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Stobadine is a potent modulator of endogenous endothelin-1 in human fibroblasts
Life Sciences, Vol. 65, Nos. 18/19, pp. 1939-1941, 1999 Copy&h1 0 1999 Elswier Science Inc. Printed in the USA. All rights reserved OC24-3205/99/bee front matter
ELSEVIER
STOBADINE
PII SOO24-3205(99)00452-X
IS A POTENT MODULATOR OF ENDOGENOUS HUMAN FIBROBLASTS JanDrimal
ENDOTHELIN-1
IN
(‘), Vladimir Patoprsty (*), Vladimir Kovacik (*)
Slovak Academy of Sciences (‘) Institute of Experimental Pharmacology, (*)Institute of Chemistry, Dubravska 9, 842 16 Bratislava , Slovak Republic
Summary Binding of endothelin (ET) peptides to their respective receptors with resulting proliferation of vascular smooth muscle has been implicated in the pathogenesis of arterial hypertension and atherosclerosis. Recently it was hypothetized that endothelin-1 (ET-l) bound to its two membrane receptors (ETA and ETB ) continues to activate signal transducing proteins in cells. It was also shown that pyridoindole stobadine stabilized lysosomal membranes in myocardium in early ischemia. Therefore we decided to study the effects of stobadine on specific, subtype-selective binding and subsequent degradation of human synthetic [125II-ET-1 in human fibroblasts (HF). Our results indicate that stobadine significantly potentiated ET-l binding by reductive ETB selective degradation of ET-1 in HF. Hence, it is very plausible that stobadine may modulate endogenous endothelin and its intracellular mitogenic and chemotactic factors, principally by affecting two presumably related processes, participating in the proliferative and mitogenie response, (1) potentiation of signal trasduction from ETA receptors, and (2) subtype-ETa selective intracellular processing Key Words: endothelin-1, ET-,, receptors, ET-, receptors, ET-I degradation, ET antagonists, l&and binding, stobadine, chloroquine
An excess of endogenous peptide endothelin-1 (ET-l), potent vasoconstrictor and mitogen was recently implicated to play a role in coronary atherosclerosis (1,2). The extraction of proteolytically derived ET follows its binding to two specific receptors (ETA and ET& present in vasculature and in the myocardium. The subsequent ET processing allows degradation of the ET- 1 to be carried out in cells, conceivably in lysosomes (3). Recently it was hypothetized that ET-1 bound to receptor may continue in activating signal transducing proteins in the cells (4). Furthermore, we found that the pyridoindole stobadine stabilized lysosomal membranes in acute ischemic injury in myocardium (5). In addition to its wide spectrum of stimulatory effects in vascular smooth muscle (6), ET-l causes release of endogenous substances, inclusive its own release and gene expression (see reviews 7,8,9). The present study was therefore undertaken to characterize in vitro the ET-l mitogenicity, and the effects of stobadine on subtype-specific binding and degradation of ET- 1 in human fibroblasts.
