An adenosine A3 receptor-selective agonist does not modulate calcium-activated potassium currents in hippocampal CA1 pyramidal neurons

Prog Brain Res. Author manuscript; available in PMC 2012 September 23.

Published in final edited form as:

Prog Brain Res. 1999; 120: 275–285.

PMCID: PMC3449169

NIHMSID: NIHMS404134

An adenosine A₃ receptor-selective agonist does not modulate calcium-activated potassium currents in hippocampal CA1 pyramidal neurons

Thomas V. Dunwiddie,^1,^2,³^* Kenneth A. Jacobson,⁴ and Lihong Diao²

¹Neuroscience Program, University of Colorado Health Sciences Center, Denver, CO, USA

²Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO, USA

³Veterans Administration Medical Research Service, Denver, CO, USA

⁴Molecular Recognition section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

^*Corresponding author: Tel.: +1 303-315-4222; fax: +1 303-315-4814; Email: Tom.Dunwiddie/at/UCHSC.edu

Introduction

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et al., 1997; Macek et al., 1998), and that this effect is mediated via PKC (Macek et al., 1998; Diao and Dunwiddie, unpublished observations).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 A_2a 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.

Materials and methods

Slice preparation

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.

Electrophysiological recording

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).

I_AHP 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 (I_AHP) 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.

Statistical analysis

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.

Chemicals

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%.

Results

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).

Fig. 1

Effect of adenosine and Cl-IB-MECA on holding currents. Changes induced in current required to clamp CA1 hippocampal pyramidal neurons to −55 mV are indicated for groups of cells treated with identical drug superfusion protocols. Although not (more ...)

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 I_AHP. 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 I_AHP, suggesting that under voltage clamp conditions, this current retained the pharmacological, sensitivity that has been previously described for the slow AHP.

Fig. 2

Effects of carbachol and isoproterenol on I_AHP. Superfusion with 10 μM carbachol (A₁) or with 500 nM isoproterenol (ISO; B₁) rapidly blocked the currents evoked by a 150 ms depolarizing step to 0 mV. The amplitudes shown at the left correspond (more ...)

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 I_AHP 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 I_AHP, 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).

Fig. 3

Effects of Cl-IB-MECA and adenosine on I_AHP amplitude. The time courses of changes in the amplitude of I_AHP are shown at the left and averaged responses obtained at the indicated times at the right, as in Fig. 2. Neither concentration of Cl-IB-MECA had (more ...)

Fig. 4

Average time course of the effects of Cl-IB-MECA and adenosine on I_AHP amplitude. Groups of slices were superfused with drugs using identical treatment protocols, and the mean ± SEM response as a percentage of pre-drug response amplitude is illustrated. (more ...)

Thus, in summary, adenosine at a concentration of 50 μM induced a significant outward change in the holding current, and significantly attenuated I_AHP, 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).

Fig. 5

Average effects of drug superfusion on holding current and I_AHP The mean ± SEM response for changes in the holding current (upper) and I_AHP (lower) are shown. All of the effects observed with adenosine were completely antagonized by CPT + DMPX (more ...)

Discussion

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 I_AHP. 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 I_AHP, 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 I_AHP. Second, activation of PLC would have been expected to lead to a reduction in I_AHP, 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 I_AHP.

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 I_AHP 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 I_AHP. 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 I_AHP, A₃ receptors activate a cyclase that lacks such access. Future experiments will be required to determine which of these alternatives is the case.

Conclusions

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 (I_AHP). As has been previously reported, a variety of agents reduced the I_AHP, 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.

Acknowledgments

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.

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