British Journal of Pharmacology (2001) 132, 879 ± 888 ã 2001 Nature Publishing Group All rights reserved 0007 ± 1188/01 $15.00 A1 adenosine receptors inhibit multiple voltage-gated Ca2+ channel subtypes in acutely isolated rat basolateral amygdala neurons*,1Brian A. McCool & 1Je€ery S. Farroni 1Department of Medical Pharmacology and Toxicology, The Texas A&M University System Health Science Center, College Station, Texas, TX 77843, U.S.A.
1 The anticonvulsant properties of 2-chloroadenosine (CADO) in the basolateral amygdala rely on the activation of adenosine-speci®c heptahelical receptors. We have utilized whole-cell voltage-clamp electrophysiology to examine the modulatory e€ects of CADO and other adenosine receptor agonists on voltage-gated calcium channels in dissociated basolateral amygdala neurons.
2 CADO, adenosine, and the A1 subtype-selective agonists N6-(L-2-Phenylisopropyl)adenosine (R- PIA) and 2-chloro-N6-cyclopentyladenosine (CCPA) reversibly modulated whole cell Ba2+ currents in a concentration-dependent fashion. CADO inhibition of barium currents was also sensitive to the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX).
3 The A2A-selective agonist 4-[2-[[6-Amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2- yl]amino]ethyl]benzenepropanoic acid (CGS21680) was without e€ect.
4 CADO inhibition was predominantly voltage-dependent and sensitive to the sulphydryl- modifying reagent N-ethylmaleimide, implicating a membrane-delimited, Gi/o-coupled signal transduction pathway in the channel regulation.
5 Using Ca2+ channel subtype-selective antagonists, CADO inhibition appeared to target multiple channel subtypes, with the inhibition of o-conotoxin GVIA-sensitive calcium channels being more prominent.
6 Our results indicate that the anti-convulsant e€ects CADO in the basolateral amygdala may be mediated, in part, by the A1 receptor-dependent inhibition of voltage gated calcium channels.
British Journal of Pharmacology (2001) 132, 879 ± 888 Keywords: Basolateral amygdala; A1 adenosine receptor; calcium channel; N-ethylmaleimide; nifedipine; o-conotoxin GVIA; o-agatoxin IVA Abbreviations: BLA, basolateral amygdaloid complex; CADO, 2-chloroadenosine; CCPA, 2-chloro-N6-cyclopentyladenosine; noic acid; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine; NEM, N-ethylmaleimide; PTX, pertussis toxin; R- As part of the limbic system, the amygdala plays a Ghani et al., 1997). This anticonvulsant activity of CADO highly integrative role in the sense/memory-response is dose-dependent and blocked by ca€eine, suggesting that pathway and is believed to occupy a pivotal position in activation of adenosine heptahelical receptors in the the regulation of fear and anxiety. Rat models of fear/ basolateral amygdala may regulate neuronal excitability.
anxiety have implicated the basolateral complex (BLA), as Previous work has demonstrated that adenosine receptors being centrally important in both the acquisition and may act presynaptically in the amygdala to inhibit both expression of fear/apprehension-related behaviours (re- excitatory and inhibitory transmission (Heinbockel & viewed in Davis, 1992). Of particular relevance for the Pape, 1999; Nose et al., 1991). However, direct regulation studies outlined below, infusion of the non-selective of postsynaptic processes by amygdala adenosine receptors adenosine receptor agonist 2-chloroadenosine (CADO) has not been examined.
into the basolateral amygdala suppresses seizure activity P1 purinoreceptors are believed to mediate the e€ects following amygdala kindling (Abdul-Ghani et al., 1997; of adenosine in the central nervous system. These Pourgholami et al., 1997), the long-term decrease in receptors belong to the heptahelical family of receptors seizure threshold brought about by repeated electrical and are coupled to heterotrimeric G proteins. Several stimulation. In fact, adenosine receptor activation can subtypes of P1 receptors can be distinguished from one even prevent the acquisition of amygdala kindling (Abdul- another by receptor pharmacology or by examination of the signal transduction pathways to which the individual receptors couple. For example, the A1 adenosine receptor *Author for correspondence at: Department of Medical subtype is classically associated with the inhibition of Pharmacology and Toxicology, The Texas A&M Univ. H.S.C., 368 Reynolds Medical Building, MS 1114, College Station, TX77843- cyclic AMP production via pertussis toxin-sensitive, 1114, U.S.A.; E-mail: [email protected] inhibitory' Gi/o heterotrimeric G proteins. A1 receptors B.A. McCool & J.S. Farroni A1 receptors inhibit amygdala calcium channels also have high anity for the agonists adenosine and 2- chloro-N6-cyclopentyladenosine (CCPA; Lohse et al., 1988) (DPCPX; Martinson et al., 1987). Unlike A1 receptors, A2 adenosine receptors appear to couple to cholera toxin- Neurons were prepared from coronal brain slices of juvenile sensitive G proteins and can stimulate cyclic AMP male rats (*P17 ± P28) as previously described (McCool & accumulation. Two A2 isoforms, the A2A and A2B Botting, 2000). Brie¯y, slices were digested with 0.5 ± receptors, arise from distinct genes and are pharmacolo- 1 mg ml71 Pronase (CalBiochem) dissolved in standard gically distinguishable. The A2A has a high anity for arti®cial CSF (in mM): NaCl 125, KCl 5, NaHCO3 25, NaH2PO4 1.25, MgSO4 1, CaCl2 2.0, and 20 D-glucose, at ethylcarboamido-adenosine (CGS 21680) but intermediate/ 378C for 20 min with constant oxygenation. Following this low anity for the antagonist DPCPX. Conversely, the digestion, slices were removed to isolation bu€er' containing A2B receptor has a very low anity for CGS 21680 but (in mM): N-methyl glucamine 130, NaCl 10, MgCl2 1, a high anity for DPCPX. The most recently identi®ed HEPES 10, D-glucose 10; pH 7.4 with HCl, osmolality P1 receptor, the A3 subtype, binds 2-chloro-N6-(3- 325 mmol kg71 adjusted with sucrose; and, those regions containing primarily basolateral amygdala were carefully reviewed in Jacobson, 1998) with high anity and dissected away from the remaining tissue. Individual neurons selectivity but is not believed to be highly or widely were isolated from these tissue pieces by mechanical expressed in brain (Rivkees et al., 2000; Zhou et al., separation using ®re-polished Pasteur pipettes. The dispersed 1992; but see Dixon et al., 1996). Thus, the pharmaco- tissue was transferred to plastic coverslips (Themonox). Large logical and signal transduction characteristics can often neurons (15 ± 35 pF) with pyramidal or stellate soma were identify the receptor subtype mediating a particular utilized exclusively in these studies and had morphological adenosine-sensitive physiological response.
