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Effect of forskolin, isoproterenol and IBMX on angiotensin converting enzyme and cyclic AMP production by cultured bovine endothelial cells
Molecular and Cellular Endocrinology, Elsevier Scientific Publishers Ireland,
103
53 (1987) 103-109 Ltd.
MCE 01714
Effect of forskolin, isoproterenol and IBMX on angiotensin converting enzyme and cyclic AMP production by cultured bovine endothelial cells Cary and F.A.O. Mendelsohn
D.A. Department
Key words: Forskolin;
Angiotensin;
of Medicine, Melbourne
University, Heidelberg,
(Received
1986; accepted
Kininase
15 December
II; Isobutylmethylxanthine;
Vie. 3084, Australia
23 April 1987)
Angiotensin
converting
enzyme;
Isoproterenol
Summary The production of angiotensin converting enzyme (ACE) is known to be increased by glucocorticoids, thyroid hormones and converting enzyme inhibitors. We have recently reported that active CAMP analogues also stimulate production of the enzyme. The effect of stimulation of adenylate cyclase in cultured endothelial cells or of phosphodiesterase inhibition on ACE production was therefore evaluated. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (lop4 M), produced 10.5 k 1.3 and 1.3 + 0.1 (P < 0.01 and P > 0.1) fold increases in extracellular and cellular CAMP levels and a 1.55 & 0.10 (P < 0.0001) fold increase in ACE accumulation. The adenylate cyclase stimulator, forskolin (0.01-10 PM), acutely stimulated cellular CAMP accumulation in a dose-dependent manner, reaching a 2.8 f O.l-fold increase at 10 PM. After 48 h exposure to 10 PM forskolin, significant increases in cellular (1.90 & 0.38-fold increase, P < 0.0001) and extracellular CAMP (2.35 f 0.26-fold increase, P -C0.0001) were also observed but ACE accumulation was unchanged (108 k 10% of control, P > 0.5). The fl-adrenoceptor agonist, isoproterenol (l-1000 nM), acutely stimulated cellular CAMP accumulation, with a threshold effect at 10 nM, an ED,, of approximately 30 nM, and a plateau effect of 2.0 f 0.13-fold increase by 100 nM. After 48 h exposure to isoproterenol(l PM), extracellular CAMP levels were increased significantly (1.68 f 0.33-fold increase, P -C0.01) but ACE production was slightly inhibited (83 + 7% of control, P < 0.05). Thus chronic stimulation of adenylate cyclase by either isoproterenol or forskolin did not stimulate ACE production in these cells. These results therefore suggest that IBMX stimulates ACE production in these cells by a mechanism independent of cyclic AMP. Taken together with our previous findings, these data suggest that although CAMP analogues and IBMX stimulate ACE production in cultured endothelial cells, we are unable to demonstrate a role for endogenously generated CAMP in regulation of the enzyme.
Introduction Angiotensin converting enzyme (ACE) is an ectoenzyme (EC 3.4.15.1) located on the luminal surface of vascular endothelial cells (Caldwell et Address for correspondence: F.A.O. Mendelsohn, University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Vie. 3084, Australia. 0303-7207/87/$03.50
0 1987 Elsevier Scientific
Publishers
Ireland,
al., 1976) as well as in other cells in specialized tissues (Erdos, 1977). The enzyme is synthesized by endothelial cells in culture (Johnson and Erdos, 1977; Hayes et al., 1978; Mendelsohn and Kachel, 1981) and may be induced by glucocorticoids (Friedland et al., 1977; Mendelsohn et al., 1982; Friedland and Silverstein, 1983) thyroid hormones (Krulewitz et al., 1984) and angiotensin converting enzyme inhibitors (Larochelle et al., 1979; Ltd.
Fyhrquist et al., 1980, 1982, 1983; GronhagenRiska et al., 1983). Recently we have found that production of ACE is markedly stimulated by the phosphodiesterase inhibitor, IBMX, and modestly stimulated by a range of active CAMP analogues (Lloyd et al., 1987). The effect of dibutyryl CAMP was additive in cells maximally stimulated by dexamethasone, suggesting independent mechanisms of action of these stimulators. Adenylate cyclase activity in the vascular endothelium has been reported to be stimulated by isoproterenol (Hopkins and Gorman, 1981; Makarski, 1981; Karnushina et al., 1982) PGE, (Abdel Halim et al., 1980; Dembinska-Kiec et al., 1980; Hopkins and Gorman, 1981; Makarski, 1981; Kamushina et al., 1982) and forskolin (Leitman et al., 1986). We therefore investigated whether stimulation of adenylate in these cells would modify ACE production. methods
Materials Collagenase (Worthington, CLSII), RPM1 1640 powder medium, Hepes buffer and fetal calf serum were obtained from Flow Laboratories, Stanmore, N.S.W., Australia. Trypsin powder, versene solution and glutamine were from Commonwealth Serum Laboratories, Victoria, Australia. Tissue culture flasks (25 cm’) and 24-well plates (2.0 cm’) were obtained from Miles Chemicals, Naperville, IL. 3-Isobutyl-I-methylx~t~ne (IBMX), CAMP, dexamethasone, forskolin and isoproterenol were obtained from Sigma Chemical Co., St. Louis, MO. Hippuryl-histidyl-leucine was from Protein Research Foundation, Osaka, Japan. Adenosine 3’,5’-cyclic phosphoric acid 2’-0-succinyl-3[1251]i~otyros~e methyl ester (1251-~AMP) was purchased from Amersham Corporation, Arlington Heights, IL.
