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The most recently discovered adenosine receptor, the A3 receptor, belongs to the general class of G-protein coupled receptors, and has been linked to several putative effector mechanisms. One distinguishing characteristic of this receptor is that many adenosine agonists have affinities for the A3 receptor that are much lower than their corresponding affinities at the adenosine A1 receptor (Zhou et al., 1992), and this is true as well for the endogenous ligand, adenosine. It has been reported that A3 receptors are coupled in an inhibitory fashion to adenylyl cyclase (Zhou et al., 1992; Zhao et al., 1997), although in other systems, A3 receptor activation activates phospholipase C and leads to an elevation in inositol phosphate levels (Ali et al., 1990; Ramkumar et al., 1994; Auchampach et al., 1997), and this occurs in brain as well (Abbracchio et al., 1995). If this is the case, then increases in intracellular Ca2+ and activation of protein kinase C should occur as a consequence of agonist occupation of the A3 receptor. Recent studies in brain have suggested that A3 receptors can inhibit the function of presynaptic modulatory receptors (Dunwiddie et al., 1997; Macek et al., 1998), and that this effect is mediated via PKC (Macek et al., 1998; Diao and Dunwiddie, unpublished observations). However, other reports have proposed that postsynaptic A3 receptors in hippocampus may elicit responses that are linked to activation of PKA but not PKC (Fleming and Mogul, 1996), suggesting that a further consideration of effector pathways for this receptor is warranted. One response in hippocampal CA1 pyramidal neurons that is affected by increases in intracellular Ca2+, as well as activation of either PKC or PKA, is the K+-mediated afterhyperpolarization that follows activation of voltage dependent Ca2+ channels by a depolarizing stimulus. Previous studies have demonstrated that activation of β-adrenergic receptors will nearly abolish the AHP (Madison and Nicoll, 1982; Haas and Konnerth, 1983), an effect that can be mimicked directly by activation of PKA (Abdul-Ghani et al., 1996). Activation of PKC with phorbol esters will also inhibit this response (Malenka et al., 1986), and activation of either muscarinic receptors (Cole and Nicoll, 1984) or metabotropic glutamate receptors (Abdul-Ghani et al., 1996) reduce the AHP via increases in intracellular Ca2+ and subsequent activation of CaM-K. Furthermore, adenosine itself has been previously reported to either increase (Haas and Greene, 1984) or decrease (Dunwiddie, 1985) this current. Thus, this response appeared to be a likely candidate that would be sensitive to activation of multiple second messenger systems, but an effect of A3 receptor activation on this conductance has not been described.
The recent development of Cl-IB-MECA, an agonist that is approximately 2500-fold selective for the A3 vs. the A1 receptor, and 1400-fold selective for the A3 vs. the A2a receptor (Jacobson et al., 1995), has facilitated the study of the physiological consequences of selective activation of this receptor. Behavioral studies have demonstrated an A3 receptor-mediated depression of locomotor activity (Jacobson et al., 1993), but the effects of A3 receptor activation on neuronal activity at the cellular level are only poorly understood. A recent report has suggested that activation of A3 receptors can increase Ca2+ currents in hippocampal CA3 neurons (Fleming and Mogul, 1996), but apart from this, there have been few reports of specific actions of A3 receptor activation on neuronal activity.
Because the hippocampus and cerebellum show the highest levels of A3 mRNA in brain (De et al., 1993), and because the responses to A1 and A2 receptor activation have been well-characterized in this brain region (Dunwiddie, 1985; Greene and Haas, 1991; Cunha et al., 1994, 1995), we have investigated the electrophysiological actions of Cl-IB-MECA in this brain region.
Hippocampal slices were obtained from 6- to 8-week-old, male Sprague-Dawley rats (Sasco Animal Laboratories, Omaha, NE) using standard techniques (Dunwiddie and Lynch, 1978; Dunwiddie and Hoffer, 1980). Animals were decapitated, the hippocampus was dissected free of surrounding tissue, and 400 μm slices were cut from the middle portion of the hippocampus with a TC-2 tissue chopper (Sorvall, Norwalk, CT). Slices were immediately placed in ice-cold artificial cerebral spinal fluid (aCSF) consisting of (in mM): NaCl, 124; KCl, 3.3; KH2PO4, 1.2; MgSO4, 2.4; CaCl2, 2.5; D-glucose, 10; and NaHCO3 25.7, pH 7.4, pregassed with 95% O2–5% CO2, and were then transferred to an incubation chamber maintained at 33°C to equilibrate for at least 1 h before electrophysiological recording. When recording, the slices were transferred to a submersion recording chamber (1 ml volume) where they were placed on a nylon net and superfused with medium at a rate of 2 ml/min. The superfusion medium was gassed with humidified 95% 02–5% CO2 and maintained at a temperature of 33°C to 34°C.
