Jonathan D. Verrier

The Brain In Vivo Expresses the 2′,3′-cAMP-Adenosine Pathway

J. Neurochem. (2012) 122, 115–125.

Expression of the 2′,3′-cAMP-Adenosine Pathway in Astrocytes and Microglia

J. Neurochem. (2011) 118, 979–987.

Extracellular guanosine regulates extracellular adenosine levels

The aim of this investigation was to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels. Rat preglomerular vascular smooth muscle cells were incubated with adenosine, guanosine, or both. Guanosine (30 μmol/l) per se had little effect on extracellular adenosine levels. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) were 0.125 ± 0.020 μmol/l, indicating rapid disposition of extracellular adenosine. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) plus guanosine (30 μmol/l) were 1.173 ± 0.061 μmol/l, indicating slow disposition of extracellular adenosine. Cell injury increased extracellular levels of endogenous adenosine and guanosine, and the effects of cell injury on endogenous extracellular adenosine were modulated by altering the levels of endogenous extracellular guanosine with exogenous purine nucleoside phosphorylase (converts guanosine to guanine) or 8-aminoguanosine (inhibits purine nucleoside phosphorylase). Extracellular guanosine also slowed the disposition of extracellular adenosine in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts, and kidney epithelial cells and in human aortic and coronary artery vascular smooth muscle cells and coronary artery endothelial cells. The effects of guanosine on adenosine levels were not mimicked or attenuated by 5-iodotubericidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)-adenine (adenosine deaminase inhibitor), 5-aminoimidazole-4-carboxamide (guanine deaminase inhibitor), aristeromycin (S-adenosylhomocysteine hydrolase inhibitor), low sodium (inhibits concentrative nucleoside transporters), S-(4-nitrobenzyl)-6-thioinosine [inhibits equilibrative nucleoside transporter (ENT) type 1], zidovudine (inhibits ENT type 2), or acadesine (known modulator of adenosine levels). Guanosine also increases extracellular inosine, uridine, thymidine, and cytidine, yet decreases extracellular uric acid. In conclusion, extracellular guanosine regulates extracellular adenosine levels.

Role of CNpase in the oligodendrocytic extracellular 2'3'-cAMP-adenosine pathway

Extracellular adenosine 3′,5′‐cyclic monophosphate (3′,5′‐cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2′,3′‐cAMP (positional isomer of 3′,5′‐cAMP) is also a source of adenosine, particularly in the brain in vivo post‐injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2′,3′‐cAMP to adenosine. Here, we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2′,3′‐cAMP and their respective adenosine monophosphates (2′‐AMP and 3′‐AMP). Cells were also isolated from mice deficient in 2′,3′‐cyclic nucleotide‐3′‐phosphodiesterase (CNPase). Oligodendrocytes metabolized 2′,3′‐cAMP to 2′‐AMP with 10‐fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3′‐AMP was minimal in both oligodendrocytes and neurons. The production of 2′‐AMP from 2′,3′‐cAMP was reduced by 65% in CNPase −/− versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2′‐AMP to adenosine, and this was also attenuated in CNPase −/− oligodendrocytes. Inhibition of classic 3′,5′‐cAMP‐3′‐phosphodiesterases with 3‐isobutyl‐1‐methylxanthine did not block metabolism of 2′,3′‐cAMP to 2′‐AMP and inhibition of classic ecto‐5′‐nucleotidase (CD73) with α,β‐methylene‐adenosine‐5′‐diphosphate did not attenuate the conversion of 2′‐AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2′,3′‐cAMP‐adenosine pathway (2′,3′‐cAMP → 2′‐AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2′,3′‐cAMP to 2‐AMP in CNS cells. By reducing levels of 2′,3′‐cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury. GLIA 2013;61:1595–1606

Pharmacological Inhibition of Pleckstrin Homology Domain Leucine-Rich Repeat Protein Phosphatase Is Neuroprotective: Differential Effects on Astrocytes

Pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) inhibits protein kinase B (AKT) survival signaling in neurons. Small molecule pan-PHLPP inhibitors (selective for PHLPP1 and PHLPP2) may offer a translatable method to induce AKT neuroprotection. We tested several recently discovered PHLPP inhibitors (NSC117079 and NSC45586; benzoic acid, 5-[2-[4-[2-(2,4-diamino-5-methylphenyl)diazenyl]phenyl]diazenyl]-2-hydroxy-,sodium salt.) in rat cortical neurons and astrocytes and compared the biochemical response of these agents with short hairpin RNA (shRNA)-mediated PHLPP1 knockdown (KD). In neurons, both PHLPP1 KD and experimental PHLPP inhibitors activated AKT and ameliorated staurosporine (STS)-induced cell death. Unexpectedly, in astrocytes, both inhibitors blocked AKT activation, and NSC117079 reduced viability. Only PHLPP2 KD mimicked PHLPP inhibitors on astrocyte biochemistry. This suggests that these inhibitors could have possible detrimental effects on astrocytes by blocking novel PHLPP2-mediated prosurvival signaling mechanisms. Finally, because PHLPP1 levels are reportedly high in the hippocampus (a region prone to ischemic death), we characterized hippocampal changes in PHLPP and several AKT targeting prodeath phosphatases after cardiac arrest (CA)-induced brain injury. PHLPP1 levels increased in rat brains subjected to CA. None of the other AKT inhibitory phosphatases increased after global ischemia (i.e., PHLPP2, PTEN, PP2A, and PP1). Selective PHLPP1 inhibition (such as by shRNA KD) activates AKT survival signaling in neurons and astrocytes. Nonspecific PHLPP inhibition (by NSC117079 and NSC45586) only activates AKT in neurons. Taken together, these results suggest that selective PHLPP1 inhibitors should be developed and may yield optimal strategies to protect injured hippocampal neurons and astrocytes—namely from global brain ischemia.

Schwann Cells Metabolize Extracellular 2′,3′-cAMP to 2′-AMP

The 3′,5′-cAMP–adenosine pathway (3′,5′-cAMP→5′-AMP→adenosine) and the 2′,3′-cAMP–adenosine pathway (2′,3′-cAMP→2′-AMP/3′-AMP→adenosine) are active in the brain. Oligodendrocytes participate in the brain 2′,3′-cAMP–adenosine pathway via their robust expression of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; converts 2′,3′-cAMP to 2′-AMP). Because Schwann cells also express CNPase, it is conceivable that the 2′,3′-cAMP–adenosine pathway exists in the peripheral nervous system. To test this and to compare the 2′,3′-cAMP–adenosine pathway to the 3′,5′-cAMP–adenosine pathway in Schwann cells, we examined the metabolism of 2′,3′-cAMP, 2′-AMP, 3′-AMP, 3′,5′-cAMP, and 5′-AMP in primary rat Schwann cells in culture. Addition of 2′,3′-cAMP (3, 10, and 30 µM) to Schwann cells increased levels of 2′-AMP in the medium from 0.006 ± 0.002 to 21 ± 2, 70 ± 3, and 187 ± 10 nM/µg protein, respectively; in contrast, Schwann cells had little ability to convert 2′,3′-cAMP to 3′-AMP or 3′,5′-cAMP to either 3′-AMP or 5′-AMP. Although Schwann cells slightly converted 2′,3′-cAMP and 2′-AMP to adenosine, they did so at very modest rates (e.g., 5- and 3-fold, respectively, more slowly compared with our previously reported studies in oligodendrocytes). Using transected myelinated rat sciatic nerves in culture medium, we observed a time-related increase in endogenous intracellular 2′,3′-cAMP and extracellular 2′-AMP. These findings indicate that Schwann cells do not have a robust 3′,5′-cAMP–adenosine pathway but do have a 2′,3′-cAMP–adenosine pathway; however, because the pathway mostly involves 2′-AMP formation rather than 3′-AMP, and because the conversion of 2′-AMP to adenosine is slow, metabolism of 2′,3′-cAMP mostly results in the accumulation of 2′-AMP. Accumulation of 2′-AMP in peripheral nerves postinjury could have pathophysiological consequences.