Isolation and culture of bovine aortic endothelial cells ~do~eli~ cells were isolated from fresh bovine aortae using collagenase digestion (Booyse et al., 1975) and maintained in 30% fetal calf serum in RPM1 1640 medium supplemented with an ad-
ditional 1 mM glutamine as previously described (Mendelsohn and Kachel, 1981; Mendelsohn et al., 1982). The cells were used at early passage (less than six population doublings). Prior to each experiment the endothelial cells were grown to confluence in 25 cm* tissue culture flasks, washed twice with phosphate-buffered saline and once with RPM1 1640 medium, and were incubated in a total of 5 ml RPM1 1640 medium without added serum for a further 2 days (unless otherwise indicated) in room air. Under these conditions the cells show a linear increase in ACE activity over 2 days (Mendelsohn and Kachel, 1981). Test drugs were dissolved in RPM1 1640 medium and added to each flask at the appropriate concentration.
Angiotensin converting enzyme assay Cell monolayers were scraped from replicate flasks, combined with the medium, disrupted by brief somcation, dialysed at 4 o C against distilled water for 24 h and then against 100 mM potassium phosphate, pH 8.3, containing 300 mM NaCl for a further 24 h and stored at -2O’C prior to assay. ACE activity was determined by a fluorimetric method using the synthetic substrate hippuryl-histidyl-leucine as previously described (Mendelsohn and Kachel, 1981). Protein content of the cell homogenate was determined using a modified Lowry method (Hartree, 1972) using BSA as a standard. Cyclic AMP radioimmu~o~say Cyclic AMP (CAMP) was measured by radioimmunoassay (Brown et al., 1972) using ‘251-cAMP purchased from Amersham (Arlington Heights, IL). The antibody was a gift from Dr. N.H. Hunt (Australian National University, Canberra, Australia) and was raised against 2’-0-succinyl CAMP. The samples were acetylated, using a 2 : 1 mixture of triethylamine and acetic anhydride prior to assay. Acute CAMP responses of endothelial cells Endothelial cells were grown to confluence in 24-well (2 cm2) tissue culture plates. The medium was aspirated and the cells washed twice with serum-free RPM1 medium and a 30 min pre-incubation was performed in medium containing 1
105
mM IBMX. Test substances were added for 20 min, at the end of which the medium was aspirated and 1 ml of cold (0 o C) 95% ethanol-HCl, pH 3.0 (Dobson and Brown, 1985), was added to the cells. After 1 h at 4 o C, the supernatant was collected for CAMP assay. Several of the wells remained untreated and the cells from these were washed in serum-free medium, sonicated and dialysed against distilled water for 24 h, before protein was measured by a modified Lowry method (Hartree, 1972). Chronic CAMP responses of endothelial cells Endothelial cells were grown to confluence in 25 cm2 tissue culture flasks. The medium was removed and replaced by serum-free RPM1 medium. The flasks were then incubated for 48 h with test substances. In the case of isoproterenol, a fresh dose of the drug was administered every 24 h. At the end of the incubation the flasks were chilled on ice and a final concentration of 0.5 mM IBMX was added to the medium. The medium was aspirated from the cells, boiled for 5 min, centrifuged and the supernatant kept for CAMP assay. The cells were scraped with 1 ml of RPM1 medium containing 0.5 mM IBMX, sonicated, boiled for 5 min, centrifuged and the supernatant used for CAMP assay. Results Validation of CAMP assay Preliminary experiments were performed to assess possible interference of IBMX in the CAMP radioimmunoassay. 50 ~1 samples of RPM1 medium with or without 1 mM IBMX were acetylated and carried through the procedure as described for cell medium. Standard curves (2-200 fmol/tube CAMP) performed with the addition of 100 ~1 of the final acetylation mixture showed no difference between those in assay buffer alone, RPM1 extract or the extract derived from the IBMX-medium. The mass of IBMX (10 nmol) processed through this procedure was 20 times higher than that encountered in the highest cell or medium extracts. Chronic effect of IBMX A CE production In eight experiments
on CAMP accumulation (Fig. 1) exposure
and
of cells
RZ .z ?i
4p ~O~OcQl
4" F ir \ '= .c ELE VI\ = +JE a=
32
1 ?-I+
Control
IBMX (IOOuM)
Fig. 1. Effect of IBMX (100 CM) on ACE activity and CAMP accumulation in bovine endothelial cell cultures incubated for 48 h. Bars represent the mean + SEM of quadruplicate flasks from each of eight experiments. The levels of statistical significance were determined by paired t-tests.
to IBMX (100 PM) for 48 h was associated with significant increases in extracellular (1050 * 130% of control, P < 0.01) CAMP levels and ACE accumulation (155 _t 10% of control, P < 0.0001) but no change was detected in cellular CAMP levels (130 f 10% of control, P > 0.1). Acute effects of isoproterenol andforskolin on CAMP accumulation Acute exposure of the endothelial cells to isoproterenol for 20 min caused a significant stimulation of cellular CAMP accumulation (Fig. 2) with a threshold dose of 10 nM (146 + 7% of control, P < 0.05), EDs,, of approximately 30 nM, and a plateau effect by 100 nM (200 5 13% of control, P < 0.0001). Similarly, acute exposure of the cells to forskolin for 20 min was associated with a marked
106
Chronic effect of isoproterenol and IBMX on CAMP accumulation and ACE production
Fig. 2. Acute effect of isoproterenol on cellular CAMP accumulation in bovine endothelial cell cultures. The cells were preincubated for 30 min in 1 mM IBMX and then incubated for 20 min with isoproterenol. Bars represent mean k SEM from two experiments in which the control was measured in quadruplicate and the isoproterenol doses measured in duplicate. Levels of significance were obtained by a two-way analysis of variance.
After 48 h exposure, both isoproterenol(1 PM) and IBMX (100 PM) increased CAMP accumulation in the extracellular medium to 168 + 5% (P < 0.01) and 520 + 51% (P < 0.0001) of control respectively. CAMP levels in the cells were not altered by these treatments (Fig. 4). ACE specific activity in cells treated with isoproterenol for 48 h showed a small but significant drop (Fig. 4) to 83 * 7% of control values (P < 0.05). In these experiments, ACE was markedly increased to 193 + 7% of control in the presence of IBMX (P < 0.0001).
p>oz
stimulation of cellular CAMP (Fig. 3) with a significant effect of 209 + 20% of control, P < 0.0005 at 100 nM and graded increases up to 286 + 14% of control at 10 PM; no plateau was observed.
