AR(‘HIVES

OF RIOCHEMISTRY

AN11 RIOPHYSICS

Vol. 279, No. 1, May 1.5,pp. 37-44, 1990

Presence of Ectonucleotidases in Cultured Chromaffin Cells: Hydrolysis of Extracellular Adenine Nucleotides Magdalena

Torres,

Departamento

de Rioquimica,

Jesus Pintor,

and M. Teresa

Facultad de Veterinaria,

Miras-Portugal’

Universidad

Complutense de Madrid,

28040 Madrid,

Spain

Received September 22,1989, and in revised form December 28,1989

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a K, for ATP of 250 + 18 PM and a I’,,, value of 167 k 25 nmol/106 cells. min (1.67 pmol/mg protein. min) for cultured chromaffin cells, while the ecto-ADPase activity showed a K, value for ADP of 375 2 40 PM and a VMAX of 125 f 20 nmol/106 cells.min (1.25 bmol/mg protein. min). The ecto 5’-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The K, for AMP was 55 + 5 HM and the VMAXvalue was 4.3 + 0.8 nmol/106 cells. min (43 nmol/mg protein. min). The nonhydrolyzable analogs of ADP and ATP, cr,@-methylene-adenosine 5’-diphosphate and adenylyl-(/3,-y-methylene)-diphosphonate were good inhibitors of ecto 5’-nucleotidase activity, the K1values being 73.3 4 3.5 nM and 193 f 29 The phosphatidylinositol-specific nM, respectively. phospholipase C released the ecto-5’-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP (~1iago exocytotically released from these neural cells. Academic

Press,

Inc.

nucleotidases in chromaffin cells are poorly characterized. The enzymes responsible for nucleotide degradation have been characterized from endothelial cells (8, 9), cholinergic synapses from mammalian brain (lo), and the Torpedo electric organ (11). The best characterized of these enzymes, both kinetically and structurally, is the membrane-bound 5’-nucleotidase (12-E). The reaction product of this enzyme, adenosine, is removed from the extracellular medium by a high affinity transport system (16, 17) that terminates adenosine actions at its plasma membrane receptors. The adenosine transport in chromaffin cells has been characterized (18-20). Once inside, adenosine can be incorporated into the cellular nucleotide pool, stored in chromaffin vesicles as ATP, and released again, thus completing the ATP-adenosine cycle in these cells (18,21, 22). These cells have the advantage of being a homogeneous neural cell population free from glia. This characteristic is an important aspect of this cellular model, as controversy exists about neural or glial localization of ectonucleotidases (23). The present article deals with the extracellular hydrolysis of nucleotides by cultured chromaffin cells. The kinetic properties of these ectonucleotidases have been studied. Special attention has been paid to the ecto-5’nucleotidase activity and the anchorage of these proteins to the plasma membrane. MATERIALS

AND

METHODS

Materials ATP is released from the secretory vesicles of chromaffin cells during the secretory response (1,2). The extracellular ATP can be degraded by ectonucleotidases. ATP and its main product, adenosine, influence many biological processes (3-5), including the secretory response in chromaffin cells (6, 7). Nevertheless, the ecto-

i To whom correspondence

should be addressed

000:3-9&x1/90 $3.00 Copyright 0 1990 by Academic Press, All rights ofreproduction in any form

Inc. reserved.

All the purine bases and nucleotides as well as CMP, TMP, the e,& methylene-adenosine 5’.diphosphate (AMP-CP),’ the adenylyl-($,ymethylene)-diphosphonate (AMP-PCP), NAD+, and phosphatidylinositol-specific phospholipase C (PI-PLC) were obtained from

’ Abbreviations used: AMP-CP, a,@methylene-adenosine phosphate; AMP-PCP, adenylyl-(8,-y-methylene)-diphosphonate; PLC, phosphatidylinositol-specific phospholipase C; DMEM, co’s modified Eagle’s medium.