characteristics that were similar to both isolated BLA In the nervous system, adenosine is a potent modulator of neurons (McCool & Botting, 2000; Viana & Hille, 1996) neuronal activity, with A1 and A2 receptors often playing and BLA neurons in situ (McDonald, 1982).
contrasting roles. For example, activation of pre-synaptic A1 receptors can depress synaptic transmission in numerous preparations and can alter both long-term (de Medonca & Ribeiro, 1990) and short-term (Lovinger & Choi, 1995) All recordings were performed at ambient room temperature modi®cations in synaptic ecacy. In contrast, pre-synaptic with standard patch-clamp techniques (Hamill et al. 1981) A2 receptor activation is frequently associated with increased using the axopatch-1D ampli®er (Axon Instruments, Inc., neurotransmitter release and enhanced synaptic function Foster City CA, U.S.A.) in the voltage clamp mode.
(Cuhna & Ribeiro, 2000; Kessey & Mogul, 1998; Umemiya Gigaohm seals were formed using patch pipettes made from & Berger, 1994). In addition to these synaptic roles, A1 and borosilicate glass (World Precision Instruments, Sarasota FL, A2 receptors often regulate voltage-gated calcium channels in U.S.A.). For whole-cell patch clamp recording, patch pipettes contrasting ways. A1 receptors typically inhibit calcium typically had input resistances of 0.5 ± 2 MO. The internal channel activity (Mynlie€ & Beam, 1994; Zhu & Ikeda, solution in the patch pipette was similar to that reported 1993). Conversely, A2 receptors can facilitate calcium channel previously (McCool & Botting, 2000) and contained (in mM): function (Goncalves et al., 1997; Umemiya & Berger, 1994; CsCl 120, HEPES 10, EGTA 11, CaCl2 1, Mg-ATP 4, Tris- Mogul et al., 1993). Thus, adenosine receptors appear to GTP 0.3, pH 7.2 with cesium hydroxide; adjust to 300 ± modulate neuronal activity via a diverse array of signal 310 mmol kg71 with sucrose. Whole cell capacitance (typi- cally 15 ± 25 pF) and series resistance (typically 510 MO) The inhibition of voltage-gated calcium channels by were compensated manually after opening the cell. Currents heterotrimeric G protein-coupled receptors is believed to were online leak-subtracted using a p×n71' protocol and low- be an important means of regulating Ca2+ entry and thus pass ®ltered (three-pole Bessel ®lter) at 1 kHz with 470% has direct consequences for many Ca2+-dependent pro- compensation. Depolarizing test pulses were typically given at cesses. In this context, dihydropyridine antagonists of 0.25 Hz from a holding potential of 780 mV to prevent somatic voltage-gated calcium channels prevent kindling- prolonged channel inactivation.
related phenomena (Hassan et al., 1999), presumably by Cells were continuously bath perfused with an extracellular attenuating the elevation in intracellular calcium associated solution consisting of (in mM): NaCl 150, Dextrose 10, with this seizure-like activity (Pal et al., 1999). The HEPES 10, KCl 2.5, CaCl2 2.5, MgCl2 1.0, pH 7.4 with inhibition of somatic voltage-gated calcium channels can NaOH; osmolality adjusted to 320 ± 340 mmol kg71 with therefore dramatically in¯uence neuronal excitability and sucrose. To isolate currents mediated by the calcium potentially underlies the e€ects of CADO on amygdala channels, cells were locally perfused with the following (in seizure activity. Here we characterize the regulation voltage- mM): tetraethylammonium chloride 140, HEPES 10, Dextrose gated calcium channels by CADO in acutely isolated 15, BaCl2 5, pH 7.35 with tetraethylammonium hydroxide; basolateral amygdala neurons. The receptor mediating these osmolarity adjusted to 320 ± 330 mmol kg71 with sucrose.
e€ects is de®ned by pharmacological analyses; and its utilization of particular signal transduction pathways is determined. Finally, we examine the discriminate targeting of speci®c calcium channel subtypes during the modulatory Data was digitized at up to 10 kHz with a Labmaster DMA (Axon), stored on a computer, and analysed o€-line using British Journal of Pharmacology vol 132 (4) B.A. McCool & J.S. Farroni A1 receptors inhibit amygdala calcium channels pClamp software (Axon). Unless otherwise stated, current at least 10 s and no longer than 30 s from an array of eight amplitudes were measured as the di€erence between current HPLC-grade capillary tubes (150 mm i.d.; Hewlett Packard levels immediately prior to and within 10 ms after the Analytical Direct, Wilmington DE, U.S.A.) placed within initiation of a depolarizing test pulse. For the calcium 50 ± 100 mm of the cell of interest.
channel antagonist experiments, per cent contribution by each component following the sequential addition of channel blockers was calculated using current amplitudes during the baseline' of the experiment using the following relationship: Inhibition of voltage-gated barium currents by Blocker N  100% …1† P1 purinoreceptors Amplitude Control where blocker N' is the nth channel antagonist added during Because CADO regulation of BLA excitability (Abdul-Ghani a sequence of blockers and control' refers to current et al., 1997; Pourgholami et al., 1997) may involve the amplitudes prior to the addition of any channel antagonist.
regulation of voltage-gated calcium channels (see Magee & A similar relationship was used to calculate the per cent Carruth, 1999; Widmer et al., 1997), we tested the e€ects of inhibition by adenosine receptor agonists during these CADO and other P1 receptor agonists on barium currents in occlusion experiments. Numerical analysis was performed acutely isolated neurons. Application of CADO (1 ± 3 mM) as using the QuatroPro software package (v 5.00; Borland well as adenosine (3 ± 10 mM) caused modest inhibition of International Inc., Scotts Valley CA, U.S.A.). Concentration- whole-cell Ba2+ currents in a substantial number of cells response curves were generated from ®ts (GraphPad Prism; with only 10 out of 68 neurons failing to respond to GraphPad Software Inc., San Diego CA, U.S.A.) of data to a a purinergic agonist. CADO inhibited currents by 21+2% standard logistic equation of the form: (mean+s.e.mean; n=11) while adenosine attenuated current amplitude in a di€erent set of neurons by 25+2% (n=25). In cells where both were tested simultaneously, the inhibition by a maximally ecacious concentration CADO was not signi®- where Y=response expressed as per cent of Ymax, X=Log cantly di€erent (paired t-test, P40.1; than that ([agonist]), and HillSlope=slope of the concentration found for adenosine (22+2%; n=11). The inhibition by both response relationship. Because concentration-response data compounds was characterized by slowing of the macroscopic in each neuron were expressed as a fractional response current activation kinetics, exempli®ed by the apparent compared to the inhibition by a maximally ecacious reduction in the amount of inhibition at later times during concentration of adenosine (10 mM), Ymin=0 and Ymax=1.0.
