No abstract

A fundamental question in development is how cells assemble to form a tubular network during organ formation. In glandular organs tubulogenesis is a multistep process requiring coordinated proliferation, polarization and reorganization of epithelial cells to from a lumen, and lumen expansion. Although it is clear that epithelial cells possess an intrinsic ability to organize into polarized structures, the mechanisms coordinating morphogenetic processes during tubulogenesis are poorly understood. Here, we demonstrate that parasympathetic nerves regulate ductal tubulogenesis in the developing salivary gland. We show that the neurotransmitter vasoactive intestinal peptide (VIP) secreted by the innervating ganglia promotes ductal growth, leads to the formation of a contiguous lumen, and facilitates lumen expansion through a cAMP/PKA-dependent pathway. Furthermore, we provide evidence that lumen expansion is independent of apoptosis and involves the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl(−) channel. Thus, parasympathetic innervation coordinates multiple steps in tubulogenesis during organ formation.

Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA-and p38-MAPK-dependent pathway. Aquaporin-2 (AQP2) is the water channel mediating arginine-vasopressin (AVP)-increases in water re-absorption in renal collecting duct principal cells. 1-4 AVP binds to plasma membrane-located vasopressin V2 receptors, thereby stimulating adenylyl cyclase and elevating cAMP. cAMP activates protein kinase A (PKA), which phosphorylates AQP2 at serine 256 (S256), inducing its redistribution from intracellular vesicles into the plasma membrane. 3,4 This short-term regulation of AQP2 occurs within seconds to minutes. In the case of long-term regulation, cAMP enhances AQP2 mRNA expression, followed by a rise in the AQP2 protein level within hours. 5,6 AQP2 can be degraded in proteasomes and lysosomes. 7,8 Ubiquitination directs proteins for degradation to both compartments. Monoubiquitination (mUb) is a signal for degradation in lysosomes, whereas polyubiquitination (pUb) is mainly linked to proteasomal degradation. 9 mUb of AQP2 is induced by FSK stimulation and occurs at the apical plasma membrane. 10 In WT5 cells, a model for AQP2 regulation, increased mUb of AQP2 persists

Nitric oxide (NO) is a ubiquitous, cell-permeable intercellular messenger. The current concept assumes that NO diffuses freely through the plasma membrane into the cytoplasm of a target cell, where it activates its cytosolic receptor enzyme, soluble guanylyl cyclase (sGC). Recent evidence, however, suggests that cellular membranes are not only the predominant site of calcium-dependent NO synthesis, but also the site of its distribution and binding. Here we extend this concept to NO signalling to show that active sGC is partially associated with the plasma membrane in a state of enhanced NO sensitivity. After cellular activation, sGC further translocates to the membrane fraction in human platelets and associates with the NO-synthase-containing caveolar fraction in rat lung endothelial cells, in a manner that is dependent on the concentration of intracellular calcium. Our data suggest that the entire NO signalling pathway is more spatially confined than previously assumed and that sGC dynamically translocates to the plasma membrane, where it is sensitized to NO.

A ntidiuretic hormone (arginine vasopressin [AVP]) induces fusion of vesicles that contain the water channel aquaporin-2 (AQP2) with the plasma membrane of renal collecting duct principal cells. This "AQP2 shuttle" increases the osmotic water permeability (Pf) of the cells, facilitating water reabsorption from the collecting duct (1). The AQP2 shuttle is initiated upon binding of AVP to vasopressin-2 receptors (V2R) and triggered by the consequent cAMP elevation and protein kinase A (PKA) activation. It is the PKA phosphorylation of AQP2 that elicits redistribution of AQP2-bearing vesicles. Pivotal to this redistribution is the compartmentalization of PKA by A kinase anchoring proteins (AKAP) (2). Phosphodiesterases (PDE), which are the sole means of degrading cAMP, are poised to regulate PKA signaling (3-6). The PDE4 family has attracted great interest because of its link to stroke (7), schizophrenia (8), and the therapeutic potential of selective inhibitors for treating inflammatory diseases (9-12). The four subfamilies (PDE4A through D) are encoded by separate genes, generating approximately 20 isoforms (9,11) that can interact with scaffolding proteins, including AKAP and ␤-arrestin (12-16), positioning them for a role in compartmentalized cAMP/PKA signaling.Here we show that compartmentalization of cAMP/PKA signaling by PDE4 is involved in the regulation of the AQP2 shuttle and the Pf. This is of particular pertinence because PDE4 hyperactivity causes nephrogenic diabetes insipidus in a mouse model (17).