I-
p
4oc
r
c s
3oc
z F \ z E
p
$Y IOC
-
ri Control
00 Forskolin
il.lMi
Fig. 3. Acute effect of forskolin on cellular CAMP accumulation in bovine endothelial cell cultures. The cells were preincubated for 30 min with 1 mM IBMX and incubated for 20 min with forskolin. Bars represent the mean + SEM from two experiments in which the control was measured in quadruplicate and the forskolin doses measured in duplicate. Levels of significance were determined by a two-way analysis of variance.
2Dav Contrd
lsoproterenol IBMX IOOJJM '1UM
Fig. 4. Effect of 48 h exposure of endothelial cell cultures to isoproterenol administered daily on CAMP and ACE levels. Results are mean+SEM of quadruplicate flasks from each of three separate experiments. The levels of statistical significance were determined by a two-way analysis of variance.
Protein content of cells treated with isoproterenol remained unchanged (97 &-4% of control, P > 0.3) as did those treated with IBMX (97 f 4% of control, P > 0.3). Chronic effect of forskolin and ACE production
on CAMP accumulation
In four experiments (Fig. 5) exposure of cells to forskolin (10 PM) for 48 h was associated with significant increases in cellular (190 + 38% of control, P < 0.0001) and extracellular (235 ? 26% of control, P < 0.0001) CAMP levels but no change was detected in ACE specific activity (108 f 10% of control, P > 0.5). Chronic effect of forskolin with IBMX accumulation and ACE production
on CAMP
In two experiments (Fig. 6) the chronic effect
p40001
Fig. 5. Effect of 48 h exposure of endothelial cell cultures to forskolin (10 PM) on CAMP and ACE levels. Results are mean + SEM of quadruplicate flasks from four separate experiments. The levels of statistical significance were determined by a two-way analysis of variance.
FORSKOLIN(clMI0
0
0.01
0.1
1.0
10.0
IBMXUOOJJM) 0
+
+
+
+
+
Fig. 6. Effect of forskolin (0.01-10 PM) on ACE activity and CAMP accumulation in bovine endothelial cell cultures incubated for 48 h in the presence of IBMX (100 PM). Bars represent the mean + SEM of quadruplicate flasks from two experiments. The levels of statistical significance were determined by a two-way analysis of variance. P refers to comparisons relative to the control and P* comparisons relative to IBMX (100 PM) alone.
of forskolin (O-10 PM) was evaluated in the presence of IBMX (100 PM). In these experiments, IBMX alone was again associated with a significant rise in extracellular but not cellular CAMP accumulation and a significant increase in ACE specific activity to (127 + 18% of control, P < 0.005). In the presence of IBMX, forskolin further stimulated extracellular CAMP accumulation (reaching 214 + 9%, of IBMX alone at 10 PM, P < 0.0001) and increased cellular CAMP (by 199 + 9% of IBMX alone at 10 PM, P < 0.0001). However, ACE specific activity was not further stimulated by forskolin at any dose examined in the presence of IBMX.
108
Chronic effect of isoproterenol in the presence IBMX on ACE production
of
In three experiments, the effect of isoproterenol (1 PM) alone and in combination with IBMX (100 PM) was examined on ACE specific activity of cell cultures. In these experiments, isoproterenol alone caused a slight inhibition of ACE activity (83 5 7% of control, P < 0.05) and IBMX exerted the usual marked stimulation of ACE (193 * 7% of control, P < 0.0001). In the presence of IBMX, isoproterenol had a slight inhibitory effect on the stimulatory effect of IBMX on ACE specific activity (87 + 8% of IBMX alone, P < 0.01). Discussion We have previously reported that the phosphodiesterase inhibitor, IBMX, stimulated ACE production by cultured endothelial cells at a threshold of approximately 30 PM, increasing to a 4-fold stimulation at 1 mM (Lloyd et al., 1987). Also, several CAMP analogues which were less susceptible to phosphodiesterase cleavage, including dibutyryl CAMP, N6-monobutyryl CAMP and 8-bromo CAMP, modestly stimulated ACE production by the cells. The effect of dibutyryl CAMP on ACE accumulation was additive when combined with maximal glucocorticoid stimulation (Lloyd et al., 1987). These results therefore suggested that CAMP might be capable of modulating ACE production by cultured endothelial cells. Therefore, the effect of agents known to stimulate adenylate cyclase in endothelium, isoproterenol and forskolin was evaluated on ACE production. It was confirmed that both forskolin and isoproterenol acutely stimulated endogenous CAMP production in endothelial cells. The forskolin stimulation was nearly 3-fold at 10 PM, which compares favourably with that reported (Leitman et al., 1986). The effect of isoproterenol occurred maximally at 100 nm, where it produced a 2-fold stimulation of cellular CAMP levels, which is also similar to previous reports (Hopkins and Gorman, 1981). Forskolin also chronically stimulated cellular and extracellular CAMP levels in the cells at a dose of 10 PM, whereas the effect of isoproterenol was only evident on extracellular CAMP levels. Under chronic conditions, CAMP levels do not
