PKC-independent Pathway

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modulationof high-threshold Ca current(Zc,)by adenosine receptorswas studiedusingthe S u voltageclampmethodon acutelydissociatedguineapig hippocampalCA3pyramidalneurons.Whenthese nettronswereexposedto adenosinein thepresenceof A A and A receptorantagonists,I potentiation occurredat test potentialso – m b n a – m S p a o u t A a N 6 a T p & a p Z a f c p w 2 n c Z e a 1 Z p b i ( c0 b w n a i t ( A r a b l t d o a t Z @1 E S L K

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Adenosine is a naturally occurringbreakdownproduct of ATP utilization and is released by cells throughout the body. Acting on the various adenosine receptors, adenosine can have many systemic and cellular effects including fatigue and listlessness,decreases in intestinal motility (Dn.uy and Szent-Gyorgyi, 1929), regulation of skeletal muscle blood flow (Dobson et a 1971) and modulation of excitability in the hippocampus(Dunwiddie, 1980; Greene and Haas, 1991). More recently, reports have suggested the involvement of adenosine in long-term potentiation (LTP) of synaptic transmission (Turner e a 1992; de Mendonqaand Ribeiro, 1994). The Al and A2 adenosine receptors were first differentiated based on their ability to down- or upregulate adenylyl cyclase (Londos and Wolff, 1977; VanCalker e a 1979), respectively. The A2 receptor has been further subdivided into an Aa and A2b adenosine receptor (Bruns et a 1986; Ukena et a 1986),based on its affinityfor NECA (5’-N-ethylcarbox-

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amidoadenosine),with the A2areceptor having the higher affinity. Evidence of an A3 adenosine receptor (Ribiero and Sebastiao, 1986) has been confirmed only recently (Zhou et a 1992). Various properties of the As adenosine receptor that have been reported include its depression of adenylyl cyclase activity (Zhou e a 1992; van Galen e a 1994), its increase in phosphoinositide metabolism(Ali et a 1990; Ramkumar et a 1993; Armstrong and Ganote, 1994) and also its distribution in various areas of the body including regions throughout the brain (Salvatore et a 1993; 1993), Fozard and Carruthers, 1993; Linden et a including relative abundance in the hippocampus (De e a 1993;Jacobsonet a 1993).However, little has been presented regarding the effects of the A3 adenosine receptoron transmembraneCa current modulation.Ki for adenosine at the A3 receptor is approximately 1 VM vs 10-30 nM at the Al and AA receptors (Jacobson et a 1995), suggesting different physiological roles for these different reeeptor subtypes. The & receptor has been shown to increase potassiumcurrent in porcine coronary artery cells (Cornfield e a 1992), although the oexistence of this receptor has not yet been confirmed. dr - Activation of the9 Al adenosine receptor m has been-

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shown to depress Ca current in peripheral neurons (Henon and McAfee, 1983; Dolphin e a 1986; Macdonald e a 1986) and in hippocampal neurons (Proctor and Dunwiddie, 1983; Haas and Greene, 1984; Scholz and Miller, 1991).A previousreport showingthat adenosine in the presence of Al receptor blockade potentiated Ca current in acutely dissociated hippocampal pyramidal neurons (Mogul e a 1993) led to the hypothesis that one of the A2 receptors was solely responsible for this potentiation, largely because it was blocked by inhibitors of the cAMP-dependent protein kinase (PKA) which is up-regulated in response to A2 receptor activation. Using the voltage clamp method on acutely dissociated CA3 pyramidal neurons, we studied the modulation of Ca current by activating specific adenosine receptors using various combinations of adenosine agonists and antagonists. Most significantly, we found that A3 receptor activationusing the A3 agonist N6-2-(4-aminophenyl)ethyl-adenosine(APNEA), both alone and in the presence of Al and A2 antagonists,also produced a significantpotentiation of high-thresholdCa current using a PKA-dependent but PKC-independent pathway, with a voltage dependence different from that observed with only Al blockade. Because adenosine is found in significant quantities outside neurons of the brain, and since intracellular Ca concentrations are critical in maintaining the le~el of activity of many intracellular processes, extracellular adenosinewhich can alter the influxof Ca in neuronsmay have the potential to profoundly modulate cellular physiology. M C