5’.diPIDulbec-

37

38

TORRES,

PINTOR,

AND

Boehringer, (Boehringer-Mannheim, West Germany). Cytosine arabinoside, dipyridamole, fluoradeoxy-uridine and p-glycerol phosphate were purchased from Sigma (St. Louis, MO). HPLC columns and the guard pack module were from Kontron. High quality water was obtained from a Milli Q-Mini RO system, and the HAFT filters for HPLC solvents were from Millipore. The HPLC solvents were from Scharlau. [2,3-3H] adenosine Y-monophosphate (18.6 Ci/mmol) was from Amersham. The scintillation liquid, Ready Safe, for aqueous samples was purchased from Beckman. Culture media, fetal calf serum, and antibiotics were purchased from Flow. Cultured vessels were from Nuclon. All other reagents were obtained from Merck.

Methods Isolation and culture of chromafin cells. Chromathn cells were isolated from bovine adrenal glands according to the method of MirasPortugal et al. (24). The cells were isolated with collagenase action and then purified through a Percoll gradient, collected, and washed with Ca*+/Mg*+-free Locke’s solution (0.14 M NaCl, 4.7 mM KCl, 2.5 mM CaCl,, 1.2 mM K,HPO,, 1.2 mM MgSO*, 10 fiM EDTA, 15 mM Hepes, 10 mM glucose, 0.56 mM ascorbate). Cells were then suspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum, penicillin (5 U/ml), streptomycin (5 fig/ml), kanamycin (100 kg/ml), amphotericin (2.5 pg/ml), 10 PM cytosine arabinofuranoside, and 10 fiM fluorodeoxyuridine. Cells were plated in 24.well Costar cluster dishes at a density of 250,000 cells/well or in 3.5~cm diameter Petri dishes at a density of lo6 cells/dish. The purity of chromafhn cell preparations was tested by the selective staining with vital dye neutral red, as described by Role and Perlman (25), that is specific for monoaminecontaining cells. More than 95% of the Percoll gradient-purified chromaffin cells were stained with this dye. HPLC chromatography. The HPLC system consisted of a Waters 600 E pump with automated gradient controller; a variable volume injector U6K and a 481-LC spectrophotometer X,,, (Waters). The detector responses were recorded, as were the integrated areas and retention times, in a 745 data module integrator (Waters). Chromatography was performed with a C,s reverse phase column (C,, i. d. 3.9 mm, 30 cm long, from Kontron). The elution buffers were degassed prior to use and filtered through HAFT filters (Millipore) under vacuum. Before use, the column was equilibrated overnight at 0.2 ml/min with the selected mobile phase. The eluents were chosen according to the products to be quantified. The procedure of Rodriguez de1 Castillo et al. (26) was used with some minor modifications: (a) The quantification of adenine nucleotides was effected with buffer A as eluent. This buffer contained 10 mM potassium phosphate, 2 mM tetrabutylammoniun, final pH 7.4, and 15% (v/v) acetonitrile. The elution order was AMP, ADP, ATP, and the retention times were 3.60,8, and 17.40 min, respectively, at a flow rate of 2 ml/min. (b) Mononucleotides were analyzed by reducing the acetonitrile content ofbuffer A to 5% (v/v) to increase their retention times (buffer B). The elution order of the mononucleotides was CMP, IMP, TMP, AMP and the retention times were 2.33, 3.67, 6.07, and 7.13 min, respectively, at a flow rate of 2 ml/min. (c) The quantitation of the substrate and product of 5’nucleotidase reaction, together with other cellular metabolites derived from adenosine, was carried out using buffer C containing 10 mM potassium phosphate pH 4.8 and 5% methanol. The elution profile was AMP, hypoxanthine, inosine, adenine, and adenosine and their retention times were respectively 1.47, 3.47, 5.93, 6.73, and 16.67 min, at a flow rate of 2 ml/min. Enzymatic activity nucleotidase activities at a density of 250,000 between the third and

All ectomeasurement of ectonucleotidases. were measured in 24-well Costar cluster dishes cells/well. All the experiments were carried out the fifth day of cell culture. Trypan blue exclu-

