Covalently Binding Adenosine A3 Receptor Agonist ICBM Irreversibly Reduces Voltage-Gated Ca2+ Currents in Dorsal Root Ganglion Neurons

Abstract  Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes, in different in vivo models of chronic pain. In particular, it was demonstrated that selective A3AR agonists reduced pro-nociceptive N-type Ca2+ channels in dorsal root ganglion (DRG) neurons isolated from rats and, by this mechanism, inhibit post inflammatory visceral hypersensitivity. In the present study, we investigate the effect of a previously reported irreversibly binding A3AR agonist, ICBM, on Ca2+ currents (ICa) in rat DRG neurons. Present data demonstrate that ICBM, an isothiocyanate derivative designed for covalent binding to the receptor, concentration-dependently inhibits ICa. This effect is irreversible, since it persists after drug removal, differently from the prototypical A3AR agonist, Cl-IB-MECA. ICBM pre-exposure inhibits the effect of a subsequent Cl-IB-MECA application. Thus, covalent A3AR agonists such as ICBM may represent an innovative, beneficial, and longer-lasting strategy to achieve efficacious chronic pain control versus commonly used, reversible, A3AR agonists. However, the possible limitations of this drug and other covalent drugs may be, for example, a characteristic adverse effect profile, suggesting that more pre-clinical studies are needed.


Introduction
Current therapies for the management of pain are inadequate, and new approaches are needed to mitigate its immense societal burden [1].Opioids, anticonvulsants, nonsteroidal anti-inflammatory drugs (NSAIDs), channel blockers, and antidepressants are widely used, but their clinical efficacy is variable and often with serious adverse effects [1][2][3].
One of the promising directions for future pain therapy is the neuromodulator adenosine, which acts through G proteincoupled receptors (GPCRs).Four adenosine receptors (ARs), comprising the A 1 and A 3 subtypes coupled to G i protein and the A 2A and A 2B subtypes coupled to G s protein [4] has been implicated in pain modulation.Selective A 1 AR and A 3 AR agonists counteract pain behaviors in various acute and chronic models, reviewed by [5][6][7], while the role of A 2A AR and A 2B AR is less well established [8][9][10].Furthermore, therapeutic use of A 1 ARor A 2A AR-selective agonists is impeded by cardiovascular side effects, resulting from the stimulation of bradycardiac A 1 receptors in the heart and vasodilatory A 2A receptors [11].However, A 3 AR agonists that are already in phase 2 and 3 clinical trials, e.g., IB-MECA (1-deoxy-1- [6-[[(3-iodophenyl)methyl]amino]-9H-purine-9-yl]-N-methyl-β-D-ribofuranuronamide) and its 2-chloro derivative Cl-IB-MECA (2-chloro-N 6 -(3-iodobenzyl)adenosine-5′-N-methyluronamide), do not induce cardiac or hemodynamic effects, at least at moderate doses [12].They display no serious adverse effects in clinical trials for autoimmune inflammatory diseases, liver cancer, and non-alcoholic steatohepatitis in > 1500 human subjects [13], thus suggesting the feasibility of using A 3 AR agonists clinically for other conditions, such as pain.Activation of the A 3 AR was efficacious in reducing pain in multiple mouse and rat models [8,[14][15][16][17][18][19][20].A range of A 3 AR agonists were used in these in vivo studies, including innovative, highly selective (N)-methanocarba adenosine derivatives MRS5841 [21], MRS7220 [22], and MRS7154 [23]; for a review, see [24], among which are the versatile pharmacological probes MRS5980 [25,26] and MRS5698 [27,28] and the water-soluble prodrug MRS7476 [29].Multiple mechanisms of action (MoA) have been proposed to mediate the A 3 AR-induced pain relief, including modulation of spinal neuro-glial communication, neuroinflammation, and the GABAergic system in the spinal cord [30,31].We described an additional activity of A 3 AR agonists in 2019, namely decreased firing of isolated nociceptive dorsal root ganglia (DRG) neurons and inhibition of pro-nociceptive Ca 2+ currents (I Ca ; sensitive to the N-type Ca 2+ channel blocker PD173212, an analogue of ω-conotoxin) [32].Both Cl-IB-MECA and the more selective (> 3000-fold) A 3 AR agonist MRS5980 produced these effects.
Voltage-dependent Ca 2+ channels (VDCCs) are inhibited by gabapentinoids, a first-line treatment for chronic neuropathic pain, by their blocking Ca v 2.2 channels upon binding to the α2δ subunits [33,34].Presynaptic VDCC inhibition in the peripheral and central nervous system attenuates nociceptive neurotransmitter release.A selective N-type VDCC inhibitor, ω-conotoxin GVIA (ω-CTX), greatly reduces (by 60%) excitatory postsynaptic currents evoked by stimulating lamina I dorsal horn neurons [35], indicating that VDCCs play a key role in regulating nociceptive neurotransmitter release.Moreover, aberrant expression and/or activation of N-type VDCCs correlates with neuropathic pain [36].Consequently, the ω-CTX derivative ziconotide (Prialt, for intrathecal administration) was approved by the FDA in 2000 to treat severe and refractory chronic pain [37][38][39].Unfortunately, this drug produces severe side effects, such as hallucinations or other psychiatric symptoms, that are associated with its principal MoA.Thus, it would be preferrable to induce an indirect partial reduction of Ca 2+ influx via these widely expressed VDCCs in the CNS [36][37][38][39].
Therefore, we now extend our study of the effects of selective A 3 AR agonists on N-type Ca 2+ channels in rat DRG neurons to a specialized agonist that has a "warhead" for covalent binding with the receptor, i.e., N 6 -(3-isothiocyanatobenzyl)-5′-N-methylcarboxamidoadenosine (ICBM).It was previously demonstrated that ICBM binds irreversibly and selectively to the rat A 3 AR in transfected Chinese hamster ovary (CHO) cell membranes and in membranes from rat basophilic cells that endogenously express the receptor [40].

