Mesangium and Glomerular Functions Koide H, Endou H, Kurokawa K (eds): Cellular and Molecular Biology of the Kidney. Contrib Nephrol. Basel, Karger, 1991, vol 95, pp I-II
Effects of Endothelin on Cultured Human and Rat Glomerular Mesangial Cells 1 Michael Simonson·, Tomohiro Osanai·, Shianq Wann·, Elisabetta Baldi', Paolo Mene a, Yuichi Nakazato a, Mark Kestera,b, Christie Thomas', Michael Dunna,b Departments of"Medicine and bphysiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
Endothelins (ET) are a recently discovered family of 21-amino acid isopeptides found in at least four distinct isoforms: ET-l, ET-2, ET-3 and VIC (endothelin ~ or vasoactive intestinal contractor). These 21-amino acid peptides have extensive homology with sarafotoxin, the toxin from the venomous asp, Atractaspis engadensis. Endothelins, since their discovery by Yanagisawa et al. [I] in 1988, have provoked substantial scientific interest because of their potent actions as vasoconstrictor and mitogenic peptides. Endothelins, especially ET-I and ET-2, cause sustained hypertension and extensive renal vasoconstriction with concomitant reductions of renal blood flow and glomerular filtration rate. Endothelin has also been shown to stimulate mitogenesis in vascular smooth muscle cells, 3T3 fibroblasts and glomerular mesangial cells [2]. In this review, we will summarize our studies which have evaluated the biologic and biochemical effects of the ET isoforms, as well as sarafotoxin, on cultured rat and human mesangial cells.
Mitogenic Actions
1 We gratefully acknowledge Julie Wolfe, Jill Patterson, Guy McDermott and Edith Hanzmann for excellent technical assistance and Norma Minear for editorial assistance and for typing the manuscript. This work was supported by National Institutes of Health grants HL-22563, DK-41684 and HL-371 17.
Downloaded by: Université de Paris 193.51.85.197 - 1/20/2020 12:52:43 AM
We have tested the mitogenic actions ofET-l when added to quiescent (Go) mesangial cells [3]. Using 3H-thymidine uptake, we observed a dose-
Simonson et al.
2
dependent endothelin response with a mitogenic threshold at 0.1 nM and a 50% effective concentration of 1 nM with maximum stimulation at concentrations greater than 10 nM (fig. 1). Endothelin alone had little mitogenic effect but, when added with the comito gens such as 0.5% fetal bovine serum, mitogenesis was stimulated over 18-24 h by greater than 300%. Insulin also functioned as a cofactor to permit ET stimulation of mesangial proliferation. Cell counts confirmed the results of 3H-thymidine uptake. We also evaluated the protooncogene c-ios since many growth factors stimulate protooncogenes as a concomitant or necessary event in the mitogenic pathways. ET stimulated the expression of mRNA for c-ios at 30 min and the transcript was no longer detectable by 3 h [3]. Other investigators have also found that endothelin is a potent mitogen and induces c-ios cultured cells including 3T3 fibroblasts, vascular smooth muscle cells, and cultured glomerular mesangial cells [4-8].
Contraction We assessed the contractile actions of ET-1 using cultured rat glomerular mesangial cells grown on three-dimensional collagen gels [9]. ET-I at 0.1 11M contracted 48% of the cells as judged by a greater than 10% reduction of cross-sectional area of the cells measured by image analysis morphometry. Whereas I pM ET-1 had no effects, 1 nM contracted 21 % of the cells. The contractile action of ET-l could be mimicked by the calcium ionophore, ionomyocin, at I and 10 11M. Nifedipine, a voltage-gated calcium channel blocker, did not antagonize the ET-l-induced mesangial contraction. We also examined the effects of ET-l on the F actin microfilament bundles in cultured mesangial cells. In control cells, F action was assembled in long, linear microfilament bundles distributed evenly throughout the cytosol along the long axis of the cell. Whereas after ET-1, 0.1 11M, these bundles resembled thick stress fibers and assumed a diffuse, irregular mesh work, consistent with a motile action of ET. Ionomyocin, which increases intracellular calcium, also caused these complex rearrangements of the F actin microfilament bundles stained with rhodamine-phalloidin [9].