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Cells were freshly prepared from the hippocampusof young adult (175–225g) guinea pig. The method used is a modified version of the procedures described by Kay and Wong (1986) as reported by Mogul and Fox (1991). Briefly,the animal was decapitatedand the brain quickly removed and placed in chilled (4”C), oxygenated Liebovitz’s L-15 medium (Gibco BRL). The hippocampus was dissected and the CA3 region isolated. Slices 450 ym thick were cut using a chopper microtome (Stoelting). The slices were placed in 15 ml PIPESbuffered saline containing (in mM): PIPES (piperazineN~’-bis-[2-ethanesulfonic acid]) sesquisodium salt 20, NaCl 120, glucose 25, KC1 5, CaC12 1, MgC12 1; pH = 7.0). Trypsin (type XI, Sigma; 10.5 mg) was added to the saline. The PIPES solution was oxygenated and gently stirred at a temperature of 32°C for 90 min. The PIP~S solution was then replaced with trypsin-free solution at room temperature. The slices were kept in oxygenated PIPES solution until needed and yielded viable ,., cells for 8-10 hr. When needed, three sliceswere removed and placed in 0.5 ml Delbucco’s Modified Eagle Medium (DMEM; Gibco BRL). Slices were triturated using four pipettes

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with progressivelysmaller bores. Cells were placed in a 1 ml experimentalbath on a glass coverslipthat had been treated with Concanavalin A (Con A; Boehringer Mannheim; 125 pg/ml in 1 M NaCl solution). Con A allowedthe neuronsto plate firmlyto the coverslipwithin 5 min and was not found to affect Ca current magnitudes as compared with poly+-lysine (result not published). Although no specific study of the effects of Con A on adenosine receptors was made, the receptor subtypes present were consistent with that reported by others for this region of the brain (Zhou e a 1992; Collis and Hourani, 1993). Although lectins have been shown to inhibit calcium channels in chick sympathetic ganglia (Golard, 1995), this occurred during superfusion at bath concentrations in the micromolar scale, considerably higher than used in this study in which coverslips were washed of all but trace amounts of con A prior to cell transfer. Cells were chosen in which the cell body possessed a triangular shape characteristic of pyramidal cells that was conserved in the dissociation process. Neuronswith excessivelylarge projectionswere rejected in order to maintain space clamp control. E

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After cellswere anchored,the bath was perfusedwith a Ca-containing tyrodes solution (CaC12 5, NaCl 134, Glucose 10, HEPES (N-[2-hydroxyethyl]piperazine-N’[2-ethanesulfonicacid]) 20,KC11,MgC121; pH = 7.3). The flow rate through the gravity-flowsystem remained constantthroughoutthe experiment.Flow rates were kept high (2.4-3 ml/min) to minimize bath equilibration times. Glass micropipette electrodes A were polished to an opening of approximately 3 pm and filled with a Trisbased rinternal solution (trizma base (tris[hydroxymethyl]aminomethane) 28, EGTA (ethylene glycolbis(~-aminoethyl ether)N~~’fl’-tetraacetic acid) 11, trizma-P04 (mono[tris(hydroxymethyl)-aminomethane] phosphate) 70, TEA-Cl (tetraethylammoniumchloride) 40, Na2-ATP 2, Na2-GTP 0.3, MgC122; pH = 7.3) just prior to patching. Electrodes in solution had a resistance of 1–3 MQ. When tight access to the cell had been established, the bathing solution was switched to a Bacontaining control solution (BaC12 5, TEA-Cl 130, glucose 10, HEPES 20, TTX (tetrodotoxin) 400 nM; pH = 7.3). Ba2+was used as the charge carrier through Ca channels. Pharmacological agents were prepared as stock solutions (500 PM or 1 mM) and diluted to their required concentrations:APNEA in DMSO (dimethyl sulfoxide); CV-1808 in EtOH (ethyl alcohol); CPT, DMPX, 8-SPT, and adenosine (Research Biochemical International) were dissolved directly in control solution. Maximum concentration of non-aqueoussolvents in final solutions were 0.2% for DMSO and 0.05% for EtOH. Control experiments were conducted for both of these solvents and no effect on Ca current was observed. The cAMPdependent protein kinase inhibitor, WIPTIDE (14-24;