accurately reflect the level of endogenous CAMP in the cells or the medium, because of high levels of phosphodiesterase in endothelial cell cultures (Dembinska-Kiec et al., 1980; Brotherton et al., 1982); however, the significant increases in levels seen with both stimuli were used as an index to confirm that adenylate cyclase stimulation observed acutely was at least partially maintained for 48 h. However, no change in ACE levels was detected using forskolin alone and the stimulatory effect of IBMX was not modified by addition of forskolin. Isoproterenol slightly lowered ACE levels after chronic exposure when used alone and slightly impaired the stimulatory effect of IBMX when used in combination. Reproducible increases of ACE accumulation with IBMX were observed and this stimulator was included in all experiments as a positive control. In these experiments addition of IBMX to the cultures for 48 h was associated with markedly increased extracellular CAMP levels, presumably reflecting inhibition of phosphodiesterase activity in the medium. Cellular CAMP levels were not altered, as has been reported in other systems using theophylline (Kakiuchi et al., 1969). These results, taken together with out previous findings, suggest that although exogenous CAMP analogues can modestly increase ACE production from cultured endothelial cells (Lloyd et al., 1987) we are unable to demonstrate increased ACE production by stimulating adenylate cyclase in the cells using either forskolin or isoproterenol. The mechanism of action of IBMX in stimulating ACE in these cells is therefore unclear. The finding that IBMX stimulates ACE production and is associated with raised extracellular CAMP, fits our original hypothesis that CAMP could be a regulator of ACE production (Lloyd et al., 1987). The failure of isoproterenol or forskolin to stimulate ACE production could be due to their inability to sufficiently elevate CAMP levels in the cultured cells. However, this seems unlikely since acute stimulation of cellular CAMP by forskolin at 0.01 pM and above or by isoproterenol at 10 nM or higher, achieved greater CAMP levels than 1 mM IBMX used in the control incubations (Figs. 2 and 3). Similarly, forskolin administered chronically at doses of 1.0 and 10 PM, stimulated CAMP
109
to a greater degree than 100 PM IBMX (Fig. 5). Alternatively, it might be argued that cells treated with IBMX were already maximally stimulated. However, the effect of IBMX on ACE production was far from maximal at 100 PM (Fig. 5; Lloyd et al., 1987). Therefore, failure of further stimulation of ACE by forskolin in the presence of IBMX seems unlikely to be due to prior maximal stimulation of ACE by IBMX through a CAMP-mediated mechanism. Whilst it is still possible that differences in the pattern or extent and duration of elevation of CAMP levels by IBMX might explain it’s activity, it seems probable that IBMX stimulates ACE production via a mechanism independent of CAMP. Acknowledgements Supported by a grant from the National Health and Medical Research Council of Australia. We thank Miss. S. Younan for typing the manuscript. References Abdel-Hahm, MS., Von Holst, H., Meyerson, B., Sachs, C. and Anggard, E. (1980) J. Neurochem. 34, 1331-1333. Booyse, F.M., Sedlak, B.J. and Rafelson, Jr., M.E. (1975) Thromb. Diath. Haemorrh. 34, 825-839. Brotherton, A.F. and Hoke, J.C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 495-499. Brown, B.L., Ekins, R.P. and Albano, J.D. (1972) Adv. Cyclic Nucleotide Res. 2, 25-40. Caldwell, P.B., Seegal, B.C., Hsu, K.C., Das, M. and Soffer, R.L. (1976) Science 191, 1050-1051. Dembinska-Kiec, A., Rucker, W. and Schonhofer P.S. (1980) Naunyn-Schmied. Arch. Pharmacol. 311, 67-70. Dobson, P.R. and Brown, B.L. (1985) Methods Enzymol. 109, 293-298.
Erdos, E.G. (1977) Fed. Proc. 36,1760-1764. Friedland, J. and Silverstein, E. (1983) Cell. Mol. Biol. 29, 85-91. Friedland, J., Setton, C. and Silverstein, E. (1977) Science 197, 64-65. Fyhrquist, F., Forslund, T., Tikkanen, I. and Gronhagen-Riska, C. (1980) Eur. J. Pharmacol. 67 (4), 473-475. Fyhrquist, F., Hortling, L. and Gronhagen-Riska, C. (1982) J. Clin. Endocrinol. Metab. 55, 783-786. Fyhrquist, F., Gronhagen-Riska, C., Hortling, L., Forslund, T., Tikkanen, I. and Klockars, M. (1983) Clin. Exp. Hypertens. A5(7-8), 1319-1330. Gronhagen-Riska, C., Forslund, T., Hortland, L. and Fyhrquist, F. (1983) Acta Med. Stand. (Suppl. 1) 677, 101-104. Hartree, E.F. (1972) Anal. Biochem. 48, 422-427. Hayes, L.W., Goguen, C.A., Ching, S.F. and Slakey, L.L. (1978) Biochem. Biophys. Res. Commun. 82, 1147-1153. Hopkins, N.K. and Gorman, R.R. (1981) J. Clin. Invest. 67, 540-546. Johnson, A.R. and Erdos, E.G. (1977) J. Clin. Invest. 59, 684-694. Kakiuchi, S., Rall, T.W. and McIlwain, H. (1969) J. Neurothem. 16, 485. Karnushina, I.L., Spatz, M. and Bembry, J. (1982) Life Sci. 30, 849-858. Krulewitz, A.H., Baur, W.E. and Fanburg, B.L. (1984) Am. J. Physiol. 247, C163-C168. Larochelle, P., Genest, J., Kuchel, O., Boucher, R., Gutkowska, Y. and McKinstry, D. (1979) Can. Med. Assoc. J. 121, 209-316. Leitman, D.C., Fiscus, R.R. and Murad, F. (1986) J. Cell Physiol. 127 (2), 237-243. Lloyd, C.J., Cary, D.A. and Mendelsohn, F.A.O. (1987) Mol. Cell. Endocrinol. (in press). Makarski, J.S. (1981) In Vitro 17, 450-458. Mendelsohn, F.A.O. and Kachel, C. (1981) Clin. Exp. Pharmacol. Physiol. 8, 477-482. Mendelsohn, F.A.O., Lloyd, C.J., Kachel, C. and Funder, J.W. (1982) J, Clin. Invest. 70, 684-692. Mender&all, W. (1979) Introduction to Probability and Statistics, 5th edn. (Duxbury Press, Belmont, CA) p_ 323. Sattin, A. and Rall, T.W. (1970) Mol. Pharmacol. 6, 13-23. Steiner, A.L., Parker, C.W. and Kipnis, D.M. (1972) J. Biol. Chem. 247, 1106-1113.