Peptide agonists of contraction and cellular proliferation often activate phospholipase C thereby hydrolyzing phosphatidylinositol bisphosphate to
Downloaded by: Université de Paris 193.51.85.197 - 1/20/2020 12:52:43 AM
Phospholipase C
Effects of Endothelin on Human and Rat Mesangial Cells
..
CI>
D_
ei=
:l~ ","-
ol'pose C
t PrOlein k ni a.e C activity
I
Translation
c-Ios mRNA~C.111$ protl in
-T~-- ~~
Nucllo,
Memb,ane
o ?
CRE
SRE CRE
t~n.
X
T~
-~~r=---r~Gene~x::::J1Gene '09u1oIO'y
Fig. 5. Short-term and long-term signal transduction pathways activated by endothelin isopeptides. Detailed discussion of these mechanisms can be found in the text [2].
Downloaded by: Université de Paris 193.51.85.197 - 1/20/2020 12:52:43 AM
element
Simonson et al.
10
but also stimulation of other protein kinases which phosphorylate key intermediates. Endothelin stimulates the transient appearance of proto oncogenes that might playa role in the induction of cellular proliferation.
2 Yanagisawa M, Kurihara H, Kimura S, Tombe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-415. 2 Simonson MS, Dunn MJ: Cellular signaling by peptides of the endothelin gene family. FASEB J 1990;4:2989-3000. 3 Simonson MS, Wann S, Men€: P, Dubyak GR, Kester M, Nakazato Y, Sedor JR, Dunn MJ: Endothelin stimulates phospholipase C, Na+/H+ exchange, c-fos expression, and mitogenesis in rat mesangial cells. J Clin Invest 1989;83:708-712. 4 Muldoon L, Rodland KD, Forsythe ML, Magun BE: Stimulation of phosphat idyl inositol hydrolysis, diacylglycerol release, and gene expression in response to endothelin, a potent new agonist for fibroblasts and smooth muscle cells. J Bioi Chern 1989;264:8529-8536. 5 Takuwa N, Takuwa Y, Yanagisawa M, Yamashita K, Masaki T: A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J Bioi Chern 1989;264: 7856-7861. 6 Badr KF, Murray JJ, Breyer MD, Takahashi K, Inagami T, Harris RC: Mesangial cell, glomerular and renal vascular responses to endothelin in the rat kidney. J Clin Invest 1989;83:336-342. 7 Komuro I, Kurihara H, Sugiyama T, Takahu F, Yazaki Y: Endothelin stimulates cfos and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett 1988;238:249-252. 8 Dubin D, Pratt RE, Cooke JP, Dzau VJ: Endothelin, a potent vasoconstrictor, is a vascular smooth muscle mitogen. J Vasc Med Bioi 1989;1:150-154. 9 Simonson MS, Dunn MJ: Endothelin-l stimulates contraction of rat glomerular mesangial cells and potentiates ~-adrenergic-mediated cAMP accumulation. J Clin Invest 1990;85:790-797. 10 Van Renterghem C, Vigne P, Barhanin J, Schmid-Alliana A, Felin C, Lazdunski M: Molecular mechanism of action ofthe vasoconstrictor peptide endothelin. Biochem Biophys Res Commun 1988; 157:977-985. II Resink TJ, Scott-Burden T, Buhler FR: Endothelin stimulates phospholipase C in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1988; 157:1360-1368. 12 Highsmith RF, Pang DC, Rappoport RM: Endothelial cell-derived vasoconstrictors: mechanisms of action of vascular smooth muscle. J Cardiovasc Pharmacol 1989; 13:S36-S44. 13 Marsden PA, Danthuluri NR, Brenner EM, Ballerman BJ, Brock TA: Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization. Biochem Biophys Res Commun 1989; 158:86-93. 14 Griendling KK, Tsuda T, Alexander RW: Endothelin stimulates diacylglycerol
Downloaded by: Université de Paris 193.51.85.197 - 1/20/2020 12:52:43 AM
References
Effects of Endothelin on Human and Rat Mesangial Cells
16 17
18 19
20
21
22
23