1940
Vol. 65, Nos. 18/19, 1999
Stobadine as a Modulator of ET-I
Methods Approved by the local Ethics Committee, experiments were performed with human fibroblasts (HF), (USOL ,Prague) maintained in Dulbeco’s modified Eagle’s medium suplemented with fetal bovine serum, streptomycin, and penicillin at 37 “C in 5% CO2 in air. Mitogenic potential of ET-l was measured by incorporation of [3H]-thymidine in HF (an approximately 5 x lo5 cells/well). After ligand exposure, HF were washed, dried on Whatman filters and then counted in Scintillation spectrometer ( LKB Wallace). Whole cell binding assays were performed on HF in HEPESbuffered physiological salt solution ( in mmol/l): NaCl 135, MgC12 1.O, KHzPO 4 0.44, NaHPO,, 0.34, NaHC03 2.6, HEPES 20.0, glucose 5.56, pH = 7.4. The ligand binding procedures, the whole cell preparations as well as the myocardial membranes were described elsewhere (6,10, 11). Protein concentrations were measured by the method of Bradford (14). Chemicals used were: Endothelin-I human, synthetic (Sigma), BQ-3020, N-Acetyl-[Ala”~” J-ET-l, BQ788, PD15 1242 all R.B.I);(3-[‘251] Iodotyrosyl) ET-1,[‘251]-PDl 5 1242, (all Amersham), [3H]-Thymidine (DuPont). Data are reported as means + SEM for at least six experiments (measurements in triplicate). Data were analyzed using Sigma Stat, and Ligand software packages. Results The effects of specific binding of human, synthetic [125I]ET-1, and ETa selective agonist [125I]BQ3020 were analyzed in six groups of experiments. For these studies we used 12 increasing concentrations of radioligands (usually from 0.5 to 3.9 mnol/l) and preincubation with three different peptide-type ET antagonists (mostly in 1 and 10 nmol/l), i.e. ETA selective PD15 1242, the ET B selective BQ-788 and nonselective PD142893. The control maximal values of specific binding for [Iz5 I] ET-l were 430 5 14 f?nol/ mg of protein and affinity in low nanomolar range (0.82 k 0.01 nmol/l). After preincubation of HF with ETA antagonist PDI 5 1242 specific binding remained unchanged (Fig. 1). Preincubation with the ETB selective BQ788 significantly increased ET- 1 specific binding, suggesting a significant role of ETa receptors in ET- 1 degradation in HF.
C
ETAx
Characteristics of [*25IlET(stripped) as % of Control ) in receptor antagonist (ETA~ ), lysosomal enzyme inhibitors: IOpmol/l). * Significant increase
ETBx
mu
ST0
Fig. 1. specific binding (Density, (solid) and Affinity human 1 fibroblasts after preincubation of ETA ETB selective antagonist(ET&,( lOnmol/l)and stobadine(ST0) and chloroquine(CQ,),(both in specific binding (p < 0.05).
The lysosomal enzyme inhibitors chloroquine and stobadine, (in concentrations 5 x lO’mol/l and higher ), increased significantly the density of ET-l binding sites specifically radiolabeled on HF. In control HPLC experiments [125IlETincubated with HF was degraded primarily to [‘25 I]-
Vol. 65, Nos. 18119,1999
Stobadine as a Modulator of ET-l
1941
tyrosine (mostly after 3.0 hours ) and the remaining radioactivity was detected in 4 or 5 small peaks. HF were incubated with buffer for 1 h. and the medium was then collected and [125IlETwas added in concentration 10 nmol/l. After 1 h. of incubation there was no evidence of any fiagmentation of [l*’ I]ET- 1. In and effort to explain the mechanism of stobadine’s induced increase of Bmax in ET-l specific binding experiments, we analyzed the effect of ET-l preincubation on the mitogenic potential of HF. The mitogenicicty of ET-l was studied at 1 and 10 nmolar concentrations both, in the absence and presence of the ETA selective antagonist PD-15 1242. ET-l significantly increased incorporation of i3 Hlthyrnidme, reacting at 10 nmovl of ET-l, and PD 15 1242 significantly and concentration-dependently reduced this increase. Discussion The key findings of our study are that in human fibroblasts and in porcine myocardial membranes ET* selective downregulation and clearing predominate, and that subtype ETA induced mitogenicity and ETB selective degradation of ET-1 are important components participating on aberrant response of ET-l in cells. This study is the first to provide evidence that lysosomal enzyme inhibitor stobadine interfere with ET-1 induced ETB receptor downregulation. Both [lz5 II-ET-1 and [lz5 1]BQ3020, were bound with high aflinity to ET receptors in HF and in myocardial membranes, with affinities similar to those reported in previous studies. The specificity of the ETB receptor agonist BQ3020 for ETB receptor was shown to be more than 4000 times higher than that of ET-1 (13). The dissociation of bound ET-l is very slow, and moreover,in the group with ETB agonist BQ3020 in our experiments a low affinity was found at ET-l binding sites (nu-merically higher values of Ko = 166 + 42 %). These findings suggest that the nonselective ET-1 may have more than one binding site within the receptor binding pocket, and/or the radioligands recognized a site (or sites) similar but not identical to the classical ET-l site. Furthermore, since [125I]BQ3020 recognized only a fraction of the ET-l binding sites and the majority of [125I]-BQ3020 binding sites was downregulated after ET-l preincubation in the present study, our fin-dings demonstrated subtype ETB selective downregulation in cells and in myocardial membranes. in conclusion, our data emphasize the pathophysiological significance of ETB receptor downregulation after excess of ET-l. This will produce ETA predominance with resultant activation of proliferation and mitogenesis in cells. The excess of ET-l, the ETA predominance and mitogenesis are characteristic features of early atherosclerosis. The stobadine may modulate endogenous ET- 1 by affecting subtype-B selective processing. Acknowledgement This work was suported
by Ministry of Education SR (Gr. 2005,4025,5305). References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13.
