Extracellular Protein Kinase A Modulates Intracellular Calcium

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Extracellular protein kinase A modulates intracellular calcium/calmodulindependent protein kinase II, nitric oxide synthase and the glutamate-nitric oxide-cGMP pathway in cerebellum. Differential effects in hyperammonemia. Andrea Cabrera-Pastor, Marta Llansola, and Vicente Felipo ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00263 • Publication Date (Web): 27 Sep 2016 Downloaded from http://pubs.acs.org on September 29, 2016

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ACS Chemical Neuroscience

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.

Andrea Cabrera-Pastor, Marta Llansola, and Vicente Felipo* Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe, Valencia, Spain

* Correspondence: Vicente Felipo, Neurobiology Laboratory, Centro Investigación Príncipe Felipe. Eduardo Primo-Yufera 3, 46012 Valencia. [email protected]

Tel: 34963289680

Abbreviations: CaMKII, calcium/calmodulin-dependent protein kinase II; LTP, long-term potentiation; NO, nitric oxide; NOS, nitric oxide synthase; PKA, cAMP-dependent protein kinase; PKs, protein kinases; sGC, soluble guanylate cyclase.

Acknowledgements: Supported by Ministerio de Ciencia e Innovación (SAF2014-51851-R), Generalitat Valenciana (PROMETEO-2009-027, PROMETEOII/2014/033) and co-funded with European Regional Development Funds (ERDF).

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ABSTRACT Extracellular protein kinases, including cAMP-dependent protein kinase (PKA) modulate neuronal functions including NMDA receptor-dependent long-term potentiation. NMDA receptors 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-NOcGMP 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/activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA; 3) if the effects are different in hyperammonemic and control rats. The pathway was analysed by in vivo microdialysis. The role of extracellular PKA was analysed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analysed 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. Key words: Extracellular protein kinase A; glutamate-NO-cGMP pathway; nitric oxide synthase; calcium/calmodulin-dependent protein kinase II; phosphorylation; hyperammonemia 2 ACS Paragon Plus Environment

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INTRODUCTION The existence of extracellular (ecto-) protein kinases (PKs) and of substrates phosphorylated by them at the surface of intact neurons was reported in 1986 by Ehrlich et al, who proposed a role for extracellular protein kinases in regulation of specific neuronal functions (1). Subsequent studies have demonstrated the existence of different types of ecto-protein kinases (ecto-PKs), including ecto-protein kinase C (ecto-PKC) (2-3) and ecto-protein kinase dependent on cAMP (ecto-PKA) (4-7). These studies indicate that the powerful regulatory machinery of protein phosphorylation also operates in the extracellular environment in the brain (2). Phosphorylation of membrane proteins by ecto-PKs has been shown to modulate ion channels and Ca2+ influx (8), norepinephrine uptake (9), purinergic P2X3 receptors (3) or neurite outgrowth (2). Ecto-PKs also modulate neurotransmission and synaptic plasticity, including long-term potentiation (LTP) in hippocampus (10-13). Surface protein phosphorylation by ectoPKs is required for maintenance of hippocampal LTP (11). It has been proposed that one of the proteins phosphorylated at extracellular domains by ecto-PKs is the NMDA receptor (12) and that this phosphorylation would modulate Ca2+ influx into neurons (13). Ecto-PKs and some membrane substrates phosphorylated by ecto-PKs have been also reported in cerebellum (14,15), but their possible functional roles have not been studied. We have been studying for several years the modulation of the glutamate-nitric oxide (NO)cGMP pathway in cerebellum by different mechanisms and their alterations in hyperammonemia (16-21). Activation of NMDA receptors increases calcium in the post-synaptic neuron. Calcium binds to calmodulin and activates nitric oxide synthase (NOS), increasing the formation of nitric oxide (NO) which, in turn, activates soluble guanylate cyclase, increasing cGMP. Part of the cGMP formed is released to the extracellular fluid. This allows the analysis of this glutamateNO-cGMP pathway in vivo by microdialysis in freely moving rats (16). The function of this pathway in cerebellum in vivo is modulated by AMPA and metabotropic glutamate receptors (17-18), by GABAA and glycine receptors (19,20) or neurosteroids (21). 3 ACS Paragon Plus Environment