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on acutely dissociated pyramidal n neurons from the CA3 d All region of young adult guinea npig hippocampus. experimentswere conducted using the following voltage protocolto elicit high-thresholdCa current (except where noted): the holding potential (HP) was set to –100 mV Peninsula Laboratories) and protein kinase C inhibitor and a test potential (TP) of –10 mV was applied every (19-36; Gibco BRL) were dissolved directly into the 15 sec. The duration of the test pulse ranged from 50 to internalsolution.A minimumof 20 min waiting time was 80 msec. provided for diffusion of a protein kinase inhibitor from c m b a n n the electrode into the cell before each experimental C r protocol was initiated. Previous reports of Ca current potentiation, by C a a e o n qwas not Al or activation of an adenosine receptor that Data acquisitionwas accomplishedusing the Axopatch AZ. (Mogul e a 1993),led us to investigatethe effects 200A amplifier (Axon Instruments, Foster City, CA, of the more recently discovered A3 and & adenosine U.S.A.) and recorded on an IBM compatible 80486 receptors on high-thresholdCa current. Al and both A2 machine runningAxobasic software (Axon Instruments). receptors were blocked by applying the antagonists CPT Signals were filtered through a four-pole Bessel filter (8-cyclopentyl-1,3-dimethylxanthine; 0.5 PM) and with a comer frequency @ of 2 kHz. Data analysiswas DMPX (3,7-dimethyl-l-propargylxanthine; 50 PM). In performed using Axobasic software to determine the some cases, the non-specificA1/A2antagonist 8-SPT (8*

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@sulfophenyl]-theophylline;30 PM) was also added to provide increased assurance of blockade of these receptors.Adenosine(50 pM) was then applied,resulting in a potentiationof Ca current (Fig. 1). The increase was measured shortly after the initial potentiation had stabilized, in order to exclude the effects of subsequent rundown or desensitizationsometimes observed. Figure l(C) compares the level of potentiation of Ca current using this protocol with and without 8-SPT, yielding average increases above baseline of 34.4% without 8-SPT (see Table 1 for complete statistics) and 45.1% with 8-SPT. The differencebetween the protocols using two different combinations of antagonists is not statistically significant (p > 0.65). The data show a statisticallysignificantincrease over the baseline current value when adenosine is added in the presence of both combinationsof antagonists (see legend to Fig. 1). The voltage dependence of the Ca current potentiated by non-Al, non-A2 adenosine receptor activation was investigated.Because rundown of the potentiatedcurrent response was sometimes observed, construction of a current–voltagerelationship across a wide test potential range would have required a rapid series of step depolarizations across the entire range, so as not to include rundown as a factor in any whole-cell current changesobserved.This could create errors if Ca channels were not permitted sufficient time to recover from inactivation.Therefore, a protocolwas selected in which

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a cell was alternatelydepolarizedfrom HP = –100 mV to a TP = –40 mV or –10 mV once every 15 sec (Fig. 2). Although extensive augmentation of whole-cell Ca current was observed at TP = –10 mV in response to adenosine in the presence of CPT and DMPX, no significant modulation of current was seen at TP = –40 mV. This is in contrast to the voltage dependence of the Ca current potentiation reported in these neurons with selective A2 receptor activation (Mogul e a 1993), indicating involvement of a Ca channel with non-A1,non-A2 receptor activation different from that observed in the previouslypublished study. Because a potentiation was observed even when both the Al and A2 receptorswere blocked,the involvementof one or both of the remaining known adenosinereceptors, As or &, to the Ca current potentiationwas investigated. First, the effects on peak Ca current of only APNEA, the A1/A3 agonist, were tested using the standard voltage protocol (HP = –100 mV, TP = –10 rev). After establishing a stable baseline in the control solution, APNEA at a concentration sufficient to produce a maximal response (300 nM) was added to the bath (Fig. 3) resulting in a peak Ca current potentiation of 34.7’%0. The contribution of the adenosine & receptor to Ca current modulation was also investigated. The standard protocoldescribed abovewas followed in the presence of the putative& agonist CV-1808 (Cornfield e a 1992). CV-1808was tested at four concentrationsover the range 25 nM to 2500 nM (n = 32), but no effects on Ca currents were observed (Fig. 4).

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There is reported to be an affinity for CV-1808 at adenosine Az receptors, although at a concentration several orders of magnitude above that required for & activation (Ki = 595 nM for A2 compared to the high affinityof the putative&receptor, Ki = 20 nM; Cornfield et a 1992). However, even at high concentrations of CV-1808 in the range of the A2 receptor’s affinity, no modulationof Ca currentwas observed.This supportsthe supposition that the A2 receptor subtype(s) activated by CV-1808 may not be significantly involved in the Ca current potentiation observed at this test potential.