MIRAS-PORTUGAL sion and lactate dehydrogenase activity release were routinely employed as controls of cellular integrity. The cultured cells were washed twice with Locke’s solution before the enzymatic activities were measured. All determinations were carried out at 37°C and the incubation conditions were as follows: (a) Extracellular ATP and ADP hydrolysis. The ecto-ATPase and ecto-ADPase activities were measured in a volume of 800 ~1 per well (250,000 cells). The concentration range for both nucleotides was 25 to 750 FM in Locke’s solution, containing 10 mM glucose to maintain the energy requirements of the cells (27,28) and the presence of 1 mM Mg*+ and 2.5 mM Ca”. At defined times the medium was removed and ATP or ADP and their metabolites were measured. Routinely, samples of 10 ~1 volume were analyzed by HPLC, with buffer A as eluent. (b) 5’.Nucleotidase activity was measured in 24-well Costar cluster dishes at a density of 250,000 cells/well. The concentration range for AMP was l-100 KM in a final volume of 800 ~1 Locke’s solution per well. All determinations were carried out in the presence of 20 mM P-glycerol phosphate. IMP, CMP, and TMP were also employed as substrates. The substrates and products of this reaction were studied by HPLC. Routinely 10 ~1 of the reaction media was injected to be analyzed. Eluents were buffers B or C depending on the substrate employed. As HPLC is slow, a radioisotopic assay for 5’nucleotidase was also employed. The assay conditions were as in the HPLC method, but 0.1 &i [3H]AMP (18.6 Ci/mmol) and the required nonlabeled AMP were added per well. Volume aliquots of 50 ~1 were taken from the well mixture and spotted onto DEAE-cellulose paper (4 cm’), which was dried and then washed twice for 5 min in 1 mM ammonium formate to remove adenosine, and rinsed once with absolute ethanol. This procedure was similar to that employed for adenosine kinase in this tissue (21, 22). As tritium from AMP is counted with low efficiency when bound to DEAE filters, the nucleotide was released before radioactivity was counted by placing the filter in a counting vial with 1 ml of HCl (0.1 M) and KC1 (0.2 M) for 15 min at room temperature, with shaking. A volume of 8 ml of scintillation liquid (Ready Safe, Beckman) was then added per vial and the radioactivity was counted in a LS 3801 Beckman counter. Lactic dehydrogenase activity was assayed according the Bergmeyer procedure (29). To study the anchorage of ecto-5’. Phospholipase C treatment. nucleotidase on the plasma membranes, chromaffin cells were treated with phosphatidylinositol-specific phospholipase C from Bacillus cereus, according to the procedure described by Jemmerson and Low for Hela cells (30) and modified for chromaffin cells as follows: Cultured cells at a density of 3 X 10fi cells/Petri dish (3.5 cm diameter) were treated with 0.5 IU of PI-PLC in a final volume of 1 ml of isosmotic solution containing 0.28 M sucrose, 10 mM glucose and 2 mM TrisHCl (pH 7.4). At required times, aliquots were taken to measure the solubilized 5’.nucleotidase activity. The remaining ecto-activity associated with the cultured cells was measured at the end of the experiment (1 h). Controls were made in absence of PI-PLC. Activities were measured by the radiometric method described before. The anchorage of ecto-ATPase and ecto-ADPase enzymatic activities were studied by the same procedure as for the 5’-nucleotidase with PI-PLC. The released and the remaining cell-bound activities were measured by HPLC as described before.

RESULTS Hydrolysis of Extracellular by Cultured Chromafin

ATP and ADP Cells

ATP and ADP were rapidly degraded by cultured chromaffin cells as shown in Fig. 1. The hydrolysis of

ECTONUCLEOTIDASES

IN CHROMAFFIN

39

CELLS

as AMP (Figs. 5A and 5B) at the same extracellular concentration (50 PM) and in presence of 20 mM P-glycerol phosphate to avoid the nonspecific phosphatase activity. The hydrolysis of the pyrimidine nucleotides CMP and TMP is shown in Figs. 5C and 5D. Both compounds were more slowly hydrolyzed, under our experimental conditions, at the same extracellular concentrations as the purine nucleotides.