the depolarizing test pulse. For example in the traces in inhibition was 28 and 25% for 3 mM adenosine and 1 mM CADO, respectively, when measured 7 ms after the onset of the test pulse; when measured 65 ms after the initiation of the test Power calculations (SSD, CECOR Ltd.) to de®ne the pulse, inhibition was reduced to 17 and 18% for adenosine and minimum sample size for each experiment were performed CADO, respectively. Furthermore, inhibition mediated by using standard deviations derived from pilot experiments.
both compounds was readily reversible and exhibited no For these calculations, a=0.05 and b=0.1. Standard student apparent desensitization after repeated applications of these t-tests compared population means between two treatment maximally e€ective agonist concentrations.
groups, with a signi®cant di€erence being de®ned as P50.05 (2-tailed). In those cases where multiple treatment groups Inhibition is mediated by the A1 receptor subtype were compared, one-way ANOVA analysis using the repeated measure design examined the population means, which were To further de®ne the receptor subtype responsible for calcium considered signi®cantly di€erent if P50.05. Bonferroni's channel inhibition by 2-chloroadenosine, we tested several multiple comparison test was used in this case to examine subtype-selective agonists and an antagonist. In one group of various pairs treatment groups. All statistical analysis was neurons (n=5), R-PIA (500 nM) and CGS21680 (500 nM) performed using GraphPad Prism (GraphPad Software, Inc.).
were compared to adenosine R-PIA inhibited whole-cell barium currents by 26+4%, similar to the level of inhibition seen with adenosine (28+3%; Con- versely, the inhibition by CGS21680 (5+1%) was signi®- Adenosine (RBI) and N-ethylmaleimide (NEM; Sigma) were cantly (P50.001) less than that for either adenosine or R- prepared as concentrated stock solutions in distilled water PIA, indicating that the A2A receptor does not substantially fresh each day (adenosine) or every 3 h (NEM). o-conotoxin modulate voltage-gated calcium channels in these neurons.
GVIA (Alamone Labs), o-conotoxin MVIIC (RBI), and o- The relative amount of inhibition by R-PIA and CGS21680 agatoxin IVA (Alamone Labs), 2-chloroadenosine (CADO; was similar when lower concentrations (R-PIA, 100 nM; RBI) were similarly prepared but were stored as frozen stock CGS21680, 100 nM) were used (not shown), indicating that solutions at 7208C. Similarly, nifedipine, 2-chloro-N6- the concentration of agonists used here were sucient to cyclopentyladenosine (CCPA; RBI), 8-cyclopentyl-1,3-dipro- produce maximal inhibition. To support the hypothesis that pylxanthine (DPCPX; RBI), R(-)-N6-(2-phenylisopropyl)- the A1 subtype adenosine receptor is responsible for adenosine (R-PIA; RBI), and CGS-21680 (RBI) were made inhibition of calcium channels in isolated BLA neurons, we as concentrated stocks in dimethylsulphoxide and stored at tested the sensitivity of 2-chloroadenosine inhibition to the 7208C. Agonists and antagonists were typically applied for selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropyl- British Journal of Pharmacology vol 132 (4) B.A. McCool & J.S. Farroni A1 receptors inhibit amygdala calcium channels Figure 1 Adenosine receptor modulation of voltage-gated calcium Figure 2 Adenosine receptor modulation of voltage-gated calcium channels in dissociated basolateral amygdala neurons. (a) Both 2- channels in dissociated basolateral amygdala neurons is mediated by chloroadenosine and adenosine attenuated whole-cell, voltage-gated Ba2+ currents. Unless otherwise stated, the holding potential was 1 receptor subtype. (a) The adenosine receptor agonists adenosine (Ade), R-PIA, and CGS21680 were tested in one group 780 mV; and, the test potential was 710 to 0 mV. The inhibition by of neurons (n=5). R-PIA (500 nM) inhibited whole-cell barium both adenosine and CADO was characterized by slowing of the currents by 26+4%, similar to the 28+3% inhibition seen with macroscopic current activation kinetics, exempli®ed by the apparent adenosine (P40.05, repeated measures ANOVA, Bonferronni's post- reduction in the amount of inhibition at later times during the test). In these same neurons, the inhibition by CGS21680 (500 nM)) depolarizing test pulse. When measured 7 ms after the onset of the was 5+1%, signi®cantly less than that for either adenosine or R-PIA test pulse, inhibition was 28 and 25% for adenosine and CADO, (*P50.001, repeated measures ANOVA, Bonferroni's post-test).
respectively; inhibition was reduced to 17 and 18% for adenosine and These results indicate that the A CADO, respectively, when measured 65 ms after the initiation of the 2A subtype does not substantially contribute to the CADO modulation of calcium channels. (b) To test pulse. Dashed line=zero current level. (b) When maximally con®rm the contribution of the A1 receptor subtype, the sensitivity of ecacious concentrations of both CADO and adenosine were tested 2-chloroadenosine modulation to the selective A in the same neurons (n=11), CADO inhibited currents by 21+2% 1 receptor antagonist DPCPX was tested. Co-application of DPCPX (100 nM) with 2- (mean+s.e.mean) while adenosine attenuated current amplitude by chloroadenosine signi®cantly reduced the modulation from 32+5% 22+2%. These values were not signi®cantly di€erent (paired t-test, to 4+3% (n=4; *P50.05 paired t-test).
xanthine (DPCPX). Co-application of DPCPX (100 nM) with Signal transduction pathway for A1 adenosine 2-chloroadenosine (0.3 mM) signi®cantly reduced the inhibi- receptor inhibition tion from 32+5% to 4+3% (n=4; P50.05).
