h-89 and Acidosis

Changes in systemic acid-base homeostasis cause a series of organ-specific cellular responses, among them changes of acid-base transporter activities, and recruitment or retrieval of these transporters from intracellular pools to the plasma membrane and vice versa. The purpose of this study was to investigate the impact of protein phosphorylation in the acidosis-induced translocation of vacuolar-type H(+)-ATPase (V-ATPase) in salivary ducts and to identify molecular targets. Therefore, the human submandibular gland cell line HSG was exposed to acidosis and V-ATPase trafficking was investigated in the presence or absence of inhibitors and activators of sAC/PKA and Src/ERK signaling pathways. Putative target genes have been identified by RT-PCR and immunoblotting, and validated by loss-of-function experiments. Acidosis caused activation of cAMP/PKA and Src signaling and inhibition of either pathway significantly impaired acidosis-induced V-ATPase redistribution and incorporation into the plasma membrane. Activation of ERK1/2 was Src-independent, whereas activation of PKA caused phosphorylation of cAMP response element-binding (CREB) and activation to regulate Rab11b transcription. Loss-of-function of CREB down-regulated Rab11b transcript and protein and significantly impaired acidosis-induced V-ATPase translocation in HSG cells. These data demonstrate that the cAMP/PKA/CREB signaling pathway initiates acidosis-induced V-ATPase trafficking in salivary ducts via regulation of Rab11b expression and provide first evidence for a molecular mechanism underlying cAMP/PKA-dependent transporter trafficking that could account for accumulation and activity of transporters in other cellular systems as well.

Topics: Acidosis; Cell Line; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Extracellular Signal-Regulated MAP Kinases; Fluorescent Antibody Technique; Humans; Hydrogen-Ion Concentration; Immunoblotting; Isoquinolines; Membrane Glycoproteins; Phosphorylation; Protein Kinase Inhibitors; Protein Subunits; Protein Transport; rab GTP-Binding Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Salivary Ducts; Signal Transduction; Sulfonamides; Time Factors; Vacuolar Proton-Translocating ATPases

The etiology of skeletal growth retardation accompanying metabolic acidosis is not clear. Using ex vivo models for endochondral ossification, we showed that the cAMP/PKA pathway, probably triggered by proton sensitive G-protein-coupled receptors, is responsible for impaired skeletal growth in acidosis.. Chronic metabolic acidosis (CMA) is very often accompanied by skeletal growth retardation. We have previously shown in an ex vivo model of endochondral ossification that murine mandibular condyles subjected to acidic conditions exhibit growth retardation accompanied by a decline of insulin-like growth factor-I (IGF-I) and its receptors. PTH-induced ameliorative effects on the CMA-induced growth retardation of the mandibular condyle are partially mediated by protein kinase C (PKC). In this study we explored the mechanisms underlying the acidosis-induced growth retardation; in particular, the involvement of the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) cellular pathway in the process.. Mandibular condyles from neonatal mice or mandibular condyle derived chondrocytes (MCDCs) were incubated for 3 days under either control or acidic conditions or in the presence of cAMP-regulating factors (cAMPrf) such as forskolin, iso-butyl methyl xanthine (IBMX), or 8-Br cAMP. The effects on proliferation and differentiation of the cultures as well as on phosphorylation of cAMP responsive element binding protein (CREB) and increased expression of the alpha subunit, Gs were determined. The intracellular pH was detected using the acridine orange assay.. Our results show that, under acidic conditions, PKA levels were increased. H89 abolished the adverse effects of acidosis on condylar development and restored IGF-I and IGF-I receptors (IGF-IR) levels. The inhibitory effects of acidosis on proliferation and differentiation of cartilaginous cells were mimicked by cAMPrf. We have also shown that acidosis stimulates activation of Gs trimeric protein and CREB phosphorylation. GDPbetaS--a Gs antagonist--abolished the acidosis-induced condylar growth arrest. Using an acridine orange assay, we showed that the intracellular environment is not acidified under acidic conditions.. Our results indicate that the adverse effects of acidosis on skeletal growth centers are mediated at least in part by the cAMP/PKA cellular pathway. We speculate that high proton concentrations exerted by acidosis conditions stimulate proton sensitive G-protein-coupled receptors, which are mediated by the cellular cAMP/PKA pathway and induce skeletal growth retardation.

Topics: Acidosis; Animals; Animals, Newborn; Blotting, Western; Cell Division; Chronic Disease; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Growth Disorders; GTP-Binding Protein alpha Subunits, Gs; Immunohistochemistry; In Situ Hybridization; Insulin-Like Growth Factor I; Isoquinolines; Mandible; Mice; Phosphorylation; Protein Kinase C; Protons; Receptor, IGF Type 1; Sulfonamides