. ADO . AOP . AMP

Kinetic Parameters of Ecto-5’-nucleotidase The kinetic parameters of ecto-5’-nucleotidase activity in chromaffin cells, were measured by HPLC, as

FIG. 1. Time course of extracellular nucleotide hydrolysis by cultured chromafhn cells. 250,000 cells/well were incubated at 37°C with 1 ml Locke’s solution containing 100 pM ATP (A) or 100 fiM ADP (B) at starting conditions. At the required times, aliquots of 50 ~1 were taken up from the extracellular medium and samples of 10 ~1 were routinely injected and processed by HPLC using buffer A as eluent. Data are mean + SD (bars) values from three experiments performed in duplicate.

extracellular nucleotides suggested the presence of ectonucleotidases at the plasma membrane level. The possibility of enzymatic activities coming from the inside of the cell were discarded because there was no release of lactic dehydrogenase activity and also the cellular viability was 100% at the end of the incubation period as tested by trypan blue exclusion. The kinetic parameters for ATP hydrolysis were studied and a K,,, value of 250 + 18 PM and a I’,,, value of 167 t 25 nmol/106 cells per minute were obtained (Fig. 2). Care was taken to obtain initial rates in order to obtain reliable results for K, values. In Fig. 3 the kinetics of ADP hydrolysis is shown. The K, value obtained was 375 ? 40 PM and the V,,, was 125 i 20 nmol/lO” cells per minute. Ecto-5’-nucleotidase

Activity

of Chromafin

.E 1 g E, >

Cells

The hydrolysis of extracellular AMP by cultured chromafhn cells as a function of time is shown in Fig. 4. In the absence of dipyridamole (Fig. 4A), which is an inhibitor of adenosine transport in these cells (18), adenosine did not accumulate in the extracellular medium proportional to AMP disappearance, and inosine, the product of the adenosine deaminase enzymatic reaction gradually appeared. To prevent the possibility of adenosine deamination, outside the cell, the same experiment was made in the presence of dipyridamole (5 PM), Fig. 4B; the increase of adenosine concentration mirrors the AMP disappearance, and no significant inosine concentration was observed outside the cell. IMP, CMP, and TMP were also dephosphorylated at the outside of the cell, Fig. 5. IMP was as good substrate

B L

I 0.01

I

1

0.02

0.03

“b“)

I 0.0

(PM)

FIG. 2. Influence of substrate concentration on ecto-ATPase activity from chromafhn cells. (A) Michaelis-Menten representation of ecto-ATPase activity. 250,000 cells/well were incubated at different concentrations of ATP (25-750 pM) in 800 ~1 Locke’s solution, after 1 min at 37°C the medium was aspirated and processed by HPLC using buffer A as eluent. Results are expressed in nanomols per minute per lo6 cells. (B) LineweaverBurk plot of ecto-ATPase from Fig. 2A. Dat a are mean i- SD values of three experiments performed in duplicate.

40

TORRES,

PINTOR,

AND

MIRAS-PORTUGAL . AMP . ADO

. AMP . ADO

A

I

“-:‘;’ -l

60‘ \\

- 60. 20-

A A loo

7 'E‘ .E \ In =

0.1

z

0.06-

200

300

400

500

600

FIG. 4. Time course of extracellular hydrolysis of AMP by chromafin cells. 250,000 cells/well were incubated at 37°C in 800 ~1 Locke’s solution containing 100 FM AMP at starting conditions, in absence (A) or presence (B) of 5 pM dipyridamole. At the required times aliquots of 10 ~1 were taken up to the extracellular incubation medium and analyzed by HPLC using buffer C as eluent. This study to avoid was effectuated in the presence of 20 mM p-glycerolphosphate nonspecific phosphatase activities. Data are means + SD from four experiments performed in duplicate.

I 700

-

8 o.oa“0

tive mechanism with respect to the substrate, AMP, was observed. Concerning the ADP analog (AMP-CP) inhibition of 5’-nucleotidase is shown in Fig. 7B. A competitive mechanism can be deduced from the Dixon plot and B I 0.01

I 0.03

I 0.02 1 /[ADPI

1 0.0

(j.Uvl)

FIG. 3. Kinetic studies of ecto-ADPase activity from chromafin cells. (A) Michaelis-Menten representation. 250,000 cells/well were incubated for 1 min at 37°C with 800 ~1 Locke’s solution containing different concentrations of ADP. The substrate degradation and the product appearance were analyzed by HPLC using buffer A as eluent. (B) Lineweaver-Burk plot of ADPase activity from values of Fig. 3A. Data are mean + SD values from three experiments performed in duplicate.