Additionally, we examined the concentration-response rela- In order to determine the signal transduction pathway tionship for several P1 receptor agonists utilized by A1 adenosine receptors, we assessed the voltage- Adenosine, CADO, and 2-Chloro-N6-cyclopentyladenosine dependence of the inhibition. Using a voltage protocol similar (CCPA) inhibited whole-cell barium currents in a concentra- to that in Ikeda (1991), two test' pulses ( Vt1' and Vt2', tion-dependent manner The rank order of were separated by a large membrane depolariza- potency, CCPA (EC50=103 nM, 4adenosine tion (+60 mV) and brief recovery period. The voltage- (EC50=225 nM) &2-chloroadenosine (EC50=290 nM), was dependence of inhibition is represented in this protocol by a consistent with the A1 subtype being the primary adenosine relief' from inhibition in the second test pulse relative to the receptor mediating the barium current inhibition by ®rst test pulse. This relief' is believed to be due to the adenosine and 2-chloroadenosine.
voltage-dependent association between calcium channel British Journal of Pharmacology vol 132 (4) B.A. McCool & J.S. Farroni A1 receptors inhibit amygdala calcium channels ®ndings that inhibition was indeed partially voltage-depen- dent. In one set of neurons (n=8), the amount of inhibition by adenosine (3 mM) or 2-chloroadenosine (3 mM) in the ®rst test pulse (19+1% inhibition) was signi®cantly (P50.05, paired t-test) reduced in the second test pulse (12+2% inhibition) by the intervening depolarization To further explore this phenomena, membrane potentials were continuously ramped' from 7100 to +60 mV to evoke the bell-shaped' current that is characteristic for voltage-gated calcium channels see McCool et al., 1996).
Application of 2-chloroadenosine (1 mM) during this voltage ramp reduced the amplitude of current response but did not change the general shape of the current, indicating that adenosine receptor activation did not substantially alter voltage sensitivity of the calcium channels. The per cent inhibition by CADO during these voltage ramps' was greatly in¯uenced by the membrane potential Speci®- cally, the amount of inhibition decreased as membrane potential increased. Thus, like many other G protein coupled receptors, inhibition of whole-cell barium currents by adenosine or 2-chloroadenosine is mediated via a voltage- dependent signal transduction pathway that is likely to involve a direct interaction between channel and activated To further characterize the signal transduction pathway utilized by A1 receptors, we utilized the sulfhydryl-modifying reagent, N-ethylmaleimide (NEM). At the concentrations used here, NEM inactivates pertussis toxin (PTX)-sensitive G protein a subunits, but not the Gq- or Gs-subtypes (McCool et al., 1998), by ethylation of the same cysteine residue that is ADP-ribosylated by PTX (Asano & Ogasawara, 1986; Hoshino et al., 1990). Speci®cally, neurons responding to adenosine with robust inhibition were subsequently treated with NEM (50 mM for 2 min) during the recording and the response to adenosine again measured. NEM exposure signi®cantly reduced the amount of inhibition by adenosine from 27+6 to 8+2% (n=4; paired t-test). These results suggest that inhibition mediated by A1 adenosine receptors primarily utilizes a well characterized, membrane- delimited, Gi/o-dependent signal transduction pathway.
Calcium channel subtypes modulated by A1 adenosine receptors Two separate experiments were performed to determine the relative contribution of each calcium channel subtype to the whole cell barium currents recorded from basolateral amygdala neurons. In one set of neurons (n=6), sequential Figure 3 The agonist pro®le of P1 receptor modulation is consistent application of the L-type channel antagonist nifedipine 1 receptor-mediated inhibition. (a) Agonist concentration- response relationships for CCPA (*), CADO ( ), and adenosine (5 mM), nifedipine plus the N-type calcium channel antagonist (&). The rank order of potency, CCPA4adenosine&CADO, was o-conotoxin GVIA (1 ± 2 mM), and then nifedipine plus the consistent with the A1 subtype being the primary P1 receptor P/Q-type calcium channel antagonist o-agatoxin IVA responsible for the barium current modulation. To reduce the (0.1 mM) inhibited total whole-cell barium currents by in¯uence of cell-to-cell variability, data in each neuron was normal- ized to a maximally ecacious concentration of adenosine (10 mM).
28+3, 27+4 and 18+2%, respectively Thus, (b) EC50 values for CCPA, adenosine, and CADO were 103, 225 and in this experiment, 28+6% of the total current that is 290 nM, respectively. (c) Hill slopes of the concentration-response resistant' to antagonist exposure. o-Conotoxin GVIA relationships were 1.1+0.1, 1.9+0.5, and 2.9+0.6 for CCPA, inhibition of N-type channels is not reversible in these adenosine, and CADO, respectively.
neurons under our recording conditions (data not shown). In a second experiment (n=4), sequential application of subunits and G protein bg subunits (Herlitze et al. 1996; nifedipine, nifedipine plus o-conotoxin GVIA, and then Ikeda 1996) that are liberated during receptor activation. The nifedipine plus the mixed N-type and P/Q-type antagonist o- representative traces in generally re¯ect our conotoxin MVIIC (3 mM) inhibited whole cell currents by British Journal of Pharmacology vol 132 (4) B.A. McCool & J.S. Farroni A1 receptors inhibit amygdala calcium channels Figure 4 The modulation by CADO is partially voltage-dependent and NEM-sensitive, implicating a membrane-delimited, Gi/o- coupled signaling pathway. (a) Example of the paired-pulse' voltage protocol and resultant whole-cell Ba2+ currents used to examine the voltage dependence of the modulation. Note the large depolarization to +60 mV reduced CADO inhibition during the second test pulse (Vt2; 10% inhibition) relative to that present in the ®rst test pulse (Vt1; 20% inhibition). (b) Pooled data for CADO (3 mM) and adenosine (3 mM; n=6) shows that modulation is partially voltage dependent, with inhibition being signi®cantly reduced from 19+1% to 12+2% (P50.05, paired t-test). (c) Example of bell-shaped' whole cell currents (inset) evoked by ramping the membrane potential from 7100 to +60 mV. Per cent inhibition (*) by 2-chloroadenosine (CADO) at each sampled interval (500 ms) in this neuron decreased from *40 to *12% during the increase in membrane potential from 730 to 0 mV (black bar, inset). Inset calibration bars: x=100 ms, y=1 nA. (d) Adenosine modulation is reduced by exposure to the sulfhydryl-modifying reagent, NEM. Neurons responding initially to adenosine were subsequently treated with NEM during the recording; and, the response to adenosine was again measured. NEM treatment (50 mM for 2 min) signi®cantly reduced the amounts of inhibition from 27+6% to 8+2% (n=4; P50.05).