Whole-cell patch recording experiments were performed using patch pipettes pulled from borosilicate glass (O.D. 1.5 mm, I.D. 0.86 mm, with filament; Sutter Instrument Co., Novato, CA). Electrodes had tip resistance of 5–8 MΩ when filled with a solution containing (in mM): K-Gluconate, 125; KCl, 15; HEPES, 10; CaCl2, 0.1; KBAPTA, 1; K-ATP, 2; Tris-GTP, 0.3; pH adjusted to 7.3 with KOH, osmolarity adjusted to 280–290 mOsm. Whole-cell electrophysiological recordings were made using the “blind” patch recording technique (Blanton et al., 1989). The access resistance was continually monitored by observation of the cell membrane capacitative transients in response to a brief voltage step and was below 25 MΩ in all experiments. Pyramidal neurons were differentiated from glial cells by their ability to fire action potentials, and from interneurons by their high membrane capacitance and accommodation of cell firing in response to depolarizing current injection. Cells were voltages-clamped using an Axoclamp-2A amplifier (Axon-Instruments, Burlingame, CA) in the single-electrode voltage-clamp mode. The membrane potentials were corrected by − 11 mV for the liquid junction potential between the electrode filling solution and the bath (Neher, 1992).
IAHP was evoked every 40 s by briefly (150 ms) stepping to 0 mV from holding potentials of − 55 mV. Membrane resistance was determined by measuring the current change in response to a − 4mV command step every 40 s, and holding current was measured every 20 s. Responses were recorded using an R.C. Electronics ISC-16 A/D board and software developed in our laboratory.
At least 10 to 15 min of stable baseline responses were obtained in each experiment before drug applications began. Following acquisition of the baseline data, the effects of drugs were then tested by adding them directly to the superfusion medium with a calibrated syringe pump (Razel Scientific Instruments, Inc., Stamford, CT). The net change in flow rate using this approach was never more than 1%. The peak amplitude of the AHP current (IAHP) was determined for each response and then averaged during the pre-drug control, drug, and the post-drug washout periods. At least 10 responses were included in each average. The data were analyzed as mean percent change in the response amplitude when compared to responses obtained during the control period.
All data were analyzed between groups, using the unpaired Student’s t-test and nonparametric test (Mann-Whitney test) with a P<0.05 criterion for statistical significance.
Adenosine, K-ATP, and Tris-ATP were obtained from Sigma (St. Louis, MO); baclofen, carbachol, 8-cyclopentyl-1,3-dimethylxanthine (CPT), and 3,7-dimethyl-1-propargylxanthine (DMPX) were purchased from Research Biochemicals (Natick, MA); 2-chloro-N6-(3-iodobenzyl)-adenosine-5-N-methyluronamide (Cl-IB-MECA) was provided by Research Biochemical International (Natick, MA) as part of the Chemical Synthesis Program of the National Institute of Mental Health, contract N01MH30003. The K-gluconate, KCl, CaCl2 and HEPES used in the patch electrode recording solution were from Fluka Chemika-Biochemika (Ronkonkoma, New York), and K-BAPTA was from Molecular Probes (Eugene, Oregon). All drugs were dissolved in distilled water except CPT and Cl-IB-MECA, which were initially dissolved in 100% dimethylsulfoxide (DMSO) at greater than 1000 times final concentration and then diluted with aCSF to the desired concentration. The final concentration of DMSO in the superfusion medium never exceeded 0.05%.
