Angiotensin II binding sites on micro-organisms contaminating cell cultures

Regulatory Peptides, 44 (1993) 233-238

233

© 1993 Elsevier SciencePublishers B.V. All rights reserved 0167-0115/93/$06.00 REGPEP 01276

Angiotensin II binding sites on micro-organisms contaminating cell cultures Steven W h i t e b r e a d a, J o s e f Pfeilschifter b, H a n s p e t e r R a m j o u 6 a and M a r c de G a s p a r o a aResearch Department, Pharmaceuticals Division, Ciba-Geigy Limited, Basle (Switzerland) and bDepartment of Pharmacology, Biocenter, University of Basle, Basle (Switzerland)

(Received 2 December 1992; accepted 11 December 1992) K e y words: Acholeplasma laidlawff; Mycoplasma; Subtype; Receptor

Summary An angiotensin II (Ang II) binding site, distinct from AT 1 and AT 2, has been found in cell cultures of rat aortic smooth muscle and rat glomerular mesangium. It is characterized by a high affinity for Ang II (K d 0.75 + 0.13 nM) and Ang I (Ki 0.72 + 0.12 nM), but a very low affinity for Ang III (K i 31 + 5 #M). Ang(1-7) (K i 1.01 + 0.26 nM) and Ang(1-6) (Ki 4.54 + 0.24 nM) are very selective for this site, with affinities more than 150- and 10,000-fold greater, respectively, than for AT 1 or AT 2. The selective angiotensin receptor subtype ligands losartan and L-158,809 (AT1), P D 123319 and CGP 42112A (AT2) were inactive. Binding to this site was abolished after the cells had been treated with the antibiotic mixture BM-Cyclin, suggesting that the site is located not on the cells, but on a cell culture contaminant. This has been identified as Acholeplasma laidlawii. Caution should therefore be exercised when interpreting Ang II-related data obtained from cells that have not been checked for Mollicute contamination.

Introduction Since the original reports of the existence of angiotensin II (Ang II) receptor subtypes [1,2], much work has been done to characterize them and to determine their distribution. The AT l receptor has Correspondence to: M. de Gasparo, Ciba-GeigyLtd., K125 1015, CH-4002 Basle, Switzerland.

now been cloned, including isoforms thereof [3-5] and some groups claim to have found additional subtypes [6,7]. Much of the characterization work has been done on cells expressing one subtype only. It is generally considered that rat vascular smooth muscle cells (SMC) and glomerular mesangial cells express only the AT 1 subtype [8,9]. It was surprising therefore to find a binding site on these cells differing markedly from either of the two subtypes de-

234 scribed so far, and that the site could not be found on isolated glomeruli, or other tissues. Bergwitz et al. [ 10] reported the existence of an Ang II binding site situated on Mycoplasma hyorhinis attached to Opossum kidney cells which differed markedly from AT 1 and AT E and which corresponded quite well with the binding characteristics with the site we had found. We therefore looked into the possibility that this binding site was situated on Mycoplasma contaminating the cells rather than on the cells themselves.

Materials

ing the ACE inhibitor enalaprilat (10 #M) and p-chloromercuriphenyl sulphonic acid (PCMS, 50 mM) in the assay [14].

Detection and elimination of Mycoplasma in cell culture. Mycoplasma detection was performed by enzyme immunoassay using a Mycoplasma Detection Kit (Boehringer) specific for the detection of four species frequently found contaminating continuous cell lines (M. arginini, M. hyorhinis, A. laidlawii, M. orale). For the elimination of Mycoplasma, the cell cultures were treated with the antibiotic combination BM-Cyclin (Boehringer) for a period of 3 weeks.