18+4, 41+5 and 10+4%, respectively, leaving 30+8% subtype, CADO inhibited 46+12% of the N-type current, resistant' current. Assuming o-agatoxin IVA and o-con- 20+6% of the L-type current, 20+5% of the P/Q-type otoxin MVIIC inhibit a similar population of channels current, and 19+5% of the current resistant' to all channel following treatment with o-conotoxin GVIA, our results antagonists However, the relative amounts of are consistent, with following contributions to whole-cell inhibition across di€erent channel subtypes only approached current: 20 ± 30% L-type, 30 ± 40% N-type, 10 ± 20% P/Q- statistical signi®cance, suggesting that, while A1 receptors type, and 30% resistant' channel subtype.
may preferentially target the N-type channels, these receptors To determine whether A1 adenosine receptors modulate can inhibit a variety of calcium channel subtypes in acutely speci®c calcium channel subtypes, the inhibition mediated by dissociated basolateral amygdala neurons.
2-chloroadenosine (3 mM) was measured in the presence of nifedipine (5 mM), nifedipine plus o-conotoxin GVIA (1 mM), and then nifedipine plus o-Agatoxin IVA (0.1 mM). A representative experiment in a single neuron is shown In a population of neurons (n=6), inhibition by CADO Based on agonist pharmacology and sensitivity to the was 24+3% without channel antagonists. During sequential antagonist DPCPX, we propose that 2-chloroadenosine application of channel antagonists, this inhibition was inhibition of calcium channels in basolateral amygdala reduced to 18+3% during co-application of nifedipine, to neurons is mediated by the adenosine A1 adenosine receptor.
9+2% during nifedipine+o-conotoxin GVIA, and to 5+2% This is consistent with the distribution of adenosine receptor in the presence of nifedipine+o-agatoxin IVA. Comparing subtypes in the central nervous system. A1 receptor mRNA is these values with the relative contribution of each channel widely expressed in the forebrain (Reppert et al., 1991); and, British Journal of Pharmacology vol 132 (4) B.A. McCool & J.S. Farroni A1 receptors inhibit amygdala calcium channels A1-speci®c radioligand binding is present in the lateral/ basolateral amygdala (Fastbom et al., 1987). The lack of e€ect by CGS21680 is also consistent with the predominant expression of A2A receptors in the striatum, nucleus accumbens, and olfactory tubercle (Schi€man et al., 1990; Wan et al., 1990). However, A2A mRNA and binding are also present elsewhere in the forebrain (Cuhna et al., 1994; Johansson et al., 1993); and, the whole-cell recording conditions used here would tend to minimize any contribu- tion by di€usable second messengers that might be produced by activation of this adenosine receptor subtype (e.g. cyclic AMP). Conventional radioligand or mRNA analyses have failed to demonstrate signi®cant A2B or A3 receptor expression in the forebrain (Rivkees et al., 2000; Dixon et al., 1996); however, polymerase chain reaction-based meth- odologies suggest that both subtypes may be expressed at low levels within the amygdala (Dixon et al., 1996). While our pharmacologic analyses strongly suggest that A1 receptors represent the predominant in¯uence on voltage-gated calcium channels in dissociated basolateral amygdala neurons, we can not rule out possible contributions by other subtypes under some circumstances. It is also possible that other adenosine receptor subtypes are expressed in a population of neurons that is distinct from those examined here.
A1 receptors have been classically associated with the inhibition of cyclic AMP production. However, it is also clear that these adenosine receptors can modulate numerous signal transduction pathways. In basolateral amygdala neurons, A1 receptors appear to utilize primarily voltage-dependent, NEM-sensitive signal transduction pathways to inhibit voltage-gated calcium channels. These characteristics are very similar to Gi/o-mediated inhibition in many other systems.
NEM treatment does not alter antagonist binding to A1 receptors (Ukena et al., 1984), suggesting the NEM-sensitive inhibition described here is most likely related to the uncoupling of A1 receptors from PTX-sensitive G proteins.
However, A1 receptors may utilize both PTX-sensitive and PTX-resistant pathways to modulate calcium channels in basolateral amygdala neurons since their inhibition is only partially NEM-sensitive. In support of this, A1 receptors couple to PTX/NEM-resistant aZ-containing G proteins to modulate cyclic AMP levels in heterologous systems (Ho & Wong, 1997; Wong et al., 1992) and voltage-gated calcium channels in isolated hypothalamic neurons (Noguchi & Yamashita, 2000). Additional studies focusing on the potential interaction between A1 receptors and di€erent G protein subtypes in these particular neurons may be selective antagonists indicate that dihydropyridine-sensitive, o- conotoxin GVIA-sensitive, o-agatoxin IVA, and resistant' channels contribute 28+3, 27+4, 18+2 and 29+6% to the whole cell current, respectively, in isolated basolateral amygdala neurons (n=6). (c) Figure 5 A1 adenosine receptors modulate di€erent calcium channel Comparison of the amount of inhibition present during co- subtypes in dissociated basolateral amygdala neurons. (a) Example of application with channel antagonists with the relative contribution calcium channel antagonist e€ects of the modulation by CADO. For of each channel subtype allowed us to examine A1 receptor this neuron, CADO (*) inhibition was 19% in the absence of any modulation of speci®c classes of voltage-gated calcium channels.
calcium channel antagonist. The inhibition was reduced to 13% in CADO inhibits 20+6% of the nifedipine-sensitive current (L-type), the presence of nifedipine (5 mM) and to 3% in the presence of both 46+12% of the o-conotoxin GVIA-sensitive current (N-type), nifedipine and o-conotoxin GVIA (1 mM). Boxes indicate the 20+5% of the o-agatoxin IVA-sensitive current (P/Q-type), and duration of channel antagonist application. GVIA'=o-conotoxin 19+5% of the current resistant to all the channel antagonists (R- GVIA. Aga'=o-agatoxin IVA. (b) Calcium channel subtype- British Journal of Pharmacology vol 132 (4) B.A. McCool & J.S. Farroni A1 receptors inhibit amygdala calcium channels The utilization of multiple signal transduction pathways amygdala neurons (Foehring & Scroggs, 1994) possess larger may also be re¯ected by the apparent inhibition of multiple contributions by dihydropyridine-sensitive channels (30 ± 42% channel subtypes in isolated amygdala neurons. A1 receptors of whole cell current amplitude) and o-agatoxin IVA (31 ± appear to preferentially target o-conotoxin GVIA-sensitive 33%) channels, with a subsequent reduction in the contribu- channels in these neurons. The inhibition of this channel tion by channel antagonist resistant' currents (to *15%).