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did not also include Al or A2 receptor activation.To this end, the effects of applyingAPNEA in the presence of the Al and A2 antagonists CPT (0.5 pM), DMPX (2050 PM) and in some cases 8-SPT (30 pM) were studied (Fig. 5). The response elicited was similar to that seen with APNEA alone and also that seen with adenosine in the presence of CPT, DMPX and 8-SPT. The two protocols using different combinations of Al and A2 antagonistsshowed no statistically significantdifference in level of potentiation (p > 0.5) and yielded an average Ca current potentiation above baseline of 54.5%. To better characterize the role of A3 receptors in F c o t a A r h a on Ca current u potentiad mediating the effects of APNEA We investigated further the Ca potentiatingproperties tion, a concentration–response relationship was deterof APNEA by verifying that its action on the A3 receptor mined. The percentage increase in peak Ca current was

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evaluated with concentrationsof APNEA at 10, 30, 100, 300 and 1000 nM (Fig. 6). Each concentrationwas tested independently on different cells to preclude the effects of rundown or desensitization. The data were then tit to the Michaelis–Menten equation {Z/l~,X= [APNEA]/ ([APNEA] + K05)}. When the data were best-fit to the Michaelis–Menten equation, Z~,X= 34.3% potentiation of current and K0,5= 29.8 nM. Data were fit using Sigma Plot, Jandel Scientific(p e 0.1). The data were also modeled using the Hill equation 1/l~m = [APNEA]nH/([APNEA]nH + KM) to determine if cooperativity in binding was implicated. The best fit produced a K0,5 = 25.4 nM with a Hill coefficient,nH,of

1.91 suggesting that positive cooperativity may be a factor in the binding re;ponse of APNEA (p c 0.02). This half-maximal concentration of APNEA in which cooperativity is permitted is slightly smaller than that determinedusing the Michaelis–Mentenformalismand is extremely close to the 15.5 nM for APNEA reported by Zhou et a (1992) in binding studies of the cloned Aq receutor, that the . . . . further sumorting .. - the mo~osal

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lipase C turnover (Ali et a 1990; Ramkumar et a 1993; Armstrong and Ganote, 1994), experiments were performed to determine if either the cAMP-dependent protein kinase (PKA) or protein kinase C (PKC) were involved in mediating the Ca current potentiation.Figure 7 displays the results of experiments in which either the Walsh inhibitor peptide (WIPTIDE; 100 nM) or PKC inhibitor peptide (19–36; 50 pM) were used to block PKA and PKC, respectively.Each peptide was dissolved into the pipette solutionand sufficienttime was permitted to insure intracellular dialysis (see Materials and Methods). Cells were first exposed to CPT and DMPX to block Al and AZ activation and subsequently to APNEA to activate As receptors similar to the protocol used in Fig. 5. When the PKC inhibitorwas present (Fig. 7(A)), Ca current potentiationwas still observed (43.7%; n = 3) comparable to that seen under control conditions. However, the inclusion of intracellular WIPTIDE (Fig. 7(B)) prevented Ca current potentiationin responseto As activation (n = O responses out of 16 trials; p e 0.005). These results suggest that Ca current potentiation required modulation of PKA but was independent of PKC. D T resultsdescribedhere show a relationshipbetween adenosinereceptor activationand potentiationof voltagedependent Ca current. We have produced several lines of evidence leading to the conclusion that activation of the As adenosine receptor can result in significantpotentiation of a high-thresholdhippocampal Ca current. First, the AZreceptor has a low affinity (Ki= 595 nM; Cornfield e a 1992)for the agent CV-1808 which was used to observe putative& activation.However, even at concentrationsof CV-1808 large enough to activate the low-affinitysite there was no modulation of Ca current, suggesting no significant A2 receptor involvement, although the agonist appears to have higher affinity for the A2, than the A2breceptor (Collis and Hourani, 1993). Experimentsperformed in which Ca current potentiation was observed while neurons were exposed to pharmacological agents which inhibit both Al and AZ receptors further supports As receptor involvement.Secondly, the half-maximal concentration of APNEA to induce a Ca current potentiationdetermined by fitting the Michaelis– Menten equation (K0.5 = 29.8 nM) and the Hill equation (K0.5= 25.4 nM) are very close to the value determined by Zhou e a (1992) in binding studies of the cloned receptor (K0.5= 15.5 nM). If the binding and functional concentration–response determinations yield similar values for K0.5, it suggests that the site which binds APNEA, the As receptor, is responsiblefor potentiating Ca current. In addition, the potentiation elicited using adenosine with the Al and AZblockers is not significantlydifferent from the potentiation observed with the As agonist, APNEA (p> 0.5; Mann–Whitney Rank Sum test).

