5′-(N-Ethylcarboxamido)adenosine Inhibits Ca2+ Influx and Activates a Protein Phosphatase in Bovine Adrenal Chromaffin Cells

Journal of Neurochemistry Raven Press, Ltd ., New York 0 1995 International Society for Neurochemistry

5'-

(N-Ethylcarboxamido) adenosine Inhibits Ca 2+ Influx and Activates a Protein Phosphatase in Bovine Adrenal Chromaffin Cells

Jesus Mateo, Enrique Castro, *Jean Zwiller, *Dominique Aunis, and M. T. Miras-Portugal Departamento de Bioquimica y Biologia Molecular, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain; and * INSERM, U-338, Biologie de la Communication Cellulaire, Strasbourg Cedex, France

Abstract : We investigated the effect of the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A lb subtype adenosine receptor . NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner . Inhibition reached 25% after 30-40-min exposure to NECA . This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27 .7 ± 3 .3%), slowly developing inhibition of [Ca 2+]i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca 21 load was not affected by the treatment with NECA . Short-term treatment with NECA failed both to modify [Ca 2+]i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca 2+ /protein kinase C signaling pathway . The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol . We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca" influx pathway (e .g ., L-type Ca" channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells . Key Words : Adenosine analogue- Catecholamine release- Cytosolic calcium-Protein phosphatase-Diacylglycerol . J. Neurochem . 64, 77-84 (1995) .

P, and y-aminobutyric acid appear to modulate acetylcholine-mediated CA release from these cells (Mizobe et al ., 1979 ; Kumakura et al ., 1980 ; Simon et al ., 1988 ; Castro et al ., 1989, 1990) . ATP is one of the main components of chromaffin granules and it is released in the exocytotic process (Rojas et al ., 1985) . This nucleotide can be degraded extracellularly by the action of ectonucleotidases to form adenosine (Torres et al ., 1990) . Both adenosine and nucleotides of adenine (ATP, ADP) modulate the secretion of CAs from chromaffin cells . ATP and ADP have been shown to depress voltage-activated Ca 2+ currents in these cells (Diverse-Pierluissi et al ., 1991), whereas adenosine is reported to inhibit (Chem et al ., 1987, 1992) or, together with forskolin, to enhance (Chem et al ., 1988) CA secretion evoked by nicotinic stimulation of chromaffin cells . The mechanisms through which adenosine exerts these opposite effects have not been elucidated . It is now well documented that adenosine modulates neural functions through its interaction with adenosine A,, AZ , and A 3 receptors . The action of adenosine on extracellular receptors is terminated by transport of extracellular adenosine into the cells by high-affinity uptake system (Miras-Portugal et al ., 1986 ; Torres et al ., 1988) . A, and A 2 receptors are considered to be coupled to adenylate cyclase by an inhibitory and stimulatory manner, respectively (Williams, 1987), although it is now becoming clear that these two subtypes of adenosine receptors can be linked to other

The use of adrenal medullary chromaffin cells as a model has been largely responsible for our further understanding of neurosecretory responses and their molecular mechanism and modulation (Winkler and Carmichael, 1982 ; Bader et al ., 1986 ; Burgoyne, 1991) . Bovine adrenal chromaffin cells are highly specialized in the secretion of catecholamines (CAs) and peptides . They are under the control of acetylcholine nicotinic receptors . The activation of these receptors leads to extracellular Ca 2' entry and exocytosis of chromaffin granule content (Kilpatrick et al ., 1982) . Many substances, including opioid peptides, substance

Received December 22, 1993 ; revised manuscript received May 9, 1994 ; accepted May 16, 1994 . Address correspondence and reprint requests to Dr. J . Mateo at Departamento de Bioquimica y Biologia Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain . Abbreviations used: CA, catecholamine ; DMPP, 1,1-dimethyl-4phenylpiperazinium iodide ; fura-2 AM, fura-2 acetoxymethyl ester ; NECA, 5'-(N-ethylcarboxamido)adenosine ; PKA, cyclic AMP-dependent protein kinase; PLC, phospholipase C ; R-PIA, R(-)N`'-(2phenylisopropyl)adenosine .

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various signal transduction systems apart from that of adenylate cyclase (Bruckner et al ., 1985 ; Alexander et al ., 1989 ; Javors et al ., 1990) . A 3 receptors have been proposed to interact with Ca 2+ channels (Ribeiro and Sebastiao, 1988) . Recently, it has been demonstrated that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A Z,, subtype (Casadó et al ., 1992), which is a low-affinity form of the A Z receptor. Thus, it is to be expected that the physiological effect of adenosine would be evident only at relatively high concentrations and would lead to cyclic AMP generation . However, both the effects of adenosine and adenosine agonists on CA secretion (Chern et al ., 1988, 1992) and adenosine transport (Delicado et al., 1990) are not associated with a modification of cyclic AMP levels in chromaffin cells . Thus, alternative signal transduction systems coupled to the A 2 adenosine receptor should be studied in chromaffin cells . The purpose of the present study was to investigate how 5'(N-ethylcarboxamido)adenosine (NECA), a potent agonist at A Z-type adenosine receptors, achieves its inhibitory effect on secretagogue-evoked CA release . MATERIALS AND METHODS