103
53 (1987) 103-109 Ltd.
MCE 01714
Effect of forskolin, isoproterenol and IBMX on angiotensin converting enzyme and cyclic AMP production by cultured bovine endothelial cells Cary and F.A.O. Mendelsohn
D.A. Department
Key words: Forskolin;
Angiotensin;
of Medicine, Melbourne
University, Heidelberg,
(Received
1986; accepted
Kininase
15 December
II; Isobutylmethylxanthine;
Vie. 3084, Australia
23 April 1987)
Angiotensin
converting
enzyme;
Isoproterenol
Summary The production of angiotensin converting enzyme (ACE) is known to be increased by glucocorticoids, thyroid hormones and converting enzyme inhibitors. We have recently reported that active CAMP analogues also stimulate production of the enzyme. The effect of stimulation of adenylate cyclase in cultured endothelial cells or of phosphodiesterase inhibition on ACE production was therefore evaluated. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (lop4 M), produced 10.5 k 1.3 and 1.3 + 0.1 (P < 0.01 and P > 0.1) fold increases in extracellular and cellular CAMP levels and a 1.55 & 0.10 (P < 0.0001) fold increase in ACE accumulation. The adenylate cyclase stimulator, forskolin (0.01-10 PM), acutely stimulated cellular CAMP accumulation in a dose-dependent manner, reaching a 2.8 f O.l-fold increase at 10 PM. After 48 h exposure to 10 PM forskolin, significant increases in cellular (1.90 & 0.38-fold increase, P < 0.0001) and extracellular CAMP (2.35 f 0.26-fold increase, P -C0.0001) were also observed but ACE accumulation was unchanged (108 k 10% of control, P > 0.5). The fl-adrenoceptor agonist, isoproterenol (l-1000 nM), acutely stimulated cellular CAMP accumulation, with a threshold effect at 10 nM, an ED,, of approximately 30 nM, and a plateau effect of 2.0 f 0.13-fold increase by 100 nM. After 48 h exposure to isoproterenol(l PM), extracellular CAMP levels were increased significantly (1.68 f 0.33-fold increase, P -C0.01) but ACE production was slightly inhibited (83 + 7% of control, P < 0.05). Thus chronic stimulation of adenylate cyclase by either isoproterenol or forskolin did not stimulate ACE production in these cells. These results therefore suggest that IBMX stimulates ACE production in these cells by a mechanism independent of cyclic AMP. Taken together with our previous findings, these data suggest that although CAMP analogues and IBMX stimulate ACE production in cultured endothelial cells, we are unable to demonstrate a role for endogenously generated CAMP in regulation of the enzyme.
Introduction Angiotensin converting enzyme (ACE) is an ectoenzyme (EC 3.4.15.1) located on the luminal surface of vascular endothelial cells (Caldwell et Address for correspondence: F.A.O. Mendelsohn, University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Vie. 3084, Australia. 0303-7207/87/$03.50
0 1987 Elsevier Scientific
Publishers
Ireland,
al., 1976) as well as in other cells in specialized tissues (Erdos, 1977). The enzyme is synthesized by endothelial cells in culture (Johnson and Erdos, 1977; Hayes et al., 1978; Mendelsohn and Kachel, 1981) and may be induced by glucocorticoids (Friedland et al., 1977; Mendelsohn et al., 1982; Friedland and Silverstein, 1983) thyroid hormones (Krulewitz et al., 1984) and angiotensin converting enzyme inhibitors (Larochelle et al., 1979; Ltd.
Fyhrquist et al., 1980, 1982, 1983; GronhagenRiska et al., 1983). Recently we have found that production of ACE is markedly stimulated by the phosphodiesterase inhibitor, IBMX, and modestly stimulated by a range of active CAMP analogues (Lloyd et al., 1987). The effect of dibutyryl CAMP was additive in cells maximally stimulated by dexamethasone, suggesting independent mechanisms of action of these stimulators. Adenylate cyclase activity in the vascular endothelium has been reported to be stimulated by isoproterenol (Hopkins and Gorman, 1981; Makarski, 1981; Karnushina et al., 1982) PGE, (Abdel Halim et al., 1980; Dembinska-Kiec et al., 1980; Hopkins and Gorman, 1981; Makarski, 1981; Kamushina et al., 1982) and forskolin (Leitman et al., 1986). We therefore investigated whether stimulation of adenylate in these cells would modify ACE production. methods
Materials Collagenase (Worthington, CLSII), RPM1 1640 powder medium, Hepes buffer and fetal calf serum were obtained from Flow Laboratories, Stanmore, N.S.W., Australia. Trypsin powder, versene solution and glutamine were from Commonwealth Serum Laboratories, Victoria, Australia. Tissue culture flasks (25 cm’) and 24-well plates (2.0 cm’) were obtained from Miles Chemicals, Naperville, IL. 3-Isobutyl-I-methylx~t~ne (IBMX), CAMP, dexamethasone, forskolin and isoproterenol were obtained from Sigma Chemical Co., St. Louis, MO. Hippuryl-histidyl-leucine was from Protein Research Foundation, Osaka, Japan. Adenosine 3’,5’-cyclic phosphoric acid 2’-0-succinyl-3[1251]i~otyros~e methyl ester (1251-~AMP) was purchased from Amersham Corporation, Arlington Heights, IL.
Isolation and culture of bovine aortic endothelial cells ~do~eli~ cells were isolated from fresh bovine aortae using collagenase digestion (Booyse et al., 1975) and maintained in 30% fetal calf serum in RPM1 1640 medium supplemented with an ad-
ditional 1 mM glutamine as previously described (Mendelsohn and Kachel, 1981; Mendelsohn et al., 1982). The cells were used at early passage (less than six population doublings). Prior to each experiment the endothelial cells were grown to confluence in 25 cm* tissue culture flasks, washed twice with phosphate-buffered saline and once with RPM1 1640 medium, and were incubated in a total of 5 ml RPM1 1640 medium without added serum for a further 2 days (unless otherwise indicated) in room air. Under these conditions the cells show a linear increase in ACE activity over 2 days (Mendelsohn and Kachel, 1981). Test drugs were dissolved in RPM1 1640 medium and added to each flask at the appropriate concentration.