subtype by G protein-coupled receptors is voltage-dependent These data may indicate that expression of di€erent calcium in most systems, suggesting a common signal transduction channel subtypes in the amygdala is developmentally pathway regardless of the tissue. However, the inhibition of regulated well into adulthood.
dihydropyridine-sensitive channels and antagonist-resistant The implications associated with A1 receptor inhibition of channels by G protein-coupled receptors is a novel ®nding for voltage-gated calcium channels will certainly depend upon the basolateral amygdala neurons. For example, somatostatin circumstances responsible for the release of adenosine. In the receptors appear to modulate primarily o-conotoxin GVIA- hippocampus for example, adenosine release during hypoxia/ and o-agatoxin IVA-sensitive channels (Viana & Hille, 1996).
hypoglycemia depresses synaptic transmission (Coelho et al., It is therefore likely that di€erent G protein-coupled 2000; Fowler, 1993). In the amygdala, increases in extra- receptors expressed by basolateral amygdala neurons may cellular adenosine can arise from either the degradation of inhibit speci®c populations of calcium channel subtypes. The synaptically-released adenine nucleotides via ecto-nucleoti- inhibition of overlapping, yet distinct, populations of calcium dases' or by direct release of adenosine from the intracellular channels by muscarinic and adenosine receptors in striatal space, probably via reversal of nucleoside transporters cholinergic interneurons (Song et al., 2000; Yan & Surmeier, (reviewed by Brundege & Dunwiddie, 1997) since the 1996) appears to support this idea. Regardless, it remains to amygdala possesses among the highest levels of adenosine- be determined if the di€erent calcium channel subtypes are like immunoreactivity (Braas et al., 1986) and ATPase modulated by A1 receptors via identical signal transduction activity (Mohanakumar & Sood, 1985) in the forebrain.
Furthermore, both spontaneous release of adenosine, prob- Using central amygdala neurons from young rats (5P19), ably via degradation of extracellular' nucleotide (MacDonald Yu & Shinnick-Gallagher (1997) ®nd a distribution of & White, 1985), and depolarization-evoked adenosine release channel subtypes that is similar to the basolateral neurons (White & MacDonald, 1990) are present in synaptosomes used here, with whole cell currents being 30 ± 31% o- prepared from amygdala. Regardless, A1 receptor inhibition conotoxin GVIA-sensitive, *28% resistant' to antagonists, of voltage-gated calcium channels is likely to in¯uence both 22 ± 27% dihydropyridine-sensitive, and 18% agatoxin IVA- neuronal excitability and local synaptic transmission within sensitive. Furthermore, the relative contributions of each the amygdala. This may be especially relevant during times of channel to whole-cell current is consistent with the expression heightened neuronal activity when increases in extracellular of their mRNAs, with prominent expression of CaV a12.2 (N- adenosine are probable.
type or a1B; see Ertel et al., 2000 for nomenclature) and CaV a12.3 mRNA (R-type or a1E; Ludwig et al., 1997; Williams et al., 1994; Fujita et al., 1993) and lower levels of CaV a12.1 We would like to thank Dr Jerry Trzeciakowski for his review of 1A), a11.2 (a1C), and a11.3 (a1D) mRNA expression (Ludwig this manuscript and helpful comments. This work is supported in et al., 1997) in the amygdala. Compared to our juvenile part by a Pharmaceutical Research and Manufacturers of America animals, whole cell calcium currents from adult basolateral Foundation Starter Grant (B.A. McCool).
CUHNA, R.A., JOHANSSON, B., VAN DER PLOEG, I., SEBASTIAO, The protective e€ect of 2-chloroadenosine against the develop- A.M., RIBEIRO, J.A. & FREDHOLM, B.B. (1994). Evidence for ment of amygdala kindling and on amygdala-kindled seizures.
functionally important adenosine A2a receptors in the rat Eur. J. Pharmacol., 326, 7 ± 14.
hippocampus. Brain Res., 649, 208 ± 216.
ASANO, T. & OGASAWARA, N. (1986). Uncoupling of gamma- DAVIS, M. (1992). The role of the amygdala in conditioned fear. In aminobutyric acid B receptors from GTP- binding proteins by N- The Amygdala: Neurobiological Aspects of Emotion, Memory, and ethylmaleimide: e€ect of N-ethylmaleimide on puri®ed GTP- Mental Dysfunction. ed. Aggleton, J.P. pp. 255 ± 306. New binding proteins. Mol. Pharmacol., 29, 244 ± 249.
BRAAS, K.M., NEWBY, A.C., WILSON, V.S. & SNYDER, S.H. (1986).
DE MENDONCA, A. & RIBEIRO, J.A. (1990). 2-Chloroadenosine Adenosine-containing neurons in the brain localized by im- decreases long-term potentiation in the hippocampal CA1 area of munocytochemistry. J. Neurosci., 6, 1952 ± 1961.
the rat. Neurosci. Lett., 118, 107 ± 111.
BRUNDEGE, J.M. & DUNWIDDIE, T.V. (1997). Role of adenosine as a DIXON, A.K., GUBITZ, A.K., SIRINATHSINGHJI, D.J.S., RICHARD- modulator of synaptic activity in the central nervous system. Adv.
SON, P.J. & FREEMAN, T.C. (1996). Tissue distribution of Pharmacol., 39, 353 ± 391.
adenosine receptor mRNAs in the rat. Br. J. Pharmacol., 118, COELHO, J.E., DE MENDONCA, A. & RIBEIRO J.A. (2000).
1461 ± 1468.
Presynaptic inhibitory receptors mediate the depression of ERTEL, E.A., CAMPBELL, K.P., HARPOLD, M.M., HOFMANN, F., synaptic transmission upon hypoxia in rat hippocampal slices.
MORI, Y., PEREZ-REYES, E., SCHWARTZ, A., SNUTCH, T.P., Brain Res., 869, 158 ± 165.
TANABE, T., BIRNBAUMER, L., TSIEN, R.W. & CATTERALL, CUHNA, R.A. & RIBEIRO, J.A. (2000). Adenosine A2A receptor W.A. (2000). Nomenclature of voltage-gated calcium channels.
facilitation of synaptic transmission in the CA1 area of the rat Neuron, 25, 533 ± 535.
hippocampus requires protein kinase C but not protein kinase A activation. Neurosci. Lett., 289, 127 ± 130.