and Methods

Compounds. [125I]Angiotensin II, [12SI]angiotensin(1-7) and [~25I]angiotensin I were obtained from Anawa, Wangen, Switzerland. Ang(1-5), Ang(1-6), losartan, L-158,809 [11], PD 123319 [12] and CGP 42112A were synthesized at Ciba-Geigy. All other peptides were from Bachem (Bubendorf, Switzerland). Enalaprilat was from Merck Sharp & Dohme. Membrane preparation. Human uterus membranes were prepared as described previously [13]. Cells (harvested using a rubber policeman and suspended in phosphate-buffered saline [PBS]) were homogenized (Polytron) in ice-cold 20 mM sodium bicarbonate and centrifuged at 60,000 g for 30 min. The pellet was resuspended in 50 mM Tris-HC1 buffer (pH 7.4), containing 125 mM NaC1, 6.5 mM MgC12, 1 mM E D T A and 2 mg/ml bovine serum albumin and proteinase inhibitors [1]. Binding assays. In a total incubation volume of 250 #l, 10-20 #g membrane protein was incubated with 175 pM radioactive tracer _+unlabelled competitors for 1 h at 25 ° C. The reaction was terminated by immediate filtration through Whatman G F / F filters, which were washed twice with 4 ml cold PBS. Non-specific binding was determined in the presence of 1 # M unlabelled Ang II. In some experiments, losartan (10 #M) was used to block AT1 receptors. Degradation of [125I]Ang I, as determined by thin layer chromatography [ 13 ], was prevented by includ-

Results

The AT 1 specific antagonist losartan (10 #M) was unable to fully inhibit binding of [125I]Ang II to rat aortic SMC and mesangial cell membranes (not shown). The residual binding, which ranged from 5 to 95 ~o, was also insensitive to the two AT E ligands C G P 42112A and PD 123319. This binding site was therefore studied in experiments where the AT t receptor was blocked by losartan (10/aM). Most of the 0.25

© 0.20

B

/ F

0.15

0.10 0.05

© 0.00 0.00

I 0.05

I 0.10

I 0.15

0.20

B [nM]

Fig. 1. Scatchard analysis of [ 125I]AngII bindingto membranes of rat mesangial cells contaminated with Acholeplasmalaidtawii; AT1receptorswere blocked with 10 #M losartan. Kd = 0.55 nM; B m a × = 2290 fmol/mgprotein.

235 TABLE I [125 I]Ang II binding to Acholeplasma attached to rat mesangial cell membranes.Ki values (nM + S.E.M., n = 4) (Ang II: Kd) of selected Ang II analogues, in comparison with their affinitiesto the AT~ and AT2 receptors.

AngI AnglI Ang III Ang(1-5) Ang(1-6) Ang(1-7) SarllleSAng II

ATI (SMC)

AT2 (Hum. uterus)

A choleplasma laidlawii

170+59 0.57-+0.06 2.93+_0.62 > 100,000 > 100,000 352 + 30 0.50_+0.13

483_+34 0.38+0.02 0.32+-0.03 > 100,000 18675+ 1025 244 + 62 0.19+0.02

0.72+0.12 0.75+0.13 31160+-4960 47.5 + 3.4 4.54 + 0.24 1.01 + 0.26 3.93+1.36

experiments were carried out on rat mesangial cell m e m b r a n e s as the n e w site a p p e a r e d to be m o r e a b u n d a n t in these cells.

Characterization

Bma x was variable, ranging from 700 to 5000 fmol/mg protein. Binding to this site could n o t be f o u n d on isolated rat glomeruli or rat k i d n e y cortex (only AT~ found [15]). The K i values o f various A n g II analogues were d e t e r m i n e d in c o m p e t i t i o n experiments a n d are c o m p a r e d with those o b t a i n e d in A T x ( S M C ) a n d A T 2 ( h u m a n uterus) (Table I). In c o n t r a s t to the results o b t a i n e d in A T 1 and A T 2, A n g I I I ( d e s - A s p 1A n g II) was virtually inactive, whereas A n g II peptide fragments lacking the C-terminal a m i n o acids a n d A n g I h a d relatively high affinities. The IC50 values o f various peptides and nonpeptides were d e t e r m i n e d to c o m p a r e with the d a t a o f Bergwitz et al. [ 10] o b t a i n e d in Mycoplasma hyorhinis a t t a c h e d to O p o s s u m kidney cells (Table II). In general the o r d e r o f potencies was similar, but A n g ( 1 - 7 ) a n d s a r l A n g II were a b o u t 40, and saralasin even 200-fold m o r e potent in the present study. A l s o in that study, A n g ( 4 - 8 ) was found to be 4.3-fold m o r e active t h a n A n g ( 3 - 8 ) , whereas we found it to be 2.3-fold less potent.