accumulation and activates protein kinase C in cultured vascular smooth muscle cells. J Bioi Chern 1989;264:8237-8240. Lee T-E, Chao T, Hu K-Q, King GL: Endothelin stimulates a sustained 1,2diacylglycerol increase and protein kinase C activation in bovine aortic smooth muscle cells. Biochem Biophys Res Commun 1989; 162:381-386. Simonson MS, Dunn MJ: Ca2+ signaling by distinct endothelin peptides in glomerular mesangial cells. Exp Cell Res 1991 ;in press. Simonson MS, Osanai T, Dunn MJ: Endothelin isopeptides evoke Ca 2+ signaling and oscillations of cytosolic free [Ca 2+] in human mesangial cells. Biochim Biophys Acta 1990; in press. Ambar I, K100g Y, Sokolovsky M: Solubilization of endothelin/sarafotoxin receptors in an active binding form. Eur J Pharmacol 1989; 170: 119-120. Masuda Y, Miyazaki H, Kondah M, Watanabe H, Yanagisawa M, Masaki T, Murakami K: Two different forms of endothelin receptors in rat lung. FEBS Lett 1989;257:208-210. De Nucci G, Thomas R, D'Orleans-J uste P, Antunes E, Walder C, Warner TD, Vane JR: Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc NatI Acad Sci USA 1988;85:9797-9800. Reynolds EE, Mok LL, Kurokawa S: Phorbol ester dissociates endothelin-stimulated phosphoinositide hydrolysis and arachidonic acid release in vascular smooth muscle cells. Biochem Biophys Res Commun 1989;160:868-873. Resink TJ, Scott-Burden T, Buhler FR: Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1989; 158:279-286. Sugiura M, Inagami T, Harem GMT, Johns JA: Endothelin action: inhibition by a protein kinase C inhibitor and involvement of phosphoinositols. Biochem Biophys Res Commun 1989;158:170-176.
Michael J. Dunn, MD, Department of Medicine, Division of Nephrology, Case Western Reserve University, 2074 Abington Road, Cleveland, OH 44106 (USA)
Downloaded by: Université de Paris 193.51.85.197 - 1/20/2020 12:52:43 AM
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11
Effects of Endothelin on Cultured Human and Rat Glomerular Mesangial Cells 1 Michael Simonson·, Tomohiro Osanai·, Shianq Wann·, Elisabetta Baldi', Paolo Mene a, Yuichi Nakazato a, Mark Kestera,b, Christie Thomas', Michael Dunna,b Departments of"Medicine and bphysiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
Endothelins (ET) are a recently discovered family of 21-amino acid isopeptides found in at least four distinct isoforms: ET-l, ET-2, ET-3 and VIC (endothelin ~ or vasoactive intestinal contractor). These 21-amino acid peptides have extensive homology with sarafotoxin, the toxin from the venomous asp, Atractaspis engadensis. Endothelins, since their discovery by Yanagisawa et al. [I] in 1988, have provoked substantial scientific interest because of their potent actions as vasoconstrictor and mitogenic peptides. Endothelins, especially ET-I and ET-2, cause sustained hypertension and extensive renal vasoconstriction with concomitant reductions of renal blood flow and glomerular filtration rate. Endothelin has also been shown to stimulate mitogenesis in vascular smooth muscle cells, 3T3 fibroblasts and glomerular mesangial cells [2]. In this review, we will summarize our studies which have evaluated the biologic and biochemical effects of the ET isoforms, as well as sarafotoxin, on cultured rat and human mesangial cells.
Mitogenic Actions
1 We gratefully acknowledge Julie Wolfe, Jill Patterson, Guy McDermott and Edith Hanzmann for excellent technical assistance and Norma Minear for editorial assistance and for typing the manuscript. This work was supported by National Institutes of Health grants HL-22563, DK-41684 and HL-371 17.
Downloaded by: Université de Paris 193.51.85.197 - 1/20/2020 12:52:43 AM
We have tested the mitogenic actions ofET-l when added to quiescent (Go) mesangial cells [3]. Using 3H-thymidine uptake, we observed a dose-
Simonson et al.