shown in Fig. 6. The Km value was 55 f 5 PM for AMP and the’ V,,, was 4.3 + 0.8 nmol/106 cell. min. These values were obtain measuring the linear period of this enzymatic reaction. When the radiometric assay, with labeled [3H]AMP as substrate, was employed the Km and VMAx was exactly the same. The inhibitory effects of the nonhydrolyzable ATP and ADP analogs adenylyl-a&3-methylene-diphosphonate and a&methylene-adenosine 5’-diphosphate on the 5’-nucleotidase reaction were studied. Using a Dixon plot to obtain the inhibition constants, Fig. 7, the ATP analog, AMP-PCP, showed a KI of 193 + 29 nM (mean f SD of three experiments in duplicate), and a competi-

20

40

60

‘120

time

20

40

60 ‘%

(min)

FIG. 5. Extracellular hydrolysis of mononucleotides by 5’.nucleotidase from chromafin cells as a function of time. 250,000 cells/well were incubated at 37°C in 800 ~1 Locke’s solutions with 50 /.LM of AMP (A), IMP (B), CMP (C), and TMP (D), in the presence (A) or in absence (0) of 20 mM fl-glycerolphosphate. At the required times aliquots of 10 ~1 were taken up and processed by HPLC with buffer C as eluent. Points are values of a representative and reproducible experiment.

ECTONUCLEOTIDASES

IN CHROMAFFIN

41

CELLS

the PI-PLC activity and an isosmotic buffer with SUcrose instead of NaCl was employed (30). The glucose was absolutely necessary to guarantee the energetic requirements of chromaffin cells and avoid cellular lysis. In this experimental situation the viability of cells was 100% after 1 h of incubation and no lactic dehydrogenase release or trypan blue staining of the cells was observed. In Fig. 8A a typical experiment of PI-PLC capacity to release Y-nucleotidase from chromaffin cells is shown. Generally with 0.5 IU/ml of PI-PLC after 1 h of incubation about 56% of the total activity present at the plasma membranes was released. Fig. 8B shows that after 1 h of incubation with PIPLC (0.5 IU), chromaffin cells present a 50% lower ca-

1 0.04

1

0.08 l/~AMPl

I

1

1

0.12

0.16

0.2

(jOA)

FIG. 6. Kinetics analysis of ecto-5’.nucleotidase activity from cultured chrornaffin cells. (A) Michaelis-Menten representation. 250,000 cells/well were incubated for 1 min at 37°C with 800 11 Locke’s solution with different concentrations of AMP (l-100 pM). Aliquots of 10 ~1 were processed by HPLC with buffer C as eluent. (B) LineweaverBurk plot of 5’.nucleotidase activity from values of Fig. 6A. Data are mean t_ SD values from four experiments performed in duplicate. 200

the KI value obtained was 73.3 ? 3.5 nM (mean 2 SD of three experiments in duplicate). The radiometric method of 5’-nucleotidase was employed in this study. Release of 5’-Nucleotidase from Chromafin Cells by Phosphatidylinositol-Specific Phospholipase C Phosphatidylinositol-specific phospholipase C from B. cereus effectively releases the ecto-5’-nucleotidase from cultured chromaffin cells. The culture media and Locke’s solution were not a good incubation media for

100

100

200

inhibitor

300

400

concentration

500 (nM)

FIG. 7. Dixon plot of AMP-PCP (A) and AMP-CP (B) inhibitory effects on ecto-5’.nucleotidase activity from chromaffin cells. Costar wells containing 250,000 cells each were preincubated in 250 ~1 Locke’s solution with variable concentrations of AMP-PCP or AMP-CP for 15 min. 0.1 PCi [2,3-“HIAMP and nonlabeled AMP were then added to reach 25 pM (O), 50 pM (A), and 75 pM (m), this being the final concentration. The AMP hydrolysis by 5’.nucleotidase was stopped by aspirating the medium after 1 min at 37°C. Aliquots of 50 pM were then put on DEAE-81 filters and processed as described under Materials and Methods. Data are mean 2 SD values from three experiments performed in duplicate.