S c a t c h a r d analysis o f [leSI]Ang II binding revealed one site with a K~ o f 0.56 + 0.12 n M (Fig. 1). 100

TABLE II [ lzsI]Ang II binding to Acholeplasma attached to rat mesangial cell membrane ICso (nM + S.E.M., n = 4). Comparison of competition binding results with those of Bergwitz et al. [ 10] on Mycoplasma

hyorhinis.

Ang I Ang II Ang III Ang(1-7) Ang(3-8) Ang(4-8) SarlAng II Saralasin Losartan GGP 42112A Arg8 AVP Bacitracin

80-

60

%

A choleplasma laidlawii

Mycoplasma hyorhinis

0.945 + 0.146 0.992 + 0.182 39,075 _+6305 1.32 + 0.37 8125 + 941 13,500 + 1440 12.1 + 1.6 123 + 5 0% 10 -4 M 0% 10 -4 M 12,860 + 2040 44,000 _+8385

1.6 ~.1 330,000 57 6030 1395 500 30,000 0% 10 -5 M 0~o 10 -5 M 0% 10-4 M 35,000

BOUND

40-

20

0" I 10

I 9

I 8

I 7

I 6

I 5

I 4

[ I N H I B I T O R ] - log M

Fig. 2. [125I]Ang I binding to membranes of rat mesangial cells contaminated with Acholeplasma laidlawii; AT 1receptors were not blocked. Competition curves of Ang I (open squares; ICso = 1.05 nM), Ang II (filled circles; 0.71 nM), Ang III (filled squares; 56 #M), Ang(l-6) (filled triangles; 3.42 nM), Ang(1-7) (open triangles; 1.1 nM) and losartan (open circles; no effect at 100 #M).

236 3500

(not shown). Dithiothreitol (DTT) partially inhibited (30%) binding with an IC50 of 0.6 mM. The high affinity and selectivity of Ang I for this site was confirmed using [125I]Ang I (0.175 nM) as radioligand, in the absence of any blocker. This radioligand bound only the Ang III insensitive site, as losartan was unable to inhibit (Fig. 2). [125I]Ang(17) can also be used to specifically and selectively label the new binding site. However, degradation of this ligand was quite high (20% after 1 h incubation) which, combined with the fast dissociation rate, is reflected in a drop in binding after reaching a maximum at 40 min incubation (Fig. 3).

3000 2500 BOUND 2 0 ~

CPM

1500 1000 500 I

I

I

I

I

20

40

60

80

100

I

120

I N C U B A T I O N T I M E (MIN.)

Evidencefor Mycoplasma binding A check for Mycoplasmacontamination of our cells showed the presence ofAcholeplasmalaidtawiiin both

Fig. 3. [125I]Ang(1-7) binding to membranes of rat mesanglal cells contaminated with Acholeplasma laidlawii; AT 1 receptors were not blocked. Association and dissociation kinetics (Kobs = 0.104 rain- 1 and K l = 0.029 min- 1). Dissociation was initiated by adding 1 #M unlabelled ligand.

SMC and the mesangial cells. Binding experiments were performed on membranes from contaminated SMC and SMC treated with the antibiotic BMCyclin. Ang(1-6) was chosen as a probe for the Ang III insensitive binding site due to its high selectivity, and virtual inactivity at the AT~ and AT 2 receptors. In the untreated cells neither the AT~ specific antag-

The non-hydrolyzable analogue of GTP, GTP ?S (10 #M) had no effect on [x25I]Ang II binding, suggesting that this site is not coupled to a G-protein

80-

80

60-

60

% BOUND 40

40 L__

A

20"

B

0" q

I

I

I

I

I

11

10

9

8

7

6

[ I N H I B I T O R ] - log M

11

I

I

I

I

I

10

9

8

7

6

[ I N H I B I T O R ] - log M

Fig. 4. (A,B) [ 125I]Ang II binding to membranes of rat aortic smooth muscle cells; AT 1 receptors were not blocked. Competition curves for Ang II (filled circles), Ang(1-6) (filled triangles) and L-158,809 (open circles). (A) Control cells, contaminated with Acholeplasma laidlawii; (B) cells treated with the antibiotic mixture BM-Cyclin.