When hippocampal brain slices were superfused with adenosine (50 μM), a rapid outward current developed that showed little desensitization during a 10 min superfusion period, and reversed readily upon return to control buffer (Fig. 1A). This response was completely blocked by the selective adenosine A1 receptor antagonist N6-cyclopentyl-theophylline (CPT; 1 μM), and by CPT + DMPX (a moderately selective A2 antagonist; 50 μM). In either case, there was no significant effect of adenosine superfusion on the holding current (Fig. 1A). These latter conditions are nearly identical to those under which both adenosine and N6-2-(4-aminophenyl)ethyl-adenosine (APNEA) have been reported to increase Ca2+ currents in isolated hippocampal CA3 pyramidal neurons, an effect attributed to activation of A3 receptors (Fleming and Mogul, 1997). These results suggested that under resting conditions, activation of A3 receptors by adenosine did not have any significant effect upon the holding current in CA1 pyramidal neurons voltage clamped at −55 mV. This observation was confirmed by superfusion of slices with either 100 nM or 1 μM Cl-IB-MECA, also in the presence of CPT + DMPX to block any possibility’ of activation of A1 or A2 receptors; in this situation, there was also no significant change in the holding current required to clamp neurons to −55 mV (Fig. 1B).
It has been suggested that activation of protein kinase C (PKC) in hippocampal pyramidal neurons is linked to a reduction in the magnitude of afterhyperpolarizations (AHPs) induced by depolarizing current injection (Malenka et al., 1986; Engisch et al., 1996). Because previous studies have shown that A3 receptors can also, be linked to activation of phospholipase C (PLC), and hence activation of PKC, we characterized the effects of adenosine and the selective A3 agonist Cl-IB-MECA on IAHP. A 150 ms command step to 0 mV typically elicited a slow, outward current that lasted 10 s or more, and which was usually between 50–500 pA in amplitude (Fig. 2A2, 2B2). The time course of this response was consistent with the time course of the slow AHP activated by depolarizing current injection in current clamped neurons (Madison and Nicoll, 1982; Haas and Greene, 1984). To confirm the identity of this current, and to verify its previously reported sensitivity to other pharmacological agents, we examined the effects of carbachol (10 μM; Fig. 2A), and isoproterenol (500 nM; Fig. 2B), both of which have been reported to inhibit this current. Bath superfusion with either of these agents virtually abolished the IAHP, suggesting that under voltage clamp conditions, this current retained the pharmacological, sensitivity that has been previously described for the slow AHP.
In subsequent experiments, slices were super-fused with either 100 nM (Fig. 3A) or 1 μM (Fig. 3B) Cl-IB-MECA, and the amplitude of IAHP was monitored. Neither concentration of this A3 selective agonist had any significant effect on AHP amplitude. On the other hand, as we have previously noted (Dunwiddie, 1985), adenosine itself produced a rapid and highly significant reduction in the amplitude of IAHP, and this effect reversed readily upon washing (Fig. 3C). The effect of adenosine, which was highly significant by itself (t = 4.92, P < 0.01), did not appear to be mediated via A3 receptors, because this action was successfully antagonized by the combination of CPT + DMPX (t = 1.66, n.s.; Fig. 4A). On the other hand, neither concentration of Cl-IB-MECA had a significant effect when tested on seven to 10 cells under the same conditions (Fig. 4B).
Thus, in summary, adenosine at a concentration of 50 μM induced a significant outward change in the holding current, and significantly attenuated IAHP, and both of these effects were completely blocked by the combination of the A1 selective antagonist CPT and the somewhat A2 selective antagonist DMPX (Fig. 5); these observations are consistent with previous work suggesting that A1 receptor agonists can elicit both of these effects (Dunwiddie, 1985; Gerber et al., 1989). On the other hand, Cl-IB-MECA had no significant effect on either one of these parameters, and this was true both when it was tested alone, and in the presence of CPT + DMPX (Fig. 5).