A. LERMAN, S.H. KUB0,L.K. TSCHUMPERLIN, J.Am.Coll.Cardiol. 20 848-853 (1992) O.A. SALOMONE, P.M. ELLIOTT, R.CALINO, J.Am.Coll.Cardiol. 28 14-19 (1996) B.M.JACOT-GUILLAMOND, J.P.MAIRE, Biochem.Int. 27 755-761 M.CHUN, U.K.LIYANAGE,H.F.LODISH, J.Biol. Chem.270 1085-1094 (1995) M.KETTNER, O.ONDREJICKA, T.MACICKOVA,J.DRIMAL, Cs.Physiol.30 46-7 (1981) J.DRIMAL+, V.KOPRDA, Physiol.Res. 45 5 l-58 (1996) HKANAIDE, Gen. Pharmac.27 559-563 (1996) L. BENATI, MSFABRINI, CPATRONO, AnnN.Y.Acad.Sci. 714 109-121 (1994) G.A.GRAY, D.J.WEBB, Pharmacol Ther. 72 109-148 (1996) J.DRIMAL, Gen. PhysioLBiophys. 8 341-350 (1989) J.DRIMAL, Gen Physiol. Biophys. 11 555-565 (1992) M.BRADFORD, Annal Biochem. 72 248-254 (1976) I.T. SAEKI,M.IHARA,T.FUKURODA, Biochem. Biophys.Res.Comm.179 286-292(1991)
ELSEVIER
STOBADINE
PII SOO24-3205(99)00452-X
IS A POTENT MODULATOR OF ENDOGENOUS HUMAN FIBROBLASTS JanDrimal
ENDOTHELIN-1
IN
(‘), Vladimir Patoprsty (*), Vladimir Kovacik (*)
Slovak Academy of Sciences (‘) Institute of Experimental Pharmacology, (*)Institute of Chemistry, Dubravska 9, 842 16 Bratislava , Slovak Republic
Summary Binding of endothelin (ET) peptides to their respective receptors with resulting proliferation of vascular smooth muscle has been implicated in the pathogenesis of arterial hypertension and atherosclerosis. Recently it was hypothetized that endothelin-1 (ET-l) bound to its two membrane receptors (ETA and ETB ) continues to activate signal transducing proteins in cells. It was also shown that pyridoindole stobadine stabilized lysosomal membranes in myocardium in early ischemia. Therefore we decided to study the effects of stobadine on specific, subtype-selective binding and subsequent degradation of human synthetic [125II-ET-1 in human fibroblasts (HF). Our results indicate that stobadine significantly potentiated ET-l binding by reductive ETB selective degradation of ET-1 in HF. Hence, it is very plausible that stobadine may modulate endogenous endothelin and its intracellular mitogenic and chemotactic factors, principally by affecting two presumably related processes, participating in the proliferative and mitogenie response, (1) potentiation of signal trasduction from ETA receptors, and (2) subtype-ETa selective intracellular processing Key Words: endothelin-1, ET-,, receptors, ET-, receptors, ET-I degradation, ET antagonists, l&and binding, stobadine, chloroquine
An excess of endogenous peptide endothelin-1 (ET-l), potent vasoconstrictor and mitogen was recently implicated to play a role in coronary atherosclerosis (1,2). The extraction of proteolytically derived ET follows its binding to two specific receptors (ETA and ET& present in vasculature and in the myocardium. The subsequent ET processing allows degradation of the ET- 1 to be carried out in cells, conceivably in lysosomes (3). Recently it was hypothetized that ET-1 bound to receptor may continue in activating signal transducing proteins in the cells (4). Furthermore, we found that the pyridoindole stobadine stabilized lysosomal membranes in acute ischemic injury in myocardium (5). In addition to its wide spectrum of stimulatory effects in vascular smooth muscle (6), ET-l causes release of endogenous substances, inclusive its own release and gene expression (see reviews 7,8,9). The present study was therefore undertaken to characterize in vitro the ET-l mitogenicity, and the effects of stobadine on subtype-specific binding and degradation of ET- 1 in human fibroblasts.
