Regulation of extracellular adenosine in rat hippocampal slices is temperature dependent: role of adenosine transporters

doi:10.1016/S0306-4522(99)00404-2

Neuroscience

Volume 95, Issue 1, November 1999, Pages 81-88

https://doi.org/10.1016/S0306-4522(99)00404-2 Get rights and content

Abstract

While a great deal is known about stimuli that can induce the release of adenosine from brain tissue, relatively little is known about the regulation of the basal extracellular concentration of adenosine that is present in the absence of stimulation. Under normal conditions, enough adenosine is present to tonically activate a significant portion of the high-affinity adenosine A₁ receptors. The present experiments demonstrated that the estimated basal concentration of extracellular adenosine in rat hippocampal slices maintained at 21°C (430 nM) is approximately twice that at 32°C (220 nM). The sensitivity of presynaptic modulatory adenosine A₁ receptors was not significantly different at 21°C or at 32°C. Slices maintained at 21°C also showed a reduced ability to inactivate extracellular adenosine, which reflects a reduction in adenosine transport across cell membranes. This effect appears to be primarily due to a reduction in the function of the equilibrative, dipyridamole-sensitive (ei) adenosine transporter; the nitrobenzylthioinosine-sensitive equilibrative transporter (es transporter) appears to be relatively less affected by temperature than is the ei transporter. These experiments demonstrate that extracellular concentrations of adenosine in the brain are sensitive to temperature, and suggest that some of the neurological effects of hypothermia might be mediated via increased concentrations of adenosine in the extracellular space.

Section snippets

Experimental procedures

Transverse hippocampal slices (400 μm-thick) were prepared from six to eight-week-old male Sprague–Dawley rats (Sasco Animal Laboratories, Omaha, NE), and maintained in an incubation chamber at either 32°C or 21°C (room temperature), as has been previously described.11., 23. In all cases, the temperature at which recordings were made was identical to the temperature at which slices were incubated prior to recording. Animals were treated in such a way as to minimize suffering, using experimental

Results

In initial experiments, it was noted that the amplitude of excitatory synaptic responses elicited by stimulation of the Schaffer collateral and commissural inputs to the CA1 region in rat brain slices declined significantly when the temperature of the incubation medium was reduced from 32° to 21°C. As illustrated in Fig. 1, there was an increased latency to onset of the presynaptic fiber spike (indicating a slowed conduction speed in the presynaptic axons), and a concomitant decrease in the

Discussion

Brain microdialysis experiments in vivo,3., 26., 31. as well as biochemical experiments in vitro,21., 22. have demonstrated the presence of significant concentrations of extracellular adenosine in normal brain. Consistent with this, excitatory synaptic responses in many brain regions, including the CA1 region of the hippocampus, are tonically inhibited by extracellular adenosine. When there are changes in the level of this tonic inhibition, as occurred in the present experiments, these could

Conclusions

These experiments demonstrate that when rat hippocampal slices are incubated at reduced temperatures (21°C), the concentration of adenosine in the extracellular space is approximately twice that of slices incubated at 32°C. This increase in the concentration of adenosine appears to reflect a diminished activity of adenosine transporters, and in particular, the activity of the dipyridamole-sensitive ei transporter appears to be particularly sensitive to temperature, relative to the

Acknowledgements

This work supported by grant R01 NS 29173 from the National Institute of Neurological Disorders and Stroke and the Veterans Administration Medical Research Service.