The slow AHP that can be elicited in hippocampal pyramidal neurons by brief depolarization is a response that has been shown to be quite sensitive to activation of multiple second messenger systems, and appears to be inhibited by activation of PKA, PKC, and CaM-K. For these reasons, it appeared to be an appropriate candidate to determine whether activation of A3 receptors was linked to changes in the activity of any of these kinases. However, the results of these studies demonstrated that neither the selective A3 receptor agonist, Cl-IB-MECA, nor the non-selective ligand adenosine (in the presence of A1- and A2-selective blockers), had any significant effect on the amplitude of IAHP. Furthermore, these results also suggest that A3 receptor activation does not inhibit the voltage gated Ca2+ channels that mediate the Ca2+ influx that activates this conductance. In the absence of antagonists, adenosine elicited a reduction in the amplitude of IAHP, but these effects were blocked by the combination of the A1 (CPT) and A2 (DMPX) selective antagonists. This is most probably the result of a direct reduction in Ca2+ channel activity mediated via A1 receptors, or the activation of inwardly rectifying K+ channels via A1 receptors, which could indirectly reduce the Ca2+ influx. In an intact system such as the brain slice, it is not possible to effectively clamp CA1 pyramidal neurons during a depolarizing step to 0 mV; the effectiveness of the clamp, and hence the net Ca2+ influx, can be affected by the activation of other conductances, such as the inwardly-rectifying K+ channels that are linked to activation of A1 receptors.
At least three kinds of responses might have been expected based upon the known actions of A3 receptors. First, the enhancement of Ca2+ currents that has been associated with A3 receptor activation in CA3 neurons (Fleming and Mogul, 1997) might have been expected to lead to an enhancement of IAHP. Second, activation of PLC would have been expected to lead to a reduction in IAHP, either via activation of PKC, or via the IP3-dependent release of intracellular Ca2+. Finally, because it has been proposed that A3 receptors may activate PKA in hippocampal neurons (Fleming and Mogul, 1997), this might also have been expected to lead to a decrease in IAHP.
The fact that none of these effects were observed suggests several possibilities. It is possible that there were offsetting effects, such that an enhancement of Ca2+ influx combined with second messenger mediated down-regulation of the IAHP led to no significant change in the current. Although possible, it seems unlikely that such precisely offsetting responses could occur in every slice; the lack of change even in the variability of the response (Fig. 4B) is not consistent with this conclusion. A somewhat more likely alternative perhaps is that the effects on Ca2+ currents are confined solely to CA3 neurons; little work has been done on the localization of A3 receptors, and it is certainly possible that the CA1 and CA3 neurons differ in terms of receptor expression and/or coupling. Alternatively, A3 receptors may be present on CA1 pyramidal neurons, but localized in such a way that they do not directly affect IAHP. Much recent work on adenylyl cyclase has emphasized the localization or anchoring of this enzyme near appropriate substrates, and it is possible that while β-receptors activate adenylyl cyclase in a subcellular compartment with ready access to the channels that mediate IAHP, A3 receptors activate a cyclase that lacks such access. Future experiments will be required to determine which of these alternatives is the case.
The adenosine A3 receptor is found in brain, and has been linked to a number of physiological responses, but the cellular mechanisms that underlie these effects are still unclear. In the present experiments, the effects of a selective A3 agonist, 2 - chloro - N6 - (3 - iodobenzyl) - adenosine - 5′ - N - methyluronamide (Cl-IB-MECA), were characterized on hippocampal CA1 pyramidal neurons. More specifically, we examined the possibility that activation of A3 receptors could lead to activation of protein kinase A (PKA), protein kinase C (PKC), or calcium/calmodulin dependent protein kinase II (CaM-K), which might then induce significant changes in the activity of hippocampal pyramidal neurons. CA1 pyramidal neurons were recorded in the whole cell mode using patch electrodes, and the effects of adenosine agonists were determined on the holding current, and on the amplitude of the slow outward calcium-activated K+ currents activated following a depolarizing step in the command voltage (IAHP). As has been previously reported, a variety of agents reduced the IAHP, including the (β-adrenoceptor agonist isoproterenol, the muscarinic agonist carbachol, and adenosine itself; however, CI-CB-MECA had no significant effects on this current. Cl-IB-MECA by itself also had no effect on the holding current, and had a weak but not statistically significant antagonistic effect on the outward currents induced by activation of A1 receptors on pyramidal neurons. These results suggest that either A3 receptors are not present on the cell bodies of hippocampal CA1 pyramidal neurons, that they are present but not linked to PKA, PKC, or CaM-K activation, or that they only activate these kinases in cell compartments that are not accessible to the ion channels that underlie the AHP response.
This work was supported by grant R01 NS 29173 from the National Institute of Neurological Disorders and Stroke, and by the Veterans Administration Medical Research Service.