase (sAC) as the source of cAMP downstream of NGF and a mediator of NGF-induced activation of the monomeric GTPase Rap1 in PC12 cells (Stessin et al., 2006). sAC is a ubiquitously expressed cAMP source in mammalian cells, distinct from the classically described trans-membrane adenylyl cyclases (tmACs) in its subcellular localization and biochemical profile. In this report, we demonstrate that the NGFsAC signaling axis is also required for fast migration. We also show that stimulation of PC12 cells with PMA results in cAMP elevation via tmACs, but, unlike sAC-generated cAMP, tmAC-generated cAMP does not contribute to the fast migration phenomenon. These results reveal the existence of distinct pools of cAMP, which are generated by independent sour...

Cyclic Nucleotide Phosphodiesterase 1 Regulates Lysosome-Dependent Type I Collagen Protein Degradation in Vascular Smooth Muscle Cells

“…As shown in Supplemental Figure VIIIA, rat aortic VSMCs express sAC at a level that is comparable to PC12 cells, in which sAC has been shown to be functionally important. 25,26 To specifically target sAC, we used sAC siRNA. We found that downregulation of sAC with sAC siRNA (Figure 5D, right) significantly attenuated the effects of IC86340 on collagen I protein, similar to KH7 (Figure 5D, left).…”

Soluble Adenylyl Cyclase Controls Mitochondria-dependent Apoptosis in Coronary Endothelial Cells

“…mitochondria and nuclei (10), permits cAMP to mediate various and independent cellular effects. Indeed, a recent study has demonstrated that cAMP produced by tmAC and sAC leads to different effects on PC12 cell migration (36). Furthermore, using pulmonary EC, Sayner et al (37) observed opposite effects of tmAC and sAC activation on the microvascular EC barrier.…”

Regulation of Epithelial Na+ Transport by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells

“…Interestingly, however, forskolin had no stimulatory effect on the measured I sc following a 30-min pretreatment with KH7. Because forskolin stimulation of tmAC is not directly inhibited by KH7 (20,36,37,39), these results suggest that the cAMP generated by forskolin-stimulated tmACs is not sufficient to overcome sAC inhibition and that sAC activity may be required for the increased transepithelial I sc caused by stimulation of tmACs as well as basal Na ϩ transport. Because the enzymatic product of sAC is cAMP, we next tested whether treating polarized mpkCCD c14 cells with the cell-permeant cAMP analog 8-Bromo-cAMP could override the inhibition of Na ϩ current by the sAC inhibitor KH7.…”