237 onist L-158,809 [9] nor Ang(1-6) was able to block all of the binding (Fig. 4A). After treatment with BM-Cyclin, L-158,809 blocked completely, whereas Ang(1-6) was inactive (Fig. 4B), suggesting that the binding sensitive to Ang(1-6) was on the Acholeplasma, not the cells. A similar result was obtained with mesangial cells (not shown).

face is activated by the attachment of Acholeplasma. However, Bergwitz et al. [ 10] also performed Ang II binding on Mycoplasma hyorhinis monocultures. The binding specificity was similar to that of Mycoplasma attached to cells, although the affinities of Ang I and Ang II were lower by a factor of 7 and 3, respectively. Furthermore, this binding site was found in six different mammalian cell lines only after infection with

M. hyorhinis. Discussion and Conclusions

Micro-organisms of the class Mollicutes are well known as contaminants of cell cultures. Some are known to cause pathological conditions in animals and humans, e.g., Mycoplasma pneumoniae [ 16,17]. Certain Mycoplasma species have been reported to enhance HIV replication [ 18]. Acholeplasma species have most often been found in insects, but also in plants and in mammals. A. laidlawii, which was originally isolated from the sewers of London, has been found in decaying plant and animal tissue [19], and it has been found in the human female lower genital tract [20]. Throat swabs of humans commonly reveal Mycoplasma species and a recent report shows that even freshly isolated cells can be infected from this source [21]. Our results of binding to Acholeplasma compare quite well with those of Bergwitz et al. [ 10] who reported Ang II binding to Mycoplasma hyorhinis attached to Opossum kidney cells (Table II). The differences observed in the IC50 values could be due to various reasons. Firstly, this could represent a real difference between the binding sites of the two Mycoplasma species. Secondly, these compounds could have been partly degraded by proteinases in the M. hyorhinis study; in the present study it was found that [x25I]Ang(1-7) was prone to degradation. Finally, the earlier binding study was performed under rather different conditions: whole cell binding performed in tissue culture dishes for 80 min at 4°C. From this study alone it cannot be concluded that the new Ang II binding site is on Acholeplasma membranes. It could be that a latent site on the cell sur-

A similar binding site has also been reported recently by Chaki and Inagami [7] in a mouse neuroblastoma cell line (very low affinity for Ang III, no effect of GTPTS, partial inhibition by DTT, AT 1 and AT 2 specific ligands inactive). This receptor, called AT 3 [22], stimulates soluble guanylate cyclase by a nitric oxide-mediated process [23]. This finding might suggest that such a site could exist on the cell membrane. However, in these papers no evidence was provided to show that the cell line was not contaminated by Mycoplasma and no data was given on the existence of such a receptor in membranes prepared from tissues. In conclusion, the common cell culture contaminant Acholeplasma laidlawii expresses an Ang II binding site which differs markedly from both AT 1 and AT 2. It is characterized by a very low affinity for Ang III, but a high affinity for Ang(1-7) and Ang I. Losartan, L-158,809 (both AT 1 specific) and CGP 42112A (AT 2 specific) are inactive at this site. This binding site is closely related to that described by Bergwitz et al. [10] from Mycoplasma

hyorhinis. Caution should be exercised when describing new binding sites from cells only. A check for Mycoplasma contamination should always be performed.

Acknowledgements

We would like to thank M. Dominique Monna, M. Mich~le Mele, Mr. Oliver Peter and Mr. Erwin Hermes for excellent technical assistance.

238

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