Adenine nucleotides undergo rapid, quantitative conversion to adenosine in the extracellular space in rat hippocampus

Abstract

There are multiple mechanisms by which adenine nucleotides can be released into the extracellular space in brain. Adenine nucleotides are converted extracellularly to adenosine, which then acts on adenosine receptors to elicit physiological responses, but the rate at which this conversion takes place is unknown. In the present experiments, adenine nucleotides were applied to individual hippocampal neurons, and the subsequent activation of a postsynaptic K+ conductance by adenosine A1 receptors was used to determine the rate of adenosine formation. None of the adenine nucleotides tested (cAMP, AMP, ADP, and ATP) activated A1 receptors directly at the concentrations tested (</=200 microM). AMP, ADP, and ATP were all rapidly converted to adenosine, with a T1/2 for ATP conversion to adenosine of approximately 200 msec, and the last step in this pathway (transformation of AMP to adenosine by 5'-nucleotidase) seems to be the rate-limiting step. As we have reported previously, cAMP is converted to adenosine as well, but on a much slower time scale than any of the other nucleotides tested. These experiments demonstrate that fast, localized release of AMP, ADP, or ATP can result in a transient activation of adenosine receptors but that this is unlikely to occur with cAMP. The existence of a highly active ecto-nucleotidase pathway in brain provides a mechanism for the rapid generation of adenosine after the release of adenine nucleotides into the extracellular space.

Fig. 2.

Local drug application protocol. This photomicrograph shows the relative positioning of the patch recording electrode (large arrow) and the drug application pipette (small arrow) during recording from a CA1 pyramidal neuron. After the initiation of whole-cell recording, the drug application pipette was lowered through the tissue under visual control while periodically testing for adenosine responses. Adenosine and adenine nucleotides were ejected by applying brief pressure pulses to the drug application pipette (typically 10 psi/10 msec). Responses could be obtained when the drug pipette was in close proximity to the neuron (2–10 μm), but they were generally undetectable when the pipette was moved farther than 20 μm from the cell being tested. Stable responses could be evoked with this protocol at 30 sec intervals for periods >2 hr. Despite the proximity of the drug pipette to the cell, pressure ejection artifacts were rarely encountered. Scale bar, 13 μm.

Fig. 1.

Effects of bath superfusion with adenine nucleotides on field potentials. Synaptic responses were evoked at 30 sec intervals, and the peak field EPSP (fEPSP) amplitude was plotted as a function of time. Superfusion of slices with increasing concentrations of adenine nucleotides inhibited the fEPSP in a dose-dependent manner. Adenosine A₁ receptors show no desensitization under these conditions (Dunwiddie and Fredholm, 1984), so accurate cumulative dose–response curves can be obtained in this manner. The competitive adenosine receptor antagonist theophylline (THEO) completely reversed the effects of ATP, AMP, and cAMP (A, B, C, respectively), demonstrating that the inhibitory effect was in each case mediated via adenosine receptors. cAMP was significantly more potent than adenosine itself in eliciting this response, but its effects were completely blocked by adenosine deaminase (Brundege et al., 1997), indicating that it is converted to adenosine before it acts on the receptor.

Fig. 3.

Outward currents evoked by local application of adenosine. Application of adenosine (Ado) (200 μm concentration in the drug pipette) to CA1 pyramidal neurons elicited slow outward currents that reached a peak within ∼2–3 sec and typically lasted between 10 and 20 sec. The eight records at the top are consecutive individual evoked responses, low-pass-filtered at 1 kHz, elicited by pressure application of adenosine (10 psi/10 msec), and the upper of the two records at the bottom is an average of 51 such responses shown at 5 × gain (calibration = 50 pA for individual responses, 10 pA for averages). The smooth line superimposed on the average is the best fit (r² = 0.976) to the response using the double exponential equation described in Materials and Methods, with parameters r = 14.9 pA, τ_on = 2.38 sec, τ_off = 4.18 sec, andT₀ = 344 msec. The lowest record is a similar average of responses to local application of AMP, with the best fit line (r² = 0.975) corresponding to parameters r = 39.3 pA, τ_on = 9.09 sec, τ_off = 3.09 sec, and T₀ = 280 msec. The thin vertical line on theleft indicates the time at which adenosine/AMP were applied, and T₀ corresponds to the delay between drug application and the point at which the exponential function crosses the abscissa (i.e., the onset of the response). Thedashed lines at the bottom indicate the pre-response baseline.

Fig. 4.