Cell culture
All animal experiments satisfied the regulatory requirements of the European Parliament (Directive 2010/63/EU), European Union Council (September 22, 2010) and Italian Animal Welfare Law (DL 26/2014).The protocol received approval from the Institutional Animal Care and Use Committee (Univ.Florence) and the Italian Ministry of Health.Animal suffering and the animal number required for reproducibility were minimized.Male and female Wistar rats (age 3-4 weeks) were obtained from Envigo, Udine, Italy, and housed under temperature-and humidity-control (12-h dark/light cycle) and allowed free food and water access) and euthanized by cervical dislocation.Primary DRG neurons were isolated and cultured as reported [32].In brief, ganglia were excised bilaterally and treated with type 1A collagenase and trypsin (Sigma-Aldrich, Milan, Italy, 2 and 1 mg/ml in Hank's balanced salt solution, respectively, followed by incubation at 37 °C for 25-35 min.Cells were centrifuged and the pellet resuspended and mechanically digested in Dulbecco's Modified Eagle's Medium (DMEM) containing 20% heat-inactivated FBS, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and L-Gln (2 mM).The processed neurons were further centrifuged at 1200 g for 6 min and then re-suspended in supplemented DMEM, additionally containing mouse nerve growth factor (100 ng/ml) and cytosine-β-D-arabino-furanoside as the free base (2.5 mM).The neurons were plated on glass coverslips (13-mm) coated with poly-L-lysine (8.3 mM) and laminin (5 mM) and cultured for 1-2 days prior to immunohistochemical or electrophysiological experiments.
Neurons were introduced in a 1-ml platform-mounted recording chamber in an inverted microscope (Olympus CKX41, Milan, Italy), and superfusion was controlled using a 3-way perfusion valve (Harvard Apparatus, Holliston, MA, USA) to maintain a 2 ml/min flow rate.Electrodes (borosilicate glass, Harvard Apparatus) were formed to provide a to a final tip resistance of 2-4 MΩ using a P-87 Micropipette Puller (Sutter Instruments, Novato, CA, USA).Electrophysiological experiments were conducted at 21 ± 1 °C (ambient temperature).Signals were passed through an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA), with 10 kHz low-pass filtering, and the stored recordings were analyzed using pClamp 9.2 software purchased from Axon Instruments, Inc. (Union City, CA).Recording of fast hyperpolarizing voltage pulses (from − 60 to − 70 Mv, 40-ms duration) enabled analysis of series resistance (Rs), membrane resistance (Rm), and membrane capacitance (Cm).The analysis was limited to cells that displayed stable Cm and Rs levels at all experimental stages.VDCC currents were evoked with 0-mV step depolarizations (200 ms, Vh = − 65 mV) every 30 s under Cs + -replacement conditions, an interval compatible with reproducible and stable I Ca recording and with minimal Ca 2+ current run down.The Ca 2+ current-to-voltage relationship was captured by eliciting 10 depolarizing voltage steps (each 200 ms duration in 10 mV increments at 5 s intervals) from − 50 to + 50 Mv starting from − 65 mV (Vh).
Averaged currents expressed as pA/pF were obtained by normalizing cell capacitance and the neuronal diameter was also approximated from the cell capacitance by assuming a roughly spherical cell shape according to the calculated Cm for all biological membranes of 1 µF/cm 2 and the equation for the total surface area of a sphere (A = 4 πr 2 ).