42

TORRES,

PINTOR,

AND

MIRAS-PORTUGAL

pacity of AMP hydrolysis, when compared with control cells. Thus the existence of an anchoring domain for 5’nucleotidase through phosphatidyl inositol can be suggested. ATPase and ADPase activities were not released from the chromaffin cells in culture by the action of PI-PLC. In our experimental conditions there were no measurable activities released into the extracellular media, and the cultured chromaffin cells showed the same activities as the control cells after 1 h of treatment with the PI-PLC. DISCUSSION

time

(min)

FIG. 8. Phosphatidylinositol phospholipase C effect on ecto-5’nucleotidase activity from chromafhn cells. (A) Hydrolysis of AMP by the ecto-5.nucleotidase released under the treatment of phosphatidylinisitol-specific phospholipase C. Petri dishes with 3 X 10” chromafhn buffer (pH 7.4) containcells were incubated in 1 ml of 2 mM Tris/HCl ing 0.28 M sucrose and 10 mM glucose, in the presence (A) or absence (0) of 0.5 IU PI-PLC. At the required times 100 ~1 of incubation medium were taken up and assayed for 5’.nucleotidase activity. The 5’nucleotidase activity was measured as follows: 100 ~1 of incubation medium from the wells were mixed with 100 pl of 25 FM AMP that contain 0.1 PCi [2,3-3H]AMP in 50 mM Tris/HCl (pH 7.4), 20 mM MgSO, and 20 mM fi-glycerolphosphate. (The final volume being 200 ~1.) The incubation period was 10 min and 50 ~1 of the reaction mixture was spotted on DEAE-filters as described under Materials and Methods. (B) Time course of AMP hydrolysis by the PI-PLC treated (A) or nontreated (0) chromafhn cells. 3 X 10s cells were preincubated for 1 h in presence (A) or absence (0) of 0.5 IU of PI-PLC. Cells were then washed twice with Locke’s solution and incubated with 1 pCi [2,33H]AMP and nonlabeled AMP to reach 25 pM substrate concentration at starting conditions. At the required times aliquots of 50 ~1 of incubation medium were taken up and spotted on DEAE-81 filters. The filters were processed as described under Materials and Methods. Results are the means + SD of three experiments in duplicate.

The work reported here shows that cultured chromaffin cells obtained from bovine adrenal medulla can hydrolyze adenine nucleotides at the extracellular side of their plasma membrane. Thus, the cycle ATP-adenosine can be accomplished in these aminergic neural cells, in a similar way to that found in cholinergic synapses (lo), and this is a possible regulatory mechanism of extracellular adenosine concentration and its action. The ectonucleotidase activities found in these cells can be compared with the bibliographical data in two main ways, by comparison of the activity per lo6 cells or per milligram of protein, taking into account that lo6 chromaffin cells contain 0.105 mg of protein (18). The ecto-ATPase activity present in chromaffin cells was 2 times higher than that found in isolated endothelial cells from pig aorta (9,31), 5 times higher than that found in endothelial of piglet lung (31), and 20-30 times higher than that found at the cholinergic synapse of Torpedo (32). (Comparison being made by milligrams of protein.) The ecto-ATPase from chromaffin cells has a similar affinity to that found in endothelial cells, in the presence of 2.5 mM Cazt and 1 mM Mg’+. A 2-3 times higher affinity for the ecto-ATPase in synaptic terminal of Torpedo, at the optimal concentration of Ca2+ and Mg”+, has been reported (32). The ecto-ADPase activity in chromaffin cells was about 10 times higher than that found in endothelial cells when activity per cell was considered. Nevertheless, their affinity constants were very similar (9,31). The ecto-5’-nucleotidase presented in chromaffin cells showed 2-3 times higher activity than that found in endothelial cells (per lo6 cells) with the same or a very similar K,,, (9, 31). The same affinity but an activity one order of magnitude lower (per lo6 cells) were reported in mouse sperm (33). The blockage of adenosine transport by dipyridamole showed that the 5’-nucleotidase reaction was extracellular and no adenosine deaminase reaction occurs at the external surface of the cell. One interesting and controversial aspect is the location of 5’-nucleotidase in neural tissues. The inmunocytochemical techniques showed a clear association of this enzyme with the Schwann cell membranes in Torpedo