Angiotensin converting enzyme assay Cell monolayers were scraped from replicate flasks, combined with the medium, disrupted by brief somcation, dialysed at 4 o C against distilled water for 24 h and then against 100 mM potassium phosphate, pH 8.3, containing 300 mM NaCl for a further 24 h and stored at -2O’C prior to assay. ACE activity was determined by a fluorimetric method using the synthetic substrate hippuryl-histidyl-leucine as previously described (Mendelsohn and Kachel, 1981). Protein content of the cell homogenate was determined using a modified Lowry method (Hartree, 1972) using BSA as a standard. Cyclic AMP radioimmu~o~say Cyclic AMP (CAMP) was measured by radioimmunoassay (Brown et al., 1972) using ‘251-cAMP purchased from Amersham (Arlington Heights, IL). The antibody was a gift from Dr. N.H. Hunt (Australian National University, Canberra, Australia) and was raised against 2’-0-succinyl CAMP. The samples were acetylated, using a 2 : 1 mixture of triethylamine and acetic anhydride prior to assay. Acute CAMP responses of endothelial cells Endothelial cells were grown to confluence in 24-well (2 cm2) tissue culture plates. The medium was aspirated and the cells washed twice with serum-free RPM1 medium and a 30 min pre-incubation was performed in medium containing 1
105
mM IBMX. Test substances were added for 20 min, at the end of which the medium was aspirated and 1 ml of cold (0 o C) 95% ethanol-HCl, pH 3.0 (Dobson and Brown, 1985), was added to the cells. After 1 h at 4 o C, the supernatant was collected for CAMP assay. Several of the wells remained untreated and the cells from these were washed in serum-free medium, sonicated and dialysed against distilled water for 24 h, before protein was measured by a modified Lowry method (Hartree, 1972). Chronic CAMP responses of endothelial cells Endothelial cells were grown to confluence in 25 cm2 tissue culture flasks. The medium was removed and replaced by serum-free RPM1 medium. The flasks were then incubated for 48 h with test substances. In the case of isoproterenol, a fresh dose of the drug was administered every 24 h. At the end of the incubation the flasks were chilled on ice and a final concentration of 0.5 mM IBMX was added to the medium. The medium was aspirated from the cells, boiled for 5 min, centrifuged and the supernatant kept for CAMP assay. The cells were scraped with 1 ml of RPM1 medium containing 0.5 mM IBMX, sonicated, boiled for 5 min, centrifuged and the supernatant used for CAMP assay. Results Validation of CAMP assay Preliminary experiments were performed to assess possible interference of IBMX in the CAMP radioimmunoassay. 50 ~1 samples of RPM1 medium with or without 1 mM IBMX were acetylated and carried through the procedure as described for cell medium. Standard curves (2-200 fmol/tube CAMP) performed with the addition of 100 ~1 of the final acetylation mixture showed no difference between those in assay buffer alone, RPM1 extract or the extract derived from the IBMX-medium. The mass of IBMX (10 nmol) processed through this procedure was 20 times higher than that encountered in the highest cell or medium extracts. Chronic effect of IBMX A CE production In eight experiments
on CAMP accumulation (Fig. 1) exposure
and
of cells
RZ .z ?i
4p ~O~OcQl
4" F ir \ '= .c ELE VI\ = +JE a=
32
1 ?-I+
Control
IBMX (IOOuM)
Fig. 1. Effect of IBMX (100 CM) on ACE activity and CAMP accumulation in bovine endothelial cell cultures incubated for 48 h. Bars represent the mean + SEM of quadruplicate flasks from each of eight experiments. The levels of statistical significance were determined by paired t-tests.
to IBMX (100 PM) for 48 h was associated with significant increases in extracellular (1050 * 130% of control, P < 0.01) CAMP levels and ACE accumulation (155 _t 10% of control, P < 0.0001) but no change was detected in cellular CAMP levels (130 f 10% of control, P > 0.1). Acute effects of isoproterenol andforskolin on CAMP accumulation Acute exposure of the endothelial cells to isoproterenol for 20 min caused a significant stimulation of cellular CAMP accumulation (Fig. 2) with a threshold dose of 10 nM (146 + 7% of control, P < 0.05), EDs,, of approximately 30 nM, and a plateau effect by 100 nM (200 5 13% of control, P < 0.0001). Similarly, acute exposure of the cells to forskolin for 20 min was associated with a marked
106
Chronic effect of isoproterenol and IBMX on CAMP accumulation and ACE production
Fig. 2. Acute effect of isoproterenol on cellular CAMP accumulation in bovine endothelial cell cultures. The cells were preincubated for 30 min in 1 mM IBMX and then incubated for 20 min with isoproterenol. Bars represent mean k SEM from two experiments in which the control was measured in quadruplicate and the isoproterenol doses measured in duplicate. Levels of significance were obtained by a two-way analysis of variance.
After 48 h exposure, both isoproterenol(1 PM) and IBMX (100 PM) increased CAMP accumulation in the extracellular medium to 168 + 5% (P < 0.01) and 520 + 51% (P < 0.0001) of control respectively. CAMP levels in the cells were not altered by these treatments (Fig. 4). ACE specific activity in cells treated with isoproterenol for 48 h showed a small but significant drop (Fig. 4) to 83 * 7% of control values (P < 0.05). In these experiments, ACE was markedly increased to 193 + 7% of control in the presence of IBMX (P < 0.0001).
p>oz
stimulation of cellular CAMP (Fig. 3) with a significant effect of 209 + 20% of control, P < 0.0005 at 100 nM and graded increases up to 286 + 14% of control at 10 PM; no plateau was observed.