Immunocytochemical analysis
DRG neurons were cultured on 13-mm diameter coverslips and fixed (10 min at ambient temperature) with paraformaldehyde (4%) in 0.1 M PBS (Pan-Biotech, Milan, Italy).Cells were then PBS-washed twice and incubated in PBS solution containing 0.25% Triton X-100 (Merck Life Science S.r.l., Milano, Italy; PBST).After triply washing with PBS, the neurons were incubated with 10% goat serum (Merck Life Science S.r.l) in PBST (PBST-GS) for 30 min, to block nonspecific antibody binding, and then incubated at ambient temperature for 2.5 h in PBST-GS containing primary rabbit anti-A 3 R antibody (Alomone Labs, Jerusalem, Israel, diluted 1:100) plus mouse anti-β3-tubulin primary antibody (Cell Signaling Technology, Danvers, MA, USA, diluted 1:400).The cells were washed PBS (3X) and incubated (1 h at ambient temperature) with specific secondary antibodies: Alex-aFluor488-labeled anti-mouse and AlexaFluor555-labeled anti-rabbit (AbCam, Cambridge, UK), each diluted 1:500 in PBST-GS.Coverslips were mounted with Fluoroshield (Merck Life Science S.r.l) containing the DNA stain DAPI to identify cell nuclei.Immunocytochemical images were captured using a TSC SP8 confocal microscope (Leica Microsystems, Mannheim, Germany), equipped with a × 63 oil-immersion objective (NA 1.40).The collected images were then analyzed using open-source software (ImageJ, version 1.49v, National Institutes of Health, Bethesda, MD,  [40] and was used as a selective A 3 AR agonist.Stock solutions of ICBM in DMSO were prepared and stored as small aliquots at − 20 °C and warmed to RT immediately before use, to avoid decomposition associated with freeze-thaw cycles [41].The K i values of this compound were described in rat cloned A 1 AR, A 2A AR and A 3 AR stably transfected in CHO cells (K i values are 145, 272, and 10.0 nM, respectively).

Statistical analysis
Shapiro-Wilk normality test was performed to check data distribution.As all data resulted normally distributed, statistical analysis was made uniformly with parametric tests.Data are expressed as mean ± SEM.Student paired or unpaired t tests and one-way analysis of variance (ANOVA) followed by Bonferroni analysis were performed, as appropriated, to determine statistical significance (set at P < 0.05).Data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA) software.