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Although APNEA has been reported to have an affinity for the Al receptor (Armstrong and Ganote, 1994), our experiments which included Al and A2 antagonists provide further evidence that the effect observed was not mediated by either the Al or A2 adenosinereceptors. Variability in the magnitude and recovery time course of Ca current potentiationwas observedin responseto A3 receptor activation, either with APNEA or adenosine. Because there were considerable differences in the baseline level of Ca current recorded before receptor activation in different cells, some of this variability in magnitude may have been a consequence of differences in Ca channel density between different dissociated pyramidal neurons obtained from the CA3 region. In addition, because the response of Zc, to A3 activation involveda diffusiblesecond messenger,the variability in the recovery time course may have been an artefact of intracellular dialysis using the whole-cell-patch technique. Future experiments using perforated patch electrodes will be one way to test for this. Activation of the Al adenosine receptor has been shown to decrease both adenylyl cyclase activity as well as Ca current (Dolphin e a 1986; Macdonald e a 1986), although these two effects do not appear to be linked (Gross e a 1989).Although the A3 receptor has I been shown to decrease adenylyl cyclase activity in Chinese hamster ovary (CHO) cells (Zhou e a 1992; van Galen e a 1994), it has also been shown to stimulate phosphoinositide turnover (Ali e a 1990; Ramkumar e a 1993; Armstrong and Ganote, 1994) leading to an increase in diacylglycerol levels and consequently an increase in protein kinase C. Although recent reports have demonstrated that activation of PKC by phorbol esters leads to a potentiation of Ca current (Yang and Tsien, 1993; Swartz, 1993; Zhu and Ikeda, 1994) by increasing Ca channel open probability, evidence for PKA modulation of high-threshold Ca current (Trautwein and Hescheler, 1990; Gross e a 1990;Hartzell e a 1991;Surmeier e a 1995)has also been reported. The mechanism behind the PKA involvement in Ca current potentiation that we observed is not yet known. Recent cloning of eight adenylyl cyclases has indicated that each species may be uniquely regulated by a variety of sources, including by-products of phosphoinositide turnover (Cooper e a 1995). Alternatively, while upregulation of adenylyl cyclase has been demonstrated to increase the phosphorylationof voltage-dependentLtype Ca channels, resulting in an increase in whole-cell current, work on the effects of dopamine D1 receptors in rat neostriatalneurons suggeststhe possibilitythat levels of PKA might proportionally regulate levels of protein phosphatase (Surrneier e a 1995), such that downregulation of PKA could increase certain Ca currents by decreasing dephosphorylation of N- and P-type Ca channels. This could be tested by selective blockade of these different channel types to test whether such blockade occludes an A3-mediated ZQ potentiation.

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However, although reports concerning the regulation by PKA of Ca channels is still controversial, its regulation by adenosineAs receptors, directly or indirectly,appears to have a significant effect on whole-cell Ca current in hippocampal pyramidal neurons. Whether PKA inhibition by WIPTIDE blocked the prior expression of the calcium channel that was potentiated by A3 activation under control conditions, or whether it interrupted the transduction pathway, is not currently known. Further investigation of calcium channel modulation by protein kinases will assist in addressing this question. Although we did not fully characterize the Ca channel type(s) being modulated, our results suggest a different channel with different voltage dependence (Fig. 2) from that reported by Mogul et a (1993). Because that report utilized a protocol that would have activated, in addition to A3 receptors, both the A2, and A2b receptors, it is possible that more than one adenosine receptor subtype may mediate Ca current potentiation. Evidence further suggestive of this is that potentiation of Ca current by activation of a non-A1 receptor using a nonspecificA2 agonist (DPMA) has previously been reported to be blocked by inhibiting the cAMP-dependent protein kinase (Mogul e a 1993) whose up-regulation is associated with the A2 receptor. We have attempted to prevent this possibility in our experiments through the use of selective agonists and antagonistsin exploringthe role of the A3 receptor. Adenosineis found throughoutthe body and is released in significantquantitiesby cells in the brain includingthe hippocampuswhich has been shown to play an important role in memory and learning as well as involvement in clinical pathologies such as Alzheimer’s disease and various epilepsies. Excessive levels of A3 receptor activation have been implicated in hippocampal cell damage during cerebral ischemia (von Lubitz et a 1994). Adenosine levels outside neurons rise with increased frequency of neuronal firing and under conditions of hypoxia. Our research demonstrates that a mechanism exists whereby Ca2+ influx may b unregulated significantly in response to changes in the extracellular concentration of adenosine with particular relevance to brain physiology, depending upon receptor and channel localizationwithin a neuron or brain region. A M 3

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