Secretory response of chromaffin cells CA release was measured by direct electrochemical detection (656 Electrochemical Detector, Metrohm) of CA eluting from a superfused cell bed (10 6 cells), in a monitoring system similar to that described by Castro et al . (1989) . The thermostated perfusion chamber was formed by a Millex GS filter (0 .22 pm pore size, 25 mm diameter) and cells were perfused at 1 .5 ml/min with Locke's solution (composition in mM : NaCl 140, KCl 4 .4, CaC1 2 2 .5, MgS0 4 1 .2, KH2PO 4 1 .2, NaHC0 3 4 .0, glucose 5 .6, and HEPES 10, pH 7 .5) . The cell bed was stimulated by injection of chemical stimuli into the flow stream through a loop injector (Rheodyne 7010, Cotati, CA, U .S .A .) . When considering the filter volume and the flow speed, the maximum concentrations of the secretagogue in the cell bed were five times lower than the secretagogue concentration in the injected solution . This ratio was measured experimentally for each cell bed, by injecting an adrenaline standard under the same experimental conditions and referring peak height to the signal produced by continuous perfusion with the standard solution of adrenaline. The concentration of secretagogue was corrected to give the desired concentration at cell bed . The voltage of the electrochemical detector was adjusted to +500 mV, and provided a continuous signal in proportion to the concentration of CAs in the perfusate . None of the drugs used in our experiments gave electrochemical signals in the absence of cells in the perfusion chamber . ]

Materials l,l-Dimethyl-4-phenylpiperazinium iodide (DMPP), bradykinin, histone type II-A, cyclic AMP-dependent protein kinase (PKA), cyclic AMP, dioctanoylglycerol, cytosine arabinofuranoside, fluorodeoxyuridine, phosphatidylserine, and ATP were supplied by Sigma (St . Louis, MO, U .S .A .) . Collagenase, dithiothreitol, and R(-)N 6 -(2-phenylisopropyl)adenosine (R-PIA) were supplied by Boehringer (Mannheim, Germany) ; NECA by Research Biochemicals Inc . (Natick, MA, U .S .A .) . lonomycin and diacylglycerol kinase were obtained from Calbiochem (San Diego, CA, U .S .A .) and the [,Y-32p]ATP (4,500 Ci/mmol) from ICN Radiochemicals (Irvine, CA, U .S .A .) . Culture medium (Dulbecco's modified Eagle's medium), fetal calf serum, and antibiotics were supplied by Flow Laboratories (Irvine, CA, U .S .A.) . Culture plates were obtained from Costar (Cambridge, MA, U .S .A .) . Fura-2 acetoxymethyl ester (fura-2 AM) was from Molecular Probes (Eugene, OR, U .S .A .) . All other reagents were supplied by Merck (Darmstadt, Germany) .

Measurement of cytosolic [ Ca 2+ Cytosolic Ca 2+ concentration was determined by the fluorescent indicator fura-2 . Chromaffin cells were collected by centrifugation and resuspended in Locke's solution . Cells were incubated with the Ca 21 indicator fura-2 AM (2 .5 yM) for 45 min at 37°C . After incubation, cells were washed, centrifuged, and resuspended in fresh Locke's medium at a density of 10 6 cells/ml . The recordings were made in a ]ml sample containing 10 6 cells in thermostated and stirred cuvettes, by a Perkin-Elmer LS-50 fluorometer . Fluorescence intensity was determined using an excitation wavelength of 340 nm and an emission wavelength of 510 nm . At the end of each experiment, the cells were lysed in 0.4% Triton X-100 and the dye content calibrated from measurements with 2.5 mM Ca 2' and 7 .5 mM EGTA/45 mM Tris (final [Ca `+] < 0 .2 nM) . [Ca 2+]i was derived from fluorescence traces following the equation of Grynkiewicz et al . (1985) . Additions to the cuvette were made using Hamilton syringes from at least 100-fold concentrated stock solutions, to avoid large volume variations .

Preparation of chromaffin cells Bovine adrenal chromaffin cells were isolated by collagenase digestion and purified on a self-generating Percoll gradient as described by Miras-Portugal et al . (1986) . The cells were suspended in culture medium supplemented with 10% fetal calf serum containing 10 pM cytosine arabinofuranoside, 10 pM fluorodeoxyuridine, penicillin (50 U/ml), streptomycin (50 pg/ml), kanamycin (100 pg/ml), and amphotericin (2 .5 pg/ml) . A fraction of chromaffin cells was dispersed and maintained in suspension cultures at 37°C in 5% C02 and 95% air, at a density of 0 .5 X 10 6 cells/ml with continuous stirring for experiments measuring cytosolic Ca 2+ concentration, CA secretion, and diacylglycerol kinase assays . Another fraction of these cells was plated in petri dishes (35-mm diameter) at a density of 5 X 10 6 cells/plate for phosphatase activity experiments, in the same conditions .