2
dependent endothelin response with a mitogenic threshold at 0.1 nM and a 50% effective concentration of 1 nM with maximum stimulation at concentrations greater than 10 nM (fig. 1). Endothelin alone had little mitogenic effect but, when added with the comito gens such as 0.5% fetal bovine serum, mitogenesis was stimulated over 18-24 h by greater than 300%. Insulin also functioned as a cofactor to permit ET stimulation of mesangial proliferation. Cell counts confirmed the results of 3H-thymidine uptake. We also evaluated the protooncogene c-ios since many growth factors stimulate protooncogenes as a concomitant or necessary event in the mitogenic pathways. ET stimulated the expression of mRNA for c-ios at 30 min and the transcript was no longer detectable by 3 h [3]. Other investigators have also found that endothelin is a potent mitogen and induces c-ios cultured cells including 3T3 fibroblasts, vascular smooth muscle cells, and cultured glomerular mesangial cells [4-8].
Contraction We assessed the contractile actions of ET-1 using cultured rat glomerular mesangial cells grown on three-dimensional collagen gels [9]. ET-I at 0.1 11M contracted 48% of the cells as judged by a greater than 10% reduction of cross-sectional area of the cells measured by image analysis morphometry. Whereas I pM ET-1 had no effects, 1 nM contracted 21 % of the cells. The contractile action of ET-l could be mimicked by the calcium ionophore, ionomyocin, at I and 10 11M. Nifedipine, a voltage-gated calcium channel blocker, did not antagonize the ET-l-induced mesangial contraction. We also examined the effects of ET-l on the F actin microfilament bundles in cultured mesangial cells. In control cells, F action was assembled in long, linear microfilament bundles distributed evenly throughout the cytosol along the long axis of the cell. Whereas after ET-1, 0.1 11M, these bundles resembled thick stress fibers and assumed a diffuse, irregular mesh work, consistent with a motile action of ET. Ionomyocin, which increases intracellular calcium, also caused these complex rearrangements of the F actin microfilament bundles stained with rhodamine-phalloidin [9].
Peptide agonists of contraction and cellular proliferation often activate phospholipase C thereby hydrolyzing phosphatidylinositol bisphosphate to
Downloaded by: Université de Paris 193.51.85.197 - 1/20/2020 12:52:43 AM
Phospholipase C
Effects of Endothelin on Human and Rat Mesangial Cells
..
CI>
D_
ei=
:l~ ","-
ol'pose C
t PrOlein k ni a.e C activity
I
Translation
c-Ios mRNA~C.111$ protl in
-T~-- ~~
Nucllo,
Memb,ane
o ?
CRE
SRE CRE
t~n.
X
T~
-~~r=---r~Gene~x::::J1Gene '09u1oIO'y
Fig. 5. Short-term and long-term signal transduction pathways activated by endothelin isopeptides. Detailed discussion of these mechanisms can be found in the text [2].
Downloaded by: Université de Paris 193.51.85.197 - 1/20/2020 12:52:43 AM
element
Simonson et al.
10
but also stimulation of other protein kinases which phosphorylate key intermediates. Endothelin stimulates the transient appearance of proto oncogenes that might playa role in the induction of cellular proliferation.