ECTONUCLEOTIDASES

(23), the outermost membranes of myelinatedperipheral nerves from the rat (34), and the enrichment in myelin fractions from bovine brain (35). Nevertheless in other cells of neural origin, such as the pineal gland phototransducer in lower vertebrates (36), the enzyme is located at the plasma membrane. The presence of 5’nucleotidase in cultured chromaffin cell plasma membranes is supported by three main lines of evidence: (i) There is no glial contamination in this tissue. (ii) The possible contamination by cells from the vascular endothelium is lower than 5% of the total. (iii) The activity present in chromaffin cells is higher than that reported for endothelial cells. As for ecto-5’-nucleotidase from rat heart, the nonmetabolizable ADP and ATP analogs were competitive inhibitors of this enzyme in chromaffin cells. The cu,pmethylene adenosine 5’-diphosphate showed a KI in the nM range, but it was one order of magnitude higher than that reported for the rat heart enzyme (13,37). The adenylyl fl,y-methylene diphosphonate also showed a KI in the nM range for ecto-5’-nucleotidase in the chromaffin cells. These results suggest that the extracellular adenosine formation is highly controlled by the other adenine nucleotides, allowing a gradual adenosine formation in the extracellular space. This observation is important to assure the modulatory effects of adenine nucleotides and adenosine over longer periods of time. Since the pioneering work of Low and Finean (38), it has been recognized that an extremely diverse group of proteins is anchored to the plasma membrane through a covalently attached glycosyl-phosphatidylinositol moiety. These include various enzymes and other surface glycoproteins (39-41). Also in PC 12 pheochromocytoma cells, the tumor homolog of chromaflin cells, PIPLC, can release a large number of proteins not yet characterized (42). In chromaffin cells from adrenal medulla, the ecto-5’-nucleotidase is released by the action of phosphatidylinositol-specific phospholipase C. A similar release from plasma membrane was seen for 5’-nucleotidase from other mammalian tissues (41,43). There are no data, so far, about the possible attachment of ecto-ATPase and ecto-ADPase to plasma membranes. In our experimental conditions the PI-PLC was unable to detach these enzymatic activities from the chromaffin cell plasma membranes. In conclusion chromaffin cells seem to be self suficient in recycling the ATP released exocytotically, the three necessary enzymatic nucleotidases being present at the plasma membrane, and show high activities when compared with other mammalian cells. The high capacity of adenosine transport (18) assures the quick recovery of the storage granules and the full functioning of the chromaffin cells. ACKNOWLEDGMENTS We are indebted to GIPISA slaughterhouse of Madrid for materials. This investigation was supported by a research grant from the Spanish

IN CHROMAFFIN

43

CELLS

Ministry of Education and Science, Comision Interministerial de Ciencia y Tecnologia No. PB 86-0009 and from The Fondo de Investigaciones Sanitarias de la Seguridad Social No. 88-0925. We thank Mr. Christian Salleroli for English corrections. REFERENCES 1. Burgoyne, R. D. (7984) Biochim. Biophys. Acta 779,201-216. 2. Phillips, J. H., and Pryde, J. G. (1987) Ann. NY. Acad. Sci. 493, 27-40. 3. Gordon, J. L. (1986) Biochem. J. 233,309~319. 4. Reilly, W. M., and Burnstock, G. (1987) Eur. J. Pharmacol. 138, 319-324. 5. Williams, M. (1987) Annu. Reu. Pharmacol. Toricol. 27, 315-345. 6. Chern, Y. J., Herrera, M., Kao, L. S., and Westhead, E. W. (1987) J. Neurochem. 48,1573-1576. 7. Chern, Y. J., Kim, K. T., Slakey, L. L., and Westhead, E. W. (1988) J. Neurochem. 50,1484&1493. 8. Pearson, J. D., Carleton, J. S., and Gordon, J. L. (1980) Biochrm. J. 190,421-429. 9. Gordon, E. L., Pearson, J. D., and Slakey, L. L. (1986) J. Riol. Chem. 261,15,496-15,504. 10. Richardson, P. J., Brown, S. J., Bailyes, E. M., and Luzio, J. P. (1987) Nature (London) 327,232-234. 11. Keller, F., and Zimmermann, H. (1983) Life Sci. 33,2635-2642. 12. Harb, J., Mefilah, K., Duflos, Y., and Bernard, S. (1983) Eur. J. Riochem. 137,131&138. 13. Naito, Y., and Lowenstein, J. M. (1985) Biochem. J. 226, 645651. 14. Thompson, L. F., Ruedi, J. M., and Low, M. G. (1987) Riochim. Biophys. Res. Commun. 145, 118-125. 15. Casado, V., Mallol, J., and Bozal, J. (1988) Neurochem. Res. 13, 349-357. 16. Paterson, A. R. P., Lau, E. V., Dahlig, Mol. Pharmacol. 18,40-44. 17. Woffendin, C., and Plagemann, Acta 903,18-30.