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Exogenous adenosine produces potent synaptic inhibition in spinal substantia gelatinosa (SG), a region involved in nociceptive and thermoreceptive mechanisms. To examine the possibility that endogenous adenosine tonically modulates excitatory synaptic transmission in spinal SG, whole-cell, voltage-clamp recordings were made from SG neurons in adult rat spinal cord slices. In all SG neurons sensitive to exogenous adenosine, the adenosine uptake inhibitor, NBTI, mimics adenosine's inhibitory actions on dorsal root evoked EPSCs (eEPSCs) and miniature spontaneous EPSCs (mEPSCs). These inhibitory effects were antagonized by A1 adenosine receptor antagonist, DPCPX. DPCPX also potentates eEPSCs in those SG neurons in which adenosine or adenosine A1 receptor agonists (CHA, CCPA) suppressed eEPSCs. DPCPX often increases mEPSC frequency without altering mEPSC amplitude, suggesting presynaptic action on adenosine A1 receptors. Selective A2 (DMPX) and A2a (ZM 241385) adenosine receptor antagonists had no or minimal effects upon either eEPSCs or mEPSCs. The adenosine degrading enzyme, adenosine deaminase, mimicked the effects of DPCPX on the mEPSC frequency. We conclude that the excitatory synaptic transmission in the spinal SG is under an inhibitory tone of endogenous adenosine through the activation of A1 receptors. The present results suggested that the background activity of A1 receptors in the spinal SG might be contributed to setting the physiological “noceceptive thresholds”.
Inhibition of the equilibrative nucleoside transporter 1 and activation of A<inf>2A</inf> adenosine receptors by 8-(4-chlorophenylthio)-modified cAMP analogs and their hydrolytic products
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Similar to PC12 cells, 8-Br-cAMP and 8-Br-ado had no effect on [3H]thymidine uptake in hENT1-overexpressing HEK 293 cells (Fig. 5B), supporting the suggestion that 8-Br-cAMP and 8-Br-ado did not inhibit ENT1. Adenosine transport inhibition may increase the amount of endogenous adenosine available to bind cell surface adenosine receptors, leading to adenosine receptor activation (22, 23). In PC12 cells, the Gs protein-coupled A2AR is the only functionally relevant adenosine receptor expressed endogenously (24).
Cyclic AMP analogs containing hydrophobic modification of C⁸ at the adenine ring such as 8-(4-chlorophenylthio)-cAMP (8-pCPT-cAMP) and 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT-2′-O-methyl-cAMP) can penetrate membranes due to their high lipophilicity and directly activate intracellular cAMP effectors. Therefore, these cAMP analogs have been used in numerous studies, assuming that their effects reflect the consequences of direct activation of cAMP effectors. The present study provides evidence that 8-pCPT-modified cAMP analogs and their corresponding putative hydrolysis products (8-(4-chlorophenylthio)-adenosine (8-pCPT-ado) and 8-(4-chlorophenylthio)-2′-O-methyl-adenosine (8-pCPT-2′-O-methyl-ado)) inhibit the equilibrative nucleoside transporter 1 (ENT1). In PC12 cells, in which nucleoside transport strongly depended on ENT1, 8-pCPT-ado, 8-pCPT-2′-O-methyl-ado, and, to a smaller extent, 8-pCPT-2′-O-methyl-cAMP caused an increase of protein kinase A substrate motif phosphorylation and anti-apoptotic effect by an A_2A adenosine receptor (A_2AR)-dependent mechanism. In contrast, the effects of 8-pCPT-cAMP were mainly A_2AR-independent. In HEK 293 showing little endogenous ENT1-dependent nucleoside transport, transfection of ENT1 conferred A_2AR-dependent increase in protein kinase A substrate motif phosphorylation. Together, the data of the present study indicate that inhibition of ENT1 and activation of adenosine receptors have to be considered when interpreting the effects of 8-pCPT-substituted cAMP/adenosine analogs.

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Regulation of extracellular adenosine in rat hippocampal slices is temperature dependent: role of adenosine transporters

Abstract

Section snippets

Experimental procedures

Results

Discussion

Conclusions

Acknowledgements

Neurosci. Lett.

Trends Pharmac.

Brain Res.

Int. Rev. Neurobiol.

Neuroscience

Prog. Neurobiol.

Neurosci. Lett.

Brain Res.

Neurochem. Int.

Neurochem. Int.

Neuropharmacology

J. biol. Chem.

Gen. Pharmac.

Neuron

Neurosci. Lett.

Neuroscience