I-
p
4oc
r
c s
3oc
z F \ z E
p
$Y IOC
-
ri Control
00 Forskolin
il.lMi
Fig. 3. Acute effect of forskolin on cellular CAMP accumulation in bovine endothelial cell cultures. The cells were preincubated for 30 min with 1 mM IBMX and incubated for 20 min with forskolin. Bars represent the mean + SEM from two experiments in which the control was measured in quadruplicate and the forskolin doses measured in duplicate. Levels of significance were determined by a two-way analysis of variance.
2Dav Contrd
lsoproterenol IBMX IOOJJM '1UM
Fig. 4. Effect of 48 h exposure of endothelial cell cultures to isoproterenol administered daily on CAMP and ACE levels. Results are mean+SEM of quadruplicate flasks from each of three separate experiments. The levels of statistical significance were determined by a two-way analysis of variance.
Protein content of cells treated with isoproterenol remained unchanged (97 &-4% of control, P > 0.3) as did those treated with IBMX (97 f 4% of control, P > 0.3). Chronic effect of forskolin and ACE production
on CAMP accumulation
In four experiments (Fig. 5) exposure of cells to forskolin (10 PM) for 48 h was associated with significant increases in cellular (190 + 38% of control, P < 0.0001) and extracellular (235 ? 26% of control, P < 0.0001) CAMP levels but no change was detected in ACE specific activity (108 f 10% of control, P > 0.5). Chronic effect of forskolin with IBMX accumulation and ACE production
on CAMP
In two experiments (Fig. 6) the chronic effect
p40001
Fig. 5. Effect of 48 h exposure of endothelial cell cultures to forskolin (10 PM) on CAMP and ACE levels. Results are mean + SEM of quadruplicate flasks from four separate experiments. The levels of statistical significance were determined by a two-way analysis of variance.
FORSKOLIN(clMI0
0
0.01
0.1
1.0
10.0
IBMXUOOJJM) 0
+
+
+
+
+
Fig. 6. Effect of forskolin (0.01-10 PM) on ACE activity and CAMP accumulation in bovine endothelial cell cultures incubated for 48 h in the presence of IBMX (100 PM). Bars represent the mean + SEM of quadruplicate flasks from two experiments. The levels of statistical significance were determined by a two-way analysis of variance. P refers to comparisons relative to the control and P* comparisons relative to IBMX (100 PM) alone.
of forskolin (O-10 PM) was evaluated in the presence of IBMX (100 PM). In these experiments, IBMX alone was again associated with a significant rise in extracellular but not cellular CAMP accumulation and a significant increase in ACE specific activity to (127 + 18% of control, P < 0.005). In the presence of IBMX, forskolin further stimulated extracellular CAMP accumulation (reaching 214 + 9%, of IBMX alone at 10 PM, P < 0.0001) and increased cellular CAMP (by 199 + 9% of IBMX alone at 10 PM, P < 0.0001). However, ACE specific activity was not further stimulated by forskolin at any dose examined in the presence of IBMX.
108
Chronic effect of isoproterenol in the presence IBMX on ACE production
of
In three experiments, the effect of isoproterenol (1 PM) alone and in combination with IBMX (100 PM) was examined on ACE specific activity of cell cultures. In these experiments, isoproterenol alone caused a slight inhibition of ACE activity (83 5 7% of control, P < 0.05) and IBMX exerted the usual marked stimulation of ACE (193 * 7% of control, P < 0.0001). In the presence of IBMX, isoproterenol had a slight inhibitory effect on the stimulatory effect of IBMX on ACE specific activity (87 + 8% of IBMX alone, P < 0.01). Discussion We have previously reported that the phosphodiesterase inhibitor, IBMX, stimulated ACE production by cultured endothelial cells at a threshold of approximately 30 PM, increasing to a 4-fold stimulation at 1 mM (Lloyd et al., 1987). Also, several CAMP analogues which were less susceptible to phosphodiesterase cleavage, including dibutyryl CAMP, N6-monobutyryl CAMP and 8-bromo CAMP, modestly stimulated ACE production by the cells. The effect of dibutyryl CAMP on ACE accumulation was additive when combined with maximal glucocorticoid stimulation (Lloyd et al., 1987). These results therefore suggested that CAMP might be capable of modulating ACE production by cultured endothelial cells. Therefore, the effect of agents known to stimulate adenylate cyclase in endothelium, isoproterenol and forskolin was evaluated on ACE production. It was confirmed that both forskolin and isoproterenol acutely stimulated endogenous CAMP production in endothelial cells. The forskolin stimulation was nearly 3-fold at 10 PM, which compares favourably with that reported (Leitman et al., 1986). The effect of isoproterenol occurred maximally at 100 nm, where it produced a 2-fold stimulation of cellular CAMP levels, which is also similar to previous reports (Hopkins and Gorman, 1981). Forskolin also chronically stimulated cellular and extracellular CAMP levels in the cells at a dose of 10 PM, whereas the effect of isoproterenol was only evident on extracellular CAMP levels. Under chronic conditions, CAMP levels do not