1940
Vol. 65, Nos. 18/19, 1999
Stobadine as a Modulator of ET-I
Methods Approved by the local Ethics Committee, experiments were performed with human fibroblasts (HF), (USOL ,Prague) maintained in Dulbeco’s modified Eagle’s medium suplemented with fetal bovine serum, streptomycin, and penicillin at 37 “C in 5% CO2 in air. Mitogenic potential of ET-l was measured by incorporation of [3H]-thymidine in HF (an approximately 5 x lo5 cells/well). After ligand exposure, HF were washed, dried on Whatman filters and then counted in Scintillation spectrometer ( LKB Wallace). Whole cell binding assays were performed on HF in HEPESbuffered physiological salt solution ( in mmol/l): NaCl 135, MgC12 1.O, KHzPO 4 0.44, NaHPO,, 0.34, NaHC03 2.6, HEPES 20.0, glucose 5.56, pH = 7.4. The ligand binding procedures, the whole cell preparations as well as the myocardial membranes were described elsewhere (6,10, 11). Protein concentrations were measured by the method of Bradford (14). Chemicals used were: Endothelin-I human, synthetic (Sigma), BQ-3020, N-Acetyl-[Ala”~” J-ET-l, BQ788, PD15 1242 all R.B.I);(3-[‘251] Iodotyrosyl) ET-1,[‘251]-PDl 5 1242, (all Amersham), [3H]-Thymidine (DuPont). Data are reported as means + SEM for at least six experiments (measurements in triplicate). Data were analyzed using Sigma Stat, and Ligand software packages. Results The effects of specific binding of human, synthetic [125I]ET-1, and ETa selective agonist [125I]BQ3020 were analyzed in six groups of experiments. For these studies we used 12 increasing concentrations of radioligands (usually from 0.5 to 3.9 mnol/l) and preincubation with three different peptide-type ET antagonists (mostly in 1 and 10 nmol/l), i.e. ETA selective PD15 1242, the ET B selective BQ-788 and nonselective PD142893. The control maximal values of specific binding for [Iz5 I] ET-l were 430 5 14 f?nol/ mg of protein and affinity in low nanomolar range (0.82 k 0.01 nmol/l). After preincubation of HF with ETA antagonist PDI 5 1242 specific binding remained unchanged (Fig. 1). Preincubation with the ETB selective BQ788 significantly increased ET- 1 specific binding, suggesting a significant role of ETa receptors in ET- 1 degradation in HF.
C
ETAx
Characteristics of [*25IlET(stripped) as % of Control ) in receptor antagonist (ETA~ ), lysosomal enzyme inhibitors: IOpmol/l). * Significant increase
ETBx
mu
ST0
Fig. 1. specific binding (Density, (solid) and Affinity human 1 fibroblasts after preincubation of ETA ETB selective antagonist(ET&,( lOnmol/l)and stobadine(ST0) and chloroquine(CQ,),(both in specific binding (p < 0.05).