Theophylline reversibility of responses to adenosine, AMP, ADP, and ATP. Application of AMP, ADP, and ATP (B–D) (each at 200 μm in the drug pipette) elicited responses that were qualitatively and quantitatively similar to those elicited by 200 μm adenosine (A). Responses to adenosine as well as to the nucleotides were antagonized by bath superfusion with 200 μm theophylline (Theo), a competitive antagonist at adenosine receptors, but showed full recovery when theophylline was washed from the bath (not shown). In this and in subsequent figures, each response is the average of 5–20 responses low-pass-filtered at 1 kHz, and the time of drug application is denoted with an arrow. The theophylline antagonism of these responses in all slices tested for each of the nucleotides is summarized in E; numbers below thebars indicate the number of cells tested with each combination of drugs. A subset of the control data for adenosine, AMP, and cAMP has been published previously (Brundege et al., 1997). Calibration: 1 sec and 3 pA (A, B); 1.5 pA (C); and 1 pA (D). *p < 0.02; **p < 0.002.

Fig. 7.

Comparison of paired applications of AMP or cAMP versus adenosine. When applied alternately from adjacent barrels of a drug pipette, very similar responses were elicited by AMP and adenosine (A). Both adenosine and AMP responses were blocked by superfusion with theophylline (not shown). In contrast, cAMP did not elicit a detectable current when it was applied alternately with AMP (B). C, Responses from another pyramidal neuron when adenosine and AMP were applied alternately from a two-barrel drug pipette. Both responses were well fit by the product of two exponential functions (solid lines superimposed on the averaged responses). At thebottom the fit lines are shown superimposed; thesolid line corresponds to the adenosine response, and the dashed line the AMP response. The fit parameters for the adenosine and AMP responses (respectively) werer = 18/25 pA, τ_on = 1.28/1.95 sec, τ_off = 4.52/3.71 sec, and T₀ = 110/260 msec. In this example, T₀ was the only parameter that differed significantly for the two responses. Time of drug ejection is indicated by the vertical arrow, and the calibration bar in all cases is 3 pA. The time scaleat the bottom applies to all the records.

Fig. 5.

Effect of superfused theophylline versus theophylline in the drug application pipette. Double-barrel pipettes were filled with adenosine alone (200 μm) in one barrel and adenosine + theophylline (also 200 μm) in the other, and the two solutions were applied alternately to the same cell.A, The response to concurrently applied adenosine and theophylline (Ado/Theo) was virtually identical to the response to adenosine alone (Ado); i.e., there was no antagonism by theophylline, even though bath superfusion with this concentration of theophylline was sufficient to nearly abolish the adenosine response (compare Fig. 4A).B, The effect of bath superfusion of 200 μm theophylline on the adenosine + theophylline response from A. The degree of antagonism of the adenosine + theophylline response by bath-superfused theophylline (C) was equivalent to that observed with application of adenosine alone (not shown). This experiment was replicated in another cell with identical results.

Fig. 6.

GMP and AOPCP antagonize responses to nucleotides but not adenosine. Responses to local application of adenosine (A) (200 μm) were completely insensitive to bath superfusion of the slice with 2 mmguanosine monophosphate (+GMP), which is an inhibitor of 5′-nucleotidase, the enzyme that converts AMP to adenosine. GMP concentrations up to 5 mm had no effect on the response to adenosine; however, responses to AMP (B), ADP (C), and ATP (D) (all at 200 μm) were blocked nearly completely by superfusion with 2 mm GMP. In C and D, separate averages of pre-GMP responses and responses obtained after GMP washout are illustrated and are virtually superimposable. InD, there was a small, very slow inward current response to ATP that persisted in 2 mm GMP, and this was abolished by increasing the GMP concentration to 5 mm (not shown). Calibration bars indicate 1 sec and 3 pA for each set of averages. Summary data for all slices tested with GMP are shown inE, as well as for slices tested with 250 μm AOPCP. The dose–response curve for bath-superfused GMP versus 200 μm ATP (local pressure ejection) is illustrated in F. Each point represents an individual slice tested with a single concentration of superfused GMP; the solid line represents the best fit to the points using the logistic equation, with an EC₅₀ of 0.74 mm.

Fig. 8.

Comparison of the kinetics of adenosine and ATP responses. Application of ATP (200 μm) (light line) from one barrel of a drug pipette elicited outward current responses that were qualitatively and quantitatively similar to those elicited by the ejection of adenosine (also 200 μm) (heavy line) from the adjacent barrel. The only period during which there was a statistically significant difference between these two averaged response waveforms, as determined by lack of overlap in their associated 95% confidence limits, is during the segment bracketed by the vertical lines. For the adenosine response, the raw data were fit by the product of two exponential functions, as described in Materials and Methods. For the ATP response, a somewhat modified function was used. It was assumed that ATP had no direct effect on the receptor, that a constant fraction of the ATP that was present was converted to adenosine per unit time (i.e., first order kinetics), and that the time course of the response to adenosine formed from ATP was governed by the kinetic parameters corresponding to the best fit to the direct response to adenosine. When theT_1/2 for the conversion of ATP to adenosine was allowed to vary as a free parameter, the best fit to the ATP response was obtained with a T_1/2 of 170 msec. The fit lines corresponding to these two functions are thesmooth lines superimposed on the responses. Time of drug ejection is indicated by the vertical arrow.