E., and Cass, C. E. (1980)

P. G. W. (1987) Biochim. Riophys.

M. T., Torres, M., Rotllan, 18. Miras-Portugal, (1986) J. Biol. Chem. 261,1712-1719. 19. Torres, M., Molina, Lett. 201, 124-128.

P., and Miras-Portugal,

P., and Aunis,

D.

M. T. (1986) F,Q?S

20. Torres, M., Bader, M. F., Aunis, D., and Miras-Portugal, M. T. (1987) J. Neurochem. 48,233-235. 21. Rotllan, P., and Miras-Portugal, M. T. (1985) J. Neurochem. 44, 1029-1036. 22. Rotllan, P., and Miras-Portugal, 151,365-371.

M. T. (1985) Eur. J. Biochem.

23. Grondal, E. J. M., Janetzko, A., and Zimmermann, H. (1988) Neuroscience 24, 351-363. 24. Miras-Portugal, M. T., Rotllan, P., and Aunis, D. (1985) Neurothem. Int. 7,89-93. 25. Role, L. W., and Perlman, R. L. (1980) J. Neurosci. Methods 2, 253-265. 26. Rodriguez de1 Castillo, A., Torres, M., Delicado, E. G., and MirasPortugal, M. T. (1988) J. Neurochem. 51, 1696-1703. 27. Millaruelo, A., Sagarra, M. It., and Miras-Portugal, M. T. (1982) J. Neurochem. 38,470-476. 28. Millaruelo, A., Sagarra, M. R., Delicado. E., Torres, M., and Miras-Portugal. M. T. (1986) Mol. Gel. Riochem. 70,67?76. 29. Bergmeyer, H. U. (1974) Methods of Enzymatic Analysis, Vol. 2 pp. 574-576, Academic Press, San Diego.

44

TORRES,

PINTOR,

AND

30. Jemmerson, R., and Low, M. G. (1987) Biochemistry 26, 57035709. 31. Pearson, d. D., and Coade, S. B. (1987) in Topics and Perspectives in Adenosine Research (Gerlach, E., and Becker, B. F., Eds.), pp. 1455153. Springer-Verlag. Berlin/Heidelberg. 32. Zimmermann, Ii., Keller, F., and Grondal, E. J. M. (1984) Regulation of transmitter function (Vizi, E. S., and Magyar, K., Eds.), pp. 87-96. Elsevier Science. 33. Monks, N. J., and Fraser, L. R. (1988) J. Reprod. Fertil. 83,389399. 34. Cammer, W., and Tansey, F. (1987) Exp. Neural. 97,758-762. 35. Casado, V., Mallol, J., and Bozal, J. (1988) Neurochem. Res. 13, 359-368. 36. Falcon, J., Besse, C., Guerlottir, J., and Collin, J. P. (1988) Cell Tissue Res. 251,495-502.

MIRAS-PORTUGAL 37. Collinson, A. R., Peuhkurinen, K. J., and Lowenstein, J. M. (1987) in Topics and Perspectives in Adenosine Research (Gerlach, E., and Becker, B. F., Eds.), pp. 133-144. Springer-Verlag, Berlin/ Heidelberg. 38. Low, M. G., and Finean, B. (1978) Biochim. Biophys. Acta. 508, 565-570. 39. Hooper, N. M., and Turner, A. J. (1988) FEBS Lett. 229, 340344. 40. Littlewood, G., Hooper, N. M., and Turner, A. J. (1989) Biochem. J. 257,361-367. 41. Low, M. G. (1987) Biochem. J. 244,1-13. 42. Margolis, R. K., Goossen, B., and Margolis, R. U. (1988) Biochemistry 27,3454-3458. 43. Thompson, L. F., Ruedi, J. M., and Low, M. G. (1987) Biochem. Biophys. Res. Commun. 145,118-125.

Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides.

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma me...
786KB Sizes 0 Downloads 0 Views