British Journal of Pharmacology vol 132 (4) B.A. McCool & J.S. Farroni A1 receptors inhibit amygdala calcium channels FASTBOM, J., PAZOS, A. & PALACIOS, J.M. (1987). The distribution MARTINSON, E.A., JOHNSON, R.A. & WELLS, J.N. (1987). Potent of adenosine A1 receptors and 5'-nucleotidase in the brain of adenosine receptor antagonists that are selective for the A1 some commonly used experimental animals. Neuroscience, 22, receptor subtype. Mol. Pharmacol., 31, 247 ± 252.
MCCOOL, B.A. & BOTTING, S.K. (2000). Characterization of FOEHRING, R.C. & SCROGGS, R.S. (1994). Multiple high-threshold strychnine-sensitive glycine receptors in acutely isolated neurons calcium currents in acutely isolated rat amygdaloid pyramidal from adult rat basolateral amygdala. Brain Res., 859, 341 ± 351.
cells. J. Neurophysiol., 71, 433 ± 436.
MCCOOL, B.A., PIN, J.-P., BRUST, P.F., HARPOLD, M.M. & LOVIN- FOWLER, J.C. (1993). Purine release and inhibition of synaptic GER, D.M. (1996). Functional coupling of rat group II transmission during hypoxia and hypoglycemia in rat hippo- metabotropic glutamate receptors to an o-conotoxin GVIA- campal slices. Neurosci. Lett., 157, 83 ± 86.
sensitive calcium channel in human embryonic kidney 293 cells.
FUJITA, Y., MYNLIEFF, M., DIRKSEN, R.T., KIM, M.S., NIIDOME, T., Mol. Pharmacol., 50, 912 ± 922.
MAN, K.A. & LOVINGER, D.M. (1998). Rat group I metabotropic (1993). Primary structure and functional expression of the glutamate receptors inhibit neuronal Ca2+ channels via multiple omega-conotoxin-sensitive N-type calcium channel from rabbit signal transduction pathways in HEK 293 cells. J. Neurophysiol., brain. Neuron, 10, 585 ± 598.
79, 379 ± 391.
GONCALVES, M.L., CUNHA, R.A. & RIBEIRO, J.A. (1997). Adenosine MCDONALD, A.J. (1982). Neurons of the lateral and basolateral A2A receptors facilitate 45Ca2+ uptake through class A calcium amygdaloid nuclei: a Golgi study in the rat. J. Comp. Neurol., channels in rat hippocampal CA3 but not CA1 synaptosomes.
212, 293 ± 312.
Neurosci. Lett., 238, 73 ± 77.
MOGUL, D.J., ADAMS, M.E. & FOX, A.P. (1993). Di€erential HAMILL, O.P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, activation of adenosine receptors decreases N-type but potenti- F.J. (1981). Improved patch-clamp techniques for high-resolution ates P-type Ca2+ current in hippocampal CA3 neurons. Neuron, current recording from cells and cell-free membrane patches.
10, 327 ± 334.
P¯ugers Archiv., 391, 85 ± 100.
MOHANAKUMAR, K.P. & SOOD, P.P. (1985). Inhibitory action of HASSAN, H., GRECKSCH, G., RUTHRICH, H. & KRUG, M. (1999).
morphine on adenosine triphosphatase content in the whole and E€ects of nicardipine, an antagonist of L-type voltage-dependent individual nuclei of mouse brain during tolerance-dependence calcium channels, on kindling development, kindling-induced development and its reversal by naloxone. J. fur Hirnforsh., 26, learning de®cits and hippocampal potentiation phenomena.
Neuropharmacol., 38, 1841 ± 1850.
MYNLIEFF, M. & BEAM, K.G. (1994). Adenosine acting at an A1 HEINBOCKEL, T. & PAPE, H.C. (1999). Modulatory e€ects of receptor decreases N-type calcium current in mouse motoneur- adenosine on inhibitory postsynaptic potentials in the lateral ons. J. Neurosci., 14, 3628 ± 3634.
amygdala of the rat. Br. J. Pharmacol., 128, 190 ± 196.
NOGUCHI, J. & YAMASHITA, H. (2000). Adenosine inhibits voltage- HERLITZE, S., GARCIA, D.E., MACKIE, K., HILLE, B., SCHEUER, T.
dependent Ca2+ currents in rat dissociated supraoptic neurones & CATTERALL, W.A. (1996). Modulation of Ca2+ channels by via A1 receptors. J. Physiol., 526, 313 ± 326.
Gprotein beta gamma subunits. Nature, 380, 258 ± 262.
NOSE, I., HIGASHI, H. INOKUCHI, H. & NISHI, S. (1991). Synaptic HO, M.K. & WONG, Y.H. (1997). Functional role of aminoterminal responses of guinea pig and rat central amygdala neurons in serine16 and serine27 of G alphaZ in receptor and e€ector vitro. J. Neurophysiol., 65, 1227 ± 1241.
coupling. J. Neurochem., 68, 2514 ± 2522.
PAL, S., SOBATI, S., LIMBRICK, D.D., DELORENZO, R.J. (1999). In HOSHINO, S., KIKKAWA, S., TAKAHASHI, K., ITOH, H., KAZIRO, Y., vitro status epilepticus casuses sustained elevation of intracel- KAWASAKI, H., SUZUKI, K., KATADA, T. & UI, M. (1990).
lular calcium levels in hippocampal neurons. Brain Res., 851, Identi®cation of sites for alkylation by N-ethylmaleimide and pertussis toxin-catalyzed ADP-ribosylation on GTP-binding POURGHOLAMI, M.H., ROSTAMPOUR, M., MIRNAJAFI-ZADEH, J.
proteins. FEBS Lett., 276, 227 ± 231.
& PALIZVAN, M.R. (1997). Intra-amygdala infusion of 2- IKEDA, S.R. (1991). Double-pulse calcium channel current facilita- chloroadenosine suppresses amygdala-kindled seizures. Brain tion in adult rat sympathetic neurones. J. Physiol. (Lond.), 439, Res., 775, 37 ± 42.
IKEDA, S.R. (1996). Voltage-dependent modulation of N-type (1991). Molecular cloning and characterization of a rat A1- calcium channels by G-protein beta gamma subunits. Nature, adenosine receptor that is widely expressed in brain and spinal 380, 255 ± 258.
cord. Mol. Endocrinol., 5, 1037 ± 1048.
JACOBSON, K.A. (1998). Adenosine A3 receptors: novel ligands and RIVKEES, S.A., THEVANANTHER, S. & HAO, H. (2000). Are A3 paradoxical e€ects. Trends Pharmacol. Sci., 19, 184 ± 191.
adenosine receptors expressed in the brain? Neuroreport, 11, JOHANSSON, B., GEORGIEV, V., PARKINSON, F.E. & FREDHOLM, 1025 ± 1030.