The lysosomal enzyme inhibitors chloroquine and stobadine, (in concentrations 5 x lO’mol/l and higher ), increased significantly the density of ET-l binding sites specifically radiolabeled on HF. In control HPLC experiments [125IlETincubated with HF was degraded primarily to [‘25 I]-
Vol. 65, Nos. 18119,1999
Stobadine as a Modulator of ET-l
1941
tyrosine (mostly after 3.0 hours ) and the remaining radioactivity was detected in 4 or 5 small peaks. HF were incubated with buffer for 1 h. and the medium was then collected and [125IlETwas added in concentration 10 nmol/l. After 1 h. of incubation there was no evidence of any fiagmentation of [l*’ I]ET- 1. In and effort to explain the mechanism of stobadine’s induced increase of Bmax in ET-l specific binding experiments, we analyzed the effect of ET-l preincubation on the mitogenic potential of HF. The mitogenicicty of ET-l was studied at 1 and 10 nmolar concentrations both, in the absence and presence of the ETA selective antagonist PD-15 1242. ET-l significantly increased incorporation of i3 Hlthyrnidme, reacting at 10 nmovl of ET-l, and PD 15 1242 significantly and concentration-dependently reduced this increase. Discussion The key findings of our study are that in human fibroblasts and in porcine myocardial membranes ET* selective downregulation and clearing predominate, and that subtype ETA induced mitogenicity and ETB selective degradation of ET-1 are important components participating on aberrant response of ET-l in cells. This study is the first to provide evidence that lysosomal enzyme inhibitor stobadine interfere with ET-1 induced ETB receptor downregulation. Both [lz5 II-ET-1 and [lz5 1]BQ3020, were bound with high aflinity to ET receptors in HF and in myocardial membranes, with affinities similar to those reported in previous studies. The specificity of the ETB receptor agonist BQ3020 for ETB receptor was shown to be more than 4000 times higher than that of ET-1 (13). The dissociation of bound ET-l is very slow, and moreover,in the group with ETB agonist BQ3020 in our experiments a low affinity was found at ET-l binding sites (nu-merically higher values of Ko = 166 + 42 %). These findings suggest that the nonselective ET-1 may have more than one binding site within the receptor binding pocket, and/or the radioligands recognized a site (or sites) similar but not identical to the classical ET-l site. Furthermore, since [125I]BQ3020 recognized only a fraction of the ET-l binding sites and the majority of [125I]-BQ3020 binding sites was downregulated after ET-l preincubation in the present study, our fin-dings demonstrated subtype ETB selective downregulation in cells and in myocardial membranes. in conclusion, our data emphasize the pathophysiological significance of ETB receptor downregulation after excess of ET-l. This will produce ETA predominance with resultant activation of proliferation and mitogenesis in cells. The excess of ET-l, the ETA predominance and mitogenesis are characteristic features of early atherosclerosis. The stobadine may modulate endogenous ET- 1 by affecting subtype-B selective processing. Acknowledgement This work was suported
by Ministry of Education SR (Gr. 2005,4025,5305). References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13.
A. LERMAN, S.H. KUB0,L.K. TSCHUMPERLIN, J.Am.Coll.Cardiol. 20 848-853 (1992) O.A. SALOMONE, P.M. ELLIOTT, R.CALINO, J.Am.Coll.Cardiol. 28 14-19 (1996) B.M.JACOT-GUILLAMOND, J.P.MAIRE, Biochem.Int. 27 755-761 M.CHUN, U.K.LIYANAGE,H.F.LODISH, J.Biol. Chem.270 1085-1094 (1995) M.KETTNER, O.ONDREJICKA, T.MACICKOVA,J.DRIMAL, Cs.Physiol.30 46-7 (1981) J.DRIMAL+, V.KOPRDA, Physiol.Res. 45 5 l-58 (1996) HKANAIDE, Gen. Pharmac.27 559-563 (1996) L. BENATI, MSFABRINI, CPATRONO, AnnN.Y.Acad.Sci. 714 109-121 (1994) G.A.GRAY, D.J.WEBB, Pharmacol Ther. 72 109-148 (1996) J.DRIMAL, Gen. PhysioLBiophys. 8 341-350 (1989) J.DRIMAL, Gen Physiol. Biophys. 11 555-565 (1992) M.BRADFORD, Annal Biochem. 72 248-254 (1976) I.T. SAEKI,M.IHARA,T.FUKURODA, Biochem. Biophys.Res.Comm.179 286-292(1991)