accurately reflect the level of endogenous CAMP in the cells or the medium, because of high levels of phosphodiesterase in endothelial cell cultures (Dembinska-Kiec et al., 1980; Brotherton et al., 1982); however, the significant increases in levels seen with both stimuli were used as an index to confirm that adenylate cyclase stimulation observed acutely was at least partially maintained for 48 h. However, no change in ACE levels was detected using forskolin alone and the stimulatory effect of IBMX was not modified by addition of forskolin. Isoproterenol slightly lowered ACE levels after chronic exposure when used alone and slightly impaired the stimulatory effect of IBMX when used in combination. Reproducible increases of ACE accumulation with IBMX were observed and this stimulator was included in all experiments as a positive control. In these experiments addition of IBMX to the cultures for 48 h was associated with markedly increased extracellular CAMP levels, presumably reflecting inhibition of phosphodiesterase activity in the medium. Cellular CAMP levels were not altered, as has been reported in other systems using theophylline (Kakiuchi et al., 1969). These results, taken together with out previous findings, suggest that although exogenous CAMP analogues can modestly increase ACE production from cultured endothelial cells (Lloyd et al., 1987) we are unable to demonstrate increased ACE production by stimulating adenylate cyclase in the cells using either forskolin or isoproterenol. The mechanism of action of IBMX in stimulating ACE in these cells is therefore unclear. The finding that IBMX stimulates ACE production and is associated with raised extracellular CAMP, fits our original hypothesis that CAMP could be a regulator of ACE production (Lloyd et al., 1987). The failure of isoproterenol or forskolin to stimulate ACE production could be due to their inability to sufficiently elevate CAMP levels in the cultured cells. However, this seems unlikely since acute stimulation of cellular CAMP by forskolin at 0.01 pM and above or by isoproterenol at 10 nM or higher, achieved greater CAMP levels than 1 mM IBMX used in the control incubations (Figs. 2 and 3). Similarly, forskolin administered chronically at doses of 1.0 and 10 PM, stimulated CAMP
109
to a greater degree than 100 PM IBMX (Fig. 5). Alternatively, it might be argued that cells treated with IBMX were already maximally stimulated. However, the effect of IBMX on ACE production was far from maximal at 100 PM (Fig. 5; Lloyd et al., 1987). Therefore, failure of further stimulation of ACE by forskolin in the presence of IBMX seems unlikely to be due to prior maximal stimulation of ACE by IBMX through a CAMP-mediated mechanism. Whilst it is still possible that differences in the pattern or extent and duration of elevation of CAMP levels by IBMX might explain it’s activity, it seems probable that IBMX stimulates ACE production via a mechanism independent of CAMP. Acknowledgements Supported by a grant from the National Health and Medical Research Council of Australia. We thank Miss. S. Younan for typing the manuscript. References Abdel-Hahm, MS., Von Holst, H., Meyerson, B., Sachs, C. and Anggard, E. (1980) J. Neurochem. 34, 1331-1333. Booyse, F.M., Sedlak, B.J. and Rafelson, Jr., M.E. (1975) Thromb. Diath. Haemorrh. 34, 825-839. Brotherton, A.F. and Hoke, J.C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 495-499. Brown, B.L., Ekins, R.P. and Albano, J.D. (1972) Adv. Cyclic Nucleotide Res. 2, 25-40. Caldwell, P.B., Seegal, B.C., Hsu, K.C., Das, M. and Soffer, R.L. (1976) Science 191, 1050-1051. Dembinska-Kiec, A., Rucker, W. and Schonhofer P.S. (1980) Naunyn-Schmied. Arch. Pharmacol. 311, 67-70. Dobson, P.R. and Brown, B.L. (1985) Methods Enzymol. 109, 293-298.
Erdos, E.G. (1977) Fed. Proc. 36,1760-1764. Friedland, J. and Silverstein, E. (1983) Cell. Mol. Biol. 29, 85-91. Friedland, J., Setton, C. and Silverstein, E. (1977) Science 197, 64-65. Fyhrquist, F., Forslund, T., Tikkanen, I. and Gronhagen-Riska, C. (1980) Eur. J. Pharmacol. 67 (4), 473-475. Fyhrquist, F., Hortling, L. and Gronhagen-Riska, C. (1982) J. Clin. Endocrinol. Metab. 55, 783-786. Fyhrquist, F., Gronhagen-Riska, C., Hortling, L., Forslund, T., Tikkanen, I. and Klockars, M. (1983) Clin. Exp. Hypertens. A5(7-8), 1319-1330. Gronhagen-Riska, C., Forslund, T., Hortland, L. and Fyhrquist, F. (1983) Acta Med. Stand. (Suppl. 1) 677, 101-104. Hartree, E.F. (1972) Anal. Biochem. 48, 422-427. Hayes, L.W., Goguen, C.A., Ching, S.F. and Slakey, L.L. (1978) Biochem. Biophys. Res. Commun. 82, 1147-1153. Hopkins, N.K. and Gorman, R.R. (1981) J. Clin. Invest. 67, 540-546. Johnson, A.R. and Erdos, E.G. (1977) J. Clin. Invest. 59, 684-694. Kakiuchi, S., Rall, T.W. and McIlwain, H. (1969) J. Neurothem. 16, 485. Karnushina, I.L., Spatz, M. and Bembry, J. (1982) Life Sci. 30, 849-858. Krulewitz, A.H., Baur, W.E. and Fanburg, B.L. (1984) Am. J. Physiol. 247, C163-C168. Larochelle, P., Genest, J., Kuchel, O., Boucher, R., Gutkowska, Y. and McKinstry, D. (1979) Can. Med. Assoc. J. 121, 209-316. Leitman, D.C., Fiscus, R.R. and Murad, F. (1986) J. Cell Physiol. 127 (2), 237-243. Lloyd, C.J., Cary, D.A. and Mendelsohn, F.A.O. (1987) Mol. Cell. Endocrinol. (in press). Makarski, J.S. (1981) In Vitro 17, 450-458. Mendelsohn, F.A.O. and Kachel, C. (1981) Clin. Exp. Pharmacol. Physiol. 8, 477-482. Mendelsohn, F.A.O., Lloyd, C.J., Kachel, C. and Funder, J.W. (1982) J, Clin. Invest. 70, 684-692. Mender&all, W. (1979) Introduction to Probability and Statistics, 5th edn. (Duxbury Press, Belmont, CA) p_ 323. Sattin, A. and Rall, T.W. (1970) Mol. Pharmacol. 6, 13-23. Steiner, A.L., Parker, C.W. and Kipnis, D.M. (1972) J. Biol. Chem. 247, 1106-1113.