Diacylglycerol determination Before these experiments, chromaffin cells were collected and resuspended in Locke's medium at a density of 3 X 10 6 cells/ml for the assay . Cells were incubated with each agonist for a few seconds (preliminary experiments determined the optimum stimulation time to obtain the peak in diacylglycerol generation) . An aliquot (0 .5 ml) of the cellular suspension (3 X 10 6 cells/ml) was added to 0 .5 ml of a mixture of chloroform/methanol (95 :5) to stop the reaction and rapidly frozen at -80°C, until all samples were collected . After thawing, the phases of samples were separated by centrifugation in a microcentrifuge for 1 min at 4°C and the lower phase collected and evaporated by placing the tubes in a water bath at 60°C . The diacylglycerol was estimated as the amount of `P incorporated from [ y-'2 P1 ATP into phosphatidic acid in the

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presence of diacylglycerol kinase (Loomis et al ., 1985) . The diacylglycerol-containing tubes were incubated in medium A [NaCl 100 mM, ß-mercaptoethanol 2 mM, MgCI= 50 mM, Nonidet P-40 1%, phosphatidylserine 0 .2 mg/ml, and piperazine-N,N'-bis(2-ethanesulfonic acid) 50 mM buffer at pH 6 .8] and the reaction was started by the addition of 1 U of diacylglycerol kinase, 50 uM ATP, and 0 .1 pCi [, Y -3ZP] ATP and incubated at 30°C for l h . The phosphatidic acid formed during the reaction was extracted by adding 0 .7 ml of chloroform methanol (95 :5) . The lower phase containing the [ 32p] phosphatidic acid was evaporated and the radioactivity measured after the addition of 2 ml of scintillation cocktail . Dioctanoylglycerol was used as standard to construct a calibration curve . Determination of protein phosphatase activity Histone type 11-A was phosphorylated with PKA as described by Zwiller et al . (1988) . Before the protein phosphatase activity experiments, cells plated at a density of 5 x 10' cells/plate were washed and were then submitted to an additional preincubation period in Locke's solution containing the corresponding drug . Cells were then scraped off and homogenized in 0.5 ml of ice-cold homogenizing buffer composed of 5 mM Tris-HCI, pH 7 .5/1 mM EDTA . The homogenate (0 .5 ml) was centrifuged at 100,000 g for 20 min . Phosphatase activity was determined in both the supernatant and the membrane fraction by measuring the release of 3zP,, using phosphohistone as substrate. The protein phosphatase assay mixture contained the following (final concentration in a volume of 80 fi1) : 50 mM Tris-HCI, pH 7 .5/0 .5 mM dithiothreitol/l mM EDTA/0 .1 mM EGTA/[ 3ZP]histone (2 I-tM in phosphate content) plus cellular extract. Dephosphorylation reaction was allowed to continue for 10 min at 30°C . The reaction was stopped by the addition of 0 .1 ml of 1 M H2SO4 containing l mM potassium phosphate . The 32 P; released was extracted as phosphomolybdate complex by the addition of 20 1A of 7 .5% ammonium molybdate in H2SO4 and measured according to Killilea et al . (1978) . The dephosphorylation was kept below a limit of 20% conversion from the phosphorylated form to ensure reaction linearity . Statistical analysis Data are presented as mean ± SEM of at least four determinations in different cell cultures. Significant differences were determined by two-tailed Student's t test . When appropriate, single experiment traces are presented in the figures . They are representative of at least six other experiments with equivalent results . RESULTS Catecholamine secretion Figure I A shows a typical record of CA release from a chromaffin cell bed monitored on line . NECA (20 pM) did not modify by itself the basal secretion rate (data not shown) but reduced the secretion evoked by stimulation with the nicotinic agonist DMPP (10 ttM) . Higher concentrations of NECA failed to produce further inhibitory effects . At 100 pM NECA elicited a 105-110% response with respect to 20 NM NECA . In our system there was a slight fade in DMPP response with time . Control experiments showed that DMPP response decayed linearly in the time range

FIG . 1 . Effects of NECA on CA release evoked by DMPP . A : The trace is an example of the procedure used to follow CA secretion . After basal current was stabilized, brief (50 pl) pulses of 10 pM DMPP were delivered to the cells every 5 min (arrowheads) . After three to five pulses of DMPP, NECA 20 pM was introduced into the system with the perfusion flow and DMPP effect tested again during exposure to NECA and after withdrawal of this drug . B : Time-dependent inhibitory effect of NECA on 10 N.M DMPP-evoked CA secretion . Control response was corrected for spontaneous fading of DMPP response . Points represent mean ! SEM of six experiments .

used in these experiments . In NECA experiments three to five DMPP challenges were delivered before introduction of NECA and the rate of response fading calculated by linear regression . Only cell beds showing none or