2 Yanagisawa M, Kurihara H, Kimura S, Tombe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-415. 2 Simonson MS, Dunn MJ: Cellular signaling by peptides of the endothelin gene family. FASEB J 1990;4:2989-3000. 3 Simonson MS, Wann S, Men€: P, Dubyak GR, Kester M, Nakazato Y, Sedor JR, Dunn MJ: Endothelin stimulates phospholipase C, Na+/H+ exchange, c-fos expression, and mitogenesis in rat mesangial cells. J Clin Invest 1989;83:708-712. 4 Muldoon L, Rodland KD, Forsythe ML, Magun BE: Stimulation of phosphat idyl inositol hydrolysis, diacylglycerol release, and gene expression in response to endothelin, a potent new agonist for fibroblasts and smooth muscle cells. J Bioi Chern 1989;264:8529-8536. 5 Takuwa N, Takuwa Y, Yanagisawa M, Yamashita K, Masaki T: A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts. J Bioi Chern 1989;264: 7856-7861. 6 Badr KF, Murray JJ, Breyer MD, Takahashi K, Inagami T, Harris RC: Mesangial cell, glomerular and renal vascular responses to endothelin in the rat kidney. J Clin Invest 1989;83:336-342. 7 Komuro I, Kurihara H, Sugiyama T, Takahu F, Yazaki Y: Endothelin stimulates cfos and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett 1988;238:249-252. 8 Dubin D, Pratt RE, Cooke JP, Dzau VJ: Endothelin, a potent vasoconstrictor, is a vascular smooth muscle mitogen. J Vasc Med Bioi 1989;1:150-154. 9 Simonson MS, Dunn MJ: Endothelin-l stimulates contraction of rat glomerular mesangial cells and potentiates ~-adrenergic-mediated cAMP accumulation. J Clin Invest 1990;85:790-797. 10 Van Renterghem C, Vigne P, Barhanin J, Schmid-Alliana A, Felin C, Lazdunski M: Molecular mechanism of action ofthe vasoconstrictor peptide endothelin. Biochem Biophys Res Commun 1988; 157:977-985. II Resink TJ, Scott-Burden T, Buhler FR: Endothelin stimulates phospholipase C in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1988; 157:1360-1368. 12 Highsmith RF, Pang DC, Rappoport RM: Endothelial cell-derived vasoconstrictors: mechanisms of action of vascular smooth muscle. J Cardiovasc Pharmacol 1989; 13:S36-S44. 13 Marsden PA, Danthuluri NR, Brenner EM, Ballerman BJ, Brock TA: Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization. Biochem Biophys Res Commun 1989; 158:86-93. 14 Griendling KK, Tsuda T, Alexander RW: Endothelin stimulates diacylglycerol
Downloaded by: Université de Paris 193.51.85.197 - 1/20/2020 12:52:43 AM
References
Effects of Endothelin on Human and Rat Mesangial Cells
16 17
18 19
20
21
22
23
accumulation and activates protein kinase C in cultured vascular smooth muscle cells. J Bioi Chern 1989;264:8237-8240. Lee T-E, Chao T, Hu K-Q, King GL: Endothelin stimulates a sustained 1,2diacylglycerol increase and protein kinase C activation in bovine aortic smooth muscle cells. Biochem Biophys Res Commun 1989; 162:381-386. Simonson MS, Dunn MJ: Ca2+ signaling by distinct endothelin peptides in glomerular mesangial cells. Exp Cell Res 1991 ;in press. Simonson MS, Osanai T, Dunn MJ: Endothelin isopeptides evoke Ca 2+ signaling and oscillations of cytosolic free [Ca 2+] in human mesangial cells. Biochim Biophys Acta 1990; in press. Ambar I, K100g Y, Sokolovsky M: Solubilization of endothelin/sarafotoxin receptors in an active binding form. Eur J Pharmacol 1989; 170: 119-120. Masuda Y, Miyazaki H, Kondah M, Watanabe H, Yanagisawa M, Masaki T, Murakami K: Two different forms of endothelin receptors in rat lung. FEBS Lett 1989;257:208-210. De Nucci G, Thomas R, D'Orleans-J uste P, Antunes E, Walder C, Warner TD, Vane JR: Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc NatI Acad Sci USA 1988;85:9797-9800. Reynolds EE, Mok LL, Kurokawa S: Phorbol ester dissociates endothelin-stimulated phosphoinositide hydrolysis and arachidonic acid release in vascular smooth muscle cells. Biochem Biophys Res Commun 1989;160:868-873. Resink TJ, Scott-Burden T, Buhler FR: Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1989; 158:279-286. Sugiura M, Inagami T, Harem GMT, Johns JA: Endothelin action: inhibition by a protein kinase C inhibitor and involvement of phosphoinositols. Biochem Biophys Res Commun 1989;158:170-176.
Michael J. Dunn, MD, Department of Medicine, Division of Nephrology, Case Western Reserve University, 2074 Abington Road, Cleveland, OH 44106 (USA)
Downloaded by: Université de Paris 193.51.85.197 - 1/20/2020 12:52:43 AM
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