Results
Present data were collected from 57 DRG neurons isolated from 12 rats.Averaged membrane resistance (Rm) was 984.17 MΩ and membrane capacitance (Cm) was 22.51 pF (n = 57).
First, we confirmed by confocal microscopy that isolated rat DRG neurons express the A 3 AR (Fig. 1) and that the prototypical A 3 AR agonist, Cl-IB-MECA, significantly decreases I Ca (Fig. 2A-B) in these cells, as previously demonstrated by us [32].Consistent with previous data, the Cl-IB-MECA effect peaked within 5 min of application and was partially reversed after 10 min of drug washout (Fig. 2A).In another set of experiments, we applied a newly synthesized batch of the highly selective and irreversibly binding A 3 AR agonist ICBM [40].Similarly, to Cl-IB-MECA, ICBM significantly decreases peak I Ca , but, unlike the prototypical agonist, its effect was not reversible after ≤ 15 min washout (Fig. 2C-D).
The inhibitory effect of ICBM (0.01-3 µM) on I Ca was concentration-dependent (Fig. 3A), with an EC 50 of 11.7 nM (confidence limit 4.1-33.5 nM; Fig. 3B) and was prevented by the selective A 3 AR antagonist MRS1523 (1 µM; Fig. 3A  and C).As shown in Fig. 3D, the averaged ICBM-mediated I Ca inhibition was, unlike the Cl-IB-MECA-mediated effect, not reversible after 9-11 min washout.Hence, functional data are consistent with previous binding experiments demonstrating irreversible displacement of radioligand binding by ICBM on A 3 AR-transfected CHO cells [40].
To corroborate the hypothesis of irreversible A 3 AR-mediated I Ca inhibition after acute exposure to ICBM, we added efficacious concentrations of Cl-IB-MECA (30 nM) or ICBM (1 µM) to the culture medium of DRG neurons for 10 min, then the A 3 AR agonist was removed by replacement with control medium for 15 min.Then, cells were transferred to a recording chamber for whole-cell patch recordings and the effect of a subsequent 7 min Cl-IB-MECA application on I Ca was evaluated (Fig. 4A).As shown in Fig. 4B, the application of 30 nM Cl-IB-MECA after a previous incubation with Cl-IB-MECA (Cl-IB-MECA preincubation 10 min, 30 nM) was still able to decrease I Ca in DRG neurons (P = 0.0064; Fig. 4B).In contrast, no effect of Cl-IB-MECA was observed in ICBM-pre-incubated (10 µM; 10 min) cells (P = 0.1343; Fig. 4C, D).
It is also worth to note that the peak I Ca amplitude evoked in ICBM-pre-incubated DRG neurons during baseline (bsl; i.e., before the 7 min Cl-IB-MECA application; Fig. 5A) was significantly smaller than that measured in non-incubated cells (Fig. 5B, P = 0.0073, One-way ANOVA, Bonferroni post-test).These results indicate that, after a 10 min ICBM pre-incubation, I Ca inhibition by A 3 AR activation persists, thus occluding the effect of a subsequent Cl-IB-MECA treatment.

