Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.
Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.
Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo-Yufera 3, 46012 Valencia, Spain.
Andrea
A
Cabrera-Pastor
Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo-Yufera 3, 46012 Valencia, Spain.
Marta
M
Llansola
Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo-Yufera 3, 46012 Valencia, Spain.
Vicente
V
Felipo
D064751:Ammonium Compounds; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D004032:Diet; D004195:Disease Models, Animal; D005110:Extracellular Space / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D022124:Hyperammonemia / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010766:Phosphorylation; D017208:Rats, Wistar; D015398:Signal Transduction; D046509:Tissue Culture Techniques
D064751:Ammonium Compounds; D009569:Nitric Oxide; D018698:Glutamic Acid; D019001:Nitric Oxide Synthase; D017868:Cyclic AMP-Dependent Protein Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D006152:Cyclic GMP
Extracellular protein kinase A; calcium/calmodulin-dependent protein kinase II; glutamate−NO−cGMP pathway; hyperammonemia; nitric oxide synthase; phosphorylation