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Patients with liver cirrhosis and hepatic encephalopathy (HE) show cognitive impairment and motor alterations (22). In these patients ammonia is not properly detoxified in the liver, leading to chronic hyperammonemia, which is a main contributor to the neurological alterations in HE (22,23). A main animal model of chronic hyperammonemia is rats fed an ammonium containing diet (24). These hyperammonemic rats show reduced function of the glutamate-NO-cGMP pathway in cerebellum in vivo (16). Modulation of the glutamate-NO-cGMP pathway by AMPA and metabotropic glutamate receptors, by GABAA and glycine receptors or neurosteroids is also altered in hyperammonemia (16-20). The glutamate-NO-cGMP pathway in cerebellum modulates the ability to learn a task in the Y maze. The reduced function of this pathway in cerebellum in hyperammonemia leads to reduced learning ability, which is restored by treatments that restore the glutamate-NO-cGMP pathway function such as phosphodiesterase inhibitors (25,26) or anti-inflammatories (27). The function of the pathway is reduced in hyperammonemic rats due to reduced NOS activity, which is a consequence of increased phosphorylation at Ser847 by calcium/calmodulin dependent protein kinase II (CaMKII), which phosphorylation at Thr286 and activity are increased in hyperammonemic rats (28). Inhibiting CaMKII with KN62 normalizes phopsphorylation and activity of NOS and the function of the glutamate-NO-cGMP pathway in cerebellar slices from hyperammonemic rats (28). In the course of the above studies we obtained some results suggesting that the function of this pathway in cerebellum could be also modulated by an extracellular PKA and that its effects could be different in hyperammonemic rats. The aims of the present work were: 1) assess whether the function of the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an ecto-PKA; 2) assess the role of phosphorylation/activity of CaMKII and NOS in the modulation of the pathway by ecto-PKA;


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3) assess if the effects and/or mechanisms involved are different in hyperammonemic than in control rats. The function of the pathway was analysed in freely moving rats by microdialysis in cerebellum. We choose the cerebellum because we have previously shown that the function of the glutamateNO-cGMP pathway in cerebellum modulates the ability to learn the Y maze task. The studies were performed in control and hyperammonemic rats as two models with different grades of glutamate-NO-cGMP pathway function. The role of ecto-PKA was analysed by inhibiting it with a cell membrane impermeable PKA inhibitor. The mechanisms involved were analysed in freshly isolated cerebellar slices.

RESULTS AND DISCUSSION Inhibition of extracellular protein kinase A increases extracellular cGMP, restores the glutamate-NO-cGMP pathway in hyperammonemic rats and impairs it in control rats in cerebellum in vivo. Figure 1A shows the levels of extracellular cGMP in cerebellum of control and hyperammonemic rats. Fractions 1-5 show the basal levels. Starting from fraction 5, the inhibitor of PKA was administered to one half of the control and hyperammonemic rats. Fractions 5-9 show the cGMP levels in the presence or absence of the cell membrane impermeable PKA inhibitor. NMDA was administered to all rats during fraction 10. Fractions 10-13 show the effects of NMDA on extracellular cGMP levels. The PKA inhibitor induced a rapid and significant increase in extracellular cGMP. The accumulated increase, calculated as described in



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the legend to Fig. 1 was 210±70% of basal in control rats and 459±95% in hyperammonemic rats (Fig. 1B). NMDA was administered through the microdialysis probe during fraction 10 to activate the glutamate-NO-cGMP pathway. The time-course of NMDA-induced increase in cGMP is also shown in Fig. 1A (fractions 10-13). In the absence of the PKA inhibitor, the NMDA-induced increase in cGMP, calculated as described in the legend to Fig. 1, was 1385±295% of basal in control rats and 503±119% in hyperammonemic rats (Fig. 1C). Administration of the PKA inhibitor reduced NMDA-induced increase in cGMP in control rats to 199±126% (p

Extracellular Protein Kinase A Modulates Intracellular Calcium

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