Discussion
In the present work, we investigated the functional effect of ICBM, a new A 3 AR agonist designed for covalent binding to the receptor, on DRG neurons by means of electrophysiological recordings.We demonstrated that ICBM inhibits I Ca evoked by membrane depolarization in isolated rat DRG neurons, an effect similar to that observed in the presence of the non-covalent prototypical A 3 AR agonist, Cl-IB-MECA [32].However, unlike Cl-IB-MECA, ICBM-mediated inhibition of I Ca persisted after drug removal, indicating a long-lasting effect of this compound.
The A 3 AR is expressed in diverse tissues at relatively low levels, compared to other adenosine receptors.Genomic analysis of the expression of the A 3 AR gene in various human tissues shows highest levels in testes, the spinal cord, and various brain regions, bladder, lung, adipose tissue, and whole blood 1 3 [42,43].Here, we also confirmed by histochemical analysis that rat DRG neurons express the A 3 AR (Fig. 1).Moreover, the A 3 AR structure displays notable interspecies variations in ligand recognition [44].For all these reasons, as well as the diverse pharmacological profiles of the species homologs, it has been challenging to characterize A 3 AR ligands.
In 1994, Ji et al. synthesized a new A 3 AR agonist (ICBM) containing a chemically reactive isothiocyanate group that was found to bind this adenosine receptor subtype selectively and irreversibly [40].Isothiocyanate groups on A 1 AR and A 2A AR ligands were previously shown to induce irreversible receptor binding.ICBM showed a high affinity for rat A 3 AR in membranes of transfected CHO cells (10.0 ± 2.3 nM) and RBL basophilic cells.Furthermore, preincubation of transfected CHO cell membranes induced a concentration-dependent, irreversible antagonism that was demonstrated by the failure of repeated washing to regenerate the A 3 AR binding sites.Moreover, a preincubation with 100 nM ICBM followed by washing resulted in diminished B max , representing a loss in density of binding sites of 41% [40].We have extended the earlier findings of irreversible binding of ICBM in rat A 3 AR-expressing cell membranes to introduce a promising potential therapeutic direction for a covalent A 3 AR agonists, i.e., in pain therapy.
Here, we examined the functional effects of this isothiocyanate derivative on voltage-dependent I Ca in DRG neurons.In line with previous data, ICBM mimicked the effect of Cl-IB-MECA in inhibiting I Ca in these cells.Furthermore, the irreversible binding of ICBM to the A 3 AR was demonstrated as follows: (i) when cells were pre-incubated with ICBM, washed for 10 min, and then challenged with acute Cl-IB-MECA exposure, Cl-IB-MECA was ineffective in inhibiting I Ca ; (ii) the amplitude of I Ca in DRG neurons preincubated with ICBM was significantly smaller than untreated and Cl-IB-MECApreincubated cells.Thus, we confirmed, at a functional level, that the covalent binding of ICBM to the A 3 AR produces an irreversible inhibition of I Ca .It is well recognized that A 3 AR undergoes rapid agonist-induced desensitization and internalization in model cell systems [45].Following these events, the receptor undergoes recycling with re-sensitization of receptor responsiveness, while after prolonged (≤ 24 h) agonist exposure the receptor undergoes downregulation [45].However, activation of many GPCRs, including A 3 AR, can lead to β-arrestin binding and sequestration of the receptor from the cell surface, but some receptors can still signal to the cAMP pathway even after receptor internalization [46], as found for other receptor subtypes [47][48][49].Stoddart and co-authors demonstrated that the highly conserved tryptophan (W6.48) in TM6 is essential for the active conformation of A 3 AR to interact with β-arrestin2, and necessary for it to undergo receptor internalization [46].In addition, their data showed that individual agonists elicit different changes in the position of this residue, with consequent implications for their ability to activate G i -coupling and receptor internalization [46].Nevertheless, the prolonged activation of the A 3 AR by ICBM is striking, and further studies will be informative about the cellular location and signaling properties of the covalently ligated receptor.Future studies could also probe the site of covalent reactivity of the isothiocyanate group on the receptor protein.Also, other electrophilic groups might be suitable for incorporation in A 3 AR agonists in addito an isothiocyanate.From a pre-clinical perspective, it is recognized that A 1 AR and A 3 AR agonists inhibit I Ca in DRG neurons and play an important anti-algetic role in several pain models [5,32,50,51].It should be noted that A 3 AR activation is known to selectively inhibit N-type Ca 2+ channels, as the effect of the prototypical A 3 AR agonist Cl-IB-MECA is prevented by the ω-CTX analogue PD173212 but not by the L-type blocker lacidipine [32].Hence, A 3 AR stimulation on DRG neurons would selectively decrease N-type Ca 2+ currents and, in turn, neurotransmitter/ neuropeptide release at the synapse, since it was demonstrated that the block of these currents prevents sensory neurons from releasing of the pain-related neuropeptides, such as substance P and calcitonin gene-related peptide [52,53].Ziconotide, the preferred ω-CTX analogue, is currently being used clinically in the USA as an intrathecal pain reliever for chronic pain [37,54].It should be noted that a direct inhibition of N-type Ca 2+ channels, such as that accomplished by ziconotide or ω-CTXs, is related to adverse side effects (psychological and neuropsychiatric symptoms including depression, cognitive impairment, and hallucinations; anxiety; panic attacks; ataxia; asthenia; headache; and dysesthesia) [55].Interestingly, an "indirect" VDCC modulation, as that accomplished by A 3 AR activation, could represent a suitable approach to pain control with milder adverse effects.In addition, the A 1 AR has shown cardiovascular side effects in clinical trials [14].On the other hand, unlike A 1 AR, activation of the A 3 AR in humans by potent, selective, and orally accessible A 3 AR agonists is not linked to cardiac or hemodynamic adverse effects [14], therefore representing a promising treatment for chronic pain of different etiologies.Moreover, A 3 AR agonists proved relatively free from adverse effects in phase II/III clinical trials for other pathologies [14,56] and are considered to be encouraging therapeutic candidates in advanced phases of clinical research, possibly due to the lower expression of A 3 AR in peripheral tissue [57].It has been demonstrated in in vivo models of neuropathic or chronic pain that A 3 AR stimulation has profound anti-hyperalgesic effects, by central and/or peripheral mechanisms of action [3,5,19,20,30,32,[58][59][60].Based on the present data, ICBM might be an interesting compound to be investigated as a non-narcotic pain reliever, because by irreversibly binding to the A 3 AR, it produces long-lasting A 3 AR activation able to provide pain control over a longer time span after drug administration.
From a translational perspective, irreversibly binding A 3 AR agonists such as ICBM may represent an innovative, beneficial strategy to achieve efficacious chronic pain control as A 3 AR agonists, i.e., Cl-IB-MECA and IB-MECA, already proved safe and secure in clinical trials for other pathologies.Irreversibly binding drugs containing a covalent warhead are now coming into focus in the pharmaceutical industry as a viable drug discovery approach [61].ICBM could be viewed as a prototypical covalent A 3 AR agonist, on which future molecules can be designed.Of note, possible limitations of this drug may consist, for example, of a longer-lasting effect which is difficult to manage in case of over dosage.Furthermore, a different adverse effect profile might be hypothesized in comparison to prototypical (reversible) A 3 AR agonists such as IB-MECA and Cl-IB-MECA.Of note, the fact that this compound irreversibly binds to the A 3 AR might be advantageous to defer the time or decrease the dosage of drug administration, especially in the case of chronic diseases, such as neuropathic pain.Hence, more pre-clinical studies are needed to define the functional effect/s of ICBM in animal models.