The Many Roles of Adenosine in Traumatic Brain Injury

Many secondary mechanisms regulate the evolution of damage and repair after traumatic brain injury (TBI). Bench-to-bedside studies from our team have shown that adenosine plays a key role in several secondary injury pathways in the brain, namely, excitotoxicity, neuroinflammation, and cerebral blood flow (CBF) dysregulation. Studies of adenosine A1-receptor (A1R) knockout (KO) mice strongly support a key role for adenosine-dependent effects at this receptor in attenuating posttraumatic excitotoxicty. A1R KO mice develop lethal status epilepticus after experimental TBI. Evidence also supports a role of the A1R in human TBI since A1R gene variants are associated with posttraumatic seizures in patients with TBI. A1R also modulates the neuroinflammatory response to TBI. A1R KO mice exhibit enhanced microglial proliferation early after TBI. Adenosine acting at A2A and/or A2B receptors also modulates posttraumatic CBF. Local injection of either the nonselective AR agonist 2-chloroadenosine or the A2AR agonist CGS21680 markedly increases CBF in naive or brain-injured rats. However, whether the effects of adenosine on CBF after TBI are beneficial or detrimental is controversial. Mitigation of ischemia by adenosine produced from breakdown of ATP after TBI has been suggested, and consistent with this hypothesis, we reported increases in brain interstitial adenosine levels in patients with severe TBI during episodes of ischemia. However, in studies of CBF in patients after severe TBI, we also noted an association between cerebrospinal fluid (CSF) adenosine levels and uncoupling of CBF and metabolism (cerebral metabolic rate for oxygen [CMRO2]). Uncoupling of CBF and CMRO2 is associated with intracranial hypertension. Thus, adenosine may represent a two-edged sword after TBI. Additional evidence supporting a protective role of adenosine in TBI comes from clinical studies showing a powerful association between CSF caffeine levels and favorable outcome after TBI—given the interplay between caffeine and adenosine in neuroprotection. Finally, our work also suggests that the newly discovered 2,3-cyclic AMP pathway represents an important component of the adenosine response to TBI.

Role of CD73 in renal sympathetic neurotransmission in the mouse kidney

Adenosine formed during renal sympathetic nerve stimulation (RSNS) enhances, by activating A1 receptors, the postjunctional effects of released norepinephrine and participates in renal sympathetic neurotransmission. Because in many cell types CD73 (ecto‐5′‐nucleotidase) is important for the conversion of 5′‐AMP to adenosine, we investigated whether CD73 is necessary for normal renal sympathetic neurotransmission. In isolated kidneys from CD73 wild‐type mice (CD73+/+; n = 17) perfused at a constant rate with Tyrodes solution, RSNS increased perfusion pressure by 17 ± 4, 36 ± 8, and 44 ± 10 mm Hg at 3, 5, and 7 Hz, respectively. Similar responses were elicited from kidneys isolated from CD73 knockout mice (CD73−/−; n = 13; 28 ± 11, 43 ± 10, and 44 ± 10 mm Hg at 3, 5, and 7 Hz, respectively); and a high concentration (100 μmol/L) of α,β‐methyleneadenosine 5′‐diphosphate (CD73 inhibitor) did not alter responses to RSNS in C57BL/6 mouse kidneys (n = 5; 21 ± 5, 36 ± 8, and 43 ± 9 at 3, 5, and 7 Hz, respectively). Measurements of renal venous adenosine and inosine (adenosine metabolite) by liquid chromatography‐tandem mass spectrometry demonstrated that the metabolism of exogenous 5′‐AMP to adenosine and inosine was similar in CD73−/− versus CD73+/+ kidneys. A1 receptor mRNA expression was increased in CD73−/− kidneys, and 2‐chloro‐N6‐cyclopentyladenosine (0.1 μmol/L; A1 receptor agonist) enhanced renovascular responses to norepinephrine more in CD73−/− versus CD73+/+ kidneys. We conclude that CD73 is not essential for renal sympathetic neurotransmission because in the absence of renal CD73 other enzymes metabolize 5′‐AMP to adenosine and because of compensatory upregulation of postjunctional coincident signaling between norepinephrine and adenosine.