B.B. (1993). The binding of the adenosine A2 receptor selective SCHIFFMANN, S., LIBERT, F., VASSART, G., DUMONT, J.E. & agonist [3H]CGS 21680 to rat cortex di€ers from its binding to VANDERHAEGHEN, J.-J. (1990). A cloned G protein-coupled rat striatum. Eur. J. Pharmacol., 247, 103 ± 110.
protein with a distribution restricted to striatal medium-size KESSEY, K. & MOGUL, D.J. (1998). Adenosine A2 receptors neurons. Possible relationship with D1 dopamine receptor. Brain modulate hippocampal synaptic transmission via a cyclic-AMP- Res., 519, 333 ± 337.
dependent pathway. Neurosci., 84, 59 ± 69.
SONG, W.-J., TKATCH, T. & SURMEIER, D.J. (2000). Adenosine LOHSE, M.J., KLOTZ, K.N., SCHWABE, U., CRISTALLI, G., VITTORI, receptor expression and modulation of Ca2+ channels in rat S. & GRIGANTINI, M. (1988). 2-Chloro-N6-cyclopentyladeno- striatal cholinergic interneurons. J. Neurophysiol., 83, 322 ± 332.
sine: a highly selective agonist at A1 adenosine receptors. Naunyn UKENA, D., POESCHLA, E., HUTTEMANN, E. & SCHWABE, U.
Schmiedebergs Arch Pharmacol., 337, 687 ± 689.
(1984). E€ects of N-ethylmaleimide on adenosine receptors of LOVINGER, D.M. & CHOI, S. (1995). Activation of adenosine A1 rat fat cells and human platelets. Naunyn Schmiedebergs Arch.
receptors initiates short-term synaptic depression in rat striatum.
Pharmacol., 327, 247 ± 253.
Neurosci. Lett., 199, 9 ± 12.
UMEMIYA, M. & BERGER, A.J. (1994). Activation of adenosine A1 LUDWIG, A., FLOCKERZI, V. & HOFMANN, F. (1997). Regional and A2 receptors di€erentially modulates calcium channels and expression and cellular localization of the alpha(1) and beta glycinergic synaptic transmission in rat brainstem. Neuron, 13, subunit of high voltage-activated calcium channels in rat brain. J.
1439 ± 1446.
Neurosci., 17, 1339 ± 1349.
VIANA, F. & HILLE, B. (1996). Modulation of high voltage-activated MACDONALD, W.F. & WHITE, T.D. (1985). Nature of extrasynapto- calcium channels by somatostatin in acutely isolated rat somal accumulation of endogenous adenosine evoked by K+ and amygdaloid neurons. J. Neurosci., 16, 6000 ± 6011.
veratidine. J. Neurochem., 45, 791 ± 797.
WAN, W., SUTHERLAND, G.R. & GEIGER, J.D. (1990). Binding of the MAGEE, J.C. & CARRUTH, M. (1999). Dendritic voltage-gated ion adenosine A2 receptor ligand [3H]CGS 21680 to human and rat channels regulate the action potential ®ring mode of hippocam- brain: Evidence for multiple anity sites. J. Neurochem., 55, pal CA1 pyramidal neurons. J. Neurophysiol., 82, 1895 ± 1901.
1763 ± 1771.
British Journal of Pharmacology vol 132 (4) B.A. McCool & J.S. Farroni A1 receptors inhibit amygdala calcium channels WHITE, T.D. & MACDONALD, W.F. (1990). Neural release of ATP YAN, Z. & SURMEIER, D.J. (1996). Muscarinic (m2/m4) receptors and adenosine. Ann. N.Y. Acad. Sci., 603, 287 ± 298.
reduce N- and P-type Ca2+ currents in rat neostriatal WIDMER, H., AMERDEIL, H., FONTANAUD, P. & DESARMENIEN, cholinergic interneurons through a fast, membrane-delimited, M.G. (1997). Postnatal maturation of rat hypothalamoneurohy- G-protein pathway. J. Neurosci., 16, 2592 ± 2604.
pophysial neurons: evidence for a developmental decrease in YU, B.J. & SHINNICK-GALLAGHER, P. (1997). Dihydropyridine- and calcium entry during action potentials. J. Neurophysiol., 77, neurotoxin-sensitive and -insensitive calcium currents in acutely dissociated neurons of the rat central amygdala. J. Neurophysiol., WILLIAMS, M.E., MARUBIO, L.M., DEAL, C.R., HANS, M., BRUST, 77, 690 ± 701.
P.F., PHILIPSON, L.H., MILLER, R.J., JOHNSON, E.C., HARPOLD, ZHOU, Q.-Y., LI, C., OLAH, M.E., JOHNSON, R.A., STILES, G.L. & M.M. & ELLIS, S.B. (1994). Structure and functional characteriza- CIVELLI, O. (1992). Molecular cloning and characterization of an tion of neuronal alpha 1E calcium channel subtypes. J. Biol.
adenosine receptor: The A3 adenosine receptor. Proc. Natl. Acad.
Chem., 269, 22347 ± 22357.
Sci. U.S.A., 89, 7432 ± 7436.
WONG, Y.H., CONKLIN, B.R. & BOURNE, H.R. (1992). Gz-mediated ZHU, Y. & IKEDA, S.R. (1993). Adenosine modulates voltage-gated hormonal inhibition of cyclic AMP accumulation. Science, 255, Ca2+ channels in adult rat sympathetic neurons. J. Neurophy- siol., 70, 610 ± 620.
(Received July 19, 2000 Revised November 27, 2000 Accepted December 5, 2000) British Journal of Pharmacology vol 132 (4)


El artículo 14 de la nueva Ley Peruana de Arbitraje: Reflexiones sobre el contrato de arbitraje – realidad Eduardo Silva Romero* 1. Quienes elaboran las normas jurídicas deben, en términos generales, mantener un equilibrio (bastante frágil) entre los valores de la flexibilidad (y, por ende, adaptabilidad) del Derecho a situaciones futuras y la previsibilidad del mismo;

V8 final summary ua 2011 en-nov 27.pdf

Progress rePort summary 2011 Foreword 1. Introduction 2. Key findings: Update on the HIV epidemic 3. Key findings: Selected health sector interventions for HIV prevention 4. Key findings: Knowledge of HIV status 5. Key findings: Scaling up treatment and care for people living with HIV