1 3 USA
). Incubation of fixed cells in the presence of 2˚ antibodies and DAPI, alone, was used as a control to exclude nonspecific binding.Drugs 2 -C h l o r o -N 6 -( 3 -i o d o b e n z y l ) -a d e n o s i n e -5 ′ -Nmethyluronamide (Cl-IB-MECA; Merck Life Science S.r.l.) was used as a selective A 3 AR agonist.N 6 -(3-Isothiocyanatobenzyl)-5′-N-methylcarboxamidoadenosine (ICBM) was synthesized as reported

Fig. 1
Fig. 1 A 3 AR expression on cultured rat DRG neurons.Confocal imaging analysis showing 20X (upper panels) and respective magnification of A 3 AR-like immunofluorescence (red) on β-III-tubulin (green)-expressing DRG neuronal cultures.Cells nuclei are marked with DAPI (blue)

Fig. 2
Fig.2The new, irreversible, A 3 AR agonist ICBM inhibits N-type I Ca in rat DRG neurons.A, C Time courses of peak Ca 2+ current amplitude (I Ca peak) evoked by a 0 mV step depolarization in a typical DRG neuron once every 30 s before, during, and after the application of the prototypical competitive A 3 AR agonist Cl-IB-MECA (100 nM, A) or the irreversible A 3 AR agonist ICBM(1 µM, C).Upper panels: original current traces recorded in respective cells before and during A 3 AR agonist application (Cl-IB-MECA, upper panel in A; ICBM, upper panel in C).Scale bars: 200 pA, 100 ms.B, D Pooled data (mean ± SEM) of peak I Ca measured before (baseline, bsl) or during Cl-IB-MECA (30 nM, n = 10; B) or ICBM (1 µM, n = 6; D) application.P values are referred to paired Student's t-test

Fig. 3
Fig. 3 The effect of ICBM is concentration-dependent, sensitive to the A 3 AR antagonist MRS1523, and, differently from Cl-IB-MECA, not reversible after washout.A Pooled data (mean ± SEM) of I Ca inhibition in DRG neurons superfused with different concentrations of the irreversible A 3 AR agonist ICBM (0.01-3 µM), by a maximal concentration (30 nM) of the prototypical A 3 AR agonist Cl-IB-MECA, by a maximal concentration (1 µM) of ICBM in the presence of the A 3 AR antagonist MRS1523 (1 µM) or by vehicle (0.1% DMSO).** P < 0.01; **** P < 0.0001 vs DMSO, one-way ANOVA, Bonferroni post hoc test.B Concentration-response curve of the effect of ICBM

Fig. 4
Fig. 4 Pre-exposure of DRG neurons to the new A 3 AR agonist ICBM irreversibly blocks I Ca and prevents the effect of a successive application of Cl-IB-MECA.A, C Experimental protocol used to compare the long-term effects of the prototypical A 3 AR agonist Cl-IB-MECA (30 nM) or the new, irreversible, A 3 AR agonist ICBM (1 µM) on I Ca inhibition in DRG neurons.The protocol consisted of a 10-min exposure of DRG cultures to the A 3 AR agonist (added to the culture medium), followed by a 15-min washout (removal of the A 3 AR agonist-containing medium and perfusion of DRG neurons with the extracellular patch-clamp solution in the recording chamber) during which a stable baseline of peak I Ca amplitude (peak I Ca )